Diagnostic Laboratory Medicine

blood constitutes 6-8% of total body weight fluid plasma comprises 45-60% of total blood volume there is, on average, 5600 ml of blood in a 70K male (or approximately 5.5 liters) the normal pH of blood ranges from 7.35 to 7.45 there are three accepted "sources" of blood collection which include skin puncture, venous puncture and arterial puncture.

Skin Puncture blood collected by skin puncture is a mixture of arterial and venous blood and is often erroneously considered "capillary blood" this blood is often mixed with interstitial and intracellular fluids skin puncture is generally the method of choice in the pediatric patient there is a genuine risk associated with venous puncture in the pediatric patient which includes cardiac arrest, hemorrhage, thrombosis, venous constriction with subsequent gangrene, and damage to organs or tissues which are accidentally punctured or secondarily infected skin puncture is also occasionally utilized in the adult patient situations where this would be appropriate include extreme obesity, fragile veins, severe burns and thrombotic tendencies it is also utilized in home testing for blood glucose levels Technique selection of an appropriate site is crucial in infants the lateral or medial plantar heel is usually chosen with older infants and adults, the palmar surface of the second, third or fourth fingers is more appropriate - other sites may include the thumb, ear lobe or hallux.

warming the area with a moist towel ( no hotter than 42C) will increase blood flow to the area the area should be prepped with alcohol - which must be allowed to completely dry to prevent dilution or contamination of the specimen the puncture should be made with a sterile lancet or lancet pen theoretically, the first drop of blood should be discarded (however, this is not always practical) the puncture site should not be "milked" as this may cause hemolysis or introduce excessive tissue fluid into the specimen when utilized for hematocrit, the specimen is often collected directly into a s mall capillary tube for future centrifugation

Venous Puncture one must keep in mind that venous blood is oxygen deficient relative to arterial blood composition of venous blood varies dependent on the metabolic activity of perfused organs it differs from arterial blood in pH, carbon dioxide content and packed cell volume venous blood is usually a darker red than arterial blood as it has passed through the capillaries where oxygen is transferred from the blood to surrounding tissues the ease of obtaining venous blood makes it the primary source for clinical studies Technique assemble all the necessary equipment (needles, collection tubes, tourniquet, alcohol wipes, etc.) prior to beginning ask the patient to make a fist - generally this will distend the veins and make them more readily palpable

venipuncture is most easily accomplished in the antecubital fossa - the median cubital and cephalic veins are preferred other suitable veins include wrist, ankle and hand veins veins on the flexor surface of the forearm are often used to place an IV line this location has the advantage of allowing the patient to move the arm more freely without compromising the IV line also, if the IV fails, the line can always be placed more proximally however, these veins are often difficult to hit because of their tendency to roll. Once identified, the vein should be stabilized between the thumb and index forefinger - proximal and distal to the puncture site in the elderly patient veins are often difficult to find - the use of a blood pressure cuff may enhance venous distention and allow for a less difficult insertion of the needle cleanse the area with an alcohol wipe - be certain to let the alcohol dry prior to the needle puncture apply a tourniquet several inches above the intended puncture site anchor the vein above and below the puncture site the needle should be delivered at a 15 angle through the skin with the bevel of the needle up the needle should be delivered quickly through the skin but do not "bury" it into the skin direct the needle along the proximal path of the vein release the tourniquet when the blood begins to flow the patient should relax his/her fist when all the blood has been drawn apply pressure with a sterile cotton ball or 2x2 to prevent hematoma or extravasation of blood into the subcutaneous tissues (this may cause a nasty looking bruise if blood is accidentally injected into the surrounding tissue. It is also uncomfortable for the patient. Should this occur, warm, moist compresses may alleviate some of the symptoms. Careful technique is the best safeguard against these complications.)

Arterial Puncture arterial blood is uniform in composition throughout the body arterial blood is utilized for blood as measurements the radial artery is the most common site but the presence of collateral circulation must be verified with the Allen test the modified Allen test ascertains the patency of the ulnar and radial arteries. (Compress the radial and ulnar arteries until the palm of the hand becomes blanched. Release pressure from the ulnar artery and the hand should become flushed. If the hand remains blanched, do not puncture the radial artery.) radial artery puncture is quite uncomfortable the femoral artery and brachial artery at the antecubital fossa provide alternative sources. In the infant, scalp arteries are used. Technique it may be easier if the puncture site is first infiltrated with a local anesthetic prepare the needle and use a heparin collection tube (green top) prepare the skin with alcohol and let it dry thoroughly the artery is identified with a finger and the needle delivered, bevel up, distal 5 to 10 mm distal to where the pulse is felt quickly withdraw the needle and apply firm compression at the site for a minimum of five minutes expel air bubbles from the specimen and mix gently to stir the anticoagulant into the specimen the specimen is transported immediately to the lab for analysis
GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

Collection Tubes for Whole Blood serum is whole blood collected in a tube without any anticoagulant red top no anticoagulant whatsoever the blood will clot within the tube and the serum will be separated via centrifugation it is used for blood chemistries and blood bank tiger top (serum separator tube SST) polymer gel with a silica activator the silica will enhance the speed of clot formation and the polymer gel forms a thin barrier between the serum and the cells on the bottom of the tube it is utilized for chemistries

plasma is anti-coagulated whole blood in clinical hematology and serology, several anti-coagulants are utilized. These include: trisodium citrate - blue top - coagulation studies EDTA - lavender top - blood counts, lymphocyte markers green top lithium or sodium heparin - blood gases, cortisol, porphyrins, vitamin assays gray top - sodium fluoride and potassium - glucose, lactate (fluoride stops glycolysis) yellow top - sodium polyanethol sulfonate and sodium chloride - HLA tissue typing, paternity testing, DNA studies

Principles of Interpreting Laboratory Values never rely on a single lab value, but rather look for trends "Osler's Rule" - in patient's less than 60 years of age, one should seek to attribute all abnormal lab values to a single underlying pathology all abnormal lab values should be repeated

Coagulation Studies blood should be collected in a sodium citrate (blue top) tube citrate was first utilized as an anticoagulant in 1914 it acts via chelation, binding with calcium to prevent the coagulation cascade the mixture of citrate phosphate dextrose was formulated in the 1950's - this increases red blood cell viability adenine may also be added which provides a substrate for red cells to maximize ATP synthesis and, therefore, increase red cell viability coagulation testing is critically dependent upon the quality of the plasma specimen obtained

Prothrombin Time this test evaluates the time it takes for a fibrin clot to form following the addition of tissue extract and a phospholipid - typically an extract from brain or lung - in citrated plasma - in the presence of excess calcium it evaluates the extrinsic clotting pathway reflects the activity of the vitamin K dependent clotting factors (factors II, VII, IX, X , protein C and protein S) these factors are synthesized in the liver - decreased production of these factors could result in a prolonged prothrombin time
GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

common conditions where PT would be prolonged include vitamin K deficiency, impaired fat absorption, severe liver disease and coumadin therapy reference values: 11-13 seconds or within 2 seconds of control labs will frequently report PT via the "international normalized ratio" or INR this was developed in an attempt to minimize variations in reported PT values between different clinical laboratories it is used to characterize prothrombin time only and is a measure of the activity of the vitamin K-dependent factors it is calculated as follows: INR = observed PT x ISI* control PT
*

international sensitivity index - this is a calibration factor. It is a value determined by the manufacturers of the thromboplastin reagent

the American College of Chest Physicians recommends and INR between 2.0 and 3.0 for prophylaxis against pulmonary embolism coumadin prophylaxis for patients with mechanical heart valves should be maintained with an INR between 2.5 and 3.5

Partial Thromboplastin Time this is considered to be the best singular screening test for coagulation it evaluates both the intrinsic and final common clotting pathway (factors XII, XI, IX, VIII [X, V, II and fibrinogen]) factor Xa is where the intrinsic and extrinsic pathways join to form the common pathway (this factor is inhibited by enoxyparin) a standardized phospholipid preparation is added to it as a platelet substitute in the excess of calcium - this will speed up coagulation is reflected as aPTT or "activated" it is used to screen for disorders of fibrin formation and to monitor heparin therapy reference values: PTT - 60-85 seconds aPTT - 30 - 40 seconds or within 5 seconds of control Antifactor Xa Assay this is used to monitor therapeutic levels of low-molecular weight heparins and danaparoid since they do not affect PTT monitoring for LMWH and danaparoid however is rarely warranted as they have very predictable doseresponse effect renal disease may result in higher than desirable levels it may also be ordered in certain factor deficiencies (for example, factor XII) or in patients with lupus anticoagulants resulting in a prolonged baseline PTT

reference values:

not on anticoagulants 0 units/mL heparin 0.3 - 0.7 units/mL LMWH 30 mg twice daily 0.4 - 1.1 units/mL LMWH 40 mg once daily 1 - 2 units/mL Danaparoid (Orgaran) 0.5 0.8 units/mL

Thrombin Clotting Time (TCT) this is a measure of the rate of conversion of fibrinogen to fibrin when thrombin has been introduced by adding thrombin, it bypasses both the extrinsic and intrinsic clotting pathways and assesses the terminal steps of the common pathway the time taken for citrated plasma to clot after calcium and a known amount of thrombin have been added is evaluated
GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

thrombin induced clotting is very rapid (thrombin powders and gels may be used in the surgical setting to encourage coagulation) - and is generally adjusted to desired levels between 10-15 seconds TCT will be prolonged when: fibrinogen levels are below 100 mg/dl when there are circulating anticoagulants such as heparin that interfere with thrombin's action when there are circulating fibrin degradation products in the plasma which competitively inhibit the thrombin/fibrinogen interaction

Fragment D-Dimer Assay D-dimer is a protein that is released into the circulation during the process of fibrin blood clot breakdown D-dimer represents an area of cross linked fibrin degradation product that originated from the breaking down of the fibrin clot network during the bodys repair mechanisms it will elevate with deep venous thrombosis, pulmonary embolism and disseminated intervascular coagulation the ELISA method is the standard for evaluating D-dimer concentration there is a wide variation in testing validity depending upon the manufacturer of the antibody used to detect the D-dimer Bleeding Time this is often used as a screening test for platelet disorders there is a good correlation between bleeding time and platelet count it should not be performed if the platelet count is less than 40,000/ml Lee-White Clotting Time the oldest but least accurate test of coagulation it is the measurement of how long it takes for blood to clot in a test tube 4 - 8 minutes is normal Complete Blood Count - CBC this includes basic data on red and white blood cells, white blood cell differentials, hemoglobin concentration and hematocrit measurement. It will also tell you information about the size, shape and structure of the red blood cells, as well as the presence of immature red cells or reticulocytes it is collected in lavender top tube which utilizes EDTA (ethylenediaminetetracetic acid salts - 1 to 2 mg/ml of blood) cell counts are valid up to 24 hours if the lavender top tube is refrigerated slide mounts are accurate for cell morphology when performed 2 to 3 hours after collection EDTA prevents platelet clumping and is the anticoagulant of choice for platelet counts Red Blood Cell Count red cell counts are expressed as cells per unit volume the normal red blood cell is a biconcave disc, 6-8 m in diameter staining is deeper in the periphery of the cell and gradually lessens as the center of the cell approaches this pale central portion of the cell is normal and occupies approximately one-third of the diameter normal life span of the red blood cell is approximately 120 days in the normal individual, 0.8-1.0% of all red cells turn over daily

reference values (red cells/l):

male female

4.6-6.2 x 10
6

4.2-5.4 x 10

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

Reticulocytes a reticulocyte circulates around for approximately 24 hours before it completely matures into a red blood cell normal reticulocyte count - ~1% (the range from 0.5 to 1.8%) reticulocyte count will increase in proportion to the severity of certain types of anemia reticulocyte count is calculated as follows: Corrected Reticulocyte Count = measured reticulocyte count x patients hematocrit 45 Hemoglobin [Hb] Content hemoglobin is the main component of the red blood cell it is the vehicle for transportation of oxygen and carbon dioxide fully saturated, each gram of hemoglobin holds 12.34mL of oxygen its main function is to transport oxygen from the lungs - where oxygen tension is high, to the tissues - where it is low reference values for hemoglobin are: Males 13.5-18 g/dl Females 12-16 g/dl

a qualitative estimation of hemoglobin concentration may be obtained by assessing the specific gravity of whole blood the depth of staining is a rough guide to the amount of hemoglobin in the red cells

Hematocrit [Hct] it represents the percentage of a given volume of blood occupied by packed red cells, therefore it is a measure of packed red cell volume. it is expressed as a percentage or decimal fraction, the units L/L are implied reference values for hematocrit: Male 40-54% Female 38-47 Red Blood Cell (Corpuscular) Indices these calculations were introduced by Wintrobe to help determine the size, content and Hb concentration of red cells they may be useful in the characterization of various anemia and are calculated from RBC's, Hb and Hct Mean Cell Volume - MCV this is the volume of the average red cell it is calculated from the hematocrit and the red cell count as follows: MCV = (Hct x 1000)/RBC

MCV is expressed in femtoliters or cubic micrometers. One femtoliter (fl) = 10 L = 1 cubic micrometer m ) reference values for MCV are: Males 80-98 fl Females 81-99 fl

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Mean Cell Hemoglobin - MCH this represents the hemoglobin content (or weight) of the average red cell
GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

it represents the amount of oxygen-binding protein in the cells and therefore, the ability of the red cells to delivery oxygen to organs and peripheral tissues. it is calculated from the Hb concentration and the red cell count as follows: MCH = Hb/RBC

MCH is expressed in picograms. One picogram (pg) = 10-12 g = 1 micromicrogram g). reference values for MCH are: Males 26-32 pg Females 26-32 pg

Mean Cell Hemoglobin Concentration this is a calculation representing the average concentration of hemoglobin in a given volume of packed red cells it is calculated from the hemoglobin concentration and the hematocrit as follows: MCHC = Hb/HCT

reference values for MCHC are:

Males Females 3

32-36% 2-36%

RDW - Red Blood Cell Distribution Width it is a measure of the variation in red blood cell size it is beneficial in characterizing anemias that share similar mean corpuscular hemoglobin values RDW seems to be the first indicator to become elevated in iron deficient anemia secondary to chronic blood loss or renal dialysis. This occurs secondary to a marked variation in cell volume (size). RDW is generally normal in the anemia of chronic disease Reference values: 11.6 - 14.6% with an average of 13.1% Erythrocyte Morphology Color the "color" or a red blood cell is a reflection of the hemoglobin concentration normochromic refers to normal intensity of staining which represents normal hemoglobin parameters

hypochromia reflects a decreased amount of hemoglobin - the normally pale center of the erythrocyte becomes larger and even more pale concomitant with this decreased central staining, the MCH and MCHC are usually decreased iron deficient anemia is traditionally regarded as a hypochromic anemia. hyperchromia occurs when red cells have an increased hemoglobin content - the cells are generally larger and thicker than normal red cells they absorb the stain more deeply with less central pallor this is a characteristic finding in megaloblastic anemias. anisochromia or sometimes termed dimorphic anemia, reveals the presence of both hypochromic and normochromic red blood cells polychromatophilia refers to a blue-gray tint to the red cells and represents a combination of the affinity of hemoglobin for acids stains as well as the affinity of RNA for basic stains the presence of residual RNA in the red cell indicates that it is young and has been in the circulation for only one to two days these red cells are larger than mature cells and may lack central pallor

Size

microcytes are abnormally small red cells macrocytes are abnormally large red cells 7

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

anisocytosis refers to any abnormal variation in red cell size - a feature of most anemias over time, the terms microcytic and macrocytic have become a reflection of actual cell volume rather than cell diameter, and today these terms are reflected quantitatively by Mean Cell Volume when this is noted on the basis of a peripheral smear it is a reflection usually of the cell diameter on which an inference of cell volume can be made therefore, microcytic red cells are also usually hypochromic macrocytic red cells are often hyperchromic

Shape poikilocytosis refers to variation in shape. Any abnormally shaped cell is called a poikilocyte. these cells may be oval, pear-shaped, tear drop-shaped, saddle-shaped, helmet-shaped and irregularly shaped - all of which may be seen in one singular case of megaloblastic anemia.

elliptocytes are most abundant in hereditary elliptocytosis - here, the majority of the cells are elliptical they can, however, be seen in a normal blood smear but should account for less than 10% of the red blood cells they are more commonly encountered in iron deficient anemia, myelofibrosis, megaloblastic anemias and sickle cell anemia. spherocytes are nearly spherical red cells in contradistinction to normal biconcave disks their diameter is smaller than normal and they either lack the normal central pale area or it is eccentrically placed they can be encountered in hereditary spherocytosis (passed on by autosomal dominant transmission), some cases of acquired hemolytic anemia and in some conditions where there has been a direct physical or chemical insult to the cells they are the most common red cell abnormality in hemolytic disease they can be the hallmark of a splenic condition it is the common shape of red blood cells in birds and reptiles target cells are red cells that are thinner than usual and stain as peripheral rim of hemoglobin with a dark, central, hemoglobin containing area. target cells are found in obstructive jaundice, in postsplenectomy and in any hypochromic anemia, especially in beta-thalassemia where the cells are packed full with hemoglobin C. the most common cause of target cells is liver disease schistocytes are cell fragments and indicate the presence of hemolysis causes of schistocytosis can include megaloblastic anemia, severe burns or microangiopathic hemolytic anemia which is associated with either small vessel disease or fibrin within the small vessels frequently seen concomitantly are helmet cells and triangularly shaped cells all of these processes are associated with intravascular fragmentation acanthocytes are irregularly spiculated red cells in which the ends of the spicules are either rounded or bulbous present in abetalipoproteinemia where there are described as bizzarely spiculated crenated cells or echinocytes are regularly contracted cells these are a common artifact of preparation of the blood smears

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

Poikilocytosis poikilocytosis spherocytes

Sph

Target Cells

Acanthocytes

Reticulocyte

Characterization of Anemias Anemia in General moderate anemia hemoglobin 10-13 g/dl severe anemia hemoglobin 7-8 g/dl Normocytic Anemias these generally occur secondary to an increase in blood loss or a decrease in red cell production reticulocyte count provides a convenient means of differentiating between these two causes anemias into two general categories increased red cell destruction or loss - high reticulocyte count decreased red cell production - low or normal reticulocyte count Increased Red Cell Loss of Destruction acute blood loss - this is generally quite obvious, however, in the case of occult loss such as a retroperitoneal space bleed, it may be more difficult to discern splenomegaly - spleen is three to more times its normal size this occurs most commonly with portal hypertension, chronic infections and myeloproliferative disorders hemolytic disorders - concomitant findings generally include an elevated indirect (unconjugated) bilirubin levels and decreased serum haptoglobin levels
GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

congenital hemolytic anemias include disorders of the cell membrane, hemoglobinopathies and red cell enzyme deficiencies hereditary spherocytosis - confirmed with osmotic fragility tests hereditary elliptocytosis - 15% of all patients with this condition have hemolysis hemoglobin abnormalities- Hemoglobin SS (sickle cell anemia), Hemoglobin SC, Hemoglobin S-beta thalassemia, Hemoglobin C -a mild to moderate anemia

glucose-6-phosphate dehydrogenase deficiencies - disorder asymptomatic unless patients subjected to oxidant injury X-linked transmission, affects 2-3% of the world population oxidized hemoglobin is precipitated within the red cells in the form of inclusions called Heinz bodies affected males are more vulnerable to oxidant injury - female carriers are generally asymptomatic cells are further damaged as the splenic phagocytes attempt to extract the foreign bodies from the red cells by taking a little bite out of them - these are burr cells drug mediated causes of anemia include the antimalarials, sulfonamides, nitrofurantoin, phenacetin, aspirin

Acquired Hemolytic Disorders mechanical hemolysis is demonstrated by the presence of schistocytes on a peripheral smear warm-reactive IgG antibodies may be associated with the collagen-vascular diseases a blood spear will show rouleaux formation with may spherocytes and large polychromatophilic reticulocytes cold agglutinin disease may be associated with mycoplasmal or viral infection or with certain lymphomas; however, the majority are idiopathic Decreased Red Blood Cell Production occurs when the corrected reticulocyte count is not elevated primary marrow disease affecting all cell lines if likely aplastic anemia - the marrow is primarily hypocellular human parvo virus B19(HPV) is considered a major culprit - patients present with a nonspecific viral syndrome (fever, malaise, headache, myalgia and a fleeting rash. in children the most common inciting factor is fifth disease and is augmented in persons with a pre-existing hemolytic anemia other considerations - declining erythropoietin production by the kidneys with shortened red cell survival (the anemia of chronic renal failure) patients with hypothyroidism usually have a normocytic anemia patients with liver disease usually have a normocytic anemia Anemia of Chronic Disease a chronic inflammatory process of greater than one months duration will often cause an anemia typically, the Hb will be between 9 - 11 g/dl; MCHC - ~32 it is generally a normocytic, normochromic anemia although may occasionally border on the microcytic side of things it is primarily due to a defective RBC production and secondarily due to abnormal incorporation of iron into the RBCs (up to 50% of normal) Macrocytic Anemias must first determine if the anemia is megaloblastic or non-megaloblastic megaloblastic - if oval macrocytes, hypersegmented neutrophils or giant platelets are present in the peripheral smear Megaloblastic Anemias generally caused by a B or folate deficiency

12

B binds with intrinsic factor and subsequently absorbed in the ileum

12

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

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a decrease in B is referred to as pernicious anemia and is often caused from atrophy of the gastric musosa
12

which results in decreased intrinsic factor secretion it may occur possibly secondary to an autoimmune reaction against the gastric parietal cells this more commonly occurs in the elderly (>60 year of age) the Schilling test is the definitive test for pernicious anemia following a fast of twelve hours, a small oral dose (0.5 to 2 mcg) of radioactive vitamin B12 is administered. Two hours later, a larger dose (1 mg) of nonradioactive vitamin B12 is given IM, as a parenteral flush. A 24 hour urine specimen is collected and the amount of radioactive material in this urine ismeasured Ideally 7% of the radioactive B12 is excreted in those with pernicious anemia less that 3% is excreted Additional tests may include the presence of antiparietal cell antibodies and intrinsic factor

References: Oh, RC; Brown DL. Vitamin B12 Deficiency; Am Fam Physician 2003;67:979-86,993-4.

folate is absorbed in the upper small intestine - a deficiency of folate manifests more quickly as body stores only last a few months drug therapy may cause a depletion in folate stores - phenytoin, anti-metabolites (especially methotrexate), trimethoprim and sulfamethoxazole, pyrimethamine

Clinical Features of the Megaloblastic Anemias glossitis and a variety of gastrointestinal symptoms (indigestion, bloating, anorexia, diarrhea) the neurologic disease points to a B12 deficiency and not a folate deficiency alone

degeneration of the dorsal and lateral columns - the legs affected more severely than the arms

Nonmegaloblastic Macrocytic Anemias presence of large polychromatophilic reticulocytes (shift cells) will cause elevation of MCV Microcytic Anemias severely anemic patients will often be symptomatic at rest and unable to tolerate significant exertion when the hemoglobin concentration falls below 7.5 g/dl these include iron-deficiency, siderolastic and the thalassemias they collectively represent a decrease in the availability or synthesis of one of the three major constituents of the hemoglobin molecule - iron, porphyrin and globin since hemoglobin makes up over 90% of the content of the RBC, it is not surprising that these anemias result in microcytes the anemias of chronic disease and malignancy may be slightly microcytic - this results from a decrease in the availability of iron (however, these anemias are more commonly normocytic) measurement of serum iron and iron-binding capacity are useful in distinguishing between these anemias Iron Deficient Anemia this is the most common type of anemia world-wide it is characterized by low serum ferritin - <12g/L elevated TIBC
GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

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the vast majority of serum iron (~80%) recycled from broken down RBCs iron is shuffled through the blood stream on transferrin a bilobed glycoprotein with two iron-binding sites the turnover time of transferring-bound iron is very quick usually within 60-90 minutes erythropoiesis does not increase as long as transferrin saturation is maintained between 20-60%. iron absorption takes place in the proximal small intestine Daily iron needs: 1 mg/day male 1.4 mg/day females

Physical findings pallor is the most common feature tachycardia (due to increase in resting cardiac output), a wide pulse pressure vertigo and headache White Blood Cells normal circulating blood contains approximately 4,000-11,000 white cells per microliter there are three distinct white blood cell populations which are the granulocytes, the lymphocytes and the monocytes granulocytes and monocytes arise from the myeloid line of stem cells lymphocytes arise from the lymphoid line of stem cells

White Blood Cell Count Differential White blood cell types are generally reported in two ways - as a percentage and as an absolute count Absolute cell count is calculated by multiplying the total white cell count by the percent of a specific cell reported. Examples of this calculation: total number of WBC's x (% neutrophils + % band cells) = Absolute Neutrophil Count (ANC) total number of WBC's x % lymphocytes = Absolute Lymphocyte Count Granulocytes the granulocytes are composed of the neutrophils, eosinophils and basophils the term granulocyte refers to the presence of "granules" within the cells the staining characteristics of these granules further subdivide this group into: neutral staining granules or neutrophils reddish (eosinophilic) staining granules or eosinophils, and bluish (basophilic) staining granules or basophils Neutrophils the average neutrophil is 12 m in diameter the nucleus is irregular and stain deeply reference values: segmented neutrophils - 56% band neutrophils - 3% ANC - 1800-7000 cells/mm3

what appears to be multiple nuclei are actually segments of nuclear material connected by delicate filaments these are also commonly referred to as polymorphonuclear leukocytes (PMN's or polys) as the nuclear material takes on so many (poly-) possible forms (morph-) a segmented neutrophil has at least two of its lobes separated by a filament 12

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

a band neutrophil has either a strand of nuclear material thicker than a filament connecting the lobes of nuclear material or is a U-shaped nucleus of uniform thickness. This band shaped nuclear configuration is characteristic of a less mature neutrophil. the number of lobes in a normal neutrophil ranges from two to five with three being the most common

Function the function of a neutrophil is closely aligned to the defense systems of the body interaction with antibody production and activation of the complement system enhance their ability to phagocytize and remove debris, particulate material and the digestion and eradication of bacteria. primary neutrophilic granules which appear in the promyelocyte stage, contain myeloperoxidase and acid phosphatase as well as other acid hydrolytic enzymes secondary neutrophilic granules which appear at the myelocyte stage and predominate in mature neutrophils possess alkaline phosphatase and lysozyme a "shift to the left" occurs when there are increased bands and fewer mature neutrophils in the blood or the overall percentage of PMN's exceeds 75% Elevations of Neutrophils - Neutrophilia causes for neutrophilia include: infection - generally systemic. There is a characteristic pattern in response to infection which includes progressive neutrophilic leukocytosis, increased numbers of band cells and a fall in eosinophils. As the infection begins to subside, the total number of leukocytes goes down and the number of monocytes increases. This phase is then replaced by a slight absolute lymphocytosis and eosinophilia as recovery proceeds. drug and chemical reasons include lead, mercury, digitalis, epinephirine, corticosteroids tissue trauma including burns, surgical operations, crust injuries, fractures and neoplastic disease with extensive tissue necrosis Decreased Neutrophil Count - Neutropenia The more severe the neutropenia, the greater patient risk is for bacterial infection and delayed healing. Mild neutropenia - ANC 1000 -1500 cells/mm3 Moderate neutropenia - ANC 500-1000 cells/mm3 Severe neutropenia - ANC < 500 cells/mm3 causes for neutropenia can be congenital or acquired Eosinophils eosinophils average 13 m in diameter reference values: 3% of total leukocyte population Absolute eosinophil count - 100-300 cells/mm3 eosinophils are easily recognized by their bright red (acid staining) larger round or oval granules eosinophils generally have only two nuclear lobes, very rarely three Function the biologic role seems to be the modulation of cellular and chemical activities in immunologically mediated inflammation via inactivation of mast cell products and reduction of granulocytic migration eosinophils are capable of phagocytosis, but they are not bactericidal; they are more sluggish and inefficient than the PMN at phagocytosis. release a toxin known as major basic protein (MBP) histamine and the slow reactive substance of anaphylaxis (SRS-A) is released by the eosinophilic granules eosinophils have a unique ability to damage larvae of certain helminth parasites

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

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Elevations in Eosinophils - Eosinophilia absolute count - >450 eosinophils/g the mneumonic CHINA may be helpful in remembering possible etiologies Collagen vascular diseases Occasionally may elevate with psoriasis and eczemas With many immune-complex mediated disease processes Helminthic disease Ccn directly injure larvae Idiopathic hypereosinophic syndrome Neoplasm lymphomas, gastric and lung carcinomas Allergic asthma, hay fever, drug reactions, allergic vasculitis Basophils they may resemble neutrophils but their nucleus is less segmented (usually just indented or partially lobulated) they are 10 12 in diameter reference values: less than 1% of total leukocyte population Absolute basophil count - 40-100 cells/mm3 possess large, coarse cytoplasmic granules which stain deep blue with basic dyes granules possess acid-mucopolysaccharides, hyaluronic acid and a large amount of histamine and SRS-A (slow reaction substance of anaphylaxis) Function basophils in the circulation serve no known function and are found in very low numbers mast cells, however, are very similar to basophils there are two kinds of mast cells (1) the mucosal mast cell (MMC) and (2) the connective tissue mast cell (CTMC) tissue basophils have IgE receptors which react with allergens Elevation in Basophils - Basophilia absolute basophils count is >100 basophils/l myeloproliferative disorders chronic hypersensitivity states Lymphocytes constitute the second most abundant leukocyte in the peripheral blood comprise the essential constituents of the immune defense system mononuclear cells without specific cytoplasmic granules cells possess a single, sharply defined, generally round, nucleus containing heavy blocks of chromatin Reference values: ~ 30% of total white cells Absolute lymphocyte count - 1700-3500 cells/mm3 small lymphocytes range from 6 to 10 m - most of the circulating lymphocytes are less than 10 m in diameter larger lymphocytes range from 12 to 15 m in diameter and possess less densely staining nuclei. Large lymphocytes comprise less than 10% of the circulating lymphocytes. They may represent natural killer-cytotoxic lymphocytes and active, antigenically stimulated cells. there is, however, a continuous range of lymphocytes, ranging from 6 to 15 m Function the primary function is to interact with antigens and mount the immune response
GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

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it is the primary mediator of the humoral, cell mediated, and cytotoxic responses the absolute quantities of these cells is important but equally important is the ratio or proportion of their subtypes important subtypes are the T-lymphocytes - which constitute 75-80% of the circulating lymphocytes in healthy adults and the B-lymphocyte which comprises 10-15% of the circulating lymphocytes both T and B cells secrete cytokines, specifically termed lymphokines when secreted by lymphocyteslymphokines are potent chemical messengers secreted by the immune system that can: recruit other cells needed for the immune response promote cell activation encourage cell growth, and destroy target cells - including cancer cells due to their role as messengers between white blood cells, they are also known as interleukins

B lymphocytes the B cell possesses an antigen-specific receptor which is a sample of the antibody it is prepared to manufacture it is capable of recognizing an antigen in its native state to be transformed into an antibody secreting plasma cell, it will require activation via a T helper cell once the B cells binds to a matching antigen, it will begin to process it, combining a fragment of the antigen with a class II protein. (A class II protein is generally derived from exogenous antigens, such as soluble proteins or extracellular organisms and will combine only with T helper cells) this complex is then recognized and bound by a T helper cell which carries a matching recetor this binding will active the B cell - via secretion of interleukins - to transform into an antibody-secreting plasma cell T cell lymphocytes all T cells express a surface molecule called CD3 which is specific only to T cells. Expression of the CD3 surface marker is specific for T lymphocytes, and is often used to characterize T cells like B cells, T lymphocytes also carry an antigen specific surface receptor T4 helper cells (CD4) these cells possess antigen specific receptor molecules similar to but more complex than B lymphocytes these cells require an antigen-class II protein complex for activation the cell binds with this complex which causes secretion of interleukins which mature the T cell

at least two types of lymphocytes are killer cellscytotoxic T cells and natural killer cells. cytotoxic T cells need to be presented with a specific antigen to destroy invaders cytotoxic T cells require the T helper cell to mature and become activated these cells respond to and kill cells which are infected with intracellular pathogens natural killer or NK cells do not require any specific antigen to be activated both types contain granules filled with potent chemicals, and both types kill on contact

Lymphocytosis Increased Absolute Lymphocyte Count (>4500/mm3) Non-activated Lymphocytes are often elevated with viral infectionsInfluenza, Pertussis, Tuberculosis, Mumps, Varicella, Herpes Simplex Virus, Rubeola, Brucellosis, Chronic Lymphocytic Leukemia, Acute Lymphoblastic Leukemia Activated or Atypical Lymphocytes are elevated with: Cytomegalovirus Infection, Infectious Mononucleosis, Infectious Hepatitis, Toxoplasmosis, Post-transfusion, and with some medications (mephenytoin, dilantin, para-aminosalicylic acid)

Decreased lymphocyte counts occur with: AIDS, bone marrow suppression, aplastic anemia, neoplasms, steroids, adrenocortical hyperfunction and and believed to be auto-immune mediated neurological disorders such as multiple sclerosis, myasthenia gravis and Guillain-Barre syndrome 15

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

Lymphocyte Assays CD3 marker the most major structural component and universal antigen receptor other cell markers: CD5 is also present on some subpopulations of B cells CD2 can be found on some natural killer (NK) cells CD7 is weakly expressed on some mature T cells

the major T cell subsets are CD4 (helpers) and CD8 (which can be further subdivided) CD8 cells are suppressor cells broken down into the so-called true suppressor cells - (CD8+ and CD11+) which influence B cell antibody function and the cytotoxic T cells (CD8+ and CD11-) Diagnosis of HIV Seroconversion HIV ab (ELISA) with Western blot confirmation CBC with CD4 and CD8 lymphocyte subsets

CD4 Counts CD4 levels are useful in AIDS prognosis and monitoring treatment - the higher the CD4 count, the more intact the immune system. These tests should be repeated every 3 to 6 months. a CD4 count between 500 and 1500 cells/mm3 suggests immune competence. less than 500 cells/mm3 is generally considered the "threshold" in therapeutic management when the CD4 count begins to drop below 250 opportunistic infections and complications increase - elective foot surgery is not an option. to help further evaluate the integrity of the immune system in HIV positive patients, viral load is now being measured. Many treatment regimens are defined on the basis of CD4 counts and viral load.

Viral Load viral load or viral burden is the quantity of HIV-RNA that is present in the plasma it is a useful marker in evaluation of disease progression and if therapy is working viral load should ideally be below 4500 to 5000 copies/ml initiation of therapy should begin when the viral load exceeds 30,000 copies/ml a viral load below 400 copies/ml is considered to be "undetectable". This does not imply, however, that the patient is disease free or unable to transmit the virus. initial testing should include two tests, taken two to three weeks apart, to establish a baseline. It should then be repeated every 3 to 6 months along with a CD4 count to monitor the disease Monocytes this is the largest cell of normal blood reference values: 4% of total leukocytes Absolute monocyte count: 200-600 cells/mm3 it is usually two to three times the diameter of a red blood cell - ranging from 14 to 20 &m it contains a single nucleus, which is partially lobulated, deeply indented or horseshoe- shaped the nucleus stains less densely than that of other leukocytes Function it is the monocyte which is capable of transforming into a macrophage macrophages are versatile cells - they act as scavengers, secrete powerful leukotrienes and play an essential role in activating T cells. with transformation it becomes up to 40 &m in diameter the cytoplasm becomes irregular at the cell margins and contains vacuoles which are phagocytic in nature
GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

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Elevations in Monocytes monocytosis may be present in subacute bacterial endocarditis it may also be present in mycotic, rickettsial, protosoal and viral infections elevations, however, may be most frequent secondary to a hemotologic disease such as a leukemia or lymphoma Normal Leukocyte Count, Differential and Hemoglobin at Various Ages
Age 12 mo 4 yr 6 yr 10 yr 21 yr leukocytes 11.4 9.1 8.5 8.1 7.4 PMN's 31 42 51 54 59 Bands 3.1 3.0 3.0 3.0 3.0 Segs (total) 28 39 48 51 56 Eos 2.6 2.8 2.7 2.4 2.7 Baso 0.4 0.6 0.6 0.5 0.5 lymphs 61 50 42 38 34 monos 4.8 5.0 4.7 4.3 4.0 hemoglobin 12.6 12.7 13.0 13.4 13.8

Platelet Function normal platelet counts are between 150,000 and 400,000/l (average value 250,000/l) a properly drawn specimen mixed with EDTA will remain stable up to 12 hours platelet count below 10,000 requires transfusion

Clot Retraction - when blood first clots within a test tube, the entire column of blood solidifies. With time, the serum is expressed leaving behind only the red cells within the shrunken fibrin mass. Platelets are necessary for this fibrin clot retraction and the expression of serum. this is a very simple test a normal clot at 37C generally shrinks to about one half is original size within an hour patients with throbocytopenia will have a scant amount of serum and a soft, plump, poorly demarcated clot tests results may be skewed if patient has low hematocrit (this would increase the proportion of serum) or in patients with polycythemia vera (this can cause the clot to be more densely packed and therefore, poorly retracted) Platelet Aggregation - this can be measured by bringing platelet rich plasma in contact with known aggregation inducers

PFA-100 analyser simulates high shear platelet function the test can be performed within four hours of blood collection citrated blood (blue top) is aspirated under constant negative pressure via a capillary tube through a microscopic aperture cut into a membrane coated with either collagen/epinephrine (CEPI) or collagen/ADP (CADP). The shearing force generated by pulling the platelets through the tube and tiny opening together with the platelet activators present in the membrane result in platelet adhesion, activation and aggregation. As a result a platelet plug forms within the aperture. Platelet function is recorded as the amount of time it takes for the aperture to become occluded. It is sensitive to many variables - increased or decreased platelet numbers, hematocrit, vonWillibrand factor defects. CBC should be performed with this to rule out presence of thrombocytopenia or anemia. 17

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

Typical ranges for CEPI are 79-164s and 55-112s for CADP. It may be used to evaluate platelet function in patients taking drugs to prevent platelet aggregation Clopidogrel irreversibly binds with the ADP receptors on platelets - thereby inhibiting coagulation (alcohol also affects ADP-regulated aggregation) Aspirin and nonsteroidal anti-inflammatory medications affect the arachnidonic pathywas and therefore alter thromboxane A2 synthesis and platelet aggregation.

Mean Platelet Volume (MPV) this value represents the average volume of the platelets reference range - 7.5-11.5 elevation of MPV is associated with and increased risk of stroke and heart attack Thrombocytosis thrombocytosis is an elevation in platelet count - with this, platelet function studies are generally normal thrombocythemia refers to the unregulated, uncontrolled production of platelets (for example elevations occurring in the myeloproliferative diseases) - with this, platelet function studies may be quite abnormal it is often associated with inflammatory disorders, infections, malignancy and post acute bleeding episodes sustained platelet counts of greater than one million are generally associated with primary thrombocythemia and in general, 600,000 may be the upper limit for concern it is an acute phase reactant peripheral smear may show presence of giant or large platelets - this generally indicated increased platelet turnover or abnormal production serum may reflect a spurious hyperkalemia since platelets contain intracellular potassium and when the blood clots, the potassium is released Platelet Disorders Secondary to Other Disease Processes underlying neoplasm (often gastrointestinal) underlying inflammatory or infections process severe uremia - the patients platelets behave abnormally and transfused normal platelets acquire this abnormality oozing and mucocutaneous bleeding often complicate advanced uremia presence of abnormal serum proteins multiple myeloma high molecular weight dextrans presence of fibrin-fibrinogen split products - these byproducts are normally cleared by the liver (that is the low levels of degradation associated with everyday trauma and repair) myeloproliferative syndromes often, ironically, predispose to both bleeding and thrombotic tendencies References: Tefferi A, Hanson CA, Inwards DJ; How to Interpret and Pursue an Abnormal complete Blood Cell Count in Adults. 2005 Mayo Clin Proc, 80(7):923-936. Harrison P, Platelet function analysis; 2005 Blood Reviews 19:111-123. Erythrocyte Sedimentation Rate - ESR must use well-mixed venous blood principle - determination of the rate at which erythrocytes fall to the bottom of a vertical tube of anticoagulated blood within a specified period. Measurement of the distance from the top of the column of sedimented erythrocytes to the top of the fluid level in a given period determines the erythrocyte sedimentation rate.

ESR normally increases with age two methods exist for detection of ESR the Wintrobe method which uses undiluted anticoagulated blood the blood stands for one hour in a tube 100 mm tall and 2.8 mm in diameter 18

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

reference values for the Wintrobe method:

Males Females

8 mm/hr 15 mm/hr

the Westergren technique - this is the usual method performed because of its simplicity and reproducibility utilizes a straight pipette 30 cm long and 2.5 mm in internal diameter - it holds about one ml of blood the tube is placed exactly vertical in a rack after 60 minutes, the distance to the top of the column of red cells is recorded in millimeters as the ESR value reference values for Westergren technique: Males 10 mm/hr Females 20 mm/hr the rate at which red cells fall is greatly influenced by the ability of the erythrocytes to form rouleaux rouleaux formation is a function of the surface attraction - or zeta potential - of the red cells and reflects their ability to "clump" together sedimentation rate is dependent upon red blood cell factors and plasma factors red blood cell factors influencing sedimentation rate, causing an increase rate and hence a lower recorded value include: anemia red cell surface area (i.e. macrocytes sediment more quickly than microcytes) rouleaux formation which decreases the surface area of the red cells by aggregation plasma factors which influence sedimentation time include: factors which reduce the zeta potential, that is, enhance the ability of the red cells to clump together an increased ratio of globulins to fibrinogen - which occurs in most inflammatory processes (regardless of etiology) and in pregnancy erythrocyte sedimentation rate really has three main uses: as an aid in detecting inflammatory processes as a monitor of disease course or activity (e.g. monitoring treatment for osteomyelitis) as a screen for occult inflammatory or neoplastic conditions overall, it is relatively nonsensitive and nonspecific

Sodium Heparin - GREEN TOP heparin (0.1 to 0.2 mg/ml of blood) does not affect cell size or hematocrit heparin prevents coagulation by directly inhibiting thrombin; it prevents the conversion of fibrinogen (clotting factor I) to fibrin by augmenting a natural anti-coagulant molecule, anti-thrombin III, to neutralize thrombin. It does no influence the calcium concentration in the plas best anticoagulant for prevention of hemolysis and to detect osmotic fragility it is not satisfactory for leukocyte or platelet counts because of cell clumping it is used to transport blood gases may be used for CD4 count

GMS 715 Podiatric Medicine III Diagnostic Laboratory Medicine 2011 - Jacqueline Buchman, D.P.M.

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