VPT-73-Midterms-Coverage.pdf

VPT 73: Veterinary Clinical Pathology Absin, LAS Unit 1. Introductory Concepts Samples must be analyzed within minutes, usually...

VPT 73: Veterinary Clinical Pathology Absin, LAS Unit 1. Introductory Concepts Samples must be analyzed within minutes, usually w/in hours rarely w/in days. Introduction Plasma Fluid component of blood that is harvested after Clinical pathology: sub-specialty of pathology that centrifugation of an anticoagulated blood sample. deals w/ the use of laboratory methods for the diagnosis and T/x of disease. Contain the anticoagulant that can interfere with In general terms, it is the study of disease in the some assays. clinical environment by use of laboratory assays. Anticoagulants Laboratory tests should be used w/ other diagnostic EDTA (NaEDTA, KEDTA) procedures; two diagnostic procedures are Preferred anticoagulant for almost all routine imperative: hematologic tests, including the complete blood o Obtain a complete history, and count (CBC) assays. o Perform a complete physical examination. Citrate (as Sodium Citrate) With the knowledge gained from these two basic Preferred anticoagulant for most tests of the procedures, a diagnostician can select diagnostic coagulation system. Ca2+ is added to the citrated procedures to clarify or classify identified problems. plasma to override the effects of citrate and enable Importance of Analyzing Samples via Laboratory coagulation enzymes to function. Procedures Citrate’s anticoagulant activity is achieved by its forming an ionic bond with Ca2+. To detect an unidentified pathologic state. Because it has low toxicity, citrate is also preferred To define, classify, or confirm a pathophysiologic for collection of whole blood to be used for disorder or disease state. transfusions. To eliminate (rule out) a possible cause of the Oxalates (as lithium, ammonium, and potassium salts) animal’s illness. Few laboratory tests: for example, oxalate is the To assess changes in a pathologic state either due to anticoagulant in sodium fluoride tubes that are used natural progression of the disease or because of for glucose and lactate assays; distort morphologic medical or surgical therapy. features of leukocytes and erythrocytes and thus are unsuitable for hematologic samples. Samples Oxalate’s anticoagulant activity is achieved by it forming an ionic bond with Ca2+. Most clinical laboratory assays are designed to detect or quantify substances or cells in blood Heparin (as lithium, ammonium, potassium, or sodium samples; the substance or cell of interest is called salts); the analyte. Inhibits coagulation factor (including thrombin) Obtaining useful results for the analyte requires Used for several special laboratory assays (such as appropriate samples. blood gas analysis) and can be used for many clinical Whenever there is doubt about the appropriate chemistry assays. sample for a particular test at a particular laboratory, Major disadvantages: the laboratory should be contacted prior to sample o Alters morphologic features and staining of collection. leukocytes. o Allows clotting as effects are slowly Blood overridden by the coagulation system. Blood and its major components are frequently used o Allows platelet clumps to form. as samples for laboratory assays; must be collected Serum and processed properly. Composed of blood cells and plasma. Serum is the fluid component of blood that is Immediately mixed w/ an anticoagulant to prevent harvested after centrifugation of a coagulated initiation of clot formation and to maintain cells and (clotted) blood sample. other components in suspension. To get maximal amount of serum from the clotted Analysis or processing of whole blood must be sample, centrifugation should not be started prior to relatively rapid because the cells die w/in a few the retraction of the clot (w/c typically takes at least hours; unacceptable for analysis. 30 min if a clot activator is not present in the tube). If samples are centrifuged prior to clot retraction, some serum will be trapped in a soft fibrin clot. 1 VPT 73: Veterinary Clinical Pathology Absin, LAS Same composition as plasma except serum does not frequently break during transit, so if you are using contain most of the coagulation major protein (on a them, wrapping them in bubble wrap or other weight/ volume basis) that is absent in serum but cushioning material will protect them. present in plasma is fibrinogen. Add cool packs: Ship fluid samples on cool packs. The samples should not be in direct contact w/ the cool but wrapped in paper towels. Direct contact of cells w/ an ice pack will cause freezing of the sample and cell lysis. Submission Avoid formalin: Do not ship formalin containers in the same package as the slides or tubes. Formalin readily leaks out of containers and affects the quality of the samples. Ship ASAP: The quicker the sample gets to the Examples of commercial blood collection vials. From the left, laboratory, the fewer the false changes in results due the red stopper indicates no anticoagulant (“no tube”), the to storage. lavender one (often called “purple top”) has EDTA, the green Provide a good history: This includes signalment has heparin, and the yellow and blue plastic capped tubes are (species, age, breed, sex of patient), history that may specialized tubes for specific tests. be relevant (e.g., travel, access to toxins, current medications), pertinent clinical signs (e.g., epistaxis) and, for cytology, a good description of the aspirated lesions (including imaging findings), e.g., multiple hypoechoic masses in the liver. Major Types of Laboratory Assays Clinical hematology assays Clinical chemistry assays Clinical microscopy Basic Laboratory Safety Sharps Handling and Storage Chemicals Biologic materials Separation: For hemostatis and chemistry testing, PPE (Personal Protective Equipment) the plasma or serum should be separated from cells as soon as possible after sample collection, w/c is done by centrifugation. Labeling: All body fluid samples taken from a patient should be correctly labeled w/ the patient name or identification and the type of specimen (e.g., serum, plasma, synovial fluid, peritoneal fluid) Storage: All fluid samples should be stored at 4°C until submission. In contrast, slides should not be refrigerated (the cells lyse with storage). Submission Label properly: Make sure all tubes and slides are labeled w/ patient identification and the contents (e.g., blood, urine). Package appropriately: Make sure the samples are protected from breakage. Cardboard slide boxes 2 VPT 73: Veterinary Clinical Pathology Absin, LAS Unit II. Erythrocytes Plasma color and Transparency Normal plasma is clear and colorless (dog and cat) to Erythrogram light (horse and cow). Icteric plasma is yellow and clear. Counts of red blood cells (RBC) Hemoglobinemic plasma is pink to red and clear. RBC mass (hematocrit) Hemoglobin concentration Lipemic plasma is whitish to pink and opaque. RBC indices (cell size, hemoglobin content) Buffy Coat RBC size variation (red cell distribution width or A white layer bet. the RBCs and plasma, is comprised RDW) of leukocytes and platelets. Reticulocyte counts (percentage or absolute) Measurement of its width has been used to estimate Nucleated RBC count (per 100 WBC or absolute) white blood cell counts. RBC morphologic features, including presence of Microfilaria may be detected by microscopic parasites. examination of the plasma just above the buffy coat layer. Packed Cell Volume (PCV) Hemoglobin determination Hematocrit is the % of blood composed of erythrocytes; to separate blood is thru centrifugation The most accurate direct indication of oxygen (platelets, plasma, and leukocytes are trapped transport capacity of blood and approximately 1/3 of between red cells) the PCV if erythrocytes are of normal size. PCV, buffy coast, and plasma Acid-hematin method. Microhematocrit and microhematocrit. Oxyhemoglobin (Tallqvist Hgb scale and Dare Red blood cell packing is species-dependent—it chemoglobinometer) takes longer for ruminant RBCs to pack compared to Cyanmethemoglobin dogs, cats, and horses. Therefore, the Avian erythrocytes must be lysed and the specimen microhematocrit tubes are spun for 10 mins. In must be centrifuged to remove free nuclei before ruminants vs. 3 mins. In other species. Hgb concentration. RBC counts Performed with a hemocytometer, have a large degree of error; hemocytometer-derived RBC counts are of limited value, except in avian spp. Automatic counters, if standardized for mammalian blood, allow for more accurate RBC counts. Automated counters are not well validated for avian blood, because all nucleated cells (RBCs, WBCs, and thrombocytes) are counted. The primary value of the RBC count is that it allows determination of MCV and MCH. Mean Corpuscular Values (MCV) MCV = (Hematocrit x 10)/ RBC in millions = femtoliters (fl) o Ex. PCV: 45%, RBC count: 5 M/μL. ▪ 450/5.0 = 90 fl MCV (↑) in macrocytic anemias (B12, Folic acid deficiencies). Reticulocytsis causes transitory increase. MCV (↓) in iron deficiency. Mean Corpuscular Hemoglobin (MCH) MCH = (Hgb x 10)/ RBC in millions = picograms (pg) o Ex. Hgb: 15 g., RBC count: 5 M/μL 3 VPT 73: Veterinary Clinical Pathology Absin, LAS ▪ 150/5.0 = 30 pg Feline Erythrocytes MCH (↓) in iron deficiencies. Slight increase/normal in reticulocytosis Smaller and more variable in size and shape than MCH (↑) in vitro and in vivo during hemolysis. those of dogs. Mean Corpuscular Hemoglobin (MCHC) Little to no central pallor. MCHC = (Hgb x 100)/Hematocrit = grams/deciliter Polychromatophilic cells are very few in health (