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Bernard F. Feldman, DVM, PhD Department of Biomedical Sciences and Pathobiology Virginia-Maryland Regional College of Veterinary Medicine Virginia Polytechnic Institute and State University Blacksburg, Virginia, USA 24061-0442 THE TWENTY-FIRST CENTURY HEMOGRAM
Interpretation Revisited Introduction - The following is considered a COMPLETE hemogram: Examining red blood cells
Examining platelets
The Relationship Between Hemoglobin, Hematocrit, and the Red Cell Indices plus the Histogram of Red Cell Volume Distribution The hemoglobin concentration is probably the most accurate (and the red cell count the least accurate) of the three analytes needed to calculate the red cell indices. The packed cell volume is the number derived from packing red cells by centrifugation in a glass tube. It is similar to but not necessarily the same as the hematocrit which is a calculated number. If the red cell indices are within the reference interval (this is the new jargon for normal range), then the relationship between hemoglobin and hematocrit is steady. That is to say, the hemoglobin multiplied by 3 should approximate the hematocrit or the hematocrit divided by 3 should approximate the hemoglobin. Any significant departure from this ratio suggests laboratory error. If the red cell indices are not within the reference interval the ratio is not valid. It is important to recall the red cell indices are MEAN values. Therefore it takes a lot of abnormally sized cells to move the MCV out of range. It takes a significant decrease in hemoglobin production to drop the MCHC. Abnormal indices provide free useful information. However normal indices require further examination of the hemogram. Responsive anemias, responsive to hemolysis or blood loss, usually become macrocytic and hypochromic. Macrocytic normochromic anemias suggest aberrant red cell maturation and are often the first signs of impending marrow dysplasia or neoplasia. Most clinical anemic presentations are normocytic and normochromic requiring additional examination of the hemogram and, perhaps, additional testing. Microcytic hypochromic anemias suggest blood loss. In younger animals this often is associated with parasitism. In older animals ulceration and/or neoplasia is suggested. Histograms of red cell volume distribution is a relatively new measurement allowing visualization of erythrocyte volume variability. Disturbances of red cell production that result in altered red cell size are more readily detected. These histogram abnormalities alert the technologists or clinicians to the presence of unusual red cell volumes when the mean values of the red blood cell indices are within the reference interval. Reticulocytes and Nucleated Red Cells Reticulocytes are an index of marrow erythroid production and effective delivery to the vascular space. Reticulocytes are observed in blood smears stained with vital stains. Polychromasia, virtually the same as reticulocytes, is observed in Romanowsky (Wright's) stained smears. Reticulocytes, to be interpreted properly, in dogs and cats, must be adjusted for packed cell volume and erythropoietin production. The reticulocyte production index is one attempt at understanding how responsive is bone marrow erythropoiesis. Metarubricytes are the only nucleated red cells that should ever be observed in peripheral blood smears. Red cell precursors younger than these often suggest marrow dysplasia or neoplasia. Since nucleated red cells inflate the white cell count, whenever nucleated red cells are present the white cell count should be corrected. Nucleated red cells are never an indication of red cell response unless accompanied by reticulocytes in numbers exceeding nucleated red cells (sans unitage). Clinicians should be capable of developing a diagnostic differential scheme for the presence of nucleated red cells and/or reticulocytes in nonanemic patients as well as anemic patients. Red Cell Morphology It is almost too mundane to think of red cell morphology in only terms such as polychromasia, poikilocytosis, anisocytosis and occasional spherocytosis. There are many red cell shapes and sizes that are diagnostically useful. A few include codocytes and stomatocytes which also may be observed in immune-mediated hemolysis, burr cells in advanced glomerulonephritis, acanthocytes in some forms of cholestatic disease, schistocytes associated with disseminated intravascular coagulation, Heinz bodies when hemoglobin is oxidized, and blister cells observed in hypersplenic diseases. There are many more helpful red cell changes. Red Cell Cytograms The typical erythrocyte cytogram plots each red cell by the size of the cell and the hemoglobin content. The cytogram is compared to appropriate cytograms for the species in question. This technology allows for the earlier detection of iron deficiency anemia and documentation of active erythropoiesis These are just several examples. Total Protein, Fibrinogen, Erythrocyte Sedimentation Rate, Icterus Index The only way red cell mass can be viewed critically is with total protein quantitation. Dehydrated patients may mask mild anemias. Blood loss is often clinically obvious and is supported by lowered proteins. Younger animals have lower proteins (less immunoglobulins) than do older animals. Fibrinogen and erythrocyte sedimentation rate are helpful additions to our diagnostic armamentaria as regards inflammation. They often increase, preceding changes in the leucogram and return towards normal before the leucogram does. Decreases in fibrinogen are associated with disseminated intravascular coagulation. Marked increases in fibrinogen quantitation and erythrocyte sedimentation rate are associated with renal or neoplastic diseases. Icterus index is a way of making an objective assessment of patient jaundice. Plasma color is compared to potassium dichromate standards and given a number value. Comparative aspects become evident on patient reexamination or when speaking to a colleague about the patient. Regularly inspecting plasma color and clarity often reveals free hemoglobin or lipemia. Platelet Count, Platelet Morphology, and Mean Platelet Volume Platelet numbers should be critically assessed. It is simply not enough to receive a report that platelet numbers are increased, "adequate," or decreased. In patients that have primary hemostatic defects, diagnostic and therapeutic decisions must be based on absolute numbers. In addition, large platelets suggest young platelets. Platelet granularity is observably affected by some diseases and some drugs. Oval to cigar-shaped platelets often suggest occult bleeding. Mean platelet volume (MPV) is a new measurement designed to enable us to view platelet size distribution. The detection of microthrombocytosis in immune mediated thrombocytopenia is but one example of the advantage of determining MPV. Another example is the detection of macrothrombocytes and active response to thrombocytopenia. White Cell Count, Distribution, and Morphology White cells counts must be corrected for increased numbers of nucleated red cells. Additionally, the white cell count is use to calculate absolute numbers of individual cell types. Percentage distribution is not informative and often leads to misdiagnoses. Included among the categories of cells in the white cell differential should be cells called "unclassifiable" and "degenerated." These additional categories are an alert to the presence of neoplastic cells and/or toxic cells. The difference between a stress leucogram - mature neutrophilia, monocytosis, lymphopenia, and eosinopenia - and an inflammatory leucogram - left shift - must be grasped. Regenerative and degenerative left shifts are useful diagnostic academic terms which affect therapies. The neutrophil to lymphocyte ratio is 3:1 in dogs and 2:1 in cats. These differences allow quantification of response comparatively between the two species. Dogs often mount an elegant response to a lesion or process. Cats, given the same degree of insult, mount a lesser response. Ratio inversions are significant. Nonspecific evidence of systemic toxicity is revealed when neutrophil morphology changes. Vacuolation, toxic granulation, and hypersegmentation, when observed, require additional patient examination in order to determine the cause of these morphological changes. Leucocyte Cytograms There are several leucocyte cytograms now available. These are the peroxidase cytogram and the basophil cytogram. The peroxidase cytogram plots each leucocyte by cell size and peroxidase activity and the computer draws lines around cell clusters that are distinctly different from each other and allows an automated leucocyte differential count. The basophil cytogram strips away leucocyte cytoplasm and plots naked leucocyte nuclear size and density. Interpretation of the basophil cytogram is based on the shape of the cell cluster. The shape can determine individual leucocyte types and numbers, detect left shifts automatically, detect toxicity, detect modest eosinophilias, and detect blast cells in early leukemic patients. Summary Time spent critically reviewing all aspects of the complete hemogram is diagnostically rewarding. This, coupled with newer automated technology better enables clinicians to detect sample errors and potential errors as well as graphically depicting cells. Automated technology also improves the identification of appropriate and abnormal cells, and may reduce labor time as well as providing more objective quantitative information about blood samples. Recommended Reading Tvedten H. Advanced hematology analyzers interpretation of results. Vet Clin Path 1993; 22:72-80. Weiser MG. Sizing of animal erythrocytes using sulfate-based diluent. Vet Clin Path 1985; 14:7-9. Weiser MG. Modification and evaluation of a multichannel blood cell counting system for blood analysis in veterinary hematology. J Amer Vet Med Assoc 1987; 190:411-415. Northern J, Tvedten HW. Reports of retrospective studies. Diagnosis of microthrombocytosis and canine immune-mediated thrombocytopenia in dogs with thrombocytopenia: 68 cases (1987-1989). J Amer Vet Med Assoc 1992. 200: 368-372. Kocher WD. Autoimmune hemolytic anemia in: Atlas of automated cytochemical cytology. Edited by Simson, Ross, Kocher. Tarrytown NY, Technicon Instruments 1988. 26-27. BFFts100698.1 THE EXPANDED HEMATOCRIT
There is a wealth of information available to the trained eye and mind in the centrifuged microhematocrit tube. So starting from the top down ... Icterus Index: Comparison of plasma color to potassium dichromate standards allows an objective comparison (a number assignation). This is an inexpensive method of ascertaining progression or regression in diseases producing icterus. A numerical comparison of plasma color is also useful when talking to a colleague over the telephone. Less than 7.5 is normal for dogs and cats. Less than 20 is normal for horses and cattle. (Jain NC: Schalm's Veterinary Hematology, Fourth Edition, Lea and Febiger, Philadelphia, 1986.) Total Protein: By breaking the microhematocrit tube in the plasma layer and placing several drops of plasma on the glass plate of a refractometer (total solids meter) total protein may be determined. Fibrinogen: The difference between total protein in the plasma column before and after heat precipitation (56oC, 3-5 minutes+) and centrifugation is a clinically satisfactory method to quantitate fibrinogen increases. No, you cannot subtract total protein of serum from that of plasma as other proteins come down in the clot and fibrinogen is always overestimated. (Sorry folks!) Heartworms: Heartworms may be found most easily by examining the area of the interface of plasma and buffy coat under 100X (low power) magnification. These parasites may be visualized elsewhere in the buffy coat and the red cell pack. Trypanosomes: Trypanosomes concentrate at the interface of the buffy coat and the plasma layer. Direct examination for these parasites may be accomplished (see Heartworms above) or the microhematocrit tube may be broken just above the buffy coat and the buffy coat/plasma interface spilled onto a glass slide, smeared and stained for microscopic examination. Platelet Quantitation: At the interface of the buffy coat and the plasma is a cream-colored layer (you may need a magnifying glass), much different from the grey buffy coat. These are platelets. Presence of this layer almost invariably suggests adequate platelet numbers. This layer is especially visible in feline blood. White Blood Cell Quantitation: The first single percent (1% of the microhematocrit tube) of the buffy coat represents 10,000 white blood cells/µl. The second single percent represents 20,000 white blood cells/µl. For example a buffy coat that is 3% of the microhematocrit tube represents a 50,000 white blood cell count/µl, 10,000 cells from the first percent and 20,000 cells for each of the remaining two percents. There is good correlation between buffy coat counts and automated white blood cell counts. A buffy coat less than 0.5% generally represents leucopenia while a buffy coat of greater than 1.5% represents leucocytosis. Buffy Coat Smears: Breaking the microhematocrit tube just above the buffy coat and smearing and staining the buffy coat is routine whenever Ehrlichia inclusions in white blood cells are sought. A buffy coat smear is also used to examine large numbers of white blood cells when there is clinical concern about an occasional unclassifiable cell form noted on the blood smear or when a myeloproliferative process is suspected. The uses of buffy coat smears are virtually endless. Reticulocytes and Nucleated Red Blood Cells (NRBCs): Any pinkish tinge to the lower portion of the buffy coat suggests the presence of red blood cells of low specific gravity. Included among red cells with low specific gravity are leptocytes, reticuloytes and nrbcs. Noting a pinkish tinge in the buffy coat suggests examination of the peripheral blood smear for the presence of the cells listed above and their quantitation. Red Blood Cell Age: Individual red blood cell position in the red blood cell pack is determined by intracellular specific gravity. Therefore the youngest red blood cells are found in or just below the buffy coat and the oldest red blood cells are at the bottom of the red blood cell pack. In general, the oldest red blood cells are more vulnerable to red blood cell parasitism than younger cells. Examination of concentrated older red blood cells, cells obtained and smeared by breaking the microhematocrit tube near the bottom, allows examination for Hemobartonella, Eperythrozoon and, Cytauxzoon. Examination of Erythrocyte Glycolytic Pathway Intermediates: The age difference between erythrocytes is a consideration when attempting to ascertain erythrocyte glycolytic pathway deficiencies. Pyruvate kinase deficiency, a nonspherocytic hereditary hemolytic anemia, has been described in Basenjis, Beagles and West Highland White Terriers. In order to correctly quantitate erythrocyte pyruvate kinase in a suspect patient, blood from the middle of the red blood cell pack to the bottom must be used. Pyruvate kinase will be deficient in those cells which have lost their nuclei weeks to months previously. Younger cells, recently having lost their nuclei, often will have "normal" to increased concentrations of pyruvate kinase, masking the deficiency. Many pyruvate kinase deficient patients have enormously high reticulocyte counts (some exceed 50%). Unless these cells are removed from examination, diagnosis will be extremely difficult. ...and, the microhematocrit may be determined. CLINICAL HEMOSTATIC DISORDERS
Introduction Appropriate hemostasis represents physiological balance. Any imbalance results in hemostatic dysfunction. Too much hemostasis results in thrombosis, too little hemostasis results in hemorrhage. The relationship between the endothelial cell and the platelet is unique. The endothelial cell performs a number of critical functions including
When platelets are appropriate in terms of numbers and function (quantitatively and qualitatively), endothelial cells function correctly. However, if platelet numbers are inappropriate - thrombocytopenia or thrombocytosis - or are dysfunctional, endothelial cells are perturbed resulting in either hemorrhage or thrombosis. OVERVIEW OF HEMOSTASIS
Hemostasis is composed of primary, secondary, and tertiary components. PRIMARY HEMOSTASIS Vascular component: The first portion of primary hemostasis is vascular constriction. This is the first response when a vessel is damaged. This constrictive effort (strategy) is effective with small vasculature but ineffective with larger vessels. Vascular disorders can result from trauma, lack of muscular support for the vascular tree, or immunologic or septic damage of the vasculature. Platelet component: As the result of endothelial cell damage, subendothelial collagen is exposed. Adhesive molecules on platelets and on subendothelial collagenous fibers are mutually attractive. The most important of the adhesion molecules moderating platelet adhesion to subendothelial collagen is von Willebrand's factor (vWf). VWF is produced by endothelial cells and secreted into the subendothelial space where it binds to collagen and into the plasma where it is adsorbed to platelet surfaces. Megakaryocytes, platelet marrow precursors, also produce vWf. As a result platelets have vWf on their surface (note: this includes the platelet canalicular system which is in direct contact with the platelet phospholipid cellular membrane). To date, the two sources of vWf are immunologically and functionally indistinguishable. Platelet vWf adheres to subendothelial vWf by disulfide bridging. Deficiency or dysfunction in vWf results in no platelet adhesion or poor platelet adhesion. This results in hemorrhage. Platelets adhered to subendothelial collagen release cytokines attractive to other platelets. The most prominent among these substances are thromboxane A2 (TxA2) and adenosine diphosphate (ADP). The result of the release from platelets of these substances is recruitment of platelets into the damaged area, a process known as platelet aggregation. The platelet aggregate physically fills the damaged area in small vessels. It is not an effective response in large vessels where hemorrhage occurs. In addition many drugs including most of the nonsteroidal antiinflammatory drugs interfere with the prostaglandin synthetic path, specifically with function of cyclooxygenase. As a result of this interference hemorrhage occurs due to lack of platelet aggregation. The platelet plug is limited to the area of damage by other prostaglandins produced by endothelial cells. The most active inhibitor of platelet aggregation is prostaglandin I 2 (prostacyclin; PGI2). PGI2 prevents the platelet plug from expanding upstream or downstream - formation of thrombosis. The combination of vascular constriction, platelet adhesion, and platelet aggregation is termed primary hemostasis. SECONDARY HEMOSTASIS
Coagulation component: Platelet adhere and aggregate in the damaged area. They also carry - on their surfaces and in their organelles - at least six platelet factors (PFs), and all of the coagulation proteins (called coagulation factors). Among the PFs is PF3 (note: this is always written as Arabic number 3), which is, in fact, the platelet phospholipid surface. PF3 catalyzes coagulation - the events which lead to fibrin formation and platelet plug stabilization. Fibrin not only stabilizes the platelet plug, but is the skeletal superstructure upon which endothelial cell healing will take place. The formation of fibrin is called secondary hemostasis or coagulation. The word coagulation is used only when describing secondary hemostasis. Dysfunctional factors (dysplastic factors), deficiency of coagulation factor quantity, or immune activity against coagulation factors often results in hemorrhage or thrombosis. Almost all of the coagulation factors are inherited in an autosomal manner. Most are autosomal recessive. Factors in the intrinsic path are XII, XI, IX, and VIII. This pathway is activated by factors intrinsic to the vascular tree. Factor VII is in the extrinsic path and is activated by tissue factor (Factor III; tissue thromboplastin), factors extrinsic to the vascular tree. Both the intrinsic an extrinsic paths come together at a common point - the common path - made up of factors X, V, II (always called prothrombin when inactive and thrombin when active) and factor I (always called fibrinogen). Factor XII (Hageman factor) is the focal point for the initiation of inflammation. Activated Factor XII (by convention we indicate an activated factor by adding "a" to the Roman numeral; activated factor XII is written XIIa) initiates coagulation culminating in thrombin activation (IIa) and fibrin formation - clot stabilization. XIIa also initiates fibrinolysis by activating tissue plasminogen activator (tpa) and thus activating plasminogen to plasmin - resulting in fibrin(ogen) degradation (split) products (FDPs/FSPs. XIIa also activates the complement cascade and the chemoattraction of white cells into the area of cell damage. XIIa also activates the kinin system resulting in the formation of bradykinin causing pain and vasodilation. Factor VIII is activated by exposure of subendothelial collagen. Factor XII activation is accelerated by portions of the kinin system. Factor XII is called a "contact activation factor." A significant percentage of cats (estimates range from 5 to 18 percent) are factor XII deficient and, while these cats do not bleed, they are unable to initiate an appropriate inflammatory response and may be more susceptible to infection, especially retroviral infection. Factor XI (hemophilia C factor) is important in bovine medicine. This factor is intrinsic to appropriate absorption and function of zinc. XI deficient calves are also zinc deficient calves manifesting all of the signs of this mineral deficiency. XI deficiency is also involved with appropriate platelet function. Factor XI is also called a a "contact activation factor." Factors IX and VIII are called hemophilic factors. Both of these factors are inherited as sex-linked recessive factors. Factor IX is called Hemophilia B (or Christmas disease, named after the first family in which the disease was described). Factor VIII is called Hemophilia A (or "classic" hemophilia - the hemophilia associated with European and Russian aristocracy). Actually the factor VIII molecule is complexed with another molecule - von Willebrand's factor. Factor VIII and vWF in normal individual always are associated. This combination of factors is termed "the factor VIII complex." There is much terminology associated with factor VIII: Factors II, VII, IX, X, and Protein C (and its subsequent product Protein S) are dependent on vitamin K for activation. Activation of the inactive protein occurs by carboxylating a single terminal glutamic acid residue on each factor. All of the factors in the coagulation paths save fibrinogen and factors V and VIII are serine proteases inhibited by a natural plasma inhibitor antithrombin III (ATIII) incorrectly written as AT3. The natural inhibitor of the two cofactors (factors V and VIII) is Protein C. TERTIARY HEMOSTASIS
Fibrinolysis The fibrinolytic mechanism is vital to the prevention of intravascular clotting and the reestablishment of vascular patency. The principle factor in this system is plasminogen, a serine protease which must be activated to plasmin to acquire enzymatic activity. Plasmin attacks soluble fibrinogen or fibrin and enzymatically degrades the clot producing a variety of fibrin(ogen) degradation or split products (FDPs; FSPs). The degradation of fibrin(ogen), fibrinolysis, is called tertiary hemostasis. The pathogenesis of defective hemostasis (hemorrhage or thrombosis) results from vascular disorders, quantitative or qualitative platelet disorders, defects in coagulation proteins, or too much or too little fibrinolysis. BLEEDING: A LOCAL PROCESS OR A BLEEDING TENDENCY?
While most hemorrhaging patients have a local problem, a bleeding tendency is potential when there is
PETECHIA, PURPURA, ECCHYMOSIS
Petechia are small punctate areas of bleeding. Purpura are coalescing areas of petechial hemorrhage. Ecchymosis is a bruise. Petechial, purpuric, or ecchymotic hemorrhage are specific indications of defects in primary hemostasis. These signs are specific clinical findings. HEMATOMA, HEMARTHROSIS
Hematoma formation and/or hemorrhage into synovial cavities are specifically associated with defects in secondary hemostasis - defects in coagulation. These signs are specific clinical findings. EPISTAXIS, HEMATEMESIS, HEMATURIA, HEMATOCHEZIA ETC., can be associated with primary or secondary hemostatic defects. These signs are not specific clinical findings. CLINICAL APPROACH
Medical History Obtaining a complete medical history is essential. Is the hemorrhage associated with primary, secondary, or tertiary hemostasis? Is there any family history of a bleeding tendency? Has this patient had any historical hemorrhage? Have any drugs been used in the past month? Vaccines? Physical examination Again, examine the patient for any evidence of hemorrhage remembering that hemorrhage can be occult. Retroperitoneal hemorrhage is difficult to ascertain. Patients hemorrhaging in this area often splint their abdomens, are febrile, and are anorectic. Bleeding into body cavities or into joint spaces in some cases may only be ascertained by direct examination - aspiration. Laboratory Examination Screening Procedures Determining fibrinogen Microhematocrit - With the microhematocrit tube we many obtain screening hemostatic information. Fibrinogen (factor I) may be determined by heat precipitation. Total protein is determined on one microhematocrit tube. A second, already centrifuged tube is then heated in a 56 degree Celsius water bath for 5 minutes and recentrifuged. Total protein is determined on this plasma. Total protein of the first tube minus total protein of the second, heated, tube, equals fibrinogen in milligrams. Platelet count may be estimated if a platelet layer is observed above the buffy coat. This is a cream colored layer on top of the grey buffy coat. Sometimes visualizing this layer requires a magnifying glass. Observation of this layer suggests platelet number sufficiency. Sometimes platelets do become intermixed with the buffy coat negating the value of this observation. Hemogram - Adequacy of platelet numbers may be estimated by examining monolayered areas for platelets. In small animals, platelet sufficiency is suggested by at least twelve platelets per oil immersion field. In cats, platelet clumping usually suggests platelet number adequacy. Platelet shapes are useful too. If platelets approach red cell size or are even larger (platelet anisocytosis), immature platelets are suggested. Elongated, cigar shaped platelets suggests platelet shape change induced by an agonist that is, is indicative of patient bleeding. Of course, platelet counts should be routinely ascertained in hemograms. The presence of fragmented red cells on the hemogram, schistocytes, suggests microvascular fibrin formation and cell cleavage. Examination of Primary Hemostasis - Primary hemostasis consists of vascular constriction, platelet adhesion and platelet aggregation. It must be distinguished from secondary hemostasis - coagulation (fibrin formation) and tertiary hemostasis (fibrinolysis). Primary hemostasis has been partially examined in the microhematocrit and hemogram above in terms of determining platelet quantitative adequacy. Specific examination of the primary hemostatic reaction is accomplished with the buccal mucosal bleeding time (BMBT). The BMBT is accomplished with a bleeding time device and has been described. The BMBT is sensitive to platelet dysfunctional states primarily and to vascular dysfunction secondarily. The BMBT is not performed when thrombocytopenia is pronounced - the result will be obvious, a prolonged BMBT. Examination of Secondary Hemostasis - Secondary hemostasis (coagulation) is mediated by coagulation proteins or factors. It is important to recall that all of the coagulation factors are circulating in the plasma. The tests of coagulation - PT, APTT, ACT, PIVKA There are numerous universal tests of coagulation. The most common of these includes the prothrombin time (sometimes called the one-stage prothrombin time, PT or OSPT), the activated partial thromboplastin time (APTT) and the activated coagulation time (ACT). The PT examines the extrinsic and common paths. The APTT examines the intrinsic and common paths. The ACT examines the same paths as the APTT. While all of these tests are relatively easy to do and do not require expensive equipment most veterinary hospitals and emergency centers only do the ACT. The ACT is, in essence, a less sophisticated APTT. In fact if the ACT is prolonged there is no reason to do the APTT. However, because of the relative insensitivity of this test, if there is clinical suspicion of a secondary bleeding disorder and the ACT is normal, the APTT must be performed. Since there is no phospholipid (platelet substitute) added to the ACT (the APTT includes this step), it is also potentially possible (but rare) to have a prolonged ACT due to severe and prolonged thrombocytopenia, while APTT is normal. The PIVKA test (proteins induced by or involved in vitamin K antagonism or absence) is a specific test of vitamin K deficiency. In fact, the PIVKA test often prolongs as much as 24 hours before clinical signs may be observed. The PIVKA plasma is deficient in three of the four vitamin K coagulation proteins, factors II. VII, and X. The test is simple and reproducible. It can be accomplished in-hospital and requires only the deficient plasma, calcium chloride, and a hot water bath. Understanding test abnormalities Recognizing test abnormalities may be enhanced by following a few rules. There are a number of anticoagulants readily available. Included are heparin (potentiating action of ATIII), EDTA, and citrate (both of which chelate calcium). Calcium chelation by citrate is reversible while calcium chelation by EDTA is unpredictable. Citrate is used as the anticoagulant for testing the hemostatic system, specifically secondary hemostasis - coagulation. Citrated plasma samples should not have observable hemolysis as hemolysis induces major unpredictable changes. Hemolysis has the potential to cause a normal patient to appear abnormal or a abnormal patient to appear normal. The ratio of citrate to blood must be observed. Consult your laboratory concerning their requirements. In our laboratory 3.8 percent citrate is used. Hence a ration of 9 parts of blood is mixed with 1 part of citrate. Submission of a normal (control) plasma taken at the same time and in the same way as the patient sample is helpful. A patient value that exceeds the control value by more than thirty percent should be considered abnormal. That is, a patient to control ratio of greater than 1.3 suggests a prolonged patient value. Examination of Tertiary Hemostasis - Tertiary hemostasis, fibrinolysis, may also be examined in-hospital. Examination of the retracted clotted whole blood (not anticoagulated blood) left in a graduated centrifuge tube at hourly intervals may be revealing. Normally the clot should appear tight and firm. If the clot begins to appear tight and firm but later appears ragged, excessive fibrinolysis is suggested. The presence of schistocytes among red cells indicated fragmentation due to microvascular fibrin formation and fibrin(ogen) degradation. Fibrinolysis may also be suggested by the presence of increasing concentrations of fibrin(ogen) degradation (split) products (FDPs). There are several simplistic tests available to examine for FDPs. The test that is commonly used is the Thrombo Wellco test produced by Burroughs Wellcome (now Glaxo Wellcome). This test kit is inexpensive and easily and quickly performed. The test kit contains positive and negative controls. This is a latex agglutination reaction. Antibodies to fibrin(ogen) split products are annealed to latex beads. A positive test results in marked clumping as visualized on a glass plate. Other Hemostatic Procedures There are other simple and commonly used procedures associated with hemostatic testing. However, these procedures are performed in specialized laboratories. These include testing for
Defective hemostasis causing bleeding can result from vascular disorders, thrombocytopenia, thrombocytosis, defective coagulation proteins, or excessive fibrinolysis. TECHNIQUES IN EXFOLIATIVE CYTOLOGY: AVOIDING THE PITFALLS
Exfoliative cytology (diagnostic fine needle biopsy) is a simple and cost effect adjunct to other laboratory diagnostic procedures. The only limiting factor is creativity. If you are capable of putting a needle into a site - in most instances this is a relatively atraumatic and innocuous procedure - you can accomplish cytology. Simple equipment is required after the potential aspiration site has been appropriately prepared: a 22 gauge or smaller needle, a scalpel blade, cotton tipped applicators, a 3 milliliter syringe, cover slips, and cleaned glass microscope slides. Today's modern microscopes require that stained materials be cover slipped after staining. Glass slides should be meticulously cleaned prior to usage, even if the box is labeled "precleaned." OBTAINING THE SPECIMEN
A needle 22 gauge or smaller (the needle length is dependent upon the aspiration site) is required. The larger the needle lumen the more probable will be hemorrhage and red cell contamination. A small 3 milliliter syringe is attached to the needle. In effect, the syringe is utilized as a handle. Use of a larger syringe generally results in too much vacuum and resultant cellular distortion and destruction. For solid tissue aspiration the needle should be introduced into the tissue and the needle redirected numerous times without removing the needle from the tissue. If aspiration is attempted, care should be taken not to exceed 1 to 2 milliliters of aspiration pressure and to release the syringe plunger prior to removing the needle. For aspiration from body cavities care should be taken to aspirate gently and handle to test tube contents gently. Body cavity fluids should always be handled by 1) making direct smears, and 2) making a concentrated preparation. The concentrated preparation is prepared by removing the supernatant and resuspending the cell button by gently flicking the test tube bottom. For protein poor fluids, resuspending the cell button in a milliliter of species specific plasma will result in better cell adherence to the glass slides and more appropriate cytological morphology. Some tissues do not exfoliate cells readily and biopsy is required. Biopsied tissue should be gently blotted with a paper towel to remove excess blood. A scalpel blade may be used to scrape the tissue and the materials accumulated on the blade transferred gently to a glass slide. Alternatively, after removing excess blood, the tissue may be inverted and impressed on a glass slide or held with a forceps as a glass slide is repeatedly touched to the tissue. Formalin should not be in the same area as the tissue and cytological preparation. Formalin tends to cause individual cell distortion. Preparations may be made and then the tissue taken to another area where it is placed in formalin. If a cotton tipped applicator is used, the cotton tip should be moistened and excess moisture squeezed out. The applicator may be rolled gently over the surface in question followed by rolling the applicator on the surface of the glass slide. SMEAR PREPARATION
The ultimate goal of smear preparation requires a monolayered cellular area on the slide. It is this area that cellular detail may be adequately examined. Once a smear is made, if a fluorescent light is viewed through the unstained smear, a monolayered area will give a rainbow effect. Smears without this rainbow effect are too thick and are cytologically useless. A number of techniques should be used with each patient's preparation. A standard preparation as is used in making a blood smear is generally the poorest way to make a preparation. In this type of preparation the cellular distribution is affected by the relative weight of cells and distribution will be uneven. A preparation made by allowing materials to spread between two slides results in a good monolayered area. The cartwheel technique of dropping a glass slide on top of a drop of material on another slide, and picking the second slide almost off of the first, twisting the slide, dropping it again, and repeating this procedure several times usually results in a number of adequately monolayered areas. The spider technique of taking an applicator stick and drawing out "spider legs" is an excellent technique to achieve a monolayered area. STAINING THE PREPARATION
Stains should be regularly filtered. This is accomplished by pouring the stain through a funnel enclosing a folded paper towel. This should be accomplished at least daily and can be accomplished more often. Stains tend to become old fairly quickly. Instead of simply replenishing a stain, throw out the old stain, scrupulously clean the stain bottle, then replace the old stain with new stain. Stain contaminated with infectious materials should be replaced. Stain precipitate looks like bacterial organisms and should be minimized by filtration. EXAMINING THE PREPARATION
Preparations should be examined in a light, clean, and odor-free area. Ideally the microscope used for fecal and urine samples should be used for just those specimens. Another microscope should be used for examination of blood and cytological specimens. Fecal and urinary enzymes severely damage the objective lens and prevent fine cellular examination. A chair which is adjustable, which has a back, and which is comfortable, lends itself to the microscopist's comfort and effectiveness. The preparation must have some cells to be read. Preparations which are submitted to an outside laboratory should at least be examined to see if there are cells on the slide. That requires that at least one slide be stained and that the slide be examined. This accomplishes several important things: it insures that the submitted preparation includes diagnostic material, and it insures that the submitter will have examined the material - a built-in continuing education system. Submission of materials requires a complete history and more than several slides if exfoliative cytology is to be effective. All slides should be individually and adequately identified. This is the beginning of an effective clinical exfoliative cytology diagnostic program... Clinical Biochemistry and Hepatic Function
Clinical Signs of Hepatic Disease
Anion Gap = (Na + K) - (HCO3 + CL)
Osmolality = 2xNa + Urea/3 + Glucose/18 Compare actual osmolality to calculated osmolality
1-8. Cat, Female, 1 year old; not eating 5 days; pale, yellow mucous membranes Hematology: Severe responsive anemia; Heinz bodies Urinalysis: 1.035, ^protein, ^bilirubin, ^blood, ^WBC, ^rbc
2-9. Persian Cat, Male, 4 years old; hair loss, anorexia, lethargic, thin; hepatosplenomegaly Hematology: Severe nonresponsive microcytic anemia, leucopenia, neutropneia Urinalysis: SG 1.045
3-10 Greyhound, Spayed female,11 years old; lost for 5 days, returned weak and depressed Hematology: Increased PCV (64), Hb, RBC, lymphopenia Urinalysis: SG 1.068
4-13 Springer Spaniel, Spayed female,12 years old; weight loss, poor appetite, weak Hematology: Modest NN nonresponsive anemia; PCV 24; leucopenia, neutropenia Urinalysis: SG 1.025; 3+ protein Prot. Electro: low albumin; ^ gamma globulin (monoclonal)
5-17 Terrier, Spayed female,4 years old; weak, distended abdomen, febrile Hematology: PCV 56; marked leucopenia (neutropenia-672); left shift; thrombocytopenia (120,000) Urinalysis: Normal; SG 1.047
6-28 Doberman, male, 2 years old; red colored urine, anorexia Hematology: Modest leucocytosis, neutrophilia, left shift Urinalysis: ^Protein, ^ Blood, ^ Bilirubin, ^ WBC, ^ Bacteria
7-29 Dog, Beagle, neutered male, 9 years old; anorexia, quiet, enlarged liver is asymmetric, mass on ultrasound; abdominal fluid Hematology: Marked leucocytosis, neutrophilia, monocytosis,left shift, toxic neutrophils
8-32 Labrador, spayed female, 12 years old; anorexia, quiet, 1 week history of dark-colored urine Hematology: Slight leucocytosis, neutrophilia, left shift, lymphopenia
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