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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.

This is an example of an electrophoresis on normal blood.

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.

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.

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.

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.

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).

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+.

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 intrathechal 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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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%).

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Patient safety when performing a capillary blood collection includes positive patient identification prior to performing the procedure. The accepted policy in most healthcare 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.

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.

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 Step Comment Caution Positively identify patient Always 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 appropriately Position 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 site Use 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 needed Use 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 equipment Only have needed equipment at hand. Keep track of ALL equipment to prevent patient injury. Cleanse the puncture site Use 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 container Puncture 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 blood Use 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 container Allow 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 specimen Specimen must belabeled in the presence of the patient. Unlabeled specimens will be rejected by the laboratory.

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: Container Additive Use Lavender top EDTA For hematology blood counts Green top Lithium heparin Tests that require a heparinized plasma sample __ Other tubes containing anticoagulants Varied Red or gold top Clot activator Tests that require a serum sample Red top No additive Tests that require a serum sample but clot activator and/or gel may affect test

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 Reminder Reason Comment Gloves are always necessary Blood 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 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 injury Often, capillary procedures are performed on very young children who are curious and may grab something that could cause injury.

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. NCCLS. 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-A3. NCCLS.Wayne, PA: 2004.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.Stevens B, Yamada J, Ohlsson A. Sucrose for analgesia in newborn infants undergoing painful procedures. Available at http://www.nichd.nih.gov/cochrane/Stevens/Stevens.HTM accessed January 12, 2010.

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.

In addition to the puncture device, additional equipment may be required when performing a successful dermal puncture.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 healthcare 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.

There are some instances where a dermal puncture is prohibited or not recommended.Mastectomy patientsAs 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 siteDermal punctures should not be performed on previously punctured sites or swollen sites. Excess tissue fluid may contaminate the specimen.Dehydrated patientIf 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.

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.

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 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.

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.

In some instances, the healthcare 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.

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.

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.

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.

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. .

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.

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.

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.

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%)

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.

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.

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

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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).

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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?

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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)

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%.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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 any Medicare patients that the phlebotomist might draw, 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 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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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's 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.

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.

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)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 lower 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. 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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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).

?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.

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.

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)

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.

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

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

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).

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.

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.

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.

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.

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

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.

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.

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.

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)

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.

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.

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.

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.

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

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.

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.

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).

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.

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.

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.

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

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

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.

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.

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

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.

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

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.

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

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.

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).

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

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.

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.

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

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.

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.

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.

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.

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.

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

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.

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

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

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.

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.

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.

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

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.

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).

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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.

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

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.

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

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.

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.

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

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.

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.

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.

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.

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.

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.

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!

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

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

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

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

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

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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").

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.

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.

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.

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

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.

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 healthcare professional that has been trained to perform this duty. In this course, we will refer to the person performing the venipuncture as the phlebotomist.

Preanalytical Error What is it? How does it happen? What is the result? Hemolysis Red 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 specimen Clumped or clotted cells in specimen that requires anticoagulated or whole blood Insufficient 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 volume Too 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.

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

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.

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)

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. NCCLS. 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. NCCLS. 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-A3. NCCLS.Wayne, PA: 2004.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.Lowe B. Reinforcing safety sticklers. Advance for Medical Laboratory Professionals. May 2004; 16:2A-3A.The Joint Commission. National Patient Safety Goals. Available at: http://www.jointcommission.org/standards_information/npsgs.aspx. Accessed July 2, 2012.

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.

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 color Additive Function of Additive Common laboratory tests Light-blue 3.2% Sodium citrate Prevents blood from clotting by binding calcium Coagulation Red or gold (mottled or "tiger" top used with some tubes is not shown) Serum tube with or without clot activator or gel Clot activator promotes blood clotting with glass or silica particles. Gel separates serum from cells. Chemistry, serology, immunology Green Sodium or lithium heparin with or without gel Prevents clotting by inhibiting thrombin and thromboplastin Stat and routine chemistry Lavender or pink Potassium EDTA Prevents clotting by binding calcium Hematology and blood bank Gray Sodium fluoride, and sodium or potassium oxalate Fluoride inhibits glycolysis, and oxalate prevents clotting by precipitating calcium. Glucose (especially when testing will be delayed), blood alcohol, lactic acid

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.

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.

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. .

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

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.

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.

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.

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 typing ABO Forward Group ABO Reverse Group Rh anti-A anti-B A1 cells B cells anti-D 0 0 4+ 4+ 3+ Antibody screen Cells Gel IAT* Screen Cell I 3+ Screen Cell II 2+ Screen Cell III 2+ * IAT = indirect antiglobulin test

The laboratory obtained post-transfusion blood specimens in order to perform a serological investigation. Pretransfusion and post-transfusion DATs were performed. Patient cells DAT CC Pretransfusion 0 2+ DAT = direct antiglobulin test with polyspecific antiglobulin serumCC = IgG sensitized RBC

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.

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).

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.

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.

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.

Transfusion of blood components has the potential for both benefit and risk to the patient. According to the FDA Annual Summary of Fiscal Year 2009, 74 fatalities were reported following blood transfusions; forty-four of those fatalities were transfusion-related. Medical errors that could result in transfusion reactions include: Patient misidentification Sample labeling error Wrong blood type issued Transcription error Technical error Storage error 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.Reference: U.S Food and Drug Administration. (2009). Fatalities Reported to FDA Following Blood Collection and Transfusion: Annual Summary for Fiscal Year 2009. Retrieved from http://www.fda.gov/downloads/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/TransfusionDonationFatalities/UCM205620.pdf. Accessed April 26, 2011.

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 hemoglobulinemia and hemglobinuria 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.

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 pre-transfusion 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 pre-transfusion 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 which affected the blood, such as inappropriate warming or faulty infusion pump, should be suspected. If bacterial contamination is suspected, the unit can be cultured. A positive culture indicates a reaction due to bacterial contamination.

".....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 April 26, 2011.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.

Acute hemolytic transfusion reactions (AHTR) are caused when red cells are transfused to a patient with a pre-existing antibody that destroys the transfused incompatible red cells through intravascular or extravascular hemolysis. Life threatening acute hemolytic reactions most commonly occur 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. AHTRs feature rapid destruction 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. Fever is the most initial symptom followed by the chills. These reactions are mostly associated with the transfusion of ABO-incompatible red cells. Causes include clerical errors, such as mislabeled patient samples and mislabeled blood products. Although acute hemolytic reactions are rare with an incidence of 1 to 9 in 100,000 transfusions, they are the most dangerous and are severely life threatening.

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.

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 febrile nonhemolytic transfusion reactions (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.

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 and 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.

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.

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.

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 adminstered slowly when possible, particularly in patients at risk for TACO.

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.

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

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 (RBCs) 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/uL. 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.

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.The disadvantages are that they are not widely available at this time and are more expensive than the TST.

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.

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.

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.

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.

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.

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.

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.

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.

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.