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Blood Information and Courses from MediaLab, Inc.

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

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Laboratories Individuals

Cerebrospinal Fluid
True or false: most of the chemical elements in CSF have levels similar to blood levels.View Page
Which of the following criteria may invalidate CSF results?View Page
Which of the following characteristics are present if blood is due to brain hemorrhage?View Page
Location of CSF

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

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Initial Specimen Examination

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

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

When blood is present in a CSF specimen, it is necessary to determine whether the blood is due to a traumatic puncture or to a pathologic condition. There are several clues to help make this distinction: Traumatic tap:More blood is present in tube 1 than in tubes 2, 3, or 4.When sample is centrifuged within one hour, supernatant is clear.Blood clots on standing.Subarachnoid or cerebral hemorrhage:Blood is evenly distributed in all tubes.When sample is centrifuged within one hour, supernatant is pink or yellow.Blood does not clot on standing.

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

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

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Cells Seen in CSF

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

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Mature Peripheral Blood Cells

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

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Tissue Cells

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

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Monocytes

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

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

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

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Clinical Significance cont'd

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

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Clinical Significance cont'd

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

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Clinical Significance

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

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Clinical Significance of Positive Urine Ketone Result

Ketone bodies are usually absent in urine. High levels of ketones are present in the urine of individuals with uncontrolled diabetes. In diabetes the ketones are present because the body's ability to metabolize carbohydrates is defective. Detecting the presence of ketones in the urine is a valuable aid to managing and monitoring individuals with diabetes mellitus. Ketonuria is an indication that the insulin dose needs to be increased. It is also an early indicator of insulin dosage problems in juvenile diabetes or in diabetics experiencing other medical problems. Electrolyte imbalance and dehydration occur when ketones accumulate in the blood. If these conditions are not corrected, the patient may develop acidosis and ultimately diabetic coma. Low levels may be detected during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake or increased loss of carbohydrates.

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Hematuria

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

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The Test for Blood

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

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False Positive Results

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

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False Negative Results

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

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Clinical Significance

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

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

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

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CLIA Blood Banking Review
Which of the following activities will put an employee at risk for exposure to a Bloodborne Pathogen (BBP)?View Page
Which BBP is not covered in the OSHA Bloodborne Pathogen Standard?View Page
What should you do if your lab coat or gown has dried or caked-on blood on it?View Page
What type of Personal Protective Equipment (PPE) is necessary when opening a centrifuge (chance for splashing)?View Page
Why would a unit of group O blood never be administered to a Bombay patient:View Page
Which of the following blood group antigens are most susceptible to destruction by the action of enzymes:View Page
The classification of Du refers to:View Page
Which of the following is used as a source for irradiation of blood products:View Page
Based on the following reactions indicate the correct blood group for each set of reactions:View Page
Which of the following patients represents an acceptable donor.View Page
Which one of the following is not a benefit of using packed RBCs:View Page
Which of the following is most commonly associated with febrile non-hemolytic transfusion reactions:View Page
If a potential donor has been transfused blood products, he must be deferred from blood donation for:View Page
Which of the following tests must be repeated by the lab on homologous blood received from the Red Cross or other community blood sources:View Page
All of the following cellular antigens are important to an immunohematologist except:View Page
Deglycerolized red cells are most effectively used to:View Page
If an R1 r patient received R2R2 blood, which of these antibodies could be produced :View Page
Which of the following blood group antigen-antibody reactions is enhanced by using enzymes:View Page
Before testing all cord cells should be thoroughly washed in order to:View Page
Which of the following blood groups reacts least strongly with Anti-H:View Page
Which of the following are not appropriate indications for the use of fresh frozen plasma:View Page
Match the blood groups on the left with corresponding results of forward typing on the right.View Page
Therapeutic hemapheresis may be used to treat all of the following except:View Page
All of the following are benefits of autologous donation except:View Page
Which of the following types of whole blood would be the least satisfactory to transfuse to a type AB patient:View Page
The following steps must be followed in preparation of a platelet concentrate:View Page
Match each blood type with the corresponding antibody you would find in its serum:View Page
Type B blood is found in higher frequency in:View Page
Which of the following options gives in order from most to least important, the factors you would use to select blood for a transfusion:View Page
Which of the following statements is not true about the Lewis blood group:View Page
The use of cells with known blood groups to confirm ABO typing is known as:View Page
The generally accepted age range for homologous blood donation is:View Page
Fresh frozen plasma :View Page
An urticarial reaction is characterized by:View Page
Which of the following is responsible for causing graft-versus-host reactions:View Page
The shelf-life of whole blood collected in CPDA-1 is:View Page
The use of the direct antiglobulin test is indicated in all the following except:View Page
After transfusion, a red cell sample from the donor unit, and the recipient's blood sample, must be kept for:View Page
Which of the following refers to the most common procedure for donating whole blood for use by the general population:View Page
Which of the following is the proper storage temperature for whole blood:View Page
Which of the following is the most prevalent blood type found in the United States:View Page
Autologous blood must be tested for which of the following before transfusion:View Page
Gamma irradiation of cellular blood components is required in which of the following situations:View Page
A severe hemophiliac, with a Factor VIII activity of less than 1%, is actively bleeding due to a serious accident. The blood product of choice is:View Page
Match substance(s) secreted with blood groups:View Page
What percentage of glycerol is generally used when freezing red cells of rare phenotypes:View Page
Which of the following blood components will provide the best source of fibrinogen for a patient with hypofibrinogenemia:View Page
The accepted interval between blood donations is:View Page
How long may blood be stored using CPDA-1 preservative prior to transfusion?View Page
The most severe acute hemolytic transfusions reactions are the result of which of the following:View Page
The chief purpose of performing a standard crossmatch is to :View Page
Which of the following contains all the possible phenotypes that could be the result of parents who are type O and type A:View Page
A refrigerator used to store whole blood must be able to maintain a temperature in the ranges of:View Page
A false-negative reaction while performing the DAT technique may be the result of:View Page
A patient's serum reacts with all reagent red cell samples. The autocontrol is negative. An alloantibody to a high incidence antigen is suspected. Which of the following would be most likely to be a compatible donor:View Page
ABO blood groups were discovered by:View Page
In an extreme emergency , if the ABO and Rh type are unknown which of the following should be given to the patient?View Page

CLIA Chemistry / Urinalysis Review
Match collection tube colors and additive type on the right with clinical usage on the left.View Page
Which of the following anticoagulants will not produce a significant effect on calcium levels in plasma:View Page
Increases in blood ammonia levels would be expected in which of the following conditions:View Page
Which of the following blood additives is most useful for serum collection:View Page
The measurement of total glycosylated hemoglobin A1c is an effective means of assessing the average blood glucose levels:View Page
Carbon dioxide is predominately found in blood in the form of:View Page
Respiratory acidosis is associated with:View Page
Which one of the following serum constituents is increased following strenuous exercise:View Page
Which one of the following statements about lead poisoning is false:View Page
The primary mechanism responsible for glomerular filtration is:View Page
The renal threshold is best described as:View Page
Which one of the following statements about urea is false:View Page

CLIA General Laboratory Review
When drawing blood for coagulation studies, one should always:View Page
Which one of the following statements about Hepatitis is true?View Page
Which of the following is not appropriate for a routine blood specimen:View Page
Standard precautions means that:View Page
Which one of the following statements about the hepatitis B vaccine is correct?View Page
A smear that is prepared from equal parts of methylene blue and whole blood will be used for:View Page
Which of the following immunoglobulin classes is chiefly responsible for the degranulation of mast cells and basophils:View Page
The Kleihauer-Betke test is used to:View Page
Classic automated blood cell counters are based on:View Page
A definitive diagnosis of malaria can be made by:View Page
Hematocrit is:View Page

CLIA Hematology / Hemostasis Review
The RBCs found in this illustration are the result of:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Identify the cell in this illustration indicated by the arrow:View Page
Identify the cell in this illustration indicated by the arrow:View Page
What is the cell indicated by the arrow in this illustration:View Page
Found frequently in a newborn's blood the cells indicated by arrow in this illustration are:View Page
The cell indicated by the arrow in this illustration is called:View Page
The abnormal RBC shape seen in this illustration is:View Page
Which of the two WBCs indicated by the arrows on this illustration is normally the most numerous in peripheral blood and what is its name:View Page
The impedance principle shown in this illustration is best described by the following statement:View Page
Which of the following conditions is frequently associated with these cells?View Page
Expected life span of a neutrophil in the peripheral blood of an adult is:View Page
Which of the following major cellular elements does not develop solely in the bone marrow:View Page
What is another name used to designate a fully committed B-lymphocyte:View Page
Which of the following is not primarily a hemolytic process?View Page
Which blood cell is found in the largest numbers in the peripheral blood of a normal adult:View Page
Which of the following cells is most common in adult bone marrow:View Page
Hypochromia can best be described as:View Page
If greater than 50% lymphocytes were found on the peripheral blood smear of a 5 month old child you would suspect which of the following conditions:View Page
Which of the following may interfere with the accurate measurement of hemoglobin:View Page
The reticulocyte count is used to assess which of the following:View Page
The ratio of whole blood to anticoagulant is very important in the PT assay; at which hematocrit level should the standard anticoagulant volume be adjusted:View Page
When three tubes of cerebrospinal fluid are received in the laboratory they should be distributed to the various laboratory sections as follows:View Page
Which of the following would best describe what you might observe after a traumatic CSF tap:View Page
Which of the following blood smears these illustrations would be best suited for performing a differential count:View Page

CLIA Microbiology / Serology Review
This parasite is found in blood.View Page
This suspicious form was recovered in blood.View Page
Which of the following media contains the X and V factors necessary for the growth of Haemophilus influenzae:View Page
Which of the following organisms is best visualized by use of a darkfield microscope:View Page
With regard to blood cultures, which blood to broth ratio is most conducive to growth:View Page
On sheep blood agar Haemophilus influenzae may exhibit satellite formation around all but which of the following organisms:View Page
Match type of hemolysis on the right with best description:View Page
Which of the following is the most suitable specimen for the isolation of Bordetella pertussis:View Page
Which of the following specimens would not be considered suitable for anaerobic culture:View Page
Match type of media on the right with media on the left:View Page
Match the organisms on the right with culture medium:View Page
Which of the following parasites is not commonly found in the peripheral blood:View Page
Which of the following specimens is the most sensitive for detecting active CMV infection:View Page
Which of the following media is a selective medium for Campylobacter jejuni:View Page
Sheep blood agar contains inhibitors to which of the following organisms:View Page
The adult worms of which of the following reside in the intestine or its blood vessels:View Page
Which one of the following statements about E.coli O157:H7 is false:View Page
What is the best term to describe the clear areas seen around the colonies on this blood agar plate:View Page

Confirmatory and Secondary Urinalysis Screening Tests
Urine Bilirubin

Bilirubin is formed as a result of the breakdown of hemoglobin from erythrocytes in the reticuloendothelial system. It becomes bound to albumin and transported through the blood to the liver. This free or unconjugated bilirubin is insoluble in water and cannot be filtered through the glomerulus of the kidney. In the liver, bilirubin becomes conjugated with glucuronic acid to form bilirubin diglucuronide. This conjugated bilirubin, which is also called direct bilirubin, is water soluble and is excreted by the liver through the bile duct and into the duodenum.

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Urine Bilirubin

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

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The Ictotest®

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 Pyridium. Because of this it is important to verify positive bilirubin results with a confirmatory test such as the Ictotest®.

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The Presence of Glucose in the Urine

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

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Causes of Ketonuria

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 ketone bodies is often the cause of acidosis and coma in diabetics.

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The Acetest®

Urine to be screened for ketone bodies should be tested immediately or refrigerated in a closed container since acetone is lost to the air if the sample is left standing at room temperature for any length of time. The Acetest® can be used for the semiquantitation of ketones in urine, serum, or whole blood, however the reaction times differ depending on the type of specimen tested. The same substances which interfere with the dipstick tests for ketones will also interfere with Acetest® because the same reaction is involved.

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Current Topics in Clinical Microbiology
The patient was admitted to the hospital. The sputum specimen was inoculated to sheep blood agar. Based on the colony morphology seen in the accompanying photograph, the most likely identification is:View Page
The oxacillin screen test alone is not sufficient for determining the susceptibility to penicillin for S. pneumoniae isolates recovered from blood and CSF.View Page
MIC susceptibility tests should also be performed against other select beta lactam antibiotics on important S. pneumoniae isolates from blood cultures and other sterile body fluids.View Page
Clinical History

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

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

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

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The gram stain shown in the photograph was prepared from a positive anaerobic blood culture bottle after 36 hours incubation. Based on the morphology of the bacterial cells (some with spores--blue arrows), the most likely identification is:View Page
Colony Morphology

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

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

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

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Spleen Specimen

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

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Cellulitis Skin

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

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The Gram stain prepared from the positive blood culture is shown in the photograph. The appropriate report is:View Page
Group A Strep A Disk/SXT

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

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Beta hemolytic colonies grew from the blood culture bottle after 18 hours incubation (see photograph). The following tests would be helpful in making a preliminary identification:View Page
Colony Morphology

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

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Each of the following is related to the virulence of Listeria monocytogenes except:View Page
Review 3

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

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Eikenella BAP

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

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Descriptive Statistics
Discrete and Continuous Data

There are two main types of data that you might encounter.  The first is discrete data, which is a count of whole events, objects or persons.  For example, the number of people with a certain illness is a discrete quantity.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.

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Samples and Populations

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

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Table Specifications

Here are the criteria for the preparation of tables, as specified by the Journal of Clinical Laboratory Science: Write table titles at the top of the table. Number tables sequentially with Roman numerals. Include the following information in a title, whenever possible: who, what, where, why and when. Put the independent variable in the left column, and the dependent variable in the right, if you are listing data with independent and dependent variables. Label each column with the appropriate units. Adequately space tables that appear on the same page. Example:Table I Patient specimens analyzed for blood urea nitrogen on the Dimension RxL and the Vitros 250 at City Hospital Sample # RxL (mg/dL urea) Vitros 250 (mg/dl) urea 1 8.8 8.8 2 11.2 10.0 3 12.4 13.6 4 16.2 13.2 5 20.0 21.2 6 25.0 20.0 7 28.8 26.2 In this case, the Dimension RxL is the "reference method" and is considered the independent variable, while the Vitros 250 is the "test method" and is considered the dependent variable.

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Using Frequency Distributions

A frequency distribution is a chart that groups data into different classes, and then graphically shows how many data points fall into each class. A frequency distribution allows the reader to see easily the approximate center and spread of the data. Table II shows the frequencies of different hemoglobin concentrations. Figure 2 is a histogram of the data. Table II Frequency distribution of blood hemoglobin levels from healthy women determined on the Coulter Gen S Hemoglobin (gm/dL) Number of Women 6 - 8 1 8 - 10 2 10 - 12 10 12 - 14 25 14 - 16 9 16 - 18 1 Figure 2 Frequency Distribution Blood Hemoglobin Levels from 48 Healthy Women Determined on the Coulter Gen S

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Erythrocyte Inclusions - Wright Stained Smears
More on Pappenheimer bodies

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

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Reticulocytes

Although the nucleus has been extruded, the reticulocyte is still considered immature because it retains numerous organelles needed for hemoglobin production, such as ribosomes, mitochondria, and fragments of the Golgi apparatus. The reticulocyte is slightly larger (10 microns) than the mature erythrocyte. A reticulocyte normally remains in the bone marrow for one or two days before entering the circulation and its final 24 hours of maturation. The red cell is mature when hemoglobin production is complete and the organelles have disintegrated. Reticulocytes normally make up 0.5 - 1.5% of the peripheral blood red cells. They appear blue/gray on the Wright's stained smear. The residual RNA in the cytoplasm causes the blue/gray color. The terms, polychromasia or polychromatophilic, are used to describe these cells on a Wright's stained preparation. A supravital stain such as new methylene blue N or brilliant cresyl blue is used to stain reticulocytes for an actual count.

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Stress Reticulocytes

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

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First Aid
Applying Direct Pressure

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

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Elevate the Wound

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

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Fundamentals of Hemostasis
An Introduction to the Fundamentals of Coagulation

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

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An Introduction to the Fundamentals of Coagulation

Blood flow 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 damage Ability of vasoconstriction to occur Availability of platelets & their functionality Availability of clotting factors & their functionality Absence of inhibitors & anticoagulants

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An Introduction to the Fundamentals of Coagulation

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

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Primary Hemostasis – The Vascular System

Our blood circulates freely through undamaged, intact vessels. The design of the vasculature, or blood vessels, is such that the walls of the vessels are chemically inert to both coagulation factors and platelets under normal conditions. Damage to a vessel breaks that inert epithelial lining, exposing the subendothelium and collagen, and releasing chemical signals that trigger subsequent hemostatic mechanisms.

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Primary Hemostasis – The Vascular System

Overview of Vascular System Involvement in Primary Hemostasis: Vasoconstriction Reroute blood flow Platelet aggregation Contact activation of coagulation system (start of secondary hemostasis at this point)

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Primary Hemostasis – The Vascular System

The first specific, recognizable hemostatic mechanism is a process known as vasoconstriction, which is initiated by chemical signals stemming from a breach of the vasculature. 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. A short process in terms of the overall time elapsed, the entire vascular response typically lasts less than one minute! Though fleeting, vasoconstriction is an exceedingly important hemostatic mechanism as it prepares the damaged vessel for subsequent repair activities.

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All of the following processes occur during primary hemostasis except:View Page
Summary of Primary Hemostasis

In summation, we have covered the following sequence of events which comprise primary hemostasis. The process begins with damage to a vessel wall, as blood flows outside the vasculature. The body responds with vasoconstriction, decreasing blood flow to the affected area. Platelets begin sticking to the damaged vessel walls. As the platelets stick, they release chemicals which signal other platelets to respond. As other platelets arrive, they begin sticking to one another, clumping together, forming a plug to fill in the breach. 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. Construction of the fibrin strands occurs during secondary hemostasis, our next topic to be covered.

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Secondary Hemostasis – The Extrinsic Pathway

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

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The Fibrinolytic System

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

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The Fibrinolytic System

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

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Collecting Blood Specimens for Coagulation Testing

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

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Fibrin/Fibrinogen Degradation Products and D-dimers

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

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Which of the following statements is incorrect?View Page
Coagulation Disorders - Acquired

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

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Anticoagulation Therapy - Heparin Therapy

The use of heparin is prophylactic. It is used either to prevent thromboembolism (a condition in which a blood clot forms inside a vessel), or used to limit a previous thromboembolism. Heparin inhibits thrombin. The degree of inhibition is dosage dependant. 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) and/or activated clotting time is used to monitor unfractionated heparin therapy.

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

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

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

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Potentially infectious body fluids

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

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Risk factors associated with increased HIV infection

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

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

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

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Overview

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

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Face and Eye Protection

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

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Gloves

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

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Introduction to Bioterrorism
Agent: Pneumonic plague (Bacterium)

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

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Early symptoms of inhaled Anthrax includeView Page
Types of Chemical Agents

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

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Blood Agents

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

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Laboratory Response - Chemical, Level 2

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

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Laboratory Response - Chemical, Level 1

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

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

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

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Preparation of Direct Smears

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

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Basic Structure and Function of Bone Marrow

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

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Supporting Cells

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

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Location of Cells within Cord

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

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Lymphocytes

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

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Summary

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

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

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

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Increase Marrow Iron Stores

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

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Evaluation of Bone Marrow

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

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

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

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Introduction to the ABO Blood Group System
Match the blood type on the left with the appropriate description on the right.View Page
An individual with type AB blood will demonstrate the complete absence of which of the following antigen sites?View Page
If an individual has blood type O, which of the following are possible genotypes?View Page
Use the pull down boxes to match the blood types on the left with the correct phenotype on the right.View Page
In what way are the ABO serum antibodies unique among blood group systems?View Page
Why is it dangerous to transfuse a person with type O blood with a unit of A blood?View Page
The History of the ABO System

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

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Table 1: ABO Blood Group System

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

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

In routine practice, specially prepared blood grouping sera containing anti-A, anti-B, (and optionally anti-A,B) are used to identify the four types of red cells. These sera will agglutinate cells with the corresponding antigen. This is called forward typing. ABO Blood Group Patient Red Cells Tested with Known Antisera Anti-A Anti-B Anti-A,B A 4+ 0 4+ B 0 4+ 4+ O 0 0 0 AB 4+ 4+ 4+ + = agglutination (graded 1+ to 4+)0 = no agglutination

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

It has been demonstrated that antibodies occur predictably in the sera of all normal adults in association with the ABO antigens. Demonstration of these antibodies is therefore 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 Group Patient Serum Tested with Known Reagent Cells A Cells B Cells A 0 4+ B 4+ 0 O 4+ 4+ AB 0 0 + = agglutination (graded 1+ to 4+)0 = no agglutination or hemolysis

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Importance of Understanding the ABO System

While the predictability of ABO antibodies in persons lacking the corresponding antigen makes the ABO blood group system an easy one for testing purposes, it can be treacherous as far as transfusion is concerned. If a patient receives cells containing A or B antigens and his/her serum contains the corresponding antibody, the donor cells will be destroyed almost immediately with severe and sometimes fatal transfusion reaction. It is, therefore, of utmost importance to thoroughly understand the ABO blood group system. Compatibility of the ABO system is essential for all other pre-transfusion testing.

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

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

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Bombay Blood Group Genes

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

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Inherited Genes

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

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

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

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

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

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A1 and A2

The most common classifications are A1 and A2. These account for over 99% of group A bloods. Of this 99%, A1 compromises approximately 80%. Commercial anti-A typing serum does not differentiate between A1 and A2 cells. A1 cells contain “A” antigen and “A1” antigen. A2 is not really a unique antigen. It is thought to be simply “A” antigen with no “A1” antigen. Several preparations are available that will react with A1 cells, but not other subgroups of A. An extract of the seeds of the plant, Dolichos biflorus has specific anti-A1 activity. “Absorbed anti-A” serum can also be prepared. To do this, the anti-A from group B people is absorbed with A2 cells. Anti-A is removed and a second antibody that reacts only with A1 cells remains. Anti-A1 can also be found as a separate antibody in the sera of A2 and A2B individuals.

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Agglutination Reactions

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

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Example of an ABO discrepancy

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

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel
Description of Specialties (1)

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

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Description of Specialties (2)

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

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Description of Specialties (3)

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

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Description of Specialties (4)

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

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

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

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Avoiding Systems Failure

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

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Direct Error Detection Even perfect systems designs cannot avert human limitations. Medical errors occur and they have to be detected before they can be resolved. Sometimes people directly observe and immediately report these mistakes.View Page
JCAHO Patient Safety Goals JCAHO adopted national patient safety goals for laboratories and many other healthcare organizations. 2006 Laboratory Services National Patient Safety Goals These goals are directly quoted.View Page
Errors of Commission

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

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

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

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

Medicare Compliance for Clinical Laboratories
Case Study 10

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

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Case Study 3

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

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