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Blood Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Blood and links to relevant pages within the course.

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Advances in Noninvasive Prenatal Testing For Down Syndrome and other Trisomies
Screening Tests, continued: Maternal Serum Screening

Maternal serum screening tests may be performed during the first and second trimester of pregnancy. These serum tests involve the measurement of specific biomarkers in the maternal blood. These five biomarkers are typically used to assess the risk for Down syndrome: Maternal Serum Alpha-fetoprotein (MS-AFP) AFP is a fetal protein that is initially produced in the fetal yolk sac and liver; by the end of the first trimester of pregnancy, most if not all of the AFP is produced by the fetal liver. The concentration of AFP peaks in fetal serum at 10-13 weeks gestation. The fetal AFP normally diffuses across the placental barrier and into the maternal circulation so that MS-AFP rises throughout pregnancy to about 250 ng/mL at 32 weeks gestation. Lower MS-AFP values may be associated with increased risk for trisomy 21 (Down syndrome) or trisomy 18 (Edwards syndrome).Unconjugated estriol (uE3-estriol) Estriol is a female sex hormone that increases during pregnancy. It is produced by the placenta and passes into the maternal bloodstream during pregnancy. Its roles during pregnancy may be linked to the proper functioning of the uterus, softening of the cervix, and assistance in the lactation process. A biologically active form of estriol called uE3-estriol increases in the maternal circulation during pregnancy by the seventh to ninth weeks of gestation and continues to increase throughout pregnancy. uE3-estriol levels in maternal serum is typically decreased in the Down syndrome pregnancy. Human chorionic gonadotropin, total or free beta subunit (beta-hCG) HCG is a glycoprotein hormone produced by the placenta during pregnancy. It is present in blood and urine around 7-13 days following fertilization of the ovum. HCG has two subunit chains, alpha and beta. The beta subunit confers its specificity. A specific smaller part of the hormone, called free beta hCG may be used as a screening test during the first trimester of pregnancy. An increase in maternal serum free beta hCG (ie, greater concentration than in other pregnancies) may indicate an increased risk for Down syndrome. Total beta-hCG is tested as part of a triple or quad screen during the second trimester of pregnancy. An increase in total beta hCG in the maternal serum is also associated with increased risk of Down syndrome.Dimeric inhibin A (DIA) Inhibins are a family of glycoproteins mainly secreted by the ovaries and testicles. The beta subunits of the inhibins exist in two forms, the A and B forms. DIA is secreted by the ovaries and is designed to inhibit the production of the hormone FSH by the pituitary gland. The level of DIA is increased in the blood of mothers of fetuses with Down syndromePregnancy Associated Plasma Protein A (PAPP-A) PAPP-A is produced by the covering of the newly fertilized egg. It is thought to be involved in local proliferative processes such as wound healing and bone remodeling. Unexplained low levels of PAPP-A in the maternal serum during pregnancy may indicate increased chance for intrauterine growth restrictions, premature delivery, preeclampsia, and stillbirth. In the first trimester, low levels of this protein are seen in Down syndrome pregnancies.In the first trimester, serum screening is typically done in combination with an ultrasound to screen for nuchal translucency. Serum screening in the first trimester usually involves the measurement of two biomarkers in the maternal serum, free beta-hCG and PAPP-A. Combining the results of these two biomarkers with an ultrasound improves the screening process. In the second trimester during weeks 16-18, maternal serum assays for 3-4 levels of biomarkers are typically performed. The screening for these biomarkers has been established as a triple or quadruple (quad) screen, since the benefit of the screening lies in the combined use of the three to four biomarkers. The biomarkers used in the screening process may include MS-AFP, uE3-estriol, total beta HCG, and DIA. Increased serum levels of MS-hCG and DIA combined with decreased levels of UE3-estriol and MS-AFP suggests an increased risk of Down syndrome. Maternal age, family history, weight, race, and diabetic status are also used to determine a numeric risk for Down syndrome. It is important to understand that for women who have positive triple or quad screening results, only a very small number of them have babies who actually have a chromosomal abnormality.

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Directed DNA Analysis

Directed DNA analysis involves selective sequencing of relevant chromosomes. In directed DNA analysis, specific cfDNA fragments from maternal blood are used for selective sequencing. Digital analysis of selected regions (DANSR), a recently developed technique, uses directed DNA analysis and digitally analyzes sequencing results from targeted, selected genomic regions on particular chromosomes. This technique is touted as being potentially more efficient than MPSS because it uses fewer genetic fragments.

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Commercially-Available cfDNA Prenatal Tests for Aneuploidy

Currently, there are three major commercially-available cfDNA NIPTs for detection of aneuploidy:MaterniT21™Plus Test MaterniT21 PLUS test, developed and validated by Sequenom CMM, is a laboratory-developed test (LDT) that analyzes circulating cell-free DNA extracted from a maternal blood sample. This test is offered by Sequenom Center for Molecular Medicine and utilizes the MPSS technology to identify increased numbers of chromosomes. In the test, circulating cell-free DNA is purified from maternal plasma and the DNA is analyzed for chromosomal material and converted into a genomic DNA library for the determination of chromosome 21, 18, 13 and Y representation based on massively parallel DNA sequencing. The MaterniT21 Plus test has a reported performance characteristics of 99.1% sensitivity and 99.9% specificity for the detection of Down syndrome trisomy. Test results are reported as "negative" or "positive" for trisomies.Verifi™ Prenatal Test This test, offered by Verinata Health Inc., also utilizes MPSS technology. Verifi™ Prenatal Test uses a normalized chromosome value (NCV) calculation for each chromosome tested. This NCV calculation removes variation within and between sequencing to optimize the test precision. Test results are reported as “no aneuploidy detected”, “aneuploidy suspected" for borderline cases, and “aneuploidy detected” for the respective chromosome tested. The test claims to have a >99.9% sensitivity and 99.8% specificity for detection of Down syndrome. Harmony Prenatal Test™ This test is offered by Ariosa Diagnostics and utilizes directed DNA analysis sequencing of specific cell-free DNA (cfDNA) fragments using digital analysis of selected regions (DANSR). It is a laboratory-developed test that analyzes cfDNA in maternal blood. As with other cfDNA tests, the Harmony prenatal test is best performed anytime after the tenthweek of pregnancy. Using the DANSR methodology, the Harmony Prenatal Test™ analyzes sequencing results of selected, targeted genomic regions on chromosome 21 (for Down syndrome assessment). The test results will yield an assessment of the risk for trisomy 21. Using a specific algorithm, the test results are reported out as an individualized risk for each trisomy. The test claims to have a >99% sensitivity and 99% specificity for Down syndrome detection.All of the aforementioned cfDNA tests are considered laboratory-developed tests and are not FDA-approved. Moreover, it must be stressed that at this time the cfDNA tests are not considered diagnostic and a “low-risk” test result does not suggest an unaffected pregnancy while a “high-risk” test result still requires an invasive confirmatory test.In addition, all cfDNA tests are typically intended for use in women 18 years or older with a singleton pregnancy at a minimum of 10 weeks gestation and who have been determined by their physician to be at risk for fetal trisomy. Such risks include advanced maternal age, positive results of prenatal screening tests (serum and/or NT ultrasound), the presence of ultrasound soft or hard markers, previous family history of genetic disorders, or a previous affected pregnancy for fetal aneuploidy.It is important to remember that at present, invasive prenatal diagnostic tests, such as CVS and amniocentesis, should be considered the most accurate and comprehensive way to assess fetal abnormalities.

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Alpha Thalassemia
Case History

A 29-year-old female of Philippine descent was seen by her physician for fatigue. The patient states that a relative told her that their family has a long history of anemia.She presented with sclera icterus and her spleen was palpable. Routine blood work was initially ordered.

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Alpha Thalassemia Major

In alpha thalassemia major, anemia is actually fatal. Red blood cell (RBC) count is increased while hemoglobin is severely decreased. Both the MCV and MCHC are decreased. Red cell distribution width (RDW) is increased. RBC morphology shows slight hypochromic microcytosis with codocytes, schizocytes, nucleated RBCs. Reticulocytes are increased.Hemoglobin electrophoresis demonstrates abnormal pattern on cord blood: Hb A - absentHb Bart's - 80-90%Hb Portland - 0-20%Note: Bone marrow demonstrates marked erythroid hyperplasia.

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Normal Hemoglobin Electrophoresis

This is an example of an electrophoresis on normal blood.

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Antibody Detection and Identification (retired 12/6/2013)
Naturally occurring antibodies found in the ABO blood group system may be due to exposure to which of the following?View Page
Course Introduction

Antibody screening and antibody identification are critical components in blood bank testing. Clinically significant antibodies must be identified so that appropriate blood products are selected for transfusion and the risk of adverse reaction is minimized. Clinically significant antibodies are capable of causing transfusion reactions, hemolytic disease of the newborn and in severe cases, death.This course will discuss the techniques that are used by blood bank technologists to detect and identify various types of antibodies.

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Antibodies to Low- and High-Incidence Antigens

Low-incidence antigens are antigens that occur in less than 1% of the population.Antibodies to low-incidence antigens Low-incidence antigens are not usually found on screen cell and antibody panels. Antibodies are hard to test for, but it is usually not difficult to find compatible blood. Suspect this antibody if an AHG crossmatch is incompatible and other causes have been ruled out, such as a positive donor DAT or ABO incompatibility. Examples of low-incidence antigens include: Cw, V, Kpa, Jsa. When going through the process of Ruling Out, antibodies like anti-V, anti-Cw, anti-Lua, anti-Kpa, and anti-Jsa usually fall into the "unable to rule out" category. High-incidence antigens are antigens that occur in greater than 99% of the population. Antibodies to high-incidence antigens Antibodies are rare and may be difficult to identify due to lack of negative panel cells for other high-incidence antigens (difficult to rule out). Reactions with screen and panel cells will all be positive (same strength and same phase). Auto control will be negative. Difficult to find antigen-negative compatible blood. Examples of antibodies to high-incidence antigens are: anti-k, anti-Kpb, anti-Jsb, and anti-Lub. If an antibody to either a high- or low-incidence antigen is present, it may be difficult to identify and may require further testing in a reference blood bank.

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Authentic and Spurious Causes of Thrombocytopenia
Preanalytic Variable Leading to False Thrombocytopenia

The platelet count is only as good as the sample collected. Blood samples that are collected in EDTA or sodium citrate tubes for coagulation purposes should be inverted 5 - 10 times for proper mixing of the anticoagulant and the blood. If the tube is not mixed, small fibrin clots may form, causing a falsely decreased platelet count.

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Pseudo-thrombocytopenia: Platelet Satellitism and Platelet Clumping

Platelet satellitism and platelet clumping can cause pseudo-thrombocytopenia. Platelet satellitism was first reported in the early 1960's. It is a rare condition that occurs when an IgG antibody forms in the presence of EDTA, the anticoagulant that is used for the collection of hematology blood specimens. The IgG antibody is directed against the glycoprotein IIb/IIIa complex on the platelet membrane. As the antibody coats the platelets, the platelets rosette around segmented neutrophils, bands, and sometimes around monocytes. Antibody-coated platelets that are huddled around white blood cells (WBCs) will not be counted as platelets by automated equipment and the platelet count will be falsely decreased. If a peripheral blood smear is reviewed, platelets will be observed attached to WBCs. The image on the right illustrates platelet satellitism with platelets adhering to a neutrophil. Platelet clumping can also occur in the presence of EDTA and the platelet count again will be falsely decreased. The count will probably be flagged by the analyzer for platelet clumps or giant platelets. If either platelet satellitism or platelet clumping is observed on the peripheral smear, the sample could be recollected using sodium citrate as the anticoagulant. Platelets can then be counted using the automated method. The platelet count from a tube that contains liquid sodium citrate will need to be corrected for the dilutional effect of the citrate. This can be accomplished by multiplying the platelet count that is obtained from the automated analyzer by 1.1.

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Which of these conditions is associated with a pseudo-thrombocytopenia?View Page
Definition of Thrombocytopenia

Thrombocytopenia is a decrease in the number of platelets in the peripheral blood. Most laboratories use a reference range for platelets that is approximately 150 - 450 x 109/L. Thrombocytopenia occurs when the platelet count falls below the lower limit of this range. The image shows how a normal platelet count would appear on a peripheral blood smear (Wright-Giemsa stain; 1000x original magnification). Representative platelets are indicated by arrows.

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Laboratory Findings

These laboratory findings are associated with TTP and HUS:Thrombocytopenia -- Platelet count is often less than 20 x 109/L in TTP, but may not be as low in HUS. Schistocytes (red blood cell fragments, as indicated by the arrows in the image to the right) may be observed on the peripheral blood smear. Schistocytes are the result of erythrocytic membrane damage caused by sheering of red blood cells as they pass through a fibrin mesh of clot formation occurring in the blood vessels. LDH, serum bilirubin, and reticulocyte counts are elevated. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are usually normal. Proteinuria and hematuria may be present.

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Thrombocytopenia in Neonates

Transplacental ITP may occur in newborn infants who are born to mothers with ITP. If the mother has had one baby born with thrombocytopenia, it is usually an indication that all subsequent infants will also be born with thrombocytopenia. A very small percentage of babies born with ITP will have severe thrombocytopenia. Neonatal alloimmune thrombocytopenia (NAIT) is caused by platelet destruction that is the result of alloantibodies stimulated by foreign antigens during pregnancy or blood transfusions. Platelet destruction by alloantibodies may occur in neonates if the mother lacks the platelet-specific antigen but the baby has inherited the antigen from its father. When maternal IgG antiplatelet antibodies cross the placenta, immune destruction of the neonate's platelets occurs. The major concern with both of these conditions is intracranial bleeding if the neonate's platelet count is less than 50 X 109/L. NAIT has a high mortality rate due to bleeding into the central nervous system. Prompt diagnosis of the condition and treatment is critical. The thrombocytopenia lasts on average 3 - 4 weeks postnatal until the maternal antibodies have cleared the newborn's system.

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Basic Tissue Orientation and Paraffin Embedding Technique
Fallopian tubes, vas deferens, and blood vessels should be embedded on their SIDES.View Page

Basics of Lean and Six Sigma for the Laboratory
Additional Lean Processes and Concepts

In addition to 5S and the waste walk, other tools are considered essential in a Lean system. These tools are listed below:Lean Process/ConceptDefinitionLaboratory ApplicationPull systemA system where supply and production are dependent on demand. Instead of ordering the same amount of supplies on a fixed time interval, a laboratory utilizing a pull system would review data from the analyzer test counter or take inventory to match the order to the testing demand. Continuous flowOne-for-one process. No waiting between steps. Linking the rate of demand to the rate of production. Patient moves from registration to blood draw area to doctor without waiting.Blood sample arrives in the laboratory, is received, processed, and tested immediately rather than waiting to batch. Set up reductionPreparation of the work area and supplies in advance so that the process proceeds smoothly. Set up supplies into ready-to-use kits. Perhaps these can be set up by the prior shift during slow times. Error-proofing A system that doesn't let a worker make a mistake. Can be used in combination with visual signals. Many point-of-care instruments have automatic lockout of testing if quality controls are not tested, or if they fail, prior to patient testing. Value stream mapA picture that illustrates the flow of material, inventory, and product. Value stream maps are used for value stream analyses, which can allow an organization to identify waste in its processes through Lean thinking. The value stream map should start from the supplier and end at the customer. In a clinical laboratory the start would be when the physician place the order or when the courier drop off the sample. And the end would be when the results are sent to the client or physician. Different processes: preanalytical, analytical and postanalytical within the laboratory are included in-between. The value stream map can help to identify unneeded or non-value-adding steps and provide a high level review of the entire process.Takt time and level loadingTakt time is a measurement of the process flow over time that will help determine how to combine work to achieve the best flow. Based on the takt time, level loading helps to balance the volume and variety of work among employees so that work progresses at a more even pace. Cross-training allows a laboratory worker to help in another department when there is a lull in their department's workflow.

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Six Sigma Level of Quality in Health Care

Is there any process in health care that can run at or above Six Sigma level? Transfusion medicine has actually achieved above and beyond Six Sigma, confirming that a Six Sigma level of quality can actually be achieved not only in aviation or manufacturing, but in health care as well. Transfusion medicine is one of the most regulated and earliest adapters of a quality system approach to quality management. Let us look at how transfusion medicine measures up when we translate this statistic into Six Sigma.Based on the FDA Center for Biologics Evaluation and Research (CBER) "Fatalities Reported to FDA Following Blood Collection and Transfusion" annual summary, there were 52 transfusion-related fatalities in the fiscal year of 2007 (October 1, 2006, through September 30, 2007). According to the National Blood Collection and Utilization Survey conducted by AABB, a total of 30,044,000 units of blood components were transfused during 2006. Three opportunities for defect (pre-analytical, analytical and post-analytical) will be used to determine transfusion safety in the United States. By using the formula from the previous page, we can determine both the DPMO and process sigma.DPO = 52/(30,044,000 x 3) = 0.0000005769DPMO = 0.0000005769 x 106 = 0.58Process Sigma = 6.36Transfusion medicine did not achieve Six Sigma level of quality overnight, and other industries who are operating at Six Sigma level, such as the aviation industry, have taken decades to achieve this level of quality.

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Pareto Chart

A Pareto chart is a line and bar graph that provides a visual illustration of a problem. Pareto charts can be created using Microsoft Excel or Open Office. The Pareto chart ranks the problem based on occurrences from left to right. The Pareto chart is used extensively in quality control; it can help a team focus improvement efforts on the most critical problem. Here is an example of a Pareto chart that ranks different types of errors that occurred in a blood donor center. From the chart, the most frequent error is "no collector initials." While this information is helpful, it is probably not specific enough. To achieve more meaningful data one can create another Pareto chart, breaking the "no collector initials" further down by site (mobile blood drive, donor center location) or by procedure type (apheresis, whole blood collection, autologous donation, etc).

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Beta Thalassemia
Beta Thalassemia Major

Children with beta thalassemia major, also called Cooley's anemia, usually develop clinical signs during their first year of life. They appear to be malnourished and may exhibit abdominal girth expansion. They show bone marrow expansion and skeletal deformations, which are a result of increased erythropoiesis due to low hemoglobin levels. A common finding is facial bone changes caused by this bone marrow expansion (sometimes referred to as Mongoloid facial features). Other clinical signs include frequent infections, hepatomegaly, splenomegaly, gall stones, leg ulcers, iron toxicity, and poor growth and sexual development. In addition, cardiac failure due to increased burden of the heart attempting to oxygenate the tissues, can lead to serious complications and death if the condition is not treated.In general, death usually occurs by the time these patients are in their early twenties unless treated with blood transfusions along with iron-chelating agents. If no chelating agent is used during treatment life will only be prolonged by about a decade.The different genotypes associated with beta thalassemia major are: B0/B0, B0/B+, or B+/B+.

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Case History

A 45 year old Asian woman had blood work done as part of a routine employment physical. Her only complaint was occasional fatigue. Upon review of her CBC, additional blood work was ordered to investigate her microcytosis.

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This is a representative field from the patient's peripheral blood smear.What RBC morphology is prominent on this patient's smear?View Page

Blood Banking Question Bank - Review Mode (no CE)
Which of the following activities will put an employee at risk for exposure to a Bloodborne Pathogen (BBP)?View Page
Which BBP is not covered in the OSHA Bloodborne Pathogen Standard?View Page
What should you do if your lab coat or gown has dried or caked-on blood on it?View Page
What type of Personal Protective Equipment (PPE) is necessary when opening a centrifuge (chance for splashing)?View Page
Why would a unit of group O blood never be administered to a Bombay patient:View Page
Which of the following blood group antigens are most susceptible to destruction by the action of enzymes:View Page
The classification of Du refers to:View Page
Which of the following is used as a source for irradiation of blood products:View Page
Based on the following reactions indicate the correct blood group for each set of reactions:View Page
Which of the following patients represents an acceptable donor.View Page
Which one of the following is not a benefit of using packed RBCs:View Page
Which of the following is most commonly associated with febrile non-hemolytic transfusion reactions:View Page
If a potential donor has been transfused blood products, he must be deferred from blood donation for:View Page
Which of the following tests must be repeated by the lab on homologous blood received from the Red Cross or other community blood sources:View Page
All of the following cellular antigens are important to an immunohematologist except:View Page
Deglycerolized red cells are most effectively used to:View Page
If an R1 r patient received R2R2 blood, which of these antibodies could be produced :View Page
Which of the following blood group antigen-antibody reactions is enhanced by using enzymes:View Page
Before testing all cord cells should be thoroughly washed in order to:View Page
Which of the following blood groups reacts least strongly with Anti-H:View Page
Which of the following are not appropriate indications for the use of fresh frozen plasma:View Page
Match the blood groups on the left with corresponding results of forward typing on the right.View Page
Therapeutic hemapheresis may be used to treat all of the following except:View Page
All of the following are benefits of autologous donation except:View Page
Which of the following types of whole blood would be the least satisfactory to transfuse to a type AB patient:View Page
The following steps must be followed in preparation of a platelet concentrate:View Page
Match each blood type with the corresponding antibody you would find in its serum:View Page
Type B blood is found in higher frequency in:View Page
Which of the following options gives in order from most to least important, the factors you would use to select blood for a transfusion:View Page
Which of the following statements is not true about the Lewis blood group:View Page
The use of cells with known blood groups to confirm ABO typing is known as:View Page
The generally accepted age range for homologous blood donation is:View Page
Fresh frozen plasma :View Page
An urticarial reaction is characterized by:View Page
Which of the following is responsible for causing graft-versus-host reactions:View Page
The shelf-life of whole blood collected in CPDA-1 is:View Page
The use of the direct antiglobulin test is indicated in all the following except:View Page
After transfusion, a red cell sample from the donor unit, and the recipient's blood sample, must be kept for:View Page
Which of the following refers to the most common procedure for donating whole blood for use by the general population:View Page
Which of the following is the proper storage temperature for whole blood:View Page
Which of the following is the most prevalent blood type found in the United States:View Page
Autologous blood must be tested for which of the following before transfusion:View Page
Gamma irradiation of cellular blood components is required in which of the following situations:View Page
A severe hemophiliac, with a Factor VIII activity of less than 1%, is actively bleeding due to a serious accident. The blood product of choice is:View Page
Match substance(s) secreted with blood groups:View Page
What percentage of glycerol is generally used when freezing red cells of rare phenotypes:View Page
Which of the following blood components will provide the best source of fibrinogen for a patient with hypofibrinogenemia:View Page
The accepted interval between blood donations is:View Page
How long may blood be stored using CPDA-1 preservative prior to transfusion?View Page
The most severe acute hemolytic transfusions reactions are the result of which of the following:View Page
The chief purpose of performing a standard crossmatch is to :View Page
Which of the following contains all the possible phenotypes that could be the result of parents who are type O and type A:View Page
A refrigerator used to store whole blood must be able to maintain a temperature in the ranges of:View Page
A false-negative reaction while performing the DAT technique may be the result of:View Page
A patient's serum reacts with all reagent red cell samples. The autocontrol is negative. An alloantibody to a high incidence antigen is suspected. Which of the following would be most likely to be a compatible donor:View Page
ABO blood groups were discovered by:View Page
In an extreme emergency , if the ABO and Rh type are unknown which of the following should be given to the patient?View Page

Body Fluid Differential Tutorial
Leukemic Fluids

Leukemia and lymphoma cells can be found in cerebrospinal, pleural, peritoneal and pericardial fluids at the time of diagnosis as well as anytime during the course of the disease. They can be found in any of these fluids, even when there are not abnormal cells present in the peripheral blood. Just as there are many morphological variations of leukemia and lymphoma in the peripheral blood and bone marrow, there are as many variations of leukemia/lymphoma in cytospins from fluids.Once the diagnosis is made from the initial tissue samples, there may be frequent spinal taps performed to monitor disease status and to instill intrathecal chemotherapy to prevent the development of central nervous system involvement by the leukemia or lymphoma.Cytospins are made for each sample and must be meticulously scrutinized. If any display abnormalities indicative of a disease state, or if there are any suspicious cells, then they should be sent for hematology or pathology review in compliance with your hospital's policy.

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Peripheral Morphology vs Cytospin Morphology

Most technologists working in the clinical hematology setting are familiar with the morphology of blood cells found in peripheral blood smears. Many of the same blood cells found in the peripheral blood are also found on cytospin preparations of body fluids. While the morphologies are similar between the two sources, there are changes to the "comfortable/familiar" peripheral blood morphology that are introduced by the cytospin technique. It is paramount to fully conceptualize the appearance of both normal and abnormal blood cell morphologies in body fluids in order to accurately perform fluid differentials.The cytopsin process works by wicking fluid into a filter while fluids samples are spun into a central column and deposited in a mono-layer onto a defined area of a slide. This allows the cells to be concentrated for appropriate identification. This technique can cause pronounced changes to morphology and staining characteristics, as well as cellular destruction if the cytospin malfunctions.The cytospin technique is known to stretch and distort cellular and nuclear morphology and allow nucleoli to appear more prominent than what one would normally see in peripheral smears. Cytospinning, however, does not change nuclear:cytoplasmic ratios nor does it alter relative chromatin textures or clumping patterns. Though this technique can make cells appear larger that on the peripheral blood smear, it can not change cytoplasmic textures and granulation. Focusing on these steadfast features can make cytospin morphology less intimidating.

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Bone Marrow Contamination in Cerebrospinal Fluid

When a spinal tap is performed correctly, the cell types found in the cytospin present a "snapshot" of the cerebrospinal fluid cell content. When there is difficulty obtaining a clean tap, the cell types present will reflect the type of fluids that are co-mingled with the cerebrospinal fluid. In traumatic taps, the cell types and proportions of red and white blood cells present will mirror that found in the patient's peripheral blood, as there has been a contamination of peripheral blood (from the trauma) mixed into the cerebrospinal fluid during the sample collection.In patients with softer bones (infants and osteopenic patients), it is not uncommon for the contamination to come, not from peripheral bleeding, but from bone marrow found inside the softened spinous process that was penetrated inadvertently during the procedure. When this occurs, any or all of cell types found within the bone marrow can be present in the cytospin.In the image shown here there are early nucleated RBC and WBC precursors, typical of contamination with bone marrow.

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Choroid Plexus Cells

Choroid plexus cells are involved with ependymal cells in the production of cerebrospinal fluid, or CSF. They are found in the ventricles and are part of the blood brain barrier.Like ependymal clumps they are infrequently seen in cerebrospinal fluid cytospins. They tend to be observed in clusters, but it is evident that they are individual cells rather than a single multinucleated mass like ependymal clumps.

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Lymphocytes and Atypical Lymphocytes

Similar to peripheral blood, lymphocytes in fluids come in all sizes. A larger lymphocyte is not necessarily an abnormal or atypical lymphocyte, however. In the image shown here there is one atypical lymphocyte (blue arrow) adjacent to the monocyte at the top of the smear. The atypical lymphocyte is almost as big as the monocyte, however the nucleus is more regular. The amount of cytoplasm is similar between the two cells, but the atypical lymphocyte has a deeper blue shading at the edge of the cytoplasm. Though there are nucleoli present in this atypical lymphocyte, it does not necessarily mean that this is a malignant cell. Cyto-spinning can expose nucleoli in cells that would not normally appear to have nucleoli in the peripheral blood. The balanced amount of cytoplasm in comparison to the nucleus and the overall size of the cell are consistent with the range of variation found with atypical lymphocytes.In this smear, there is also a plasmatoid lymph in the lower left of the cluster (green arrow) and a basophil in the lower right (orange arrow).

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Macrophages are actually lymphocytes that have entered the tissues and body fluids via diapedesis.View Page
Neutrophils

Neutrophils in a cytospin will look the same as in peripheral blood. The cytospin technique will accentuate the segmentation in the nucleus. Toxic vacuolation and toxic granulation will be the same as on a peripheral smear.

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The cytospin technique perfectly preserves the morphology of blood cells in a fluid sample.View Page
Central Nervous System (CNS) Toxoplasmosis

This image represents a cerebrospinal fluid (CSF) cytospin preparation from a patient who recently received a bone marrow transplant for recurrent central nervous system (CNS) Burkitt's lymphoma. The patient was admitted to the hospital two weeks after transplant due to rapidly altering mental status. When the CSF cell count demonstrated a high white blood count (WBC) count, the first concern was a possible CNS relapse of the Burkitt's lymphoma. However, the cytospin showed many neutrophils in spite of the patients peripheral blood neutropenia. No malignant cells were identified.On closer examination of the neutrophil clusters, ovoid inclusions were noted (see arrow) as well as free banana shaped organisms (see circled area). The ovoid inclusions in the neutrophils and the free forms have lavender cytoplasm with a centrally placed cluster of reddish granules.

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Central Nervous System (CNS) Toxoplasmosis continued

This is a higher power view of this same smear demonstrating a neutrophil that is filled with Toxopasma gondii tachyzoites (blue arrow).There are a few free organisms in this image well, indicated by the red arrows. Again, the typical morphology for toxoplasmsa organisms is lavender cytoplasm with a red granular cluster in the center of each parasite.This patient was negative for Toxoplasma gondii prior to a transplant but had received 15 units of blood products due to cytopenias.It is believed that a donor for one of the transfused units had been exposed to Toxoplasma gondii either through cats or contaminated food and had transient circulating Toxoplasma gondii in his or her blood when the donation was made. In this case, the recipient was profoundly immunocompromised, which lead to rapidly developing systemic disease.

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Match the following blood cell types to the respective body fluids where they may reside.View Page

Bone Marrow Aspiration Part I: Normal Hematopoiesis and Basic Interpretive Procedures
Introduction to Bone Marrow Aspirates and Biopsies

Bone marrow aspiration and biopsy are standard tools used in the hematology laboratory to aid in the evaluation and diagnosis of peripheral blood abnormalities. Some of these abnormalities include: cytopenias (such as neutropenia), thrombocytopenia and anemias. Bone marrow aspiration and biopsies are also used by hematology/oncology specialists in the diagnosis of leukemias, dysplastic syndromes, and proliferative syndromes. A bone marrow aspiration and biopsy may also be part of the evaluation of fever of unknown origin (FUO), failure to thrive(FTT) in the pediatric setting, as well as some metabolic and genetic disorders.A bone marrow aspirate sample is obtained by inserting a needle into the bone marrow space and withdrawing 5- 10 milliliters (mL) of marrow in several different syringes. These samples are then transferred to evacuated blood collection tubes containing the anticoagulants required for the types of assays desired. A portion of this liquid marrow is smeared for staining and evaluation under light microscopy. It can be sent for various types of laboratory assessment including : immunophenotyping, cytogenetic evaluation, and molecular analysis.While bone marrow aspirations and biopsies are usually obtained by the hematologist or oncologist, they are evaluated and interpreted by a hematopathologist with the assistance of the laboratory technologists who prepare and stain the smears. In many laboratory settings the technologists also perform the bone marrow differentials.

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Bone marrow Differentials

For the clinical laboratory professionals who are only familiar with peripheral blood morphology, the first few observations of bone marrow aspirate smears can be overwhelming. The difference in cellularity between the two sample types, not to mention the wider variety of cell types, can lead to mental and visual overload. It is important to step back and break it down into more manageable pieces, starting on low power. Use low power (10x) to look at the distribution on the slide and the quality of the stain. Find areas where the spread/distribution of cells are thin enough (monolayer) to read easily and where you like the color balance and intensity of the stain. Next, add oil and move up to 50x and/or 100x power on the microscope.* Remember that there are several different cell types that are normally present and develop in the bone marrow before heading out into the peripheral blood. Most hematology technologists are familiar with the myeloid maturation sequence from peripheral differentials, even if immature cells are less commonly seen. However, there are additional cell types that are not seen on the peripheral blood differential, since they reside only in the bone marrow. Becoming more familiar with these cell types and the maturation sequences of the myeloid, erythroid, and megakaryocytic cells found in normal bone marrows will make performing these differentials less intimidating.One important concept to grasp is the continuum of cellular maturation sequences. There is no such thing as a magical switch that flips causing cells to jump to the next "textbook photo stage" as cell lines mature. Rather, each cell matures at its own pace. The maturation and morphology will vary from cell to cell and bone marrow to bone marrow. Understanding both nuclear and cytoplasmic normal morphology can aid in the identification of cells. *As counter-intuitive as it sounds for most applications, higher magnification does not always help with morphology. Reserve 100x for ultra fine detail.

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Rules for Bone Marrow Differentials

Bone marrow differentials have significant differences from peripheral blood differentials that need to be considered as they are reviewed and counted.One of the most important facts to consider is the large variability in cellularity and cell distribution depending on the type of preparation that is used. Choosing where to count and when to use which of the smear types available to you, takes time and experience and can be directed by a pathologist's preference.Regardless of how many, or what types of smears you have available to choose from, you will always start with a simple visual inspection of your smears. Begin by recording the patient identification information as well as date of sample, and any other mandatory patient identifying information necessary for your laboratory. Record aspiration site information when provided. Many patients will have bilateral bone marrow aspirates performed as part of a diagnostic or staging workup. Standard aspiration sites are: posterior iliac crest (PIC), anterior iliac Crest (ANT), sternum (S), spinous process (SP) and sometimes in very young children, bone marrow is obtained from the tibia (T). Be aware, that while a bilateral bone marrow aspirate usually involves an aspirate of the same site from opposite sides of the body, e.g., L-PIC and R-PIC, in some situations, a bilateral staging aspirate will be from two different compartments on the same side, e.g. R-AIC, R-PIC. Observe the appearance of the bone marrow smears. Do any have feather edges? Are there fragments or spicules present on any of the smears available? If so, they should be your first choice to view, since they are more representative of what the biopsy will show if one was obtained. Once you select your smears, scan using 10X magnification on the microscope. Are some of the fragments/smears so thick that you cannot see good morphology? If so, reject these areas/slides. Are some of the fragments/smears so thin that everything is smashed? These areas/smears cannot be used either. Are there areas in the vicinity of any of the fragments that have good staining characteristics as well as readable morphology? This is where you should begin your differential.

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Rules for Bone Marrow Differentials, continued

Bone marrow smears can be very cellular and it can be difficult to keep track of where you are on the smear while keeping your correct hand position on the keyboard . Having a good strategy to use when counting cells and performing differentials can make this less difficult. On peripheral blood differentials, it is easy to observe and count each cell individually as the stage is moved to bring the next field into view. However, with bone marrows, the total number of keys that need to be used on the differential counter is greater than the number that need to be used with a peripheral blood smear and the number of cells per field is also increased dramatically, making it easy to lose track of the cells on the smear or one's hand/keyboard placement. It can be simpler and less stressful to work on the quadrant system. There are two different ways to do this: Divide the field into quadrants. Count the individual cells in each quadrant separately. This decreases the number of cells into more manageable bites. However, you still have the increased number of cell types to deal with and possible keyboard frame-shifts. Divide your keyboard into quadrants. Search your field for a limited number of cell types and tally all you see before moving on to the next grouping of cell types. Once you tally all your groups then move on to the next field (e.g., lymphocytes, monocytes, macrophages, eosinophils, basophils, plasma cells, erythroids, segmented neutrophils, bands, etc). You can make these small groupings for any cells as long as you cover the entire list of cell types that your laboratory reports in its bone marrow differential protocol. Remember that blasts are identified by cell type and there will usually be a separate key for pronormoblasts, myeloblasts, lymphoblasts, and possibly monoblasts and plasmablasts. It is possible to combine both methods, using the keyboard quadrant technique with a restricted portion of the total microscope field. This is useful when you are getting close to your total tally and do not want to alter the balance by only counting one cell type for the last few cells.

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Biopsy Touch Preparation Technique

While smears from the bone marrow aspirate are the most common preparations, touch preparations (touch preps) made from the bone marrow biopsy core may also be useful or necessary. When aspirates are difficult or a dry tap occurs, the only sample available to be evaluated in the hematology laboratory may be the bone marrow biopsy. To create a fresh biopsy touch preparation, the fresh bone marrow core is gently rolled between two slides, then gently rolled between five or six pairs of coverslips. There should be enough cellular elements present when using this method for the laboratory professional to evaluate. The imprints will be wet and cellular at first but as the surface dries it will eventually become less cellular. At this point the core is placed in fixative and sent to pathology for evaluation. The number of touch preps you can make is dependent on how wet/ bloody or fibrotic the core is to begin with, but even one set can be enough to aid in diagnosis.While it is not practical to practice making touch preps from a real biopsy core, it is possible to practice the technique by using a length of applicator stick soaked in either blood or stain to simulate a real biopsy core. To do this, simply break off a short, 0.5 inch piece of a standard plain or cotton tipped applicator stick and soak it in the fluid of your choice. As you roll it between slides or coverslips you will see the pattern it leaves behind. Think of the motion of a teeter-totter (seesaw) as you roll. There should be very little downward force on the core as you coax it to roll. If the core will not roll then you can just touch the slides or coverslips to the surface of the core few times on each slide.Note: If the biopsy is placed directly into fixative and sent to pathology, it must first be decalcified before it can be sectioned and stained. This process will take at least 24 hours depending on the lab and if additional stains are required, it may be at least 48 hours before a result is released.

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Bone marrow Smear Preparation: Selecting Fragments

Most bone marrow slides are made simply by placing a drop of bone marrow on a slide and using a smear preparation technique. However, in order to obtain consistently high quality smears, it is necessary to select or concentrate the fragments on these smears. Selecting or concentrating fragments can be performed with different methodologies. At the patient bedside, some clinicians will use the touch-preparation or pull-preparation method, while tilting the slide to allow excess blood to roll off. This leaves more of the bone marrow spicules on the slide. This can be wasteful and rather messy but does not require a high level of skill.A less wasteful method is to pour a portion of the marrow aspirate into a small petri dish and swirl it about, then tilt the dish to reveal the marrow spicules. These can then be extracted using a capillary pipette with a micro-pipette bulb and transferred to the slide for use in making smears. This technique allows the laboratory professional to make numerous smears containing fragments rather than relying on the random luck of the drop. Any excess marrow can be saved and returned to the EDTA tube for further testing. This capillary pipette concentration technique can be coupled with any of the smear preparation techniques but does require practice to perfect and maintain proficiency. When coupled with the coverslip method, it is possible to make 2-3 dozen quality smears from as little as a 0.25 - 0.50 mL of marrow aspirate, making it ideal in small sample volume situations.

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Manual Staining of Bone Marrow Preparations: Wright's and Wright-Giemsa Stain

Wright's or Wright-Giemsa stains are usually the preferred staining method for bone marrow aspirate smears. These are methanol-based staining solutions with similar dye composition to the diff-quick stain but require longer stain contact time for adequate staining. The Wright's and Wright-Giemsa stains have a buffer step as well. Since Wright's stains are methanol based they do not require a fixation step prior to staining, although you might prefer to do so first to reduce water artifact that can occur on humid days or with aged stain.In the dip method of staining, the smears are first dipped in methanol to fix the specimens and then placed in Wright's or Wright-Geimsa stain for 10-15 minutes to stain. The smears are next moved to a mixture of stain and 6.8 pH phosphate buffer (usually one part stain to 2-3 parts buffer) and allowed to stain for at 20-30 minutes. After staining, they are given a quick rinse in distilled water and allowed to air dry before mounting or cover-slipping.When using a staining rack, the marrow slides or coverslips are first flooded with enough stain to cover the slide and stained for 10-15 minutes. Then, a 6.8 pH buffer is carefully added without overflowing and gently mixed by blowing until a green metallic sheen forms. This is allowed to stand for 20-30 minutes and then rinsed off with distilled water. The slides or coverslips are then air dried and mounted.Staining times can be extended for extremely cellular marrows; however, care must be taken when using the rack staining method. Extended times can lead to evaporation of the stain and cause excessive precipitation. Both the stain and buffer can be topped up if necessary to prevent this from occuring, while additional rinse time may be needed.Wright's and Wright-Giemsa stains, when performed properly, give sharp and clear nuclear, cytoplasmic, and granule detail. There can be variation in the quality of the stain from batch to batch, dependent on the manufacturer's quality control, storage, and shipping conditions. Many manufacturers age their stains for a minimal amount of time before shipping and assume that there will be additional standing time at the distributor before it reaches your lab. This may work for peripheral blood staining, but it is not ideal for bone marrow staining. It is advisable, if possible, to keep a separate stock of Wright's stain for bone marrow staining which is kept at least 6 months before use. Like a fine wine, the older Wright's stain gets, the better the quality and clarity of the final stain.

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Bone Marrow Collection: Patient Bedside

When the technologist accompanies the clinician to assist with the bone marrow aspiration procedure to make smears at the bedside, it is necessary to understand the role of the clinician and the technologist.The clinician is responsible for patient positioning and sterile preparation, pain control, and performing the aspirate and biopsy. The clinician often hands off sample syringes to the technologist, once collected. The clinicians are responsible for providing the procedure kit and fixative for the biopsy, all labels, and obtaining the requisitions and a copy of the clinical history for the hematopathologist. The technologist will set up a mini workspace near the bedside where the samples are split into the required tubes. Smears are then prepared from the aspirate as well as biopsy touchpreps before the biopsy is placed in fixative. In this setting the technologist will usually deliver the samples and requisitions to pathology and continue the processing procedure.The kit the technologist brings to the bedside usually contains mini petri dishes, coverslips, slides, microcapilary tubes or Pasteur pipettes, micro-pipette bulb and the various evacuated blood collection tubes and media flasks required for the standard bone marrow draw.Most institutions will have a standard draw and testing protocol designed to ensure that enough sample is obtained to cover all of the usual testing requirements. An example would be a three-syringe-draw with the first two syringes containing no anticoagulant and the third syringe rinsed with preservative-free heparin. The first dry pull would be split between a green and a purple top evacuated blood collection tube and would be used for morphology (EDTA) and flow cytometry and cytogenetics (green) if needed. The second dry pull is split into two additional purple top tubes plus a green top tube and would be used for molecular assays such as SNP array, Flt-3, JAK2, MPL mutation, etc. The final heparinized syringe could be used for other treatment protocol requirements or to provide sample for additional assays.

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Bone Marrow Delivery

In some institutions, the laboratory technologist does not assist the clinician at the bedside with the bone marrow aspiration procedure. Instead the clinician delivers the bone marrow sample to the laboratory, similarly to other laboratory specimens. When this is the case, the bone marrow sample may be delivered in one of two manners with the laboratory's responsibilities dependent on which method is used. A clinician may deliver to the laboratory a specified number of smears, made at bedside, along with the bone marrow sample. Samples may also be designated for flow cytometry, cytogenetics, or molecular diagnostics. A clinician may deliver a standard package of bone marrow aspirate to the laboratory in various evacuted blood collection tubes. In this situation the laboratory will usually have a standard order set that directs the distribution of the marrow samples based on diagnosis. The hematology laboratory will use these samples to prepare the bone marrow smears, while the other tubes would be distributed for flow cytometry, cytogenetics, molecular diagnostics, etc. based on the direction of the hematopathologist.

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Myeloblast

Under normal circumstances, the segmented neutrophil is the most common nucleated cell in the peripheral blood. These bacterial-infection-fighting cells are produced in the bone marrow and arise from their precursor cell, the myeloblast. The myeloblast is the youngest cell in the myeloid lineage. It is approximately 12-20 microns in size with very basophilic cytoplasm. The nucleus takes up around 2/3 of the total cell volume with a soft, finely stranded chromatin with very little clumping. The nucleus is eccentrically placed and ovoid, but can also be slightly flattened. Myeloblasts will typically have two or more nucleoli with well defined nucleolar membranes. In a well-stained preparation, you should be able to observe the outline and blue color of the nucleoli.The myeloblast's cytoplasm is basophilic and can have a hint of background "ground glass" graininess. This graininess is separate from any primary granules that develop as the cell progresses toward the progranulocyte stage. The cytoplasmic membrane tends to be regular without much denting, bumps, pseudopods, or shredding.The cell in the first image on the right shows the relative size, nucleus, and gritty basophilic cytoplasm of a classic myeloblast. Note that there is a small cluster of red primary granules present which, in addition to its other features, help to identify this cell as a myeloblast.The second image shows a myeloblast (blue arrow) at a later stage that is not quite a promyelocyte but is very close. The nucleoli are still prominent, the size has not changed much, and the cytoplasm is still only about 1/3 of the cell. There are a few more primary granules but they are not prominent enough to consider this cell a progranulocyte.While the myeloid sequence tends to be the predominant cell type found in normal bone marrows, myeloblasts should make up less than 5% of the bone marrow's nucleated cells.

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Orthochromic Normoblast

Orthochromic normoblasts are the last nucleated stage of erythroid maturation. In this stage, the nuclei of the cells completely shrink to a pyknotic remnant. The cytoplasm color approaches the color of a peripheral RBC as it becomes fully hemoglobinized. This is the stage that is most commonly seen when NRBCs are found in the peripheral blood. In the top image on the right there are many orthochromic normoblasts scattered across this section of bone marrow. Note the pyknotic-appearing nuclei which make them easy to spot, even at lower magnification. It is also evident that the cytoplasm is well hemoglobinized and the color is just slightly more blue than the non-nucleated red bloods cells present.In the higher magnification (second image), notice the orthochromic normoblast (blue arrow) to the right of the basophilic normoblasts. The color of the cytoplasm of the orthochromic normoblast is almost identical to the background RBCs. Notice how condensed the nucleus has become as well. You can actually observe the nucleus in the early stages of extrusion/elimination from the cell. Once the nucleus has been extruded, the slight blue color, also known as polychromasia, will begin to fade and the now non-nucleated RBC will be indistinguishable from any other circulating RBC.

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Erythrocytic Cells: Introduction

When performing bone marrow cell identification, it is necessary to differentiate the stages of erythrocyte development. This differs from a peripheral blood differential, where the term "nucleated red blood cells" ("NRBCs") is used to describe all stages of circulating normoblasts. As with the myeloid sequence, there is a continuum in the erythroid maturation process in terms of nuclear and cytoplasmic morphology. Becoming familiar with the range of variation in each nucleated erythrocyte stage will make bone marrow differentials less intimidating.The image to the right shows several different stages of erythroid maturation with several clusters of NRBCs all maturing together.

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When performing bone marrow differentials it is not necessary to distinguish the precursor forms of the erythroid sequence.View Page
Monoblast

Monocytes progress through maturational stages in a similar fashion to the myeloid series before entering the peripheral blood circulation. The final stage of monocyte maturation into macrophages occurs after they have migrated out of the peripheral blood and into the surrounding tissues via diapedesis. Mature macrophages are also found in the bone marrow. The monocyte lineage does not maintain a maturational pool in the bone marrow as large as the myeloid pool. As a result, the monoblast stage is infrequently noted in most normal bone marrows.Monoblasts are the largest blasts of all the hematopoeitic cell lines present in the bone marrow. They have a large, round, centrally-placed nucleus with soft, fine-stranded chromatin. They normally have a single, large, prominent nucleolus. The cytoplasm is very generous and has a fine, grainy texture. In the monoblast stage, the cytoplasm will be basophilic, similar to other blasts, but will possess a slightly lighter shade of blue. In the monoblast, the color will shift to blue-gray as the cell matures into a monocyte.The top image on the right shows a single monoblast. Notice the large, round nucleus, the single large nucleolus and the generous blue, grainy cytoplasm. The second image shows a group of monocyte precursors. The large cell at the top is a monoblast (see red arrow). Notice the round and flat look of the nucleus in the blast compared to the other stages. Observe the nuclear shape becoming more folded and three-dimensional as the cell matures.

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Monocyte

The monocyte is the final stage of monocyte maturation found in the peripheral blood before it migrates into tissues and further develops into a macrophage (histiocyte).When seen in the bone marrow, a mature monocyte will look identical to its peripheral counterpart. It will have fine, lacy chromatin pattern with varying degrees of nuclear folding and condensation. The cytoplasm will be blue-gray in color with a slightly grainy texture. The cytoplasm may have a light sprinkling of fine pink cytoplasmic granules. The mature monocyte will be larger than mature segmented neutrophils, but not quite as large as promyelocytes or early myelocytes. The top image to the right shows several monocytes with varying degrees of nuclear folding (see red arrows). Notice that the chromatin clumping is not as dense as that found in neutrophils. Notice also that the cytoplasm is blue-gray and grainy, not the pink/tan of a neutrophil. Observe that the mature monocytes are slightly smaller than the promyelocytes in the image.The lower image to the right shows a monocyte (red arrow) adjacent to a segmented neutrophil (blue arrow). The monocyte is clearly larger. Notice the increase in size of the two monocytes below (green arrows) as they begin to transform into macrophages (histiocytes). The vacuolation is an indication of this transformation occurring.

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Lymphocyte

Lymphocytes mature in the lymph nodes rather than in the bone marrow and therefore are not routinely assessed when deciding if a marrow has "trilinear" (myeloid, erythroid, megkaryocytic) maturation. However, they are normally present in the bone marrow and, when clustered in a lymphoid follicle, can be very prominent. Since lymphocytes mature in the lymph nodes, they will appear identical to peripheral blood lymphocytes when viewed in the bone marrow. They will have the same range of variation in size and cytoplasm and will demonstrate the same types of viral transformations noted in the peripheral blood. Viral/atypical lymphocytes are combined together with normal lymphocytes in a bone marrow differential count and not placed into their own category, as they are in a peripheral blood differential. However, the hematopathologist may include this information in the interpretation, if these changes are noted.Lymphocytes can be found scattered throughout the bone marrow and must be distinguished from early erythroid precursors, which they can closely resemble. Lymphocytes are frequently found in and around early NRBC clusters. In the top image on the right, notice the medium-sized lymphocyte (red arrow) next to the two basophilic normoblasts (blue arrow). The color and texture of the scant lymphoid cytoplasm is almost identical to the NRBC, which can be a bit confusing. However, observe the differences in the nuclei between the two cell types. The lymphocyte has a less distinct chromatin clumping pattern than the basophilic normoblasts and the lymphocyte does not have any "nuclear pores." Also, the lymphocyte has an irregularly-shaped nucleus that is hugging the cytoplasmic border, while the NRBC has a round and regular, centrally-placed nucleus. Identify the three lymphocytes circling the NRBCs in the second image (see red arrows). Notice the chromatin of the lymphocytes; the lymphoid smudgy/clumpy pattern is certainly not as dense and clumped as what is noted in the NRBCs. This nuclear difference becomes more pronounced as the erythroids mature. The cytoplasmic differences should be more apparent as well, since lymphocytes will never produce hemoglobin.

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Megakaryocyte: Immature

The megakaryocyte lineage is the cell line responsible for the production of platelets found in the peripheral blood. Unlike the other bone marrow lineages that decrease in size as they mature, the megakaryocyte starts smaller and increases in size as it matures. The megakaryocyte begins as a mononuclear cell that has the same physical size and nuclear/cytoplasmic proportions as a lymphoblast. Eventually the megakaryocyte matures into a multinucleated giant cell with vast amounts of cytoplasm. As this cell matures, it can actually increase to more than ten times the size of other nucleated cells found in the bone marrow.In the early stages of development, the cytoplasm of a megkaryocyte is basophilic without any obvious platelet granules. The cytoplasm will be darker near the edges of the cell and may have a "foamy" look in the golgi area adjacent to the nucleus. Cytoplasmic granule development is not usually noticeable until the cell's cytoplasm color begins to lighten.Notice the sizes of the early-intermediate stage megakaryocytes (red arrows) in comparison to the background bone marrow cells present in the two images to the right. The megakaryocyte nucleus makes up the largest part of the cell at this early stage. Notice the increasing lobulation as the cell increases in size and how the cytoplasm becomes more foamy and slightly more granular as well.

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Osteoblast

Osteoblasts are the cells responsible for the production and deposition of bone. They may not be apparent in normal cellular bone marrow, since they appear in low frequency. In situations where the total bone marrow cellularity is decreased, they become more visible.Osteoblasts are individual cells but tend to travel in small groups or clusters. They are quite large compared to the normal background blood cells and resemble giant plasma cells. They are oval-shaped cells and tend to have quite basophilic cytoplasm. An osteoblast has a single round nucleus with a fairly open chromatin texture. Notice in the images to the right how the nucleus of the osteoblast is eccentrically placed. On some smears it will almost appear as if the nuclei are in the process of being extruded from the cells. This effect is more commonly seen on extremely hypocellular bone marrows and is less pronounced in bone marrows with a higher cellularity. Notice the large size of the osteoblasts in comparison to the background bone marrow elements.

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Cardiac Biomarkers (retired 12/6/2013)
The Human Heart

The human heart is a muscular organ that is formed into four chambers with an interconnecting vascular system. Venous blood from the body enters the right atrium, is pumped into the right ventricle and from there is pumped to the lungs for reoxygenation. Oxygenated blood from the lungs accumulates in the left atrium and is pumped into the left ventricle and out to the body. Myocytes (muscle cells) require large amounts of energy and oxygen to accomplish this. The capacity of the heart to pump adequately is regulated by the volume of blood, systemic blood pressure, and the force of contraction achieved in the left ventricular wall.

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Atherosclerosis

Atherosclerosis is one of the leading causes of heart disease and its presence is an important risk factor for events leading to acute myocardial infarction (AMI). In the past, atherosclerosis was described as a cholesterol and lipid storage event. Now we know it is a chronic inflammatory disorder of the arterial vessels with lipid components. Atherosclerosis begins with damage to the cells that line the blood vessels. Some possible causes of this cell injury are bacterial infection, hyperlipidemia, hypertension, glycosylated products of diabetes, cytokines from adipose tissue, or exposure to toxins such as pollution and second-hand smoke. Monocytes and lymphocytes adhere to the injured site; macrophages enter and ingest proteins and, along with modified lipoproteins, create foam cells. An inflammatory milieu results as cytokines and other inflammatory molecules become involved; foam cells and white blood cells begin secreting cytokines and metalloproteinases. Myeloperoxidase is also released by degranulated white blood cells and macrophages. As inflammation and accumulation of these products continues, fatty dots and streaks are formed on the vessel lining and the formation of plaque begins. As the atherosclerotic process continues, involved cells proliferate forming a complex extracellular matrix and a fibrous cap. If development continues, possibly over decades, the plaque formations are distributed throughout various vessels, become calcified or collagenized and make the vessel walls rigid. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.

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Fibrous Cap

If the atherosclerotic process continues and other risk factors are involved, the lipid core of the plaque grows and pushes the arterial wall out. The myeloperoxidase and metalloproteinases degrade the cellular matrix, thinning the fibrous cap making it capable of tearing. Once there are tears, platelet aggregation and the coagulation cascade begin; thrombi or blood clots become part of the plaque. Further plaque growth and repair leads to occlusion and necrosis of vessels. Varying degrees of pain, cerebral or pulmonary infarction, and/or ischemic heart disease result.

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Which heart chamber pumps oxygenated blood out to the body?View Page
Infarction is an inadequate blood supply that decreases the availability of oxygen.View Page
Congestive heart failure (CHF) ordinarily results when the left ventricular myocardium is weakened.View Page
Lipids

Even though atherosclerosis is primarily a chronic inflammatory process, lipids are involved in atherosclerotic plaque formation. Lipoproteins are components of the foam cells that eventually develop into plaque if the inflammation in blood vessels continues. Lipids are transported in circulatory system in complexes composed of lipids, phospholipids, and protein. Cholesterol and triglyceride are the primary lipids transported in lipoproteins. There are four major lipoproteins: High-Density Lipoprotein (HDL) Low-Density Lipoprotein (LDL) Very-Low Density Lipoprotein (VLDL) Chylomicron (CM)

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Terminology Describing Ischemic Heart Disease and Heart Failure

Descriptions of cardiac biomarkers and their use require knowledge of several terms. These terms and their definitions follow.Acute Coronary Syndrome (ACS) Includes all the ischemic events that can occur in the heart. These events range from angina (where there is no cell death or reversible cell injury) to an AMI with large areas of cell necrosis. A continuum of events that are involved in ACS is illustrated on the page that follows this glossary of terms.Acute Myocardial Infarction (AMI) Commonly referred to as a heart attack. A sudden loss of circulating blood and oxygen that causes necrosis of myocardial tissue. It is most often caused by the narrowing of coronary arteries by atherosclerosis, a thrombus, or dislodged plaque material.Angina Chest pain caused by inadequate supply of oxygen to heart myocardium. It is synonymous with angina pectoris, pectoris meaning chest.Congestive Heart Failure (CHF) Usually, a left ventricular dysfunction resulting from aging, hypertension, atherosclerosis or muscle damage from an AMI or repeated AMIs. In CHF, the heart is not able to effectively pump blood through its chambers and to the body. Fluid accumulates in the lungs and tissues causing edema because less blood leaves through the arteries than what entered the heart from the veins. Electrocardiogram (ECG or EKG) The tracings of the electrical current that passes through the myocardium. The heart contractions are stimulated by this current. In areas of myocyte necrosis, the current does not pass and the tracings display abnormal patterns.Infarction An area of tissue death that occurs due to lack of oxygen. Clogging of an artery will cause dead muscle tissue or infarction.IschemiaAn inadequate blood supply that decreases availability of oxygen. Atherosclerosis is the main cause of myocardial ischemia.

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Optimal Cardiac Biomarkers

An ideal marker for cardiac disease should have these qualitites: Should be specific to myocardial tissue Be in low concentrations in normal peripheral blood Be rapidly released after myocardial injury Should be detected in low quantities with little interference from like compounds Should remain in circulation for a sufficient length of time for detection The plasma concentration of the marker should be directly related to the extent of injury. The test for the biomarker should be easily automated and relatively inexpensive to run, and results should be obtained rapidly.

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History

In the past, an AMI was primarily diagnosed by evaluating symptoms at patient presentation, ECG measurement, and results of enzyme assays that were considered cardiac enzymes. The enzymes, creatine kinase (CK), lactate dehydrogenase (LD), and aspartate aminotransferase (AST) were assayed several times a day often for several days to observe peak concentration and return to normal level for each enzyme. The first assay result was the baseline level or baseline concentration. Isoenzymes of CK and LD were later added for AMI diagnosis. All three of these enzymes are found in other tissues, making the diagnosis difficult and lengthy. In the 1980s, CK isoenzyme, CK-MB, though not totally cardiac specific, became the benchmark marker for an AMI. None of these enzymes are in any of the current recommendations except for CK-MBCurrent diagnosis, monitoring, and screening relating to heart disease includes measurement of lipids, proteins, enzymes, and other biomolecules. Risk stratification for cardiac and vascular disease is an additional role for measurement of these analytes. The physiological changes in the development of heart disease are better understood and the role of the clinical laboratory is greatly expanded.Today's markers are significant because of their location in the myocyte, the kinetics of their release in myocyte damage, and their rate of clearance from peripheral blood.

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cTnI

cTnI is encoded by a separate gene and after translation, a 31-amino acid chain is added to the amino terminal end. This chain makes troponin I cardiac specific. It is measured as an early indicator of an AMI because it is usually released and detectable within 4 - 6 hours following myocardial damage and peaks around 24 hours. It remains elevated in peripheral blood 3 - 7 days and can be elevated as long as 14 days.

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Biomarker Sampling Guidelines

Blood samples should be collected and assayed in intervals. ESC/ACC guidelines stress the importance of serial sampling. They recommend sampling at presentation of chest pain (baseline), then at 6 - 9 hours, and if these samples are negative and necrosis is still suspected, repeat samples at 12 - 24 hours. Some studies recommend sampling at presentation, 2 - 4 hours, 6 - 8 hours, and at 12 hours.Biomarker measurement should be available 24 hours a day and the results presented within 30 - 60 minutes after sample collection.

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Select the correct statements regarding troponins.View Page
Function of BNP

Once released, BNP stimulates natriuresis and diuresis. This action works against aldosterone which conserves sodium and water. BNP causes loss of sodium and water through the kidneys to relieve the ventricular wall stretch. It also increases renal blood flow and glomerular filtration rate.

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Homocysteine

Homocysteine is a sulfur-containing amino acid in the blood plasma. Elevation of homocysteine has been linked to a higher risk of cardiovasular disease (CVD). This elevation is significant in those where family history places them at risk for CVD.Folic acid, Vitamin B6, and VitaminB12 help to prevent elevated homocysteine levels. Recent data shows that folate fortification of foods has reduced the average level of homocysteine in the United States. Laboratory testing for plasma homocysteine levels may improve the assessment of cardiac risk, particularly in patients with a personal or family history of CVD, but with no well-established risk factors present such as smoking high cholesterol, or high blood pressure.

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Case Studies in Clinical Microbiology
The patient was admitted to the hospital. The sputum specimen was inoculated to sheep blood agar. Based on the colony morphology and the alpha hemolysis seen in the image to the right, the most likely identification is:View Page
The oxacillin screen test alone is not sufficient for determining the susceptibility to penicillin for S. pneumoniae isolates recovered from cerebrospinal fluid (CSF).View Page
Minimum inhibitory concentration (MIC) susceptibility tests should be performed against other beta lactam antibiotics on important S. pneumoniae isolates from blood cultures and other sterile body fluids when a MIC for penicillin is performed.View Page
Clinical History

A 67-year-old man entered the hospital with cough, right lower chest pain accentuated by deep breathing, and fever. He had a history of chronic obstructive pulmonary disease secondary to a long history of smoking. The temperature on admission was 39.2C, and auscultation of the chest revealed rales in the right lower lung field. The admission white blood count was 13,500/ml with 80% segmented neutrophils and a shift to the left. A blood culture was obtained.

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The colonies shown in the blood (BAP) agar (upper) and MacConkey (MAC) agar (lower) biplate are a 24 hour growth from an aerobic blood culture bottle that became positive at 12 hours after inoculation. The appearance of the colonies on MAC agar rules out the following two bacterial species:View Page
Isolates of Escherichia coli, Klebsiella pneumoniae, K. oxytoca and clinically significant isolates of Proteus mirabilis may possess extended-spectrum beta-lactamase (ESBL) activity.View Page
The bacterial species shown growing on 5% sheep blood agar was recovered from the spun sediment of a midstream urine specimen after 24 hours incubation at 35C. Each of the following tests would be useful in supporting the presumptive identification of Enterococcus species except: (Choose all that apply)View Page
The spot test that is helpful in separating Enterococcus species (positive as shown in the image) from the viridans streptococci and S. pneumoniae (both negative) is:View Page
Case History

A 63-year-old man was seen in the emergency room with the complaints of sudden onset of fever, chills, and abdominal pain, accompanied by mild diarrhea. The blood pressure was 140/84, the pulse rate 82/minute, and the body temperature 39.8C. A blood sample was drawn for a complete blood count, and a blood culture. A second blood culture was drawn from the opposite arm, with 10 mL of blood being placed into each an aerobic and an anaerobic bottle, following customary practice. The complete blood count revealed a hemoglobin of 15.8 mg/dL, a hematocrit of 45%, and a white blood count of 4.2/L. The neutrophils were 39%, lymphocytes 45%, monocytes 10%, eosinophils 4% and basophils 2%. The platelet count was 255/L. The patient was admitted to the hospital for further work-up and empiric antibiotic therapy. Within 24 hours after admission, the body temperature had decreased to 38.2C, although the mild diarrhea persisted. A stool toxin test for Clostridium difficile was negative and neither enteric pathogens nor Campylobacter species were recovered in stool culture after 24 hours incubation. Fecal neutrophils were not seen on direct examination. The anaerobic blood culture became positive 36 hours after inoculation.

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The Gram stain shown in the image was prepared from a positive anaerobic blood culture bottle after 36 hours incubation. Based on the morphology of the bacterial cells (some with spores, noted by the blue arrows), what the most likely identification?View Page
Colony Morphology

The growth observed on the anaerobic blood agar plate after 48 hours incubation (see upper image), revealed a spreading colony. The spreading nature of the colony is better observed in the lower image. No growth was observed on subcultures incubated aerobically indicating that this isolate is truly an anaerobe (although aerotolerance studies would be needed for confirmation). The spreading nature of the colony and the lack of hemolysis are highly suggestive of Clostridium septicum. However, biochemical confirmation is necessary.

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It is important to establish a species identification of C. septicum in blood culture isolates because of its close association with carcinoma of the colon.View Page
Staph on Blood Agar

The image of the surface of a 5% sheep blood agar illustrates the colonies that grew out of the foot drainage after 24 hours at 35C. They are entire, convex, smooth, and have a slight yellow pigmentation. Hemolysis is not observed. A Gram stain was prepared from one of the isolated colonies.

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Spleen Specimen

A 23-year-old man had complained of right lower quadrant abdomonal pain for approximately one week. Initially the pain was sharp and localized to a small area just above the right iliac crest. The pain subsided for approximately two days, but then recurred more diffusely over the lower abdomen, accompanied by cramping and mild diarrhea. The onset of fever and vomiting promted a visit to the emergency room. His temperature was 101 F, pulse was 90/minute, and palpation of the right lower abdomen elicited severe pain. The white blood count was 23,000/mm with a distinct left shift, including 5% metamyelocytes. Emergency surgery was performed for a large peri-appendiceal abscess. During surgery, multiple abscesses were noted in the spleen, which was removed (see image). Recovery was uneventful following five days of adjuvant clindamycin therapy.

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Cellulitis Skin

A 40-year-old woman with a long history of diabetes mellitis developed swelling and erythema of the left lower leg following superficial abrasion of the skin after a fall. The patient developed high fever and mild prostration. The cellulitis of the lower leg is shown in the image. Note in the photograph that the acute inflammation is most evident as red areas of streaking at the sites of abrasion. Blood cultures were obtained that turned positive in 18 hours.

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The image shows the Gram stain prepared from the positive blood culture. What is the appropriate report?View Page
Group A Strep A Disk/SXT

In follow up to the previous question, the upper image again illustrates the colonies recovered from the blood culture bottle. The colonies are small, transluscent, gray-yellow, and surrounded by a wide zone of beta hemolysis. The size of the colonies compared to the zones of hemolysis suggests a group A streptococcus. The susceptibility to bacitracin (zone of inhibition around the "A" disk in the lower image) is virtually diagnostic of a group A streptococcus. The absence of a zone of inhibition around the SXT disk indicates resitance to sulfamethoxazole/ trimethoprim. SXT resistance is also shared by group B streptococci, which are, however, resistant to bacitracin. The resistance to SXT is used for the primary recovery of groups A and B streptococci from specimens with mixed culture. Their resistance allows them to selectively grow out from contaminating bacteria that are inhibited by this antibiotic.

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Beta hemolytic colonies grew from the blood culture bottle after 18 hours incubation (see image). Which of following tests would be helpful in making a preliminary identification? (Choose all that apply)View Page
Colony Morphology

Image of the surface of blood agar after 24 hours incubation at 35C in 10% CO2, on which are growing tiny, translucent, gray colonies surrounded by a narrow zone of "soft" beta hemolysis. There was no growth on the MacConkey plate.

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Which of the following is NOT related to the virulence of Listeria monocytogenes? View Page
Review 3

Rouquette C. Berche P. The pathogenesis of infection by Listeria monocytogenes Microbiologia. 12:245-58, 1996 Listeria monocytogenes is a Gram-positive bacterium responsible for severe infections in human and a large variety of animal species. It is a facultative intracellular pathogen which invades macrophages and most tissue cells of infected hosts where it can proliferate. The molecular basis of this intracellular parasitism has been to a large extent elucidated. The virulence factors, including internalin, listeriolysin O, phospholipases and a bacterial surface protein, ActA, are encoded by chromosomal genes organized in operons. Following internalisation into host cells, the bacteria escape from the phagosomal compartment and enter the cytoplasm. They then spread from cell to cell by a process involving actin polymerisation. In infected hosts, the bacteria cross the intestinal wall at Peyer's patches to invade the mesenteric lymph nodes and the blood. The main target organ is the liver, where the bacteria multiply inside hepatocytes. Early recruitment of polymorphonuclear cells lead to hepatocyte lysis, and thereby bacterial release This causes prolonged septicaemia, particularly in immunocompromised hosts, thus exposing the placenta and brain to infection. The prognosis of listeriosis depends on the severity of meningoencephalitis, due to the elective location of foci of infection in the brain stem (rhombencephalitis). Despite bactericidal antibiotic therapy, the overall mortality is still high (25 to 30%).

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Eikenella BAP

Image of the surface of a 5% sheep blood agar plate after 48 hours incubation at 35 degrees C in 10% CO2. The colonies shown are small, flat, entire, dull gray, and show superficial pitting of the agar (see yellow arrows). A slight discoloration of the agar surrounding the colonies is seen. A bleach-like odor is detected. Similar growth was seen on a chocolate agar plate set up in parallel. Growth was not observed on the MacConkey plate.

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Cerebrospinal Fluid (retired 7/17/2012)
True or false: most of the chemical elements in CSF have levels similar to blood levels.View Page
Which of the following criteria may invalidate CSF results?View Page
Which of the following characteristics of cerebrospinal fluid are present if blood is due to subarachnoid hemorrhage (SAH)?View Page
Location of CSF

Most cerebrospinal fluid originates in the choroid plexus. The choroid plexus is composed of a mass of tiny blood vessels which are located in the third lateral and fourth ventricles. The remaining CSF, about 30%, is formed in other sites such as the subarachnoid space and the ependymal lining of the ventricles.

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Initial Specimen Examination

The technologist is responsible for examining CSF samples as they are received. If any of the following conditions are present, the results of testing could be uninterpretable: Tubes are not labeled.Tubes are not numbered.Specimen contains a blood clot.Specimen contains less than 0.5 ml CSF.

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Which of the following conditions can cause a sample to be uninterpretable?View Page
Bloody Specimen

When blood is present in a CSF specimen, it is necessary to determine whether the blood is due to a traumatic puncture or to a pathologic condition. There are several clues to help make this distinction: Traumatic tap:More blood is present in tube 1 than in tubes 2, 3, or 4.When sample is centrifuged within one hour, supernatant is clear.Blood clots on standing.Subarachnoid or cerebral hemorrhage:Blood is evenly distributed in all tubes.When sample is centrifuged within one hour, supernatant is pink or yellow.Blood does not clot on standing.As mentioned earlier in the course, distinguishing between a traumatic tap and a subarachnoid hemorrhage (SAH) may be complicated if a traumatic tap is overlying an SAH. The presence of blood in the last tube could be due to an SAH, even though the blood may be visibly less than what is observed in the first tube.

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Which of the following situations indicate a clot is most likely due to a traumatic tap?View Page
WBC Correction for Traumatic Tap

A calculation is used to correct CSF WBC counts which are falsely increased due to a traumatic tap: WBCs added = WBC(blood) x RBC(CSF) / RBC(blood)The blood WBC count is multiplied by the ratio of the cerebrospinal fluid RBC count to blood RBC count.The result is the number of artificially introduced WBCs. The true CSF white cell count is then calculated by subtracting the artificially introduced WBCs from the actual CSF WBC count. If the patient's peripheral WBC and RBC counts are within normal limits, some laboratories use the following formula: Subtract one white cell from the CSF WBC count for each 750 RBC counted in the spinal fluid.

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Cells Seen in CSF

Cells that may be seen in cerebrospinal fluid may be divided into four categories:mature peripheral blood cellsimmature hematopoietic cellstissue cellsmalignant cells

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Mature Peripheral Blood Cells

In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes. Up to 2% neutrophils is also considered within normal limits when a cytospin smear is used for the differential. In children, normal CSF cells are 70% monocytes, up to 20% lymphocytes and up to 4% neutrophils. When any of these normal cell abundances are increased, the term pleocytosis is used. Neutrophil pleocytosis is an increase in neutrophils and usually indicates the presence of a bacterial infection.

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Tissue Cells

Tissue cells that are never seen in peripheral blood but are often seen in spinal fluid samples are presented in the table below: Cells Causes macrophages RBC's in CSF viral meningitis tubercular meningitis ependymal normal - due to shedding of cells that line the ventricles pia arachnoid mesothelial cells (PAM) normal - due to shedding of cells lining the arachnoid space These cells are important because they must be differentiated from tumor cells and blast cells.

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Monocytes

The arrow in this slide is pointing to a monocyte. The nucleus has an open chromatin pattern which gives it a spongy appearance. The other two nucleated cells could be classified as macrophages (histiocytes) because the nucleus in each cell is oval or kidney bean-shaped and the cytoplasm is very irregular. After circulating in the blood for one to three days, monocytes enter the tissues. The tissue form of the monocyte is called a macrophage or histiocyte.

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Chemical Screening of Urine by Reagent Strip
Chemical Urinalysis Reagent Strips

A chemical urinaylsis reagent strip, also called a dipstick, for screening urine is a narrow band of paper which has been saturated with chemical indicators for specific substances or properties. Depending on the product being used, chemical urinalysis reagent strips may include test indicators for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocyte esterase. The results obtained from urine screening using chemical urinalysis strips can indicate the patient's carbohydrate metabolism status, kidney and liver function, urinary tract infection, and acid-base balance. Most chemical urinalysis reagent strips can be read visually and do not require instrumentation for automatic reading, though many laboratories utilize instruments for this purpose. When performing chemical urinalysis reagent strip analysis, the directions must be performed exactly. Accurate timing is paramount in order to achieve appropriate and optimal results. In addition, the reagent strips must be stored properly in their containers with the lid tightly closed to maintain reagent reactivity. It is always essential to utilize well-mixed urine which has been collected within 2 hours of analysis.Always read the package insert for your particular brand of chemical urinalysis reagent strip, as each manufacturer may have slightly different instructions and interpretations.

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Precautions in Urinalysis Chemical Reagent Strip Analysis

The following precautions should be observed when working with urinalysis reagent strips: Store strips according to the manufacturer's recommendation.DO NOT expose strips to moisture, volatile fumes, or direct sunlight. Remove only enough strips for immediate use and immediately recap the bottle.Avoid contamination of test strips. Do not touch the test areas with fingers or do not lay the test strips directly on the workbench.DO NOT use discolored strips. Compare the color of the unused strip to the negative area on the color chart provided by the company. The color should be similar.Check the expiration date. Re-label the container with a revised expiration date if the manufacturer states a shortened usage period once the container has been opened.Procedural PrecautionsAlthough the procedure is simple to perform, accurate results depend on careful adherence to manufacturer's directions and adequate quality control.Normal and abnormal controls should be tested whenever a new lot of strips is opened, and at the frequency defined by the laboratory's procedure.If quality control results do not correspond to the published control values, the problem must be resolved before patient samples are tested.High levels of ascorbic acid (Vitamin C) in the urine may inhibit some reagent strip reactions, such as glucose, blood, bilirubin, nitrate and leukocyte esterase. The urine dipstick's package insert will provide information about potential interfering substances, including ascorbic acid.Intensely colored urine may make it difficult to correctly interpret color reactions on the dipstick, as illustrated below. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

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Clinical Significance of Urine Protein

The presence of an increased amount of protein in a urine specimen is often the first indicator of renal disease. Proteinuria may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria. Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dL. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

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Clinical Significance of Urine Protein (continued)

Individuals with diabetes mellitus may excrete small amounts of albumin in the urine (microalbumin) which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Both type I and type II diabetes mellitus are leading causes of renal failure. Microvascular damage caused by excessive renal exposure to glucose can lead to diabetic nephropathy. By the time the urine protein level reaches the 30 mg/dL level that is necessary for detection by routine reagent strips, damage to the kidneys may have already occurred. Reagent strips are available that use a dye-binding technique rather than the traditional protein-error of indicators principle. These strips are more sensitive and specific for albumin, detecting levels as low as 8 mg/dL.Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms.Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness. Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, that occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein.

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Clinical Significance of Glucose in the Urine

In a healthy individual, almost all of the glucose filtered by the renal glomerulus is reabsorbed in the proximal convoluted tubule. The amount of glucose reabsorbed by the proximal tubule is determined by the body's need to maintain a sufficient level of glucose in the blood. If the concentration of blood glucose becomes too high (160-180 mg/dL), the tubules no longer reabsorb glucose, allowing it to pass through into the urine. It is important to note that glucose may appear in the urine of healthy individuals after consuming a meal that is high in glucose. Fasting prior to providing a sample for screening eliminates this problem. Conditions in which glucose levels in the urine are above 100 mg/dL and detectable include: diabetes mellitus and other endocrine disordersimpaired tubular reabsorption due to advanced kidney diseasepregnancy - glycosuria developing in the 3rd trimester may be due to latent diabetes mellituscentral nervous system damagepancreatic diseasedisturbances of metabolism such as, burns, infection or fractures

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Clinical Significance of Positive Urine Ketones

Ketone bodies are usually absent in urine. The presence of ketones in the urine most likely indicates that the body is using fats rather than carbohydrates for energy. For example, high levels of ketones may be present in the urine of individuals with uncontrolled diabetes because the body's ability to metabolize carbohydrates is defective. Detecting the presence of ketones in the urine is a valuable aid to managing and monitoring individuals with diabetes mellitus. Ketonuria is an indication that the insulin dose needs to be increased. Electrolyte imbalance and dehydration may occur when ketones accumulate in the blood. If these conditions are not corrected by adjusting the dose of insulin, the patient may develop ketoacidosis and ultimately diabetic coma. Low levels of ketones may also be detected in the urine during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake of carbohydrates or increased loss of carbohydrates.

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Introduction to Hematuria

The term hematuria is used to describe the presence of intact red blood cells in the urine. The urine may be cloudy/red or pink in color and red blood cells are visible upon microscopic examination.If the red blood cells have been destroyed, hemoglobin will be excreted in the urine. The term, hemoglobinuria, is used to describe this condition. The color of the urine will be pink or red but clear rather than cloudy. The presence of only five red blood cells per microliter of urine is considered to be clinically significant. For this reason, a chemical test is needed to detect quantities of blood too small to change the color of the urine. Microscopic examination is used to differentiate between hematuria and hemoglobinuria if the chemical reagent strip is positive for blood.

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Reagent Test Pad for Blood

The test for blood on the urine chemical reagent strip is based on the peroxidase-like activity of hemoglobin which catalyzes the reaction of cumene hydroperoxide and 3, 3', 5, 5' tetramethylbenzidine. The test is sensitive to free hemoglobin, myoglobin and a minimum of 5 intact red cells per microliter of urine.

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False Positive and Negative Results

False Positives:A false positive result for blood on the urine chemical reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can also result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity.The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test. False Negatives:False negative results may occur in some analysis methods when the concentration of ascorbic acid is greater than 5 mg/dL. The sensitivity of the blood portion of the test strip is decreased in specimens with a high specific gravity and increased protein. High levels of nitrites may delay the reaction, causing a false negative to be reported. If the pH of a urine sample is below 5, hemolysis of red cells as part of the test reaction is inhibited which results in a false negative reaction. An improperly mixed specimen may test negative if the red blood cells are in the sediment.

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Clinical Significance of Blood in Urine

Blood is normally not present in the urine of healthy individuals, apart from blood during menses that may be detected in urine samples from females, Hematuria is associated with renal or genital disorders in which the bleeding is the result of irritation to the involved organs or some type of trauma. Examples include:Renal calculiPyelonephritisGlomerulonephritisTumorsTraumaExposure to toxic chemicals or drugsStrenuous exerciseHemoglobinuria may be due to the lysis of red blood cells within the urinary tract. This can be caused by intravascular hemolysis, as the hemoglobin is filtered through the glomeruli. In a healthy, normal individual, the hemoglobin molecule attaches to haptoglobin and bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, hemoglobin passes into the urine. Hemoglobinuria may be associated with:Hemolytic anemiaSevere burnsTransfusion reactionInfection Strenuous exercise

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Which of the following substances can cause a false positive result for blood on the urine chemical reagent strip?View Page
Match the following positive urine blood results with the possible conditions which cause them to occur.View Page
Introduction to Urobilinogen

Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal levels of urobilinogen and the patient should be evaluated further. It is important to note that the chemical reagent strip cannot determine the absence of urobilinogen, so a negative result is impossible.

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Chemical Screening of Urine by Reagent Strip (retired March 2012)
Which of the following tests included on a urine reagent strip would never be reported out as negative?View Page
Match the following reagent strip tests to the disease or disorder that would most likely cause a positive test result.View Page
Procedural Considerations

Although the procedure is simple to perform, accurate results depend on careful adherence to manufacturer's directions and adequate quality control. Normal and abnormal controls should be tested whenever a new lot of strips is opened, and at the frequency defined by the laboratory's procedure. If quality control results do not correspond to the published control values, the problem must be resolved before patient samples are tested. High levels of ascorbic acid (Vitamin C) in the urine may inhibit some reagent strip reactions, such as glucose, blood, bilirubin, nitrate and leukocyte esterase. The urine dipstick's package insert will provide information about potential interfering substances, including ascorbic acid. Intensely colored urine may make it difficult to correctly interpret color reactions on the dipstick, as demonstrated in the image on the right. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

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Clinical Significance cont'd

Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dl. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

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Clinical Significance cont'd

Individuals with diabetes mellitus may excrete small amounts of protein in the urine which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms.Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness.

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Clinical Significance

In the healthy individual, almost all of the glucose filtered by the renal glomerulus is reabsorbed in the proximal convoluted tubule. The amount of glucose reabsorbed by the proximal tubule is determined by the body's need to maintain a sufficient level of glucose in the blood. If the concentration of blood glucose becomes too high (160-180 mg/dL), the tubules no longer reabsorb glucose, allowing it to pass through into the urine. It is important to note that glucose may appear in the urine of healthy individuals after consuming a meal that is high in glucose. Fasting prior to providing a sample for screening eliminates this problem.

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Clinical Significance of Positive Urine Ketone Result

Ketone bodies are usually absent in urine. The presence of ketones in the urine probably indicates that the body is using fats rather than carbohydrates for energy. High levels of ketones may be present in the urine of individuals with uncontrolled diabetes because the body's ability to metabolize carbohydrates is defective. Detecting the presence of ketones in the urine is a valuable aid to managing and monitoring individuals with diabetes mellitus. Ketonuria is an indication that the insulin dose needs to be increased. Electrolyte imbalance and dehydration occur when ketones accumulate in the blood. If these conditions are not corrected by adjusting the dose of insulin, the patient may develop ketoacidosis and ultimately diabetic coma. Low levels of ketones may be detected during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake of carbohydrates or increased loss of carbohydrates.

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Hematuria

The term hematuria is used to describe the presence of intact red cells in the urine. The urine may be cloudy/red or pink in color and red cells are visible upon microscopic examination. If the red cells have been destroyed, hemoglobin will be excreted in the urine. The term, hemoglobinuria, is used to describe this condition. The color of the urine will be pink or red but clear rather than cloudy. The presence of only five red blood cells per microliter of urine is considered to be clinically significant. For this reason, a chemical test is needed to detect quantities of blood too small to change the color of the urine. Microscopic examination is used to differentiate between hematuria and hemoglobinuria if the reagent test strip is positive for blood.

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The Test for Blood

The test for blood on the urine reagent strip is based on the peroxidase-like activity of hemoglobin which catalyzes the reaction of cumene hydroperoxide and 3, 3', 5, 5' tetramethylbenzidine. The test is sensitive to free hemoglobin, myoglobin and a minimum of 5 intact red cells per microliter of urine.

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False Positive Results

A false positive result for blood on the reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity. The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test.

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False Negative Results

False negative results may occur with some methods when the concentration of ascorbic acid is greater than 5 mg/dL. The sensitivity of the blood portion of the test strip is decreased in specimens with a high specific gravity and increased protein. High levels of nitrites may delay the reaction, causing a false negative to be reported. If the pH of a urine sample is below 5, hemolysis of red cells as part of the test reaction is inhibited which results in a false negative reaction. An improperly mixed specimen may test negative if the red blood cells are in the sediment.

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Clinical Significance

No blood is found in the urine of healthy individuals although samples from menstruating females, frequently, but not always, test positive for blood. Hematuria is associated with renal or genital urinary disorders in which the bleeding is the result of irritation to the involved organs or trauma. Examples include renal calculi, pyelonephritis, glomerulonephritis, tumors, trauma or exposure to toxic chemicals or drugs and/or strenuous exercise. Hemoglobinuria may be due to the lysis of red cells within the urinary tract. If it is caused by intravascular hemolysis, the hemoglobin is then filtered through the glomeruli. In the normal individual, the hemoglobin molecule attaches to haptoglobin and in this way bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, as in cases of hemolytic anemia, severe burns, transfusion reaction, infection or strenuous exercise, hemoglobin passes into the urine.

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Which of the following substances cause a false positive result for blood on the urine reagent strip? (Choose ALL of the correct answers)View Page
Which of the following substances may cause a false negative result for blood on the urine reagent strip? (Choose ALL of the correct answers)View Page
Urobilinogen

Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal and the patient should be evaluated further. It is important to note that the reagent strip cannot determine the absence of urobilinogen.

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Chemistry / Urinalysis Question Bank - Review Mode (no CE)
Match collection tube colors and additive type on the right with clinical usage on the left.View Page
Which of the following anticoagulants will not produce a significant effect on calcium levels in plasma:View Page
Increases in blood ammonia levels would be expected in which of the following conditions:View Page
Which of the following blood additives is most useful for serum collection:View Page
The measurement of total glycosylated hemoglobin A1c is an effective means of assessing the average blood glucose levels:View Page
Carbon dioxide is predominately found in blood in the form of:View Page
Respiratory acidosis is associated with:View Page
Which one of the following serum constituents is increased following strenuous exercise:View Page
Which one of the following statements about lead poisoning is false:View Page
The primary mechanism responsible for glomerular filtration is:View Page
The renal threshold is best described as:View Page
Which one of the following statements about urea is false:View Page

Confirmatory and Secondary Urinalysis Screening Tests
Urine Bilirubin

Bilirubin is a degradation product of hemoglobin. When red blood cells (RBCs) have reached the end of their normal life span (approximately 120 days), they are destroyed in the spleen and liver. Hemoglobin that is freed in the process is further broken down into iron, protein, and protoporphyrin. Protoporphyrin is converted to bilirubin and released into the circulation. Bilirubin binds to albumin and is transported in the blood to the liver. This unconjugated bilirubin is insoluble in water and cannot be filtered through the glomerulus of the kidney. Bilirubin is then conjugated with glucuronic acid in the liver. This conjugated bilirubin is water soluble and is excreted by the liver through the bile ducts and into the duodenum; bilirubin does not normally appear in the urine. However, if the normal degradation cycle is disrupted, as happens with cirrhosis, hepatitis, and other conditions that damage the liver, conjugated bilirubin will appear in the urine. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma bilirubin level is increased.

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Urine Bilirubin

Normally, small amounts of conjugated bilirubin, regurgitate back from the bile duct and enter the blood stream, so small amounts of conjugated bilirubin can be found in the plasma. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma level is increased. Normally, no detectable amount of bilirubin (sometimes referred to as "bile") is found in the urine.

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Confirmation of Urine Bilirubin Result

The reagent strip test for bilirubin may not be sensitive enough to detect small amounts of bilirubin in urine, which may be present in the earliest phases of liver disease or viral hepatitis. The Ictotest® reagent tablet is more sensitive and is recommended when bilirubin in the urine is particularly of interest. False-positive results can occur in screening procedures for bilirubin due to color interference from large amounts of blood in the urine, very concentrated urine, or drugs that discolor the urine, such as phenazopyridine (Pyridium). Because of this, it is important to verify positive or questionable bilirubin results with a confirmatory method, such as the diazo tablet test, available commercially as the Ictotest®. Ictotest® will detect as little as 0.05-0.10 mg bilirubin/dL urine, making it the procedure of choice for confirming bilirubin in urine specimens.

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Urine Glucose

The presence of significant amounts of glucose in the urine is called glycosuria (or glucosuria). The amount of glucose present in urine is dependent upon the blood glucose level, the rate of glomerular filtration, and the degree of tubular reabsorption of the sugar. Usually glucose will not be present in the urine until the blood level exceeds 160-189 mg/dL, which is the normal renal threshold for glucose. The main reason for glycosuria is an elevated blood glucose level (hyperglycemia). Diabetes mellitus is the most common cause of hyperglycemia. However, stress, obesity, brain injury, myocardial infarction, hyperthyroidism, pregnancy, and a lowered renal threshold due to kidney damage can all cause glycosuria.

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Causes of Ketonuria

Ketonuria occurs when fatty acids are moved from triglyceride stores in the body in response to inadequate intake or availability of carbohydrates. Under conditions of abnormal carbohydrate metabolism, such as occurs in diabetes mellitus, ketones accumulate in the blood (ketonemia) and are excreted in the urine (ketonuria). The accumulation of ketones is often the cause of acidosis and coma in diabetics. Ketonuria is also associated with:StarvationDigestive disturbancesDietary imbalance (high fat/low carbohydrate diet)EclampsiaProlonged vomiting and diarrheaGlycogen storage diseasesSevere, sustained exerciseFeverProlonged exposure to cold temperaturesKetones are mildly toxic to the body, tending to interfere with the excretion of uric acid, produce mild depression of the central nervous system, and cause acidosis.

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Acetest®

Acetest® tablets can be used for the semiquantitation of ketones in urine, serum, or whole blood. However, an assay for serum/plasma beta-hydroxybutyrate (BHB) is very important in the assessment of diabetic ketoacidosis as BHB is the predominant ketone body and the most sensitive marker for detection of acidosis. Because the nitroprusside method (reagent strips and Acetest) do not measure BHB, a specific test for this ketone body is needed.Urine to be screened for ketone bodies using the Acetest method should be tested immediately or refrigerated in a closed container since acetone is lost to the air if the sample is held at room temperature for a prolonged period.

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Dermal Puncture and Capillary Blood Collection
Patient Identification

Patient safety when performing a capillary blood collection includes positive patient identification prior to performing the procedure. The accepted policy in most health care facilities is to use two forms of identification, including a unique number if possible, such as a hospital number or medical record number.Ideally, the patient (or the parent/guardian if the patient is a small child) should be asked to spell his/her name and state his/her date of birth. This may not always be possible, but it will aid in positive patient identification whenever it can be done.The phlebotomist should LOOK at the patient's paperwork while they LISTEN to the patient's response. For inpatients, the patient identification bracelet, which must be attached to the patient's wrist or ankle, should be used to verify patient identity. A hospital number recorded on the bracelet may be used as a second identifier in the case of an inpatient.Paying close attention to these details and correcting any discrepancy discovered will greatly reduce the risk of misidentifying a patient. Always follow the policy of your facility for identification and never shortcut the patient identification procedure.

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Site Preparation

Once the phlebotomist has successfully identified the patient, the next step of the dermal puncture procedure is to locate and determine a site suitable for puncture. If a heelstick in an infant is being performed, the phlebotomist should apply a warming device for approximately 3-5 minutes to the heel to increase blood flow to the area, which will facilitate the collection of the capillary specimen. The use of a warming device is also recommended when a fingerstick is performed, if the hands are cool to the touch.A heat-standardized, pre-packaged, chemically activated heel warmer, or comparable heating agent should always be used to warm the heel of an infant to prevent scalding or burning. The temperature of the heating device should not exceed 42°C.Caution-- do not use a cloth that has been moistened and warmed in a microwave oven. This may have hot spots that could cause injury to the patient. It is also not advisable for the phlebotomist to hold a patient's hand under hot running water. This again could cause an injury. If feasible, the patient could be instructed to warm his or her own hands under running water, but allow the patient to adjust the water temperature.

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Let Gravity Be Your Friend

To encourage the flow of blood to the hand when performing a capillary collection, allow gravity to work in your favor! Position yourself so that you are holding the patient's hand in such a manner that the finger is pointed downward. Gravity will draw the blood to the fingertip and allow the procedure to be completed more efficiently.

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Finger Puncture

Procedural StepCommentCautionGreet and positively identify patientAlways use at least two patient identifiers to ensure positive patient identification.Never rely on name placards that are placed on or near the patient's bed or crib to identify the patient.If there is a discrepancy in identification, do not proceed until the discrepancy is resolved. Explain the procedureIf the patient is a small child, be at eye level when explaining the procedure. Also explain the procedure to the parent(s). If the patient is aware of what will be happening there is less chance of the patient suddenly jerking away his/her hand when the puncture occurs.Position patient appropriatelyAn outpatient who is a small child should sit on the parent's lap. If necessary, seek assistance for finger puncture if the patient is a small child. Cleanse hands and put on glovesUse soap and water or alcohol-based gel to cleanse hands. Cleanse hands before donning gloves and after removing gloves.Warm puncture site if neededUse the method that is approved by the laboratory for prewarming the puncture site.Never use a moist cloth that has been heated in a microwave as this may cause injury to the patient.Gather appropriate equipmentOnly have needed equipment at hand.Keep track of ALL equipment to prevent patient injury.Cleanse the puncture siteUse 70% isopropanol unless the patient is sensitive to alcohol.Allow the site to air dry. Performing the puncture before the alcohol has dried may hemolyze the blood specimen. Securely grasp and puncture fingerPuncture the side edge of the fleshy pad of fingertip.Avoid extreme side and tip of finger.Discard puncture device into appropriate containerPuncture device should be discarded into a sharps container that is puncture-proof, has rigid sides, and has a lid. Do not discard puncture devices into regular trash or biohazard bags; injury to personnel who handle these bags may occur. Wipe away the first drop of bloodUse slight pressure to facilitate blood flow.The first drop of blood contains tissue fluid that may contaminate or dilute the blood specimen and affect test results.Collect blood into containerAllow blood to flow freely into the collection device. Tap the container gently on a hard surface to move blood further down into the tube if necessary. Do not "milk" the finger or scrape the collection device across the finger to obtain specimen; both actions may cause the specimen to hemolyze. Mix specimen immediately upon completion of the collection. Apply pressure to the puncture site to stop the bleeding.Use gauze to apply pressure to the puncture site.It is not advisable to apply an adhesive bandage over the skin puncture site if the child is less than two years old as the child may place the bandage in his/her mouth. Label specimenSpecimen must be labeled in the presence of the patient.Unlabeled specimens will be rejected by the laboratory.

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Heel Puncture

The heel of the foot is the preferred site for dermal puncture and capillary blood collection for infants less than 12 months old. CAUTION: In premature infants, the bone may be as close as 2.0 mm under the skin of the plantar surface of the heel. The bone may be even closer--maybe half this distance-- on the back curve of the heel. Any puncture more than 2.0 mm may risk a puncture of the bone causing severe consequences to the infant. Only use approved preemie puncture devices on small infants.Procedural StepCommentCautionPositively identify patientAlways use at least two patient identifiers to ensure positive patient identification.Never rely on name placards that are placed on or near the infant's crib to identify the patient.If there is a discrepancy in identification, do not proceed until the discrepancy is resolved. Position patient appropriatelyPosition the infant so that the heel can be easily accessed.If necessary, seek assistance to stabilize baby's foot during the blood collection.Cleanse hands and put on gloves and any other required PPE.Use soap and water or alcohol-based gel to cleanse hands. Cleanse hands before donning gloves and after removing gloves.Choose puncture siteUse the area of heel that is not striped (the white area) in the image on the left.Do not use the center portion of the heel, the arch of the foot, or toes as any of these sites may cause injury to nerves, tendons, and cartilage.Warm puncture site if neededUse only approved warming device.Never use a moist cloth that has been heated in a microwave as this may cause injury to the patient.Gather appropriate equipmentOnly have needed equipment at hand.Keep track of ALL equipment to prevent patient injury.Cleanse the puncture siteUse 70% isopropanol.Allow the site to air dry. Performing the puncture before the alcohol has dried may hemolyze the blood specimen. Securely grasp and puncture the heel.Choose either side of the fleshy part of heel.Avoid center of heel and arch of the foot.Discard puncture device into appropriate containerPuncture device should be discarded into a sharps container that is puncture-proof, has rigid sides, and has a lid Do not discard puncture devices into regular trash or biohazard bags. Injury to personnel who handle these bags may occur.Wipe away the first drop of bloodUse slight pressure to facilitate blood flow.The first drop of blood contains tissue fluid that may contaminate or dilute the blood specimen and affect test results.Collect blood into containerAllow blood to flow freely into the collection device. Tap the container gently on a hard surface to move blood further down into the tube if necessary. Do not "milk" or squeeze the heel excessively. Do not scrape the collection device across the heel to obtain specimen; these actions may cause the specimen to hemolyze. Mix specimen immediately upon completion of the collection to prevent clots. Apply pressure to the puncture site to stop the bleeding.Use gauze to apply pressure to the puncture site.Use a bandage only if this is an acceptable procedure in your facility.Label specimenSpecimen must belabeled in the presence of the patient.Unlabeled specimens will be rejected by the laboratory.

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Properly Filling and Mixing a Microcollection Container

It is important that the container is filled to the appropriate level when blood is collected into a microcollection container that has an anticoagulant. The device should then be capped and the blood mixed well immediately following collection of the specimen. The manufacturer of the containers that are used specifies what is considered adequate mixing and the laboratory's collection procedure should be based on these recommendations. Mixing involves a gentle tapping on a hard surface to move the blood further down into the tube during collection and then capping the tube upon completion of the collection so that the tube can be mixed end-over-end for the specified number of times as shown in the image on the right. The correct fill is also important. A container that is overfilled will not be properly anticoagulated and clots may form that will affect the laboratory test results. A container that is underfilled may not contain sufficient specimen to perform the test(s) or the excess anticoagulant may interfere with the test. For example, excess anticoagulant could cause morphologic changes in blood cells.

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Order of Draw

The order of draw for a capillary blood collection is slightly different than the order of draw for a venous blood collection.If capillary blood gases are ordered, they are drawn first to avoid introduction of room air as much as possible. A specimen for blood count is collected before tubes containing other anticoagulants and additives. This is to ensure that the blood will not begin to clot before this specimen is collected; clots will affect the accuracy of the blood count. The following order of draw is commonly used: ContainerAdditiveUseLavender topEDTAFor hematology blood countsGreen top Lithium heparinTests that require a heparinized plasma sample__Other tubes containing anticoagulantsVariedRed or gold topClot activatorTests that require a serum sampleRed top No additiveTests that require a serum sample but clot activator and/or gel may affect test

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Positioning the Puncture Device for a Fingerstick

The fingerstick device should be held firmly against the puncture site. To obtain the best capillary specimen using the finger, align the puncture device perpendicular (horizontal) to the whorls of the fingerprint. This cross-cut of the fingerprint whorls causes the blood to bead at the puncture site, allowing the phlebotomist to efficiently collect the drops of blood into the container. This image illustrates the correct position of the cut in relation to the fingerprint lines.If the puncture is made parallel to the fingerprint whorls (as shown below), the blood will not bead but rather it will travel down the channels between the lines of the fingerprint. This makes it difficult to collect the blood into the container. The phlebotomist may inadvertently "scrape" the blood from the skin while filling the container, resulting in hemolysis and/or clotting of the specimen.The tip of the finger should be avoided. Puncturing the fingertip may cause unnecessary discomfort to the patient.

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Which of these methods should be used to verify the identification of an infant in the nursery prior to collecting a blood specimen?View Page
A lavender top microcollection container that has EDTA as an anticoagulant is used to collect a capillary hematology specimen for a complete blood count (CBC). If additional specimens are also collected with the same dermal puncture into a green top and a red top container, at what point should the lavender top for the CBC specimen be collected?View Page
Protect Yourself and Your Patient

It is important to remember that the collection of a specimen by dermal puncture may involve the potential of exposure to bloodborne pathogens as well as other safety considerations for both the phlebotomist and the patient. Some important safety reminders are listed in the table below.Safety ReminderReasonCommentGloves are always necessaryBlood contaminates the skin during a capillary blood collection. Gloves protect the phlebotomist from potential exposure to bloodborne pathogens.Gloves must remain intact to be an effective barrier against exposure to potential pathogens. Wear additional personal protective equipment (PPE), such as lab coat or gown when appropriate or required. Safety goggles and surgical mask may be needed if there is a potential for splashes or sprays of blood.May be needed to protect the phlebotomist or may be required to protect the patient from potential infection in some cases.Safety goggles and mask should both be worn to adequately protect the eyes and mucous membranes from exposure to bloodborne pathogens if there is the potential for splashes or sprays of blood. Only have the equipment needed for this procedure at hand and additional equipment out of reach of the patient. Protects the patient from accidental injuryOften, capillary procedures are performed on very young children who are curious and may grab something that could cause injury.

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Which of these pieces of personal protective equipment (PPE) is always required when a dermal puncture is performed to collect a capillary blood specimen?View Page
References

Bersch C. ed. The ABCs of pre-, neo-, and post-natal testing. MLO. September 2009;41.Clinical and Laboratory Standards Institute (CLSI). Procedures and Devices for the Collection of Diagnostic Capillary Blood Specimens; Approved Standard. Fifth ed. CLSI document H4-A5. CLSI. Wayne, PA: 2004.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third ed. CLSI document H18-A4. CLSI. Wayne, PA: 2010.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.Garza D. Becan-McBride K. Phlebotomy Handbook. 7th ed. Upper Saddle River, NJ: Pearson Prentice Hall: 2005.RHL: The WHO reproductive health library. Sucrose for analgesia in newborn infants undergoing painful procedures. Available at: http://apps.who.int/rhl/newborn/cd001069_murkis_com/en/. Accessed April 9, 2014.

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Using the Heel for Dermal Puncture

A dermal puncture of the heel should only be performed on an infant or small child prior to the age of walking. The age limit for a heel puncture is approximately 12 months of age. After that time, the skin becomes very thick which could prevent the phlebotomist from obtaining a quality specimen for testing. The fleshy bottom of the heel toward the sides are acceptable sites for dermal puncture. Note in the illustration that the white areas are acceptable sites for heel puncture; any area that is red-striped in the image should not be used for blood collection.DO NOT puncture the central area of the bottom of the foot (arch of the foot), the back curvature of the heel, or the toes. These sites must be avoided to prevent damage to nerves, tendons, and cartilage.

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The recommended finger for capillary blood collection is the index finger (finger next to the thumb).View Page
Venous, Arterial, and Capillary Blood Specimens

Venous BloodVenous blood is deoxygenated blood that flows from tiny capillary blood vessels within the tissues into progressively larger veins to the right side of the heart. Venous blood is the specimen of choice for most routine laboratory tests. The blood is obtained by direct puncture to a vein, most often located in the antecubital area of the arm or the back (top) of the hand. At times, venous blood may be obtained using a vascular access device (VAD) such as a central venous pressure line or Hickmann Catheter or an IV start. Most laboratory reference ranges for blood analytes are based on venous blood.Arterial BloodDeoxygenated blood is pumped from the right side of the heart to the lungs where it takes up oxygen. The now oxygenated blood is pumped through the left side of the heart via arteries.The most common reason for collection of arterial blood is the evaluation of arterial blood gases. Arterial blood may be obtained directly from the artery (most commonly, the radial artery) by personnel who are trained to perform this procedure and are knowledgeable about the complications that could occur as a result of this procedure. Arterial blood may also be obtained from a vascular access device (VAD) inserted in an artery such as a femoral arterial line or Swan-Gantz catheter. Capillary BloodCapillary blood is obtained from capillary beds that consist of the smallest veins (venules) and arteries (arterioles) of the circulatory system. The venules and arterioles join together in capillary beds forming a mixture of venous and arterial blood. The specimen from a dermal puncture will therefore be a mixture of arterial and venous blood along with interstitial and intracellular fluids.Capillary blood is often the specimen of choice for infants, very young children, elderly patients with fragile veins, and severely burned patients. Point-of-care testing is often performed using a capillary blood specimen. Specimen Type Method of Collection Common Use Venous Direct puncture of vein by venipuncture; vascular access device Routine laboratory tests Arterial Direct puncture of artery; vascular access device Arterial blood gases Capillary Dermal puncture of fingertip or heel Infants and young children Elderly patients with fragile veins Severly burned patients Point-of-care testing

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Dermal Puncture vs Venipuncture

In some situations, the phlebotomist will make the decision if a blood specimen will be obtained by dermal puncture or venipuncture. The patient's condition, the age of the patient, the amount of blood needed for testing, and the risks associated with the procedure will help the phlebotomist determine the best method for collection.A dermal puncture requires less precision, therefore it is less critical for the patient to be still or immobilized. However, if the puncture is not performed correctly, or an approved site is not used, the puncture may cause more discomfort, or even injury to the patient.The risk of accidental needlestick injury to the patient and phlebotomist is minimal since the puncture device is designed to retract the needle once the puncture is made. The puncture is quick and standardized for puncture depth. However, the procedure takes longer to complete. This delay in collection of the blood specimen could result in hemolysis or clotting of the blood or tissue fluid contamination of the specimen and specimen rejection by the laboratory.The dermal puncture minimizes the amount of blood taken from the patient. This will be important to consider, especially with infants in an intensive care nursery. However, some laboratory tests require larger amounts of blood for testing; in these cases, capillary collection is not an option.If a patient is dehydrated or has poor peripheral circulation, an adequate blood collection from a dermal puncture may not be possible.

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Which of these patients may not be a candidate for capillary blood collection by dermal puncture?View Page
Miscellaneous Equipment

In addition to the puncture device, additional equipment is required to perform a safe and successful dermal puncture and to collect an acceptable specimen. This may include any of the items discussed below.Plastic microcollection devices: Plastic microcollection devices are small plastic tubes designed to collect capillary blood from a dermal puncture wound. Each small collection tube is color-coded in the same manner as blood collection tubes used for venipuncture. The color of the cap of each container tube corresponds to the type of additive inside the tube, most often an anticoagulant. The additive coats the inside of the tube. Examples of microcollection devices are shown below. Heel warmer: It is best practice to warm the heel of an infant with a warming device known as a heel warmer. The heel warmer, when activated, is designed to warm its contents to a standardized temperature. This temperature will be hot enough to effectively warm the heel and facilitate blood flow to the area without causing heat injury to the patient. It is unacceptable to warm a cloth using a microwave. There may be "hot spots" on the cloth that could potentially burn the patient. Keep in mind, what may feel warm to you, the phlebotomist, may feel hot to your patient!Plastic or Mylar-wrapped capillary tube: In some facilities blood from a capillary puncture is collected directly into a capillary tube. These tubes are very delicate and must be used with great caution. As soon as the tube is two thirds to three-fourths filled, one end is sealed to prevent blood from leaking out.Glass microscope slides: In some facilities, the person collecting the capillary specimen may also be required to prepare a blood smear for laboratory examination. A drop of blood is placed directly on a glass slide and spread to create an area for cell examination. If you are required to prepare blood smears, remember that the slide is considered infectious until fixed or stained. It is also important to remember that glass is a sharps hazard. If not used correctly, the glass may cause injury to both the patient and the phlebotomist. Be as cautious with a glass slide containing blood as you are with a contaminated needle. Dispose of glass slides that will not be used for testing in approved sharps containers.Alcohol and gauze pads: Alcohol is the disinfectant of choice for dermal puncture. The alcohol must be allowed to air dry, which will prevent hemolysis of the specimen and discomfort for the patient. A piece of clean or sterile gauze is used to wipe away the first drop of blood. Gauze is also used to apply pressure to the wound after the specimen collection is complete to stop the wound from bleeding.Iodine or other approved cleaning agents may be used as an alternative to alcohol.Bandage: It may be necessary to apply a bandage to the puncture wound on a finger or heel if the site continues to bleed. However, it is NOT recommended to bandage the finger of a child who is 2-years-old or younger since the bandage may become a choking hazard if the child puts that finger in his/her mouth.Personal protective equipment (PPE): All health care professionals that may come in contact with blood and/or body fluids while performing a laboratory procedure are required to wear intact gloves. It is against safety guidelines to alter gloves in any way that may compromise the integrity of the gloves. Eye protection, such as safety goggles, is recommended if there is the possibility of a splash of blood while collecting a capillary blood specimen. In many facilities, special gowns are required in some patient areas such as special-care nurseries. Always follow the policies of your facility in regard to PPE.

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Oh No...The Blood Has Stopped Flowing

On occasion, blood may stop flowing from the punctured site before the required amount of blood is obtained. When this happens, it is not recommended to squeeze harder. This only serves to cut off the supply of blood to the capillary bed. Additionally, squeezing with too much force, especially on the heel of an infant, may cause injury to the patient. The phlebotomist should never scrape the skin with the collection device in an attempt to scoop up the blood that is laying on the surface of the finger or heel. This could cause the blood specimen to hemolyze, making the specimen unacceptable for some laboratory tests. Always allow the drop to flow freely into the collection tube.If a clot has formed, an attempt could be made to dislodge it and re-establish blood flow by wiping the puncture site again with a new alcohol pad, massaging the finger or heel gently, and attempting to recollect the specimen once the alcohol has dried. If blood is not flowing freely from the initial puncture, it may be necessary to perform a second puncture to obtain enough blood for the testing required. If a second puncture must be performed, do not repuncture the same site.

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Inappropriate Sites/Patients for Capillary Puncture

There are some instances where a dermal puncture is prohibited or not recommended.Mastectomy patients As a general rule, a dermal puncture, or a venipuncture, should not be performed on the side affected by a mastectomy. The body's ability to fight infection is compromised if lymph nodes were removed. A physician's permission must be obtained before performing a blood collection procedure on the same side as a mastectomy. Edematous site Dermal punctures should not be performed on previously punctured sites or swollen sites. Excess tissue fluid may contaminate the specimen.Dehydrated patient If the patient is dehydrated or has poor circulation, it may be impossible to get a quality specimen. Fingerstick on a newborn or young infant Dermal punctures must never be performed on the fingers of a newborn or very young infant (usually defined as under 12-months-old). There is very little distance between the skin and the bone. Therefore, the bone could be easily pierced during the puncture, causing injury to the bone, infection, or gangrene.

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Infants and Geriatric Patients: Monitor the Amount of Blood Obtained

The collection of a capillary blood specimen is often used on newborns and geriatric patients. These two groups are most susceptible to blood depletion. Therefore, a dermal puncture is preferred over venipuncture where too much blood may be inadvertently collected.In some facilities, the amount of blood obtained from a patient will be charted or recorded after every procedure. This may become part of the patient's medical record and is usually entered by the nursing staff. In these cases, the nurse will interact with the laboratory staff to advise them of the safe amount of blood that can be obtained.

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Protect Me From the Light

Some specimens routinely collected for testing by using a capillary puncture are adversely affected by exposure to light. One example is a specimen collected for bilirubin testing that is obtained from a newborn. When obtaining the specimen for this testing, it is important for the phlebotomist to recognize the effect of light on the specimen. Room light or sunlight can metabolize the bilirubin in the specimen to a different compound. This will cause a falsely lower bilirubin level. A neonatal bilirubin specimen should be obtained in a dark-colored (amber) container. Alternately, a clear or white container can be immediately wrapped in aluminum foil following the blood collection, preventing the blood from exposure to light.

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If blood has stopped flowing from the finger puncture site, you should repuncture the same site to re-establish the blood flow.View Page
The Need for Metabolic Testing on Newborns

Many state governments in the United States mandate that all newborns be tested for metabolic disorders very soon after birth. This required testing is used to determine if the infant has a metabolic disorder that could adversely affect a child's development. If discovered early, many of the effects of the metabolic disorder can be alleviated or averted. Not every state tests or screens for the same disorders, so the phlebotomist must be certain to understand the requirements for the state in which they reside. There is a movement to standardize testing throughout the United States.Typically, the method used to screen for the presence of newborn metabolic disorders is collection of capillary blood on a filter paper card. It is imperative that the phlebotomist follows the very specific directions for the collection of these samples. If a specimen is submitted to the state laboratory for testing and deemed unacceptable, the specimen would have to be re-collected. The infant would then have to be subjected to a second invasive puncture procedure, causing stress and trauma to the infant as well as the parents. More importantly, the need to obtain a second specimen can also cause a delay in treatment.

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Capillary Blood Collection for Metabolic Testing

The collection of these specimens requires the same attention to detail as with any phlebotomy procedure. Gather all necessary equipment Be certain to choose a device that punctures the heel to a depth appropriate to the size of the infant. Only use the filter cards provided by your state to collect the specimen. These cards are calibrated to the exact specifications needed for testing of metabolic disorders. An alternate or homemade card must not be used. Put on all necessary personal protective equipment Gloves are always required. Gowns and eye protection may also be required. Positively identify the patient Use two identifiers. The infant who is in the nursery should have an identification band attached to the ankle or wrist. In special care nurseries an alternate form of identification may be used. However, a crib card should never be used as a form of identification. Follow the practice for your facility. Position the infant Be certain that the heel can be easily accessed. Follow all nursery requirements that apply to safe handling of newborns. Warm the heel using an approved warming device Clean the site with alcohol or the approved disinfectant. Allow the site to air dry before proceeding with collection of the specimen. Grasp the heel firmly but not tightly, activate the puncture device, wipe away the first drop of blood, and begin collection of the specimen.Allow the blood to wick onto the card. Completely saturate the circle with one continuous drop of blood. Avoid touching the card to the skin. Apply the blood only to one side of the card. Do not layer the blood by applying a second drop on top of the first. Repeat the procedure to completely fill each circle on the card. Each circle should be completely and uniformly saturated as shown in the bottom image on the right. Follow the policy of your institution or state to determine how many circles must be completely filled. Apply pressure to the puncture site using a sterile gauze Gently raising the infant's leg above the level of the heart will also aid in clotting the puncture site. Bandage according to site-specific policy.

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Processing Filter Cards after Collecton

Once the required circles on the filter card have been completely saturated with blood and the care of the infant is complete, all necessary patient demographic information on the card must be completed.Every item included in the demographic section must be completed accurately. The age of the infant is important and is often recorded in hours and/or days. Contact information for the parent or guardian as well as the primary care giver is also important so that this information can be used by public health officials to initiate and track the follow-up treatment for the patient. When completing the demographic section of the card, it is advisable to use a ball point pen. Soft or felt tip pens can be absorbed by the filter paper and can possibly affect the test results.Cards should be allowed to air dry completely. Never stack cards in such a way that will allow the blood drops of the cards to come in contact with each other. This could result in transfer of one patient's blood to another patient's card. Many facilities have special racks in which to place cards while drying to avoid the contamination of specimens. After the cards are dry, they should be delivered in a timely manner to the state testing facility.

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Hematology Specimens

In some institutions, the phlebotomist is responsible for collecting specimens that will be directly tested to yield results for hematology studies.Blood Smear FilmsIf it is the practice of the institution, the phlebotomist may make a blood film slide directly from the blood flowing at a dermal puncture site. In this case, a drop of blood is allowed to fall directly onto the glass slide. The image below illustrates the approximate size of the drop that should be used.Using a second glass slide, the phlebotomist should spread the blood by first aligning the edge of the spreader slide in front of the drop of blood, pulling back into the drop so that it is evenly distributed behind the spreader slide as shown in the image below. Then spread the blood forward, maintaining an angle of approximately 20° between the slides. The finished slide should be at least 2.5 cm in length, there should be a gradual transition in thickness from thick to thin, ending in a feather edge. The blood smear should be made at the beginning of the dermal puncture procedure to avoid formation of microclots. Remember that the glass slides used to make the blood smear are considered sharps and can cause accidental puncture injury to both the patient and the phlebotomist. Dispose of the spreader slide in a sharps container. Also, until the smear is stained or fixed, the blood film is considered potentially infectious so bloodborne pathogen precautions must be followed.Microhematocrit collectionIn some institutions, capillary blood specimens are collected directly into heparinized capillary tubes, which are then analyzed to determine packed cell volume. These results can be used to indicate the presence of anemia. At least two capillary tubes should be filled for microhematocrit testing. The capillary tubes should be filled with blood to about two- thirds the length of the tube. One end of each tube should then be sealed to prevent blood from escaping. The sealant may be sealing clay or commercially-provided covers that are made specifically for the microhematocrit system that is in use. Capillary tubes should be plastic or mylar-wrapped glass tubes. Plain glass capillary tubes should not be used to prevent the possible transmission of bloodborne pathogens if the tube broke and punctured through the glove and skin of the phlebotomist.It is imperative that the specimens are labeled appropriately with patient information. This can be accomplished by inserting the capillary tubes into a second larger blood collection tube that is labeled with the patient name and second identifier, such as hospital or medical record number and capping the large tube. Taping the capillary tubes individually to a paper requisition with the patient information is an alternate method.

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Capillary Blood Gases

In some instances, the health care provider may request an analysis of the capillary blood for blood gases. This is most often requested on infants. Collection of this specimen requires a skilled phlebotomist and specialized equipment. The patient must be positively identified. All appropriate PPE must be used. The procedure for site selection, preparation and puncture is identical to other infant dermal punctures, however, capillary blood gases are always drawn first if other capillary blood specimens will be collected.Blood specimens for capillary gases are always collected in long, large-bore heparinized glass tubes. Blood should be drawn into the tube using capillary action. The phlebotomist should start filling the tube using a large well-formed drop of blood, drawing continuously as the blood flows. Each tube must be filled completely end to end as shown in the image on the right. Every effort must be made to avoid drawing air bubbles or air gaps into the tubes as these could adversely affect the results of the test. Before sealing both ends of the tube, the phlebotomist will insert a tiny metal "flea" into the blood-filled tube and slide a magnet lengthwise back and forth on the outside of the tube. The magnet will cause the flea to move back and forth inside the tube mixing the specimen with the anticoagulant coated on the inside of the tube. This technique should also prevent the blood from clotting, which could result in specimen rejection by the laboratory.The properly filled glass tubes must be delivered to the analyzing laboratory in a timely manner. Delay in specimen delivery may adversely affect the quality of the patient results.

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Using Sucrose as an Analgesic Prior to Heel Puncture and Capillary Blood Collection

Recent research has indicated that an appropriate dilution of sucrose solution when administered to an infant may serve as a pain relief measure. In some institutions, the nursing staff may require that an infant receive several drops of sucrose immediately prior to the puncture of a heel. This may release endorphins to relieve pain and reduce crying by the infant. Excessive crying may adversely affect some test results such as white blood cell count and capillary blood gases.If it is the policy of your institution to administer a sucrose solution, coordinate the timing of the dermal puncture with the administration of the sucrose solution by the clinical staff. Outpatients would also require the intervention of a nursing staff member to provide the sucrose solution. Phlebotomists are not licensed to administer medications or drugs. Therefore, it is typically NOT the responsibility or duty of the phlebotomist to administer sucrose solution. There may be contraindications for sucrose administration with some infants. Therefore, the clinical person in charge of the patient's care must determine if it is safe to administer the solution. As with all procedures, follow the policies and guidelines of your facility.

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Lead: An Important Public Health Concern

Lead may be found on surfaces touched by children and adults. Lead may be present in the paint that was used in older homes or apartments, and it has even been detected in the paint used on some toys.Elevated lead levels in children can cause developmental delays. Many state governments closely monitor the presence of lead in children. To accomplish this, government agencies require official forms be completed and submitted for each patient at the time of specimen collection for lead testing. It is the responsibility of both the phlebotomist and healthcare provider to submit the completed form with the specimen. If an elevated lead level is obtained, the government authority can then track and monitor follow-up treatment for the patient. When the phlebotomist determines that a capillary puncture on the finger will be used to collect a specimen for lead testing, it is imperative that the patient's hands be washed with soap and water prior to the start of the collection to ensure the skin is free of any contaminant that could falsely elevate the test result. The patient should thoroughly wash his or her hands or if the patient is a child, the parent or guardian could be asked to assist the child. If necessary, wash the patient's hands yourself.It is important to note that washing hands with soap and water aids in removing surface lead but is not a substitute for the cleaning step in the blood collection procedure. The finger must still be cleansed with alcohol and allowed to dry before a dermal puncture is made.

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Descriptive Statistics
Discrete and Continuous Data

There are two main types of data that you might encounter. The first is discrete data, which is a count of whole events, objects or persons. For example, the number of people with a certain illness is a discrete quantity, ie, countable.The other type of data is continuous data, which is the measure of a quantity such as length, volume, or time, which can occur at any value. For example, the concentration of glucose in the blood is a continuous quantity. Even if the instrument you are using rounds off values to whole numbers, these quantities are still continuous; ie, not countable.

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Samples and Populations

A population is the entire group of persons or objects about which you want to make inferences. A sample is a small portion of that population that you actually test and examine in order to collect data and make those inferences.For example, suppose you wanted to test the average fasting blood glucose value of diabetics in the United States. It would be impossible to test all of them, so you would choose a small sample of them, usually through some random process. Then you would test only that sample, and from that, make an inference about the average glucose value of the whole country's diabetic population.Choosing a sample that is representative of the population, however, is not an easy task. No matter how large a sample is, or how precisely the tests on that sample are carried out, the results are worthless if your sample is biased.

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Table Specifications

Here are the criteria for the preparation of tables, as specified by the Journal of Clinical Laboratory Science:Write table titles at the top of the table. Number tables sequentially with Roman numerals. Include the following information in a title, whenever possible: who, what, where, why and when. Put the independent variable in the left column, and the dependent variable in the right, if you are listing data with independent and dependent variables. Label each column with the appropriate units. Adequately space tables that appear on the same page. Example:Table I Patient specimens analyzed for blood urea nitrogen on the Dimension RxL and the Vitros 250 at City HospitalSample #RxL (mg/dL urea)Vitros 250 (mg/dl) urea18.88.8211.210.0312.413.6416.213.2520.021.2625.020.0728.826.2In this case, the Dimension RxL is the "reference method" and is considered the independent variable, while the Vitros 250 is the "test method" and is considered the dependent variable.

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Using Frequency Distributions

A frequency distribution is a chart that groups data into different classes and then graphically shows how many data points fall into each class. A frequency distribution allows the reader to see easily the approximate center and spread of the data. Table II shows the frequencies of different hemoglobin concentrations. Figure 2 is a histogram of the data. Table II Frequency distribution of blood hemoglobin levels from healthy women determined on the Coulter Gen S Hemoglobin (gm/dL)Number of Women6 - 818 - 10210 - 121012 - 142514 - 16916 - 181Figure 2 Frequency Distribution Blood Hemoglobin Levels from 48 Healthy Women Determined on the Coulter Gen S

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Detecting and Evaluating Coagulation Inhibitors and Factor Deficiencies
Preanalytical Variables That Can Cause Falsely Elevated PT or aPTT Results

Improper collection of the blood specimen that is used for testing can cause false prolongation of PT or aPTT results. The following table covers several preanalytical variables that may affect PT or aPTT test results Preanalytical Variable Cause of False Elevation of PT and or aPTT Test Result Corrective Action Blood collection tube is inadequately filled. Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Recollect specimen ensuring proper fill to achieve a blood to anticoagulant ratio of 9:1. Patient has a hematocrit level above 55% Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Prepare a specimen collection tube that contains less anticoagulant. Refer to your laboratory's procedure for the proper amount of anticoagulant. Specimen is clotted. Coagulation factors have been activated; insufficient levels left in the plasma. PT and aPTT results will be affected. Recollect the specimen. Specimen collected from an arm with a heparin lock or from a heparinized vascular access device (VAD). Heparin contamination will prolong the aPTT. Collect the blood from a vein rather than a VAD. If blood must be drawn from the VAD, flush it first with 5 mL of saline, and discard the first 5 mL of blood before collecting the specimen. Patient is receiving heparin therapy. Heparin will prolong the aPTT If the patient is being evaluated for possible factor deficiencies or coagulation inhibitors, use a heparin digesting enzyme as a pretreatment before testing the PT or aPTT. .

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Which of the following may produce a falsely prolonged aPTT test result?View Page
When To Perform a Mixing Study

A mixing study should be considered when a patient has a prolonged PT and/or aPTT along with: no history of heparin or warfarin therapy no history of liver disease It is also essential to first verify that a correctly collected sample (free from clots) with a proper blood to anticoagulant ratio has been obtained.

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Mixing studies may help to determine the presence of which of the following?View Page
Introduction: Coagulation Inhibitors

As the name implies, coagulation inhibitors (also called circulating anticoagulants) interfere with normal blood coagulation. Coagulation inhibitors may be congenital or acquired (developing in patients during the course of a disease) and are almost always immunoglobulins, either IgG or IgM. There are two types of inhibitors: those directed toward a coagulation factor (or multiple factors) and the lupus anticoagulant. Lupus anticoagulant is one of the more commonly encountered coagulation inhibitors. It is also known as antiphospholipid antibody because it is directed toward phospholipids. Lupus anticoagulant is usually an IgG antibody. It differs from factor-specific inhibitors in that lupus anticoagulant causes thrombosis and abnormal clotting while factor-specific inhibitors cause serious bleeding.

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Diabetes and the Current American Diabetes Association Guidelines
Introduction

Diabetes is a metabolic disorder caused by impaired pancreatic function, resulting in decreased insulin concentration and activity. This causes the patient with diabetes to have elevated blood glucose concentrations (hyperglycemia). Hyperglycemia leads to serious risk factors and life-threatening complications for the individual. Because of these risks and the ensuing chronic illness for diabetic patients, ongoing medical care and education for self-management are required. Diabetes is a national and international healthcare issue due to its high incidence and healthcare costs. According to the World Health Organization (WHO) in 2000, there were 171 million individuals worldwide with diabetes. That number is projected to increase to 366 million by 2030.

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Organizations and Agencies

This course will primarily focus on recommendations made by the American Diabetes Association (ADA) that are related to the diagnosis and monitoring of diabetes. The ADA states on its website, "Our mission is to prevent and cure diabetes and to improve the lives of all people affected by diabetes."*Other important agencies and studies referred to in this course are: International Diabetes Federation (IDF): An alliance of 200 diabetes associations; acts as a global advocate for individuals with diabetes.World Health Organization (WHO): An arm of the United Nations; provides programs for prevention, treatment, and care of those with diabetes worldwide.Diabetes Control and Complications Trial (DCCT): A major clinical study 1983-1993; proved the correlation between control of glucose blood level and lowered onset and severity of the complications of diabetes. *Reference: American Diabetes Association. Available at: http://www.diabetes.org/about-us/. Accessed April 14, 2010.

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Case A

A 50-year-old male with a family history of diabetes visits his physician for routine physical. He reports that he feels his health is excellent. He exercises regularly, but often his diet is high in calories and fat.Physical Examination: Slightly overweight; blood pressure and pulse normal.A basic metabolic panel and PSA are ordered. All results are within reference range except the plasma glucose. The patient's physician orders a hemoglobin A1C (HbA1C) the following week.Laboratory results:Fasting plasma glucose (FPG)= 110 mg/dL (Reference interval 75 - 100 mg/dL)One Week Later:Hb A1C= 6.0% (Reference interval 4 - 6%)

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Blood Glucose and Hormonal Control

Several hormones regulate blood glucose concentration. Insulin, the main regulatory hormone, is produced by and secreted from the pancreatic beta-cells. Insulin stimulates the uptake of glucose and the movement of glucose from blood to cells for energy production. Insulin also stimulates glycogenesis, inhibits glycogenolysis, and regulates protein synthesis.Other hormones that are also involved in carbohydrate metabolism include: Pancreatic glucagon- stimulates glycogenolysis and gluconeogenesis Adrenal gland cortisol- promotes gluconeogenesis Epinephrine- a neurotransmitter that increases glycogenolysis

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Diabetes - A Metabolic Disorder

Diabetes results when insulin concentrations are absent, reduced, or when insulin action is impaired (referred to as insulin resistance). Without cellular uptake of blood glucose for energy, the balance of metabolizing carbohydrates, fats, and proteins for energy is lost. Hyperglycemia and excess use of fats and proteins for energy result. The latter causes excess acetyl-CoA which is converted to ketone bodies or to cholesterol.Polydipsia, polyuria, and unexplained weight loss are symptoms of diabetes. Polydipsia and polyuria occur as the body tries to lower blood glucose concentrations with increased urinary excretion of glucose. Weight loss results from increased utilization of proteins and fats for energy. The image on the right represents impaired metabolism in diabetes. The thicker arrows represent the pathways that are imbalanced. In normal carbohydrate metabolism, the opposing arrows would be of the same size, representing a normal pathway and a balanced metabolism.

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ADA Recommended Criteria for Diagnosis of Diabetes

Assay Description Criteria for Diabetes HbA1C Performed in laboratory by method NGSP certified and standardized to DCCT assay > 6.5 % Fasting plasma glucose At least 8 hour fast > 126 mg/dL Casual plasma glucose Symptoms of diabetes; Blood glucose measured at any time of day > 200 mg/dL Two-hour plasma glucose Following a glucose load of 75g anhydrous glucose dissolved in water > 200 mg/dL

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Case A (continued)A 50-year-old male with a family history of diabetes visits his physician for routine physical. He reports that he feels his health is excellent. He exercises regularly, but often his diet is high in calories and fat.Physical Examination: Slightly overweight; blood pressure and pulse normal.A basic metabolic panel and PSA are ordered. All results are within reference range except the plasma glucose. The patient's physician orders a HbA1C the following week.Laboratory results:Fasting plasma glucose (FPG)= 110 mg/dL (Reference interval 75 - 100 mg/dL)One Week Later:HbA1C= 6.0% (Reference interval 4 - 6%)Which of the following statements is most accurate regarding the patient in Case A?View Page
HbA1C versus Blood Glucose Measurement

Advantages of utilization of HbA1C over blood glucose measurement include: Fasting is not required Greater specimen stability Less fluctuations in day-to-day levels caused by stress and illness Disadvantages of utilization of HbA1C over blood glucose measurement include: Cost per test is higher than blood glucose. Conditions that shorten red blood cell (RBC) survival e.g., hemolytic anemia, homozygous sickle cell trait, pregnancy, or recent significant blood loss, will reduce exposure of RBCs to glucose, thereby lowering the HbA1C test value. Specimens with >10% fetal hemoglobin (HbF) may have a falsely decreased HbA1C test result. If onset of diabetes is rapid, blood glucose levels will more correctly reflect glycemia than HbA1C levels.

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Risks and Complications of Diabetes

The diabetic patient is at risk for many serious complications and often experiences a diminished quality of life. Angiopathy, damage to basement membranes of vessels, injures the linings of blood vessels and leads to microvascular and macrovascular damage.

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Other Complications

Ketoacidosis is always a serious complication for type 1 diabetics. Due to lack of uptake of glucose into cells by insulin, proteins and fats are utilized as energy sources. This results in excess acetyl CoA which is converted to ketone bodies. A serious acidosis results and if untreated or not resolved by the body, coma and death can occur.Most often the acetyl CoA in a type 2 patient is converted to cholesterol and results in hyperlipidemia and heart disease in these patients.The elderly type 2 diabetic is at risk for a hyperosmolar nonketotic coma. The patient becomes dehydrated due to increased urine excretion to lower the blood glucose. If reduced renal or cardiac function is also present, glucose excretion is impaired and blood glucose concentrations can become extremely high. Ketones are not produced in excess, thus the patient remains nonketotic. Insufficient hydration, elevated blood glucose, and decreased renal excretion of waste products result in an increased osmolality and total concentration of all plasma components.

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Clinical Testing

A large number of assays related to carbohydrate management and diabetes monitoring are performed in clinical laboratories, hospital nursing units, nursing homes, physician offices, clinics, and by patients at home, school, or work.Assays that will be discussed are: Blood Glucose Urine Glucose Ketones Microalbuminuria Insulin and C-Peptide Insulin Antibodies Glycosylated Proteins

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Blood Glucose

Serum, plasma, and whole blood glucose levels are among the most common laboratory assays. Due to self-monitoring of blood glucose (SMBG), blood glucose is also the most common assay performed by patients themselves or their caretakers. Fasting, timed, and casual serum or plasma specimens are run in hospital laboratories for screening, diagnosis, and monitoring of patients.

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Whole Blood Glucose Testing

In the past twenty years there have been significant improvements in the accuracy of handheld glucose meters. Patient use has resulted in substantial improvements in diabetic control and insulin therapy. Capillary whole blood is easily obtained and glucose concentration is derived on simple to use, portable meters. Since whole blood glucose is lower than plasma glucose, the meters are programmed to correct the value before presenting the result; therefore, the whole blood glucose meter result correlates to serum or plasma results.Clinical and Laboratory Standards Institute (CLSI) has set standards for correlation between glucose meter and laboratory measured glucose levels. If the laboratory measured glucose is > 75 mg/dL, the glucose meter result should be within 20%. For laboratory measured values < 75 mg/dL, the glucose meter result should be within 15 mg/dL.

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Urine Glucose

Before glucose meters were available, urine glucose was frequently used to approximate diabetic glucose levels. Blood glucose levels can be related to urine glucose concentration because of urinary excretion of glucose. Physician offices, clinics, and patients at home tested urine with reagent strips for a semi-quantitative measurement of urine glucose and adjustments in insulin therapy were made. Monitoring a diabetic carbohydrate management is seldom performed this way today. Portable meter measurement of blood glucose is a much better management method. Urine glucose measurement is neither sensitive nor specific and does not give information about blood glucose below the renal threshold (usually 180 mg/dL).As a semiquantitative measurement, urine glucose is a routine assay on urinalysis test and an abnormal result would be investigated with blood levels. If quantitative measurements are needed, a timed urine specimen is collected and measured for glucose by blood glucose methods.

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Urinary Albumin Excretion

Screening for early occurrence and low amounts of albumin in urine detects microvascular disease before impaired renal function and insufficiency occur. Regular screening of urinary albumin excretion (UAE) is recommended for individuals with both type 1 diabetes and type 2 diabetes as an early indicator of renal disease. It is recommended at the time of initial diagnosis and annually thereafter for patients with type 2 diabetes, and commencing annually 5 years after the initial diagnosis of type 1 diabetes. Control of blood pressure and blood glucose concentrations can slow the rate of renal function decline.

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Ketones

Acetyl CoA is converted to acetone, acetoacetate, and beta-hydroxybutyrate. These are acids and when dissolved in body fluids in excess lower the blood pH. Increased ketones can result in a metabolic acidosis referred to as ketosis, ketoacidosis or diabetic acidosis. Type 1 diabetic patients are especially at risk for ketoacidosis. Urine and serum ketones are measured semiquantitatively and a diabetic in ketosis is monitored for ketones and blood pH.

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Insulin and C-Peptide

Insulin is secreted by the pancreatic beta-cells as a prohormone composed of fragments: C-peptide and insulin. The C-peptide fraction is cleaved off the prohormone. The insulin fraction becomes active. C-peptide is inactive but provides structure to the prohormone and has a much longer half-life. Both of these hormones can be quantitated in blood.Insulin levels are not measured to diagnose or monitor diabetes but can give information about a patient and is an important assay in hypoglycemia. C-peptide is also measured in evaluating hypoglycemia and is used to distinguish between endogenous and exogenous insulin; it would be present in circulation in endogenous insulin secretion. It is also often used to monitor pancreatic surgery and transplant because of its longer half-life.

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HbA1C

Hemoglobin A comprises the majority of normal adult hemoglobin (Hb) and includes the minor hemoglobins, Hb A1a, Hb A1b, and Hb A1c. Sometimes these three are referred to as Hb A1 but A1C is the major fraction and composes 80% of Hb A1. Following synthesis of Hb A, a nonenzymatic reaction adds glucose to the N-terminal valine on either beta chain forming glycated Hb. The pre-A1C molecule is a labile Schiff base and this reaction is reversible. As the red blood cells circulate, an irreversible Amadori rearrangement of the pre-A1C base occurs forming a stable ketoamine, A1C. Over the life span of the red blood cells (120 days) this process continues and the concentration of A1C is proportional to the concentration of the blood glucose. The concentration of A1C then relates to an individual's average glucose over time and can be used as an index relating to the extent of carbohydrate control during a 2 - 3 month period. There is also a direct relationship between the concentration of HbA1C and risk of complications in diabetic patients. Therefore, the ADA has recommended using HbA1C measurements to monitor glycemic control.

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Oral Glucose Tolerance Test

There remains some disagreement on the use of the oral glucose tolerance test (OGTT) in diabetes testing and diagnosis. Those that recommend using OGTT assert that the OGTT better detects diabetics who are at risk for developing complications associated with diabetes.The ADA discourages the use of the OGTT for at-risk individuals unless blood glucose and HbA1C concentrations remain below diagnostic ranges for diabetes but patient displays symptoms of diabetes. The OGTT is utilized to diagnose gestational diabetes. Those at risk for gestational diabetes are screened with FPG, casual, and sometimes a 50-g oral glucose load. Definitive diagnosis of gestational diabetes is made with a glucose challenge test of 100-g or 75-g glucose and timed blood glucose measurements (OGTT).

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A clinical laboratory scientist is reviewing the results of comparison studies between laboratory plasma glucose results and patients' self-monitoring (whole-blood) blood glucose (SMBG) results. Which SMBG results are acceptable?View Page
The Laboratory's Role in Diagnosis and Monitoring of Diabetes

Even though most diabetics, physician offices, clinics, nursing homes, and nursing units use glucose meters for monitoring glucose levels, the laboratory's role in diagnosis is vital. The function of the laboratory is crucial in diagnosis, monitoring, and management of diabetes. Diabetic patients can go into severe metabolic imbalances that are life threatening. These metabolic conditions include: diabetic ketoacidosis, hyperosmolar nonketotic coma, and hypoglycemia. Laboratory testing is essential in diagnosing and monitoring these conditions.Laboratory blood glucose and HbA1C levels are used to demonstrate the level of hyperglycemia required for diagnosis. If an OGTT is needed for classification or characterization of hyperglycemia, a patient is sent to a hospital or clinical laboratory for the test. Detection of elevated microalbumin levels that can signal early stages of renal impairment is accomplished through laboratory testing. There are many other disease states and complications associated with diabetes. Clinical laboratories detect these diseases and monitor the complications that result. Important among these assays are urea, creatinine, and serum lipids. If a diabetic does have a pancreatic transplant, serum C-peptide and insulins levels monitor transplant success and viability of transplanted organ.

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Electrophoresis
Routine Electrophoresis

Routine electrophoresis is a generic term for the traditional clinical laboratory electrophoresis performed on a rectangle-shaped slab gel. Routine electrophoresis is mostly used for separation of proteins and has some use in separating nucleic acids. Generally several patient specimens and control(s) can be placed on one gel and solutes separated in one run. This type of electrophoresis is sometimes called zone electrophoresis.A serum sample with normal plasma proteins yields five zones or bands of separated proteins: albumin, alpha-1-globulins, alpha-2-globulins, beta-globulins, and gamma-globulins. Proteins in CSF and urine proteins are also separated with routine electrophoresis. Using whole blood treated with a reagent to lyse red blood cells, variant and glycosylated hemoglobins can be detected. With different visualization methods, isoenzymes and lipoproteins in a serum sample can be identified.A manual agarose gel electrophoresis of eight serum samples is pictured below. After electrophoresis, the gel was stained with Ponceau S.

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Currently there has been a revitalization in the clinical usage of electrophoresis. Previously, methods were primarily used to separate proteins in blood and other body fluids. From the following statements, select the statements that correctly describe newer applications of electrophoresis.View Page

Emerging Cardiovascular Risk Markers (retired 12/6/2013)
Introduction

We are all aware of the clinical laboratory's role in assessing overall health and we are also aware that measuring a patient's serum lipids will provide some insight into their cardiovascular health. The traditional measurements of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides are the 'classic' cardiovascular risk markers.Laboratorians, and even the general public are now well-aware that LDL-C ('bad' cholesterol) concentrations should be low while HDL-C ('good' cholesterol) concentrations should be high. Triglycerides should be kept in check as well. Optimal levels are shown in the table below. So what is the risk if these values are not within optimal ranges?Cardiovascular risk can be simply defined as increasing the odds of having a pathology which affects blood flow and/or the heart. The most common cardiovascular pathology is atherosclerosis. Other cardiovascular pathologies whose odds increase as serum lipids and other cardiovascular markers become suboptimal are myocardial infarction (heart attack), stroke, congestive heart disease and coronary artery disease. Other diseases such as diabetes and the metabolic syndrome are also strongly associated with the classic cardiovascular risk markers LDL-C, HDL-C and triglycerides.

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Atherosclerosis continued

If a plaque ruptures it will expose sub-endothelial tissue to blood cells and in so doing stimulate the formation of a clot. The clot is the body's attempt to seal off the crack but the clot itself can cause further obstruction to blood flow. This sudden increase in the blockage caused by the raised ruptured plaque and associated clot can transform a mild blockage into a critical one within a matter of hours. If it occurs within the blood vessels of the heart, the decrease in blood flow leads to severe and prolonged chest pain known as unstable angina. Such a patient is at obvious risk for a myocardial infarct should the blockage become any worse.Atherosclerosis typically begins in early adolescence, and is found in most major arteries but since it is asymptomatic during the early half of life we need cardiovascualr risk markers to help assess patient risk. If an at-risk patient is identified early, the hope is that medication, lifestyle changes or medical procedures can be used to avert a serious cardiovascular event. So, although the vast majority of us have some degree of atherosclerosis, risk markers can help identify those among us who are in more imminent danger or who have increased risk of an adverse cardiovascular event.

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Atherosclerosis

Atherosclerosis is a clogging, narrowing and hardening of the body's large and medium-sized blood vessels. Atherosclerosis can lead to hypertension, stroke, myocardial infarction (heart attack), renal problems, etc. Not surprisingly, cardiovascular risk markers tend to reflect a person's degree of atherosclerosis.Atherosclerosis is actually a chronic inflammatory response within the walls of arteries. Small lipoproteins like LDL are able to diffuse through the endothelial wall of blood vessels and accumulate. The inflammatory component of atherosclerosis results from the migration of leukocytes (mainly macrophages) that enter the blood vessel walls. These macrophages seek to remove the deposited LDL as well as intermediate-density lipoproteins (IDL). As macrophages phagocytose these lipoproteins, they become foam cells that get trapped in the endothelial space. This eventually leads to "hardening" or "furring" of the arteries and plaque formation. Arteriosclerosis is a general term describing any hardening (loss of elasticity) of medium or large arteries whereas atherosclerosis is a hardening of an artery specifically due to plaque. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.

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Transport of Lipophilic Substances

Many lipophilic substances, including fat-soluble vitamins, cholesterol, and triglycerides are essential for life. The body needs to be able to absorb and transport these substances. However, lipophilic substances are not water-soluble, and, since blood is aqueous, this presents a challenge. The body addresses this need by using 'carriers' which can bind or sequester lipophilic molecules to aqueous 'vehicles' and thus transport them through the aqueous environment of the blood. Small lipid-soluble hormone molecules like estrogen, testosterone or cortisone are carried through the blood by binding to carrier proteins. Cholesterol and triglycerides are carried through the body in small spherical particles which trap the lipophilic molecules in their centers. These particles have an outer shell that is polar on the surface so that the particles are soluble in the blood but they have a lipophilic core which can hold fat-soluble molecules.

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Apolipoproteins

Lipoproteins differ in size and density as well as in their content (what they tend to carry). They also can differ in their origination (where they are made). Another significant difference between lipoprotein molecules is the proteins they have on their surfaces. These proteins, known as apolipoproteins, are the major identifying characteristics of a lipoprotein. There are many different apolipoproteins and we are continually learning more about them. Apolipoproteins have multiple roles. One role is that these amphipathic (detergent-like) proteins increase the overall solubility of the lipid particle, helping it to dissolve in the aqueous environment of the blood. Apolipoproteins can also function as enzyme co-factors (receptor ligands) and facilitate the transfer of their lipid cargo to specific cells. Thus, the apoliproteins are the smart or working-end of the lipoprotein particle. The apolipoproteins dictate where the particles will dock and where they can bind, and in so doing the apolipoproteins regulate lipid metabolism in the body.

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What are apolipoproteins?View Page
References

Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.html Accessed March 25, 2013.Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919.Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615.Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165.Interpretive Handbook, (MC0440rev0407) Mayo Clinic, RochesterMN;2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24.Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217.NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed March 25, 2013.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: BurtisCA, Ashwood ER. BrunsDE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565.Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42.Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46.Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360.Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164.Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033.Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952.

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Erythrocyte Inclusions
What are Erythrocyte Inclusions?

Erythrocyte inclusions are elements that may be present in red blood cells (RBCs). The appearance, composition, and associated physiology of the inclusions are specific for each type of inclusion. Identification and reporting of these inclusions are important because their presence may indicate diseases or disorders.Many erythrocyte inclusions can be visualized on a Wright-stained smear. However, some erythrocyte inclusions can only be observed by using a special stain. For example, to confirm the presence of Heinz bodies, hemoglobin H bodies, or reticulocytes, smears must be prepared after staining an aliquot of fresh whole blood with a supravital stain such as new methylene blue or brilliant cresyl blue. The image on the right is a peripheral blood smear prepared after staining the blood sample with a supravital stain; Heinz bodies, which are clumps of precipitated hemoglobin, are indicated by the arrows. To detect siderosomes, a stain must be used that is specific for iron, such as Prussian blue.

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Stress Reticulocytes

When the large reticulocytes normally found in the bone marrow are present in the peripheral blood, they are referred to as shift or stress reticulocytes. These cells may be up to twice the size of normal mature red cells and are an indication of the bone marrow's response to severe anemia.

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Pappenheimer Bodies, continued

Pappenheimer bodies, while visible on a Wright's stained smear, should be confirmed with an iron stain, such as Prussian blue stain. The image on the right is a Prussian blue stain that confirms the presence of Pappenheimer bodies. Wright stain does not stain the iron, but rather the protein matrix that contains the iron.Pappenheimer bodies are seen in certain types of anemia that are characterized by an increase in the storage of iron, such as sideroblastic anemia and thalassemia. These inclusions are also seen in the peripheral blood following a splenectomy. In a healthy person with a normal spleen, Pappenheimer bodies are destroyed before the erythrocytes enter the peripheral circulation.

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Siderocytes and Ringed Sideroblasts in Bone Marrow

20 to 60% of red cell precursors seen in bone marrow slides normally contain siderotic iron granules visible with Prussian blue stain. The presence of sideroblasts and siderocytes in the bone marrow indicates that the red cell precursors have an ample supply of iron. Neither sideroblasts nor siderocytes are present in normal peripheral blood.When a red cell precursor contains too much iron, the siderotic granules form a ring around the nucleus. If five or more siderotic granules form a ring around at least half the periphery of the nucleus of a nucleated red blood cell, the cell is referred to as a ringed sideroblast. The ringed sideroblast is an abnormal (pathological) form of a sideroblast. The ringed sideroblast is associated with sideroblastic anemias and myelodysplasias. Ringed sideroblasts are indicated by the arrows in the image on the right.

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Cabot Rings

Cabot rings appear as thin, red-violet-staining strands in the shape of rings, figure eights, or shapes of the letter B on Wright-stained smears. They are rarely seen in peripheral blood. The rings are probably microtubules from the mitotic spindle that remain behind after the rest of the erythrocyte nucleus is extruded. Cabot rings have been observed in megaloblastic anemia, lead poisoning, severe anemia, leukemia, myelodysplastic syndromes, and other cases of dyserythropoiesis.

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Erythrocyte Inclusions (retired 7/10/2012)
More on Pappenheimer bodies

Pappenheimer bodies, while visible on a Wright's stained smear, should be Perls' Prussian blue stain, which is specific for iron. Pappenheimer bodies are seen in certain types of anemia characterized by an increase in the storage of iron, such as sideroblastic anemia and thalassemia. These inclusions are also seen in the peripheral blood following a splenectomy. In a healthy person with a normal spleen, Pappenheimer bodies are destroyed before the erythrocytes enter the peripheral circulation.

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Reticulocytes

Although the nucleus has been extruded, the reticulocyte is still considered immature because it retains numerous organelles needed for hemoglobin production, such as ribosomes, mitochondria, and fragments of the Golgi apparatus. A reticulocyte normally remains in the bone marrow for one or two days before entering the circulation and its final 24 hours of maturation. The red cell is mature when hemoglobin production is complete and the organelles have disintegrated. The reticulocyte is slightly larger (10 microns) than the mature erythrocyte. Reticulocytes appear blue-gray on the Wright-Giemsa-stained smear and are referred to as polychromatophic red cells (image on the left). The residual RNA in the cytoplasm causes the blue-gray color. A supravital stain such as new methylene blue N or brilliant cresyl blue is used to stain reticulocytes for an actual count as seen in the image on the right.

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Stress Reticulocytes

When the large reticulocytes normally found in the bone marrow are present in the peripheral blood, they are referred to as shift or stress reticulocytes. These cells may be up to twice the size of normal mature red cells and are an indication of the bone marrow's response to severe anemia. In addition to recognizing their appearance as polychromatophlic cells on Wright's stained smears, it is now possible to quantify stress reticulocytes using a fluorescent stain. They are classified as high, medium, or low using a fluorescent-sensitive flow cytometer.

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First Aid
Applying Direct Pressure

Wear disposable latex gloves if available.Place a thick, clean compress (consisting of gauze or soft clean cloth) directly over the wound. The compress will absorb blood and help the clotting process.Apply pressure to the victim's wound by placing your palm directly over the compress and pressing firmly.If blood soaks through, do not remove the compress. Instead, add more cloth pads over it as needed. Removing the compress may reopen the wound and result in further bleeding.

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Elevate the Wound

Elevate the wound if it is on the hand, arm, or leg and there is no bone fracture.Elevating the wound reduces the circulation to the wounded area and decreases blood loss.

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Fundamentals of Hemostasis
Introduction to the Fundamentals of Coagulation

The ability of the body to maintain a state of homeostasis, or physiological equilibrium, is absolutely essential for effective, efficient functionality of all body systems. Hemostasis is the cessation of free blood flow, external to the vascular system, when a vessel wall has been breached.With the maintenance of homeostasis in mind, it is vital that the body be able to rapidly repair vascular damage, arresting blood flow in the process, while simultaneously maintaining blood in a fluid state within the vascular compartment.

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Introduction to the Fundamentals of Coagulation, continued

When a vessel wall is damaged, blood flow out of the vessel is arrested by way of a complex series of interrelated physiological and biochemical processes. There are a wide variety of factors that influence the effectiveness of hemostatic processes including the following: Type of, and degree of, vessel damageAbility of vasoconstriction to occurAvailability of platelets & their functionalityAvailability of clotting factors & their functionalityAbsence of inhibitors and anticoagulantsThe image on the right illustrates vessel size as related to time required for clotting to occur, amount of coagulation products used (platelets and clotting factors), and size of the corresponding bleed.

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An Introduction to the Fundamentals of Coagulation

As we will discover later in the course, there are other variables which impact the effectiveness of hemostatic mechanisms as well, such as acquired disease states, and inborn metabolic pathway defects.For now, however, our focus will be on the mechanisms, processes, and components which work together to achieve coagulation, or the cessation of blood flow from a damaged vessel.Note: The terms coagulation and hemostasis are used interchangeably throughout this course.

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Primary Hemostasis: The Vascular System

Our blood circulates freely through undamaged, intact vessels. The design of the vasculature, or blood vessels, is such that the walls of the vessels are chemically inert to both coagulation factors and platelets under normal conditions. Damage to a vessel will break the inert epithelial lining, exposing the subendothelium and collagen, while releasing chemical signals that trigger subsequent hemostatic mechanisms. The first hemostatis mechanism is termed "primary hemostasis". Overview of the Primary Hemostasis Process:VasoconstrictionPlatelet adhesionPlatelet activationPlatelet granule secretionPlatelet aggregationPrimary hemostatic plug formation

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Primary Hemostasis: The Vascular System

Overview of Vascular System Involvement in Primary Hemostasis:VasoconstrictionReroute blood flowPlatelet aggregationContact activation of coagulation system (start of secondary hemostasis at this point)

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Primary Hemostasis: The Vascular System, continued

The first specific, recognizable hemostatic mechanism is a process known as vasoconstriction. Vascoconstriction is initiated by chemical signals stemming from a damaged area of a blood vessel. Vasoconstriction, or vascular constriction, immediately reduces the quantity of blood flowing through the damaged area. Its action is the physical decrease in the size of the vessel and the redirection of blood flow around, and away from, the damaged area. Vasoconstriction is akin to putting a clamp on a pliable piece of plastic tubing. Vasoconstriction also allows for a closer interaction of the coagulation proteins and platelets with the damaged vessel wall. In addition, the epithelial cells lining the vessel also contract, which allows plasma to leak from the injured area. This is part of our normal inflammatory reponse and is the reason for swelling at the site of an injury. Vasoconstriction is an exceedingly important hemostatic mechanism as it prepares the damaged vessel for subsequent repair activities. These activities will be discussed next.

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Which of the following processes does NOT occur during primary hemostasis?View Page
Summary of Primary Hemostasis

In summation, we have covered the following sequence of events, which comprise primary hemostasis. The process begins with damage to a vessel wall, as blood flows outside the vasculature. The body responds with vasoconstriction, decreasing blood flow to the affected area. Platelets begin sticking to the damaged vessel walls (platelet adhesion). As the platelets stick, they change their shape (platelet activation) and release chemicals which signal other platelets to respond (platelet secretion). As other platelets arrive, they begin sticking to one another, clumping together, forming a plug to fill in the breach (platelet aggregation). This plug, while strong, is a temporary fix, and must be reinforced with fibrin strands to effectively fill the breach during the vessel repair process (secondary hemostasis).

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Secondary Hemostasis: The Extrinsic Pathway, continued

Once a vessel has been breached, tissue factor is exposed to circulating factor VII, and the two substances bind to form a complex. The newly formed tissue factor/factor VII complex is thought to be the primary physiological stimulus for blood coagulation. In other words, more hemostatic activities are initiated by the extrinsic pathway than the intrinsic. This complex leads to the activation of factor VII (factor VIIa) which is now ready to catalyze the conversion of factor X to factor Xa as part of the common pathway.

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The Fibrinolytic System

There is a very close relationship between the formation of fibrin, and its eventual degradation, or lysis. A fibrin clot serves as a temporary seal, intended to prevent continued blood loss from the damaged vessel while repair activities are performed. The breakdown of the clot begins almost as soon as the clot is formed! The process by which fibrin is broken down and removed from the clot, ultimately leading to complete dissolution of the clot, is called fibrinolysis.

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The Fibrinolytic System, continued

Fibrin strands woven into the clot structure are cleaved into soluble fibrin fragments and then removed by macrophages. The action of fibrinolysis also serves to restore blood flow into the area that had been sealed off, helping to promote further healing. Fibrinolysis is mediated by a proteolytic enzyme called plasmin (plasminogen is the inactive precursor form of plasmin that is found in plasma). Plasmin takes on fibrinolytic properties after activation, digesting both fibrin and fibrinogen. Inhibitors act to control the process, serving as a check and balance system for fibrinolytic activities.

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Collecting Blood Specimens for Coagulation Testing

Venous blood specimens for coagulation assays should be collected into a tube containing 3.2% buffered sodium citrate tube (blue top tube), yielding a whole blood sample with a 9:1 blood to anticoagulant ratio. Inadequate filling of the collection tube will decrease this ratio, and may affect test results.A blue top tube used for coagulation testing should be drawn before any other tubes containing additives. This includes tubes containing other anticoagulants and/or plastic serum tubes containing clot activators. A serum tube that does not contain an additive can be collected before the blue top tube.If a winged blood collection set is used in drawing a specimen for coagulation testing, a discard tube should be drawn first. The discard tube must be used to fill the blood collection tubing dead space to assure that the proper anticoagulant/blood ratio is maintained, but the discard tube does not need to be completely filled. The discard tube should be a nonadditive or a coagulation tube.If a blood specimen used for coagulation testing must be collected from an indwelling line that may contain heparin, the line should be flushed with 5 mL of saline, and the first 5 mL of blood, or 6 times the line volume (dead space volume of the catheter), be drawn off and discarded before the coagulation tube is filled.

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Fibrin/Fibrinogen Degradation Products and D-Dimers

The presence of D-dimers in plasma or whole blood indicates that fibrin has been formed and degraded (fibrinolysis). Plasmin can also degrade intact fibrinogen, generating fibrinogen degradation products that are detected in fibrin/fibrinogen degradation products (FDP) assays. D-dimers and FDP can become elevated whenever the coagulation and fibrinolytic systems are activated. The presence of D-dimer confirms that both thrombin and plasmin have been generated, since it can only be produced as the result of the plasmin degradation of fibrin. D-dimer is a sensitive, but non-specific marker of fibrin formation and fibrinolysis that occurs with the formation of blood clots.The D-dimer test can be useful in the diagnosis of deep venous thrombosis (DVT) or pulmonary embolism (PE), two forms of venous thromboembolism (VTE). When the test is being used for this purpose, it is important that the D-dimer method has been validated by medical literature and D-dimer levels are accurately measured and accurately reported because of the serious nature of this clinical decision. If the test is positive in a patient suspected to have DVT or PE, clinicians proceed with further diagnostic tests. If the test is negative, depending on the clinical situation and the sensitivity of the D-dimer assay, DVT or PE is considered unlikely and further diagnostic tests for DVT or PE might not be pursued. D-dimer is also a sensitive, but non-specific diagnostic test for disseminated intravascular coagulation, and an indicator of increased risk of future myocardial infarction in patients evaluated for chest pain.

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Which of the following statements regarding D-dimer are true?View Page
Coagulation Disorders - Acquired

Disseminated Intravascular Coagulation (DIC) is best described as a disorder of consumption, because clotting factors are depleted from the blood. Basically, clotting occurs randomly throughout the body, as opposed to just in the localized areas where vascular damage has occurred, consuming clotting factors and other components such as platelets in the process. Symptoms may range from a mild bleed, to severe, profuse bleeding, primarily dependant upon the availability of clotting factors. As more and more coagulation factors and components are consumed, the disorder progresses and symptoms worsen. Most heavily impacted are the levels of factors I, V, and VIII as well as the number of available platelets. Clinically, DIC is detected via an elevated (positive) FDP, positive D-dimer test, a prolonged PT and APTT, plus the manifestation of hemorrhagic episodes. DIC is diagnosed as two primary types, acute and chronic. Acute DIC manifests in a few hours or a few days, has a high mortality rate, and is seen in infections, obstetric complications, liver disease, and tissue injury. Chronic DIC is a secondary condition to some other disease state. Once you treat the primary disease, this type of DIC will go away. Treatment is often factor replacement therapy through the use of fresh frozen plasma and/or cryoprecipitate.

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Heparin Therapy

The use of heparin is prophylactic. It is used either to prevent thromboembolism (a condition in which a blood clot forms inside a vessel) or used to limit a previous thromboembolism. Heparin inhibits thrombin. The degree of inhibition is dose-dependent. Low doses of heparin inhibit initial thrombin formation in the coagulation cascade and act to slow down overall thrombin generation. At higher doses, heparin can inhibit thrombin entirely, making blood coagulation impossible. Heparin is a potent anticoagulant. Accurate monitoring is essential. The activated partial thromboplastin time (aPTT), activated clotting time (ACT), and/or anti-Xa assays are used to monitor unfractionated heparin therapy.

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Fundamentals of Molecular Diagnostics (retired 2/12/2013)
Targets

Molecular based clinical diagnostic test methodologies differ according to the target of interest. For example, patients suspected of having different diseases will require the identification of different targets. These targets might be found in different cells of the body and may therefore require different specimens to provide the answers. Patient A suspected of having Disease 1: Requires the identification of a target of missequenced DNA - might require specimen of whole blood Patient B suspected of having Disease 2: Requires identification of a target of antibody production -methodology might require specimen of serum Using this specific approach of disease diagnosis based on unique target identification, tests can provide answers that are more:Rapid Sensitive Specific

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Pre-analytical Variables

Pre-analytical variables are those that affect the specimen before the actual testing begins. Some of the pre-analytical variables to consider with molecular testing include those that are applicable to all clinical specimens but should be emphasized when discussing molecular methodologies. Some of these include but are not limited to:Receipt of valid orderProper patient identificationProper venipuncture procedure for blood collection Use of correct anticoagulant Collection of correct specimen type (eg - plasma, serum, whole blood)Order of drawProper storage Proper transportProcedures if there is a delay in testing and/or transport

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Specimen Collection and Handling

Some global specimen collection and handling issues to consider include:Specimens that contain nucleated cells will be of interest in DNA methodologies while specimens lacking nucleated cells are more useful in RNA methodologies.rRNA is more stable than mRNA, which is labile and sensitive to contamination.DNA is relatively stable and can be obtained from nonviable sources.Serum or plasma obtained by standard routine venipuncture procedures can be used as long as proper site selection and decontamination occur.Standard anticoagulants such as ethylenediaminetetraacetic acid (EDTA) and acid citrate dextrose (ACD) can be used; however, avoid the use of heparin as an anticoagulant as it interferes with some polymerase chain reaction (PCR) methodologies.When using fluorescence, fasting serum or whole blood specimens should be used to decrease the interference by lipids.

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When collecting blood samples, one anticoagulant to avoid, especially when performing PCR is:View Page

General Laboratory Question Bank - Review Mode (no CE)
When performing a routine venipuncture in which you are collecting a serum separator tube for immunology, a green top tube for chemistry, a blue top tube for coagulation, and a lavender top tube for hematology, which tube should be collected first?View Page
Which one of the following statements about Hepatitis is true?View Page
Which of the following is not appropriate for a routine blood specimen:View Page
Standard precautions means that:View Page
Which one of the following statements about the hepatitis B vaccine is correct?View Page
A smear that is prepared from equal parts of methylene blue and whole blood will be used for:View Page
Which of the following immunoglobulin classes is chiefly responsible for the degranulation of mast cells and basophils:View Page
The Kleihauer-Betke test is used to:View Page
Classic automated blood cell counters are based on:View Page
A definitive diagnosis of malaria can be made by:View Page
Hematocrit is:View Page

Hematology / Hemostasis Question Bank - Review Mode (no CE)
The RBCs found in this illustration are the result of:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Identify the cell in this illustration indicated by the arrow:View Page
What is the cell indicated by the arrow in this illustration:View Page
Found frequently in a newborn's blood the cells indicated by arrow in this illustration are:View Page
The cell indicated by the arrow in this illustration is called:View Page
The abnormal RBC shape seen in this illustration is:View Page
Which of the two WBCs indicated by the arrows on this illustration is normally the most numerous in peripheral blood and what is its name:View Page
The impedance principle shown in this illustration is best described by the following statement:View Page
Which of the following conditions is frequently associated with these cells?View Page
Expected life span of a neutrophil in the peripheral blood of an adult is:View Page
Which of the following major cellular elements does not develop solely in the bone marrow:View Page
What is another name used to designate a fully committed B-lymphocyte:View Page
Which of the following is not primarily a hemolytic process?View Page
Which blood cell is found in the largest numbers in the peripheral blood of a normal adult:View Page
Which of the following cells is most common in adult bone marrow:View Page
Hypochromia can best be described as:View Page
If greater than 50% lymphocytes were found on the peripheral blood smear of a 5 month old child you would suspect which of the following conditions:View Page
Which of the following may interfere with the accurate measurement of hemoglobin:View Page
The reticulocyte count is used to assess which of the following:View Page
The ratio of whole blood to anticoagulant is very important in the PT assay; at which hematocrit level should the standard anticoagulant volume be adjusted:View Page
When three tubes of cerebrospinal fluid are received in the laboratory they should be distributed to the various laboratory sections as follows:View Page
Which of the following would best describe what you might observe after a traumatic CSF tap:View Page
Which of the following blood smears these illustrations would be best suited for performing a differential count:View Page

Hemoglobinopathies: Hemoglobin S Disorders
Vaso-occlusive Crisis

HbSS blood may contain reticulocytes with an abnormal presence of CD36 on their membranes, allowing platelets to form a bridge between these young sickle cells and endothelial cells in post-capillary venules. This initial slow down of blood flow creates an environment in which cells containing HbSS can easily form sickled cells.The rigid cells, which are formed as a result of the sickling process, can collect in and plug small blood vessels. This can then cause tissue damage and organ infarction. In addition, the polymerized hemoglobin is thought to disrupt the RBC membrane, exposing phosphatidylserine that can trigger hemostasis. Subsequently, white blood cells (WBCs) may adhere to endothelium in response to recruitment in the inflammatory process. This leads to further occlusion as neutrophils capture additional RBCs in the post-capillary venules.Contributing further to vaso-occlusion is the decreased level of L-arginine in patients with sickle cell disease. L-arginine is a substrate needed to produce nitric oxide (NO). NO has vasodilatory properties as well as anti-inflammatory and anti-platelet properties. Thus a decrease in NO may lead to increased cellular adherence to endothelium.

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Hemolytic Crisis

Sickle cell anemia, in addition to being a hemoglobinopathy, is a hemolytic anemia. Hemolysis is both intravascular (about one-third) and extravascular (about two-thirds). Common markers of hemolysis include elevated LDH, bilirubin, and reticulocyte levels.The hemoglobin that is released into the plasma during intravascular hemolysis combines with nitric oxide (NO). The resulting decrease in NO availability contributes to the vaso-occlusive crisis by stimulating vasoconstriction, endothelial adhesiveness, and thrombosis.Hemolytic crisis also involves splenic sequestration, which occurs in an effort to remove damaged red blood cells. This can result in hypovolemia, which may lead to shock, especially in children. Children can also exhibit splenomegaly due to intrasplenic pooling of blood.Adults in hemolytic crisis may experience autosplenectomy. This occurs when the spleen has multiple infarctions, followed by fibrosis, which renders the spleen nonfunctional.

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In HbSS blood, an increased amount of which of the following surface antigens on young sickle cells (reticulocytes) may allow platelets to form a bridge between the reticulocytes and endothelial cells, ultimately leading to vaso-occlusion?View Page
Which of these blood levels will increase during hemolysis?View Page
References

Afenyi-Annan, A., Kail, M., Combs, M.R., Orringer, E.P., Ashley-Kock, A., & Telen, M.J. Lack of Duffy antigen expression is associated with organ damage in patients with sickle cell disease. Transfusion. 2008;48:917-924. Ataka, K. I. et. al.Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia. Blood: 2008; 11(8) 3991-3997.Ballas, S.K., Sickle Cell Anaemia: Progress in Pathogenesis and Treatment. Drugs 2002: 62(8); 1143-1172.Bianchi, N., Zuccato, C., Lampronti, I., Borgatti, N., and Gambari, R. Fetal Hemoglobin Inducers from the Natural World: a novel approach for the identification of drugs for the treatment of B-thalassemia and Sickle-cell anemia. eCAM: 2009; 6(2)141-151.Centers for Disease Control and Prevention. Sickle cell disease: Symptoms and treatments. Available at: http://www.cdc.gov/ncbddd/sicklecell/symptoms.html. Accessed January 21, 2010.Harmening, Denise M., Clinical Hematology and Fundementals of Hemostatis 4th., F.A. Davis, 2001.Inati, A., Koussa, S. Taher, A., & Perrine, S. Sickle cell disease: New insights into pathophysiology and treatment. Pediatr Ann. May 2008.Kaushansky, K., Lichtman, M.A., Beulter, E., Kipps, T.J., and Prchal, J.T. Williams Hematology 8th Ed. McGraw Hill 2010.Lotspeich-Steininger, Stiene-Martin and Koepke, Clinical Hematology Principles, Procedures, Correlations, Lippincott 1992. McKenzie, Shirlyn B., Textbook of Hematology 2nd ed., Williams and Wilkins 1996. Miale, John B, Laboratory Medicine Hematology 6th ed., Mosby 1982. Niscola, P., Sorrentino, F., Scaramucci, L., de Faritiis, P., & Cianciulli, P. Pain syndromes in Sickle Cell Disease: An update. American Academy of Pain Medicine. 2009:470-480.Rodak, Bernadette, Diagnostic Hematology, W.B.Saunders Co., 1995.Yoon, S.L. & black, S. Comprehensive, integrative management of pain for patients with Sickle-Cell Disease. Journal of Alternative and Complementary Medicine. 2006: 12; 995-1001.

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The Sickling Process, Continued

The sickling process progresses through stages of nucleation, growth and alignment. Nucleation involves the aggregation of a small number of hemoglobin tetramers into clusters or short linear polymers. This stage is also called the lag phase. These clusters may be reversed upon re-oxygenation. With each repeated occurrence, the polymers continue to grow. In this polymerization stage, the internal cellular viscosity increases.Eventually the fibers that are formed become irreversible crystalline structures, which align into bundles, thus creating the pointed elongated and typically crescent shaped morphologies associated with sickle cells (drepanocytes) as seen in the peripheral blood field below.

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Normal Blood Flow

Normally, red blood cells (RBCs) traverse the vasculature without incident. The biconcave disc shape of the red blood cell allows for its deformability to enter and move through capillaries. In addition, red blood cells do not normally have cause to adhere to blood vessel endothelium.

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Blood Flow During Sickling

RBCs with sickle hemoglobin (HbSS) undergo changes during deoxygenation that may make them less deformable in the smaller blood vessels. The lack of deformability and cellular interactions cause a slowing down of the blood flow in affected blood vessels. Eventually, occlusion may result.

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Blood Tranfusions

The use of blood transfusions for stroke prevention in children with sickle cell disease (SCD) has become a standard of care. The goal is to keep the level of HbS under 30%. Transfusions are not normally needed for SCD unless patients develop a sudden worsening of anemia due to splenic sequestration or infection. Of concern is the occurance of iron overload. Iron chelators are helpful in the management of iron overload and include desferrioxamine and deferasirox.Transfusing phenotypically matched blood (especially C, E, and K) is highly recommended to prevent alloimmunization.

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Transplantation and Gene Therapy

Bone marrow transplants may be a cure but currently the risks are too high. Impediments to transplantation include the lack of matched sibling donors and prior transfusions, which have exposed the patient to donor antigens.The best candidates for bone marrow transplants are children less than 16 years old. Two umbilical cord blood transplantations have been performed that reportedly have not remanifested with sickle cell disease.Gene therapy may become an option in the future that may alter the expression of the sickle gene.

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Solubility Test

Most hemoglobins are soluble in a high-molarity phosphate buffer; hemoglobin S is not. The buffer is made of of dibasic and monobasic potassium phophates, Saponin and dithionate. Kits are available, which consist of this reagent, pipets and a reading rack. A 1:100 dilution of blood into buffer is made, incubated for 5 minutes, and turbidity is observed against a white background with black lines.A positive result (A below) is indicated by a turbid solution. A negative result (B below) is obtained when lines are visible through the solution.The solubility test should only be used as a screening test as it is not reliable for diagnosing sickle cell disease.

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Solubility Test Errors

False-positive results using the solubility test may be obtained in persons with increased red blood cell counts as occur in polycythemias; extremely high white counts, as in some leukemias; and extremely high platelet counts. Increased levels of lipids or globulins may also cause a false-positive. Hemoglobins that show a positive solubility test include HbC-Harlem, HbC-Georgetown, and HbC-Ziquinchor.False-negatives in the solubility test may be obtained on persons with severely decreased hemoglobins/hematocrits and those recently transfused. Infants less than six months old may also demonstrate false-negative results due to higher levels of HbF.False results in the solubility test can occur due to problems with technique: too much or too little blood added will result in false-positive and false-negative respectively; use of incorrect size test tube, holding the test tube too close to the background, and using deteriorated reagents can all lead to false-negative results.

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Densitometer Tracing of Various Hemoglobins

The densitometer tracing shown below demonstrates the pattern for a blood containing some A2 and S, along with greater amounts of F and A. Other hemoglobins that migrate with A2 and S are indicated.

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Hemolytic Disease of the Fetus and Newborn
Advance Organizer

Before beginning the course take some time to review and think about what you already know about HDFN. For example, jot down brief notes to answer the following questions: Which antibody causes the most severe HDFN? Antibodies in which blood group system are the most common cause of positive direct antiglobulin tests (DATs) in newborns but rarely cause clinically significant hemolysis? Should DATs be performed on all newborns regardless of maternal ABO and Rh blood groups? What is Rh immune globulin (RhIg), its source, constituents, purpose, and mechanism of action? Which tests are used to determine postnatal RhIg dosage? Which type of D variant can produce anti-D? What follow-up tests are typically indicated if a pregnant female has a positive antibody screen when initially tested? Which laboratory findings would suggest that an infant may have ABO HDFN? How can the clinical status of fetuses at risk for HDFN be monitored? What are the characteristics of red cells suitable for intravenous transfusion to fetuses suffering from severe HDFN due to anti-D?

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Postnatal Treatment: Exchange Transfusion

Whenever possible, a hallmark of HDFN treatment is to induce labor as early as possible once lung maturity has been attained so that the newborn will be able to survive. Once the infant is born, the main treatment for severe HDFN due to anti-D (and other antibodies causing severe disease) is exchange transfusion. In exchange transfusions, up to 85–90% of the infant's blood can be exchanged with donor blood by a process of removing 5–20 mL of blood at a time, and injecting an equivalent amount until the exchange is complete. An exchange transfusion accomplishes the following: Removes bilirubin and thus helps prevent kernicterus; Removes sensitized red cells that have not been broken down yet; Removes circulating maternal antibody; Provides antigen-negative red cells that will not be destroyed by the maternal antibody, thus will survive and provide oxygen to the tissues.

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Fetal Monitoring: Doppler Ultrasonography

Fetal monitoring is used to assess the severity of HDFN and determine whether antenatal transfusion Is warranted.Monitoring can be accomplished by: Doppler ultrasonography Amniocentesis CordocentesisDoppler sonography Doppler sonography is a type of ultrasound that detects and measures blood flow. As related to HDFN, Doppler sonography can be done beginning at 18 weeks. It measures the peak velocity of systolic blood flow in the fetal middle cerebral artery and is used to predict severity of fetal anemia. The hypothesis is that a faster rate of blood flow indicates a more severely anemic fetus, with severe anemia being an indicator of fetal hydrops.Because Doppler sonography is noninvasive and a safer alternative to amniocentesis, it has largely replaced serial amniocentesis for predicting severity of HDFN.

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Fetal Monitoring: Cordocentesis

Cordocentesis, also known as percutaneous umbilical blood sampling or PUBS, can be done after 18 weeks gestation. PUBS is a prenatal procedure in which a fetal blood sample is removed from the umbilical cord. The sample is confirmed to be of fetal origin by a rapid alkaline denaturation test. The fetal blood can then be analyzed using routine diagnostic tests, e.g., blood group, DAT, reticulocyte count, platelet count, hemoglobin/hematocrit, and more.Cordocentesis / PUBS can also be used to deliver intravenous transfusions (IVTs).

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ABO HDFN - Etiology and Symptoms

ABO HDFN is the most common type of HDFN, in that anti-A is the antibody most often found bound to the red cells of a newborn. While the disease is usually so mild as to not require treatment, severe HDFN is possible. EtiologyABO HDFN is caused by maternal IgG anti-A or anti-B, which can be produced as a result of prior pregnancy or prior inoculation (some common inoculations contain A or B substances). In Caucasians, most often the mother is group O and the child is group A, although other combinations are possible. Group O people tend to produce IgG ABO antibodies more commonly than other blood groups.Just as in other types of HDFN, maternal IgG antibody crosses the placenta and destroys fetal red cells.SymptomsTypical symptoms of ABO HDFN include mild anemia and especially jaundice appearing in the first 24 hours. In rare severe cases the infant can have the more severe symptoms of Rh HDFN, except that prenatal death is unlikely. Rationales to explain the mild nature of ABO HDFN include Fewer A and B antigens on fetal cells Poorly developed fetal A and B antigens Presence of A and B antigens on cells and tissues other than red cells

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ABO HDFN - Diagnostic Tests

Before ABO HDFN is considered as a possible cause of jaundice and anemia in the newborn, other causes should be considered, for example, erythrocyte membrane defects or red cell enzyme deficiencies. The diagnosis of ABO HDFN in the laboratory differs from diagnosing Rh and other types of HDFN in which clinically significant antibodies must be identified. Diagnosis may be difficult, because the DAT on the newborn's red cells is unreliable. Indeed, many labs do not routinely do a DAT on infants born to Rh positive females, since many will be positive in the absence of clinically significant hemolysis. Cord blood is often retained (e.g., for 7 days) should the infant develop signs of HDFN and required testing.If ABO HDFN is possible, based on incompatible ABO blood groups and a positive DAT, and the mother's antibody screen is negative, many laboratories do not investigate the positive DAT as would be done for unexpected antibodies like anti-D or anti-K (the laboratory does not perform an elution on the newborn's red cells). Instead, the infant's plasma is tested against group A1 (or B cells) and group O screen cells using the indirect antiglobulin test (IAT). A positive reaction with A1 or B cells, but not group O cells, would suffice to report a case of possible ABO HDFN.

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ABO HDFN - Treatment

Prenatal treatment Prenatal management and treatment of ABO HDFN is not routinely done because: Titers of anti-A and anti-B do not correlate well with severity of disease; The risks of fetal monitoring (e.g., amniocentesis, cordocentesis) and fetal transfusion are greater than the risk of ABO HDFN since it is usually mild and subclinical. However, if a woman has a history of infants with moderate to severe ABO HDFN requiring treatment, she may be monitored so that the infant can be treated for possible HDFN as soon as possible. Postnatal TreatmentTreatment of ABO HDFN usually consists of phototherapy in which the newborn is placed under a "blue light" that chemically alters bilirubin in the surface capillaries to a harmless substance.For more severe cases, exchange transfusion may be performed. Donor RBC for exchange transfusion in cases of ABO HDFN must meet these criteria: Group O; Rh compatible with infant; Less than or equal to 7 days old (or fresher); Reconstituted with AB FFP to obtain a prescribed hematocrit; CMV negative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from sickling under conditions of reduced oxygen concentration in the newborn; Irradiated to prevent graft-versus-host disease. Exchange transfusion is also discussed later in the course in the section related to HDFN due to anti-D and other antibodies. Red Blood Cells are crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

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All of the following criteria for donor RBC to be used for an exchange transfusion relate to both ABO HDFN and HDFN due to anti-D:Less than or equal to 7 days old (or fresher) Reconstituted with AB FFP CMV negative Negative for hemoglobin S Irradiated View Page
ABO HDFN - Expected Findings

Diagnosis of ABO HDFN is supported by these findings: ABO incompatibility between mother and child, with mother typically group O; Maternal antibody screen negative; Cord DAT weakly positive or negative; Newborn hyperbilirubinemia with jaundice occurring in first 24 hours; Increased spherocytes and reticulocytosis in the newborn; Presence of IgG anti-A or anti-B in cord plasma / serum.

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Which procedure used to obtain a fetal blood sample to monitor severity of HDFN can also be used to deliver intravenous transfusions?View Page
Choosing Donor RBC for IUT and IVT

Donor RBC for IUTs and IVTs have these criteria: Group O Rh negative*; Crossmatched with maternal serum; Fresh: less than or equal to 7 days (or fresher); High hematocrit, e.g, 85–90% (0.85–0.90) to prevent volume overload; CMV seronegative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from hypoxia-induced sickling in the fetal circulation; Irradiated with a minimum dose of 25 Gray (Gy) to prevent graft-versus-host disease.* Some laboratories use red cells that are also K-negative since the K antigen is very immunogenic. This also applies to exchange transfusions.

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Criteria for Transfused Red Blood Cells

The Red Blood Cells (RBC) that are chosen for exchange transfusion must meet these criteria: ABO-compatible with mother and infant (usually group O) and lack antigens to any maternal IgG antibodies; If mother has anti-D, RBCs are group O Rh negative; No greater than 7 days old; Reconstituted with AB Fresh Frozen Plasma (FFP) to obtain a prescribed hematocrit, e.g., 45–60% (0.45–0.60); CMV negative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from hypoxia-induced sickling; Irradiated with a minimum dose of 25 Gray (Gy) to prevent graft-versus-host disease.RBC are normally crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

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Routine Serologic Tests - Mother

Tests done routinely as part of perinatal testing programs vary from country to country and within countries. Below is one example of routine serologic tests typically done when pregnant females lack clinically significant antibodies. Other test protocols exist.Tests on Mother ABO, Rh*, and antibody screen at first prenatal visit; Test for weak D, if initial Rh typing appears to be D-negative (Optional -not mandated by blood safety standards); D-negative females: Tested again (ABO, Rh, and antibody screen) at ~ 28 weeks gestation prior to administration of RhIg (depending on the country) and again at delivery.* The mother, putative father, and fetus can be typed for D using DNA methods, if available.

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Follow-up Investigative Tests (Mother)

If a pregnant woman is found to have an unexpected clinically significant antibody, routine antenatal serologic tests on the mother include Antibody identification to detect clinically significant antibodies. Antigen typing: Once the antibody is identified, the mother is tested for the corresponding antigen, which she should lack. Antibody titration: Laboratories have different protocols. Depending on the antibody titer, titration may be performed at 2 or 4 week intervals after 18 weeks gestation.Notes (titration): Maternal antibody titer is an unreliable indicator of fetal disease and is mainly done to determine if clinical fetal monitoring is warranted, e.g., Doppler ultrasonography of fetal cerebral blood flow or, more rarely, invasive monitoring such as amniocentesis. Careful quality control is needed for titrations. QC includes using red cells from donors with the same phenotype or likely genotype (e.g., R2r or R2R2) and titrating the new sample in parallel with the prior sample. A two-tube rise or more in a doubling dilution is considered a significant rise in titer. In the case of anti-D, a predetermined critical titer (often 16 or 32 for anti-D depending on the method) indicates the need for clinical fetal monitoring.

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Follow-up Investigative Tests (Fetus)

If a mother has a clinically significant antibody, fetal blood for phenotyping can be obtained by several procedures, depending on gestational age and the antigen involved. These include Amniotic fluid sampling* Chorionic villus sampling* Cell-free fetal DNA in maternal plasma* Percutaneous umbilical cord blood sampling (PUBS) / cordocentesis** * molecular genotyping / ** serologic testsAs noted, typing the fetus is warranted when the father's blood type is unknown or the father tests as heterozygous positive.

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Molecular Genotyping - Introduction

The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. Molecular methods exist for typing Rh (RHD and RHCE), Kell (K & k), Duffy (Fya & Fyb), and Kidd (Jka &Jkb) loci.In perinatal testing programs, molecular typing can determine the Rh type of the mother, father, and fetus and may be done if the mother has anti-D or another antibody known to cause HDFN. More specifically, if available, DNA methods are typically used in these circumstances: For women who type as weak D in serologic tests, to determine the Rh genotype of the mother to identify if she is partial D or weak D; For women who have made anti-D, to determine the Rh genotype of the father to see if fetal monitoring is needed; For women who have made anti-D, to determine the Rh type of the fetus if the father is heterozygous for RhD or unavailable for testing. Fetal blood typing can be done using fetal DNA from cells obtained by amniocentesis or by testing cell-free, fetal-derived DNA present in maternal plasma at 5 weeks gestation and later. Like all diagnostic methods, DNA typing has limitations and is not 100% sensitive and specific. For example: The blood group's molecular basis may be unknown; Not all alleles in ethnic populations are known; Rare mutations in the RHD and other genes may not be detected; Silencing changes (switching off of a gene) may affect antigen expression; Fetal typing using amniotic fluid may give false-negative results because of maternal cell contamination.

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Immunogenicity

Immunogenicity is the ability of an antigen to provoke an immune response in an antigen-negative recipient. Why some antigens are more immunogenic than others is unknown. Not considering antigens in the ABO system, Rh(D) is the most immunogenic red cell antigen, followed by K in the Kell blood group system. Other immunogenic antigens include c and E in the Rh system. In routine blood banking, assessments of an antigen's immunogenicity are typically based on the prevalence of the corresponding antibody and do not take into account the frequency of the antigen in the general population. For example, k in the Kell system may be very immunogenic but anti-k is rare since 99.8% of Caucasians are k+ and cannot make anti-k.

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Newborn Serologic Testing Protocols

Protocols for testing newborns vary internationally and within countries. The table below summarizes some of the more common protocols. Scenario Typical Newborn Testing Protocol Comments Mother is D-negative with no unexpected antibodies Newborn is tested at delivery for: ABO and Rh Test for weak D (mandatory) if initial Rh typing appears to be D-negative Direct antiglobulin test (DAT)* A positive DAT does not always mean that the newborn has clinically significant hemolysis. A positive DAT commonly occurs due to ABO incompatibility, yet infants seldom require treatment. Infants born to mothers who received antenatal RhIg sometimes have a positive DAT that does not cause clinically relevant hemolysis. Mother is Rh positive and a blood group other than group O Routine testing not performed Cord blood retained for a specified period of time (e.g., seven days) in the event that the mother has an unexpected antibody at delivery or the newborn develops signs of red cell hemolysis. Routine testing would result in many positive DATs due to ABO incompatibility- not clinically significant. Mother is group O Rh positive Newborn is tested- especially important if women and their infants are discharged within 24 hours since hyperbilirubinemia due to ABO HDFN may develop later. Optional only if there is appropriate surveillance and risk assessment before discharge and provided there is follow-up (American Academy of Pediatrics). *Policies for DAT testing of newborns whose mothers have received antenatal RhIg vary internationally. For example, the British Committee for Standards in Haematology guidelines state that a DAT should not be performed on cord blood routinely since in some cases it may be positive due to antenatal RhIg prophylaxis. A DAT is recommended only if HDFN is suspected because of a low cord blood hemoglobin or the presence of unexpected maternal antibodies. However in North America, DATs are always performed on infants born to Rh negative mothers who are RhIg candidates.

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RhIg Dosage

In North America, a standard dose of RhIg is considered to be 1500 IU (300 µg). Note: 1 µg of anti-D = 5 IU.300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood (15 mL of packed rbc). If gestational age is known to be less than 12 weeks, a 600 IU (120 µg) dose may be sufficient.Depending on the gestation of the fetus, recommended dosages vary from country to country and within countries. Samples of recommendations that may change over time: USA: American Congress of Obstetricians and Gynecologists (1999, reaffirmed 2007): Antenatal RhIg dose of 300 µg (1500 IU) at 28 weeks and another 300 µg after delivery of a D-positive infant. Canada: Society of Obstetricians and Gynaecologists of Canada (2003): Antenatal RhIg dose of 300 µg (1500 IU)at 28 weeks (alternatively, 2 doses of 100–120 µg, one at 28 weeks and one at 34 weeks). After delivery of a D-positive infant, another 300 µg (alternatively, 120 µg IM or IV). UK: Royal College of Obstetricians and Gynaecologists (2002): Antenatal RhIg does of 100 µg (500 IU) at both 28 weeks and 34 weeks of gestation, and another 100 µg after delivery of a D-positive infant. All recommendations require testing to detect larger fetal bleeds, e.g., FMH larger than 30 mL of whole blood (for 300 µg doses) and FMH over 12 mL of rbc for 100 µg doses.

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A 300 µg dose of RhIg can suppress immunization to how many mL of D-positive whole blood?View Page
RhIg 'Failures'

Numerous studies have shown that, if administered correctly, RhIg is effective at preventing D immunization. To work, RhIg must be given in sufficient dose, and it must be given before Rh immunization has begun.Unfortunately, despite RhIg's proven efficacy, some women continue to make anti-D in the perinatal period. Such 'failures' are mainly (but not totally) due to human error. Examples of how women may still produce anti-D some 40+ years after the implementation of RhIg prophylaxis: Immunization to D occurred before the administration of RhIg, e.g., before 28 weeks gestation*; Immunization to D occurred after the administration of RhIg at 28 weeks and before delivery because an antenatal fetomaternal hemorrhage (FMH) occurred that was too large for residual passive anti-D to give protection; Female was already immunized from a prior pregnancy but anti-D was too weak to be detected in antibody screen tests prior to RhIg administration; RhIg dosage was insufficient to clear a larger fetal bleed at delivery (e.g., FMH screen was not done or a false negative occurred); Incorrect calculation of RhIg dosage; RhIg administered too late , e.g., well after 72 hours of delivery; Antenatal RhIg not given, e.g., mother had no, or limited, access to prenatal care, or did not seek it, and a FMH occurred during pregnancy; Failure of physician to carry out prenatal blood testing; RhIg not given due to laboratory clerical or technical error in Rh typing the mother or child; RhIg not given in cases such as abortions, ectopic pregnancies, and trauma (e.g., car accidents).* Because anti-D production before 28 weeks is rare (the order of 0.24% to 0.31%), RhIg's use earlier in pregnancy is not recommended. It is not cost effective and would expose most women to an unneeded blood product.

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Introduction

Fetomaternal hemorrhage (FMH) greater than 30 mL of whole blood occurs in only about 0.3% of cases but must be detected to prevent the mother from producing anti-D. Once the mother has become immunized to D, RhIg is of no use.A typical test protocol is to first screen for a large FMH and then quantitate the bleed if the screen is positive. Some laboratories proceed directly to a test that can quantitate the size of the FMH.Once the size of the FMH is determined, a formula is used to determine how much RhIg is needed. Recall that A standard vial of RhIg contains 1500 IU (300 µg) of IgG anti-D; 300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood.

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Calculating RhIg Dosage

Using the estimated volume of fetal bleed determined by the Kleihauer-Betke test or flow cytometry, the number of vials of RhIg (300 µg) to inject is calculated as follows: Number of vials of 300 µg RhIg = volume of fetal bleed/30 mLIn the interests of safety some American organizations recommend the following to deal with decimal points: If the number to the right of the decimal point is <5, round down and add 1 vial (e.g., 1.4 = 1 +1 = 2 vials) If the number to the right of the decimal point is greater than or equal to 5, round up and add 1 vial (e.g., 1.7 = 2 +1 = 3 vials) Sub-calculations: Volume of fetal bleed: % fetal cells x maternal blood volume Maternal blood volume: 70 mL/kg x weight (kg) (assume 5,000 mL if maternal information is unknown) Note: RhIg dose calculators are available (see Further Reading: Paxton A. Bringing new rigor to RhIG calculations).

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Rosette Test

The rosette test is a screening test for FMH that detects fetal D+ red cells in maternal Rh negative blood. If the rosette test is positive, follow-up testing is done to quantitate the FMH, e.g, a Kleihauer-Betke acid elution test or flow cytometry.Note: The rosette test cannot be done if the fetus is weak D as false negatives may result. In such cases, a Kleihauer-Betke test or flow cytometry can be done.General description (example only): Incubate a maternal 3-5% red cell suspension with IgG anti-D at 37°C. The anti-D will bind to any infant D+ cells that are present.After washing to remove unbound anti-D, add indicator red cells. Indicator cells are ficin-treated R2R2 cells that will bind to the antibody-coated infant RBCs causing agglutination (“rosettes”) that can be detected microscopically.A specified number of agglutinates (e.g., 3 or more in 10 fields or 7 or more in 5 fields) is designated a positive and suggests a significant FMH (>30 mL) requiring more RhIg. The top image on the right illustrates a negative rosette test. The bottom image is representative of a field that would meet the criteria for a positive rosette test, if the same number of agglutinates, or more, are counted in the required number of fields, as discussed above.These images were provided courtesy of Mount Sinai Blood Transfusion Laboratory, Toronto, Ontario and can be found on Canada's Transfusion Safety Officer's Website. Available at: http://www.transfusionsafety.ca/library/kb-ros.html. Accessed September 26, 2011.

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The appropriate dosage of Rh immune globulin (RhIg) to administer post-delivery to an Rh-negative mother delivering an Rh-positive child is calculated based on the estimated volume of fetal bleed.What is the value of x in the formula given below that is used to calculate RhIg dosage?Number of vials of 300 µg RhIg = volume of fetal bleed/x mLEnter the number in the box below that is represented by x in the formula; do not spell out the number.(e.g., use "5" and not "five").View Page
Main Learning Goals

This course reviewed some of the key learning goals relevant to HDFN and its investigation, prevention, and treatment. More specifically, the course reviewed the following topics: Historical aspects of HDFN due to anti-D and its prevention; HDFN due to antibodies in the ABO, Rh, and other blood group systems; Clinical symptoms and associated laboratory test results in HDFN; Best practices related to perinatal testing programs to prevent and diagnose HDFN; Characteristics and uses of RhIg; Interpretation of typical serologic test results when investigating HDFN.Before taking the final quiz, for each of the above topics, list as many of the key learning points that you can recall, then review topics that need more study. As well, re-read the learning objectives at the start of the course as these determine assessment questions.It's also worthwhile to read the literature and online resources in Further Reading as these reinforce key points, add to the depth of learning, and enrich the course materials.

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Literature and Online Resources

The following published literature and online resources, while useful, should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.References indicated by * provide a broad overview of HDFN and are highly recommended.LITERATUREAvent ND, Reid ME. The Rh blood group system: a review. Blood 2000 Jan 15;95 (2):375-87.Bowman J. Thirty-five years of Rh prophylaxis. Transfusion 2003 Dec;43(12):1661-6.* Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematology 2006;22(4):188–195. (scroll to article)Eder, AF, Manno, C.S. Alloimmune hemolytic disease of the fetus and newborn. In Wintrobe's Clinical Hematology, 11th ed. (Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader BE, (eds). Philadelphia, PA: Lippincott, Williams & Wilkins, 2004.Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol. 2006 Mar-Apr;13(1-2):4-12. Kennedy MS, McNanie J, Waheed A. Detection of anti-D following antepartum injections of Rh immune globulin. Immunohematology 1998;14(4):138-40.Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis. BJOG. 2009 Sep;116 (10): 1307-14. Epub 2009 Jun 17.* Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ. 2005 May 28;330(7502):1255-8. (UK perspective but much valuable information relevant to all)* Murray NA, Roberts IAG. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007 Mar; 92(2): F83–F88. Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G; DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006 Jul 13;355(2):156-64.Ramsey G. Inaccurate doses of Rh immune globulin after Rh-incompatible fetomaternal hemorrhage: survey of laboratory practice. Arch Pathol Lab Med 2009 Mar; 133(3):465-9. Reid ME. The Rh antigen D: a review for clinicians. Blood Bulletin 2008 Apr; 10(1).Sandler SG. Effectiveness of the RhIg dose calculator. Arch Pathol Lab Med 2010 Jul;134(7): 967-8.Shulman IA, Calderon C, Nelson JM, Nakayama R. The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies. Transfusion 1994 Aug;34(8):666-70.Tamul KR. Determining fetal-maternal hemorrhage with flow cytometry. Advance 2000. Posted online June 5, 2000.Westhoff CM, Sloan SR. Molecular genotyping in transfusion medicine. Clin Chem 2008;54(12): 1948-50.ONLINE RESOURCESPaxton A. Bringing new rigor to RhIg calculations. CAP TODAY. May 2008. Accessed January 18, 2011.*Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010. Accessed January 18, 2011.

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Hereditary Hemochromatosis (retired 2/13/2014)
References

1. Beutler E. Iron storage disease: Facts, fiction and progress. Blood Cells Mol Dis. 2007;39:140-7.2. Higgins T, Beutler E, Doumas BT. Hemoglobin, iron, and bilirubin. In: Burtis CA, editor. Teitz Fundamentals of Clinical Chemistry. 6th ed. Saunders Elsevier, 2008.3. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia and inflammation. Blood 2003;102(3):78-8.4. Andrews NC, Schmidt PJ. Iron homeostasis. Annu Rev Physiolo. 2007;69:69-85.5. Murtagh LJ, Whiley M, Wilson S, et al. Unsaturated iron binding capacity and transferrin saturation are equally reliable in detection of HFE hemochromatosis. Am J Gastroenterol. 2002;97(8):2093-9.6. Haddy TB, Castro OL, Rana SR. Hereditary hemochromatosis in children, adolescents, and young adults. Am J Pediatr Hematol Oncol 1988;10:23-4.7. Edwards CQ, Ajoika RS, Kushner JP. Hemochromatosis: A genetic definition. In Barton JC, Edwards CQ, eds. Hemochromatosis: Genetics, Pathophysiology, Diagnosis and Treatment. Cambridge, UK:Cambridge Univ Pr 2000:8-11.8. Whitlock EP, Garlitz BA, Harris EL , et al. Screening for Hereditary Hemochromatosis: A Systematic Review for the U.S. Preventive Services Task Force. Ann Intern Med. 2006; 145: 209-23.9. Wallace DF, Subramaniam VN. Non-HFE haemaochromatosis. World J Gastroenterol. 2007;13(35):4690-8.10. Tavill AS. Diagnosis and management of hemochromatosis. Hepatology. 2001;33:1321-811. Qaseem A, Aronson M, Fitterman N, Snow V, Weiss KB, Owens DK, et al. Screening for hereditary hemochromatosis: a clinical practice guideline from the American College of Physicians. Ann Intern Med. 2005;143:517-21.12. Phatak PD, Bonkovsky HL, and Kowdley KV. Hereditary Hemochromatosis: time for targeted screening. Ann Intern Med. 2008; 149(4): 270 – 2.13. Brissot P, deBels F. Current approaches to the management of hemochromatosis. Hematology Am Soc Hematol Educ Program. 2006:36-41. 14. Guidance for industry: Variances for blood collection from individuals with hereditary hemochromatosis. http://www.fda.gov/cber/gdlns/hemchrom.htm Accessed 12/17/08.

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Storage Iron

Storage forms normally comprise approximately 27% of total body iron. Stored iron provides a source of iron when physiologic demand is high, such as in blood loss, pregnancy, and periods of rapid growth. Storage compounds include ferritin and hemosiderin. Ferritin is a protein-bound, water-soluble, mobilizable storage compound and is the major source of stored iron. Hemosiderin is a water-insoluble form that is less readily available for use. When the amount of total body iron is relatively low, storage iron consists predominately of ferritin. When iron stores are increased, hemosiderin predominates. Unlike ferritin, hemosiderin stains with the Prussian blue stain (Perls reaction) and may be observed in tissues. The image on the right shows iron deposits in a liver section that was stained with Prussian blue.Image is courtesy of John Woosley, MD.

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What is the protein that carries iron in the blood plasma?View Page
Development of Iron Overload

The amount of time needed for iron to increase to levels causing organ damage is variable and may be partially dependent on gender, dietary or other environmental factors, and unknown genetic factors. Blood loss through menstruation and pregnancy are thought to delay the onset of iron overload, and therefore symptoms of HH, in women. Similarly, regular blood donation may confer some degree of protection. The loss of hemoglobin within intact erythrocytes reduces the amount of iron available for recycling.As levels of storage iron increase, clinical features of iron overload, including hepatic dysfunction or failure, diabetes, hypogonadism, arthritis, cardiomyopathy, hyperpigmentation, and fatigue, may become evident.Symptomatic patients typically present in middle age between the ages of 30 and 60, although this is quite variable. Persons as young as 20 may show clinical signs and symptoms of HH.(6) In the US, males are more than twice as likely as females to be diagnosed with HH, and the majority of cases are found in Caucasians.

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Transferrin Saturation

Transferrin saturation (TS) is usually reported along with the serum iron (SI) and total iron binding capacity (TIBC). TS indicates the percent of iron binding sites on transferrin that are carrying iron. TS is derived from a calculation using the formula:TS =(SI/TIBC) x 100TS results are reported as percentages. Typical reference intervals for TS are 20% to 55% for males and 15% to 50% for females. TS is currently considered to be a good test for screening persons for hereditary hemochromatosis (HH) due to its sensitivity and specificity for iron overload. It may be elevated prior to significant deposition of tissue iron. TS levels increase as additional iron is accumulated.A drawback to using the TS is that it is dependent on performing both the SI and TIBC. The unsaturated iron-binding capacity UIBC may be a lower cost alternative.The optimal TS criterion for detecting HH is controversial. Using a TS of >60% for males and >50% for females has been found highly accurate in detecting abnormal iron metabolism in persons with HH. Others studies suggest using lower TS levels, e.g. 45%, as a criterion indicating further testing is warranted. Current guidelines from the American College of Physicians include a TS cutoff level of >55% for identifying iron overload. (11)Patients with initially increased TS should be followed by performing a second TS from a fasting morning specimen. The patient should also be advised not to take vitamins supplemented with iron or oral contraceptives for several days prior to the repeated test. TS levels may be affected by diurnal variation, dietary factors, and co-existing disease states such as inflammation and hepatitis. Patients with HH may have falsely normal TS if chronic blood loss or inflammatory disease is present.

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Molecular Tests

DNA tests for HFE mutations associated with hereditary hemochromatosis (HH) are available in some clinical laboratories and reference laboratories. Testing for the presence of the C282Y is essential, although most labs also test for H63D and S65C mutations. Molecular testing is most appropriate for confirmatory testing of symptomatic individuals with altered iron studies (increased TS and SF), in pre-symptomatic individuals (increased TS, normal SF and liver function tests), and in family members of individuals diagnosed with HH. The use of genetic tests alone for routine screening of asymptomatic persons is not recommended for several reasons. A positive test indicating the presence of HFE mutations does not guarantee that an individual will develop clinically significant iron overload or predict severity of symptoms. A negative result (no HFE mutations present) does not rule out a diagnosis of iron overload because of genetic heterogeneity. Compared to biochemical analyses for iron, molecular assays are expensive. Finally, molecular testing may result in the diagnosis of a genetic disease, thus opening up the possibility for discrimination in health insurance coverage. Using molecular methods, DNA is extracted from leukocytes in whole blood samples or from buccal cells and analyzed for specific HFE mutations using polymerase chain reaction (PCR) with melt curve analysis. Currently there are no FDA-cleared products for HFE testing, and testing laboratories are using "home brew" reagents. This situation is expected to change as manufacturers submit products for FDA approval.

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Quantitative Phlebotomy

An alternative to liver biopsy as a means of documenting iron overload may be provided by quantitative phlebotomy performed during treatment. The removal of 4 to 5 grams of iron through documented successive phlebotomies (16 to 20 phleblotomies) without development of anemia is indicative of iron overload. (One unit, or 450 mL, of blood is assumed to contain approximately 200 to 250 mg of iron.) Quantitative phlebotomy is useful in patients for whom liver biopsy is contraindicated or patients who refuse the procedure.

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Initial Treatment

Phlebotomy is considered the treatment of choice for patients with iron overload due to hereditary hemochromatosis (HH). Each unit of blood contains approximately 200 to 250 mg of iron. As erythrocytes are removed by phlebotomy, iron stores are mobilized and utilized in the production of new, circulating erythrocytes. Through periodic phlebotomies, stored iron is removed until iron-deficient erythropoiesis is induced. The initial, or iron reduction, phase of treatment typically consists of removing one unit (450 mL) of whole blood once or twice weekly. Prior to beginning phlebotomy, the patient's hemoglobin and hematocrit must be checked to ensure that the patient is not anemic. A sample for serum ferritin is also collected at this time.Initial treatment goals include inducing iron deficient hematopoiesis without the development of debilitating symptoms of anemia. A hemoglobin concentration of 10.0 to 12.0 g/dL is often used as a target range. The initial treatment phase continues until excess stored iron is removed and ferritin levels decrease to approximately 50 ng/mL. (13) Ferritin and hemoglobin levels are periodically monitored during this phase. The number of phlebotomies needed to reduce iron levels and induce anemia is related to the degree of initial iron overload. Patients may be referred to a hematologist or gastroenterologist during the initial treatment phase. Many patients receive therapeutic phlebotomy services in a hospital or doctor's office, but patients may also undergo phlebotomy at a blood center. Blood collected from persons with HH may be used for transfusion or as blood products if it has been collected from a facility with an approved variance from the US Food and Drug Administration. Not all blood centers have applied for or been granted this variance.(14)The initial treatment phase continues until excess stored iron is removed and ferritin levels decrease to approximately 50 ng/mL. Removal of excess stored iron may take from one month to three years.

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What is a typical schedule for phlebotomy during the initial treatment phase for hereditary hemochromatosis (HH)?View Page
Maintenance Therapy

Lifelong treatment of hereditary hemochromatosis (HH) is needed to keep iron at low levels. Without regular treatment, iron stores will re-accumulate. The primary care physician may manage patient care during long-term maintenance. Long-term maintenance typically consists of removal of an average of 2 to 6 units of whole blood yearly, although this number is variable. Monitoring of hemoglobin and serum ferritin levels determine the frequency of phlebotomy. Serum ferritin levels should be maintained at concentrations of no more than 50 ng/mL. (10,13))

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HIPAA Privacy and Security Rules
Case Study: Administrative Safeguards You are the technologist in charge of the hematology section in a hospital laboratory. The laboratory manager and the pathologist who oversee the laboratory's Quality Management Program have asked you to review blood count results for 100 patients as part of an internal quality assurance project. You review only the clinical findings in the electronic medical record that are relevant to this study and correlate the findings with the laboratory results. The QA report that you develop does not include any personal information, such as patient names. The following week, you get a call from your hospital security officer. She says that a routine computer system audit has revealed that you accessed the records of 100 patients and she would like to know why. What would be a correct response to her concern, considering the reason you accessed the information?View Page
Case Study: Minimum Necessary Use & Disclosure You are a phlebotomist at a specimen collection center. A patient arrives with orders for a blood glucose test and a lipid profile. You get the patient's address, phone number, health insurance coverage, and ask how long ago he ate his most recent meal. You then ask him about his recent auto accident, his wound infection, and his family. You write down all the extra information. Under the HIPAA Privacy Regulations, which of the following information requests is acceptable?View Page
Case Study: Incidental disclosures and safeguards. As a manager, you guided a group of high school students through your clinical laboratory during a field trip. You did not explain the laboratory's privacy policy to the teacher and students, because you thought they would have little access to PHI. However, during the tour, the students overheard names of patients and associated blood tests, saw laboratory reports laying on desks, and viewed test results on computer screens. True or false: This is acceptable under the HIPAA Privacy Regulation since these were incidental disclosures that could not reasonably be prevented.View Page

Histology Special Stains: Connective Tissue
What is Connective Tissue?

Connective tissue offers structural and metabolic support structure for organs and tissue. It is the most abundant tissue type in the body and can be found throughout. Cells and extracellular material called connective tissue matrix make up connective tissue. Fibroblasts, mast cells, macrophages, adipose cells, blood leukocytes, and plasma cells can all be found to some degree in connective tissue. In addition to cells, the matrix has 3 different fibers present:Reticular fibers - Support soft organs and the network around nerve fibers, fats cells, lymph nodes, and muscle fibers.Collagenous fibers - Found in ligaments, tendons, cartilage, and bone.Elastic fibers - Allow tissue to expand and are typically located in skin and blood vessel walls.

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Classifying Connective Tissue

Connective tissue is classified by cellular patterns and the composition of the matrix. There are 4 categories of connective tissue in adults:Connective tissue proper - The matrix is composed of collagen, elastin, and reticulin fibers as well as adipose and mast cells.Cartilage - The matrix is composed of firm, gel-like matrix is made up of water, collagen fibers and chondrocytes.Osseous tissue (bone) - Bone is comprised of a rigid, mineralized matrix of collagen fibers and osteocytes.Vascular tissue (blood) - The matrix is a liquid called plasma, which consists of red and white blood cells, leukocytes and platelets.

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Functions of Connective Tissue

The primary functions of connective tissues include:Transportation of nutrients and metabolites through direct diffusion between organs and connective tissue properImmunological defense (fights invading cells via inflammation)Structural supportTissue repair (after injury) Additional functions found in certain body sites include:Energy storage (fat)Heat generation (brown fat)Haematopoiesis/haemopoiesis (blood formation)

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Where are reticular fibers found?View Page

HIV Safety for Florida
Which of the following is not considered a potentially infectious body fluid for transmitting HIV?View Page
The type of health-care occupational exposure with the greatest risk of HIV transmission is:View Page
Occupational Exposures

HIV transmission, due to occupational exposure, occurs by: Percutaneous injury, such as a needlestick or a cut with a sharp object; Contact of mucous membrane or abraded skin with HIV-infected blood or body fluids. The risk of HIV transmission after a percutaneous exposure to HIV-infected blood is 0.3%.The risk of HIV transmission after a mucous membrane exposure to HIV-infected blood is .09%.The risk of HIV transmission after contact of abraded skin with HIV-infected blood is estimated to be less than .09%.

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Potentially infectious body fluids

These substances are considered potentially infectious for an occupational exposure: blood cerebrospinal fluid synovial fluid pleural fluid peritoneal fluid pericardial fluid amniotic fluid any body fluid visibly contaminated with blood semen or vaginal fluid tissues removed during surgery.

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Risk factors associated with increased HIV infection

The risk factors that increase the risk of an exposure leading to HIV infection are: larger quantity of blood from source person, and blood from source person in terminal stage of HIV disease.

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Which of the following does not pose a significant risk for transmitting HIV?View Page
Evaluation and Treatment

Your supervisor will refer you for an immediate evaluation and any necessary treatment. Confidentiality will be maintained.Your blood will be tested only with your consent.

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Overview

Prevention of HIV exposure is the best line of defense to prevent occupational transmission of HIV as there is no vaccine available to develop specific immunity and the postexposure prophylaxis is toxic. Following appropriate workplace practices in the laboratory focus on preventing needlesticks or other sharps injuries and exposure of mucous membranes and abraded skin to HIV-infected blood or body fluids.

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Face and Eye Protection

Face shields, masks, and safety glasses protect your eyes and the mucous membranes of your nose and mouth.They must be worn whenever it is reasonably anticipated that splashing or spraying of blood or other contaminated materials may occur.Employees who wear prescription eyewear may be protected with a face shield, goggles, or with side shields attached to their glasses.

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Gloves

Gloves must be worn: when there is a reasonable chance of exposure to blood, other infectious body fluids, mucous membranes, or nonintact skin. during vascular access procedures, including phlebotomy. when handling contaminated items or surfaces.Wear only flat rings under gloves as large rings may tear gloves.Replace gloves: Between patient contacts If they are damaged or contaminated Before leaving the work area. Wash hands after removing gloves.Never wash disposable gloves.

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Introduction to Bioterrorism
Agent: Pneumonic plague (Bacterium)

Most likely means of dissemination: AerosolPrimary route of entry: InhalationGeneral signs and symptoms: High fever, chills, headache, coughing up of blood (hemoptysis), and toxemia, progressing rapidly to difficulty in breathing (dyspnea), and bluish discoloration of the skin and mucous membranes (cyanosis).There is another form of the disease called “bubonic plague”. While it is caused by the same organism, it is not transmissible through human contact. Pneumonic plague is transmissible through human contact.

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Early symptoms of inhaled Anthrax includeView Page
Types of Chemical Agents

There are four primary agents that could possible be used in a chemical attack: Lung-damaging or choking agents Blood agents Blister agents Nerve agentsOthers that might be used include: incapacitating agents, riot-control agents, heavy metals, volatile toxins, pesticides, dioxins, explosive nitro compounds and oxidizers, flammable industrial gases and liquids, plus corrosive industrial acids and bases.

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Blood Agents

Example: Hydrogen cyanidePhysical Properties: Highly volatile gas with a bitter almond odor.General Signs and Symptoms: Violent convulsions, stoppage of breathing, cardiac failure.Relative Rate of Action: Incapacitation within minutes and death within 15 minutes.

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Laboratory Response - Chemical, Level 2

In addition to the responsibilities listed for Level 3, over 40 laboratories also participate in Level 2 activities. At this level, laboratory personnel are trained to detect exposure to a limited number of toxic chemical agents in human blood or urine, the analysis of cyanide and toxic metals in human samples, for example.

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Laboratory Response - Chemical, Level 1

At present, 5 laboratories participate in Level 1 activities. At this level, technical personnel are trained to detect exposure to an expanded number of chemicals in human blood and urine. This includes all Level 3 and 2 laboratory analyses, plus analyses for mustard agents, nerve agents, and other toxic chemicals.

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Introduction to Bone Marrow
The two main compartments of the bone marrow are the venous sinuses/blood vessels and hematopoietic cords.View Page
Collection of the Aspirate

The marrow aspiration is usually performed before a biopsy is done. A syringe is attached to the needle, the plunger is pulled and 1.0-1.5 ml. of marrow particles and blood from marrow sinuses is withdrawn. If additional bone marrow samples are needed, a separate syringe must be used each time. If more than 2 cc. per syringe is taken out, the blood to marrow ratio will be too high and the preparations will not accurately reflect the marrow contents. As the marrow is aspirated into the syringe the patient will feel some pain and pressure even though local anesthetic has been administered.

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Preparation of Direct Smears

The sample in the first syringe is quickly delivered into a watchglass or onto a slide. After the technologist verifies the presence of white-gray marrow particles in the sample, push smears and/or coverslip smears from this unanticoagulated sample are made immediately. All films should be rapidly air dried. The appearance of fat as irregular holes in the films also give the assurance that marrow and not just blood has been obtained. This type of smear is referred to as a direct smear and is usually used to evaluate morphology. Although some evaluation of cellularity and M:E ratio is possible, particle smears or biopsy sections provide a more accurate representation of these factors.

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Basic Structure and Function of Bone Marrow

Before learning to examine bone marrow microscopically, it is important to understand the basic structure and function of the bone marrow. The bone marrow is one of the largest organs in the body. The normal adult marrow on a daily basis produces approximately 2.5 billion red cells, 2.5 billion platelets and 1.5 billion granulocytes per kilogram of body weight. The main function of this organ is the formation and development of blood cells. Hematopoiesis begins in the yolk sac in the first weeks of embryonic life; stem cells from the yolk sac travel first to the liver and then to the spleen. These organs are the only blood forming sites during the first three months of fetal life. At the beginning of the fourth month the bone marrow begins its life-long function of cell production.

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Supporting Cells

Reticular cells (adventitial cells) provide structural support for the endothelial cells that line the venous sinus and the developing blood cells within the hematopoietic cord. The cytoplasm of the reticular cells is capable of extending itself in fiberlike strands deep into the hematopoietic cords. These strands provide a meshwork for the blood cells. Other types of cells which furnish support in the cord include macrophages and fat cells.

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Location of Cells within Cord

Within the hematopoietic cords each cell line has a specific location for development. Erythroid precursors are located near a venous sinusoid and cluster around a macrophage. This is referred to as an erythroblastic island. Developing red cells obtain iron needed for hemoglobin production from macrophages. Megakaryocytes are also located close to a venous sinus. They extend their cytoplasm in fingerlike projections through the sinus wall in order to release their platelets directly into the blood in the sinus. Immature granulocytes lie within the hematopoietic cords. The metamyelocyte stage is the first stage of the granulocyte series that is motile and able to move toward the sinus area. Mature neutrophils, eosinophils and basophils enter the sinusoidal blood through the basement membrane. As maturing erythrocytes also move toward the sinus wall any remaining nuclei are lost as the red cells move through small openings in the cells lining the sinus wall.

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Lymphocytes

Lymphocytes are often located in nodules and these nodules are unevenly distributed throughout the marrow so the lymphocyte count may vary in bone marrow samples from different sites. Plasma cells are often found clustered around blood vessels. Monocytes seem to congregate about arterioles in the center of the cord.

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Summary

The bone marrow is structured to provide a suitable environment for developing cells as well as mechanisms for delivering mature cells to the circulating blood. The bone marrow is also capable of increasing production in one or more cell lines when needed.

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The bone marrow begins to produce blood cells in the ________ month of gestation.View Page
Which of the following statements are true for the blood vessel/sinus compartment of the bone marrow? (Choose ALL of the correct answers)View Page
Sinuses/Blood Vessels

Circulating blood enters the bone through the central artery which branches out into small arterioles. These arterioles are interspersed in the cords of hematopoietic tissue. The arterioles drain into venous sinuses (space or cavity). Sinuses have a basement membrane which is lined by endothelial cells within the sinus and surrounded by reticular (e.g. adventitial) cells on other side. Blood from several venous sinuses may combine in a collecting sinus which leads to a central vein. The venous sinuses alternate with hematopoietic cords in a spokelike pattern with the central vein as the core.

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Increase Marrow Iron Stores

Markely increased stainable iron is present in this biopsy. Iron stores may be increased in sideroblastic anemia, chronic infections, hemochromatosis, hemosiderosis due to numerous blood transfusions, chronic hepatitis, cirrhosis, and uremia.

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Evaluation of Bone Marrow

Evaluation of the bone marrow provides both diagnostic and prognostic information for a number of hematologic disorders. Indications for performing a bone marrow include an increase or decrease of any blood cellular element.

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The peripheral blood red cell count in this patient will likely be:View Page
The peripheral blood leukocyte count in this patient will likely be:View Page
The peripheral blood platelet count in this patient will likely be:View Page
Low Power View of Biopsy

This low power view of a hematoxyln and eosin stained bone marrow biopsy shows fat cells as clear circles, and the darker intervening areas as blood cell precursors. This biopsy is about 25% cellular, or mildly hypocellular. A normal marrow in a middle aged adult is about 50% cellular.

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Introduction to Flow Cytometry: Blood Cell Identification
Case One

A 50-year old woman presented to her physician reporting increasing fatigue over the past several weeks. Routine blood tests were ordered. Her white blood cell count was elevated with a slightly abnormal absolute lymphocyte count. To evaluate the patient's lymphocytosis, a peripheral blood sample was submitted for flow cytometry.The following cytograms and histograms represent data from the blood sample that was analyzed using flow cytometry.

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What cell population is gated in this peripheral blood sample case analyzed by flow cytometry?View Page
If 100% of the cells in the gated lymphocyte population are positive for CD45 (i.e., they are leukocytes) and 7% are B cells (represented by CD19 and CD20 positivity), approximately what percentage of T cells would be expected?View Page
T Cell Markers

Until now, the interpretation has been single color analysis. Ruling out a monoclonal B cell population does not guarantee that this is a normal peripheral blood. The T-cell lymphocyte population must also be evaluated. In order to do so, it is important to not only determine how many T cells are present, but also what subsets they represent. Dual color analysis can be used to distinguish C4 from C8 subsets in the C3 population.C2= 91%CD5= 90%CD7= 91%Image #1 CD3 is plotted with CD4. CD3/CD4 dual positive (+) cells = 55% Add Q1 and Q2 percentages together to get total CD3 = 88%Image #2 CD8 plotted with CD3. CD8/CD3 dual + cells = 33%Add Q1 and Q2 percentages together to get total CD3 = 88%Since this is the same sample and the same gated population, the total CD3 values should be very close from image to image. In the two images on the right, the total CD3 values are within one-tenth of each other (both round to 88%). C2= 91% CD5= 90% CD7= 91% CD3= 88% CD4= 55% CD8= 33%

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Final Interpretation of Case #1

The cell representations are listed below. B cellsCD19= 7%CD20= 7%CD10= 0%HLA-DR= 10%CD19/CD5 dual + = 0%Kappa=4%Lambda=3%Interpretation of the above data (refer to the decision tree to assist with interpretation of the case): There is a good mix of kappa and lambda; neither one is predominant. The B cells do not express CD5 (a B cell that expresses CD5 is abnormal because CD5 is normally a T cell marker) CD10 is not present on the B cells. Conclusion: B cells are NORMALT cellsCD2= 91%CD5= 90%CD7= 91%CD3= 88% The pan T cell markers all are within a few percentage points of one another, which translates to the sample having all markers that should be there and no values are skewed toward one marker. CD4 plus CD8 equals the total CD3 (55 + 33 = 88), therefore, there is good representation of both T helper and cytotoxic T cells (a neoplastic process could have predominantly one or the other or dual positive CD4 and CD8). Conclude: T cells are NORMALFinal conclusion: this is a normal peripheral blood lymphocyte population and there is no indication of a neoplastic process. The B cells that are present, though a small percentage of the total lymphocytes, have no abnormal markers. Most of the lymphocytes in this sample are T cells, yet they are normal as well. The CD markers that should be there are present and there is a good representation of both helper and cytotoxic cells.

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Flow Cytometry Course Introduction

Flow cytometry is a laboratory method that allows the simultaneous measurement of multiple physical characteristics of individual cells. A flow cytometer is used to isolate and fluorescently label blood cells based on molecules of interest associated with each cell. Next, the instrument analyzes and stores information about the cells based on the amount of light scatter and the fluorescent light that is emitted.Flow cytometry has many applications including: Immunophenotyping HIV disease assessment Transplant cellular distribution determination and CD3 suppression Leukemia/lymphoma diagnosis, staging, and minimal residual disease testing CD34 quantitation/Stem cell quantitation Fetal hemoglobin detection on red blood cells (RBCs) DNA ploidy determination Research, bacterial identification, population filtering, etc. This course will focus on leukemia/lymphoma assessment and analysis.

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Acceptable Samples

Flow cytometry is used most often to analyze white blood cells. Some appropriate samples are: Peripheral blood Bone marrow Lymph nodes and other tissues Fluids (cerebrospinal fluid, peritoneal, pleural, etc.)Since size and granularity are utilized to help sort cell populations, it is imperative to keep the cells intact. Thus, flow cytometry is best conducted on viable cells. Dead cells can complicate result interpretation by: Non-specifically binding monoclonal antibodies and emitting false fluorescent signals. Falling in inappropriate portions of the light scatter graph and falsely elevating event/cell counts.

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Detection of Extrinsic Cellular Characteristics: Surface Antigens

Monoclonal antibodies (MoAbs) are labeled with fluorescent particles. These MoAbs will bind specifically to corresponding cell surface antigens. Since blood cells may have multiple surface antigens, a single cell can bind to more than one type of labeled MoAb; depending upon the MoAbs that are used.Different combinations of antibodies can be used within the same staining tube, as long as the fluorochromes will emit signals that can be separated from one another; keeping in mind that compensation for spectral overlap may be necessary. For example, if two separate monoclonal antibodies are labeled with a red and orange fluorochrome respectively, it is important to compensate for the closeness of the two colors on the color spectrum and to make sure that the red-tagged MoAb signal is captured as red and the orange-tagged MoAb signal is captured as orange (and red isn't registered as orange and vice versa).In the image shown on the right, the cells were stained with MoAb-green fluorochrome and MoAb-orange fluorochrome. The cell that is illustrated expresses both antigens and the antibodies that are bound to the respective antigens. The laser beam excites the fluorochromes and fluorescent signals are emitted. The signals are captured and separated from other fluorescent signals by filters and mirrors.

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Flow Cytometry and Human Immunodeficiency Virus Clinical Application

When the scientific community discovered that human immunodeficiency virus (HIV) attacks and destroys T-helper cells, physicians began monitoring this cell population in affected patients. Flow cytometric analysis helps determine the absolute T-helper cell counts in a patient blood sample.A patient that is infected with HIV is considered HIV-positive until the absolute T-helper cell count crosses a threshold of 200 cells/mm3. After the T-helper cell count falls below this threshold, the patient's physician may change the diagnosis to acquired immunodeficiency syndrome (AIDS).

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Sample Preparation

Sample preparationSamples for flow cytometric analysis are prepared based on the following procedural steps. Prepare the sample according to cell type. Bone marrow (BM) and peripheral blood (PB): Prepare a blood smear, stain with Wright's stain, and scan under the microscope to identify basic cell distribution and morphology. BM can contain spicules; these samples need to be filtered. Tissue: Mince and filter tissues in a sterile cell culture media to release cellular components from the solid tissue form. Fluid: Filter fluid, prepare a cytospin, and scan under a microscope to identify cellular components. Obtain a white blood cell (WBC) count. Unless red blood cells are the population of interest, they should be lysed with a mild agent that will preserve the integrity of the cells targeted for analysis. If the red blood cells are not lysed, they lead to false analytic results. Adjust the WBC count by concentrating the WBCs to optimize for ‘staining' with monoclonal antibodies (MoAbs). Incubate the prepared cell concentration with assorted monoclonal antibody concentrations to allow antigen-antibody complexes to form. Lyse red blood cells (RBCs) with ammonium chloride or equivalent that will preserve cellular viability and integrity. The purpose is to eliminate RBCs while maintaining WBC integrity. If RBCs remain in the sample, they will interfere with the cell scatter plot and skew the results. Centrifuge to precipitate cells. Pour off supernatant. Wash in phosphate buffered saline (PBS) or equivalent to eliminate cellular debris and unbound MoAbs, centrifuge, and decant. Resuspend cells in PBS. Analyze cells using flow cytometer.

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Case Two

A 60-year-old man presented to the emergency department with complaints of fatigue and weakness which began during the previous week. He also stated that he had been experiencing shortness of breath without chest pain, cough, or dizziness. Upon physical examination, he had normal vital signs. However, his spleen was mildly enlarged. Laboratory tests were ordered. The white blood count (WBC) and absolute lymphocyte counts were both elevated:WBC= 28.5 x 109/L (normal 4 - 10) Lymphocyte count= 20.0 x 109/L (normal 1.2 - 4.0)A manual differential confirmed the elevated lymphocyte count. Smudge cells were also noted, as shown in the image.To evaluate the patient's lymphocytosis, a blood sample was submitted for flow cytometry analysis.

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T Cell Analysis

Though the percentages of gated lymphocyte populations (T or B cells) are important, it is still necessary to determine if the cells present have a normal or abnormal CD marker representation. A population will also need to be scrutinized to evaluate whether it is 10% or 99% of the total lymphocytes.Remember that lymphocytes will either be T cells or B cells and mature T cells will either be helper cells or suppressor/cytotoxic cells. With this in mind, normal mature T cells should express CD2, CD3, CD5, CD7, and either CD4 or CD8. CD4 marks T-helper cells CD8 marks cytotoxic T cells The CD markers present in the peripheral blood sample in Case Two are: CD2 = 17% CD5 = 97% CD7 = 17% CD3 = 18% CD4 = 10% CD8 = 8% Note the markedly increased CD5 percentage in relation to the other T cell markers. If this were a normal T cell population, it would have CD5 at roughly 17%. In order to determine if the elevated CD5 percentage is due to abnormal T cells or to abnormal B cells that are marking with CD5, analyze a sample that has been stained with both CD5 and CD19 monoclonal antibodies. Two areas of interest would be CD5+ only and whether or not there is a CD19+/CD5+ dual marking population. Note that on the image shown on the right, CD5+ only is 16.5% (rounds to 17%) and is in alignment with a normal T cell population in this sample.T-cell analysis conclusion: T-cells are normal and the CD5+ cells that are also positive for CD19 represent abnormal B cells that express the T-cell marker, CD5.

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Introduction to the ABO Blood Group System
Match the blood types from the drop-down boxes with the appropriate descriptions to the right of the boxes.View Page
An individual with type AB blood will demonstrate the complete absence of which of the following antigen sites?View Page
If an individual has blood type O, which of the following are possible genotypes?View Page
Use the drop-down boxes to match the blood types (phenotypes) that will be expressed with the genotypes listed to the right of the boxes.View Page
In what way are the ABO serum antibodies unique among blood group systems?View Page
Why is it dangerous to transfuse a person with type O blood with a unit of A blood?View Page
The History of the ABO System

In 1900, a German scientist, Karl Landsteiner, discovered that blood groups differ from one individual to another. He took blood samples from five associates and himself, allowed them to clot, and then separated the serum from the cells. Landsteiner found that when he mixed the serum and red cells from different individuals, some samples clumped and some didn’t. Our present day classification of the ABO system is based on Landsteiner’s realization that agglutination occurred because of highly reactive antigens present on the red blood cell which corresponded to antibodies present in the serum. Landsteiner isolated and named the red cell antigens “A” and “B” and the corresponding antibodies “Anti-A” and “Anti-B.” If the red cells contained neither antigen, he called these cells “O,” representing zero antigens present. The fourth type of red cells, “AB” was discovered in 1902 by Von Decastello and Sturli, associates of Landsteiner. “AB” cells contained both A and B antigens on their surface.

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Table 1: ABO Blood Group System

Antigen on Red Cells Antibodies in Serum ABO Blood Group A Anti-B A B Anti-A B Neither A nor B Anti-A, Anti-B, Anti-A,B O A and B Neither Anti-A nor Anti-B AB

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Table 2: Testing the Patient Red Cells with Known Antisera (Forward Typing)

In routine practice, specially prepared blood grouping sera containing anti-A, anti-B, (and optionally anti-A,B) are used to identify the four types of red cells. These sera will agglutinate cells with the corresponding antigen. This is called forward typing.ABO Blood GroupPatient Red Cells Tested with Known AntiseraAnti-AAnti-BAnti-A,BA3-4+03-4+B03-4+3-4+O000AB3-4+3-4+3-4++ = agglutination (graded 1+ to 4+)0 = no agglutination

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Table 3: Testing the Serum with Known Red Cells (Reverse Typing)

Antibodies occur predictably in the sera of all normal adults in association with the ABO antigens. Demonstration of these antibodies is necessary for definitive classification of an individual's ABO cell type. The individual's serum is therefore tested against reagent red cells containing known antigens. Patient ABO Blood GroupPatient Serum Tested with Known Reagent CellsA CellsB CellsA0 3-4+ B3-4+ 0 O3-4+ 3-4+ AB0 0 + = agglutination (graded 1+ to 4+)0 = no agglutination or hemolysis

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Importance of Understanding the ABO System

The predictability of ABO antibodies appearing in serum lacking the corresponding antigens makes ABO typing a simple process in most cases. However, the importance of getting it right cannot be stressed enough when a patient will be transfused with blood from a donor. If a patient receives donor cells containing A or B antigens and the transfused patient's serum contains the corresponding antibody, the donor cells will be destroyed almost immediately, causing a severe (hemolytic) and sometimes fatal reaction. Therefore, it is of utmost importance to thoroughly understand the ABO blood group system. In addition to red cells, ABO antigenic determinants (epitopes) are found in many tissues, body fluids, and other cells, including endothelial cells and platelets. Because ABO antigens are so widely expressed, ABO antigens are also a major consideration in solid organ and bone marrow transplants.

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Match the blood types in the drop down boxes with the characteristics on the right.View Page
The Bombay Blood Group

Homozygous "hh" individuals do not form H substance and thus have no way for late sugars to attach. The blood group resulting from the homozygous "hh" condition is called the Bombay blood group (Bombay phenotype). Due to the presence of anti-H in the serum of a person with the Bombay phenotype, only blood from another person with the Bombay phenotype may be transfused.

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Bombay Blood Group Genes

As mentioned previously, the A and B genes cannot act directly on the precursor substance. Thus, since individuals with the Bombay phenotype have only the precursor substance and no H antigen, they cannot have A or B antigens, even if they have the A and/or B gene.

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Inherited Genes

The A, B, and H antigens, like many other blood group antigens, are the expression of genes inherited from the previous generation. If the antigen is demonstrated, the gene controlling it must have been inherited from one or both of the parents.  As previously mentioned, the genes A, B, and O are allelic genes. Assuming the production of H substance, these three genes, in various possible combinations of two, account for the four recognized ABO groups: A, B, AB, and O. Each individual inherits two ABO genes, one from each parent, and these genes determine which ABO antigen will be present on that individual’s red cells. These genes exhibit co-dominance, meaning that if both A and B genes are present, both will be expressed.

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O blood cell membranes contain which the following?View Page
ABO Antibodies

In most other blood group systems, antibody may be formed after an individual has been immunized by an antigen that is missing from his or her red cells; perhaps as the result of pregnancy or transfusion. In the ABO system, when the antigen is missing from the cells, the corresponding antibody will predictably be found in the serum and must be found before determining the ABO type. There are few exceptions to this rule and any exception must be explained before the true ABO blood type can be determined.

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What is present in the blood of an individual with the Bombay phenotype which will cause it to agglutinate with any non-Bombay individual's blood?View Page
Which of the following is NOT a way in which "immune" ABO antibodies may be formed?View Page
Strength of the A Antigen

The strength of the A antigen can vary considerably, and although most A cells react strongly with anti-A and anti-A1B, some cells have been found that are very weakly reactive. The blood group has been divided into subgroups and is classified not only by the strength of the A antigen but also by certain other serologic characteristics.

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A1 and A2 Subgroups

The most common subgroups of group A phenotype are A1 and A2. These account for over 99% of individuals who are classified as Group A. Of this 99%, A1 comprises approximately 80%. Commercial anti-A typing serum does not differentiate between A1 and A2 cells. A1 cells contain "A" antigen and "A1" antigen. A2 is not really a unique antigen. It is thought to be simply "A" antigen with no "A1" antigen. Several preparations are available that will react with A1 cells, but not other subgroups of A. The most commonly used reagent is Anti-A1 lectin, an extract of the seeds of the plant, Dolichos biflorus, which has specific anti-A1 activity.Approximately 4% of individuals who are subgroup A2 have naturally occurring anti-A1 in their serum.

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Agglutination Reactions

Antibodies of the ABO system cause agglutination of saline-suspended red cells at 4°C to 20°C. Heating to 37°C weakens the reaction. "Naturally" occurring ABO antibodies may not be strong enough to agglutinate cells without centrifugation. Thus, testing serum for the presence of anti-A or anti-B has classically been performed using the tube system in which serum and cells added to a test tube are centrifuged and then evaluated for agglutination. A slide test has also been performed for forward reactions. Although tube tests are still in wide use, newer systems utilizing other technology such as gel agglutination are becoming more prevalent. The image on this page illustrates agglutination reactions observed with the tube system, from 4+ in the topmost image, to 0 in the lowest image. ABO reactions should be strong. Weak or missing reactions occur, but must be "resolved" before blood products can be released.4+ agglutination: Red blood cell button is a solid agglutinate; clear background.3+ agglutination: Red blood cell button breaks into several large agglutinates; clear background.2+ agglutination: Red blood cell button breaks into many medium-sized agglutinates; clear background; no free red blood cells.1+ agglutination: Red blood cell button breaks into many small clumps barely visible macroscopically; background is turbid; many free red blood cells.Negative: No agglutinated red blood cells present; red cells are observed flowing off the red blood cell button during the process of grading.Other reaction which may occur are the mixed-field reaction, in which mixtures of agglutinated and unagglutinated red blood are present; and hemolysis, in which red cells are hemolyzed by the antibody. Both of these patterns are considered positive reactions.

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Example of an ABO discrepancy

The composite image shown on the right illustrates the ABO typing reactions that were obtained for a patient. This particular case illustrates an ABO discrepancy. An ABO discrepancy occurs when the results of forward and reverse typing do not match. The reactions shown are described below in descending order:Patient red cells with reagent anti-A: negative reaction.Patient red cells with reagent anti-B: 4+ agglutination.Patient red cells with reagent anti-D: 4+ agglutination.Patient serum with reagent A1 red cells: negative reaction.Patient serum with reagent B red cells: negative reaction.This patient forward types as a group B, but reverse types as a group AB. (A group B patient should have anti-A. This patient demonstrates neither anti-A nor anti-B, similar to an AB patient). Further workup is necessary to determine the ABO type since the forward and back typing do not match. In this case, incubation at 40 C demonstrated the presence of weakened anti-A. The patient was therefore typed as group B. This case is an example of an ABO discrepancy which was due to a "missing" anti-A antibody. This could be due to old age, severe illness, or immunosuppression. Although evaluation of ABO discrepancies is beyond the scope of this course, it is important to note that all ABO discrepancies must be resolved before blood products can be released for transfusion.This patient is Rh (D) positive, as evidenced by the strong agglutination of his cells with reagent anti-D antibody.

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Laboratory Ergonomics
Scenario #3Jim spends most of his workday sitting on a stool at the technical workbench. The image on this page illustrates how he routinely sits. Lately, he has been experiencing lower back and leg discomfort that continues to bother him when he leaves work. He has been having trouble sleeping because of the pain in his legs. Eventually, the pain progresses to the point where he cannot work an entire day. What may have caused the problem and what could have been done to prevent the MSD from developing? Consider what could be the problem based on your observation of Jim's normal sitting position. Then click on the blue text below to see the ergonomic evaluation and possible solutions.View Page
Repetitive Motion Injuries

Repetitive motions can cause a variety of disorders that affect nerves, tendons, and muscles. Symptoms can include tingling or numbness in the fingers or hands, decreased range of motion, decreased grip strength, sleep interrupted by numbness or discomfort in the hands, pain in fingers, hands, or wrist, or pain shooting up into the forearms or arms.Some common afflictions that could affect laboratory workers due to the nature of their jobs are listed in the table below. Condition Symptoms Cause Carpal tunnel syndrome Pain that radiates up the arm, numbness or tingling in the thumb, index, or middle finger and weakness in the wrist and hand Compression of the median nerve that runs from the forearm into the hand Thoracic outlet syndrome Numbness and tingling in the hand, intensified with overhead activities Compression of the nerves and blood vessels between the neck and shoulder Radial tunnel syndrome Elbow pain, pain near the base of thumb, or pain anywhere in between. A common symptom is wrist weakness. Compression or entrapment of the radial nerve; may be caused by repetitive wrist and finger extension or repetitive forearm turning. Tendinitis Stiffness, tightness, and burning sensation; may experience a deep nonspecific pain. Grip impairment. Occurs most often in the tendons of the fingers, thumb, forearm, elbow, and shoulder. Repetitive motions or maintaining an awkward position that stresses tendons beyond their strength. Friction from overuse can cause inflammation. Tenosynovitis Pain, swelling, difficulty moving the joint in the affected area Inflammation of the tendon sheath

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel (retired 9/1/2010)
Description of Specialties (1)

Specialists in microbiology perform testing to diagnose and stop the spread of infectious organisms, including bacteria, viruses, and parasites. Specialists should be able to isolate and identify a wide variety of these organisms. Testing procedures include direction examination and antigen detection methods. Specialists in serology and immunology measure antibodies to infectious organisms. Specialists should be familiar with all serology techniques (except those specific to immunohematology). This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in hematology must be able to identify and evaluate cells in blood and bone marrow and identify disorders of these cell. Specialists should be familiar with routine and special tests to determine the number, morphology, and function of cells in body fluid.

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Description of Specialties (2)

Specialists in immunohematology perform all testing prior to blood transfusions and work to prevent transfusion infections. They also investigate any post-transfusion reactions. This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in clinical chemistry analyze body fluids such as blood, urine, and spinal fluid to determine the chemical makeup, including the amount of carbohydrates, proteins, enzymes, and trace elements. The special covers urine microscopics and chemical evaluation of the liver, kidneys, lungs, heart, and other vital organ systems. This specialty also covers all testing performed in the specialties of radioassay and blood gas analysis. Specialists in blood banking can perform all immunohematology testing as well as testing from the specialties of clinical chemistry, hematology and serology/immunology that relates to donor blood. Clinical laboratory personnel who are licensed in the specialties of immunohematology, clinical chemistry, hematology, and serology / immunology may perform all tests in the blood banking specialty.

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Description of Specialties (3)

Specialists in radioassay use radionuclides to determine the chemical makeup of body fluids such as blood and urine. Specialists in blood gas analysis evaluate lung and breathing function by levels of oxygen, carbon dioxide, pH, and hemoglobin with automated tests. Specialists in histology examine cellular and tissue samples using fixation, dehydration, embedding, microtomy, frozen sectioning, staining, and other similar techniques. Histology specialists licensed as technicians can perform specimen processing, embedding, cutting, staining, and frozen sectioning only under the general supervision of a director, supervisor, or technologist. Specialists in cytology process and interpret samples relating cytopathological disease. Non-gynecological cytology preparations can be screen by a specialist in cytology but final review and interpretation must be done by a physician.

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Description of Specialties (4)

Specialists in cytogenetics detect chromosome abnormalities and genetic disorders. Cytogenetics counseling may only be performed by an individual licenses in the cytogenetics specialty at the director level. Specialists in molecular genetics analyze DNA and RNA to find disease-related genotypes, mutations, and phenotypes in order to detect or predict disease and identify carriers. Specialists in histocompatibility test to determine tissue compatibility, prevent infections, and investigate and post-transplant problems. Techniques include blood typing, HLA typing, HLA antibody screening, disease markers, flow cytometry, crossmatching, HLA antibody identification, lymphocyte immunophenotyping, immunosuppressive drug assays, allogenic, isogeneic and autologous bone marrow processing and storage, mixed lymphocyte culture, stem cell culture, cell mediated assays, and assays for the presence of cytokines. Specialists in andrology and embryology examine gametes and embryos, including production, morphology, number, and motility, to address issues of fertility and infertility.

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Medical Error Prevention (retired)
System failure can be avoided by ________ the procedure for identifying patients who have blood samples drawn for crossmatching.View Page
Which statement describes an Adverse Event?View Page
Which statement(s) describe potential causes of medical errors involving the blood bank?View Page
Sentinel Event Categories

Sentinel Events are sentinels--they function as guards or watchkeepers. They indicate serious situations that require immediate attention: Patient deathParalysisComaPermanent loss of functionAny procedure on the wrong patient, the wrong side of the body, or the wrong organ Hemolytic transfusion reaction involving major blood group incompatibility

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Avoiding Systems Failure

Standardized systems should be used in virtually every circumstance to reduce errors. For example, medical errors can be avoided by using the standardizing procedure for preparing a venipunture site before drawing a blood alcohol specimen. A standardized system for this procedure is developed, published, trained, and posted. Everyone learns one protocol. Encouraging them to review and use the procedure for drawing blood alcohol tests avoids the error of using alcohol wipes to prepare the venipuncture site.

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Direct Error Detection Even perfect systems designs cannot avert human limitations. Medical errors occur and they have to be detected before they can be resolved. Sometimes people directly observe and immediately report these mistakes.View Page
Joint Commission Patient Safety Goals Joint Commission adopted national patient safety goals for healthcare organizations, including specific goals for laboratories. 2009 Laboratory Services National Patient Safety Goals These goals are directly quoted.View Page
Errors of Commission

There are two types of medical errors: Errors of commissionErrors of omission These are examples of errors of commission--medical errors involving actions. Mislabeling a test specimenDrawing a blood sample from the wrong patientIncubating a test at an incorrect temperature

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Types of Medical Errors Medical errors usually belong to one or more of these categories:View Page
Which occurrence is a medical error?View Page
Near Misses

Near misses are also related to medical errors: Near misses are medical events that avert unwanted consequences.Someone or something identifies and corrects harmful influences before they cause adverse events.The medical community sometimes calls near misses “close calls.” For example, a transfusion is stopped when the nurse discovers that the identification number on a unit of blood does not match the unit number on the requisition. This is a near miss for the patient receiving a transfusion of incompatible blood. Near misses often provide important insight into new ways of preventing medical errors. In this case, a flaw in Blood Bank cross-checking systems is discovered so it can be prevented from causing a medical error.

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Factors that Contribute to Medical ErrorsView Page
These statements describe sources of laboratory-related errors.View Page

Medicare Compliance for Clinical Laboratories
Case Study 5

The setting is a nursing home where a phlebotomist from the laboratory goes to draw blood samples each day. The phlebotomist picks up the requisitions for blood tests at the nursing station and then goes to the various rooms to draw blood from the patients. She notices that every requisition has an Advanced Beneficiary Notice (ABN) attached to it that is signed by the patient, even when the tests that were ordered don't need them. She asks the nurse at the station but she informs the phlebotomist that she doesn't know anything about it because it is done on the night shift.She lets the phlebotomist know that she will inform the nursing supervisor about it when she arrives at 9:00 AM. The phlebotomist completes her blood draws and returns to the laboratory. What should the phlebotomist do, if anything, in addition to her letting the nurse know about the problem?Correct Answer: The phlebotomist should report the incident to her supervisor upon returning to the laboratory.Discussion: Since the laboratory is submitting the claims for Medicare patients from whom the phlebotomist collects blood specimens, the problem is the lab's problem. However, it is not going to change the fact that the ABNs were already signed by the patients, if the phlebotomist refuses to collect specimens from these patients or if the nursing personnel are required to remove the ABNs. By contacting the supervisor, an appropriate representative from the laboratory can follow up with the nursing supervisor to ensure they understand the laws and regulations that govern ABNs.

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Case Study 6

It is 11:00 PM and the specimen processing department is finishing up the night's accessioning and test order entry. A specimen processor is working on a requisition that has an order for a Hepatic Profile but there are two tubes of blood with the order, one of which is a lavender top tube. This is the fourth requisition from this same doctor's office and all of them have had a lavender top tube and serum tube with an order for a chemistry test and a CBC. No CBC is marked on the requisition or written on the tube. The specimen processor figures the office just forgot to mark the test and knows that the results will be delayed and the sample might not be any good if he doesn't order the CBC now. He is also under pressure from the technical departments to finish processing on time so they can get their work done on time for result printing in the morning. What should the processor do?Correct Answer: Look up the laboratory's policy for handling such a situation and follow the policy.Discussion: The laboratory is not permitted to change a doctor's order in any way. By ordering the CBC the processor is ordering a test that the doctor did not specifically order and therefore makes the laboratory subject to a violation of the False Claims Act. By reviewing and following the laboratory policy, the processor assures that the laboratory, the physician, and the patient's best interests are met.

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Medicare Compliance for Clinical Laboratories (retired)
Case Study 10

The setting is nursing home where a phlebotomist from the laboratory goes to draw blood samples each day. The phlebotomist picks up the requisitions for blood test orders at the nursing station and then goes to the various rooms to draw blood from the patients. She notices that every requisition has an Advanced Beneficiary Notice (ABN) attached to it that is signed by the patient, even when the tests that were ordered don't need them. She asks the nurse at the station but she informs the phlebotomist that she doesn't know anything about it because it is done on the night shift.She lets the phlebotomist know that she will inform the nursing supervisor about it when she arrives at 9:00 AM. The phlebotomist completes her blood draws and returns to the laboratory. What should the phlebotomist do, if anything, in addition to her letting the nurse know about the problem?Correct Answer: The phlebotomist should report the incident to her supervisor upon returning to the laboratory.Discussion: Since the laboratory is submitting the claims for any Medicare patients that the phlebotomist might draw, the problem is the labs problem. However, it is not going to change the fact that the ABNs were already signed by the patients if the phlebotomist refuses to draw them or if the nursing personnel are required to remove them. By contacting the supervisor, an appropriate representative from the laboratory can follow up with the nursing supervisor to ensure they understand the laws and regulations that govern ABNs.

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Case Study 3

It is 11:00 PM and the specimen processing department is finishing up the night's accessioning and test requesting. A specimen processor is working on a requisition that has an order for a Hepatic Profile but there are two tubes of blood with the order, one of which is a lavender top tube. This is the fourth requisition from this same doctor's office and all of them have had a lavender top tube and serum tube with an order for a chemistry test and a CBC. No CBC is marked on the requisition or written on the tube. The specimen processor figures the office just forgot to mark the test and knows that the results will be delayed and the sample might not be any good if he doesn't order the CBC now. He is also under pressure from the technical departments to finish processing on time so they can get their work done on time for result printing in the morning. What should the processor do?Correct Answer: Look up the laboratory's policy for handling such a situation and follow the policy.Discussion: The laboratory is not permitted to change a doctor's order in any way. By ordering the CBC the processor is ordering a test that the doctor did not specifically order and therefore makes the laboratory subject to a violation of the False Claims Act. By reviewing and following the laboratory policy the processor assures that the laboratory, the physician and the patient's best interests are met.

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Metabolic Syndrome
Variation in Diagnostic Criteria

Metabolic syndrome may vary in definition and diagnostic criteria depending on the organization that is consulted. Health-related organizations that have developed diagnostic criteria for metabolic syndrome include: National Heart, Lung, and Blood Institute (NHLBI)/American Heart Association (AHA) World Health Organization (WHO) American Association of Clinical Endocrinologists (AACE) International Diabetes Foundation (IDF) European Group for Study of Insulin Resistance (EGIR)Each organization's set of criteria is slightly different in its parameters and its details for diagnosis. All of the above organizations agree that defining glucose intolerance, obesity, hypertension, and dyslipidemia is important. However, there are varied opinions in how important each risk factor is in relation to the others. Varied criteria to determine obesity is utilized: waist to hip ratio, BMI, and waist circumference. One organization does not include a measurement of obesity. Glucose intolerance is determined by measuring plasma insulin and/or glucose levels. Lack of standardization in insulin measurement and assay availability makes criteria using insulin levels impractical.

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Adult Treatment Panel III

The National Heart, Lung, and Blood Institute (NHLBI) initiated the National Cholesterol Education Program (NCEP) in 1985. The goal was to reduce the number of Americans with elevated cholesterol and thus reduce illnesses and deaths in the United States due to coronary heart disease. Three adult treatment panels have been published since then with clinical practice guidelines for managing cholesterol levels in adults. The most recent panel, Adult Treatment Panel III (ATP III), was published in 2001 and updated in 2004. The NCEP: ATP III also includes criteria for the diagnosis of metabolic syndrome. This criteria is the most frequently used criteria in the United States.

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NCEP: ATP III Diagnostic Criteria for Metabolic Syndrome

Presence of three or more of these componentsComponentCriteriaAbdominal obesity: Increased waist circumferenceMen: > 40 inchesWomen: > 35 inchesElevated triglycerides> 150 mg/dL ordrug treatment for elevated triglyceridesReduced HDL-Cholesterol (HDL-C)Men: < 40 mg/dLWomen: < 50 mg/dLElevated blood pressure> 130/85 mm Hg or drug treatment for elevated blood pressureElevated fasting glucose> 100 mg/dL ordrug treatment for elevated glucose

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At medical examination, a 50-year-old Caucasian male expressed concern regarding diabetes. There is a history of type 2 diabetes, hypertension, and cardiovascular disease in his family. He has gained a few pounds each year and his physician notes abdominal obesity. His physician orders laboratory tests to evalute his risk of cardiovascular disease.Vital Signs and Pertinent Laboratoy Results:Blood Pressure: 128/82 mm Hg Weight: 230 lbsHeight: 5' 11'' Calculated BMI: 32.1Waist Circumference: 45 inchesFasting Blood Glucose: 120 mg/dLTriglycerides: 170 mg/dLHDL-C: 42 mg/dLWhich one of the following statements regarding this patient is true if the physician uses the guidlines of NCEP: ATP III Diagnostic Criteria for metabolic syndrome evaluation?View Page
A 45-year-old African American female has been diagnosed and treated for type 2 diabetes for the past five years. She maintains good control of her blood glucose with medication but does not exercise and has gained 12 pounds over the past year.At her next appointment, her physician orders hs-CRP along with blood assays to monitor her diabetes.Laboratory Result:hs-CRP 2.8 mg/LView Page
A physician discusses weight with an overweight 60-year-old female at her yearly physical appointment. The female exercises regularly and eats healthy most of the time. The physician suggests she decrease carbohydrate intake and decrease portion size at meals.Review patient vital signs and laboratory assay results to decide if a diagnosis of metabolic syndrome is appropriate using the NCEP:ATP lll Diagnostic Criteria shown on the right.Height: 5' 7'Weight: 192 lbsBMI: 30.1Waist Circumference: 37 inchesBlood Pressure: 108/70Fasting Blood Glucose: 92 mg/dLTotal Cholesterol: 172 mg/dLLDL-C: 112 mg/dLHDL-C: 46 mg/dLTriglycerides: 70 mg/dLhs-CRP: <1.0 mg/LWhich of these statements regarding this patient is true?View Page
References

Armstrong C. Practice guidelines AHA and NHLBI review diagnosis and management of the metabolic syndrome. Am Fam Physician. 2006;74:891-1062.D'Amore PJ. Evolution of c-reactive protein as a cardiac risk factor. Lab Med. 2005;36:234-238.Devaraj, S, Swarbrick MM, Singh U et al. CRP and adiponectin and its oligomers in the metabolic syndrome evaluation of new laboratory-based biomarkers. Am J Clin Pathol. 2008;129:815-822.Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet. 2005;365:1415-1428.Expert Panel in Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (authors). Executive summary of the third report of the national cholesterol education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). JAMA.2001;285:2486-2497.Gade W, Gade J, Collins M et al. Failures of feedback: rush hour along the highway to obesity. Clin Lab Sci. 2010;23:39-50.Gade W, Gade J, Collins M et al. Beyond obesity: the diagnosis and pathophysiology of metabolic syndrome. Clin Lab Sci. 2010;23:51-61.Grundy SM. Does a diagnosis of metabolic syndrome have value in clinical practice? Am J Clin Nutr. 2006;83:1248-1251.Grundy SM, Brewer HB, Cleeman JI, et al. Definition of metabolic syndrome: report of the national heart, lung, and blood institute/american heart association conference on scientific issues related to definition. Circulation. 2004;109:433-438.Grundy SM, Cleeman JI, Daniels SR, et al. Diagnosis and management of the metabolic syndrome: An American Heart Association/National Heart, Lung, and Blood Institute scientific statement. Circulation. 2005;112:2735-2752.Grundy SM. Obesity, metabolic syndrome, and cardiovascular disease. J Clin Endocrinol Metab. 2004;89:2595-2600.Mathew B, Francis L, Kayalar A, et al. Obesity: effects on cardiovascular disease and its diagnosis. J Am Board Fam Med. 2008;21:562-568.Metabolic Syndrome. National Heart Lung and Blood Institute. Diseases and Conditions Index. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/ms/ms_whatis.html. Accessed December 5, 2011.Mittal S. The Metabolic Syndrome in Clinical Practice. London, England. Springer-Verlag Springer Science; 2008.Molinaro RJ. Metabolic syndrome: an update on prevalence, criteria, and laboratory testing. MLO. 2007;39:24-27.Ronti T, Lupattelli G, Mannarino E. The endocrine function of adipose tissue: an update. Clin Endocrinol. 2006;64:355-365.

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Role of Adipokines

Over twenty adipokines have been identified. Some adipokines are secreted solely by adipocytes; several are secreted by adipocytes and other body cells. Their role is very widespread as they integrate with various body organs and tissues: skeletal muscle, adrenal cortex, brain and sympathetic nervous system. Adipokines function in: Energy balance Immune reactions Insulin sensitivity Angiogenesis Blood pressure maintenance Lipid metabolism Hemostasis

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Adiponectin

Adiponectin is very different from TNF-a, IL-6, and PAI-1. It is synthesized and secreted almost exclusively by the adipocytes and is an anti-inflammatory cytokine. Levels of adiponectin are decreased in weight gain, obesity and in those who are insulin resistant. Secretions of TNF-a and IL-6 reduce adipocyte secretion of adiponectin. Adiponectin is a protective adipokine. It inhibits several steps in the inflammatory process and increases insulin sensitivity by enhancing glucose transport into muscle cells. Adiponectin also decreases liver glucose production. Adiponectin slows and inhibits steps in plaque formation in blood vessels and is thus antiatherogenic.

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Angiotensinogen

Adipocyte production of angiotensinogen explains the frequent occurrence of hypertension in obesity and insulin resistance. Adipose tissue and liver cells produce angiotensinogen, a precursor of angiotensin II. Most angiotensinogen is from liver synthesis but increased levels in obesity are likely from increased adipocyte synthesis. Besides increasing blood pressure, angiotensin II may stimulate adipose cell formation and thus increase adipose mass.

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Insulin Resistance

Insulin is a pancreatic hormone that plays a vital role in carbohydrate and lipid metabolism. Insulin regulates glucose concentrations by: Promoting glycolysis - the uptake of glucose by cells for energy Stimulating glycogenesis - the conversion of excess blood glucose to glycogen storage in the liver Inhibiting glycogenolysis - the conversion of glycogen back to glucose Inhibiting gluconeogenesis - the formation of glucose from noncarbohydrates Insulin increases lipid synthesis in the liver and fat cells and inhibits lipolysis, the release of non-esterified fatty acids (NEFAs) from triglycerides in fat and muscle cells. Insulin also promotes protein synthesis.If insulin resistance occurs, carbohydrate and lipid metabolism are impaired. Insulin resistance ordinarily results in increased insulin levels as the body senses a need for more insulin action. The impaired insulin action results in elevated plasma glucose levels. The increase in lipolysis increases blood concentrations of NEFAs and causes abnormal blood lipid levels.

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Risk Factors

According to the American Heart Association, the risk factors for metabolic syndrome include:Abdominal obesity (excessive fat tissue in and around the abdomen) Atherogenic dyslipidemia (blood fat disorders – high triglycerides, low HDL cholesterol and high LDL cholesterol – that foster plaque buildups in artery walls) Elevated blood pressure Insulin resistance or glucose intolerance (the body can't properly use insulin or blood sugar) Prothrombotic state (e.g., high fibrinogen or plasminogen activator inhibitor–1 in the blood) Proinflammatory state (e.g., elevated high sensitivity C-reactive protein in the blood) Reference: Metabolic syndrome.The American Heart Association website. Available at:http://www.heart.org/HEARTORG/Conditions/More/MetabolicSyndrome/Metabolic-Syndrome_UCM_002080_SubHomePage.jsp#. Accessed December 5, 2011.

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Endothelial Dysfunction

The endothelium is the thin layer of cells at the inner lining of blood vessels. Endothelial dysfunction is a pathological state where the balance of vasodilating and vasoconstricting is lost. Endothelial dysfunction is also a preclinical stage of atherosclerosis and precursor of future cardiovascular disease. Inflammation from increased levels of inflammatory adipokines are one factor in the development of endothelial dysfunction.

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Dyslipidemia and Metabolic Syndrome

Those diagnosed with metabolic syndrome are at risk for atherogenic dyslipidemia, a state of abnormal lipids and lipid levels. A state of atherogenic dyslipidemia also enhances the development of atherosclerosis and cardiovascular disease. The increased release of NEFAs and their infiltration of the liver initiate the dyslipidemia process. Increased NEFAs in the liver result in a fatty liver and increased liver triglyceride synthesis. Increased liver synthesis and secretion of very low density lipoprotein (VLDL) follow. VLDL is the lipoprotein that transports triglycerides in circulation. Blood triglyceride concentration then increases.A fatty liver also increases low density lipoprotein-cholesterol (LDL-C) circulating in blood. The predominant lipid in LDL molecules is cholesterol. LDL molecules in the dyslipidemia state are described as small dense LDLs. The increased triglyceride presence causes depletion of the cholesterol and phospholipid content in LDL, making the molecules smaller and denser.Decreased high density lipoprotein-cholesterol (HDL-C) also results. Most researchers believe this is also caused by the increased production of triglyceride-rich VLDL. Decreasing the concentration of HDL molecules is atherogenic in that HDL is the helpful lipoprotein transporting excess cholesterol to the liver and decreasing total blood cholesterol. Higher levels of HDL-C aid in preventing atherosclerosis and cardiovascular disease.

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Which lipid group in increased concentrations probably decreases HDL-C and causes the formation of small dense LDL molecules?View Page
Why are small dense LDL molecules more atherogenic?View Page
Hyperglycemia

It was noted at beginning of this unit that the risk of type 2 diabetes is five times greater than normal for those diagnosed with metabolic syndrome. The insulin resistance state impairs carbohydrate metabolism and elevates blood glucose levels. However, diabetes is often diagnosed before a patient is evaluated for metabolic syndrome.

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Hypertension

Frequently elevated blood pressure accompanies the diagnosis of metabolic syndrome. As previously discussed, angiotensinogen secreted by the adipocytes is one cause of hypertension. Increased NEFAs released from breakdown of triglycerides in adipose tissue elevates blood pressure. Hypertension increases the risk of stroke in these individuals.

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Which of the following most likely causes the hypertension that often occurs in metabolic syndrome?View Page

Microbiology / Serology Question Bank - Review Mode (no CE)
This parasite is found in blood.View Page
This suspicious form was recovered in blood.View Page
Which of the following media contains the X and V factors necessary for the growth of Haemophilus influenzae:View Page
Which of the following organisms is best visualized by use of a darkfield microscope:View Page
With regard to blood cultures, which blood to broth ratio is most conducive to growth:View Page
On sheep blood agar Haemophilus influenzae may exhibit satellite formation around all but which of the following organisms:View Page
Match type of hemolysis on the right with best description:View Page
Which of the following is the most suitable specimen for the isolation of Bordetella pertussis:View Page
Which of the following specimens would not be considered suitable for anaerobic culture:View Page
Match type of media on the right with media on the left:View Page
Match the organisms on the right with culture medium:View Page
Which of the following parasites is not commonly found in the peripheral blood:View Page
Which of the following specimens is the most sensitive for detecting active CMV infection:View Page
Which of the following media is a selective medium for Campylobacter jejuni:View Page
Sheep blood agar contains inhibitors to which of the following organisms:View Page
The adult worms of which of the following reside in the intestine or its blood vessels:View Page
Which one of the following statements about E.coli O157:H7 is false:View Page
What is the best term to describe the clear areas seen around the colonies on this blood agar plate:View Page

Molecular Methods in Clinical Microbiology
Identification of Staphylococcus aureus with Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH)

Staphylococcus aureus, particularly methicillin resistant strains (MRSA), have represented a likely target for molecular development, particularly in blood cultures. As more institutions implement patient screening protocols for MRSA, replacement of routine culture methods with molecular assays has gained increasing attention.PNA-FISH assays provide for the definitive identification of Staphylococcus aureus from positive blood culture vials. Peptide nucleic acid fluorescent in-situ hybridization is a relatively straight forward procedure that does not involve amplification and has limited equipment requirements. Procedurally it is easy to perform with minimal hands on time.PNA is a synthetic imitator of a nucleic acid sequence in which the backbone is a pseudopeptide rather than a sugar. PNA behaves similarly to DNA and will bind to complementary nucleic acid strands. A PNA probe is constructed, utilizing a complementary, hybridizing sequence for a known nucleic acid target sequence. The probe is typically bound to a fluorescent protein as a means of visualizing/detecting the target. In one commercially available method, once a blood culture vial demonstrates gram-positive cocci in clusters, a drop of the blood culture broth is added to fixation solution on a slide. Heat or methanol is used to fix the smear. After fixation, probe that targets species-specific ribosomal RNA is added to the smear, which is then cover-slipped.Slides are then incubated at 55oC. Post incubation, slides are immersed in a preheated wash solution and coverslips gently removed. After incubation in the wash solution, smears are air dried; a drop of mounting medium is added and the slide is cover-slipped again.The slides are examined with a fluorescent microscope, utilizing specific filters. Green fluorescing cocci in clusters are identified as Staphylococcus aureus. This identification would be available, depending on the routine identification system utilized, potentially 24 hours earlier than the norm.A significant number of blood cultures that demonstrate gram-positive cocci in clusters yield coagulase negative staphylococci (CNS), which represent potential contaminants, rather than significant infection. What is the significance of differentiating blood cultures that contain S. aureus from those that are growing CNS in a much earlier timeframe?Studies have shown that IF the differentiation of CNS from S. aureus is effectively communicated to clinicians and pharmacy/antimicrobial stewardship teams, active assessment can occur utilizing defined exclusion criteria for those patients whose cultures yielded CNS rather than S. aureus. In scenarios where contamination rather than infection is indicated, vancomycin can be discontinued earlier, and length of hospital stay is also shortened. Reduced antibiotic exposure, reduced risk of development of resistance, and reduced cost are all potential benefits.

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Molecular Versus Culture - Pros and Cons

Traditional culture methods for the detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) employing mannitol salt and/or blood agar for cultivation, can take up to 72 hours for isolation and identification, depending on the identification procedures utilized. Concurrent with the development of molecular assays, improvements in culture methods have also been achieved. CHROmagar™ media, specific for MRSA, are employed by many laboratories. These media are both selective and differential, containing chromogenic substrates. MRSA strains utilize the substrates to produce colonies of a specific and characteristic color, minimizing the need for additional identification procedures.Initially these agars required 48 hours of incubation; newer formulations require only 24 hours incubation.Given the reduced incubation and identification requirements, what are the pros and cons of the molecular assays? Cost per test will be greater with the molecular assays as compared to culture methods. Will molecular methods provide for a more efficient workflow and significant improvement in availability of results? To some extent, this will depend on how they can be implemented within each different laboratory setting. Both of the previously described molecular assays require manual specimen preparation and extraction before the sample is placed into the instrument. This hands-on work may actually be greater than the effort expended in swabbing and streaking a culture plate. How much an obstacle this is for implementation will depend on both the volume of testing and the staff available. In a high volume setting, this will be a greater factor.Will tests be performed as specimens come in, or will specimens be accumulated and batched? If controls are required with each run, batching is desirable to reduce this cost. If testing will occur in batches, how many batches can be performed in one day? This will be heavily influenced by the capacity of the instrument. (For example, a single Smart Cycler unit can run up to 16 samples; multiple units would be needed in a high volume lab.) Can they be set up on more than one shift? The greater the number and frequency of batches that can be run, the greater improvement in turnaround time can be realized. Given these variables, implementation of a molecular assay for MRSA is not a given in each laboratory.

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References

BD GeneOhm™ MRSA [package insert]. Quebec, Qc, Canada: BD Diagnostics; 2009. Available at: http://www.bd.com/geneohm/english/products/pdfs/mrsa_pkginsert.pdf. Accessed February 22, 2012.Bonetta L. Prime time for real-time PCR. Nature Methods. 2005;2:305-312. Available at: http://www.nature.com/nmeth/journal/v2/n4/full/nmeth0405-305.html. Accessed February 22, 2012.Boughton B. Universal PCR Screening for MRSA May Cut Costs, Reduce Infection. In Medscape Medical News. Available at: http://www.medscape.com/viewarticle/708813. Accessed February 22, 2012.CDC Response: A Year in Review. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/yearinreview.htm. Accessed February 22, 2012.Centers for Disease Control and Prevention. Evaluation of Rapid Influenza Diagnostic Tests for Detection of Novel Influenza A (H1N1) Virus ---United States, 2009. Morbidity and Mortality Weekly Report. August 7, 2009;58(30):826-829. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5830a2.htm. Accessed February 22, 2012.Department of Biochemistry. University at Buffalo, School of Medicine and Biomedical Sciences Website. Available at: http://www.smbs.buffalo.edu/bch/Labs/SinhaLab/Protocols/RT-PCR.pdf. Accessed February 22, 2012.Desjardins M, Guibord C, Lalonde B, Toye B, Ramotar K. Evaluation of the IDI-MRSA Assay for the Detection of Methicillin-Resistant Staphylococcus aureus from Nasal and Rectal Specimens Pooled in Selective Broth. J Clin Microbiol. 2006 April;44(4):1219-1223. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448652/. Accessed February 22, 2012.Eastwood K, Else P, Charlett A, Wilcox M. Comparison C. difficile detection methods. J Clin Microbiol. 2009;doi:10.1128/JCM.01082-09. Available at: http://jcm.asm.org/cgi/content/short/JCM.01082-09v1Farley JE, Stamper PD, Ross T, Cai M, Speser S, Carroll KC. Comparison of the BD GeneOhm Methicillin-Resistant Staphylococcu aureus (MRSA) PCR Assay to Culture by Use of BBL CHROMagar MRSA for Detection of MRSA in Nasal Surveillance Cultures from an At-Risk Community Population. J Clin Microbiol. 2008;46(2):743-746. Available at: http://jcm.asm.org/content/46/2/743.full. Accessed February 22, 2012.Forrest GN, Mehta S, Weeks E, Lincalis DP, Johnson JK, Venezia RA. Impact of Rapid In Situ Hybridization Testing on Coagulase Negative Staphylocci Positive Blood Cultures. J Antimicrob Chemother. 2006;58(1):154-158. Available at: http://jac.oxfordjournals.org/content/58/1/154.full. Accessed February 22, 2012.Garcia LS, Isenberg HD, eds-in-chief. Clinical Microbiology Procedures Handbook. 2nd ed. Washington, DC: ASM Press; 2007.Hindiyeh M, Hillyard DR, Carroll KC. Evaluation of the Prodesse Hexaplex Multiplex PCR Assay for Direct Detection of Seven Respiratory Viruses in Clinical Specimens. Am J Clin Pathol. 2001;116:218-224. Available at: http://ajcp.ascpjournals.org/content/116/2/218.full.pdf. Accessed February 22, 2012.Hunt M. Real Time PCR. University of South Carolina School of Medicine Website. Available at: http://pathmicro.med.sc.edu/pcr/realtime-home.htm. Accessed February 22,2012.Interim Guidance for Influenza Surveillance: Prioritizing RT-PCR Testing in Laboratories. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/screening.htm. Accessed February 22, 2012.Interim Guidance for the Detection of Novel Influenza A Virus Using Rapid Influenza Diagnostic Tests. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/guidance/rapid_testing.htm. Accessed February 22, 2012.Levenson D. Molecular Testing for Respiratory Viruses. In Clinical Laboratory News. March 2008: Vol 34, No 3. Washington, DC: AACC Press; 2008. Available at: http://www.aacc.org/publications/cln/2008/mar/Pages/cover1_0308.aspx. Accessed February 22, 2012.Morshed MG, Lee MK, Jorgensen D, Issac-Renton JL. Molecular methods used in clinical laboratory: prospects and pitfalls. FEMS Immunol Med Microbiol. 2007;49:184-191. Available at: http://www.canlyme.com/morshed_pcr.pdf. Accessed February 22, 2012.Paillard F, Hill CS. Direct nucleic acid diagnostics tests for bacterial infectiousdiseases: Streptococcal pharyngitis, pulmonary tuberculosis, vaginitis, chlamydial and gonococcal infections. MLO-online. 2004;10-15. Available at: http://www.mlo-online.com/articles/0104/mlo0104coverstory.pdf. Accessed February 22, 2012.PCR: an outstanding method. Roche Website. Available at: http://www.roche.com/pages/facets/pcr_e.pdf. Accessed February 22, 2012.Persing DH, ed-in-chief.Molecular Microbiology, Diagnostic Principles and Practice. 2nd ed. Washington, DC: ASM Press; 2010.Pfaller MA. Molecular Approaches to Diagnosing and Managing Infectious Diseases: Practicality and Costs. Emerg Infect Dis. 2001;eid0702. Available at: http://wwwnc.cdc.gov/eid/article/7/2/70-0312_article.htm. Accessed February 22, 2012.Rossney AS, Herra CM, Brennan GI, Morgan PM, O'Connell B. Evaluation of the Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Assay Using the GeneXpert Real-Time PCR Platform for Rapid Detection of MRSA From Screening Specimens. J Clin Microbiol. 2008;46(10):3285-3290. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566096/. Accessed February 22, 2012.The 2009 H1N1 Pandemic: Summary Highlights, April 2009-April 2010. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/cdcresponse.htm. Accessed February 22, 2012.Timeline of PCR and Roche. Roche Website. Available at: http://molecular.roche.com/About/pcr/Pages/PCRTimeline.aspx. Accessed February 22, 2012.

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Multi-drug Resistant Organisms: MRSA, VRE, and Clostridium difficile
Clinical significance of Staphylococcus aureus

In general, the infection that develops is dependent on the virulence of the particular strain, the inoculum size, and immune status of the host. Staphylococcal infections are typically suppurative, producing abscesses filled with pus and damaged leukocytes surrounded by necrotic tissue. Skin infections range from superficial - boils, carbuncles and furuncles, to bullous impetigo; largely opportunistic infections that develop as a result of previous injury e.g., cuts, burns, surgical wounds - and scalded skin syndrome (extensive exfoliative dermatitis; also known as Ritter Disease). Other major infections include pneumonia, osteomyelitis (localized infection of bone), and septic arthritis. S. aureus also causes food poisoning as a result of ingestion of food contaminated with an enterotoxin producing strain (enterotoxins A&D) and the potentially fatal toxic shock syndrome, a multisystem disease most often associated with the use of highly absorbent tampons. Toxic shock syndrome is attributed to another toxin (enterotoxin F – TSST1) released by certain strains of S. aureus.Human staphylococcal infections usually remain localized by the normal host defenses. Foreign objects (fomites) such as sutures or intravenous (IV) lines - are readily colonized by S. aureus from skin and can allow the organism to spread systemically via the blood stream – bacteremia/septicemia - leading to more serious infections. Staphylococcal pneumonia is becoming a frequent complication of influenza. Whatever the mode of entry, the invasive nature of S. aureus always poses the threat of more serious deeper tissue invasion and/or bacteremia and hematogenous spread.

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Stool Culture

Stool culture is very effective in detecting C. difficile. Unfortunately, non-toxigenic strains will also grow, requiring strains to be tested for toxin production. The greatest disadvantage to culture is the length of time that is needed before results are available, which may be up to four days. However, antibiotic sensitivity testing following culture is useful for strain-typing that would provide necessary epidemiological information during nosocomial outbreaks.Colonies of C. difficile will appear white, flat, and spreading on blood agar (see top image on the right). Cycloserin-cefoxitin-fructose agar(CCFA) is a selective media that is used for isolation of C. difficile. There is however, no distinction between pathogenic and commensal strains, which all produce yellow colonies with a characteristic "ground glass" appearance. as shown in the bottom image on the right. The characteristic odor of "horse manure" aids in identification of C. difficile. Stool samples are directly inoculated onto CCFA and incubated in an anaerobic atmosphere at 37°C for 48 hours. Large, thin, gram-positive bacilli with spores will be observed on a Gram stain of a typical colony, as shown below.

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Enterococci

Enterococci are catalase-negative gram-positive cocci occurring singly, in pairs, or in chains; cells can be ovoid to coccobacillary. There are over twenty species in the genus, categorized within five major groups. Enterococcus faecalis is the most frequently isolated species; Enterococcus faecium, although less frequently encountered, is a significant pathogen.The Enterococci are widespread in nature; in humans they primarily colonize the gastrointestinal tract but are also found in the genitourinary tract. Enterococci are frequently encountered in urinary tract infections; they are also isolated from wound infections and blood cultures. They are also an important cause of endocarditis.

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Identification of Enterococcus Species From Clinical Cultures

Gram stain: Gram-positive cocci in singles, pairs, or chains; cells can be ovoid to coccobacillaryColony morphology: On blood agar after 24 hours of incubation, colonies are nonhemolytic or alpha hemolytic (rare strains may be beta hemolytic), and approximately 1 to 2 mm in diameter.Catalase: NegativePresumptive identification: Growth on bile esculin agar and in 6.5% salt broth are two characteristics that have commonly been used to identify enterococcus species to the genus level. A positive esculin in combination with a positive PYR reaction is another approach to presumptive identification.Species identification:E. faecalis and E. faecium are usually easily identified by most commercial systems. Successful identification of the other species on these systems may vary. With respect to vancomycin intermediate or resistant strains, two key characteristics are motility and pigment. E. casseliflavus is both motile and possesses a yellow pigment; E. gallinarum is also motile but non-pigmented. E. faecalis and E. faecium demonstrate neither characteristic.

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A laboratory performs MIC tests, in addition to Kirby Bauer, and a PBP 2a assay on blood culture isolates. Which of the following isolates should be reported as oxacillin-resistant?View Page

Mycology: Yeasts and Dimorphic Pathogens (retired 2/12/2013)
The colonies shown in the upper image were obtained on blood agar from a sputum specimen after 10 days incubation at 30°C. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The diagnosis is:View Page
The growth of the colonies shown in the upper image was obtained on blood agar from a sputum specimen after 8 days of incubation at 30°C. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The diagnosis is:View Page
One of the characteristics common to the dimorphic molds is the ability to convert the mold forms to the yeast forms by incubating subcultures in enriched media at 35°-37°C. The upper image illustrates a subculture of a mold colony suspected of being a dimorphic fungus inoculated to the surface of blood agar and incubated for 3 days at 37°C. Note that the colonies have a prickly appearance, suggesting an intermediate stage of conversion. The lower image is a lactophenol blue mount of a portion of one of the prickly colonies. This fungus can be identified as:View Page
The colonies growing on the surface of this brain-heart infusion with blood agar plate were "converted" from a mold colony suspected of being Histoplasma capsulatum by incubating a subculture at 37°C for 5 days. The yeast forms that must be identified in mounts made from one of these colonies to confirm the identification are:View Page
The growth of the yeast-like colonies shown in the upper image was obtained on blood agar from a skin culture only in the area overlaid by virgin olive oil. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The disease associated with this fungus is:View Page
The colonies illustrated in this photograph were recovered from a blood culture after 48 hour incubation at 30°C. The most likely source for the septicemia is:View Page
A hematology technologist observed the intracellular forms seen in the field of view of a Wright-Giemsa-stained peripheral blood smear shown in this photomicrograph. In consultation, the microbiology technologist advised that the form seen most likely represents:View Page

Normal Peripheral Blood Cells
The Cellular Components of Blood

Blood is composed of an isotonic fluid, called plasma, in which various peripheral blood cells (hemocytes) are suspended. There are three major groups of peripheral blood cells. The three major groups include:Red Blood Cells (Erythrocytes)White Blood Cells (Leukocytes)Platelets (Thrombocytes)

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Thrombocytes or Platelets

The third group of formed elements in normal peripheral blood is made up of thrombocytes, also known as platelets. Although platelets are not very large in comparison to the other cell types, their role in the process of hemostasis is critical. Platelets are the small granular bodies shown with the arrows in the Wright's stained smear below.

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Identifying Peripheral Blood Cells

All three types of peripheral blood cells have different characteristics. In order to accurately identify each type of cell, a peripheral blood film must be made, preferably from blood anticoagulated with EDTA (Ethylenediaminotetracetic Acid) or from capillary blood. EDTA, in contrast to many other anticoagulants, preserves cellular morphology. The individual characteristics of each cell type are made visible by staining the blood films with Wright's stain, and observing them under the microscope. Most laboratories utilize high powered, oil magnification for the morphologic identification of peripheral blood cells.

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The three main types of peripheral blood cells are:View Page
More on Phagocytosis in Neutrophils

Neutrophils have a relatively short life span. They are produced in the bone marrow, and when they reach the band or segmented stages are released into the peripheral blood. They remain there for approximately ten hours before randomly entering body tissues.Neutrophils in the blood stream can be divided into circulating granulocyte pool (CGP) and marginating granulocytic pool (MGP). The white blood cell count reflects the cells in the circulating pool. The cells in the marginating pool move quickly into the circulating pool when needed.During an infection the neutrophil concentration of the peripheral blood can increase almost immediately due to the shift of these cells from the marginating pool and release from the bone marrow storage pool, if needed. Neutrophils then migrate to areas of tissue damage or infection. Neutrophils do not reenter the blood stream from the tissues, thus end their life in the tissues either as a result of phagocytosis or senescence.

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Eosinophil Function and Lifespan

Eosinophils have a circulating half-life of approximately 18 hours and a tissue life span of at least 6 days. They are capable of locomotion and phagocytosis and can enter inflammatory sites, but do so less readily than neutrophils. In tissues the primary location for eosinophils is in the epithelial barriers to the outside world such as, lungs, skin and GI tract. They are capable of returning to the circulating blood and bone marrow after they enter the tissues. Eosinophils are active in parasitic infections and in allergic reactions such as asthma and hay fever, and may be present in great numbers in the peripheral blood during these conditions. Stress, shock, or burns may also cause an increase in this type of cell. Eosinophils modulate an allergic response by liberating substances which can neutralize mast cell and basophil products. The image on the right shows malarial ring forms, which are parasites. This patient showed an increased eosinophil count due to his parasitic infection.

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Basophil Function and Lifespan

Basophils serve as mediators of inflammatory responses, especially hypersensitivity reactions. IgE binds to the membrane receptors on basophils and degranulation is initiated. The enzymes released are vasoactive, bronchorestrictive and chemotactic (especially for eosinophils), so basophils seem to play a role in inducing and maintaining allergic reactions.The granules of basophils contain histamine, heparin and peroxidase. After degranulation occurs, basophils can synthesize more granules. The release of large numbers of these granules can cause anaphylactic shock and death. Basophils circulate in the blood for a short time and make up only a small percentage (0.5%) of the cells in circulation. They do not migrate to the tissues under normal conditions but may be seen when inflammation resulting from hypersensitivity to protein, contact allergy or skin allograft rejection is present. Basophils are sometimes increased in patients with chronic myeloproliferative disorders.

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Where is the main site of action for monocytes after diapedesis?View Page
What is the Function of Lymphocytes?

Lymphocytes are primarily involved in the body's immune response mechanism. This involves complex phenomena which end in the development of humoral and cellular immunity. Humoral ImmunityHumoral immunity involves the production of antibodies (immunoglobulins), and is brought about by lymphocytes which we call B-cells. B-cells are bone-marrow derived lymphocytes. After B-cells are stimulated by an antigen, they proliferate and transform into plasma cells which produce specific antibodies. Cellular ImmunityCellular immunity includes delayed hypersentivity reactions, graft rejection, graft-versus-host reactions, defense against intracellular organisms, and probably defense against neoplasms. Cellular immunity is mediated by lymphocytes which we call T-cells. T-cells are so named because they are dependent on the thymus for their production and development. The majority of T-cells are long-lived with an average lifespan of 4.4 years, but it is known that some survive for as long as 20 years or more. T-cells are capable of leaving and re-entering the circulation many times during their long life. T and B cells cannot be differentiated when viewing blood films. They are identified through the use of immunologic cell markers.

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T lymphocytes are larger and have more vacuoles than B lymphocytes.View Page
Monocytic Function

Monocytes are phagocytes which remove injured and dead cells, cell fragments, microorganisms and insoluble particles from the blood and body tissues. Monocytes also secrete substances that affect the function of other cells, especially lymphocytes. They are produced in the bone marrow, and when mature are released into the peripheral blood. Although they do serve a phagocytic role in the blood, their main site of action is the body tissues. The half-life for monocytes in the peripheral blood is approximately 8 hours. Monocytes migrate into the tissues, often to sites of inflammation, where they serve their primary purpose. Here they transform into fixed or free macrophages, and continue their function as avid phagocytes. When activated, macrophages may enlarge and have enhanced metabolism.Monocytes provide defense against mycobacteria, fungi, bacteria, protozoa and viruses. They respond to chemotactic factors, phagocytize and kill the microorganisms.

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Platelet Clumps

Occasionally platelets occur in clumps, particularly if the film was made from capillary blood or if the specimen tube was not well mixed, forming mini clots.

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Platelet Kinetics

Platelets are derived from the cytoplasm of megakaryocytes, giant cells in the bone marrow. At any given time, two thirds of the total platelets are found within the circulation while one third sequestered within the spleen. In persons with enlarged spleens 80-90% of the platelets are in the spleen resulting in a decreased concentration of circulating platelets. In individuals who have had a splenectomy all of the platelets will be in the circulating blood. The life span of the platelet is 8-10 days.

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Platelet Appearance

Platelets are anucleate cells, measuring only 1-4 microns in diameter. They are the smallest of the formed elements found in normal peripheral blood. The arrows point to platelets.Their shape varies greatly, but they are usually round, oval or rod-shaped. In addition, platelets stain light blue to purple in color, and are very granular. The cytoplasm of platelets can be divided into two areas: the chromomere and the hyalomere. The chromomere is located centrally where the granules tend to aggregate. The hyalomere surrounds the chromomere and is a clear, blue, non-granular zone. The diagram on the right illustrates the central granular chromomere, and the peripheral clear hyalomere of a platelet.

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All of the following statements describe a method by which platelets aid coagulation EXCEPT:View Page
Platelet Function

Platelets function both mechanically and biochemically in the process of hemostasis. When injury to a blood vessel occurs, platelets aggregate forming a primary hemostatic plug which helps to stop the flow of blood. Platelets also release certain substances, among them serotonin and platelet Factor 3. Serotonin causes the blood vessels in the area to constrict, thereby further stopping the flow of blood. Platelet Factor 3 catalyzes the coagulation reaction whereby a fibrin clot is formed, completing the seal. Platelets also maintain the integrity (leak-free) state of blood vessels.

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Appearance of the Erythrocyte

Erythrocytes are non-nucleated, round, biconcave, disc-shaped cells They are 6.7 to 7.7μ in diameter, 2μ thick, and have an average volume (Mean Corpuscular Volume, MCV) of 80-100μ3. In stained blood films, only the flattened surfaces of the RBC's are seen. Therefore, they appear circular with an area of central pallor corresponding to the indented area. The central pallor occupies about 1/3 of the diameter of the cell. The overall red blood cell diameter is slightly less than that of the nucleus of the small lymphocyte. The cytoplasm stains pink to brick-red, and no nucleus is present.

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Erythrocyte Function and Kinetics

Erythrocytes are produced in the bone marrow and released into the peripheral blood where they may remain for approximately 120 days before senescence. Their main function is the transport of the respiratory gases (oxygen and carbon dioxide) between the lungs and body tissues.Each erythrocyte can be thought of as an "envelope" containing hemoglobin. Each hemoglobin molecule contains iron which has a high affinity for oxygen. As a result, when an erythrocyte passes through one of the capillaries of the lungs, it picks up oxygen. The oxygen is transported through the blood to the tissues where it is released. Carbon dioxide from the tissues then diffuses into the RBC where it undergoes chemical changes. About 70% of the altered carbon dioxide diffuses into the plasma, 25% binds to the hemoglobin molecule, and 5% goes into simple solution within the red cell. In each of these three ways carbon dioxide is transported from the body tissues back to the lungs, where it is released.

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Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Glossary of Terms A through M.

Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus.

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Glossary of Terms N through Z.

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

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Introduction to Segmented and Band Nuclei

The granulocytes found in normal peripheral blood are neutrophils, eosinophils and basophils. Most have segmented nuclei, and are therefore classified as being at the "segmented" stage of development. The granulocytes that are a little less mature have unsegmented nuclei. These are classified as "bands." Generally, we differentiate between the band and segmented forms of neutrophils; however, it is not common practice to designate the band forms of eosinophils and basophils on a routine basis.Since various hematologists and textbook resources use several synonomous terms to describe these cells, various synonyms for each term will be given and may be used interchangeably throughout the course.

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Segmented Neutrophils

Segmented neutrophils may also be referred to as segs, polymorphonuclear leukocytes, polys and PMNs. Segmented neutrophils are the most mature neutrophilic granulocytes present in circulating blood. Their diameter is approximately 9-15 microns, and their N:C ratio is approximately 1:3. The abundant cytoplasm of a segmented neutrophil is of virtually the same appearance as that of the band. It stains faintly pink and contains numerous fine specific granules which are pinkish-lilac.In order to identify a segmented neutrophil, the cell must have the following characteristics:The nucleus is a deep reddish-purple color, and the chromatin has a coarse, clumped texture.The seg nucleus normally has from 2-5 lobes, with an average of 3.The lobes are connected to each other by a fine filament or strand of nuclear membrane. A filament is a thread-like strip which is so narrow that there is no visible nuclear material between the two sides.

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The most immature neutrophil found in normal peripheral blood is:View Page
Eosinophils

Eosinophils are also known as eosinophilic granulocytes, or eos. Eosinophils are easy to recognize in the peripheral blood because of their large, bright reddish-orange granules. The diameter of the eosinophil is 9-15 microns, and the nuclear to cytoplasmic (N:C) ratio is 1:3. Eosinophils are generally the largest granulocytes found in normal blood.Their cytoplasm is usually colorless or light blue. However, the color is usually masked by the large granules that are present. These granules take up the acid components of Wright's stain, and are therefore reddish-orange.

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Basophil Granules and Chromatin Pattern

When examining a blood film you may find that some basophils have many dense granules, as seen in the upper image on the right, while others appear washed out with only a few granules, as shown in the lower image. This is because the granules are water soluble and tend to wash out during the rinse phase of the staining process. The chromatin pattern of the basophil nucleus is not quite as coarse as that of the neutrophil or eosinophil nuclei. Although the nucleus is usually segmented, the lobes are often difficult to discern because they tend to crowd together and are obscured by the cytoplasmic granules.

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Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell that is indicated by the arrow:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Monocyte Appearance

Monocytes are the largest of the normal peripheral blood cells, ranging from 14-20µm in diameter with an N:C ratio of approximately 3:1. Monocytes have abundant blue-gray cytoplasm containing many fine lilac granules. These give the cytoplasm a "ground glass" appearance. However, these granules may be difficult to see if the blood film is poorly stained. Frequently, cytoplasmic vacuoles are present. These vacuoles appear as unstained areas or "holes" in the cytoplasm; an example of which can be found in the lower image to the right.Because monocytes are extremely motile cells, blunt pseudopods may be seen. These should not be confused with the apparent cytoplasmic projections produced when large lymphocytes are indented by surrounding cells. Monocytes have generally lighter staining nuclei than do other leukocytes. The nucleus stains a pale bluish-violet, and the chromatin is fine. Overall, the nucleus has a soft, spongy, three-dimensional appearance, in contrast to the hard, flat nucleus of the large lymphocyte and the densely clumped nucleus of the band. The nucleus may be round, kidney-bean shaped, folded, indented, or horseshoe, and may show "brain-like" convolutions.

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Identify the nucleated blood cell that is indicated by the arrow:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page

Normal Peripheral Blood Cells (retired 6/20/2012)
Platelets are the smallest nucleated cells seen in normal peripheral blood.View Page
Cellular Immunity

Cellular immunity includes delayed hypersentivity reactions, graft rejection, graft-versus-host reactions, defense against intracellular organisms, and probably defense against neoplasms.Cellular immunity is mediated by lymphocytes which we call T-cells.T-cells are so named because they are dependent on the thymus for their production and development.The majority of T-cells are long-lived with an average lifespan of 4.4 years, but it is known that some survive for as long as 20 years or more.T-cells are capable of leaving and re-entering the circulation many times during their long life.T and B cells cannot be differentiated when viewing blood films.They are identified through the use of immunologic cell markers.

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Where is the main site of action for monocytes?View Page
T lymphocytes are larger and have more vacuoles than B lymphocytes.View Page
The half-life of monocytes in the circulating blood is:View Page
Monocytes

Monocytes are phagocytes which remove injured and dead cells, cell fragments, microorganisms and insoluble particles from the blood and body tissues.Monocytes also secrete substances that affect the function of other cells, especially lymphocytes.They are produced in the bone marrow, and when mature are released into the peripheral blood. Although they do serve a phagocytic role in the blood, their main site of action is the body tissues.The half-life for monocytes in the peripheral blood is approximately 8 hours. Monocytes migrate into the tissues, often to sites of inflammation, where they serve their primary purpose.Here they transform into fixed or free macrophages, and continue their function as avid phagocytes.When activated, macrophages may enlarge and have enhanced metabolism.

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Platelet Clumps

Occasionally they occur in clumps, particularly if the film was made from capillary blood.

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Platelet Kinetics

Platelets are derived from the cytoplasm of megakaryocytes, giant cells in the bone marrow. At any given time, two thirds of the total platelets are in the circulation and one third are present in the spleen. In persons with enlarged spleens 80-90% of the platelets are in the spleen resulting in a decreased concentration of circulating platelets. In individuals who have had a splenectomy all of the platelets will be in the circulating blood. The life span of the platelet is 8-10 days.

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Platelets

Platelets are anucleate cells, measuring only 1-4 microns in diameter. They are the smallest of the formed elements found in normal peripheral blood. The arrows point to platelets.

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All of the following statements describe a method by which platelets aid coagulation EXCEPT:View Page
Platelet Function

Platelets function both mechanically and biochemically in the process of hemostasis. When injury to a blood vessel occurs, platelets aggregate forming a plug which helps to stop the flow of blood. They release certain substances, among them serotonin and Platelet Factor 3. Serotonin causes the blood vessels in the area to constrict, thereby further stopping the flow of blood. Platelet Factor 3 catalyzes the coagulation reaction whereby a fibrin clot is formed, completing the seal. Platelets also maintain the integrity (leak-free) state of blood vessels.

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Erythrocyte Shape

In stained blood films, only the flattened surfaces of the RBC's are seen. Therefore, they appear circular with an area of central pallor corresponding to the indented area. The central pallor occupies about 1/3 of the diameter of the cell.

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Function and Kinetics

Erythrocytes are produced in the bone marrow and released into the peripheral blood where they may remain for approximately 120 days before senescence.Their main function is the transport of the respiratory gases (oxygen and carbon dioxide) between the lungs and body tissues.Each erythrocyte can be thought of as an "envelope" containing hemoglobin.Each hemoglobin molecule contains iron which has a high affinity for oxygen.As a result, when an erythrocyte passes through one of the capillaries of the lungs, it picks up oxygen.The oxygen is transported through the blood to the tissues where it is released.Carbon dioxide from the tissues then diffuses into the RBC where it undergoes chemical changes.About 70% of the altered carbon dioxide diffuses into the plasma, 25% binds to the hemoglobin molecule, and 5% goes into simple solution within the red cell.In each of these three ways carbon dioxide is transported from the body tissues back to the lungs, where it is released.

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All of the following methods can be used to transport carbon dioxide to the lungs EXCEPT:View Page
What is Blood Composed of?

Blood is composed of an isotonic fluid (plasma) in which various cells (hemocytes) are suspended. There are three major groups of these cells.

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Thrombocytes (Platelets)

The third group of formed elements in normal peripheral blood is made up of thrombocytes (platelets). Although platelets don't look very impressive, their role in the process of hemostasis is critical. Platelets are the small granular bodies shown with the arrows in this Wright stained smear.

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Overview

All of these peripheral blood cells have different characteristics. In order to accurately identify each of them, a peripheral blood film must be made, preferably from capillary blood or blood anticoagulated with EDTA (Ethylenediaminotetracetic Acid). EDTA, in contrast to many other anticoagulants, preserves cellular morphology. The individual characteristics of each cell type are made visible by staining the blood films with the Wright stain, and observing them under the microscope.

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Glossary of Terms A through M.

Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus.

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Glossary of Terms N through Z.

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

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Eosinophils

Eosinophils are also known as eosinophilic granulocytes, or eos. Eosinophils are easy to recognize in the peripheral blood because of their large bright granules. The diameter of the eosinophil is 9-15 microns, and the nuclear to cytoplasmic (N:C) ratio is 1:3. Eosinophils are generally the largest granulocytes found in normal blood.

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Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
The most immature neutrophil found in normal peripheral blood is:View Page
Band Neutrophil

Band neutrophils are also referred to as stabs or simply as bands. The diameter of a band is approximately 9-16 microns, and its nuclear to cytoplasmic (N:C) ratio is 1:2.

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Segmented Neutrophil

Segmented Neutrophil may also be referred to as seg, polymorphonuclear leukocyte, poly and PMN. Segmented neutrophils are the most mature neutrophilic granulocytes present in circulating blood. Their diameter is approximately 9-15 microns, and their N:C ratio is 1:3.

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Identify the nucleated blood cell that is indicated by the arrow:View Page
Segmented and Band Nuclei

The granulocytes found in normal peripheral blood are neutrophils, eosinophils and basophils.Most have segmented nuclei, and are therefore classified as being at the "segmented" stage of development. Some that are a little less mature have unsegmented nuclei. These are classified as "bands." Generally, we differentiate between the band and segmented forms of neutrophils, but since eosinophils and basophils are present in such low numbers, and since their nuclei are often obscured by cytoplasmic granules, we usually don't concern ourselves with designating the band forms.Since hematologists and textbooks use several different terms for these cells, synonyms for each term will be given and then may be used interchangeably throughout the course.

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Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Basophil Granules

When examining a blood film you may find that some basophils have many dense granules while others appear washed out with only a few granules, as shown in the image on the right. This is because the granules are water soluble and tend to wash out during the rinse phase of the staining process.

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Definition of a Segmented Cell continued.

Since these recommendations have been adopted by many groups, including the College of American Pathologists and the Centers for Disease Control, we will be using them as our criteria for differentiating between bands and segs.This definition was first reported by the Committee for Clarification of the Nomenclature of Cells and Diseases of the Blood and Blood Forming Organs, in the American Journal of Clinical Pathology (18:443-450, 1948).

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Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
The Process of Phagocytosis

?Neutrophils have a relatively short life span.They are produced in the bone marrow, and when they reach the band or segmented stages are released into the peripheral blood.They remain there for approximately ten hours before randomly entering body tissues.Neutrophils in the blood stream can be divided into circulating granulocyte pool(CGP) and marginating granulocytic pool (MGP).The white blood cell count reflects the cells in the circulating pool.The cells in the marginating pool move quickly into the circulating pool when needed.During an infection the neutrophil concentration of the peripheral blood can increase almost immediately due to the shift of these cells from the marginating pool and release from the bone marrow storage pool, if needed.Neutrophils then migrate to areas of tissue damage or infection.Neutrophils do not reenter the blood stream from the tissues, thus end their life in the tissues either as a result of phagocytosis or senescence.

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Life Span and function of Eosinophils

Eosinophils have a circulating half-life of approximately 18 hours and a tissue life span of at least 6 days.They are capable of locomotion and phagocytosis and can enter inflammatory sites, but do so less readily than neutrophils.In tissues the primary location for eosinophils is in the epithelial barriers to the outside world such as, lungs, skin and GI tract.They are capable of returning to the circulating blood and bone marrow after they enter the tissues.

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Eosinophils in Parasitic Infections and Allergic Reactions

Eosinophils are active in parasitic infections and in allergic reactions such as asthma and hay fever, and may be present in great numbers in the peripheral blood during these conditions.Stress, shock, or burns may also cause an increase in this type of cell.Eosinophils modulate an allergic response by liberating substances which can neutralize mast cell and basophil products.

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Basophils in the Blood

Basophils circulate in the blood for a short time and make up only a small percentage (0.5%) of the cells in circulation.They do not migrate to the tissues under normal conditions but may be seen when inflammation resulting from hypersensitivity to protein, contact allergy or skin allograft rejection is present.Basophils are sometimes increased in patients with chronic myeloproliferative disorders.

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Where do neutrophils serve their primary function?View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell that is indicated by the arrow:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Apprearance of Cytoplasm

Monos have abundant blue-gray cytoplasm containing many fine lilac granules. These give the cytoplasm a "ground glass" appearance. However, these granules may be difficult to see if the blood film is poorly stained.

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Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page
Identify the nucleated blood cell:View Page

OSHA Bloodborne Pathogens
You Are At Risk!

As a health care worker, you come into contact with materials that may contain bloodborne pathogens. These are infectious organisms, usually viruses, that live in human blood and body fluids.The bloodborne pathogens that are of greatest concern to health care workers are:Hepatitis B virus (HBV) Human immunodeficiency virus (HIV) Hepatitis C virus (HCV)

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About This Course

This course will provide you with basic information about bloodborne pathogens, the regulations that govern safe work practices when handling blood and other potentially infectious body fluids, and necessary precautions that must be taken to minimize your risk of exposure to these infections.

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Exposure Categories

There are three exposure categories :Category I are those employees who, on a day-to-day basis, will come in contact with blood or body fluids as part of their normal job. This includes medical laboratory professionals, pathologists and operating room nurses.Category II are those employees who may come in contact with blood or body fluid during the course of their normal job. This includes housekeepers, transporters, and some technicians such as EKG techs.Category III are those persons who would not normally ever come in contact with blood or body fluids and generally includes secretaries, administrators, and gardeners.Persons may move from one category to another during the course of a workday.

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The Hepatitis B Vaccination

The hepatitis B vaccine is one of the most important ways to prevent infection with HBV. The vaccine is safe and very effective, if the series is completed. The series includes three shots in the upper arm given over a six-month period.The present recombinant vaccine uses genetically-altered bakers yeast and contains no blood components.Side effects are minimal. Symptoms such as temporary soreness at the injection site, mild fever, or joint pain may occur, but are rare.The OSHA standard requires that employers provide the vaccine free of charge to you if your occupation puts you at risk for hepatitis B infection. You may decline the vaccine. If you choose not to have it, you will be asked to sign a Declination Statement. If you initially decline, but later choose to have the vaccine while still an employee, you will be able to receive it at that time. However, if your job puts you at risk for occupational exposure to HBV, you are strongly urged to receive the vaccine when it is first offered to you unless you have previously received the complete hepatitis B vaccination series, antibody testing has revealed that you are already immune, or you have been told not to receive the vaccine for medical reasons.

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Standard Precautions

Standard precautions mean that all blood and body fluids should be handled as if they are infectious and capable of transmitting disease. Standard precautions apply to: BloodBody fluidsSecretions (except sweat)ExcretionsNon-intact skinMucous membranes

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What should you do if you accidentally stick your finger with a contaminated needle?View Page
How Can HBV Be Prevented?

You can avoid exposure to HBV by taking the appropriate precautions, such as: Receiving the immunization against Hepatitis B Following standard precautions Maintaining proper work practices Using proper techniques when handling materials, which may be contaminated with blood or other potentially infected materials

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Occupational Exposure to HBV

In the health care setting, the virus is spread most often through contact with infected blood and other potentially infectious materials (OPIM), including body fluids, infectious wastes, and cultures. Body fluids most likely to transmit HBV are: Blood Semen Vaginal Secretions Pleural Fluid Peritoneal Fluid Pericardial Fluid Cerebrospinal Fluid Synovial Fluid Amniotic Fluid Saliva contaminated with blood during dental procedures Any fluid visibly contaminated with blood Sweat is not considered infectious, unless it is contaminated with blood.Contact with HBV may occur when infected blood or OPIM is introduced: Through an opening or sore in the skin Via a puncture with a contaminated sharp such as a needle Through direct contact with mucous membranes that line the insides of the mouth, nose, and eyes

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What Causes Hepatitis B Infection?

Hepatitis B infection is caused by the Hepatitis B virus (HBV).Following introduction of the virus into a susceptible person, it travels through the bloodstream to the liver. Once in the liver, the virus will multiply and cause hepatitis (inflammation of the liver).

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Who is infected?

Patients infected with HBV or other bloodborne organisms can appear healthy, so you can't tell whose blood is infectious.So treat all:bloodbody fluidssecretions (except sweat)excretionsnon-intact skinmucous membranes as if they were infectious.

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Body Fluids Most Likely To Transmit HBV

Body fluids most likely to transmit HBV are: Blood Semen Vaginal Secretions Pleural Fluid Peritoneal Fluid Pericardial Fluid Cerebrospinal Fluid Synovial Fluid Amniotic Fluid Blood contaminated saliva in dental procedures Any fluid visibly contaminated with blood Sweat uncontaminated by blood is not considered infectious.

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Blood Needed For Transmission

The amount of blood needed to cause HBV infection is very small. One milliliter of blood contains up to 100 million infectious particles.

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How common is HBV?

There are approximately 800,000 to 1.4 million individuals with chronic hepatitis B in the United States. Worldwide it is estimated that there are 350 million people infected with HBV, which contributes to an estimated 620,000 deaths worldwide each year.*The annual number of occupational infections has decreased 95% since hepatitis B vaccine became available in 1982, from more than 10,000 in 1983 to less than 400 in 2001.*** Reference: Hepatitis B information for health professionals. CDC website. Available at: http://www.cdc.gov/hepatitis/HBV/HBVfaq.htm#overview. Accessed October 28, 2011.**Reference: Exposure to blood: What healthcare personnel need to know. CDC website. Available at: http://www.cdc.gov/ncidod/dhqp/pdf/bbp/Exp_to_Blood.pdf. Accessed October 28, 2011.

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How Easily is HIV Transmitted?

After an exposure to HIV, the chance of becoming infected is usually less than 1%. However, blood and body fluid that contains high numbers of viral particles are more hazardous. Because of the extremely serious nature of HIV, it is vital to take every precaution to avoid workplace exposure.

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Occupational exposure to bloodborne pathogens can be prevented by which of the following means?View Page
Which of the following bloodborne pathogens poses the greatest risk of infection to health care workers?View Page
Needles, safety needles, and needleless systems

Most hospitals use some form of needle/holder combination that incorporates a needle safety device. This device has a mechanism that will cover the needle after use. It must be activated as soon as the task is completed. The device that is pictured here is just one of many options that are currently available. There are also needleless systems that use special adapters that can be attached to some intravenous lines and will permit blood to be obtained without the use of needles.

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Handling Specimens

Work practice controls affect the transport of blood and other potentially infectious materials (OPIM).Proper personal protective equipment (PPE), including eye protection, gloves, and lab coats or aprons, must be used when handling blood specimens and OPIM.Spilled specimens must be cleaned up using proper PPE .

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Transporting Specimens

Place blood and other infectious specimens ... first in an appropriate sealed container and then in a secondary red or biohazard-labeled bag. Or place them in the facility-approved tray for transport within the institution.

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Small Surface Spills

If a small spill of blood or other potentially infectious materials occurs on a work surface, always be sure you put on the appropriate personal protective equipment before proceeding with decontamination and clean-up procedures. It is best to use puncture-resistant utility gloves for spill clean-up. Use the decontamination and cleaning method that is approved by your facility. Be aware of the potential for splatter and contamination.

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Contaminated Wastes

It is important to always dispose of contaminated wastes properly.Examples of contaminated wastes: Microbiology waste and pathology waste All body fluids, such as pleural fluid Contaminated sharps and blood specimens

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Labeling not Required

The following do not require biohazard labeling: Units of blood, blood components, or blood products that are released for transfusion or other clinical use and are labeled as to their contentsIndividual containers of blood or other potentially infectious materials that are placed in a labeled container during storage, transport, shipment, or disposal

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Gloves

Disposable gloves must be worn whenever there is a risk of contact with blood or other body fluids. Hypoallergenic gloves must be used if you, or the patient you are caring for, has a latex allergy. Keep hand jewelry to a minimum to protect the integrity of the gloves.Replace gloves: Between patient contacts If they are damaged or contaminated Before leaving the work area Cleanse hands after removing gloves. Disposable gloves cannot be washed.Utility gloves or heavy-duty rubber gloves are useful when cleaning up spills or when there is a risk of damage from equipment handling.Utility gloves may be decontaminated and reused if their integrity has not been compromised. They should be inspected regularly, and must be replaced if damaged.

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Gloves Must be Worn...

when there is a reasonable chance of exposure to blood, other infectious body fluids, mucous membranes, or nonintact skin. during vascular access procedures, including phlebotomy. when handling contaminated items or when touching contaminated surfaces.

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Types of gloves

To protect the worker from blood borne pathogens, either latex or a latex like product such as nitrile must be worn when handling specimens or other items possibly contaminated with blood.Utility gloves or heavy-duty rubber gloves are useful when cleaning up spills or when there is a risk of damage from equipment handling.

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Face and Eye Protection

The following protect your eyes and the mucous membranes of your nose and mouth: Face shield Mask worn with safety glasses Employees who wear prescription eyewear may be protected with a face shield, goggles, or with side shields attached to their glasses (a mask must also be worn to protect the nose and mouth).Face and eye protection must be worn whenever it is reasonably anticipated that splashing or spraying of blood or other contaminated materials may occur. A splash guard (as shown below) is an engineering control that can be used for facial protection.

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Exposure Incident

Even after taking all the proper precautions there is still a small chance of an exposure incident. An Exposure incident occurs when: Blood or another potentially infectious body fluid comes into direct contact with mucous membranes or non-intact skin. Parenteral exposure means: Exposure occurring as a result of piercing the skin barrier through needlesticks, cuts, or abrasions.

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Evaluation and Treatment

Your supervisor will refer you for an immediate evaluation and any necessary treatment. Confidentiality will be maintained. Your blood will be tested only with your consent.

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OSHA Bloodborne Pathogens (retired)
You Are At Risk!

As a healthcare worker, you come into contact with bloodborne pathogens. These are infectious organisms, usually viruses, which live in human blood and other potentially infectious body fluids.The most important ones are... Hepatitis B Virus (HBV) Human Immunodeficiency Virus (HIV)

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What Causes HBV?

Hepatitis B is caused by the Hepatitis B virus, or HBV.The virus travels through the blood stream to infect the liver.

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Who is infected?

Patients with Hepatitis B and other bloodborne infections can appear healthy, so you can't tell whose blood is infectious.So treat all:blood, body fluids, secretions (except sweat), excretions, non-intact skin, and mucous membranes as if they were infectious.That's what the term Standard Precautions means.

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Spread of HBV in the community(2)

Body fluids most likely to transmit HBV are: Blood Semen Vaginal Secretions Pleural Fluid Peritoneal Fluid Pericardial Fluid Cerebrospinal Fluid Synovial Fluid Amniotic Fluid Saliva in dental procedures Any fluid visibly contaminated with blood

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Blood needed for transmission

The amount of blood needed to cause HBV infection is very small.One milliliter of blood contains up to 100 million infectious particles.Of the persons exposed to HBV by needle stick, 30% will get the infection.

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How easily is HIV transmitted?

After an exposure to HIV by a contaminated needle, the chance of becoming infected is usually less than 1%.However, exposures from patients with high numbers of viral particles in their blood may be more hazardous.Because of the extremely serious nature of HIV, we must take every precaution to avoid workplace exposure.

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The Hepatitis B Vaccination

The Hepatitis B Vaccine is one of the most important ways to prevent infection. About 90% of people who receive it get immunity.The present recombinant vaccine is made by genetically altered bakers yeast and contains no blood components. It is very safe.Side effects are minimal. Symptoms such as temporary soreness at the injection site, mild fever, or joint pain may occur but are rare.The procedure consists of three shots in the upper arm given over a six month period.The OSHA standard requires that employers provide the vaccine free of charge to you if your occupation puts you at risk. You may decline the vaccine; but you will be asked to sign a Declination Statement.

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Handling Specimens

Work practice controls affect the transport of blood and other potentially infectious materials.

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Transporting Specimens

Place blood and other infectious specimens ... first in an appropriate sealed container and then in a secondary red or biohazard labeled bag. Or place them in a compartmentalized tray for transport within the institution.

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Small Surface Spills

Small blood spills on work surfaces may be cleaned by first laying paper towels on the spill to blot and avoid splattering, and then applying disinfectant.Larger spills will require other methods.Use an approved cleaning method.Use appropriate personal protective equipment.Be aware of the potential for splatter and contamination.

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Contaminated Wastes(1)

It is important to always dispose of contaminated wastes properly!Examples of contaminated wastes: Microbiology waste and pathology wasteAll body fluids, such as pleural fluids Contaminated sharps and blood specimens

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Labeling not Required

The following do not require biohazard labeling: Blood products in clinical use Individual specimen containers However, they are subject to Standard Precautions.

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Gloves Must be Worn

Gloves must be worn: when there is a reasonable chance of exposure to blood, other infectious body fluids, mucous membranes, or nonintact skin, during vascular access procedures, including phlebotomy, or when handling contaminated items or surfaces.

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Face and Eye Protection

The following protect your eyes and the mucous membranes of your nose and mouth: Face shields Masks and safety glasses They must be worn whenever it is reasonably anticipated that splashing or spraying of blood or other contaminated materials may occur.Employees who wear prescription eyewear may be protected with a face shield, goggles, or with side shields attached to their glasses.

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Exposure Incident

Even after taking all the proper precautions there is still a small chance of an exposure incident.Exposure incident: Blood or another potentially infectious body fluid coming into direct contact with mucous membranes or nonintact skin.Parenteral exposure: Needle stick or being cut by a contaminated sharp.

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Evaluation and Treatment

Your supervisor will refer you for an immediate evaluation and any necessary treatment. Confidentiality will be maintained.Your blood will be tested only with your consent.

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Packaging and Shipping Infectious Materials (retired July 2013)
IATA and US Postal Service Exempt Specimens

Laboratory specimens that are unlikely to cause disease and do not meet the criteria for category A or B substances are not subject to Division 6.2 regulations. Specimens for which the hazardous materials regulation (HMR) does not apply include human or animal samples (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, and body parts) being transported for routine testing not related to the diagnosis of an infectious disease. This includes specimens that are being sent for:drug or alcohol testing cholesterol testing blood glucose level testing prostate specific antibody (PSA) testing testing to monitor kidney or liver function pregnancy testing tests for diagnosis of non-infectious diseases such as cancer biopsies The US Department of Transportation (DOT) has no "Exempt Specimen" classification and there are no DOT guidelines for packaging non-regulated specimens.* According to the DOT, in the U.S., if a package is marked as "Exempt Human/Animal Specimen" the understanding is that it contains no infectious substance. However, both IATA and the US Postal Service (USPS) have these requirements for packaging exempt specimens: Packaging IssueIATAUSPSType of packaging requiredTriple packagingTriple packagingOuter containerOne dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet One dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet Quantity limits: outer containerNone NoneQuantity limits: Primary receptacleNone500 mLQuantity limits: secondary packagingNone500 mL* Non-regulated specimens may become regulated because of preservatives, such as 10% formaldehyde (class 9) or 25% formaldehyde (class 8); or 25% ethanol (class 3).

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Classification Scenario 1

A blood specimen is collected from a patient that is suspected of having Hepatitis B. The specimen will be sent via commercial carrier (e.g., UPS) to a reference laboratory for further testing. What classification should be used for appropriate packaging and labeling? Work through the Classification Decision Tree.

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Classification Scenario 2

A blood specimen is collected from a patient suspected of having Hepatitis B. The specimen will be taken to the testing laboratory by the laboratory's own courier service using an exclusive use motor vehicle. What classification should be used for appropriate packaging and labeling?Work through the Classification Decision Tree.

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Classification Scenario 3

A blood sample collected from an outpatient, will be sent by ground using a carrier who follows DOT Regulations for classification of dangerous goods. The specimen is being sent to a reference laboratory for cholesterol screening. What classification should be used for appropriate packaging and labeling?Work through the Classification Decision Tree.

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Definitions

Before further discussion of Category A and Category B, it is important to define two additional terms that are used in the classification process. CultureAn infectious substance containing a pathogen that is intentionally propagated, for example a bacterium grown on bacteriological medium as seen in the image below. Culture does not include a human or animal patient specimen.Patient specimenHuman or animal materials collected directly from humans or animals and transported for research, diagnosis, investigational acitivities, or disease treatment or prevention. Patient specimen includes excreta, secreta, blood and its components, tissue and tissue swabs, body parts, and specimens in transport media (e.g., transwabs, culture media, and blood culture bottles).* *It is important to note that this means specimens that have been collected into these transport media, but have not yet been incubated and are not actively growing in the media.

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Additional Packaging Requirements for Category A and Category B Substances

If multiple primary receptacles are placed in a single secondary packaging, they must be either individually wrapped or separated so as to prevent contact between them.The primary receptacle or the secondary packaging must be capable of withstanding, without leakage, an internal pressure producing a pressure differential of not less than 95 kPa (13.8 lbs/in2) because the package may be placed into an unpressurized storage compartment in a cargo aircraft. This must be verified when choosing packaging for shipping either category A or category B substances by aircraft. It is also recommended if shipping by ground. An evacuated blood collection tube that has remained unopened qualifies as a 95 kPa container. The smallest surface of the outer packaging must be at least 100 mm X 100mm (4 inches x 4 inches).Other dangerous goods must not be packed in the same packaging as Division 6.2 infectious substances unless they are necessary for preservation of the specimen (e.g., formalin). A quantity of 30 mL or less of formalin or other dangerous goods included in hazard Classes 3, 8, or 9 (flammable liquids such as alcohol; corrosives such as acids or bases; or miscellaneous hazardous materials) may be packed in each primary receptacle containing infectious substances. A quantity greater than 30 mL will require appropriate hazard labels on the package.OverpackWhen packages are placed in an overpack, the overpack must be marked with the word "Overpack" and the package markings must be reproduced on the outside of the overpack.

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Parasitology Question Bank - Review Mode (no CE)
Match each parasite listed here with its corresponding diagnostic stage:View Page
Arrange the following phases of the hookworm life cycle in order beginning with human contact:View Page
Arrange the following phases of the Wuchereria bancrofti life cycle in order beginning with human transmission:View Page
The Knott technique serves as a means of identifying:View Page
This parasite was found on a blood smear.View Page
This suspicious form was found on a blood smear.View Page
This suspicious form was found on a blood smear.View Page
These suspicious forms were seen on a blood smear.View Page
This suspicious form was recovered in stool.View Page
This suspicious form, found in stool, which measures 15 µm by 10 µm, is responsible for which of the following diseases?View Page
A 68-year-old female, who recently vacationed in Brazil, presented to her physician exhibiting overall weakness, fever, and enlarged lymph nodes. Blood was collected for culture and parasitic examination. The culture was negative. This suspicious form was recovered upon examining the Giemsa-stained preparation. This patient is most likely suffering from:View Page
The class of protozoa with no apparent organelles for locomotion is known as:View Page
Match each parasite listed here with its corresponding optimal specimen type from which it may be recovered: (Answers may be used more than once.)View Page
The fever and chills syndrome associated with malaria is known as a/an:View Page
Match each parasite listed here with its respective common name:View Page
Match each parasite listed here with its respective common name:View Page
Match each parasite listed here with its corresponding optimal specimen type from which it may be recovered:View Page
A 55 year old female, who recently returned from an extensive trip to China, presented to her physician complaining of diarrhea and abdominal cramps. The doctor ordered a complete blood count (CBC), chem 21 panel, and stool for culture and parasite examination (O & P). The CBC revealed pronounced eosinophilia. The chem 21 and stool culture were unremarkable. The O & P revealed suspicious forms like the one below that each measured approximately 140 µm by 80 µm. This patient is most likely infected with:View Page
A 43 year old female presented to her doctor for a routine check-up. Her only complaint was that she had been experiencing watery stools that occasionally contained pus and blood. Examination revealed tenderness in her abdomen. A stool for parasite study was sent to the lab. Two suspicious forms were seen. The oblong form on measured 53 µm by 60 µm whereas the rounder form measured 45 µm by 37 µm. Use the pulldown boxes to identify each picture:View Page
A 31 year old male missionary worker recently returned from Africa where he helped a small rural community update their sanitation practices. He presented to his physician weak and complained of recent weight loss, abdominal pain, and diarrhea that was often bloody. The doctor ordered a battery of tests including a complete blood count (CBC) and stool for parasite examination. The CBC revealed eosinophilia and anemia. This suspicious form was seen on the wet preparations. It measured 52 µm by 27 µm. What parasite is mostly likely present?View Page
A 44 year old female immigrant from Southeast Asia presented to the local clinic complaining of fever, chills, diarrhea and weakness. Patient history revealed that the woman worked in a research laboratory in her homeland. Her primary project was to develop an effective insecticide for the dreaded sandfly. The doctor decided to culture her blood for parasites. This form, measuring 14 µm, was recovered. The patient is most likely suffering from:View Page
A 58 year old male, who recently returned from an extensive overseas business trip to Africa, presented to the local clinic complaining of nausea, vomiting, and an achy feeling all over his body. At first he thought it was just the flu, but it persisted. The doctor ordered a battery of tests including blood smears for parasitic study. This suspicious form was recovered. The patient is most likely suffering from:View Page
An 18 year old immigrant from the Philippines presented to the local clinic shortly after relocating to the United States complaining of fever and chills. Examination of the young adult revealed enlarged lymph nodes. Blood was drawn and submitted for culture and parasitic examination. The culture was negative. This suspicious form was seen on the Giemsa-stained blood smear. It measures 225 µm in length. This patient is most likely infected with:View Page
A 16 year old male champion athlete went to his doctor complaining of a persistent cough, fever, bloody diarrhea and overall weakness. Upon questioning the patient, it was learned that he had recently competed in a freshwater swimming competition in the Caribbean. Examination revealed a dermatitis on the patient's right calf. A battery of tests were ordered including a CBC, chemistry profile, and a stool for culture and parasitic examination. The CBC revealed the presence of eosinophilia. The other hematology and chemistry tests were unremarkable. The culture was negative. This suspicious form was seen on all parasite preparations made from the stool sample submitted. This form measures 165 µm by 68 µm. This patient is most likely suffering from an infection with:View Page
A 54 year old Finnish male presented at the local clinic with abdominal pain, weight loss, overall weakness and digestive discomfort. Patient history revealed that the man's diet was rich in raw fish. A complete blood count (CBC) was performed and revealed macrocytic anemia. A stool for parasitic examination was ordered. This suspicious form was seen upon initial screening of the sample. It measures 77 µm by 48 µm. This patient is most likely suffering from an infection with:View Page
A 27 year old West African immigrant went to the local clinic complaining of fever, chills, and joint pain. The physician immediately ordered blood for parasitic examination. The Giemsa-stained thin blood smear revealed the three suspicious forms below. This patient is most likely suffering from an infection with:View Page
I am found in blood where I invade red blood cells. I typically contain 6 to 8 merozoites.View Page
I am typically found in blood. I measure 260 µm in length and possess a sheath.View Page
I am sheathed and measure 200 µm. I am found in blood.View Page
I may be found in blood or in lymph nodes.View Page
I am found in blood.View Page
Which of the following parasites may be recovered in the peripheral blood?View Page
The episodes of fever and chills experienced by patients suffering from malaria are known as:View Page
The presence of parasites in human blood is termed:View Page

Pharmacology in the Clinical Lab: Therapeutic Drug Monitoring and Pharmacogenomics (retired 10/15/2012)
Basic Pharmacokinetics

In order to discuss TDM and PGx we need to also introduce the concept of pharmacokinetics. Pharmacokinetics is the study of drug disposition in the body: how and when drugs enter the circulation, how long they remain in the blood, and how they are eliminated. TDM is the clinical assessment of a drug's pharmacokinetic properties. Physicians and pharmacists need to establish that a drug is present at an effective concentration but not at a toxic concentration. The next few pages will describe some of the factors that determine a drug's disposition in the body. These factors ultimately decide the need for therapeutic drug monitoring.

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Other Factors Affecting Drug Absorption and Distribution

In addition to protein availability, other factors may affect drug absorption and distribution in the body as a whole or at specific sites within the body. The following table highlights some of these other factors. Factor Discussion Regional blood flow Reduced area blood flow can be seen in diabetics and enhanced blood flow can be seen in tumors. Lipid solubility of the drug The more lipophilic a drug is, the more likely it will enter the central nervous system. The integrity of the GI tract In a diseased gut, an orally-administered drug may not be absorbed as expected. Age Drug kinetics and dispositions change throughout life. In general, metabolism of drugs is reduced in the elderly. Genetics Mutations or deletions in drug metabolizing enzymes can greatly affect a drug's disposition.

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Peak and Trough Sampling Times

To assess drug concentrations during the trough phase, blood should be drawn immediately before the next dose. To assess peak levels, the time for drawing depends on the route of administration: Oral: One hour after drug is taken (assumes a half-life of > two hours) IV: 15-30 minutes after injection/infusion Intramuscular (IM): 30 minutes - one hour after injection

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Phlebotomy
Discussion

When the results on Mr. John Ready were called to the nurse, she was very surprised that the result of his CBC was normal. The nurse explained to the lab tech that Mr. John Ready had a known diagnosis of lower GI bleeding. His hemoglobin had been very low for the past 24 hours because of the internal bleeding, and she thought it was very surprising that his hemoglobin had normalized so quickly without having received a blood transfusion. Mr. Ready's doctor decided the patient should be redrawn to ensure a correct result. The nurse further questioned if the phlebotomist could possibly have drawn the wrong patient because earlier that day Mr. Ready had been moved to room 831, and room 825 was presently occupied by a patient named Walter Redding. If Julie had checked the patient's armband, she would have realized that the patient in 825 was the wrong patient.Relevant topics:Importance of patient ID, Patient identification continued, Specimen labeling, Specimen labeling Continued, Blood bank specimens

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Case

Julie Smith was a newly certified phlebotomist and had been working at Northwood Hospital for several months. As she approached room 825, she looked on her collection list to verify this was the correct room for her first collection. Indeed it was, even though there was no patient name on the door. Her collection list told her the patient in room 825 was a 55 year old male named John Ready. After knocking several times, Julie entered the room to find a middle aged man who appeared to be sleeping. Julie approached the patient and said, "Good day Mr. Ready. My name is Julie and I am from the lab. I need to draw blood for some tests ordered by your doctor." The man awoke and seemed irritated as Julie repeated herself. The patient responded and told Julie to do whatever she needed to do so he could go back to sleep Julie then proceeded to do the venipuncture.

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What crucial step did Julie fail to perform?View Page
Discussion

A phlebotomist should never use an arm with restricted usage for the venipuncture. Even if no sign is posted, the patient may tell you not to use a particular arm for various reasons, i.e. previous mastectomy, history of phlebitis, active AV fistula, etc. Do not draw blood above an IV line. If blood is taken from a vein above an IV line it might be diluted by the IV fluid, which could cause incorrect test results. In this case, Bobby should choose a vein on the dorsum of Mrs. Grayson's hand, below the IV. A butterfly needle would facilitate drawing blood from these small hand veins.Relevant topics:Alternate sites, Sites to avoid, Signs, Arms to avoid

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Case

Bobby Jones, a phlebotomist at Georgetown Hospital, entered the room of Mrs. Mary Grayson with a physician's order to draw some blood work. After properly greeting Mrs. Grayson, identifying himself and checking her armband, Bobby prepared for the venipuncture. He suddenly notice a sign posted above the bed that read: "Restricted left arm usage. Previous mastectomy - Do no use left arm for venipuncture." Bobby set up his equipment to use her right arm and noticed an IV line in Mrs. Grayson's right arm positioned in a vein slightly above her wrist on the dorsum (top) of her forearm.

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Which site should Bobby choose for the venipuncture?View Page
Case

Marcie Moore was a phlebotomist at a community hospital in Atlanta. It was her week to collect the pediatric unit and she was on her way to the room of a newborn for which she had just received orders to draw a STAT BMP (chem-7) and bilirubin. After informing the mother of the baby about the test she needed to perform, Marcie set up to perform a heel stick on the baby. Marcie chose a site on the outer edge of the heel on the bottom of the baby's foot ( the correct area for a heel stick) and made a small incision with a Tenderfoot lancet after cleaning the site well with alcohol.She immediately began collecting the blood in the correct tube for the BMP and bilirubin. Blood flow was not strong so Marcie squeezed the baby's foot a little to help the blood come out faster – the newborn was screaming and Marcie could tell it was making the mother uncomfortable. She wanted to hurry and get done so the mother could hold the baby.After the chemistry tech ran the blood tests on the tube, she informed Marcie that the newborn had a panic potassium level which did not coincide with the previous blood work on the newborn. Also the chemistry instrument could not perform the bilirubin due to hemolysis. Marcie was asked to recollect the specimen.

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Discussion

Hemolysis can easily be caused by improper phlebotomy techniques. Hemolysis occurs when RBCs are broken up and hemoglobin is released into the plasma, causing it to become pink rather than its natural straw color. Hemolysis can occur by using too small a needle, pulling a syringe plunger too rapidly, expelling blood vigorously into a tube, or shaking a tube of blood too hard. Hemolysis can cause falsely increased potassium, magnesium, iron, and ammonia levels, and other aberrant lab results.In this case, Marcie did not properly wipe the site with gauze after cleaning it with alcohol, and alcohol contacting the blood could have caused RBCs to break up or hemolyze. Marcie also squeezed the baby's foot too hard, causing hemolysis.Relevant topics:Site selection and preparation, Heelstick: Puncture, Hemolysis, Causes of hemolysis

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What had Marcie done to hemolyze the specimen?View Page
What could have caused the clotting?View Page
Discussion

Clotting of blood specimens may be caused by several factors. Clotting usually occurs due to improper phlebotomy technique,and clotted specimens will generally be rejected for those tests that require the blood to be mixed with an anticoagulant. When a clot forms in a tube containing anticoagulant, it usually indicates that the blood and anticoagulant aren't in proper balance. That is why it is crucial to invert tubes with anticoagulant almost immediately after collection to ensure proper mixing of blood and anticoagulant. Relevant topics: Lavender top tubes, Light blue top tubes, Unsatisfactory specimens: Clots, Causes of clotting

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Case

John Wagner, a phlebotomist at General Hospital, went up to the 7th floor to draw routine blood work on a patient. As he approached the door of the patient's room he noticed a red stop sign on the door with the words "Respiratory Isolation" written on it.

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At this point, what should John know to do?View Page
Case

Julie Smith, a newly certified phlebotomist at Northlake Hospital, entered a patient's room on the third floor for a routine blood draw. The patient was an elderly woman who had very small fragile veins. Julie therefore decided to use a safety butterfly needle attached to a Vacutainer tube in order to draw the blood. When Julie was finished with the venipuncture, she detached the butterfly needle from the Vacutainer, and approached the Biohazard needle disposal box. She noticed that the disposal box was full , but decided to try to fit the butterfly into the box anyway. Holding the butterfly by the tubing, she tried to push the butterfly into the box. The needle suddenly recoiled and stuck Julie's finger. Julie left the patient's room in a panic and headed back to the lab to report the needle stick injury.

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What should Julie have done to prevent the needle stick?View Page
Discussion

Tubes are drawn in a specific order to avoid the possibility of erroneous test results caused by carryover of an additive from one tube to the next. If a blood culture is ordered, it should be drawn as the first tube. Additional tubes should follow this order of draw. Sodium citrate - coagulation tube (light-blue top) Serum tube - with or without clot activator or gel. This tube is either a red top tube or a gold top tube depending on manufacturer and tube additive. Sodium or lithium heparin with or without gel plasma separator (green top) Potassium EDTA (lavender or pink top) Sodium fluoride, and sodium or potassium oxalate (gray top)

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Case

Bobby Jones, a phlebotomist at Georgetown Hospital, was called to the pre-op area to perform a bleeding time. Bleeding times may be requested on selected preoperative patients to help assure that they will not bleed excessively during surgery. Bobby gathered the appropriate equipment, then placed the blood pressure cuff of the patient's upper arm, and pumped it to 40 mm Hg. After finding the appropriate site (a few inches below the elbow on the inside of the forearm), Bobby cleaned the site with an alcohol pad and immediately made the incision with a Surgicutt parallel to the bend of the elbow. Bobby then wiped away the first drop of blood with an alcohol pad, and blotted the incision every 30 seconds thereafter. Fifteen minutes later the patient was still bleeding.

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What did Bobby do that could have falsely prolonged the bleeding time?View Page
Discussion

The blood pressure cuff was correctly inflated to 40 mmHg. The site for the incision is indeed the inside of the forearm a few inches below the bend of the elbow, and the cut was correctly made parallel to the bend of the elbow. However, the phlebotomist did not allow the alcohol to dry, and then made the additional mistake of wiping the incision with alcohol. Alcohol will retard blood coagulation, resulting in a falsely elevated bleeding time. It is also important to ask the patient about medications taken within the past week. Certain medications, particularly aspirin, will result in an elevated bleeding time.Relevant topics:Bleeding time: introduction 1, Bleeding time: introduction 2, Bleeding time: performance, Bleeding time, Apply blood pressure cuff, Bleeding time: prepare the site

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Case

A phlebotomist at an outpatient drawing station prepares to collect blood from a patient who is scheduled for surgery the next day. The patient tells the phlebotomist that she is afraid of needles. The phlebotomist assures the patient that everything will be fine. He seats the patient in a phlebotomy chair. He talks the patient through the beginning of the venipuncture and she seemed to be doing fine. As the second of four tubes is being drawn, the patient suddenly blurts out that she fells very dizzy and is going to faint.

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What should the phlebotomist do now?View Page
Discussion

Insufficient blood volume may cause erroneous test results, and specimen rejection. When blood flow stops, it can mean several things:The bevel of the needle may be pressed against the wall of the blood vessel. If this is the case, moving the needle slightly may cause blood to begin flowing again.The vein may have collapsed due to the vacuum of the tube. If moving the needle slightly does not re-establish blood flow, you will have to recollect the patient.The needle may have gone all the way through the vein. Pulling the needle back slightly may cause blood to resume flowing. The tube you are using may have insufficient vacuum. Try another tube. Never vigorously probe the patient's arm with a needle. At the first sign of discomfort the needle should be withdrawn. The patient may then be redrawn be yourself or another phlebotomist.Relevant topics: Insufficient volume, Partial collection tubes, What if no blood flows

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Case

A phlebotomist was collecting STAT blood work on a patient when blood flow unexpectedly stopped. The light blue top tube being drawn at the time was only about one third full – less than the minimum volume required for this particular tube. A red top tube had already been drawn for a cross match, and a PT was the only other test ordered.

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What could the phlebotomist do at this point to renew blood flow?View Page
Discussion

During a finger stick procedure it is important that the lancet be positioned on the finger so that the incision is perpendicular to the fingerprint. This allows a larger amount of blood to flow. It is also important to wipe away the first drop of blood that emerges form the incision with clean gauze, since it may contain tissue fluids that can cause incorrect test results. The first drop of blood may also contain traces of alcohol remaining from the cleaning step. Alcohol may break up or hemolyze blood cells, causing incorrect results.Relevant topics:Finger-stick collections, Finger-stick: site preparation, Finger-stick: puncture, Wipe away the first drop, Finger-stick specimen collection

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Case

A phlebotomist at Memorial Hills Hospital entered the room of a 6 year old patient. The only test ordered was a CBC, so the phlebotomist decided to do a finger stick. After gathering proper supplies for the finger stick, the phlebotomist began the procedure by putting on gloves and wiping the tip and side of the patient's ring finger with alcohol. He positioned the safety lancet between the ball and the side of the finger and made a small incision. The child cried as the blood was collected.

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Case

A phlebotomist from the laboratory at Midtown Memorial Hospital was working evening shift. Her shift ended at 11 PM and it was 10:30 PM. She suddenly got orders for a STAT blood culture on the second floor. The order specified blood culture times two, 30 minutes apart. The phlebotomist went to the patient's room and decided to collect both blood cultures at the same time form the same site so she would be able to leave on time without having to come back in thirty minutes to collect the second set. She also wanted to "save" the patient from an extra stick. While the phlebotomist was preparing for the collection, she realized she didn't have any Betadine on her tray, and decided she would just clean the site twice with alcohol. She finished the blood culture collections and was able to leave by 11 PM.

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Discussion

This phlebotomist violated hospital procedures in several ways that could adversely impact patient care: Cleaning the site only with alcohol, not iodine, could result in a false-positive contaminated blood culture. This might result in the patient receiving unnecessary intravenous antibiotics, and could prolong the patients hospital stay unnecessarily. Drawing both cultures at the same time lessens the chance of recovering a bloodstream organism.Drawing both cultures from the same site might result in both of them being contaminated, making it very difficult for the physician to distinguish contamination from a "real" bloodstream infection.Relevant topics:Blood cultures: introduction, Avoid skin contamination, Blood culture site preparation 1, Blood culture site preparation 2

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What did the phlebotomist do wrong?View Page
Basic metabolic panel (BMP)

Consists of an electrolyte panel, plus: Blood urea nitrogen (BUN), which a measure of renal function. Creatinine (Creat), which also measures renal function Glucose, the most important blood sugar, and Calcium. Run on serum or plasma

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Hemogram (CBC)

Also known as Complete Blood Count (CBC) and is run on whole blood.Blood is tested for quantity and quality of different blood cell types, including: White Blood Cells (WBC Count) Red Blood Cells (RBC Count) Platelets (Platelet Count) Blood is also tested for hemoglobin & hematocrit (H&H).

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Electrolytes panel (Lytes)

Blood is tested for the most important electrolytes (salts): Sodium (Na) Potassium (K) Chloride (Cl) Carbon dioxide (CO2)Can be run on serum or plasma.

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Obstetric panel

CBC Hepatitis B surface antigen Antibody, rubellaSyphilis test (RPR) Antibody screen Blood type, Rh and ABO

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Blood collection tubes: sizes

Adult tubes generally hold from 3 to 10 ml of blood. Pediatric tubes usually hold from 2 to 4 ml.Tubes for fingersticks or heelsticks generally hold one half ml or less.

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Lavender top tubes

Contain anticoagulant Ethylendiaminetetraactic acid (EDTA) to prevent clotting. Are used mostly for hematology studies. Must be completely filled to assure a correct anticoagulant to blood ratio. Must be inverted after filling to assure proper mixture of anticoagulant with blood.

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Yellow top tubes

Contain either acid citrate dextrose (ACD), which maintains RBC viability and may be used for HLA phenotyping, DNA, paternity testing, or lymphocyte surface markers, or: Sodium polyanetholesulfonate (SPS) which is sometimes used to collect blood culture specimens.

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Butterfly needles with built-in safety features continued

Two examples of butterfly needles with built-in safety devices are shown.The Punctur-Guard™ (Bioplexus), shown above, uses an internal blunt needle which is activated after blood is drawn. The activated device showing the blunt internal needle is shown in the inset on the upper right. The Angel Wing ™ (Monoject), is activated by sliding a safety shield over the needle after venipuncture.

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Syringes

Syringes consists of:A barrel, which holds the blood. A plunger that allows suction to be appliedA tip to which the needle is connected.Syringes have ml (cc) markings to show how much blood has been collected.

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Blood collection tubes: common types

Lavender top Light blue top Green top Red top Speckled top Gray top Yellow top Royal blue top

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Multiple draw needles

Multiple draw needles are used with vacuum collection tubes.They allow the collection of blood into multiple vacuum collection tubes during a single venipuncture. They have a retractable sheath over the portion of the needle that penetrates the blood tube.

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Blood collection tubes: types

Rubber stoppers of blood collection tubes are color coded. Each type of stopper indicates a different chemical additive (usually an anticoagulant to prevent clotting), or a different tube type.

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Multiple draw needles with built-in safety features.

You will be required to use multiple draw needles with built-in Safety features. One example is the Puncture-Guard™ (BioPlexus) needle, which uses an internal blunt needle (detail above) that is activated with forward pressure on the final blood tube prior to withdrawal of the needle from the vein. Refer to your institution's and the manufacturer's procedure manuals before using these devices.

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Blood collection tubes: introduction

A blood collection tube generally consists of a glass or plastic tube with a rubber stopper. It has a vacuum so that blood will flow into the tube. Blood collection tubes may contain anticoagulants and/or other chemical additives.

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Syringes with built-in safety devices

Syringes are used for injections, as well as to collect blood. There a various syringes with built-in safety features.One example is the Monoject™ (Sherwood Services AG), Safety Syringe, shown here.

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Gloves

Gloves must be worn for all procedures requiring vascular access. Non-powdered latex gloves are most commonly used; Alternatives available for health-care workers allergic to latex include: Latex gloves sandwiched between 2 vinyl gloves. Latex-free glove liners.Do not use latex gloves or tourniquets when collecting blood from patients with latex allergy.

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Blood culture bottles

Are used to collect sterile blood samples from patients who may be septic (have bacteria or other organisms growing in their bloodstream). Different blood culture bottles are used for aerobic, anaerobic, and pediatric collections.

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Needle components

The tip of the needle consists of a: A very sharp tip for puncture.A bevel which allows for blood flow. A barrel which allows for blood flow.

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Blood transfer device

A blood transfer device allows the transfer of blood from a syringe into a blood collection tube or a blood culture bottle. The BD™ blood transfer device is shown here.

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Butterflies with built-in safety features

You will be using butterfly needles with built in safety features. Butterfly needles are the number-one cause of needlestick injuries, so proper use of their safety devices is critical. Their use is described in greater detail in the section on butterfly needle blood collection.

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Plastic holders used with the evacuated tube system

A plastic holder must be used with the evacuated tube system. The needle screws into the holder to allow blood collection.

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Introduction to phlebotomy equipment

The following section will familiarize you with the supplies & equipment you will need to collect a blood specimen.

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Phlebotomy trays

A Phlebotomy tray is used to carry blood drawing equipment to the bedside.Trays should be sanitized daily, & kept well-stocked and organized. Phlebotomy trays may be sanitized using 10% bleach solution, or other appropriate disinfectant.

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Hemogard ™ blood collection tubes

Blood collection tubes with Hemogard ™ (BD) closure protect you from blood which might splatter when the tube is opened. The rubber stopper is recessed inside the plastic shield, preventing exposure to blood present on the stopper. You will probably be using Hemogard or other tubes having protective devices.

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Blood collection tubes: inversion

All tubes (except red top tubes which contain no additives) must be gently inverted 5 to 8 times immediately after filling, to ensure proper mixing of blood and anticoagulant, or other additives.

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Blood collection tubes: expiration dates

All blood collection tubes have expiration dates. Expiration dates should be closely monitored and tube stock must be rotated.

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Light blue top tubes

These tubes contain the anticoagulant sodium citrate. They are used mostly for coagulation (clotting) studies. They must be completely filled to assure proper ratio of anticoagulant to blood.They must be inverted immediately after filling to prevent clotting.

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Green top tubes

Contain either sodium or lithium heparin.Used for tests requiring whole blood or plasma such as ammonia or whole blood potassium.

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Red top tubes

Contain no additives. Used for blood bank tests such as blood typing, type and screen, and crossmatches. Also used for other tests including toxicology, and serology.

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Speckle top tubes

Also known as serum separator tubes, tiger top tubes or red gray tubes. Contain a serum-cell separator gel which separates serum from clotted blood cells during and after centrifugation.

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Remove needle

Removing the needle:Gently release the tourniquet before the last tube of blood is filled.Remove the last tube from the needle.Withdraw the needle in a single quick movement.

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Finger stick - Specimen collection

Gently massage the finger from base to tip to collect blood into the appropriate tube.Avoid hemolysis:Do not squeeze the finger too tightly during blood collection.

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Applying the tourniquet

Tie the tourniquet just above the elbow.The tourniquet should be tight enough to stop venous blood flow in the superficial arm veins.

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Push tube onto holder

Gently push the tube onto the needle holder so that the catheter inside the needle holder penetrates the tube.Blood flow should be visible at this point.

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Blood won't flow

If you do not see blood flow, the tip of the needle:May not yet be within the vein.May have already passed through the vein.May have missed the vein entirely.May be pushed up against the inside wall of the vein.

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Adjust needle

Advance or withdraw the needle slightly, if necessary, to establish the flow of blood.

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Venipuncture Standard precautions

Treat all blood & body fluids as if they were infectious.Always wear gloves during vascular access procedures.

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Butterfly needle - Butterfly needle collections

Butterfly needles (also known as a winged infusion set), are available in smaller gauges, and are used to draw venous blood from children, and adults with difficult veins.

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Finger stick - Specimen collection continued

Collect blood into an appropriate tube.Label specimens appropriately.Make sure bleeding has stopped. Apply an adhesive bandage if necessary.Discard sharps appropriately.

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Heelstick - Neonatal Blood collection

Microlances (such as the Tenderfoot™ (ITC) or the QuikHeel™ (BD), shown here, are used to puncture the heel & collect capillary blood.These devices control the depth of incision, since going too deep into an infant's heel could injure the heel bone, and cause osteomyelitis (bone infection).

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Butterfly needle - Butterfly needles with built-in safety features

You will be using butterfly needles with built-in safety device. The safety device must be activated upon completion of the blood collection.You will be using butterfly needles with built-in safety device. The safety device must be activated upon completion of the blood collection.The Angel Wing™ (Monoject) safety butterfly is shown here.

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Finger stick - Finger stick collections

A finger-stick collection is performed by piercing the fingertip with a safety Lancet, which controls the depth of incision, and collecting capillary blood. The BD Microtainer™ Brand Safety Flow Lancet is shown here.Finger-sticks should not be performed on children under one year of age.

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Finger stick - puncture

Select a safety lancet appropriate for the size of the patient's finger.You may warm the finger prior to puncture to increase blood flow.Make the puncture perpendicular, rather than parallel, to the finger print.

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Finger stick - Wipe away the first drop

Wipe away the first drop of blood using gauze to remove tissue fluid contamination.

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Syringe - Syringe blood collections

Syringes may be used to collect blood from patients having small or delicate veins that might be collapsed by the vacuum of the evacuated tube system.Syringes may also be used to collect blood culture specimens.

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Syringe - Syringe blood collections continued

Syringes may be used in two ways:Syringes may be used in two ways:A syringe may be attached to a butterfly or winged infusion set.

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Syringe - Transferring blood to collection tubes

After collecting the blood specimen into a syringe, properly activate the appropriate safety device, and dispose of the needle in a sharps container.Attach the syringe to a blood transfer device by twisting the needle tip into the hub of the device.Push a vacuum blood collection tube into the holder of the transfer device, and let the tube fill to the appropriate level.

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Syringe - Transferring blood to collection tubes contd

It is important to transfer the blood to appropriate tubes immediately because a syringe contains no anticoagulant, and the transfer must be complete before blood starts to clot.Do not push the plunger while transferring blood into a collection tube. This may cause hemolysis, ruining the specimen.

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Heelstick - Pediatric collection procedures: Introduction

Veins of small children and infants are too small for venipuncture;Safety Lancets are used to puncture the skin and collect capillary blood.Butterfly needles may be used to collect venous blood in older children.

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Heelstick - Site selection and preparation

Firmly grasp the infants foot. Do not use a tourniquet. The heel may be warmed with a cloth to help increase blood flow. Wipe the collection site with an alcohol prep pad, and allow the alcohol to dry. Wipe the site with sterile cotton or gauze, to be sure all the alcohol has been removed.

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Heelstick - Puncture

Puncture the left or right side (outskirt) of the heel, not the bottom of the foot.Wipe away the first drop of blood since it may contain excess tissue fluid or alcohol which could alter test results.

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Heelstick - specimen collection

Collect the blood into the appropriate tube.Do not: Squeeze the infant's foot too tightly and wipe with alcohol during the collection.These actions could result in hemolysis (breakdown of the red blood cells), invalidating the test results.

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Introduction continued

Prolonged bleeding time may indicate:Reduced numbers of platelets.Poorly functioning platelets, or:Medications such as aspirin, which inhibit platelet function, have been recently taken. Abnormal blood vessels may also prolong bleeding time.

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Wick the blood

After 30 seconds, wick the flow of blood using a Whatman #1 filter paper disk.Wick the blood every 30 seconds until bleeding stops. Bring the filter paper close to the incision, but do not touch the incision with the filter paper.

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Apply blood pressure cuff

Apply a blood pressure cuff on the patient's upper arm, and inflate it to 40mm Hg.Blood pressure cuff must be maintained at 40 mm Hg for the duration of the test.

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Additional tips

Contaminated blood cultures may have very serious consequences in terms of patient care.Always draw blood cultures prior to drawing other blood tubes to minimize the risk of contamination. Do not draw blood cultures from a central line, unless cultures are being drawn to determine whether or not the line is contaminated.

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Introduction

Blood is normally sterile. Any bacterial growth in the bloodstream is abnormal, and is an important cause of fever.Blood culture means the incubation of blood in appropriate media to allow growth and identification of bacteria or other organisms that may be present in a patient's bloodstream. Blood cultures are performed on febrile patients to identify and treat bloodborne organisms with the most appropriate antibiotic.

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Collection methods

Blood for culture can be collected in several ways:Standard needle attached to a syringe.Butterfly needle attached to a syringe.Blood culture bottle attached directly to tube holder (not generally recommended).Follow you own facilities' procedure for blood culture collection.

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Avoid skin contamination

Normal skin is not sterile – it contains numerous bacteria.These normal skin bacteria can contaminate a blood culture, causing a false-positive blood culture result.Thorough decontamination of the skin puncture site is therefore essential prior to obtaining the blood culture specimen.

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Equipment

These items are needed to obtain a blood culture specimen :Gloves (sterile if available)Alcohol pads and sterile gauze padsTourniquet and iodine swabsBlood culture bottlesSyringes, needles, and/or evacuated tube system.

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Clean the bottle tops

Clean blood culture bottles while the iodine on the venipuncture site is drying. Wipe the tops of the blood culture bottles, first with a new iodine swab, then with a clean alcohol pad.

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Volume is important

Collect the volume of blood recommended by the manufacturer of the blood culture bottles It is important to collect this full volume if possible. Short draws will make the blood culture less likely to grow.

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Activate needle safety device

After collecting the blood, activate the needle safety device according to manufacturer's instructions, and place it in a sharps disposal container. If blood was collected into a syringe, insert the syringe tip into the hub of a blood transfer device, and rotate the syringe clockwise to secure it to the device. Push the blood culture bottle into the holder of the transfer device, and draw the appropriate volume of blood into the blood culture bottles.

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Additional tips continued

Good sterilization is the key to avoiding contaminates:Let the iodine dry before drawing the blood.Be sure to wipe your gloved finger with iodine if palpation is necessary after cleaning. Always remove iodine from blood culture bottle with alcohol to prevent iodine from "sterilizing" the culture, and causing a false negative result.

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Concept of Hollister and similar systems

The card has adhesive labels:for blood products,for the blood specimen, anda detachable armband stub,all with identical transfusion numbers.

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Hollister system: specimen collection and labeling

Positively identify the patient in the usual manner.Collect a venous blood specimen in a red top tube.Complete the specimen label and the detachable armband stub before removing them from the card.Initial, date, and time the stamped specimen label (shown on upper right), and attach it securely to the blood specimen.

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Hollister and similar systems

The Ident-A-Blood (™Hollister) or other similar systems (shown here) help assure that each patient gets the correct blood products. These systems consists of a card with matching numbered labels, and an armband.

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Arms to avoid

In general, do not collect blood from:Arms on the same side as a previous mastectomy.Arms with phlebitis or infection.Arms with a vascular shunt.

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Hematosis

A hematoma is a blood clot which forms within the body. It is caused by leakage of blood into the tissues from an injured vein . It will resolve spontaneously.Hematomas are caused by excessive needle trauma to a vein, for example, by a needle which passed entirely through a vein and came out the other side.Apply compression to help stabilize a hematoma.

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Blood should not be drawn from arms with IVs

Blood drawn from veins with intravenous lines (IVs) may be diluted by the IV fluids. Arms containing IVs should therefore not be used to draw blood specimens. If an arm with an IV line in place must be used for venipuncture, be sure to choose a site below the location of the IV, so that the specimen will not be diluted with IV fluids.

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Patients Refusing Blood Work

If someone hesitates to let you collect a blood specimen, explain to them that their blood test results are important to their care. However, patients have a right to refuse blood tests. If the patient still refuses, report this to the nurse or physician, and document patient refusal according to your hospital's policies and procedures.

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What if no blood flows when the needle is in place?

The needle may not be in a vein. Try slightly manipulating the needle. If no blood flows, withdraw the needle and repeat the venipuncture. Never probe the patient's arm with the needle. The bevel of the needle may be compressed against the inside of the vein wall. Slightly manipulating the needle should result in blood flow. The needle may have passed entirely through the vein. Pull it back slightly, and blood should flow.

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Insufficient volume

Insufficient blood volume (short draws) within a collection tube containing anticoagulant will result in an incorrect ratio of blood to anticoagulant, and yield incorrect test results.Short draws can be caused by: A vein collapsing during phlebotomy.The needle coming out of the vein before the collection tube is full.Loss of collection tube vacuum before the tube is full. (Always keep extra tubes on hand.)

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Causes of hemolysis

Hemolysis can be caused by: Shaking the tube too hard.Using a needle that is too small.Pulling back too hard on a syringe plunger.Pushing on a syringe plunger too hard when expelling blood into a collection device.

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Causes

The most common causes of unsatisfactory specimens are: HemolysisClottingInsufficient Blood ("short draws")Labeling Errors Each of these will be discussed in turn.

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Clots

Blood clots when the coagulation factor proteins within the plasma are activated.Blood starts to clot almost immediately after it is drawn unless it is exposed to an anticoagulant.Clots within the blood specimen, even if not visible to the naked eye, will yield inaccurate results.

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Causes of clotting

Clotting can be caused by: Inadequate mixing of blood and anticoagulant within the collection tube.Delay in expelling blood within a syringe (which contains no anticoagulant), into a collection tube with anticoagulant.

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Partial collection tubes

Filling a light blue-topped tube to its recommended volume is especially critical; if it is filled incompletely, coagulation results will be incorrectly reported as abnormal.If a short draw is anticipated, a "partial collection" tube which contains less anticoagulant and requires less blood may be used.The light blue topped collection tube shown on the left requires reduced blood volume, and is filled only to the line.

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What is a phlebotomist's role in a health care facility?

The phlebotomist collects blood & other specimens which ultimately provide doctors and nurses with laboratory test information critical to patient care.He or she therefore plays a vital role in any health care system.

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What is a phlebotomist's role in health care facility? [continued]

Phlebotomists work in a variety of settings including: Hospitals Physician Offices Nursing Homes Home Health Care Clinics, and Military facilities. A well trained phlebotomist will therefore have a variety of job opportunities available.Other medical professionals, including nurses, respiratory therapists, and medical assistants may also be trained to collect blood specimens.

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What is phlebotomy?

Phlebotomy, also known as venipuncture, means collecting blood from veins.Phlebotomists, by definition, collect venous blood, but perform a variety of other important medical tasks as well.

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What is a phlebotomist?

A phlebotomist is a medical professional who:Collects blood and other specimens.Prepares specimens for testing. Interacts with patients & health care professionals.

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What is a phlebotomist? [continued]

An experienced phlebotomist should be knowledgeable in the collection of: - Venous blood specimens - Capillary blood specimens - Blood culture specimens - Urine specimens - Throat cultures, and - Medicolegal specimens requiring chain of custody. He or she may also need to know how to: - Process specimens - Perform point-of-care tests, and - Collection specimens from IV lines and central venous lines, under appropriate supervision.

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Work-flow cycle: patient ID to specimen processing

Phlebotomist positively identifies patient. Phlebotomist draws and labels blood specimen. Specimen is transported to laboratory. Specimen is accessioned and processed in lab.

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Standard precautions continued

Potentially infectious body fluids include: Blood, Semen, Vaginal Secretion, Peritoneal, pericardial and pleural fluids, and Saliva Sweat and tears are not generally considered infectious. It is important to remember that bloodborne pathogens are not transmitted by casual contact, like a handshake.

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Personal protective equipment

An impermeable lab coat should be worn to protect clothing from blood & other body fluids. Gloves must be worn while drawing blood and during all other patient contact. Appropriate face masks must be worn during contact with patients in certain types of isolation. A sign posted on the patients door will indicate special protective equipment that may be required prior to entering a patient room.

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Needlestick safety and prevention act continued

The law requires that each institution gets input from employees actually involved in blood collection. So the actual safety devices you are required to use will vary depending on where you work.

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Cardiovascular system : structure & function

The cardiovascular system consists of the Heart, and Blood Vessels. Its main function is circulate oxygenated blood from the lungs to various organs, and return blood depleted of oxygen to the lungs, where it is reoxygenated. Illustration this screen from LifeArt Collection 2000, with permission. © Lippincott Williams & Wilkins.

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Circulation: venous portion

Deoxygenated (venous) blood flows from tiny capillary blood vessels within the tissues via progressively larger veins to the right side of the heart.Blood is routinely drawn from veins, but may also be drawn from arteries, or capillaries. Illustration this screen from LifeArt Collection 2000, with permission. © Lippincott Williams & Wilkins.

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Circulation: arterial portion

Blood is then pumped from the right side of the heart to the lungs, where it takes up oxygen. Oxygenated blood is then pumped through the left side of the heart via arteries to tiny blood vessels called capillaries.Illustration this screen from LifeArt Collection 2000, with permission. © Lippincott Williams & Wilkins.

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Circulation: capillary portion

In the capillaries, oxygen and nutrients diffuse from the blood cells into the tissues. The deoxygenated blood then returns to the veins, completing the circulatory pathway. Illustration this screen from LifeArt Collection 2000, with permission. © Lippincott Williams & Wilkins.

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White blood cells

Leukocytes, or white blood cells, help the body fight infections. Leukocytes are shown in the photomicrograph of the stained blood smear to the right.

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Blood clots

When a blood sample is left standing without anticoagulant, it forms a coagulum or blood clot. The clot contains coagulation proteins, platelets, and entrapped red and white blood cells.

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Plasma

Plasma and formed elements stay mixed in circulating blood. When centrifuged (or spun down), blood is separated into plasma, and formed elements including red blood cells. The plasma separator tube shown here has a barrier to maintain separation of plasma and cellular elements during centrifugation and storage. The red cell layer also includes a relatively small amount of platelets and white blood cells, not visible in the photo on the right.

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Plasma components

Plasma is the liquid portion of the blood. It contains many substances including:Water Electrolytes Sugars Proteins Lipids Drugs & Toxins

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Plasma water

Water (H20) makes up the majority of the blood plasma.

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Plasma water continued

Water is the largest component of plasma, and makes up about 53% of whole blood.

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Plasma sugars

Sugars are also dissolved in the plasma. By far the most important is glucose. Blood glucose is increased in diabetes mellitus, and decreased in hypoglycemia.

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Plasma proteins

Numerous types of proteins are dispersed in the plasma. These include: Coagulation proteins (blood clotting factors), which, if activated, will form a blood clot , and Serum proteins, which are left dispersed in liquid after the clot is formed. Serum proteins include: Albumin, a marker of nutrition, and Globulins, or antibodies.

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Whole blood: components

Circulating whole blood is a mixture of: Plasma (which contains fluid, proteins, and lipids), and Formed elements, consisting of red cells, white cells, and platelets.

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Serum

Serum is the fluid that is left over the coagulum after the specimen is centrifuged (spun down). Serum contains all the same substances as plasma, except for the coagulation proteins, which are left behind in the blood clot.

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Platelets

Platelets are small cell fragments present in large numbers in blood.They work together with the blood coagulation proteins to form a blood clot.

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Whole blood formed elements

Formed elements are the cells suspended in the blood. They include: Red blood cellsWhite blood cells Platelets

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Red blood cells

Red blood cells contain hemoglobin, which carries oxygen from the lungs to the tissues of the body. Hemoglobin gives blood its red color. Red blood cells are shown in the photomicrograph of a stained blood smear to the right.

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Plasma lipids

Lipids are fats dispersed in plasma. They include: Triglycerides Cholesterol Lipoproteins The amount and ratios of various lipids in the blood will determine a person's risk of getting coronary artery disease.

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Anatomy & physiology: essential to phlebotomy

Since phlebotomy involves puncture of the skin (integumentary system) and veins, (A component of the cardiovascular system), a basic knowledge of the anatomy and physiology of these systems is essential. Knowledge of blood and its components is also important.

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Collection tubes

Blood may be collected into either:Red top (clot) tubes.Speckle top tubes (serum separator tube).Gray top tubes specifically designed to preserve glucose levels. Gray top tubes contain additives such as sodium fluoride or potassium oxalate, which prevent metabolism of glucose by blood cells.

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Administration of glucose

Collect venous blood for a fasting glucose level.Give the patient a standard dose of glucose, usually in the form of a beverage such as Glucola™ (Allegiance). Always follow your own procedure manual. In general:Give a 50 gram glucose dose to screen pregnant women at 28 weeks for gestational diabetes.Give a 75 gram glucose dose to nonpregnant adults.Give a 100 gram glucose dose to confirm the diagnosis of gestational diabetes.

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One hour screening test for gestational diabetes

About 2-3% of women will develop gestational diabetes.Since women with gestational diabetes have a higher risk of losing their baby or having a baby with malformations, diagnosis and treatment of gestational diabetes is important.Pregnant women are screened for gestational diabetes at 28 weeks using a modified glucose tolerance test.Patients are given a 50 gm dose of Glucola, and blood is collected for glucose testing one hour later.If the glucose level is greater than 140 mg/dl, a 3 hour glucose tolerance test is required to confirm the diagnosis of gestational diabetes.

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Introduction

Glucose tolerance test is used to help diagnose diabetes mellitus, or gestational diabetes (diabetes occurring during pregnancy).Patients are given a standard oral dose of glucose, after which their blood is collected at standard time intervals. Blood samples are then checked for glucose levels. Abnormal glucose levels may indicate diabetes mellitus, or gestational diabetes mellitus.

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Specimen collection

To screen for gestational diabetes, collect blood after one hour.For a standard glucose tolerance test collect blood and urine at 30 minute intervals, for two hours.To confirm gestational diabetes, collect blood every hour for 3 hours.

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Introduction

Physicians need to know the blood concentration of certain drugs in order to select the best dose for their patients.As a phlebotomist, you might be asked to draw peak (highest), and trough (lowest) levels of various therapeutic drugs.

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Collection kits

Sealed collection kits are opened in the presence of the donor individual.The kit contains detailed directions and materials for urine and blood collection. Use only the materials supplied in the kit.You may have to appear in court later to testify as to how you collected the specimens, and to verify their origin, so follow directions carefully.

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Blood

Collect the blood specimen next, if required.Be sure to use the iodine swab provided in the collection kit to disinfect the venipuncture site.Do not use an alcohol swab, as this might lead to suspicion of a falsely elevated blood alcohol result.

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Blood bank specimens

Labeling of blood bank specimens is even more critical than labeling of other specimen types.If a patient gets the wrong unit of blood, a serious or even fatal transfusion reaction may occur.

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Preliminary Identification of the Primary Select Agents of Bioterrorism
Bacillus anthracis

Clinical specimens where organism may be encountered: CSF Blood Stool (rare) Vesicle fluid, skin swab, or biopsy Gram stain morphology from clinical specimens: Large, gram-positive rods with square or concave ends in short chains Spores are usually NOT present Capsule may be viewed in smears from infected tissue, but this is NOT reliable Gram stain morphology from culture material: Large, gram-positive rods with square or concave ends, often in long chains (more than 2-4 cells) Cells easily decolorize as the culture ages Does NOT form capsules in culture Central to sub-terminal, oval spores, with NO significant swelling of the cell It must be noted that spore production increases with the age of the culture. Do NOT keep these cultures in the laboratory for longer than 24 hours for this reason!

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Yersinia pestis

Clinical specimens where organism may be encountered: Blood Lymph node aspirate Respiratory secretionsGram stain morphology: Gram-negative rod Resembles other Enterobacteriaceae Can form short chains Gram stains performed from blood culture or other liquid media may show bipolar staining (displayed by the arrows)Note: Use of Wright-Giemsa staining on direct specimen may enhance demonstration of characteristic bipolar staining, also referred to as "safety-pin" morphology. Use of this staining is of limited value, as the method is not very sensitive or specific.

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Francisella tularensis

Clinical specimens where organism may be encountered: Blood Biopsy, skin scraping, or swab Lymph node aspirate Respiratory secretions - oropharyngeal aspirate, sputum, or bronchial washingsGram stain morphology: Very tiny, gram-negative coccobacillus Pale or weak staining Due to the small size, often difficult to see individual cells

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Brucella species

Clinical specimens where organism may be encountered: Blood Bone marrow TissueGram stain morphology: Very small, gram-negative coccobacilli Stains very faintly and tends to retain crystal violet, especially in blood cultures May initially be identified as gram-positive Organism is larger than F.tularensis Individual cells are evident

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Burkholderia species

Clinical specimens where Burkholderia species may be encountered: Blood Bone marrow Respiratory specimens - sputum, throat, or nasal Wounds UrineGram stain morphology:B. mallei Gram-negative coccobacillus or small rod Arranged in pairs end-to-end, parallel bundles, or Chinese letter formB. pseudomallei Small, straight, or slightly curved gram-negative rod May demonstrate peripheral or bipolar staining as they age (appear like endospores) Smooth forms are arranged in long, parallel bundles Rough forms more irregularly arranged

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Gram stains are performed on positive blood culture bottles. Match the organism that MOST closely resembles the description of the Gram stain morphology provided.View Page
Bacillus anthracis

Culture Characteristics:Growth may be noted as soon as eight hours after inoculation and occurs on most routine media, including sheep blood agar (SBA), chocolate agar (CHOC), and routine blood culture media Does not grow on MacConkey (MAC) agarColony Morphology on SBA at 35°C, 18-24 hours:Flat or slightly raised, gray to white with a "ground glass" appearance Described as "tenacious" or "sticky" like petroleum jelly, shown in the top image B. anthracis is NOT hemolytic, while B. cereus is hemolyticCharacteristic Features:After 18 hours of incubation on SBA at 35°C, the slightly undulate margin may show curling, displaying a so-called "Medusa head" or described as comma-shaped protrusions, shown in the lower image

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Yersinia pestis

Culture Characteristics: Growth at 22-25oC and at 35oC Organism prefers 25oC, so the colonies will grow faster at this temperature Growth occurs on most routine media, including sheep blood agar (SBA), chocolate agar (CHOC), MacConkey (MAC) agar, and routine blood culture media Non-lactose fermenter on MAC agar Growth is slower than other EnterobacteriaceaeColony Morphology on SBA at 35oC: At 24 hours, colonies are pinpoint and translucent with a gray-white color Colonies take on a yellow tint as they age, after 48-72 hours, referred to as a "hammered copper" appearance An irregular or "fried egg" appearance, shown in the lower image, can also be seen at 48-72 hours Characeristic Features: Growth at 22-25oC is a hallmark feature of this organism

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Francisella tularensis

Culture Characteristics: Slow growing and fastidious Growth of visible colonies on agar may require two to five days Growth is stimulated by CO2 Will grow initially on sheep's blood agar (SBA), but growth is poor or absent on subculture Prefers cystein-enriched media such as chocolate (CHOC), Thayer-Martin (TM), buffered charcoal-yeast extract (BCYE), and thioglycollate (THIO) Growth is slow in broth, so blood cultures should be held up to three weeks if F. tularensis is suspectedColony Morphology on SBA at 35oC: Can grow poorly or not at all on SBA Tiny, pin-point, translucent colonies after 18-24 hours Difficult to see individual colonies in growth that is less than 24 hours Gray-white, opaque colonies less than 1 mm after 48 hours No hemolysisColony Morphology on CHOC at 35oC: Pin-point, gray-white, opaque growth after 24 hours 1-2 mm, gray-white, smooth, shiny growth after 48 hours Individual colonies exhibit mature growth at 3 days in CO2 environment and have a greenish appearance

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Brucella species

Culture Characteristics: Slow growth on sheep blood agar (SBA) and chocolate (CHOC) Growth in commercial blood culture systems, but may require extended incubation Enriched atmosphere with CO2may enhance growth of some strains If Brucella is expected or suspected, extend incubation up to seven days Colony Morphology on SBA at 35oC: Visible growth may take 48-72 hours Small, convex, and glistening Non-hemolytic

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Burkholderia species

Culture Characteristics: B. pseudomallei grows on sheep blood agar (SBA), chocolate (CHOC) agar, and MacConkey (MAC) agar B. mallei grows on very slowly on SBA and CHOC, but little or no growth on MAC Colony Morphology on SBA at 35oC: B. mallei: Smooth, gray, translucent colonies at 48 hours as displayed in the top, right image B. pseudomallei: Smooth, creamy, white colonies at 24 hours as displayed in the lower image on the right B. pseudomallei: May become dry and wrinkled as shown in the image below, often with a purplish hue at 48-72 hours Colony Morphology on MAC at 35oC: B. pseudomallei: Pink colonies at 24-48 hours (may be colorless at 48 hours) B.mallei: No growth or light pink colonies at 72 hours B. pseudomallei on CHOC at 72h image courtesy of CDC

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Which of the following organisms display the characteristic "Medusa head" on sheep blood agar (SBA) after 18 hours of incubation at 35°C?View Page
Bacillus anthrasis

Any isolate with the following features should be immediately referred to your LRN reference laboratory: Gram stain shows large, gram-positive rods with sub-terminal or central spores (if present) Gray colonies with a ground glass appearance Non-hemolytic on sheep blood agar (SBA) Tenacious or "sticky" colonies like petroleum jelly Catalase positive Non-motile

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Yersinia pestis

Any isolate with the following features should be immediately referred to your LRN reference laboratory: Gram stain shows fat, gram-negative rods in single or short chains that may demonstrate bipolar staining Faster growth at 25oC Gray-white, translucent colonies on sheep blood agar (SBA) at 24 hours that turn slightly yellow and opaque at 48 hours Irregular colonies that have a "fried egg" and/or "hammered copper" appearance after 48-72 hours Catalase positive Oxidase negative Urea negative Indole negative

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Francisella tularensis

Any isolate with the following features should be immediately referred to your LRN reference laboratory: Gram stain shows tiny, weak staining, gram-negative coccobacilli Gray-white, opaque colonies on sheep blood agar (SBA) and chocolate (CHOC) agar a at 48 hours Slow growth in broth (up to three weeks) Oxidase negative Urea negative

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Burkholderia species

Any isolate with the following features should be immediately referred to your LRN reference laboratory:B. mallei: Gram stain that reveals pale staining straight or slightly curved gram-negative coccobacilli Cells arranged in end-to-end pairs, parallel bundles, or Chinese letter form Smooth, gray, translucent colonies on sheep blood agar (SBA) at 48 hours Catalase positive Oxidase variable Indole negative Non-motileB. pseudomallei: Gram stain shows slender gram-negative rods with bipolar staining Smooth form appears in Gram stain as long parallel bundles Rough form appears in Gram stain in an irregular arrangement Smooth, creamy, white colonies on SBA at 24 hours Dry, wrinkled colonies at 48-72 hours Catalase positive Oxidase positive Indole negative Motility positive

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Location Where Organisms Naturally Occur, Disease Produced, and Mode of Transmission

These organisms can be encountered outside of a bioterrorism event and produce human disease. It's important to be familiar with the geographic areas where these organisms naturally occur and the how disease is transmitted.Bacillus anthracis: Bacillus species inhabit the soil, water, and airborne dust. Anthrax is the disease produced, which is transmitted to humans via direct contact with infected herbivorous animals. This is where the disease is primarily encountered. Anthrax is controlled in animals in the United States, so the disease is rare. In humans, most cases are cutaneous infections found in people that handle animals and animal products, including veterinarians and agricultural workers. Anthrax is consistently present in the animal population of some geographical regions, such as Iran and Pakistan, but only small numbers of animals experience the disease at any given time. Yersinia pestis: Y. pestis is found primarily in rodents, but can also be found in several animal species, such as cats, rabbits, camels, squirrels. Animal to human transmission most commonly occurs via a flea bite, causing the most common form of the disease known as the bubonic plague. Human-to-human transmission occurs by either flea bite or respiratory droplets. This causes an overwhelming disease known as pneumonic plague, which is the most likely form that would be implicated in the event of a bioterrorist attack. Human cases of the plague continue to occur in many countries, including Africa, the southwestern United States, parts of Asia, and the former Soviet Union. Francisella tularensis: Many animals, including rodents, rabbits, deer, and raccoons act as host for this organism. Humans and domesticated animals, such as horses, cattle, cats, and dogs can become infected. The infection is transmitted to domesticated animals by ticks and biting flies. Humans are most commonly infected from the bite of an infected tick or fly. Other means of infection include direct contact with the blood of infected animals when skinning game, eating contaminated meat, drinking contaminated water, or inhaling the organisms produced by aerosols. F. tularensis carries a high risk of laboratory acquired infection and documented cases of infection have occurred. Most cases of tularemia are reported in the southern and south-central United States.

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Match the organism to the disease produced outside a bioterrorism event.View Page

Quality Control
What is a Control?

QC programs require the same sample to be tested every day testing is done. This type of sample is called a control. Controls, which are often purchased from manufacturers, use a human base to ensure the analytes being tested parallel human ranges. Manufacturers pool together many human blood samples to create the large volume needed for a lot number of control.

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Appearance of Controls

Controls must resemble as closely as possible the human samples they emulate.For hematology analyzers, controls need to have the same consistency and color as human blood. Likewise, serum controls need to have similar amounts of chemicals to those found in human serum.

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Linearity Example

Looking at the example to the right, we can see that the instrument on which we are doing a calibration curve is linear up to 1000 mg/100ml of blood for a particular analyte. Accordingly, we can be fairly certain that any results obtain up to 1000 milligrams are accurate. Above 1000 milligrams our curve begins to bend. This means that any results greater than 1000mg may not reflect a true measurement of the analyte being tested. The specimen must be diluted down to the linear range.

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What is an unassayed control?View Page

Reading and Reporting Gram Stained Direct Smears
Cerebrospinal Fluid and Specimens Collected from Other Sterile Sites

Cerebrospinal fluid (CSF) and all specimens collected from sterile sites should have a microscopic examination performed along with culture. Bacteria found in CSF, blood, tissue, and specimens from other sterile sites are always significant.CSF should be cytospun, if possible, to increase the chance of detecting a pathogen. The quantity of organisms seen and the amount and type of host cells, e.g., mononuclear or polymorphonuclear (PMN) white blood cells, is important to report. The presence of PMNs indicates bacterial infection. It is also important to determine and report whether the bacteria are found inside or outside of white blood cells.

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Significance of Specific Findings

When evaluating Gram stains of clinical samples, keep in mind the source of material from which the smear was made. Bacteria found in cerebrospinal fluid (CSF), blood, tissue and specimens from other sterile sites are always significant. Gram stains of body fluids that are normally sterile must be examined carefully. For every one organism per oil immersion field, there are about 105 organisms per mL present in the sample! Examining stained smears of CSF sediment may assist the clinician in establishing a presumptive diagnosis. The Gram stain result and the results of other special stains could also guide in the selection of culture media. If bacteria are observed in a CSF specimen, it is important to determine and report whether the bacteria are inside or outside of white blood cells (intracellular or extracellular). The quantity of organisms seen and the amount and type of host cells are also important to report. Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm.

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Reading Gram Stained Direct Smears
Significance of Specific Findings:

Epithelial cells in large numbers within sputum smears means that the specimen is predominantly oral saliva, rather than true sputum from the lung. Epithelial cells in urine smears indicate that the sample has been contaminated by organisms found on the vulva or distal urethra. Bacteria found near or on epithelial cells are usually normal contaminating bacterial flora.White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells.Red blood cells in a direct smear are not usually significant.Yeast may be present as normal flora in upper respiratory tract or genital tract. They may be significant if they predominate, or if budding yeast forms are seen.Hyphae are more likely to indicate the presence of fungal infection, but this determination requires correlation with clinical findings.Bacteria found in spinal fluid, blood, tissue and specimens from other sterile sites are always significant.Body fluids which are normally sterile must be examined carefully. If only one organism per oil immersion field is identified, then there are about 105 organisms per mL present in the sample! Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm.

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
The erythrocyte at the tip of the arrow in the image to the right is an echinocycte (burr cell).View Page
The peripheral blood picture shown in the image below is most consistent with an artifact of smear preparation.View Page
The RBC inclusions shown in the photograph represent which of the following?View Page
A 5-year-old girl was brought to the emergency department with bloody diarrhea and severe abdominal pain. A complete blood count produced these results:CBC ParameterPatient ResultReference IntervalWBC9.6 x 109/L4.3 - 10.8 x 109/LHemoglobin9.1 g/dL11.5 - 13.5 g/dLHCT28%37 - 48%MCV80 fL86 - 98 fLRDW13.111 - 15Platelets90.1 x 109/L150 - 450 x 109/LThe peripheral blood smear is represented in the image to the right. Which of the following condition(s) could be present in this patient when considering the information above and the cells indicated by the arrows on the peripheral smear?View Page
Sickle cells along with target cells, as shown in this image, confirm a diagnosis of sickle cell disease (HbSS).View Page
Case Study The image on the right is representative of the peripheral blood smear from a five-month-old immigrant from Asia. Her mother was concerned that the child was not eating well. Her spleen was palpable.These blood count results were reported:ParameterPatient ResultReference IntervalRBC5.5 x 1012/L3.1 - 4.5 x 1012/LHgb9.6 g/dL9.5 - 13.5 g/dLHCT30.4%29- 41%MCV55.4 fl74 - 108 flMCH17.5 pg25 - 35 pgMCHC31.6 g/dL30 - 36 g/dLRDW34.9%11 - 15%Reticulocyte10.9%0.5 - 4.0%Knowing that the family is from a region of Thailand where HbE carriers are prevalent, the physician ordered a hemoglobin electrophoresis. The hemoglobin electrophoresis detected HbE. Based on the blood count results and this representative microscopic field, which of the following peripheral blood findings should be reported?View Page
The arrangement of erythrocytes on this peripheral blood smear can be associated with each of the following conditions except:View Page
A blood smear represented by the photograph was submitted for hematologic review. Based on the erythrocyte morphology and the accompanying histogram, which of the following choices is the most likely situation or condition?View Page
Ovalocytes/Elliptocytes

Ovalocytes/elliptocytes are oval or elliptical red blood cells that range in shape from slightly egg-shaped to rod or pencil forms. They have normal central pallor with the hemoglobin concentrated at the ends of the elongated cells. The ends of the cells are blunt and not sharp like sickle cells.A rare ovalocyte/elliptocyte (less than 1%) may be found on almost any peripheral blood smear. However, when they comprise more than 25% of the red blood cells on the blood smear, hereditary elliptocytosis (HE) is probable. In most cases, patients are asymptomatic while having normal red blood cell life spans, although a mild anemia may occur. Resistance to malarial infection may be a beneficial attribute of HE.

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Stomatocytes

Stomatocytes are erythrocytes with a slit-like central pallor, given them the appearance of "coffee beans" or "kissing lips". In three dimensions, the stomatocyte is actually the shape of a bowl, as the cell has lost its biconcave morphology due to a membrane defect. Most cases of stomatocytosis are due to alteration in permeability, leading to an increase in red cell volume. Stomatocytes form at a low blood acidic pH as seen in exposure to cationic detergents, and in patients receiving phenolthiazine or chlorpromazine. Stomatocytosis can be an inherited or acquired condition.In hereditary stomatocytosis, mild anemia and findings of on-going hemolysis may be evident if the condition presents as a clinical problem at all. Individuals who possess the Rh null phenotype have osmotically fragile red cells, which take the form of stomatocytes. Individuals with this phenotype tend to experience varying degrees of chronic hemolytic anemia. Note: Unless 10% or more of the RBC's are stomatocytes, their presence is probably artifactual.

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The complete blood count was obtained from a patient recently admitted to the emergency room. The red blood cell indices obtained revealed an MCV of 115 femtoliters (fL) (normal range 80 - 90 fL). The patient met the criteria for a peripheral blood smear examination. A representative field is shown on the right.Which of the following conditions may be indicated by the results seen on this peripheral blood smear?View Page
Criteria for Peripheral Blood Smear Review

When an Initial analysis of red blood cells (RBCs) from an automated instrument are found to be abnormal, many laboratories will microscopically evaluate the peripheral blood morphology of the RBCs. This important step can help to establish which, if any, abnormalities are present as well as correlate possible disease states or conditions associated with the findings. Most laboratories will employ guidelines for review of the peripheral blood smear for RBC morphology. Though each laboratory will create their own guidelines, the following are a few examples that could trigger a manual, microscopic peripheral blood smear review:Hemoglobin: < 8 or >18 g/dL (<10 or > 21g/dL in a newborn)Hematocrit: <20% or > 60% in adults (<40% or >65% in a newborn)MCHC: <29 g/dLMCV: <69 femtoliters (fl) or >110flFlags generated by the hematology analyzer that indicate possible red cell abnormalities or spurious results In most laboratories, when these findings are noted, they should be followed up with a peripheral blood smear review for RBC morphology.

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Introduction to Red Blood Cell Morphology Reporting

After an automated complete blood count (CBC) analysis has determined that abnormal RBC morphology may be present, a well-made and well-stained peripheral blood smear should be prepared. When a peripheral blood smear is made for the purpose of evaluating RBC morphology, accurate recognition and identification of RBC morphologic abnormalities can be an invaluable aid in the diagnosis of a variety of disorders. It is important to understand that red blood cell morphology report formats tend to vary widely among laboratories. Despite the standardization of many laboratory technologies and test result formats, there are still various protocols in use in the area of red cell morphology reporting. Current methods of reporting and quantifying red cell morphology include descriptive terms such as 'rare,' 'occasional,' 'many,' 'slight,' or 'moderate,' as well as numerical gradings of 1+, 2+, 3+, etc. Regardless of the terminology used, consistency is of greater importance. There must be a defined, semi-quantitative scheme that dictates how many cells with a specific morphologic abnormality qualify as "rare" or "many," and so on. The report format must be clear and useful to the physician. Some morphologic abnormalities are quite specific and diagnostic, but others are ambiguous and of little diagnostic significance.A well-defined, semi-quantitative report format for RBC morphology should be based on clinical significance. Some morphologic abnormalities are significant, even when they occur in very low numbers. These include:SchistocytesSickle cellsAcanthocytesSpherocytesTeardrop cellsPolychromatophilic cells Other morphologic abnormalities are significant only when seen in considerable numbers. These include:MacrocytesMicrocytesOvalocytesBurr cells (echinocytes)Target cellsStomatocytesHypochromic cells A final category includes morphologic abnormalities that do not need to be quantified as it serves no purpose; these findings can be noted as "present." These include RBC agglutinationRouleauxDimorphic or double red cell populationAn example of a standardized reporting format is given on the following page.

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Normal Red Blood Cell (RBC) Morphology

In order to properly understand and identify the abnormal red blood cell morphologies associated with RBC disorders, the laboratorian must first become competent in normal RBC characteristics.Normally, RBCs will display a defined morphology in the peripheral blood. Mature RBCs, under normal circumstances, are round, biconcave disc-shaped, anuclear cells measuring approximately 7-8 microns in diameter with an internal volume of 80-100 fL. The term used to express RBCs of normal size is normocytic. Mature red blood cells, under normal circumstances, will also have an appropriate hemoglobin content (a normal MCH and MCHC), giving them a red-orange appearance on Wright-stained smears. These cells will display a central pallor (lighter area inside of the cell) no larger than 3 microns in diameter. This normal morphology is indicated by the term normochromic. It is paramount for RBCs to contain an adequate amount of hemoglobin for the purpose of transporting oxygen to the tissues and carbon dioxide back to the lungs. An example of a normocytic, normochromic peripheral blood picture is shown on the right.In addition, the RBC membrane plays a key role in allowing the deformability of the cell to take place in order to travel through smaller vessels. Normally functioning RBCs survive for approximately 120 days in the peripheral blood circulation before being removed by the liver or spleen. Under normal circumstances, the body produces enough RBCs each day to account for the senescent (old) cells that are removed. Certain disease states can alter the normal RBC characteristics described above. This course will illustrate and correlate various RBC morphologic changes with the specific disease states or conditions, which they are associated.

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Evaluating Initial Red Blood Cell (RBC) Morphology

Evaluating RBC morphology can occur via automated blood cell counters and through visual, microscopic examination of peripheral blood smears. Typically, after a sample has been analyzed for a complete blood count (CBC), various parameters will be visible to the laboratorian to evaluate the RBCs. These primarily include the RBC count and red blood cell indices. The important RBC indices used to classify the population of RBCs present are:MCV (Mean Corpuscular Volume)MCH (Mean Corpuscular Hemoglobin)MCHC (Mean Corpuscular Hemoglobin Concentration)These parameters will simply evaluate the overall size of the RBC population, along with the hemoglobin concentration of the red blood cells. When abnormalities are noted within the RBC indices, further evaluation may be necessary to confirm RBC morphology.

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A 49-year-old male with pneumonia was treated with high-dose intravenous penicillin. He became jaundiced with yellow sclera. The image on the right is typical of other fields that were observed on his peripheral blood smear.Since penicillin may, in some individuals, cause autoimmune hemolytic anemia, the clinician requested a direct antiglobulin test (DAT) be performed. The DAT was positive, indicating that antibodies to the drug were produced, which then attached to the drug on the surface of the red cells. Hemolysis occured due to the drug-induced antibody attachment, leaving the patient with various abnormal red blood cell morphologies. Which of the following cell types would you report for this patient?View Page
Poikilocytosis Review Table

Cell TypeImageCellular DescriptionAssociated Diseases and ConditionsTeardrop cellRed blood cells (RBCs) are shaped like a teardrop with a projection extending from one end.Myelofibrosis with myeloid metaplasia (MMM)SpherocyteRBCs smaller than normalNo central pallorRound rather than disc-shapedHereditary spherocytosisCertain hemolytic anemiasSevere burnsTarget cellRBCs with characteristic bull's-eye morphology due to hemoglobin distribution.Hemoglobinopathies (e.g., sickle cell disease)Certain thalassemiasIron deficiency anemiaSplenectomySevere liver diseaseSickle cellRBCs contain hemoglobin S.Thorn or crescent-shapedSickle cell anemiaStomatocyteRBCs with thin, elongated area of central pallor (slit-like, or coffee-bean-shaped on peripheral blood smears).Three-dimensionally, RBCs are cup-shaped.Hereditary stomatocytosisAlcohol-related diseaseLiver diseaseRh null phenotypeArtifactSchistocyte (fragmented red cells)RBC blood cell fragments or piecesVary widely in size and shapeSevere burnsHemolytic uremic syndrome (HUS)Microangiopathic hemolytic anemia (MAHA)Disseminated intravascular coagulation (DIC)Thrombotic thrombocytopenic purpura (TTP)Ovalocyte (elliptocyte)RBCs are elongated-oval, cigar, or pencil-shapedHereditary elliptocytosisMegaloblastic anemiaMyelophthisic anemiaCertain thalassemiasSevere iron deficiency Acanthocyte (Spur cell)RBCs demonstrating irregularly-spaced, spiny projections that vary in size and numberNo central pallor.AbetalipoproteinemiaSevere hepatic diseaseMyeloproliferative disordersMAHANeuroacanthocytosissyndromesEchinocyte (Burr cell)RBCs have short and evenly-spaced, rounded projections surrounding the cellCentral pallor presentUremiaHeart diseasePyruvate kinase deficiencyStomach cancersBleeding peptic ulcersBite cellRed cells that appear to have bites taken out of them (Image A)Supravital stain reveals the presence of Heinz bodies--precipitated denatured masses of hemoglobin (Image B) Disorders associated with Heinz body formation:Unstable hemoglobinsChemical poisoningG-6PDHemolytic anemia associated with severe alcoholic liver disease

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A ten-year-old boy came to a physician's attention because of recent jaundice and icteric sclerae after taking the medication Primaquine before a trip to Africa. The immediate laboratory work revealed: Hct 24%(normal 36%-47%), MCV 79.5 fl (normal 78-95fl),RDW 13%(normal 11.5-15.0%). His blood smear findings are reflected in the images to the right. The upper image is a Wright-Giemsa stained smear while the lower is a supravital-stained smear. Which condition should be considered for this patient when analyzing his symptoms, history, and laboratory results?View Page
A teenage boy is visiting his doctor uder the suspicion of a viral illness. He has always been relatively healthy without many illness-related complaints. The doctor decides to order a laboratory workup including a complete blood count. A peripheral blood smear is reviewed. The image on the right is a representative field, with a predominance of the arrowed cells throughout the smear. Which of the following conditions would be most consistent with this patient's history and peripheral blood picture?View Page

Red Cell Morphology
Another Example of Macrocytosis

This peripheral blood smear is from a patient with vitamin B-12 deficiency anemia (pernicious anemia), which results from an inability to absorb the vitamin B-12 needed for DNA synthesis. Since many cells are destroyed in the bone marrow, decreased numbers of red cells are present in the circulating blood, resulting in anemia. However, the red cells that are present are generally macrocytes and are filled with hemoglobin.

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Anisocytosis

Anisocytosis is a general term used to describe variation in size of the red cell population present on a blood smear. Notice that normal, small, and large cells can be seen in this field. Since several populations of cells are present, this abnormality will not be reflected in the mean corpuscular volume (MCV) value, which is the average size of all the red cells that are counted. However, anisocytosis will affect the red blood cell distribution width (RDW), which is a measure of red cell size variation. As the severity of an anemia increases, the amount of significant anisocytosis present may also increase.

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Elliptocytes (Ovalocytes)

Elliptocytes/ovalocytes can vary in appearance from slightly oval to thin pencil-shaped forms. Less than 1% of red cells in normal blood are oval. Many examples of elliptocytes can be seen in this smear from a patient with hereditary elliptocytosis(HE). All cases of HE are associated with weakening of membrane skeleton and defective association of proteins that hold the skeleton together. The function of elliptocytes appear to be unaffected in most cases. Notice that the cells vary in shape from slightly oval to cigar-shaped. The largest percentage of elliptocytes is seen on smears from patients with hereditary elliptocytosis. Since many of these patients have no symptoms, the presence of elliptocytes on the smear may be the only diagnostic feature.

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Conditions Associated with Spherocytes

Examples of conditions in which spherocytes can be seen include hereditary spherocytosis and immune hemolytic anemias (i.e., ABO incompatibility). Spherocytes can also form in conditions where there has been a direct physical or chemical injury to the cells. An example would be a smear from an individual who has suffered severe burns. In hereditary spherocytosis, a condition where spherocytes are numerous, the MCHC value will be at the upper limits of normal, or about 36. The identification of spherocytes on the smear of a patient with hereditary spherocytosis can aid significantly in the diagnosis of the disorder. Artifactual spherocytes can appear when blood is stored for a prolonged period of time.

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Summary

It is important to differentiate in vitro changes, which are secondary to preparing the slide, from in vivo morphology, which is the result of the pathophysiological condition of the patient. Examining erythrocytes in the critical viewing area is extremely important in making this distinction. The determination of the clinical significance of the morphology reported is the responsibility of the physician, who must correlate the blood smear findings with other laboratory parameters and with the clinical picture.

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Acanthocytes, continued

Acanthocytes can also be seen in this slide. Alcoholic cirrhosis is the most common source of acanthocytes seen in blood smears in the laboratory (10-50%). Other sources are lipid disorders and a small number following splenectomy.

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Sickle Cells, continued

Sickle cells can be seen in the peripheral blood of patients who have homozygous sickle cell anemia; however other tests are needed to make the diagnosis. Most sickled cells can revert back to the discoid shape when oxygenated. About 10% of sickled cells are unable to revert back to their original shape after repeated sickling episodes.

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Burr Cells (Echinocytes)

Echinocyte comes from the Greek word meaning "sea urchin," which relates to its shell-like appearance. Echinocytes, more commonly referred to as burr cells, are reversible, meaning that this alteration can be the result of the cell's environment, pH of the medium (including the glass slides on which blood smears are made), the metabolic state of the cell and the use of some chemical substances. Several echinocytes (burr cells) can be seen in the top image; three of them are indicated by the arrows. Notice that the projections are rounded and evenly spaced around the cell and the cells have central pallor. Acanthocytes, by contrast, have irregularly spaced thorn-like projections and little or no central pallor. Although burr cells may be associated with diseases, such as uremia or pyruvate kinase deficiency, crenated cells, that may be confused with true burr cells/echinocytes, are frequent artifacts. Crenated erythrocytes are most commonly caused by excess EDTA (underfilled collection tube), but may also be caused by slow drying, drying in a humid environment, or an alkaline pH from glass slides. When crenation is an artifact, most cells on the slide will exhibit this characteristic. True burr cells are less numerous. Corrective actions include making a new smear or re-collecting the sample, if possible. The bottom image contains crenated cells that were the result of an underfilled EDTA collection tube. These should not be reported.

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Polychromasia

The cells that are indicated by the arrows in this slide are slightly blue-gray and are examples of polychromatophilic red cells. Increased numbers of these cells (averaging 2 or more per oil immersion field) indicate increased red cell output by the bone marrow. Polychromatophilic cells are larger and younger than mature red cells, and may be larger than 9 µm in diameter. Under normal conditions, these young red cells remain in the bone marrow one or two days before release into the bloodstream. However, when the bone marrow is stressed due to blood loss or other conditions, these cells are prematurely released into the blood, resulting in a blood smear with polychromasia. If stained with a supravital stain, they would be identified as reticulocytes.

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Rh negative female with anti-D at delivery: A case study
RhIg Dosage

In North America, a standard dose of RhIg is considered to be 1500 IU (300 µg). Note: 1 µg of anti-D = 5 IU.300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood (15 mL of packed rbc). If gestational age is known to be less than 12 weeks, a 600 IU (120 µg) dose may be sufficient.Depending on the gestation of the fetus, recommended dosages vary from country to country and within countries. Samples of recommendations that may change over time: USA: American Congress of Obstetricians and Gynecologists (1999, reaffirmed 2007): Antenatal RhIg dose of 300 µg (1500 IU) at 28 weeks and another 300 µg after delivery of a D-positive infant. Canada: Society of Obstetricians and Gynaecologists of Canada (2003): Antenatal RhIg dose of 300 µg (1500 IU)at 28 weeks (alternatively, 2 doses of 100–120 µg, one at 28 weeks and one at 34 weeks). After delivery of a D-positive infant, another 300 µg (alternatively, 120 µg IM or IV). UK: Royal College of Obstetricians and Gynaecologists (2002): Antenatal RhIg does of 100 µg (500 IU) at both 28 weeks and 34 weeks of gestation, and another 100 µg after delivery of a D-positive infant. All recommendations require testing to detect larger fetal bleeds, e.g., FMH larger than 30 mL of whole blood (for 300 µg doses) and FMH over 12 mL of RBC for 100 µg doses.

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RhIg prophylaxis is typically given antenatally to Rh negative pregnant females without knowing the Rh of the fetus.View Page
A 300 µg dose of RhIg can suppress immunization to _____ mL of D-positive whole blood.View Page
RhIg 'Failures'

Numerous studies have shown that, if administered correctly, RhIg is effective at preventing D immunization. To work, RhIg must be given in sufficient dose, and it must be given before Rh immunization has begun.Unfortunately, despite RhIg's proven efficacy, some women still make anti-D in the perinatal period. Such 'failures' are mainly (but not totally) due to human error. Examples of how women may still produce anti-D some 40+ years after the implementation of RhIg prophylaxis: Immunization to D occurred before RhIg was administered, e.g., before 28 weeks gestation*; Immunization to D occurred after the administration of RhIg at 28 weeks and before delivery because an antenatal FMH occurred that was too large for residual passive anti-D to give protection; Female was already immunized from a prior pregnancy but anti-D was too weak to be detected in antibody screen tests prior to RhIg administration; RhIg dosage was insufficient to clear a larger fetal bleed at delivery (e.g., FMH screen or quantification was not done or a false negative occurred); Incorrect calculation of RhIg dosage; RhIg administered too late , e.g., well after 72 hours of delivery; Antenatal RhIg not given, e.g., mother had no or limited access to prenatal care, or did not seek it, and a FMH occurred during pregnancy; Failure of physician to carry out prenatal blood testing; RhIg not given due to laboratory clerical or technical error in Rh typing the mother or child; RhIg not given in cases such as abortions, ectopic pregnancies, and trauma (e.g., car accidents). * Because anti-D production before 28 weeks is rare (the order of 0.24% to 0.31%), RhIg's use earlier in pregnancy is not recommended. It is not cost effective and would expose most women to an unneeded blood product.

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Factors Affecting RhIg Reaction Strength

Red cell reaction strengths at delivery from an antenatal RhIg injection at 26–30 weeks (usually 28 weeks) are typically 2+ or less, although stronger reactions are possible depending on the detection method, time since injection, and other factors. Multiple variables can affect the reaction strength of passive anti-D seen post-RhIg injection: Amount of RhIg injected (the greater the number of IU of anti-D administered, the stronger reactions will be); Titers of anti-D in the plasma pool used to manufacture RhIg (occasionally a donor with an exceptionally strong anti-D may be in the pool); Maternal physical size and related blood volume (a larger volume of maternal plasma will dilute RhIg more); Time between RhIg administration and testing (passive antibody will decrease in strength over time); Sensitivity of antibody detection method (e.g., gel-IAT and PEG-IAT may give stronger reactions than LISS-IAT); Volume of FMH (amount of D-positive fetal RBC available in the mother to adsorb anti-D); Route of RhIg administration: Some RhIg products can be administered IM only, whereas others can be given both IM and IV (see later). Peak levels of RhIg are reached faster with IV compared to IM administration (within hours with IV administration compared to days with IM administration). Also, with IV administration, higher levels of IgG anti-D are achieved. Operator variability (technologist techniques vary in removing cell buttons when reading IATs). Because of these variables, many laboratories consider 2+ or less reaction strengths to be consistent with passive anti-D.

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Serologic Tests on Newborn

Based on the results of the mini-panel, the laboratory concluded that only anti-D was present and that it was consistent with administration of RhIg at 28 weeks.Patient A.D. delivered a 5 lb 13 oz female by C. section with serologic test results on cord blood as follows. Well washed cord red cells were used for ABO and Rh(D) typing to remove possible Wharton's jelly.Before proceeding to the next page, evaluate if the infant's ABO and Rh(D) types are valid. You will be asked questions that assess basic knowledge of blood grouping practices and test results for newborns. ABO Forward Group ABO Reverse Group Rh anti-A anti-B A1 cells B cells anti-D* 0 0 NT NT 3+ NT = not tested / * monoclonal IgM anti-D DAT Reagent DAT CC Polyspecific AHG w+ 2+ W+ = microscopic positiveAHG = antihuman globulin serum CC = IgG sensitized cells Note: It is the lab's policy to add IgG sensitized cells to weak antiglobulin test results.

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The newborn's Rh(D) type is invalid because the DAT is positive.View Page
Assessing FMH and RhIg Dosage

The remaining issue in this case is to determine if one vial of RhIG is sufficient or if there has been a FMH >30 mL of whole blood, requiring more than one vial of RhIg (300 µg). Recall that the incidence of FMH greater than 30 mL at delivery is rare and estimated to be about 1 in 400 deliveries (~0.3%). The laboratory used the rosette test to screen for FMH and it was negative. Accordingly, quantitation using the Kleihauer-Betke test or flow cytometry was not needed.RhIg dosageBased on the negative rosette test, the mother was injected with one vial of RhIg (300 µg). She was later discharged along with her healthy infant.

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Newborn's Clinical Status

The newborn showed no clinical evidence of HDFN or early newborn hyperbilirubinemia, with related laboratory tests as follows (laboratory's reference ranges for newborns in brackets): Test USA SI Hemoglobin 16 g/dL (13.5 - 21.0 g/dL) 160 g/L(130.5 - 226.0 g/L)* Hematocrit 52% (43-62%) 0.52 (0.43-0.62) Total bilirubin (cord blood) 2.1 mg/dL (<2.5 mg/dL) 35.9 µmol/L (<43 µmol/L) *Most countries that adopted SI do not use the official SI unit for Hb (mmol/L), but rather use g/L.

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Screening for Fetomaternal Hemorrhage

Fetomaternal hemorrhage (FMH) greater than 30 mL of whole blood occurs in only about 0.3% of cases but must be detected to prevent the mother from producing anti-D. Once the mother has become immunized, it cannot be undone and RhIg is of no use.A typical test protocol is first to screen for a large FMH and then quantitate the bleed if the screen is positive. Some laboratories proceed directly to a test that can quantitate the size of the FMH.Once the size of the FMH is determined, a formula is used to determine how much RhIg is needed. Recall that: A standard vial of RhIg contains 1500 IU (300 µg) of IgG anti-D; 300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood. Several methods are available to detect FMHs that require additional RhIg.Acceptable screening tests for FMH include Rosette method; Commercial fetal bleed screening tests; Gel agglutination fetal cell screening technique.Note: The weak D (microscopic Du) test is not a reliable screening test for FMH. The rosette method will be briefly reviewed.

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Calculating RhIg Dosage

Using the estimated volume of fetal bleed determined by the Kleihauer-Betke test or flow cytometry, the number of vials of RhIg (300 µg) to inject is calculated as follows: Number of vials of 300 µg (1500 IU) RhIg = volume of fetal bleed/30 mLIn the interests of safety some organizations recommend the following to deal with decimal points: If the number to the right of the decimal point is <5, round down and add 1 vial (e.g., 1.4 = 1 +1 = 2 vials) If the number to the right of the decimal point is greater than or equal to 5, round up and add 1 vial (e.g., 1.7 = 2 +1 = 3 vials) Sub-calculations: Volume of fetal bleed: % fetal cells x maternal blood volume Maternal blood volume: 70 mL/kg x weight (kg) (assume 5,000 mL if maternal information is unknown) Note: RhIg dose calculators are available (see Further Reading: "Bringing new rigor to RhIG calculations").

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Crossmatch Implications of RhIg-associated Passive Anti-D

Once again, policies vary from laboratory to laboratory since the issue is not directly addressed by blood safety standards. For example, AABB and other standards require a version of the following: When clinically significant red cell antibodies are detected or the recipient has a history of such antibodies, RBC components shall be prepared for transfusion that lack the corresponding antigen and are serologically crossmatch-compatible, where serologically is taken to be an IAT at 37oC. If no clinically significant antibodies were detected in antibody screen tests and the patient has no record of such antibodies, detection of ABO incompatibility is required, which can be accomplished by immediate spin crossmatch or an electronic crossmatch. The key issues are whether detectable passive anti-D from RhIg or a record of passive anti-D from RhIg should be considered clinically significant for crossmatch purposes. Because standards do not directly address these issues, TS laboratories are left to interpret what is required to meet the standards. Practices may be further complicated because of the transfusion service's laboratory information system (LIS).

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Blood safety standards such as AABB Standards directly specify that an electronic crossmatch cannot be done when an Rh negative female has an anti-D consistent with antenatal RhIg administration.View Page
Rosette Test

The rosette test is a screening test for FMH that detects fetal D+ red cells in maternal Rh negative blood. If the rosette test is positive, follow-up testing is done to quantitate the FMH, e.g, a Kleihauer-Betke acid elution test or flow cytometry.Note: The rosette test cannot be done if the fetus is weak D as false negatives may result. In such cases, a Kleihauer-Betke test or flow cytometry can be done.General description (example only): Incubate a maternal 3-5% red cell suspension with IgG anti-D at 37°C. The anti-D will bind to any infant D+ cells that are present. After washing to remove unbound anti-D, add indicator red cells. Indicator cells are ficin-treated R2R2 cells that will bind to the antibody-coated infant rbc causing agglutination ("rosettes") that can be detected microscopically. A specified number of agglutinates (e.g., 3 or more in 10 fields or 7 or more in 5 fields) is designated a positive and suggests a significant FMH (>30 mL) requiring more RhIg.

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Routine Serologic Tests - Mother

Tests done routinely as part of perinatal testing programs vary from country to country and within countries. Below is one example of serologic tests typically done when pregnant females lack clinically significant antibodies. Other test protocols exist.Mother ABO, Rh, and antibody screen at first prenatal visit; Optional (not mandated by blood safety standards): Test for weak D, if initial Rh typing appears to be D-negative; D-negative females: Tested again (ABO, Rh, and antibody screen) at ~ 28 weeks weeks gestation prior to administration of RhIg (depending on the country) and again at delivery. Note: The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. When available, the mother can be typed for D using molecular methods, but this is usually not done unless she is weak D. The purpose is to determine using molecular methods which D variant the mother has, weak D or partial D, since the latter can produce anti-D. (see Further Reading) Molecular typing is reviewed more fully in Refresher on Hemolytic Disease of the Fetus and Newborn and Its Prevention, a companion course that complements this one.

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For infants born to Rh negative females, a test for weak D is optional when initial D typing shows the newborn to be Rh negative.View Page
Routine Serologic Tests - Newborn Protocols

Protocols for testing newborns vary internationally and within countries.if the mother is D-negative and has no unexpected antibodies, newborns are always tested at delivery.Many labs do not test all newborns if the mother is Rh positive and especially do not test if the mother is a blood group other than group O. If all infants born to Rh positive women were tested, many positive DATs due to ABO incompatibility would be detected that are of no clinical significance. Instead cord blood is retained for a period (e.g., 7 days) should it be needed, for example, if the mother has an unexpected antibody at delivery or if the newborn develops signs of red cell hemolysis.However, some clinical practice guidelines, such as those of the American Academy of Pediatrics specify that testing infants born to group O Rh positive mothers is optional only if there is appropriate surveillance and risk assessment before discharge and provided there is follow-up. (See Further Reading) Not testing becomes an issue if group O women and their infants are discharged within 24 hours as occurs in some locations, since hyperbilirubinemia due to ABO HDFN may develop later. Therefore, some facilities where early discharge occurs require that all infants born to group O Rh positive mothers be tested.Typical protocols: Infants born to Rh negative mothers are tested; Infants born to Rh positive mothers who are group O are often tested, especially if early discharge is common (limiting surveillance); Infants born to Rh positive mothers who are not group O are often not tested and this is acceptable good practice. Cord blood is typically retained for a period should it be needed for testing later.

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Routine Serologic Tests - Newborn

Tests on Newborn ( mandatory if mother is Rh negative) ABO and Rh*; Mandatory: Test for weak D if initial Rh typing appears to be D-negative; DAT**. * ABO typing of the infant does not require a reverse serum group with A1 and B cells since the newborn is not expected to have ant-A or anti-B (unless of maternal origin).* If cord blood is used for ABO and Rh(D) typing, the red cells should be well washed to remove possible Wharton's jelly.** A positive DAT does not indicate that the newborn has clinically significant hemolysis. For example, a positive DAT commonly occurs due to ABO incompatibility, yet infants seldom require treatment. Also, infants born to mothers who received antenatal RhIg sometimes have a positive DAT that does not cause clinically relevant hemolysis.Also note that policies for DAT testing of newborns whose mothers have received antenatal RhIg vary internationally. For example, the British Committee for Standards in Haematology guidelines state that a DAT should not be performed on cord blood routinely since in some cases it may be positive due to antenatal RhIg prophylaxis. A DAT is recommended only if HDFN is suspected because of a low cord blood hemoglobin or the presence of unexpected maternal antibodies.However in North America, DATs are always performed on infants born to Rh negative mothers who are RhIg candidates.

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A group A Rh positive mother is about to deliver her infant. Is it acceptable good practice not to test the newborn (ABO, Rh, DAT)?Answer Y (for yes) and N (for no)View Page
Immunogenicity

Immunogenicity is the ability of an antigen to provoke an immune response in an antigen-negative recipient. Why some antigens are more immunogenic than others is unknown. Not considering antigens in the ABO system, Rh(D) is the most immunogenic red cell antigen, followed by K in the Kell blood group system. Other immunogenic antigens include c and E in the Rh system. In routine blood banking assessments of an antigen's immunogenicity are typically based on the prevalence of the corresponding antibody and do not take into account the frequency of the antigen in the general population. For example, k in the Kell system may be very immunogenic but anti-k is rare since 99.8% of Caucasians are k+ and cannot make anti-k.

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Literature and Online Resources

The following published literature and online resources, while useful, should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.References indicated by * provide a broad overview of HDFN and are highly recommended.LITERATUREAvent ND, Reid ME. The Rh blood group system: a review. Blood. 2000 Jan 15;95 (2):375-87.Bowman J. Thirty-five years of Rh prophylaxis. Transfusion 2003 Dec;43(12):1661-6.* Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematology. 2006;22(4):188–195. (scroll to article).Eder, AF, Manno, C.S. Alloimmune hemolytic disease of the fetus and newborn. In Wintrobe's Clinical Hematology, 11th ed. (Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader BE, (eds). Philadelphia, PA: Lippincott, Williams & Wilkins, 2004.Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol. 2006 Mar-Apr;13(1-2):4-12. Kennedy MS, McNanie J, Waheed A. Detection of anti-D following antepartum injections of Rh immune globulin. Immunohematology 1998;14(4):138-40.Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis.BJOG. 2009 Sep;116 (10): 1307-14. Epub 2009 Jun 17.* Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ. 2005 May 28;330(7502):1255-8. (UK perspective but much valuable information relevant to all)* Murray NA, Roberts IAG. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007 Mar; 92(2): F83–F88. Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G; DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006 Jul 13;355(2):156-64.Ramsey G. Inaccurate doses of Rh immune globulin after Rh-incompatible fetomaternal hemorrhage: survey of laboratory practice.Arch Pathol Lab Med 2009 Mar; 133(3):465-9. Reid ME. The Rh antigen D: a review for clinicians. Blood Bulletin 2008 Apr; 10(1).Sandler SG. Effectiveness of the RhIg dose calculator. Arch Pathol Lab Med 2010 Jul;134(7): 967-8.Shulman IA, Calderon C, Nelson JM, Nakayama R. The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies. Transfusion 1994 Aug;34(8):666-70.Tamul KR. Determining fetal-maternal hemorrhage with flow cytometry. Advance 2000. Posted online June 5, 2000.Westhoff CM, Sloan SR. Molecular genotyping in transfusion medicine. Clin Chem 2008;54(12): 1948-50.ONLINE RESOURCESPaxton A. Bringing new rigor to RhIg calculations. CAP Today May 2008. *Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010.

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Risk Management in the Clinical Laboratory
Federal Regulations for Risk Management

Several federal agencies share responsibilities for oversight of the healthcare industry in the United States. These agencies include: U.S. Department of Health and Human Services Centers for Medicare and Medicaid Services- Responsible for regulating clinical laboratory testing through the Clinical Laboratory Improvement Amendments of 1988 (CLIA). Food and Drug Administration (FDA)- Responsible for protecting public health through regulation of food, drugs, vaccines, blood and blood products, medical devices, and more. U.S. Department of Labor Occupational Safety and Health Administration (OSHA)- Ensures safe working conditions in healthcare as well as other industries. Some of the federal laws/regulations that affect clinical laboratories in the United States and relate either directly or indirectly to risk management include: Clinical Laboratory Improvement Amendments of 1988 (CLIA) Health Insurance Portability and Accountability Act of 1996 (HIPAA) OSHA standards for hazard communication, chemical hygiene, and bloodborne pathogens Safe Medical Devices Act of 1990 (SMDA) The Patient Safety and Quality Improvement Act of 2005

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Preanalytic Phase

A study that was published in 2002 concluded that 68 - 87% of laboratory errors occur in the preanalytic and postanalytic stages of the testing process with the majority occurring in the preanalytic phase.* Steps in the pre-analytic phase occur both inside and outside the laboratory, and are performed by both laboratory and non-laboratory personnel. While the following list is not exhaustive, some of the most common sources of error in the preanalytic phase include:Patient preparation Patient not told to be fasting; improper or no instruction to patient on proper collection of specimen such as clean catch urine. Patient injured during phlebotomy Development of hematoma resulting in no specimen obtained for testing. Requisition errors Patient information missing, illegible, or on wrong patient; wrong tests ordered. Patient identification Patient incorrectly identified. Labeling of specimen Specimen not labeled or incorrectly labeled. Preparation of specimen Specimen centrifuged too long or not long enough; specimen placed in improper preservative.Storage of specimen Specimen not refrigerated or frozen as required or refrigerated when it should be at ambient temperature. Shipment of specimen Shipped at ambient temperature when it should have been shipped frozen; delay in shipment. Accessioning process including preparation for analysis Sorted into wrong batch; incorrect labeling. Order entry Incorrect data entered during manual entry of a test requisition. Specimen sub-optimal Not enough specimen for testing; visible hemolysis. Contamination Inadequate cleansing of venipuncture site resulting in contamination during blood culture collection. *Reference: Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem. 2002;48:691-698. Available at http://www.clinchem.org/cgi/content/full/48/5/691#T2BAccessed June 23, 2010.

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The Fishbone Diagram

One of the tools that can be used when performing a root cause analysis is the cause and effect diagram, popularly referred to as the "fishbone diagram" because of its appearance. Cause and Effect (Fishbone) Diagram ExampleThis type of diagram graphically helps identify and organize known or possible causes for a specific problem or area of concern. In this theoretical example, the identified problem is a "near miss." Two units of RBCs were taken to the Dialysis unit for tranfusion of two different patients. The first unit was hung by one clinical person and started just as another clinical person noticed that the unit that he/she picked up for transfusing another patient had the wrong identifying information. The blood was stopped immediately on the first patient. Some of the benefits of constructing a "fishbone diagram" are that it: Helps determine root causes using a structured approach. Encourages group participation and utilizes group knowledge. Indicates possible variations in a process. Indicates areas where more data should possibly be collected.

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Routine Venipuncture
What is Venipuncture?

Venipuncture is the collection of blood from a vein. The person having the responsibility for the performance of the venipuncture may be a phlebotomist who is a part of the laboratory staff, or he/she may be another health care professional that has been trained to perform this duty. In this course, we will refer to the person performing the venipuncture as the phlebotomist.

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Explore the Possibilities!

The antecubital area of the arm is usually the first choice for routine venipuncture. This area contains the three vessels primarily used by the phlebotomist to obtain venous blood specimens: the median cubital, the cephalic and the basilic veins.Although the veins located in the antecubital area should be considered first for vein selection, there are alternate sites available for venipuncture. These include the top of the hand, the side of the wrist, and the forearm. These sites should only be considered after determining that the veins of the antecubital area cannot be accessed or cannot be used. Vein Location Reason for Choice Placement Direction Median Cubital Mid antecubital fossa Vertical to diagonal Musculature assists in stabilizing vein; very often largest; ease of access Cephalic Thumb side of antecubital fossa Vertical Ease of access; few nerves and tendons in area Basilic Body side of antecubital fossa Vertical to diagonal More difficult to access; proximity of artery, nerves and tendons. Use this vein only as the final alternative.

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Preanalytic Errors

Preanalytical ErrorWhat is it?How does it happen?What is the result?HemolysisRed blood cells (RBCs) break and release contents of cell into plasma.Needle incorrectly positioned in vein; cells forced to squeeze through opening. Needle gauge too small; slow blood return into tube. Vigorous mixing or shaking of tube. Alcohol on skin that has not had sufficient time to dry. Some test results may be falsely elevated. (Potassium is especially affected by hemolysis.) Patient may have to be re-drawn. Clotted specimenClumped or clotted cells in specimen that requires anticoagulated or whole bloodInsufficient mixing of blood with anticoagulant in tube. Delay in mixing tube. Slow filling tube. Inaccurate test results for cell counts and clotting studies. Patient may have to be re-drawn. Tube filled to incorrect volumeToo little or too much blood in tube.Tube removed from needle too quickly. Vacuum in tube has been compromised due to use of tube past the expiration date (Results in a short fill). Manual fill of tube may lead to over-fill. Test results may be unreliable due to dilution errors. Patient may have to be re-drawn.

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Proper Patient Identification

In order to prevent errors that affect specimen quality, the phlebotomist must pay close attention to detail during the entire venipuncture process. All steps of the phlebotomy procedure must be included for every venipuncture. This will help to maintain specimen integrity during the collection, transport, and handling of blood specimensProperly identify the patient every timeThe phlebotomist is responsible for correctly identifying the patient using two unique patient identifiers that include the patient's complete first and last name, medical record or hospital number, and/or date of birth. The patient location or room number, bed tag and chart are not reliable forms of identification and should not be used for patient identification. Every patient must verbalize his/her name to the phlebotomist, if able to do so. It is unacceptable for the phlebotomist to ask the patient to confirm his/her name that was verbalized by the phlebotomist. For example, the phlebotomist should say, "Would you please tell me (or spell) your name and birthdate. " The phlebotomist should NOT say, "Are you Sally Brown, and is your birthdate June 1, 1925?" If this is a hospital inpatient, check the information on the patient's wristband and confirm that the name and hospital number or medical record number matches the patient information on the test order. Never rely on identification attached to a bed, chart or door. NEVER draw a patient whose identity is not established or is in conflict. If there is a discrepancy, the phlebotomist must STOP and seek assistance to have the discrepancy resolved before proceeding with the venipuncture. If this is an outpatient that does not have a wristband, ask the patient (or guardian/caregiver) to state the patient's date of birth. A picture ID, such as a driver's license, can also be used for positive patient identification.

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What is a Hidden Error?

Hidden errors are those that cannot be detected or corrected by the laboratory analyst prior to testing. Most often these errors can be prevented by the phlebotomist following correct venipuncture procedure for every procedure, every time.Hidden errors include hemoconcentration, incorrect order of draw, and (the most serious of all errors) misidentification of patient or specimens. Because these errors often are unknown, the analyst may inadvertently report erroneous patient results which could be harmful to the safety and well-being of the patient. Condition What is it? How does it happen? What is the Result? Hemoconcentration Blood pools at site of venipuncture Tourniquet is applied for a prolonged period of time Test results may be inaccurate because blood components move between blood and tissues Pouring Blood between tubes Mixing contents of two or more tubes Removing top of tube to combine contents of one tube with another Inaccurate test results due to over or under dilution or incorrect anticoagulant Clots form due to lack of mixing Patient may have to be redrawn Incorrect patient identification and incorrect specimen labeling Using the wrong name to label a specimen Failure to positively identify EVERY patient using 2 unique identifiers BEFORE beginning venipuncture Failure to label EVERY specimen in the presence of the patient Failure to concentrate fully on the task Results reported to caregiver for wrong patient Compromises patient care; may be life-threatening

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Correct Fill

Fill blood collection tubes completely (until vacuum is exhausted) to ensure the correct blood to anticoagulant ratio necessary for accurate patient results. Specimens may be rejected by the laboratory if the tube is short-filled or over-filled. To avoid short-filling of tubes, the phlebotomist must ensure that the blood flow stops completely before removing the tube from the needle. When using a winged device (butterfly) to collect blood for coagulation studies (e.g., protime, aPTT), the phlebotomist must draw a light blue top "waste" tube before attaching another light blue top tube for testing. If the air in the tubing of the winged device is not displaced into a waste tube and is drawn into the tube used for testing, the tube used for testing will short-fill. The laboratory may reject the specimen because of invalid blood to anticoagulant ratio.

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Do Not Tamper With the Specimens

A phlebotomist should not uncap a blood tube and pour blood between tubes or combine two partially filled tubes of blood into one. This may lead to over-fill of tubes and more importantly, invalid patient results. Combining two tubes with the same additive into one tube will alter the blood to anticoagulant ratio by doubling the amount of anticoagulant in the tube. When blood is being transferred from a syringe to a tube, the phlebotomist must not apply pressure to the plunger to force blood into the tube. This may cause over-filling of the tube and hemolysis of blood cells. With the aid of a transfer device, the tube will draw the amount of blood required to fill the tube based on the amount of vacuum in the tube.

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Avoid Prolonged Tourniquet Time

A prolonged tourniquet time may lead to blood pooling at the venipuncture site, a condition called hemoconcentration. Hemoconcentration can cause falsely elevated results for glucose, potassium, and protein-based analytes such as cholesterol.Ideally, the tourniquet should be in place no longer than one minute to prevent hemoconcentration. If the phlebotomist takes longer than one minute to assess and locate vein of choice for venipuncture, it is best practice to release the tourniquet, assemble supplies and reapply tourniquet immediately before needle insertion.

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Order of Draw

Blood collection tubes must be filled in a specific order to avoid specimen contamination from the additive in the preceding tube. The following order of draw is an accepted laboratory standard. 1. Tubes or bottles for blood cultures 2. Light-blue top tubes (sodium citrate) 3. Serum tubes (with or without clot activator) 4. Green top tubes (sodium or lithium heparin) 5. Lavender or pink top tubes (Potassium EDTA) 6. Gray (Sodium fluoride and sodium or potassium oxalate)

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Importance of Using the Correct Blood Collection Tube

Specific anticoagulants must be used for each test that requires plasma or whole blood. If the blood is drawn into a tube with the wrong additive, patient results may be adversely affected. For example, the test for lithium usually requires a serum sample. If instead of a serum tube, the phlebotomist used a tube that contained lithium heparin, the lithium result for the patient would be falsely elevated. It is imperative that the phlebotomist use the tube with the correct additive to avoid erroneous patient results.

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Preanalytic and hidden errors can greatly affect a laboratory result.Match the error listed below with the cause from the drop-down box.View Page
Protect Yourself

The safety of both the phlebotomist and patient is of utmost concern at all times. In the unfortunate event of an accidental needlestick or if you get blood or other potentially infectious materials in your eyes, nose, mouth, or on broken skin, immediately flood the exposed area with water and clean any wound with soap and water or a skin disinfectant if available. Report this immediately to your employer and seek immediate medical attention. It is imperative that the phlebotomist follow facility protocol for reporting the incident. This ensures prompt treatment for the injury. The facility procedure must be followed whether the accidental puncture was from a clean or contaminated needle.The single most important element to prevent an accidental needlestick is for the phlebotomist to fully concentrate during every procedure. Keeping your mind on the task at hand contributes to a successful and safe result.

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References

Clinical and Laboratory Standards Institute (CLSI). Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays; Approved Guideline. Fourth ed. CLSI document H21-A4. CLSI. Wayne, PA: 2003.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard. Sixth ed. CLSI document H3-A6. CLSI. Wayne, PA: 2007.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third Edition. CLSI document H18-A4. CLSI. Wayne, PA: 2010.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.The Joint Commission. National Patient Safety Goals. Available at: http://www.jointcommission.org/standards_information/npsgs.aspx. Accessed November 19, 2013.

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Blood Collection Systems and Devices

The phlebotomist has a choice of several blood collection systems. Three that are commonly used are discussed on the following pages. Evacuated Tube SystemThe primary choice for a routine venipuncture that will be performed on an adult or an older child is a blood collection system that consists of a holder (or adapter), a needle that is pointed on both ends, and evacuated blood collection tubes. One end of the needle will pierce the vein and the other end will pierce the stopper of the evacuated tube so that blood will flow into the tube to fill the vacuum. A safety device is required on either the holder or the needle to comply with current standards for needle safety. Two examples of needle holders equipped with safety devices are shown on this page.

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Syringe

The syringe and needle combination should be the last equipment option that is considered; it is not as safe a choice as the self-contained blood collection systems because it involves more manipulation. However, the phlebotomist may choose to use a syringe to prevent vein collapse if the phlebotomist thinks that the vein is too fragile to withstand the pressure exerted by the vacuum as it pulls blood into the collection tube. A transfer device aids in the safe transfer of blood from the syringe into blood collection tubes. During blood transfer, do not manually push plunger as this may cause hemolysis of the specimen.

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Blood Tube Labeling Information

Each tube used for blood collection is labeled by the manufacturer with important information. This information includes: tube volume in milliliters (mL), expiration date, lot number and, if applicable, the type of additive that is in the tube. Tube volume: Each tube contains a vacuum that allows a specific amount of blood to enter the tube. In a tube that contains an anticoagulant, the amount of blood that is drawn into the tube will establish the correct blood to anticoagulant ratio. Tubes not filled to the correct volume (over-filled or under-filled) may cause inaccurate test results. Expiration Date: An expiration date is stamped on all blood collection tubes, as shown in the image on the right. The tube manufacturer determines this date based on its studies of vacuum maintenance and anticoagulant effectiveness. The expiration date should be checked routinely; tubes that are past the expiration date should be discarded.If a blood collection tube is used past its expiration date, the vacuum may not draw the amount of blood needed to fill the tube completely. Short-filled tubes may not be acceptable for testing and the specimen would have to be recollected. If the tube contains an anticoagulant, it may not work effectively (may not prevent the blood from clotting). Lot Number: A lot number listed on the tube identifies a specific group of tubes that were manufactured at the same time. This information is important to know if a problem is identified with several collection tubes. If the defective tubes are all part of the same lot number, the manufacturer should be notified for replacement of the tubes. Additive: Most blood collection tubes contain a type of additive or chemical that, when mixed with the blood, will yield a specimen acceptable for testing. The various types of additives that are contained in blood collection tubes are discussed on the following page.

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Blood Collection Tubes

Most blood collection tubes contain an additive that either accelerates clotting of the blood (clot activator) or prevents the blood from clotting (anticoagulant). A tube that contains a clot activator will produce a serum sample when the blood is separated by centrifugation and a tube that contains an anticoagulant will produce a plasma sample after centrifugation. Some tests require the use of serum, some require plasma, and other tests require anticoagulated whole blood. The table below lists the most commonly used blood collection tubes.Tube cap colorAdditiveFunction of AdditiveCommon laboratory testsLight-blue3.2% Sodium citratePrevents blood from clotting by binding calciumCoagulationRed or gold (mottled or "tiger" top used with some tubes is not shown)Serum tube with or without clot activator or gelClot activator promotes blood clotting with glass or silica particles. Gel separates serum from cells.Chemistry, serology, immunologyGreenSodium or lithium heparin with or without gelPrevents clotting by inhibiting thrombin and thromboplastinStat and routine chemistryLavender or pinkPotassium EDTAPrevents clotting by binding calciumHematology and blood bank GraySodium fluoride, and sodium or potassium oxalateFluoride inhibits glycolysis, and oxalate prevents clotting by precipitating calcium.Glucose (especially when testing will be delayed), blood alcohol, lactic acid

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A blood collection tube that has a light-blue top contains which of these anticoagulants?View Page
Tourniquets, Alcohol, and Gauze

A tourniquet is used by the phlebotomist to assess and determine the location of a suitable vein for venipuncture. Single-use, latex-free tourniquets are preferred but reusable tourniquets are acceptable. However, if the reusable tourniquet becomes contaminated with blood or body fluid, it must be discarded immediately to avoid the spread of harmful contaminants to other patients. Follow the guidelines established by your facility for cleaning reusable tourniquets.Proper application of a tourniquet will partially impede venous blood flow back toward the heart and cause the blood to temporarily pool in the vein so the vein is more prominent and the blood is more easily obtained. The tourniquet is applied three to four inches above the needle insertion point and should remain in place no longer than one minute to prevent hemoconcentration. If the tourniquet is used during preliminary vein selection, it is best to release the tourniquet after assessing the vein and while you are assembling your supplies. Reapply the tourniquet just before starting the venipuncture; it should then be released soon after the needle has been inserted into the vein and the blood flows into the first tube. If collecting multiple tubes, the tourniquet may remain in place until blood enters the last tube.

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Cleansing the Venipuncture Site

The product used most often to cleanse and disinfect the site prior to venipuncture is 70% isopropyl alcohol in towelette form. Alternative cleansing agents available are chlorhexadine gluconate (chloraprep) and povidone-iodine which are used mainly for collection of blood cultures, blood alcohol specimens, or when the patient is sensitive to alcohol.The alcohol should be applied using a circular target motion, as demonstrated in the image. This technique pushes the bacteria away from the inside of the venipuncture site to the outside. The alcohol must be allowed to air dry for approximately one minute prior to venipuncture to properly disinfect site, prevent hemolysis of the specimen, and avoid discomfort for the patient. Gauze should be used when applying pressure to the venipuncture site immediately after the needle is withdrawn. Adequate pressure to stop bleeding is crucial to avoid formation of a hematoma or bruise. Cotton balls should not be used to apply pressure to stop bleeding because the clot formed may be dislodged by residual cotton fibers as the cotton ball is pulled away from the site.Paper tape or a bandage is used to cover the wound after bleeding has stopped to prevent disruption of the clot.

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Unacceptable Sites for Venous Blood Collection

If the antecubital area of the patient's arm is compromised or inaccessible, an alternate site must be chosen for venipuncture such as the top of the hand or the thumb-side of the wrist. However, some sites must be avoided due to the risk of complications and/or unnecessary pain to the patient.

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Performing a Venipuncture on an Arm Containing an Intravenous Line

Blood that is drawn from a vein that has an intravenous (IV) line may be diluted by the IV fluid. This can ultimately affect the accuracy of the blood test results. Therefore, an arm containing an IV should not be used to draw blood specimens if it can be avoided. However, if there is no alternative and an arm with an IV line in place must be used for venipuncture, try to choose a site away from and below the location of the IV. Document that the venipuncture was performed distal to (below) an infusion site. If the only vein available is proximal to (above and near) the IV, these steps should be followed: Ask the patient's caregiver if the IV can be turned off for a short period of time. The IV should be discontinued for at least two minutes before the venipuncture. Apply the tourniquet between the IV site and the area of the venipuncture. Perform the venipuncture. Document that the venipuncture was performed proximal to an IV site and that the IV was discontinued for two minutes prior to specimen collection. Notify the patient's caregiver when the procedure is completed and be certain that she/he restarts the IV.

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When to Use Hand Veins to Obtain Blood

Sometimes the phlebotomist may decide that the antecubital area is not the best site for venipuncture. Reasons for this decision may include: Extensive bruising (hematomas) in the antecubital area Inability to "feel" a vein suitable for puncture Presence of an intravascular line (IV) or vascular access device Physical condition of the patientWhen the veins in the antecubital area cannot be used, the phlebotomist may choose to use a vein on the top of a hand. The veins in the hand are very near the surface and often very small and thin so the procedure must be performed carefully and cautiously. .

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Handle With Care

Equipment: To successfully enter a hand vein, the phlebotomist must choose equipment that will allow needle entry at a very small angle. A winged device with a small gauged needle of 3/4 inch length is most often used to obtain blood from a hand vein. A syringe is usually attached to the end of the tubing of this device. By using a syringe, the phlebotomist can control the amount of pressure on the vein and avoid vein collapse. Evacuated tubes may collapse a vein by exerting too much pressure on the delicate vein. If available, smaller tubes containing less vacuum may be used.Insertion angle: The angle at which the needle is inserted into a hand vein is smaller compared to the angle of needle insertion into veins of the antecubital area. When drawing from a hand, the needle should be inserted into the vein at approximately a 15 degree angle to allow easier access of the surface hand veins. By inserting the needle at this angle, the risk of the needle going "through" the vein and puncturing the bony structures underneath is reduced.

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Where should the tourniquet be placed when assessing a vein in the hand as a venipuncture site?View Page
Specimen Collection Procedure

Following the approved order of draw, connect the first blood collection tube onto the needle by pushing the tube into the holder so that the tube stopper is pierced by the exposed end of the needle. Use the flanges of the holder to stabilize the needle while connecting the tube. After tube is filled completely, remove the tube, again using the flanges of the holder to stabilize the needle. Replace with the next tube and mix the removed tube immediately if it contains an additive. Release the tourniquet when blood enters the final tube. When the last tube is filled, pull it back off the needle before removing the needle from the vein. Remember: Fill tubes in correct order and to correct volume. If you suspect that a tube did not adequately fill, try another tube.

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Julie Smith is a recently certified phlebotomist who has been working at Northwood Hospital for several months. As she approaches room 825, she looks at her collection list to verify this is the correct room for her first collection. Julie enters the room to find a middle-aged man who appears to be sleeping. Julie approaches the patient and says, "Good day Mr. Ready. My name is Julie and I am from the lab. I need to draw blood for some tests ordered by your doctor." The man awakens and Julie again states that she is from the lab and needs to draw blood for some tests ordered by the doctor. The patient tells Julie to go ahead and get it done so he can go back to sleep. Julie then proceeds with the venipuncture.What procedure did Julie not follow prior to performing the venipuncture?View Page
Scenario Conclusion

When the results on Mr. John Ready were called to the nurse, she was very surprised that the result of his CBC was normal. The nurse explained to the laboratory technologist that Mr. John Ready had a known diagnosis of lower GI bleeding. His hemoglobin had been very low for the past 24 hours because of the internal bleeding, and she thought it was very surprising that his hemoglobin had normalized so quickly without having received a blood transfusion. Mr. Ready's doctor decided the patient should be redrawn to ensure a correct result. The nurse further questioned if the phlebotomist could possibly have drawn the wrong patient because earlier that day Mr. Ready had been moved to room 831, and room 825 was presently occupied by a patient named Walter Redding. If Julie had properly identified the patient by asking him to state his name and then checking the name and identification number on the wristband, she would have realized that the patient in 825 was the wrong patient.

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Bobby Jones, a phlebotomist at Community Hospital, enters the room of Mrs. Mary Grayson with a physician's order for blood tests. After greeting Mrs. Grayson, identifying himself, and properly identifying the patient, using two methods of identification, Bobby prepares for the venipuncture.As he approaches the patient's bed, he notices a sign posted above the bed that reads: "Restricted left arm use. Do not use left arm for venipuncture." Bobby prepares to use the patient's right arm and notices an intravenous (IV) line in Mrs. Grayson's right arm positioned in a vein slightly above her wrist on the dorsum (top) of her forearm.Which site should Bobby choose for the venipuncture?View Page
A phlebotomist was collecting a STAT prothrombin time (PT) and complete blood count (CBC) on a patient when blood flow unexpectedly stopped. The lavender top tube being drawn at the time was less than one-third full. The light-blue top tube had already been drawn for the prothrombin time.Before resorting to a second venipuncture, which of the following procedures should be attempted in order to adequately fill the lavender top tube?View Page

Semen Analysis
Round Cells in Semen

Round cells in semen are of two types: immature sperm (germ cells) and white blood cells (WBCs). These cells can be differentiated by examining a stained smear at 1000X magnification. A more precise identification can be achieved by detecting peroxidase activity. The presence of immature germ cells could indicate testicular damage; increased numbers of WBCs may indicate inflammation of the accessory glands.

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Preparing Semen Smears for Evaluation of Sperm Morphology

To examine sperm morphology, prepare at least two smears after the semen has liquified (> 30 minutes). The specimen should be thoroughly mixed before pipetting the aliquot onto the slide and then mixed again before preparing the second slide. If the sample is undiluted, the technique that is used is the same technique used to make a peripheral blood smear or bone marrow smear. This technique is demonstrated on this page. It is important that the sperm be spread evenly on this slide and that the concentration be such that individual sperm can be clearly viewed. Too many sperm per slide makes evaluation difficult. Too few, makes it hard to find enough sperm for an adequate count. The smears are then labeled, air-dried, fixed, and stained. Some of the more commonly used stains include: Papanicolaou stainDiff QuikShorr stain Details of these staining methods are available in the WHO 5th edition.

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Special Topics in Phlebotomy
What To Do if the Patient Feels Faints

Fainting does sometimes occur as a result of venipuncture. A patient may experience a feeling of weakness or light-headedness or in severe cases, the loss of consciousness at any time during the venipuncture procedure. Before the procedureIf a patient is aware that he/she gets light-headed, or has in the past fainted while having blood collected, the patient may alert the phlebotomist. The phlebotomist must then take appropriate measures to safeguard the patient during the procedure. For example, the phlebotomist may instruct the patient to lie down instead of sitting upright during the procedure. This practice may lessen the risk of patient fainting and eliminate the possibility of patient injury due to falling or sliding out of a draw chair. During the procedureIf a patient faints during the venipuncture, immediately abort the procedure by gently removing the tourniquet and needle from the patients arm, apply gauze and pressure to the skin puncture site and call for assistance. If the patient is seated, place the patient's head between his/her knees. A cold compress applied to the back of the neck may help to revive the patient more quickly. The use of an ammonia inhalant (smelling salts) to rouse the patient is considered an unsafe practice. The inhalant may cause irritation and/or anaphylactic shock in some patients. A typical fainting spell is self-limited and usually the patient comes around fairly quickly. However, the phlebotomist should stay with the patient for at least 15-30 minutes to ensure the patient has fully recovered from the fainting episode. After the procedureIf the patient states that he/she feels dizzy after the blood collection is completed, again, as stated above, place the patient's head between his/her knees and apply a cold compress to the back of the neck. The phlebotomist should never direct the patient to an alternate location while the patient is experiencing dizziness. There is a great likelihood that the patient will faint while walking and be injured. It is never advisable for the phlebotomist to allow the patient to leave after the procedure until the patient is safely able to do so. It is important to review your facility's specific procedures and know how to react appropriately if a patient experiences dizziness or faints during a blood collection.

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Hematoma

A hematoma is another name for a bruise. A hematoma or bruise is a collection of blood beneath the skin. Hematomas are the most common adverse reaction to venipuncture. There are many factors that can contribute to the formation of a bruise. Venipuncture techniqueIf the phlebotomist pushes the needle too far into and through the vein, blood leaks out of that opening and into the surrounding tissue. The appearance of a blue or purple discoloration at the venipuncture site indicates the presence of a hematoma. This discoloration at the site may occur immediately or some time after the venipuncture is completed. A bruise may cause slight discomfort for the patient, but the mere sight of a bruise may generate undue anxiety and discontent for some patients. A patient may associate a bruise with a negative venipuncture experience and be hesitant to have blood tests in the future. It is not advisable for the phlebotomist to perform a venipuncture at the site of a recent bruise as this may cause discomfort for the patient and may also affect the quality of the blood sample. Bleeding disorders and anticoagulant medications:A hematoma may also form after a venipuncture, if the patient has a medical condition that impairs clot formation. A patient who is on anticoagulant therapy will experience a delay in clot formation. If the phlebotomist is aware of the condition, he/she can reduce the incidence of bruising by applying pressure to the venipuncture site for a longer than normal period of time. Also, it is best to inform the patient that bruising is likely. Communication is important to relieve patient anxiety if a hematoma appears.

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Clean Up Your Act

During a blood collection, bacteria that is present on the skin surface may adhere to the outside of the needle as it enters into the vein. This can allow bacteria to infect the puncture site. A serious infection of the blood (septicemia) or of the tissue (cellulitis) may result. To avoid an infection, it is imperative that the phlebotomist uses a technique that thoroughly cleanses the skin at the site prior to venipuncture.Once the phlebotomist locates a suitable vein for venipuncture, the site of the vein that will be punctured is cleaned with a pre-packaged wipe saturated with 70% isopropyl alcohol.The site is cleansed using a "target" motion beginning at the center of the site and moving outward in concentric circles applying enough pressure to move surface bacteria away from the puncture point. (This is demonstrated in the image on the right). It is not recommended to use a scrubbing back and forth motion to clean the site since you may drag bacteria from a dirty area back into the clean area. Allow alcohol to air dry for effective disinfection of the site. Never use non-sterile gauze to wipe dry the alcohol as this will contaminate the site.During the remainder of the procedure, the site must NOT be touched by anything that has not been cleaned in an identical manner. The phlebotomist should avoid retouching the site after cleaning. If it is absolutely necessary to re-palpate, the phlebotomist MUST clean the gloved finger in a manner identical to the above procedure. Make certain that no other piece of equipment touches the site. This includes ends of the tourniquet and gauze. If you suspect that your needle has touched the site before entry, dispose of the needle, re-clean the site and repeat the procedure using a new needle. If a patient complains that there is redness or pain at the puncture site, even hours or days after the procedure, immediately refer the patient to his/her physician for evaluation.

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Case Study Two

Stop and Think !An 18-year-old male has come to the outpatient clinic for blood work. He tells you that he has not been feeling well for several days, which is obvious from his skin pallor. He also mentions being weak and fatigued. If you are the phlebotomist, what would you do?Consider how you would handle this or a similar situation before proceeding to read the suggested solution on the following page.

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Case Study Two: Discussion

Case study:An 18-year-old male has come to the outpatient clinic for blood work. He tells you that he has not been feeling well for several days, which is obvious from his skin pallor. He also mentions being weak and fatigued. If you are the phlebotomist, what would you do?Suggested plan of action: It is important to observe and listen to the patient and assess the situation to avoid a potential adverse event. In this case, because the patient is in a weakened condition, it would be best to have him lie down for the venipuncture as a safety precaution.

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Become and Remain a Competent Professional

Phlebotomy is a skill that needs to be perfected. Because phlebotomy is an invasive procedure, it is imperative that you become and remain proficient. Many people are apprehensive when getting their blood drawn. Your professionalism will greatly decrease their fears.Individuals who collect blood specimens should be assessed for competence by a qualified mentor before being allowed to perform procedures on patients and periodically thereafter. It is important to receive feedback from the instructor/mentor so that you are ensured your phlebotomy techniques are appropriate. Any remedial training needed should be provided by qualified instructors in a controlled environment--preferably a classroom and not in the presence of patients. Training and competency assessment should again occur whenever new equipment is introduced. Training and assessment records should be kept in your employee file.Ask for assistance when unsure about a collection. Be professional at all times. You are an important part of the health care team.

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References

Clinical and Laboratory Standards Institute (CLSI). Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard. 6th ed. CLSI document H3-A6. Wayne, PA: CLSI: 2007.Clinical and Laboratory Standards Institute (CLSI). Procedures and Devices for the Collection of Diagnostic Capillary Blood Specimens; Approved Standard. 5th ed. CLSI document H4-A5. Wayne, PA: CLSI: 2004. Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third Edition. CLSI document H18-A3. Wayne, PA: CLSI: 2004.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.

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Sample Integrity

Sample integrity is critical to the safety of clinical laboratory services. If there is a problem with the sample, then test results are meaningless. Each time there is a problem with specimen integrity, patients experience wasted time in addition to anxiety and loss of faith in the expertise of the phlebotomy staff. Patients may also experience harm, if harm is defined as delay in diagnosis, therapy, hospital admission or discharge.Threats to sample integrity include:Collection of a sample from the wrong patientCollection of the wrong blood sample (eg, a blue top tube when a green top is needed)Missed venipuncture (multiple attempts)Multiple venipunctures due to improper or inadequate sample collectedMislabeled and unlabeled samplesImproperly performed venipuncture or skin puncture

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Effective Communication

Effective communication is a key component of successful phlebotomy procedures. It is important to prepare the patient adequately for the blood collection procedure, not just physically, but also mentally. Educating the patient about the process is respectful to the patient and will improve sample integrity. Allow time:For patients to ask questionsTo share information that is important to the sample collection processTo describe post-venipuncture self-care information Use simple vocabulary and not complex medical terms when explaining procedures or answering patients' questions.If an error does occur during the venipuncture procedure and is realized by the phlebotomist, the appropriate actions should be taken. For example, if a blood tube was not collected for a particular test, the phlebotomist should explain the error to the patient and perform a second venipuncture to collect the required tube. Ignoring an error or taking inappropriate actions can put a patient at risk.

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How might patient harm result from each of these problems related to phlebotomy services? Consider your answer and then click on the defined problem to reveal the potentially harmful result(s) of the action or condition.View Page
Screening for Diabetes Mellitus and Gestational Diabetes

Glucose tolerance tests are used to help diagnose diabetes mellitus or gestational diabetes, which occurs during pregnancy. The procedure basically consists of these steps:Confirm that the patient has been fasting.Collect a fasting blood glucose specimen. Have the patient drink the dose of glucose solution required by the procedure.Collect blood at standard timed intervals. Blood, or blood and urine specimens, are then checked for glucose levels. The patient should be instructed to remain in the facility and remain seated between blood collections. The phlebotomist should check on the patient periodically between blood collections, especially during the first hour. For some patients, the glucose solution may cause nausea and vomiting and the test may need to be terminated.

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Standard and Post Prandial Glucose Tolerace Testing

Screening for and diagnosis of diabetes may require a patient to drink a specified dose of glucose in water ( 50 gram, 75 gram, or 100 gram), or eat a prescribed meal, depending on the test that is given. It is critical to the accurate interpretation of the test results that the correct procedure is followed. If you are responsible for administering the glucose dose and/or collecting the blood specimens, make sure that you follow the procedural steps as required by your laboratory's specimen collection manual, including:Verification of proper patient preparation (eg, when patient last had food or drink)Administration of correct glucose doseCollection of blood specimens at the correct time in relation to glucose administration or meal consumption

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Test for Gestational Diabetes

About 2 - 3% of pregnant women will develop gestational diabetes. Since women with gestational diabetes have a higher risk of losing their baby or having a baby with malformations, diagnosis and treatment of gestational diabetes is important.All pregnant women are screened for gestational diabetes at 28 weeks gestation using a modified glucose tolerance test. A fasting blood glucose is collected and then the patient drinks a 50-gram dose of glucose solution. A blood glucose specimen is collected one hour later.If the glucose results of the screen are abnormal, a 3-hour glucose tolerance test may be required. A fasting blood glucose is collected. The patient then drinks a 100-gram dose of glucose solution. Blood specimens are collected at one, two, and three hours after consumption of the glucose beverage. Be sure to label tubes as fasting, one-hour, two-hour, and three-hour. You may also be required to put the exact time of collection on the tube label.

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Case Study Three

Stop and Think ! A pregnant woman who is 28 weeks gestation has come to the blood collection area for a three-hour oral glucose tolerance test to confirm a diagnosis of gestational diabetes. The procedure requires the collection of a fasting blood sample followed by administration of a 100-gram load of glucose, which is administered in an orange-flavored beverage. Blood specimens will then be collected at one, two, and three hours. The woman has finished drinking the beverage, which she had a difficult time finishing, and you instruct her to have a seat in the waiting room until you come get her to have the one-hour post-glucose blood sample collected. After 30 minutes, she comes to tell you that she has just vomited.Consider how you would handle this or a similar situation before proceeding to read the suggested solution on the following page.

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Case Study Three: Discussion

Case study: A pregnant woman who is 28 weeks gestation has come to the blood collection area for a three-hour oral glucose tolerance test to confirm a diagnosis of gestational diabetes. The procedure requires the collection of a fasting blood sample followed by administration of a 100-gram load of glucose, which is administered in an orange-flavored beverage. Blood specimens will then be collected at one, two, and three hours. The woman has finished drinking the beverage, which she had a difficult time finishing, and you instruct her to have a seat in the waiting room until you come get her to have the one-hour post-glucose blood sample collected. After 30 minutes, she comes to tell you that she has just vomited.Suggested plan of action:The best thing to do in this situation is to contact the patient's physician. The test may need to be terminated. It may not be appropriate to continue the test under these circumstances as the glucose test results may not be accurate. The physician would need to make this determination.

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Therapeutic Drug Monitoring

Therapeutic drug monitoring helps to ensure that a dosing regimen is appropriate for a given patient. The blood plasma concentration of the drug is measured to determine the correct dose that will achieve a therapeutic level of the drug without overdosing into a toxic range. When a drug enters the body, it reaches a peak concentration that starts to fall as the drug is eliminated.The amount of time it takes for a drug's concentration in the body to decrease by 50% is called the drug's half-life. The longer a drug's half-life, the slower it is removed from the body. Most drugs are eliminated from the body in one to three days, but some drugs with longer half-lives can still be detected in the body weeks after the initial dose.The figure on the right illustrates a typical concentration pattern for a drug that is given orally (ingested).

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Peaks and Troughs

As the prescribed drug is used or metabolized by the body, the drug level decreases. The lowest level of the drug in the patient's body is called the trough level. The peak for a drug is when the level of the drug in the patient's body is the highest.To assess drug concentrations during the trough phase, blood should be drawn immediately before the next dose.To assess peak levels, the time for drawing depends on the route of administration:Intravenous (IV): 15 - 30 minutes after injection/infusionIntramuscular (IM): 30 minutes - one hour after injectionOral: One hour after drug is taken (assumes a half-life of > two hours)

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Collection and Communication

The laboratory plays an important role in monitoring the level of therapeutic drugs. Communication with clinical personnel is critical. Blood specimens are collected at specific time intervals to determine the trough level and peak levels of the drug. The pharmacist uses these trough and peak values to adjust the dose of the drugs appropriately.It is the responsibility of the phlebotomist to obtain the specimen at the precise time ordered for the specific peak or trough drug level. With some drugs, altering the draw time by even 15 minutes can have an adverse affect on adjusting and administering the next drug dose.Obtain the specimen at the requested time. If the time is missed, ask the clinical staff if the test should still be obtained or if another draw time is desired. If the clinical staff still wants a specimen collected, make a note of the time the drug was administered in relation to when the specimen was collected.Failure to communicate could have an adverse effect on the patient who may be given too little or too much medication based on an erroneous test result.

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To assess drug concentrations during the trough phase, blood should be drawn about one hour after the administration of an oral dose of the drug.View Page
Collection From a Line

An arterial line or vascular access device (VAD) is used to provide easy access to a patient's circulatory system for administration of fluids and medications. Occasionally, blood specimens are drawn from a VAD, but phlebotomists are not usually authorized to collect these specimens. However, phlebotomists may sometimes assist when a clinical person is collecting blood from a VAD. If you are present, be certain that the person performing the collection flushes the line with at least 5 mL of saline and the first 5 mL of blood is discarded before collecting the sample. If this procedure is not followed, the specimen may be contaminated with heparin that was used to flush the line or be diluted. This could cause inaccurate test results.

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Intravenous Line

Blood specimens should not be collected from an arm into which intravenous (IV) fluid is being administered. If at all possible, the phlebotomist should draw blood from the opposite arm or hand. If an IV line is delivering fluid into the patient's vein and the specimen is drawn from that vein, the specimen may be contaminated and diluted by the IV fluid; the blood test results could then be erroneous.If the arm or hand opposite of the arm that contains the IV line is not accessible or cannot be used for another reason, a capillary collection may be an option, if only a small amount of specimen is needed. However, if a venipuncture is necessary and the arm that has the IV line in place is the only option, ask the clinical person in charge of the patient's care to turn off the patient's IV. Ensure that the fluid has stopped flowing through the line, and wait at least two minutes before performing the venipuncture. It is imperative that the phlebotomist witness that the IV has physically been turned off by the health care provider and then turned back on after the draw has been completed. A phlebotomist must not turn the IV on or off.

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Procedure for Using a Winged Blood Collection Device to Collect a Specimen for Coagulation Tests

A light-blue top tube (a blood collection tube containing 3.2% sodium citrate) that will be used for coagulation testing must be filled to completion. Under-filling the tube changes the ratio of blood to anticoagulant. This can affect the accuracy of coagulation tests that are performed using this specimen. If a winged blood collection device (butterfly) is used to collect a light-blue top tube for coagulation studies, a waste tube should be drawn first, if the coagulation tube is the first tube to be collected for patient testing. The waste tube must also be a light-blue top tube or a tube that contains no additives. This waste tube is drawn first to remove the air in the tubing of the winged collection device. Once blood flows through the tubing, the waste tube can be removed and discarded. The waste tube does not need to be completely filled. If the air is not displaced from the tubing into a waste tube, it will be drawn into the tube used for testing and cause a short-fill of the tube. Less volume of blood in the tube alters the required blood to anticoagulant ratio needed for coagulation studies.

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Blood Culture Overview

Blood is normally sterile. Any bacteria in the bloodstream is abnormal. A blood culture is collected to detect the presence of bacteria in the bloodstream. Blood is collected into appropriate media to allow for growth and identification of bacteria or other organisms that may be in the patient's bloodstream. A blood culture set usually consists of two bottles: an aerobic bottle and an anaerobic bottle. Blood cultures are usually ordered in multiple sets drawn from separate sites at different times. An improperly collected blood culture can have a serious impact on the care and treatment of a patient. If bacteria enters the culture vial from sources other than the blood, as a result of improper specimen collection, a patient may needlessly be treated for an infection that is not present. On the other hand, some collection errors may cause negative culture results when the patient actually has bacteria in his/her blood. A false-negative culture result could be a life-threatening error.

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Which of the following blood culture collection techniques could cause a false-positive blood culture result?View Page
Blood Culture Collection

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Pediatric Patients

Collecting a blood specimen from a pediatric patient can be very challenging. There are several factors that contribute to this challenge. For example:The veins of a young child are typically much smaller than those of an adults. Often a child has never had the procedure before. Fear of the unknown can increase anxiety and cause the child to struggle. Unfortunately, some adults use health care professionals as "threats" for children. This can also increase anxiety. A child may have had a previous bad venipuncture experience and is now combative. Professional phlebotomists have empathy for all patients, but often, knowing that they may be hurting a small child, even slightly, can trigger emotions that interfere with a successful procedure.

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Reducing Pain for Pediatric Patients

There are some commercial products available that are designed to alleviate pain from venipuncture.Cream: A topical cream can be applied to numb the venipuncture site. Apply well in advance to be effective. Always refer to manufacturer's instructions before use on patients. Be certain to determine that no allergy exists before using the product on a child.Mechanical device: A mechanical device can be used to stimulate nerves surrounding the venipuncture site to numb the site. This device must be used according to the manufacturer's instructions.Vein Viewer: This device enables the phlebotomist to determine the flow of blood thereby identifying the presence and direction of a vein. This device does not aid during palpation of the vein to determine vein health, diameter or depth.

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Hints For Successful Pediatric Venipuncture

While pediatric phlebotomy can be challenging, these guidelines can contribute to success.Communication: Always be honest with the child. Never lie to a child and say that it won't hurt. If asked by the child if it will hurt, you could explain that it may feel like an insect bite or it may sting, but if he/she holds really still, it will be over very soon.Correct hold of child: Ask the parent or guardian to assist. If you have determined that the child's parent is willing and able to assist throughout the procedure, have the child sit on the parent's lap . The parent can gently "hug" the child in a way to limit the child's movement and stabilize the arm that will be used for venipuncture. Alternately, the child can lie on a bed or exam table. If the parent does not choose to help, ask for assistance from a coworker. Correct hold of the child's arm: A health care professional familiar with the procedure should assist by holding the arm that will be used for the blood collection. The holder should face the child and gently position the child's arm so that the arm is straight and palm facing up. Next, the holder should place one hand underneath the child's elbow grasping lightly yet firmly to stabilize the elbow. With the other hand, the holder should hold the child's hand firmly. This hold will help prevent movement of the arm, even if the child is moving his/her body. This hold also allows the phlebotomist easy access to the venipuncture site during the procedure. Distractions: At times, the phlebotomist may employ a technique to distract the child during the procedure. For example, to help the child keep still, tell the child that the only thing he/she can move is his/her eyelashes. This places the child's focus on moving only their eyelashes and before you know it, the procedure is done!

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Case Study One: Discussion

Case study:You work in a large hospital that specializes in pediatrics. The policy of the facility is to encourage the parent or guardian to remain in the room during venipuncture to comfort the child. You have taken steps to prepare the child for the venipuncture, but the child starts to cry and becomes combative. The mother says that she does not want the test done.Suggested plan of action: Do not proceed if the mother has refused the blood collection for her child. The patient's physician or clinical person in charge of the patient should be contacted and informed of the situation. This may not be something that you would do directly. It may be your facility's policy for you to contact your supervisor.

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Patients with Needle Phobia

The phlebotomist should always carefully observe the patient for clues that indicate the patient's mental and physical readiness for the procedure prior to performing a blood collection. This alertness must continue throughout the blood collection process. When the patient expresses needle phobia or a "fear of needles," it may help to offer strategies to help the patient get through the procedure safely. Sometimes, the anticipation of the needlestick may cause anxiety, and sometimes seeing the blood filling the tubes makes a patient uneasy.It may be helpful to engage the patient in conversation during the venipuncture to keep the patient's mind off the procedure. In some instances, the phlebotomist may seek assistance from a qualified associate to distract the patient with conversation or provide comfort and support by offering to hold the patient's hand. If this is an outpatient, your observations and questioning may lead you to conclude that the best solution is to have the patient lie down during the venipuncture procedure. Remember that the patient does have the right to refuse to have blood drawn and the phlebotomist should respect that patient right.

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The Disappearing Antibody: A Case Study
Case Presentation

Mr. R.M., a 55-year old male, was admitted to a hospital emergency department with severe lower gastrointestinal bleeding. His history revealed multiple prior transfusions, the last of which he received five years earlier. Physical examination revealed hemodynamic instability (systolic BP 60 mmHg). Blood tests revealed a hemoglobin (Hb) of 8 g/dL (80 g/L) and a hematocrit (HCT) of 28% (0.28). The patient received aggressive fluid resuscitation with Ringer's lactate and was sent to the operating room (OR) for an emergency laparotomy.The physician ordered four units of Red Blood Cells to be crossmatched. Two units of uncrossmatched group O Rh-negative Red Blood Cells were also ordered and authorized for immediate emergency transfusion.

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Transfusion Service Laboratory

The transfusion service laboratory (TS) instructed clinical staff to draw blood specimens for compatibility testing before transfusing any blood components or products.Once the blood samples were collected, the clinical staff immediately began transfusing the patient with the O Rh-negative blood.

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Summary

This case study presents a scenario in which a patient had an unexpected antibody that disappeared after he was transfused with 2 units of unmatched group O Rh negative RBC. The patient developed a positive DAT with MFA but an antibody identification using the post-transfusion red cell eluate was inconclusive, making the antibody unidentifiable. Fortunately, the patient improved and further transfusion was not required. Ultimately, the patient's antibody was identified as anti-Jka, with a second antibody to a low frequency antigen (Radin) also unexpectedly present.The case illustrates the risks involved in using unmatched blood.

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Risks of transfusing unmatched RBC

We often "get away" with transfusing unmatched RBC because the incidence of unexpected antibodies in patients experiencing medical emergencies is thought to be relatively low ( ~3-5% is sometimes cited, but with little solid evidence).Antibody incidence may vary according to several factors: Genetic disposition Patient's underlying disease Number of prior transfusions Gender (females may get exposed to foreign antigens via fetomaternal bleeds as well as transfusion) Concordance of antigen phenotypes of patients vs blood donors in a given locale.In general, antibody incidence increases with the number of transfusions that are given, although most antibody producers will respond within the first 3 - 4 transfusions. Antibody incidence in transfusion-dependent patients, such as those with sickle cell anemia or thalassemia, is very high. Regardless of likelihood, transfusing uncrossmatched blood to a patient with unexpected antibodies can result in a serious hemolytic transfusion reaction.

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Balancing the risks

Life-Threatening HemorrhageDespite potential risk, sometimes immediate transfusion is necessary, even for patients with red cell antibodies. In such cases transfusion service staff should alert the medical director, who can discuss options with clinical staff.The medical director will generally talk to the staff attending the patient and indicate that, if possible, they should hold off transfusion. But if it is a case of massive bleeding where exsanguinating hemorrhage is likely, it is better to give some blood and monitor for a delayed hemolytic transfusion reaction than to let the patient bleed to death.Transfusing when bleeding is brisk will result in much of the autologous and incompatible blood bleeding out, with the possibility of a delayed hemolytic reaction once the patient's antibody rebounds and destroys still present antigen-positive donor red cells.Some transfusion services also try to minimize the risk of unmatched blood by typing their emergency supply of O Rh negative RBCs for the K antigen, since anti-K is a relatively common clinically significant antibody. See Resources for two papers that discuss the risks of transfusing un-crossmatched emergency blood.

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Literature and online resources

LiteratureDutton RP, Shih D, Edelman BB, Hess J, Scalea TM. [abstract]. Available at: Safety of uncrossmatched type-O red cells for resuscitation from hemorrhagic shock.J Trauma. 2005 Dec;59(6):1445-9. Accessed November 5, 2012.Johnson ST, Rudmann SV,Wilson, SM. Serologic problem solving strategies:a systematic approach. Bethesda, MD: AABB, 1996.Online resourcesThe following are online examples of good practice. The information should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.Transfusion reactions: Transfusion complications (Canadian Blood Services)Education website for CBS's hospital customersREACT (Sunnybrook HSC, Toronto, ON, Canada) Pocket reference card for nurseson signs and symptoms of transfusion reactionsQuick cals (online calculator of p values for Fisher's exact test) Use a one-tailed test (since we would expect an antibody to react with red cells that are positive for the corresponding antigen)

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ABO, Rh, and Antibody Screen

These ABO, Rh, and antibody screen results were obtained by the TS using the blood specimen that was collected prior to starting the emergency transfusion with O Rh-negative RBCs. ABO and Rh typingABO Forward GroupABO Reverse GroupRh anti-Aanti-BA1 cellsB cellsanti-D004+4+3+Antibody screen Cells Gel IAT*Screen Cell I 3+Screen Cell II 2+Screen Cell III 2+* IAT = indirect antiglobulin test

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The antibody screen is positive but the transfusion of the O Rh-negative RBCs is already in progress. What are the transfusion service (TS) laboratory's priorities in this case?Place the following procedures that will be followed by the TS in the appropriate order of priority.View Page
Pretransfusion Direct Antiglobulin Test (DAT) Result

The laboratory obtained post-transfusion blood specimens in order to perform a serological investigation. Pretransfusion and post-transfusion DATs were performed. Patient cellsDATCCPretransfusion02+DAT = direct antiglobulin test with polyspecific antiglobulin serumCC = IgG sensitized RBC

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Which of the following most likely accounts for the patient's post-transfusion plasma giving negative panel results?View Page
Follow-up with clinical staff

The patient's physician was notified that compatible blood was unavailable and that the patient's antibody was still being investigated.When asked whether or not the patient was experiencing a transfusion reaction due to the transfusion of the two unmatched and incompatible O Rh negative RBC, the nurse in the OR stated that the patient was undergoing surgery and completely sedated. A transfusion reaction was not apparent but they would investigate and closely monitor.Hemolytic Transfusion Reactions (HTR)Before proceeding to the next page, make a short list of signs and symptoms associated with immediate hemolytic transfusions reaction and another list associated with delayed hemolytic transfusion reactions.

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Immediate HTR - Signs and symptoms

The following signs and symptoms are associated with acute HTR due to ABO incompatibility but can be associated with other blood group incompatibilities. ABO incompatibility typically results from patient misidentification.The more serious symptoms result from intravascular hemolysis (IVH) caused by antibodies such as anti-A and anti-B that can bind complement to C9.Signs and symptoms typically appear within minutes of the transfusion but can occur anytime during the transfusion. They may include: 1. Burning sensation along the vein being transfused (IVH due to complement activation to C9)*2. Lower back pain in the area of the kidneys (renal failure with subsequent oliguria/anuria) *3. Unexplained bleeding/oozing from a surgical site (fibrinolysis following DIC)*4. Hypotension leading to hypovolemic shock (release of vasoactive substances caused by C3a and C5a)5. Tightness in substernal area of the chest (bronchial constriction due to release of vasoactive substances caused by C3a and C5a fragments)6. Other symptoms: fever, chills, skin flushing, dyspnea, wheezing, anxiety, malaise, nausea, headache. * If untreated, these complications may lead to patient death.

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Signs and symptoms - Job Aids

Some blood safety standards require that a list of common signs and symptoms of suspected adverse reactions be included in both nursing and transfusion service manuals. Several organizations have developed job aids to help clinical staff recognize the signs and symptoms of various suspected transfusion reactions and to suggest appropriate actions (e.g., see REACT in Online Resources).

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Investigating weak antibodies

In this case the patient's antibody has disappeared from the plasma by adsorbing to transfused donor red cells. It is detectable but unidentifiable in the post-transfusion red cell eluate. Several trial and error procedures exist to enhance weak antibodies. Which methods will enhance the reactivity of a given antibody depend on its characteristics. Methods to investigate weak antibodies include: Use a higher plasma to red cell ratio (add more antibody-containing plasma or eluate) Increase incubation time (if consistent with manufacturer instructions, if applicable) Use enzyme-treated panel red cells (enzymes enhance IgG antibodies in Rh and Kidd blood systems but denature some antigens, e.g., Fya, Fyb, S) Try alternative antibody detection methods, e.g., if using LISS routinely, try polyethylene glycol (PEG) or column agglutination methods such as gel, providing they have been validated for use in the TS laboratory.

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Which of the following statements about antigen phenotyping are true? (Select all that apply)View Page

The Influenza A Virus: 2009 H1N1 Subtype
Treatment Options for H1N1 Infection

Most patients who have suspected or confirmed cases of H1N1 infection have a mild, uncomplicated, self-limited illness that may not require antiviral treatment. If infected individuals have a normal immune system, they should be able to recover from the infection with symptomatic treatment only and without antiviral therapy. However, it is the decision of the patient's physician whether to treat or not to treat. The CDC provides this decision tree as a guideline if the illness is mild and uncomplicated:The CDC suggests that patients with suspected or confirmed influenza should be treated if: They are hospitalized as a result of the illness They are at risk for severe disease including these patients: Patients that have certain medical conditions, such as asthma, diabetes, heart disease, or patients with weakened immune systems that may exacerbate the infection. Children younger than 2 years old Adults 65 years or older Pregnant women or women up to 2 weeks post-partum They have a progressive or complicated illness characterized by signs of: lower respiratory tract disease such as hypoxia or abnormal chest x-ray CNS complications such as encephalitis Complications of low blood pressure including shock or organ failure Myocarditis Invasive secondary bacterial infection The treatment options indicated for the 2009 H1N1 infection include oseltamivir (brand name Tamiflu®), an oral tablet, and zanamivir (brand name Relenza®), an inhaled antiviral agent.Reference: Centers for Disease Control and Prevention. Updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the 2009-2010 season. December 7, 2009. Available at: http://www.cdc.gov/h1n1flu/recommendations.htm. Accessed January 18, 2010.

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FDA Surveillance and H1N1 Preparedness

The US Food and Drug Administration's (FDA) worked with the CDC and other health agencies, both in the United States and globally, to protect public health during the H1N1 virus outbreak. The FDA ensures the safety, effectiveness, and supply of antiviral medications and the H1N1 vaccine that is produced and/or distributed in the United States; it has the responsibility of approving medical devices for the serologic testing of the 2009 H1N1 virus. The FDA also performs other roles such as ensuring the production of an adequate supply of respiratory protection and other personal protective equipment. The FDA also monitors the safety of the blood supply, and, although no cases of transmission of H1N1 virus through blood have been reported, the guidelines for donor deferral that have been established by the FDA further ensure that this would not occur.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
Cuboidal Cells

Increased numbers of cuboidal cells are found in renal transplant rejection, acute tubular necrosis (diuretic phase), injuries that interrupt blood flow to the kidney, and acute glomerulonephritis accompanied by tubular damage. Ingestion of various drugs and chemicals may cause significant tubular shedding of these epithelial cells. Cuboidal cells are easily seen in urine in cases of salicylate intoxication.

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Specimen #4 - Adult Male

The results of this specimen are abnormal but the abnormalities correlate with each other. The turbidity can be explained by the presence of bacteria and crystals. The presence of RBCs in the microscopic explains the blood found on the dipstick. The casts, bacteria and WBCs can account for the increased protein. The results may be reported.

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Theoretical and Practical Aspects of Routine H&E Staining
Liver

This is a high power image of a liver biopsy. Notice the nuclear detail within the hepatocytes or liver cells. There are red blood cells sitting within the sinusoids, which are types of blood vessels.

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Origin of Dyes: Natural

Natural dyes are derived from substances found in nature. Some examples of natural dyes are:Indigo: A blue dye obtained from a variety of plants; but mostly from those in the genus indigofera, which are native to the tropics. Cochineal: A red dye prepared from a dried tropical insect that feeds on cacti.Orcein: A purple dye prepared from a certain colorless lichen. Lichen are organisms that grow on rocks, tree branches, or bare ground and are composed of a green alga and a colorless fungus.Brazilin: Stains red and is derived from the bark of a Brazilwood tree which is a species native to Central America.Hematoxylin: Is derived from Haematoxylon campecianum, a forest tree from Central America also know as the logwood tree. Haematoxylon literally means "blood wood" and it refers to the color of the heartwood.

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Transfusion Reactions
Risks Associated with Transfusion

Transfusion of blood components has the potential for both benefit and risk to the patient. Transfusion policies and procedures must be carefully followed to reduce transfusion reactions and prevent transfusion related death or serious injury.Several causes of transfusion-related deaths are summarized in the table below.Medical errors that could result in transfusion reactions include:Patient mis-identification Sample labeling error Wrong blood type issued Transcription error Technical error Storage error

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Categories of Transfusion Reactions

Adverse complications of transfusions can be classified into several categories:Immune-mediated transfusion reactions are those that trigger a response from the patient's immune system. Many transfusion reactions are mediated by the recipient's immune system. These reactions occur as a result of antigen-antibody interactions. Antibodies involved include those with specificity towards antigens on red cells, white cells, or platelets. In general, the immune responses occur in three stages: The immune system detects foreign material (antigen)The immune system processes the antigenThe immune system mounts a response to remove the antigen from the bodyNon-immune mediated hemolytic transfusion reactions are caused by the physical or chemical destruction of transfused RBCs, bacterial contamination, circulatory overload, or citrate toxicity. Acute reactions are those that occur during or within 24 hours after the transfusion.There is usually a rapid onset of symptoms and these reactions may be fatal. Delayed reactions occur weeks or months after the transfusion of blood or blood components.

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In Vivo Red Cell Destruction

Important events that occur in an immune-mediated hemolytic transfusion reaction (HTR) include:Antibody binding to red blood cells Antibodies may be either IgM or IgG class. IgM antibodies activate complement and lead to intravascular hemolysis where free hemoglobin is released into the plasma. IgG antibodies rarely activate complement but they are often involved in effecting phagocytosis. The concentration of the antibody is directly related to the severity of the HTR. Activation of complement The end result of complement activation is red cell lysis. Activation of mononuclear phagocytes and cytokines Sensitized red cells are removed from circulation by mononuclear phagocytes. Macrophages in the spleen and Kupffner cells in the liver are active in this process. Activation of coagulation Antibody-antigen complexes may initiate coagulation and cause disseminated intravascular coagulation (DIC). Shock and Renal Failure Hemolysis can be intravascular or extravascular. In intravascular hemolysis, free hemoglobin, RBC stroma, and intracellular enzymes are released into the blood stream. This results in hemoglobinemia and hemoglobinuria, which can lead to kidney damage. In extravascular hemolysis, there is no release of free hemoglobin. Sensitized red cells are removed from the circulation by the monocytes and macrophages in the reticuloendothelial system.

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Disease Transmission

Even though blood components are tested rigorously for certain infectious diseases, bacterial, viral, parasitic, and prion pathogens continue to evolve. If they are not detected, they can cause harm to the patient and even death. Donors must be screen to determine eligibility. Their blood samples are also tested for hepatitis B and C, human immunodeficiency virus (HIV) 1 and 2, human T-cell lymphotrophic virus (HTLV) I and II, West Nile virus and syphilis. The table to the right describes the screening tests performed on all blood donors in the United States.It is not yet possible to eliminate the risk of infectious disease transmission through transfusions. There are many other organisms that may be transmitted through transfused blood, which are not routinely tested for in the blood supply. These organisms include the Epstein-Barr virus, cytomegalovirus (CMV), bacteria, and parasites such as malaria, Babesia microti, and Trypanosoma cruzi, which is responsible for Chagas disease, and prions such as variant Creutzfeldt-Jakob disease (vCJD).Selection of eligible donors is a critical part of ensuring the safety of the blood supply. Donors with certain lifestyles, medical conditions, travel histories, immigration backgrounds, or specific physical findings are deferred, either for a specific period of time or indefinitely. This minimizes the risk that a transmittable agent will be present in the donors blood. Click here to learn more about donor eligibility criteria from the American Red Cross.Click here to learn more about Babesia microti. Click here to learn more about Chagas Disease. Click here to learn more about vCJD.Click here to learn more about malaria.

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Additional Testing

If preliminary testing suggests hemolysis or if the results are misleading, additional testing may be required. If human error has been ruled out during the clerical check, repeat ABO/Rh testing should be performed on the unit of blood or its segment and the pretransfusion sample to detect any sample mix ups and clerical errors. Antibody detection studies should be performed on the pre- and post-transfusion samples to look for any unidentified antibodies. If an antibody is identified, the donor cells should be tested for the corresponding antigen. The crossmatch should be repeated with pre-and post-tranfusion specimens using the indirect antiglobulin test (IAT). An incompatible crossmatch with the pretransfusion sample indicates an original error, either clerical or technical. Incompatibility with only the post-transfusion sample indicates a possible anamnestic response, as in a delayed hemolytic transfusion reaction (DHTR), or sample misidentification. The patient's first voided urine specimen should be examined for the presence of free hemoglobin. The patient's bilirubin levels may also be evaluated. A change from normal pale yellow serum to a post-transfusion bright or deep yellow serum should prompt an investigation for hemolysis. The maximum concentration of bilirubin following hemolysis is not usually detectable until 3 to 6 hours after transfusion. The hemoglobin and hematocrit can be tested to detect a drop in hemoglobin or failure of the hemoglobin to rise after transfusion. Important information about physical or chemical hemolysis may be gained from examining the returned unit bag. If hemolysis is present in the bag or tubing, a process that affected the blood should be suspected, such as inappropriate warming or a faulty infusion pump. If bacterial contamination is suspected, the unit should be cultured. A positive culture indicates a reaction due to bacterial contamination.

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Procedure for a Suspected Adverse Reaction

Adverse reactions after transfusion of blood components must be evaluated promptly. Most serious reactions occur within the first 15 minutes of starting a transfusion. Continuous monitoring allows reactions to be discovered in a timely manner. The transfusionist must be able to recognize the symptoms of a transfusion reaction and know the appropriate steps to take when one occurs. The first critical step is to stop the transfusion immediately, but keep the patient's line open with saline. The physician should be contacted immediately for instructions regarding patient care. The transfusion service must be notified of the reaction. They will usually provide instructions on proper documentation of the reaction, and the return of any remaining component and/or tubing. The appropriate patient samples are to be sent to the laboratory and usually include blood and urine. The transfusionist must be sure to follow all hospital policies.

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Transfusion Reactions: Introduction

".....In the past, a person with blood type O negative blood was considered to be a universal donor. It meant his or her blood could be given to anyone, regardless of blood type, without causing a transfusion reaction. This is no longer a relevant concept because of a better understanding of the complex issues of immune reactions related to incompatible donor blood cells." Reference: Mayo Clinic Health Oasis - Ask a Physician 08/09/2000. As quoted in: Blood types and compatibility. Bloodbook.com; 2005. Available at: http://www.bloodbook.com/compat.html. Accessed August 12, 2013.Transfusion of blood components is generally a safe and effective way to correct hematologic deficits. However, a transfusion reaction may occur and health care providers must be aware of the risks involved with blood transfusions and evaluate the risks against the potential therapeutic benefits. A transfusion reaction can be defined as any adverse event occurring during or after the transfusion of blood components. Adverse events can range from fever and hives to renal failure, shock, and death. Some adverse events can be prevented, but others cannot.

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References

AABB. Standards for Blood Banks and Transfusion Services. 28th ed. Bethesda, MD: AABB; 2012. Harmening, DM. Modern Blood Banking and Transfusion Practices. 5th ed.Philadelphia, PA: FA Davis; 2005.Hillyer CD, Silberstein LE, Ness PM, Anderson, KC, Roback, JR. Blood Banking and Transfusion Medicine: Basic Principles and Practice. 2nd ed. Philadelphia, PA: Churchill Livingstone; 2007.Roback JD, Grossman BJ, Harris T, Hillyer CD, eds. Technical Manual. 17th ed. Bethesda, MD: AABB; 2011.Rudman, SV. Textbook of Blood Banking and Transfusion Medicine. 2nd ed. Philadelphia, PA: Elsevier Saunders; 2005. U.S. Food and Drug Administration. Infectious Disease Tests. Available at: http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/default.htm. Accessed November 18, 2013.

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Causes of Acute Hemolytic Transfusion Reaction (AHTR)

AHTR may occur when red cells are transfused into a patient with a pre-existing antibody that destroys the transfused incompatible red cells. Life threatening AHTR most commonly occurs from the transfusion of ABO incompatible blood. Naturally occurring ABO antibodies bind complement on the red cell surface and have efficient lytic properties, which cause intravascular hemolysis. Extravascular hemolysis is characterized by antigen-antibody complexes, which do not activate complement. AHTR is characterized by rapid destruction of red cells immediately after transfusion. Rapid hemolysis of as little as 10 mL of incompatible red cells can produce symptoms of an AHTR. Signs and symptoms can occur within minutes after starting the transfusion. Clerical errors, such as mislabeled patient samples and mislabeled blood products, could result in AHTR. Therefore, a system must be in place and rigidly followed to ensure samples and blood products are correctly identified.Although acute hemolytic reactions are rare with an incidence of 1-9 in 100,000 transfusions, they are the most dangerous, often life threatening.

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Clinical Signs and Symptoms

Although there is no consistent clinical picture of an acute hemolytic transfusion reaction (AHTR), common symptoms include chills, hypotension, and fever. Some patients have experienced pain at the infusion site, flank pain, and anxiety with a feeling of doom. Red or dark urine may be the first sign of intravascular hemolysis. If patients are unconscious or in surgery, changes in vital signs, unexplained bleeding, or hemoglobinuria may be the only signs. Additional signs and symptoms include, but are not limited to: rigors, facial flushing, chest and abdominal pain, nausea and vomiting, dyspnea, oliguria/anuria, diffuse bleeding, shock, and renal failure. The severity of symptoms is related to the amount of incompatible blood transfused. Patients with underlying diseases that involve intravascular hemolysis can make diagnosis extremely difficult.

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Clinical Laboratory Tests

A post transfusion specimen should be sent to the laboratory for work-up. A clerical check should be performed to investigate possible errors in specimen labeling, blood product issuance, or patient identification. The plasma must be examined for hemolysis. A direct antiglobulin test must be performed. The patient's ABO, Rh and antibody screen should be repeated and confirmed. The blood product ABO/Rh can be confirmed. Other laboratory tests include: complete blood count (CBC), urinalysis, serum bilirubin, creatinine, coagulation profile, and disseminated intravascular coagulation (DIC) evaluation. The full laboratory work-up and details of other laboratory tests will be discussed later in the course.

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Management and Prevention

The first component of therapy is to stop the transfusion immediately. Vital signs must be closely monitored. Management involves treatment of hypotension and disseminated intravascular coagulation (DIC). It is essential to maintain blood volume and adequate renal blood flow. Diuretics, substances that increase urine output, may be administered. If the patient enters renal failure, dialysis must be initiated rapidly. It is impossible to prevent all hemolytic transfusion reactions. The purpose of pre-transfusion compatibility testing is to decrease the probability of a hemolytic transfusion reaction by performing ABO/Rh testing, detecting and identifying alloantibodies, and crossmatching compatible blood. Human error, the most common cause of hemolytic transfusion reactions, cannot be completely eliminated. Steps must be taken to reduce the possibility of human error in identification of patient samples, donor units, and recipients. Each person involved in the transfusion process, from collection of the blood sample to administration of the donor unit, must carefully adhere to each step outlined in the standard operating procedures. All appropriate protocols must be followed. Some examples are:Technologist checks blood sample to ensure proper labeling. Patient's previous transfusion records are examined and all transfusion testing is performed correctly and accurately. Technologist ensures correct unit is released from the blood bank. Transfusionist ensures the recipient is correctly identified.There must be a mechanism in place to train and assess all personnel involved in the transfusion process.

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An acute hemolytic reaction may be caused by which of the following? (Choose all that apply)View Page
Febrile Nonhemolytic Transfusion Reactions: Definition, Manifestation, and Prevalence

A febrile non-hemolytic transfusion reactions (FNHTR) is defined as a temperature increase of 1°C over 37°C occurring during or after the transfusion of blood components. FNHTRs are more common in the transfusion of platelets. Multiply-transfused patients and multiparous women make up the largest populations experiencing this type of reaction. There are two mechanisms involved in the manifestation of an FNHTR. The first involves the presence of a white cell antibody in the patient's plasma that interacts with the white cells in the blood product. These antibodies may be directed against granulocyte antigens or human leukocyte antigens (HLA).This interaction causes endotoxins to be released, which act on the hypothalamus and stimulate a fever. The second mechanism involves the generation of leukocyte cytokines during product storage. The production of cytokines usually occurs during storage in warmer temperatures, which is why non-leukoreduced platelets are commonly implicated.

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Diagnosis, Treatment, and Prevention of FNHTR

Diagnosing a febrile non-hemolytic transfusion reaction (FNHTR) involves excluding all other options that may present with fever. If this type of reaction is suspected, the transfusion should be stopped. A transfusion reaction work-up should be initiated, although the antibodies involved with these reactions are not routinely identified because of the difficulty in demonstrating their presence in vitro. Antipyretics, such as acetaminophen, should be administered to the patient and the transfusion can continue once the symptoms subside.A patient with two or more documented FNHTRs should receive leukocyte-reduced blood components.Pre-storage leukocyte reduction prevents reactions that occur due to cytokine accumulation during storage. Red cell component prevention techniques include the transfusion of fresher blood or washed blood. For platelets, residual plasma may be removed. Antipyretics can be administered prior to transfusion.

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Definition, Manifestations, and Prevalence of Allergic Reactions Related to Transfusion

Allergic reactions are grouped into three categories depending on severity: mild or uncomplicated moderate or anaphylactoid life-threatening or anaphylactic reactionsMild allergic reactions occur in about 1-3% of patients receiving blood products containing plasma. Symptoms are usually mild and include urticaria, erythema (skin redness), and itching. Hives can appear anywhere on the body and may vary in size. Symptoms usually occur within minutes after the start of the transfusion. They can often last for hours or even days. Mild allergic reactions result from a patient's hypersensitivity to soluble allergens in the plasma of the donor unit. Allergen substances may be drugs or food consumed by the blood donor. Anaphylactoid and anaphylactic reactions have similar presentations. These reactions are rare but life-threatening. Anaphylactoid and anaphylactic reactions are severe systemic reactions with symptoms such as hypotension, dyspnea, nausea, vomiting, urticaria, and diarrhea. The most life-threatening symptoms include lower airway obstruction, laryngeal edema, cardiac arrhythmia, cardiac arrest, shock, and loss of consciousness. None of these reactions present with fever.

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Pathophysiology

Mild allergic reactions result from a patient's hypersensitivity to soluble allergens in the plasma of the donor unit. The blood recipient forms antibodies to these allergens which are bound to IgE on mast cells. This causes the release of histamines. Histamines increase vascular dilation and permeability, which allows vascular fluids to escape into the tissues. Swelling occurs and itchy, raised, red welts appear. Allergen substances may be drugs or food consumed by the blood donor. Anaphylactoid and anaphylactic reactions (collectively referred to as anaphylaxis) result from the recipient's forming anti-IgA, which targets IgA proteins in the donor plasma. Recipients have a genetic IgA deficiency. It is also believed that these types of reactions may be caused by other substances in the donor blood such as a peanut allergen transfused to a patient with a peanut allergy.

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Definition and Epidemiology

Transfusion-associated acute lung injury (TRALI) is a complication of blood transfusion that results in shortness of breath due to pulmonary edema, fever, and hypotension. The pulmonary edema is noncardiogenic which means it does not originate from the heart. TRALI is a severely life-threatening adverse reaction. Symptoms manifest within 6 hours of transfusion. Products typically implicated in TRALI are Whole Blood, Red Blood Cells, Fresh Frozen Plasma, Cryoprecipitate, and Platelets, with Fresh Frozen Plasma being the most often implicated product. In combined fiscal years 2005 through 2009, transfusion-related acute lung injury (TRALI) caused the higest number of reported fatalities (48%), followed by hemolytic transfusion reactions (26%) due to non-ABO (16%) and ABO (10%) incompatibilities. Complications of microbial infection, transfusion-associated circulatory overload (TACO), and anaphylactic reactions each accounted for a smaller number of reported fatalities. TRALI has accounted for the highest number of reported transfusion-related fatalities throughout the first decade of 2000.Cases occur in all age groups and genders. Most patients that develop TRALI have no history of adverse reactions. TRALI is generally under-diagnosed and under-reported and the true incidence may be higher than stated estimates. Under-diagnosing is due to lack of recognition of the condition and that it can be easily confused with other diseases. Also, TRALI may be attributed to the underlying condition of the patient.Reference: U.S. Food and Drug Administration Website. Fatalities reported to FDA following blood collection and transfusion: Annual summary for fiscal year 2009. Available at: http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/TransfusionDonationFatalities/ucm204763.htm. Accessed April 26, 2011.

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Prevention of Transfusion-Related Acute Lung Injury (TRALI)

The AABB has made several recommendations for preventing TRALI including: Blood collection facilities should implement interventions to minimize the preparation of high-plasma-volume components from donors known to be leukocyte-alloimmunized or at increased risk for leukocyte alloimmunization. Blood transfusion facilities should work toward implementing appropriate evidence-based hemotherapy practices to minimize unnecessary transfusion. Blood collection and transfusion facilities should monitor the incidence of reported TRALI and TRALI-related mortality. Transfusion services should work with clinicians to educate providers about the risks of TRALI and about the need to work toward implementing evidence-based transfusion practices for all blood components, with special emphasis on high plasma-volume components. High-plasma-volume components include the following: FFP obtained from whole blood or apheresis Plasma frozen within 24 hours Cryoprecipitate-reduced plasma Apheresis platelets Whole bloodThere have been several other suggestions for preventing TRALI, which include: Screening of all donors for anti-neutrophil or anti-HLA antibodies. Once donors are identified, they are excluded from donating, or their blood is used for products that do not contain much plasma. This method would not prevent TRALI in recipients who have alloantibodies. Use of pre-storage leukoreduced blood. Use of younger blood products. Appropriate utilization of blood products. Using blood products only when clinically indicated may reduce the frequency of TRALI. Because TRALI can coexist with other transfusion reactions and with pulmonary complications unrelated to transfusion, the diagnosis of TRALI is difficult, but it is an important step in monitoring the effectiveness of TRALI risk-reduction strategies.

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Sources of Contamination

Possible means of blood component bacterial contamination involve the blood donor, the collection process, the collection pack, and blood processing. Most bacteremic individuals are symptomatic and would not be accepted as donors. In the United States, a person cannot donate if their temperature is higher than 37°C. Sometimes a donor may be in an incubation period or in the recovery phase of bacterial infection and this may lead to contamination of their blood products. Most of the organisms isolated from platelet concentrates are normal skin flora which entered the bag during venipuncture when skin is not disinfected properly. Some organisms may even remain viable on the skin after disinfection. The donor's skin may also contain unusual pathogens. Clostridium perfringens was linked to a donor who had recently changed a child's diaper. Blood bags can be contaminated on the outer surfaces. The bacteria can enter the unit at the time of blood donation either through suction into the needle or contamination of the phlebotomist's hands and then on the donor's skin. Contamination during blood processing can occur from thawing frozen products in a contaminated water bath. Bacteria can enter the unit through microcracks in the bags or through pooling.

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Reducing Transfusion-Associated Septic Reactions

Measures taken to reduce bacterial contamination of blood components include donor screening, improved skin disinfection, diversion of the first aliquot of blood, and pretransfusion bacterial detection. Screening of donors is done by questioning them about fever occurrence and dental or medical procedures that occurred days before donation. Donors who develop symptoms of an infection may be asked to notify the blood bank. Complete skin disinfection is not possible because of organisms living in places that are inaccessible, such as sebaceous glands and hair follicles. Factors affecting skin disinfection are the type and concentration of antiseptic, use or single or multiple antiseptics, method and steps of application, and contact time. Studies have shown that a two-stage method using a sponge scrub and ampule with tincture of iodine is the most effective method. The AABB recommends an initial 30-second scrub with a 0.7% iodophor solution followed by the application of a 10% iodophor compound, which must be allowed to dry for 30 seconds. To avoid normal flora contamination, blood may be diverted into a satellite bag at the beginning of donation. These bags are developed so that backflow is prevented. Blood contained in the satellite bag is used for blood grouping and infectious disease testing. Blood diversion is not a mandatory practice in the United States. The AABB requires that the transfusion service have a method to detect bacteria in all platelet components. Culture-based methods are used at blood collecting facilities near the time of collection. Hospital-based transfusion services use other less costly non-culture based methods such as gram staining or pH and glucose analysis prior to releasing the product for transfusion. Recently, a qualitative immunoassay for the detection of bacteria in platelets has been developed. This test detects antigens on the cell walls of the bacteria. It has been documented to be more sensitive than other non-culture based methods.

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Which type of blood component is MOST often implicated in bacterial contamination?View Page
Transfusion-Associated Circulatory Overload (TACO)

Transfusion-associated circulatory overload (TACO) is caused by the inability of the circulatory system to handle an increased blood volume. This usually occurs if the product is infused into the patient too quickly. The very young, elderly, patients with small stature, and patients with compromised cardiac function are at heightened risk for circulatory overload. The frequency is difficult to determine since many instances go unreported. The patient will present with acute pulmonary edema when cardiac output cannot be maintained. Other symptoms include, cyanosis , orthopnea, hypertension, headache, tachycardia, chest tightness, and cough. Symptoms set in near the end of the transfusion or within six hours of completion. Symptoms may be confused with transfusion-related acute lung injury (TRALI). Recently, B-type natriuretic peptide (BNP), a cardiac marker, has been used as a diagnostic tool. BNP is elevated with TACO.The transfusion should be stopped as soon as TACO is suspected. The patient should be in a sitting position and provided with supplementary oxygen. Intravascular volume may be reduced by the administering of diuretics. >Blood components should be administered slowly when possible, particularly in patients at risk for TACO.

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Physical and Chemical Mechanisms of Hemolysis

Patients can experience a transfusion reaction caused by a range of physical or chemical factors. These factors can either affect the blood component or result from a transfusion event. These reactions include physical red cell damage, depletion or dilution of coagulation factors and platelets, hypothermia, citrate toxicity, hypokalemia or hyperkalemia, and air embolism. Membrane damage and lysis can occur to red blood cells (RBCs) because of hypotonic or hypertonic solutions, heat damage from blood warmers, and mechanical damage caused by blood pumps. Platelets and coagulation factors may become depleted or diluted from a massive transfusion. Hypothermia, a core body temperature of less than 35°C, can occur from transfusions of large volumes of cold products. Hyperkalemia is caused by the intracellular loss of potassium from the red cells during storage. Hypokalemia may result from transfusion of potassium depleted cells such as washed RBCs. Signs and symptoms of physically or chemically induced reactions are non-specific. Some of the more common signs include: ChillsNumbnessNausea Vomiting Cardiac arrhythmiaAltered respiration Additional laboratory tests to investigate a reaction are electrolytes, blood pH, glucose, urinalysis, complete blood count (CBC), prothrombin time (PT) and activated partial thromboplastin time (aPTT). Treatment involves correcting the underlying cause of the symptoms. For example, a patient with hypothermia may be given a heat blanket. Attention to proper transfusion practices will help prevent these types of reactions.

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Definition and Incidence

Delayed hemolytic transfusion reactions (DHTR) are reactions that occurs 3 to 10 days after the transfusion. Usually, the blood appears serologically compatible at initial testing. Delayed reactions are common in patients who have been immunized to a foreign antigen from a previous transfusion or pregnancy, but the antibody titers decrease over time and the antibody is not detectable during pre-transfusion testing. The transfusion leads to a secondary (anamnestic) response, causing increased antibody production that sensitizes antigen-positive donor red cells. Hemolysis is extravascular. Sensitized cells are removed from circulation by the reticuloendothelial system, also called the monocyte-macrophage system. Because there is a delay in the presentation of symptoms, DHTR is not usually considered as a cause of the clinical presentation. The transfusion service usually initiates investigation of a DHTR because of serologic findings in a post-transfusion specimen. DHTRs occur more frequently than acute hemolytic reactions. Approximately 1:2500 transfusions result in a DHTR.

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Prevention

The most critical aspect of prevention is for the transfusion service to document all clinically significant antibodies. One challenge in antibody detection is finding a rapid method that is sensitive enough to detect low titers of clinically significant antibodies without being too sensitive for insignificant antibodies. Preventing severe reactions in sickle patients can be done by phenotyping the patients. This is useful in providing phenotypically matched blood and solving complex antibody identification problems.

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Definition and Incidence

Transfusion-associated graft versus host disease (TA-GVHD) is a rare but highly lethal adverse reaction. The disease has a 90% mortality rate. It is caused by the transfusion of donor lymphocytes to a recipient who is immunocompromised. The donor lymphocytes engraft and escalate an immune response against the host's tissues including organs such as the lungs, skin, intestines, and liver. The recipient is unable to destroy the foreign lymphocytes and the cells proliferate and respond to incompatible antigens in the host. Certain recipients have increased risk for developing TA-GVHD. They are: Neonates less than 4 months of age Fetuses Recipients with a congenital or acquired immunodeficiency Recipients of donor units from a blood relative

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Therapy and Prevention

Transfusion-associated graft versus host disease (TA-GVHD) is generally unresponsive to medical treatment. Hematopoetic stem cell transplantation has been successful in rare instances. Gamma-irradiation of blood components containing viable lymphocytes is effective in preventing TA-GVHD. Irradiation is recommended for all Whole Blood, Red Blood Cell, Platelet, and Granulocyte transfusions to patients at risk. Patients at risk include neonates less than four months, patients with an acquired or congenital immunodeficiency, or patients receiving a directed donation from a family member. Irradiation prevents proliferation of donor lymphocytes with a required dose of 25 Gy to the mid plane of the blood container and a minimum of 15 Gy elsewhere. The dosage must not exceed 50 Gy to prevent harm to the patient from irradiation. Irradiation of blood can result in a decreased survival of red cells and a leakage of potassium from intracellular stores. Because of this, red cell units may only be stored for up to 28 days following irradiation. No reduction in storage time is required for platelets. Because Fresh Frozen Plasma (FFP) and Cryoprecipitate do not contain cells, irradiation is not required to prevent TA-GVHD in patients at risk.

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Definition/Manifestation/Prevalence

Post-transfusion purpura (PTP) is a very rare complication of blood transfusion. It has been most commonly associated with the transfusion of red blood cells and whole blood, but has also been seen in platelet and plasma transfusions. It is characterized by a rapid onset of thrombocytopenia, or decreased platelet count, which results from the product of a platelet alloantibody. Platelet counts are usually less than 10,000/µL. Reactions occur around 7 to 14 days post-transfusion. Patients present with purpura, bleeding from the mucous membranes, gastrointesinal ,and/or urinary tract bleeding. Melena and vaginal bleeding have also been reported. The thrombocytopenia is usually self-limiting. Platelet counts and coagulation studies aid in the diagnosis. Patients can also be tested for platelet specific antibodies, human leukocyte antigen (HLA) antibodies and lymphocytotoxic antibodies. The differential diagnosis includes other causes of thrombocytopenia.

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Pathophysiology, Treatment, and Prevention of Post-Transfusion Purpura (PTP)

PTP is caused by platelet-specific antibodies in a patient who has been previously exposed to platelet antigens through pregnancy or transfusion. The most frequently identified antibody is Anti-PLA1, which reacts with platelet antigen HPA-1a. The platelet antibody binds to the platelet surface, which allows for extravascular removal through the liver or the spleen. The patient's own platelets are destroyed as well, thus aggravating the thrombocytopenia. Three theories are suggested regarding the destruction of autologous platelets. One suggests that immune complexes bind to the platelets through the Fc receptor and cause destruction. The second theory proposes that the patient's platelets absorb a soluble platelet antigen from the donor plasma. The third hypothesis, which has the most support, states that the platelet alloantibody has autoreactivity that develops when the patient is exposed to the foreign platelet antigen. Platelet transfusion is NOT a treatment option. Steroids, whole blood exchange, and plasma exchange are accepted options for treatment. According to the AABB, intravenous IgG (IVIG) is the treatment of choice. Most patients will respond to treatment within several hours to four days. PTP does not usually re-occur but it is recommended that patient's with a previous reaction be transfused with antigen-matched components. Autologous donations or directed donations from antigen-matched family members may be the best sources of blood. PTP has been known to occur even after the transfusion of deglycerolized rejuvenated or washed red cells, so these processes do not prevent a reaction.

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Which of the following patients are at risk for transfusion-associated graft versus host disease (TA-GVHD) and require irradiated cellular blood products? (Choose all that apply)View Page

Tuberculosis Awareness for Health Care Workers
Latent Tuberculosis Infection (LTBI) Detection Methods

It is important to identify and treat persons with LTBI to prevent progression to active disease. The tuberculin skin test (TST) is traditionally used for the identification of prior exposure to Mycobacterium tuberculosis. However, specificity is reduced if an individual has had the vaccination Bacille Calmette-Guerin (BCG), which is intended to prevent TB, or has been infected with nontuberculous mycobacterium (environmental mycobacterial species). In these cases, newer blood tests, collectively known as interferon-gamma release assays (IGRA), may be more effective in identifying LTBI. IGRAs are more sensitive and specific, do not require the individual to return for test interpretation, and are not subject to possible inaccuracies and bias in reading the test, as may be the case with the TST. The turnaround time is usually less than 24 hours. However, the blood sample must be processed within 8-30 hours after collection while white blood cells are still viable. IGRA tests are more expensive to perform than the TST and not as widely available. There is also limited data on the ability of IGRAs to predict the risk for progressing to tuberculosis disease.

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Blood Assay for Mycobacterium tuberculosis (BAMT)

The BAMT is a blood test that can detect LTBI.The BAMT has the advantage of no false positive results due to previous BCG vaccination or infection with nontuberculosis mycobacteria.The BAMT was approved by the FDA in 2005.

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The two step TST has no false positive reactions due to infection with nontuberculosis mycobacteria or BCG vaccination.View Page

Variations in White Cell Morphology -- Granulocytes
Degenerate Neutrophils in EDTA blood

When examining a slide made from an EDTA tube of normal blood, an occasional cell containing a round pyknotic nucleus and neutrophilic-appearing cytoplasm may be seen. Rare cells such as these do not indicate the presence of Pelger-Huet anomaly.

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Barr Body

A Barr body (see image) appears as a small drumstick-like projection on one of the lobes of some neutrophils in females. Barr bodies are attached to the nuclear lobe by a single narrow stalk, which distinguishes them from other thicker projections, sometimes referred to as "clubs." Since Barr bodies are the morphological expression of the inactivated X chromosome, Barr bodies (one per neutrophil) can be seen in up to 3% of the neutrophils on a female's peripheral blood slide. In rare chromosome disorders in which three or more X chromosomes are present, two to three Barr bodies per neutrophil can be seen. Barr bodies must also be distinguished from hair-like projections sometimes seen in the band form, following irradiation or in patients with a malignant tumor that has metastasized. Recognition of a Barr body in a neutrophil is important in order to avoid reporting it as abnormal (unless two or more per neutrophil are seen). The Barr body is considered nonpathological unless associated with rare chromosome disorders.

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May-Hegglin Anomaly

This blood smear was taken from a patient with the May-Hegglin anomaly. A May-Hegglin Dohle body is indicated by the arrow near the edge of the cytoplasm at the top of the neutrophil. In addition, notice the giant platelet that is indicated by the red arrow, another characteristic of May-Hegglin anomaly.

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Inherited Anomalies

Several rare inherited anomalies show atypical granulation in the cytoplasm of peripheral blood cells.

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Toxic Granulation

Toxic granulation is manifested by the presence of large granules in the cytoplasm of segmented and band neutrophils in the peripheral blood. The color of these granules can range from dark purplish blue to an almost red appearance. Toxic granules are actually azurophilic granules, normally present in early myeloid forms, but are not normally seen at the band and segmented stages of neutrophil maturation. These granules contain peroxidases and hydrolases. Toxic granulation is seen in cases of severe infection, as a result of denatured proteins in rheumatoid arthritis or, less frequently, as a result of autophagocytosis. Infection is the most frequent cause of toxic granulation. This phenomenon may be seen in cells which also contain Döhle bodies and/or vacuoles. Cells containing toxic granules may have decreased numbers of specific granules. Note: Cells containing only a few specific granules, with or without toxic granules, are said to be degranulated. The nucleus in degranulated cells may often be round-bilobed, smooth and pyknotic. This type of nucleus is the result of aging and will disintegrate soon. Increased basophilia of azurophilic granules simulating toxic granules may occur in normal cells with prolonged staining time or decreased pH of the stain. The blue arrow in the image points to a neutrophil with toxic granulation. Döhle bodies are also present in the cell, indicated by the red arrows.

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Alder Anomaly (Alder-Reilly Anomaly )

Alder anomaly is a rare autosomal recessive disorder in which the basic defect involves protein-carbohydrate complexes called mucopolysaccharides. The accumulation of partially degraded (broken down) protein-carbohydrate complexes within the lysosomes account for the larger than normal purple-staining inclusions seen in all types of mature white blood cells, and sometimes in earlier cells. The granules may occur in clusters, rather than diffusely, throughout the cytoplasm as in toxic granulation. These inclusions may be seen in the bone marrow more frequently than in peripheral blood. The physical characteristics associated with this disorder include gargoylism and dwarfism. The function of the cells involved is not affected. This morpholical change would be classified as pathological since the body is responding abnormally even though the function is not affected.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Match the letters representing the peripheral white blood cells with the most likely clinical conditions in which the cell would be present in increased numbers.View Page
The presence in the peripheral blood of an increased number of hypersegmented white blood cells, as shown in this image, serves as a marker for preleukemia.View Page
Match the letter representing the cell type with the condition in which increased numbers of the cell may be found in the peripheral smear.View Page
The white blood cell indicated by the arrow is representative of the atypical white blood cell associated with infectious mononucleosis.View Page
The upper photograph of a bone marrow section reveals distinct hyperplasia with total replacement of marrow fat. A bone marrow smear stained with Wright/Giemsa is displayed in the lower photograph. Calculate the M:E ratio between myeloid and erythroid cells found in the lower photograph. The total peripheral blood white blood cell count was 5,400/cumm. This bone marrow architecture may be found in each of the following conditions except:View Page
The upper photograph of this bone marrow section also reveals distinct hyperplasia with total replacement of the fat. The lower photograph is a Wright/Giemsa stain. Calculate the M:E ratio of the distribution of myeloid and erythroid cells in the lower photograph. The peripheral white blood count was 18,500/cumm. The most likely associated condition is:View Page
An automated hematology counter flagged the white blood cell count. Upon review of the peripheral blood smear, the technologist viewed many cells that appeared similar to those in this image. What should the technologist report?View Page
Peripheral Blood Smear Preparation

A reproducible blood smear review requires every peripheral smear be prepared for consistent cellular distribution and proper clarity. Well-made peripheral smears can be prepared by starting with only a drop of blood at one end of a clean glass slide. The drop is smeared lightly and quickly with a wedge technique so as to leave a thin "feather" edge where all cells may be examined individually, particularly red blood cells. After staining the slide, the examination begins. The site of examination is chosen; away from clumping, piling, or stacking of the red blood cells. This can most likely be observed at a site five or six oil fields from the end of the feathery portion (about 100 red cells per field). Such an area for examination is illustrated in the image below.

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An automated platelet count of 40.0 X 109/L was reported. Review of the peripheral blood smear (see image below) reveals single platelets in open fields as well as platelet clumps. The platelet count is likely INCORRECT.View Page
Platelet Estimates

The findings in the image to the right (peripheral blood smear) would elicit a report comment of "increased platelets" of a high magnitude, such as "marked" or "4+." Estimates of platelet counts from review of a peripheral blood should be made on each smear examined. This provides a simple estimate of "high", "low", or "normal" which usually corroborates the value generated from an automated cell counter. A formula for estimating platelet counts must be established for each laboratory. One guideline for the estimation of platelets is as follows: Count platelets on 5 fields using 1000X magnification (care should be taken to ensure the fields used for counting are not too thick or too thin) Average the platelet counts obtained Multiply by 15 X 109/L to obtain estimated platelet count (some laboratories prefer a 20 X 109 multiplier in this step if capillary blood is used)Such a counting scheme for platelets when clustered, as in the image, is probably not needed, as there are more than 100 platelets in the field. This translates into a platelet count of 1500 X 109/L or more.

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Evaluation Criteria: White Blood Cells and Platelets

In most clinical hematology laboratories, an initial blood count is performed by an automated cell counting instrument. Additionally, most of these instruments also produce a five-part differential count, indicating the percentage of neutrophils, lymphocytes, monocytes, basophils, and eosinophils. Some instruments can also provide information about cellular immaturity and abnormal cellular morphologies.Occasionally, atypical cells, similar to those shown in the image to the right, would be flagged or counted as mixed cells, at which point a smear review would be required to make an identification. In cases where there are automated instrument differential flags, mixed cell count is high, or there are other indications that atypical cells may be present, a review of the smear is indicated.

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Cells that appeared similar to those illustrated in this image were repeatedly encountered as the smear was reviewed. The peripheral white blood cell count was 51.0 X 109/L with an orderly maturation sequence. The comment "leukemoid reaction" may properly be appended to the report.View Page
A peripheral blood smear with many myeloid cells was presented for morphology review (see image on the right). Toxic granulation and vacuoles in the neutrophil most likely represent which of the following conditions?View Page
Leukemoid Reaction

The term "leukemoid reaction" is used to describe a condition where peripheral white blood cells on a stained blood smear may have some resemblance to leukemia cells. Quantatively, in a leukemoid reaction, the neutrophil count may be as high as 50.0 X 109/L with more immature cells, particularly myelocytes, than are usually present in toxic left-shift syndromes. The presence of immature cells in a leukemoid reaction awakens thoughts of leukemia. Great care must be taken to make a distinct differentiation between aberrant white blood cell proliferations (possible leukemia) and a benign but exaggerated granulocytic proliferative response (leukemoid reaction). The leukocyte alkaline phosphatase (LAP) score is low in myelocytic leukemia and high in leukemoid reaction. This particular peripheral smear represents a leukemoid reaction.

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Familial disorders: summary

Several additional familial and congenital disorders associated with atypical inclusions in WBCs are now recorded. These individual syndromes carry the following names: Fechtner, Alport, Epstein, Sebastian, and Paris-Trousseau.Fechtner syndrome( Peterson etal,Blood 65:397-406,1985)was described with 8 family members spanning 4 generations presenting with varying degrees of nephritis, deafness,and congenital cataracts. The syndrome is likely a variant of Alport syndrome with the addition of leukocyte inclusions and macrocytothemia. Several more cases involving other families have been reported. The inclusions resemble toxic Doehle bodies or those of the May-Hegglin anomaly by light microscopy, but are ultrastructurally unique.Alport syndrome is autosomal dominant, X-linked , hereditary and characterized by sensorineural deafness and hereditary nephritis. It is believed to result from abnormal glycopeptide synthesis in renal basement membranes. Recurrent hematuria and slowly progressive renal insufficiency are clinical findings. Cataracts and platelet abnormalities may be added features.Epstein syndrome is essentially Alport syndrome with the addition of macrothrombocytopenia (Seri, et al. Hum Genet 110:182-186, 2002). Neutrophil inclusions are absent in this disorder; neutrophilic inclusions are considered part of the Fechtner syndrome.The Sebastian platelet syndrome is a variant of hereditary macrothrombocytopenia combined with neutrophil inclusions that differ from Doehle bodies, but are similar to those inclusions in Fechtner syndrome. (Greinacher, et al, Blut 61:282-288, 1990).Paris-Trousseau syndrome includes large platelets containing giant alpha granules identifiable in the peripheral blood.(Breton-Gorius, Blood 85:1805,1995)

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The inclusions noted in the cytoplasm of this white blood cell are most suggestive of which of these conditions?View Page
Alder Anomaly

Alder anomaly is characterized by large azurophilic granules that stain dark-purple and are seen throughout the leukocyte cytoplasm, even covering the nucleus. The inclusions (granules) are seen in the cytoplasm of almost all mature leukocytes i.e., granulocytes, lymphocytes, and monocytes. This distinguishes Alder anomaly inclusions from toxic granulation, which is only observed in neutrophils. Another feature that distinguishes Alder anomaly from toxic changes is the lack of cytoplasmic vacuoles of toxic origin in the neutrophils of Alder anomaly.The background condition in Alder anomaly is mucopolysaccharidosis, collectively, a group of inherited disorders where a deficiency of lysosomal enzymes are lacking that are needed to degrade mucopolysaccharides. The inclusions observed in the leukocytes represent partially degraded mucopolysaccharides within lysosomes. Accompanying conditions are hepatosplenomegaly, corneal opacities, and mental retardation.

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WBC inclusions: Summary

The presence of atypical inclusions within the cytoplasm of neutrophils and other leukocytes should lead to a clinical investigation of the setting for these findings. Atypical neutrophil inclusions may be seen in the following disorders: Chediak-Higashi syndrome, May-Hegglin anomaly, Alder-Reilly anomaly, Fechtner , Sebastian, Epstein and Alport-like syndromes and in infectious and toxic conditions (in the form of Dohle bodies).Although a specific entity may not be evident from examination of the peripheral blood alone, it is important that hematology technologists include a comment reporting on the presence of these inclusions or granules. A clinical investigation with further hematologic and genetic studies may then appropriately be considered. Many of the disorders with atypical neutrophil cytoplasmic granules are also associated with platelet abnormalities, particularly giant platelets (lower image). Therefore, when atypical granules are recognized, scanning of the peripheral blood smear for atypical platelets may be revealing. These observations serve as readily identifiable markers for acquired and genetic human maladies, and as a guide for unraveling the reasons for a patient's suffering and impaired health.

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The pale-staining cytoplasmic bodies marked by the arrow in the image may be seen in each of the following conditions except:View Page
Case History

A 17-year-old female was admitted to the hospital with abdominal pain and a tentative diagnosis of appendicitis. The total white blood count was 14.5 X 109/L with a left shift and neutrophils with changes tagged by the arrow in the image (see blue arrow). The bluish-staining, blurred accumulations in the cytoplasm (Döhle bodies), are located at the cell periphery in neutrophils with toxic changes.Döhle bodies are remnants of endocytoplasmic reticulum and are products of cytokine activity in the induction and shortened activity of neutrophil activation. They are often present in conditions with increased neutrophil lysosomal activity, manifest as toxic granulation.In this case, the presence of Döhle bodies serves as markers for infection-induced leukocytosis and supports the diagnosis of acute appendicitis.

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Eosinophils

The cytoplasm of eosinophils is evenly filled by numerous orange-red granules of uniform size. They do not overlie the nucleus. The eosinophil granules contain numerous enzymes including peroxidase, phospholipase D, catalase, acid phosphatase, and vitamin B12-binding proteins. The eosinophil's ability to kill bacteria is less than that of neutrophils. Their main purpose is to counteract parasitic infections and to participate in immune allergic reactions. They may also be increased in a variety of nonimmunologic inflammatory responses from bacteria and fungi causing chronic infections. A high percentages of eosinophils may be present in the peripheral blood smears of patients with a variety of non-neoplastic conditions including:Asthma Urticaria Loeffler syndrome Parasitic infections Malignancies, collagen vascular diseases, and myeloproliferative disorders may also may be settings for prominent eosinophils.

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The image on the right represents a peripheral blood smear field. The white blood cell seen in the image is a mast cell.View Page
Basophils

A basophil and a small lymphocyte are compared in the same field in the upper image, while a single basophil is shown in the lower image.The cytoplasmic granules of the basophil are larger than the granules of toxic granulation.They contain chemical mediators of immediate hypersensitivity, and are found in the cytoplasm and overlying the nucleus (better seen in the lower image). Basophilic granules stain metachromatically with toluidine blue indicating the presence of acid mucopolysaccharide or proteoglycans, both thought to be heparin or heparin-like substances.Basophils are related to mast cells (tissue basophils), each involved in hypersensitivity responses and following anaphylactic episodes. Under the stimulation of complement components C3a and C5a, many mediators are released from the basophil granules, including histamine, heparin, and eosinophil chemotactic factors of anaphylaxis, or ECF-A.Basophils are the least common granulocytes in the peripheral blood, comprising 2% or less of the differential count. The presence of large granules of irregular size in basophils and the admixture of eosinophilic granules may indicate dysplastic changes associated with myelodysplastic disorders and leukemia. Basophils may be increased in:Myeloproliferative disordersChronic metabolic conditions Myxedema Diabetes mellitusHypersensitivity responses Tuberculosis

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A peripheral blood smear is observed during a manual differental review. The patient is a 10 year-old boy with symptoms suggesting appendicitis and an appendectomy is being considered. The total WBC is 18.5 X 1000/uL, RBC's = 5.45 X 1M/uL, hemoglobin = 16.0 g/dL, hematocrit 48.2%.WBC differential:Segs = 53%, bands = 42% (two of which are shown in the image) monocytes = 2% lymphocytes= 2% These findings support the diagnosis of appendicitis.View Page
Erythrophagocytosis

Illustrated in the image is a phagocyte devouring several erythrocytes. This uncommon phenomenon occurs in the bone marrow and in the spleen as part of the process of erythrocyte destruction. Erythrophagocytosis is found in histological sections of the spleen in cases of hemolytic anemia. This phenomenon appears also in splenic sections in lupus erythematosis, and in rheumatoid arthritis. Our example is from a patient with a myeloproliferative disorder and is a rare example of a circulating erythrophagocytic cell in the peripheral blood.

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The large blue staining cells represented here in the photographs comprise 50% of the total white blood count. This picture is most consistent with which of the following conditions? (choose all that apply)View Page
Hairy cell leukemia

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Case History Two

An 80-year-old man was seen in the emergency room with sudden onset of right-side chest pain accentuated on inspiration. His cough was productive of yellow sputum, and he was short of breath. His temperature was 101.2°F. A chest X-ray revealed right middle lobe pneumonia. A complete blood count (CBC) was ordered. The results were as follows:CBC ParameterPatient ResultReference IntervalWBC33.0 x 109/L4.0 - 11.0 x 109/LRBC4.5 x 1012/L4.5 - 5.9 x 1012/LHemoglobin15.2 g/dL13.5 - 17.5 g/dLHematocrit44%41 - 53%Platelet200 x 109/L150 - 450 x 109/LSegmented neutrophil6540 - 80%Band neutrophil100 - 5%Lymphocyte 525 - 35%Eosinophil 30 - 5%Basophil 20 - 2%Monocyte252 - 10%A peripheral smear was reviewed based on the elevated WBC and increased monocyte count. A representative field from the Wright-Giemsa stained smear (1000X magnification) is shown on the right. The cells indicated by the blue arrows are atypical monocytes. They have abundant cytoplasm that is more blue than the typical gray-blue cytoplasm of normal monoctes. A few scattered vacuoles are also present. The atypical monocytes, in company with toxic neutrophils (indicated by the red arrow), appeared to be a response to infection. The patient had a past history of tuberculosis, which may account for the monocytosis.

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Multiple Myeloma

Plasma cells are uncommonly observed in the peripheral blood smear. They are normal constituents of lymph nodes, spleen, connective tissue and bone marrow. The presence of plasma cells in the peripheral blood is indicative of a large number of conditions, mostly related to infections , immune disorders, malignancies, toxic exposures, hypersensitivity reactions and their responses.Although mature plasma cells have a distinct appearance, they still may be confused morphologically with immature plasma cells and other cells with inclusions, reactive changes or nucleated red bloods cell with altered identities. In the image to the right, a plasma cell is present. The plasma cell has an eccentric immature nucleus with a muddy chromatin pattern. Note also clumping and stacking of the erythrocytes, typical of rouleaux formation, implicating an increase in plasma gamma globulin. Further studies are in order, including a bone marrow examination, where at least 30% of bone marrow cells should be variations of mature and immature plasma cells. Serum protein electrophoresis will reveal a monoclonal globulin spike, and light chains in excess of 1.0 gm/24 hours may be seen in the urine. The presence of lytic bone lesions is a convincing clinical clue. With these findings in combination, a diagnosis of myeloma can be made with assurance.

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