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

This is an example of an electrophoresis on normal blood.

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

Children with beta thalassemia major, also called Cooley's anemia, usually develop clinical signs during their first year of life. They appear to be malnourished and may exhibit abdominal girth expansion. They show skeletal deformations, which are a result of increased erythropoiesis. A common finding is facial bone changes. Other clinical signs include frequent infections, hepatomegaly, splenomegaly, cardiomegaly, gall stones, leg ulcers, and poor growth and sexual development. Death usually occurs by the time these patients are in their early twenties unless treated with blood transfusions along with iron-chelating agents. If no chelating agent is used during treatment life will only be prolonged by about a decade.Beta thalassemia is found most often in populations of people from the Mediterranean, southern China, and India.

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

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

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

In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes. 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.

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.

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

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

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

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

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

Bilirubin is 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.

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®.

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.

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

The growth observed on the anaerobic blood agar plate after 48 hours incubation (see upper 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.

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.

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.

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.

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.

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%).

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.

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.

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

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

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

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

Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.htmlAccessed June 23, 2009.Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919.Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615.Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165.Interpretive Handbook, (MC0440rev0407) Mayo Clinic, Rochester MN;2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24.Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217.NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed June 23, 2009.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: Burtis CA, Ashwood ER. Bruns DE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565.Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42.Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46.Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360.Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164.Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033.Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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

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

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

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

An alternative to liver biopsy as a means of documenting iron overload may be provided by quantitative phlebotomy performed during treatment (See next section.) The removal of 4 to 5 grams of iron through documented successive phlebotomies (16 to 20 phleblotomies) without development of anemia is indicative of iron overload. (One unit, or 450 mL, of blood is assumed to contain approximately 200 to 250 mg of iron.) Quantitative phlebotomy is useful in patients for whom liver biopsy is contraindicated, refused, or not needed for other reasons.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The setting is 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Standard precautions mean that all blood and body fluids should be handled as if they were infectious and capable of transmitting disease. Standard Precautions apply to all: Blood Body fluids Secretions (except sweat) Excretions Non-intact skin Mucous membranes

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

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

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

There are approximately 800,000 to 1.4 million chronic hepatitis B carriers in the U.S. Worldwide it is estimated that there are 350 million people infected with HBV, which contributes to an estimated 620,000 deaths worldwide each year (CDC, 2008).The annual number of occupational infections has decreased 95% since hepatitis B vaccine became available in 1982, from more than 10,000 in 1983 to less than 400 in 2001 (CDC "Exposure to Blood" report).

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

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

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

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

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

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

Laboratory specimens that are unlikely to cause disease and do not meet the criteria for category A or B substances are not subject to Division 6.2 regulations. Specimens for which the hazardous materials regulation (HMR) does not apply include human or animal samples (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, and body parts) being transported for routine testing not related to the diagnosis of an infectious disease. This includes specimens that are being sent for: drug or alcohol testing cholesterol testing blood glucose level testing prostate specific antibody (PSA) testing testing to monitor kidney or liver function pregnancy testing tests for diagnosis of non-infectious diseases such as cancer biopsies

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Water (HĢ0) makes up the majority of the blood plasma.

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

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

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

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

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

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

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

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

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

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

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

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

Reticulocytes are red blood cells prematurely released from the bone marrow. On a Wright-Giemsa stained blood smear, they appear as polychromatic macrocytes. Their presence in the peripheral blood may suggest hemolysis or bleeding. Their presence is expressed as a percentage of the red cell count: newly born= 3-7%; up to one week of age=1-3%; >one week =0.3-1.8%. Automated or manual methods may be used to enumerate reticulocytes. In clinical context, retics must be separated from debris, precipated stain, Pappenheimer bodies, Howell-Jolly bodies, and Heinz bodies.

G6PD deficiency occurs in the same geographic distribution as malaria. It has been theorized that enzyme deficient cells are more resistant to malarial parasites than normal cells.When hemolysis is triggered, the appearance of the red blood cells is modulated by activity of the spleen.Spherocytes, schistocytes, and nucleated red blood cells may appear in the peripheral blood.Denatured hemoglobin removed by an active spleen may leave bite cells, identified by the arrows in this photomicrograph, suggesting the presence of G6PD deficiency.

Illustrated in this field is a normoblast and a myelocyte, representing leukoerythroblastosis, a term associated with the release of immature cells from a disrupted marrow. Metastatic disease in the bone marrow, particularly in patients with primary breast or prostate cancer, is usually the culprit. Leukoerythroblastosis in the absence of anemia or thrombocytopenia is a signal to search for cancer metastic to the marrow. Nucleated RBCs were not identified on the blood smear seen here but were detected by an automated analyzer.The mortality rate of elderly patients with increased NRBCs, especially following accidents or general surgery, is greater.

Two photographs of a peripheral blood smear are submitted for review . The smears are from a 9-month-old baby with a heart valve replacement. In the upper photograph is a nucleated RBC and platelets are decreased. Nucleated red cells and occasional giant platelets indicate an active marrow response. In the process of forcing blood cells through the heart valve, erythrocytes are damaged, schistocytes are formed, and platelets are destroyed leading to thrombocytopenia. In the lower field are schistocytes, acanthocytes, echinocytes (burr cells), spherocytes, and the absence of platelets. The presence of burr cells could represent an artifact of smear preparation, but with the history of valve replacement, the red cell changes are likely the result of red cell damage as the cells circulate through the new valve. This situation is described as Waring Blender Effect because of damage to blood cells passing through the new valve, looking as if they had suffered the onslaught of a blender. Target cells and mild hypochromia may reflect iron deficiency through the loss of iron from destruction of RBC's. Iron loss through red cell destruction may be reflected in some hypochromia.

Stomatocytes are erythrocytes with a slit-like central pallor. Otherwise, they resemble typical RBC's in size and shape. Unless 10% or more of the RBC's are stomatocytes, their presence is probably artifactual. Stomatocytes form at a low blood acidic pH as seen in exposure to cationic detergents, and in patients receiving phenolthiazine. Hereditary stomatocytosis has some resemblance to hereditary spherocytosis, as stomatocytes may develop into spherocytes with further metamorphosis. In hereditary stomatocytosis, mild anemia and findings of on-going hemolysis should be evident if the condition presents as a clinical problem at all.

Laboratory data must be presented to clinicians in a user friendly way to promote effective decision making. Databases must be designed to provide clear information that leads quickly to the best patient care outcome. We continue learning how to collect and retrieve laboratory data from our machines, but we are not always in tune to how entry and retrieval of data is geared to and, more directly, influences patient care outcomes. Examples of blood cell abnormalities on a peripheral blood smear that may immediately direct the physician to a specific diagnosis are: (1) presence of target cells as found in thalassemia or hemoglobinopathies and target cells in liver disease, particularly with obstructive jaundice; (2) burr cells as a signal of chronic renal disease and uremia; and (3)atypical neutrophil inclusions relating to genetic disorders. Critical appraisal of such observations could add valuable clues for a diagnosis. Laboratory professionals must establish a set of principles for orderly observation of blood cell morphology, have a clear vision of the applications of their work, and understand the potential clinical implications of their reports and interpretations. Emphasis on values and relevance focuses on patient care outcomes and their dependency on prompt availability of results and contextual interpretations.

In a study on the reporting of red blood cell morphology abnormalities conducted in Ontario, Canada (Hookey L, Dexter D, Lee DH, Laboratory Hematology 7:83-88, 2001), fewer than 50% of 33 participants used the same term to describe the quantitative frequency of peripheral blood abnormalities. Seven blood smears, each containing one of several abnormal erythrocytes-- schistocytes, teardrop cells, acanthocytes, and Howell-Jolly bodies--were evaluated by 32 participants. The participants were asked to document their evaluations from a list of quantitative terms. There was a heterogeneity in the use of terms "rare," "slight," "occasional," "few," "mild", "present," "moderate," "many," and "marked." Choices of terms were subjective without points of reference. Guidelines for establishing standardized qualitative estimations of abnormal erythrocytes in the peripheral smear are presented as follows: 1+ = 2 - 4/Oil Immersion Field (OIF) 2+ = 5 - 7/OIF 3+ = 8 - 10/OIF 4+ = >10/OIF. The terms "few," "moderate," "many," and "marked" may be substituted for the 1+ - 4+ grading system, but only when their specific points of reference are universally understood in tandem with the above guidelines. A comment should be triggered if any erythrocyte abnormalities are seen in numbers >3/OIF including, but not limited to, polychromasia, basophilic stippling, nucleated RBC's, and Howell-Jolly bodies. Rouleaux or RBC agglutination are important findings and must be documented.

This peripheral blood smear is from a patient with pernicious anemia, which results from an inability to absorb the vitamin B12 needed for DNA synthesis. Since many cells are destroyed in the bone marrow, decreased numbers of red cells are present in the circulating blood, resulting in anemia. However, the red cells that are present are generally macrocytes and are filled with hemoglobin.

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

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

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

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

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

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

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

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

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

Clinical and Laboratory Standards Institute (CLSI). Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays; Approved Guideline. Fourth ed. CLSI document H21-A4. NCCLS. Wayne, PA: 2003.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard. Sixth ed. CLSI document H3-A6. NCCLS. Wayne, PA: 2007.Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third Edition. CLSI document H18-A3. NCCLS. Wayne, PA: 2004.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.Lowe B. Reinforcing safety sticklers. Advance for Medical Laboratory Professionals. May 2004; 16:2A-3A.The Joint Commission. Patient Safety-2009 National Patient Safety Goals. Available at: http://www.jointcommission.org/PatientSafety/NationalPatientSafetyGoals/. Accessed July 18, 2009.

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

Most blood collection tubes contain an additive that either accelerates clotting of the blood (clot activator) or prevents the blood from clotting (anticoagulant). A tube that contains a clot activator will produce a serum sample when the blood is separated by centrifugation and a tube that contains an anticoagulant will produce a plasma sample after centrifugation. Some tests require the use of serum, some require plasma, and other tests require anticoagulated whole blood. The table below lists the most commonly used blood collection tubes. Tube cap color Additive Function of Additive Common laboratory tests Light-blue 3.2% Sodium citrate Prevents blood from clotting by binding calcium Coagulation Red or gold (mottled or "tiger" top used with some tubes is not shown) Serum tube with or without clot activator or gel Clot activator promotes blood clotting with glass or silica particles. Gel separates serum from cells. Chemistry, serology, immunology Green Sodium or lithium heparin with or without gel Prevents clotting by inhibiting thrombin and thromboplastin Stat and routine chemistry Lavender or pink Potassium EDTA Prevents clotting by binding calcium Hematology and blood bank Gray Sodium fluoride, and sodium or potassium oxalate Fluoride inhibits glycolysis, and oxalate prevents clotting by precipitating calcium. Glucose (especially when testing will be delayed), blood alcohol, lactic acid

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

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

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

Round cells in semen are of two types: immature sperm and white blood cells. To determine the percentage of white blood cells (specifically granulocytes) a special leukocyte screening test must be done. This test involves staining for the peroxidase enzyme present in the granulocytes.The 1999 WHO manual contains a protocol for doing this test (Appendix III). There is also at least one test kit on the market for this assessment (Leukoscreen: Bioscreen, Inc.).Laboratories with particular expertise in doing CBC and assessing granulocytes in stained blood smears may be able to do a differential count by this method rather than using a biochemical test for leukocyte screening.

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

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

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

These ABO, Rh, and antibody screen results were obtained by the TS using the blood specimen that was collected prior to starting the emergency transfusion with O Rh-negative RBCs. ABO and Rh typing ABO Forward Group ABO Reverse Group Rh anti-A anti-B A1 cells B cells anti-D 0 0 4+ 4+ 3+ Antibody screen Cells Gel IAT* Screen Cell I 3+ Screen Cell II 2+ Screen Cell III 2+ * IAT = indirect antiglobulin test

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

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

Some blood safety standards require that a list of common signs and symptoms of suspected adverse reactions be included in both nursing and transfusion service manuals. Several organizations have developed job aids to help clinical staff recognize the signs and symptoms of various suspected transfusion reactions and to suggest appropriate actions (e.g., see REACT in Online Resources).

Increased numbers of cuboidal cells are found in renal transplant rejection, acute tubular necrosis (diuretic phase), injuries that interrupt blood flow to the kidney, and acute glomerulonephritis accompanied by tubular damage. Ingestion of various drugs and chemicals may cause significant tubular shedding of these epithelial cells. Cuboidal cells are easily seen in urine in cases of salicylate intoxication.

It is important to identify and treat persons with LTBI to prevent progression to active disease. Currently there are two tests available to identify LTBI.The tuberculin skin test (TST) is performed on the inner arm.The Blood Assay for Mycobacterium tuberculosis (BAMT) is performed on a blood specimen.

When examining a slide made from an EDTA tube of normal blood, an occasional cell containing a round pyknotic nucleus and neutrophilic-appearing cytoplasm may be seen. Rare cells such as these do not indicate the presence of Pelger-Huet anomaly.

This blood smear was taken from a patient with the May-Hegglin anomaly. A May-Hegglin Dohle body is indicated by the arrow near the edge of the cytoplasm at the top of the neutrophil. In addition, notice the giant platelet that is indicated by the red arrow, another characteristic of May-Hegglin anomaly.

Toxic granulation is manifested by the presence of large granules in the cytoplasm of segmented and band neutrophils in the peripheral blood. The color of these granules can range from dark purplish blue to an almost red appearance. Toxic granules are azurophilic granules normally present in early myeloid forms, but which are not normally seen at the band and segmented stages of neutrophil maturation. These granules contain peroxidases and hydrolases. Toxic granulation is seen in cases of severe infection, as a result of denatured proteins in rheumatoid arthritis or, less frequently, as a result of autophagocytosis. Infection is the most frequent cause of toxic granulation. This type of granulation may be seen in cells which also contain Dohle bodies and/or vacuoles. Cells containing toxic granules may have decreased numbers of specific granules. Cells containing only a few specific granules, with or without toxic granules, are said to be degranulated. The nucleus in degranulated cells may often be round-bilobed, smooth and pyknotic. This type of nucleus is the result of aging and will disintegrate soon. Increased basophilia of azurophilic granules simulating toxic granules may occur in normal cells with prolonged staining time or decreased pH of the stain.

A reproducible blood smear review requires every peripheral smear be prepared for consistent openness and clarity. Consistency is maintained by uniform handling of every blood smear. Good results may be expected when the preparation is begun with only a small drop of blood at one end of a clean glass slide. The drop is smeared lightly and quickly so as to leave a thin (feathery) edge where all cells may be examined individually, particularly red blood cells. The site of examination then is chosen away from clumping, piling, or bumping of cells against each other, perhaps a site five or six oil fields from the end of the feathery portion. Such an area for examination is illustrated in the photograph.

The findings in the photograph from a peripheral blood smear would elicit a report comment of "increased platelets" of some high magnitude, such as "marked" or "4+". Estimates of platelet counts from review of a peripheral blood should be made on each smear examined. This provides a simple estimate of "high" or "low" or corroborates the value generated from an electronic cell counter. A formula for estimating platelet counts must be established in each laboratory. Following is a guideline: 5/oil power field (OPF) = 100,000/cumm; each platelet thereafter = 10,000/cumm. Thus, if an average of 10 platelets/OPF are observed, the estimated platelet count is 150,000.cumm. Such a counting scheme for platelets when clustered as in the photograph is probably not needed, as there are more than 100 platelets in the field. This translates into a platelet count of 1 million/cumm or more. This peripheral smear observation, however, would serve to corroborate an electronic platelet count of 1.2 million/ cumm.

The term leukemoid reaction is used to describe peripheral white blood cells that on the stained blood smear may have some resemblances to leukemia cells. Quantatively in a leukemoid reaction, the neutrophil count is >50,000 cumm with more immature cells, particularly myelocytes, than are usually present in toxic left shift syndromes. The presence of immature cells in a leukemoid reaction awakens thoughts of leukemia. Great care must be taken to make a distinct differentiation between aberrant white blood cell proliferations and a benign but exaggerated granulocytic proliferative response. Our material is from a 1-month-old girl with Down's syndrome. Her total white blood count was 37,000/mm3 interpreted as leukocytosis with left shift. Leukocytosis with a left shift, and leukemoid reactions with high alkaline phosphatase are conditions to be mindful of in patients with Down's syndrome. The alkaline phosphatase score is high in leukemoid reactions, low in granulocytic leukemia.

The presence of atypical inclusions within the cytoplasm of neutrophils and other leukocytes should lead to a clinical investigation of the setting for these findings.Atypical neutrophil inclusions may be seen in the following disorders: Chediak-Higashi syndrome, May-Hegglin anomaly, Alder-Reilly anomaly, Fechtner , Sebastian, Epstein and Alport-like syndromes and in infectious and toxic conditions (in the form of Doehle bodies).Although a specific entity may not be evident from examination of the peripheral blood alone, it is important that hematology technologists include a comment reporting on the presence of these inclusions or granules. A clinical investigation with further hematologic and genetic studies may then appropriately be considered.Many of the disorders with atypical neutrophil cytoplasmic granules are also associated with platelet abnormalities, particularly giant platelets (lower photograph).Therefore, when atypical granules are recognized, scanning of the peripheral blood smear for atypical platelets may be revealing. These observations serve as readily identifiable markers for acquired and genetic human maladies, and as a guide for unraveling the reasons for a patient's suffering and impaired health.

A 17-year-old young woman was admitted to the hospital with abdominal pain and a tentative diagnosis of appendicitis.The total white blood count was 14,500 cells/cumm with a left shift and neutrophils with changes tagged by the arrow in the photographs (see blue arrow).The bluish-staining, blurred accumulations in the cytoplasm (Doehle bodies), are located at the cell periphery in neutrophils with toxic changes.Doehle bodies are remnants of endocytoplasmic reticulum and are products of cytokine activity in the induction and shortened activity of neutrophil activation.They are often present in conditions with increased neutrophil lysosomal activity, manifest as toxic granulation.In this case, the presence of Doehle bodies serves as markers for infection-induced leukocytosis and supports the diagnosis of acute appendicitis.

An 80 year old man was seen in the emergency room with sudden onset of right sided chest pain accentuated on inspiration. His cough was productive of yellow sputum, and he was short of breath.His temperature was 101.2F. A chest X-ray revealed right middle lobe pneumonia. His hemoglobin was 15.2 gm/dl, HCT 44%, and RBC 4.5 m/ml. The white blood count was 35,000/cuml, with 45% neutrophils, 20% bands, 5% lymphocytes, 3% eosinophils, 2% basophils, and 25% atypical monocytes as noted in the photograph.The atypical monocytes had abundant blue-grey cytoplasm with a few scattered vacuoles, which, in company with toxic neutrophils appeared to be a response to infection.The patient had a past history of tuberculosis which may account for the monocytosis.