兽医血液学和细胞学

Hematology and Cytology: More than pretty Colors Sharon M. Dial, DVM, PhD, DACVP Arizona Veterinary Diagnostic Laboratory University of Arizona Quality from the Beginning Hematology and Clinical Chemistry data is only as good as the sample submitted Pre-analysis factors can effect all data Is the sample fasting? how was the blood drawn? how was the sample handled post-draw? Sample Handling Blood collection: Label, label, label slides and blood tubes Use frosted end slides - label in pencil add paper label after staining If submitting to lab - no paper labels Sample Handling Use largest bore needle possible decrease time for collection decreases lysis of fragile RBCs Vacutainer versus needle and syringe? whatever gives you the best sample Consider use of a butterfly catheter with Vacutainer connection. Collecting the Blood Sample Principles of good venipuncture #21 gauge or larger facilitates rapid flow, minimizes hemolysis. move plunger within unit to ensure syringe patency. enter the vein keeping bevel side of needle up. Avoid repositioning or excessive suction. 1 Collecting the Blood Sample Immediately transfer blood to an EDTA collection tube. Method 1 Remove needle and tube stopper. Gently dispense the blood into the tube. Method 2 Leave needle on and push it through the stopper allow the vacuum to draw the specimen from the syringe. Do not force the blood through the needle Hematology Instrumentation Abaxis CDC Technologies Heska Idexx LaserCyte QBC technology Hematology Automated hematology Advantages Speed “accuracy” Increased information Disadvantages Cost Maintenance Tend to forget the blood film 2 Hematology Reference laboratories Advantages Large combined profiles available CBC and Chemistry Specialty testing Serology Endocrine Experienced technologist/technicians Pathologist available for review of abnormalities Disadvantages No stat testing available in most areas Hematology CBC Necessary equipment Microcentrifuge Hemocytometer (unopette system) Microscope Refractometer Hematology Components of CBC WBC -100 cell differential PCV or HCT Platelet count Total protein Review of the blood film Hematology WBC Unopette system Estimate from blood film 3 Requires good uniform film preparation Estimate from 10X or 40x Can be used to support manual count Hematology Additional information from an automated count. RBC indices MCV MCHC RBC count Automated differential Review of the blood Film Allows rapid identification of significant hematologic abnormalities Marked leukopenia or leukocytosis Increased band neutrophils Abnormal cell types Allows evaluation of cell morphology not reported by automated systems Toxic change in neutrophils Red blood cell morphology Macroplatelets Review of the Blood Film Identification of common instrument errors Miss-sampling Small clots in sample – decrease the volume analyzed – pancytopenia Instruments cannot always detect when the sample flow is altered. Daily QC does not eliminate errors due to individual sample quality! Platelet counts commonly affected by small clusters, large platelets or small red blood cells. 4 Hematology Evaluation of blood film 10X Cellularity Estimate of WBC count – High - > 50 / 10x field Low - < 20 / 10x field Feathered edge Platelet clumps Large cells (blasts, mast cells) Microfilaria Hematology Evaluation of the blood film 40x – must use a coverslip to use this objective lens Estimate WBC count Average # of cells/10 fields X 2,000 Quick differential Hematology Evaluation of blood film Counting area 100 cell Differential WBC estimate Red cell morphology Body of film RBCs touching or just overlapping Platelet estimate 5 Hematology WBC Methods Estimate from blood film – provides a number to compare with automated count Count all leukocytes in ten 40 x fields (raw leukocyte count/# of fields counted) x 2000 = estimated leukocyte count. Corrected count = (estimated count x (actual PCV/ normal PCV)). Note: 40x objectives require a coverslip!! Hematology Example 50 cells/10 fields 5 * 2000 = 10,000 / µl PCV 22 10,000 * 22/45 = 4,888 / µl Platelet Estimates Platelet estimate Average #/oil immersion field x 20,000 Example: 7 platelets/field 7 x 20,000 = 140,000 / µl >10 platelets/oil immersion field – normal platelet mass. Evaluation of the Erythrocyte The Erythrocyte Erythropoiesis – the formation of red cells Erythrocyte function – oxygen transport 6 Red Cell Shape The normal red cell is a biconcave disc Perfect shape with just the right amount of surface area to exchange oxygen in the lung and tissue Cell membrane is ~45% protein ~45% lipid ~10% carbohydrate A protein structural “net” holds the membrane in shape Erythropoiesis The formation of erythrocytes Occurs primarily in the bone marrow Can also occur in the spleen (extra-medullary hematopoiesis EMH) RBCs are formed in sinusoids and have to move though the walls of the sinusoids to get into the blood stream (must be flexible) Oxygen Transport Hemoglobin Four molecules of Heme (each with one atom of iron) One molecule of globin (protein) 95% of the red cell dry weight is hemoglobin Each heme/iron unit transports one Oxygen molecule Schistocytes Hallmark of RBC fragmentation Shearing of RBC by intravascular fibrin strands Microangiopathy Turbulent blood flow Caval syndrome Valvular stenosis 7 Intrinsic RBC abnormalities Severe iron deficiency Chronic doxorubicin toxicosis Spherocytes Hallmark of immune-mediated anemia (often large numbers) Can be seen with RBC fragmentation (small numbers) Hypophosphatemia Toxins (zinc) Heinz Bodies Most common in cat Onions Acetaminophen Propylene glycol Metabolic disease Diabetes mellitus Renal disease Lymphoma Eccentrocytes Oxidative injury Fused inner cell membrane More commonly seen in dogs Basophilic Stippling Punctate aggregates of RNA Stain with Wrights-Giemsa Associated with regeneration Lead Poisoning 8 Howell-Jolly Bodies Micronuclei or nuclear remnants Regenerative anemia Post splenectomy Can be indicator of marrow injury Evaluation of the Erythrocyte Polycythemia – increased red cell number Relative polycythemia Absolute polycythemia Anemia – decreased red cell number Regenerative Non-regenerative Evaluating the Erythrocytes Clinical signs seen with increased RBCs - Injected (red) mucous membranes Clinical signs seen with decreased RBCs Pale mucous membranes Icteric mucous membranes Anemia Classification of anemia is based on degree of regeneration. Regenerative anemia – bone marrow is functionally responding to the decrease in erythrocytes Increased erythrocyte production Release of young erythrocytes (reticulocytes) Anemia Non-regenerative anemia – bone marrow cannot respond to the need for erythrocytes Can be primary marrow failure Can be due to non-marrow disease Suppression of erythropoiesis Lack of erythropoietin 9 Anemia Changes in the RBC indicies with Regeneration Macrocytosis - increased Mean Corpuscular Volume (MCV) Dog MCV – 60-77 fl Some breeds of dog can have an MCV greater than the normal range (Poodles) or smaller (Akitas). Cat MCV – 39-55 fl Cats can have a non-regenerative macrocytic anemia associated with FELV. Cats often have a more prominent increase in MCV during regeneration than dogs. Anemia Evidence of Regeneration from the blood film. Presence of polychromasia. Blue tinge to RBC on a Wright’s Giemsa stain. Difficult to appreciate with some quick stains. Polychromatophilic RBCs have a “muddy” blue color. Anemia Evidence of regeneration from reticulocyte counts. Why do reticulocyte counts if there is polychromasia? Not all reticulocytes appear as polychromatophilic cells. Is quantitative rather than qualitative. Anemia Reticulocytes Dog – aggregate reticulocytes only Cat – both aggregate and punctate reticulocytes. Can count both Aggregate indicates recent regeneration Punctate indicates regeneration some time in the past. 10 Anemia Reticulocyte count Count number of reticulocytes/5 fields in the body of the blood film (approx #1000 RBCs)/10 = raw % The raw % can be misleading. Must be evaluated in light of the degree of anemia The simple corrected reticulocyte count. % retics x patients PCV /45 (dog) or 35 (cat) Corrected count <1.0 = non-regenerative 1.0-4.0 = adequately regenerative >4.0 = highly regenerative (commonly hemolytic) Reticulocyte Count Example Total reticulocytes in five 100x oil immersion fields = 28 28/10 = 2.8% Corrected for degree of anemia 2.8% * 21/35 (for a cat, or 45 for a dog) = 1.7% 1.7% indicates a mildly regenerative anemia Nucleated RBCs (nRBC) Metarubricytosis Appropriate metarubricytosis Part of the regenerative process Must also see polychromasia Metarubricytosis Effects WBC count on Automated analysis NRBCs are counted as leukocytes Need to correct the WBC (100/(100 + number of NRBCs counted on differential)) X WBC count. 11 Example: 10 NRBCs noted during Differential Count, WBC count is 11,000/ / µl (100/ (100+10)) X 11,000 / µl = 10,000 / µl Metarubricytosis Inappropriate metarubricytosis Lead toxicity Myeloproliferative diseases Erythemic myelosis in the cat Hypoxia and bone marrow necrosis Extramedullary hematopoiesis (EMH) Sepsis/ endotoxemia (marrow injury, splenic compromise) Neoplasia (splenic hemangiosarcoma) Hypochromasia Visual assessment MCHC (mean corpuscular hemoglobin concentration)measured assessment of hypochromasia Decreased with iron deficiency – true decrease in hemoglobin Decreased with marked regeneration – large polychromatophilic cells have less hemoglobin/volume Increased MCHC is always an artifact What to look for if MCHC is increased Hemolysis (in vivo or artifact at collection) Heinz bodies Lipemia Paraproteinemia Regenerative Anemia Blood loss External blood loss Anemia Hypoproteinemia 12 Internal blood loss Anemia Normal proteins Usually a moderately regenerative anemia. Must be chronic to result in iron deficiency. Not common in adult animals. When seen in adults often associated with bleeding gastrointestinal lesion. Iron deficiency in young animals. Usually nutritional or due to internal or external parasites. Regenerative Anemia Hemolysis - Intravascular hemolysis Associated with hemoglobinemia and hemoglobinuria Zn toxicity, Tylenol, copper toxicity Some forms of immune-mediated anemia Some hemoparasites – babesia in acute infections. DIC – microvasculopathy Sepsis – Leptospirosis, clostridial diseases Anemia Intravascular hemolysis - relatively rare. Hemoglobinemia results in artifactually increase in MCHC. May see “ghost cells” May be difficult to differentiate from increased fragility – lyse in vitro No hemoglobinuria if the hemolysis occurs during collection of the sample. Anemia Extravascular hemolysis Most common May be associated with jaundice. Most forms of immune-mediated anemia 13 Hypersplenism Some hemoparasites – M. hemophilia, babesia No hemoglobinemia, or hemoglobinuria. Removal of altered RBCs by the Macrophage phagocyte system. Spleen, liver, bone marrow Can be associated with RBC metabolic abnormalities (Pyruvate kinase deficiency). Immune-mediated Anemia Can be associated with agglutination. Must differentiate true immune agglutination from non-specific agglutination. Non-specific due to abnormal “sticky” proteins. Saline test. Add 5 drops of blood to 2 mls of saline – mix, centrifuge, pour off supernatant. Repeat once more. Evaluate wet mount on 10X. Polycythemia Polycythemia – increased red cell count Most often associated with dehydration Dehydration = “relative polycythemia” High PCV/HCT, High Total Protein Will return to normal with fluids Can be a primary disease Polycythemia Vera High PCV / HCT, Normal Total Protein Can be secondary to hypoxia (lack of oxygen) Pneumonia, large thoracic masses 14 Leukocytes Evaluation of leukocytes Differential count 100 cells categorized by type 200 cells should be counted if the WBC count is greater than 30,000 / µl Unclassified cells should be included in differential Note abnormal morphology Toxic change Reactive lymphocytes Leukocytes Normal leukocytes and their function Granulocytes Neutrophils Eosinophils / Basophils Lymphocytes Monocytes Leukocyte dynamics Normal resting state Inflammation Stress Excitement Abnormal Leukocytes: leukemia Granulocytes Neutrophil: Most common granulocyte in circulation in dog and cat. “Neutral” staining granules. Granules contain enzymes and antibacterial substances to kill and degrade bacteria 15 Primary function. Primary defense against bacterial disease Can cause considerable “innocent by-stander” damage to tissues. Eosinophils: Function still a bit of a mystery. Granules contain proteins that bind to parasites (Major basic Protein) Very “caustic” – causes tissue necrosis Associated with complex parasites. Nematodes Fungi Some protozoa Regulate allergic reactions. Associated with immune-complex disease. Granulocytes Basophils: Function not understood. Granules are similar to mast cell granules in content. Uncommon in circulation. Tend to increase in number in association with eosinophils. Lymphocytes Lymphocytes: Second most common circulating leukocyte in dogs and cats Primary function: immunity Two basic types: B-cells (produce antibody) T-cells (regulate immune responses) Types not morphologically different Can change morphology when immune system is stimulated 16 Monocytes Monocytes: Phagocytosis Bacteria, complex organisms (fungal elements, protozoa) Cellular debris associated with tissue necrosis Regulate repair of tissues Regulate immune responses Major cell involved in red blood cell turn-over and iron recycling Very busy cell!! The Leukogram Definition: Numerical and morphologic characterization of circulating leukocytes. WBC count Differential cell count Morphological description of cells Must understand normal to recognize abnormal. Neutrophil Dynamics Describes the normal flow of leukocytes from the bone marrow to peripheral blood. Leukocyte compartments Proliferating pool Maturation/storage pool Circulating/ marginated pool Leukocytes Inflammatory leukogram Band neutrophils are the hallmark of inflammation. Neutrophil toxicity is the hallmark of sepsis. Dog – basophilic foamy cytoplasm and Dohle bodies. Cat – basophilic foamy cytoplasm. Dohle bodies are commonly seen in cat neutrophils with no evidence of toxicity. 17 Leukocytes The leukocytes Variation in band neutrophil morphology. Dog and Cat neutrophils are not as segmented as horse or human. Bands are often over estimated on differential. Horse bands are more consistent with human morphology. Human laboratories often over call band neutrophils in the dog and cat. Leukocytes Compensated versus Non-compensated left shift. Compensated – leukocytosis with greater number of mature neutrophils compared to bands. Non-compensated – Greater number of bands than mature neutrophils regardless of total count. Normal or low count with significant numbers of bands. Neutrophilia Causes Inflammation May or may not have a left shift May or may not have toxic change Stress (steroid leukogram) Does not have a left shift Associated with lymphopenia Excitement (epinephrine) Associated with increases in lymphocytes as well. 18 Stress Mature neutrophilia Increased release of neutrophils from storage pool. Neutrophils are less “sticky” and move from the marginated pool to the circulating pool. Increased retention of neutrophils in the circulation. Steroids make neutrophils less flexible, they can’t squeeze between the cells lining blood vessels. Lymhopenia Lymphocytes can’t get into blood vessels. Monocytosis Mechanism unknown – seen in the dog. Eosinopenia Mechanism unknown. Excitement Increase in blood pressure “washes” marginated neutrophils off the vessel wall into circulating pool. Increase in lymphocytes (lymphocytosis) that may exceed the neutrophilia. More prominent in cats (have a larger marginated pool). No change in the other cell compartments. Neutropenia Causes Severe inflammation Usually associated with left shift Toxic change Bone marrow injury Can be reversible Infectious disease (viral, Ehrlichia) Drugs 19 Chemotherapeutics Estrogen Antibiotics Or irreversible Often idiopathic Neoplasia also see anemia and thrombocytopenia Endotoxemia (Gram-Negative Sepsis) Makes neutrophils very “sticky”. Increases marginated pool. Increases exit from the blood vessel to tissue. Decreases recruitment from the storage pool. Decreases proliferation and maturation. Often associated with a remarkable “rebound” neutrophilia with a left shift. These changes happen FAST!! Lymphocytosis Excitement All small normal appearing lymphocytes Usually <20,000/µl Transient Post vaccination (young dogs/cats) Small normal appearing lymphocytes Occasional large immunoblasts Leukemia Chronic lymphocytic Acute lymphoblastic Chronic canine ehrlichiosis (tick fever) 20 Lymphopenia Stress Loss of lymphocytes Chylothorax Protein-losing enteropathy- Due to dilated lymph vessels in the intestine Viral disease Genetic immune deficiency diseases Monocytosis Stress Chronic inflammation Tissue necrosis Monocytopenia is not a problem. Eosinophilia Allergy Parasitism Fungal disease Neoplasia (mast cell tumor) Hypereosinophilic syndrome Eosinophilia Most common diseases Allergic dermatitis Asthma - Idiopathic or parasitic Eosinophilic enteritis - Idiopathic or parasitic Heartworm disease Atypical Circulating Cells Reactive lymphocytes Antigenic stimulation Neoplastic cells Myeloid – neutrophil origin Lymphoid – lymphocyte origin Myelomonocytic – both neutrophils and monocytes 21 Monocytioid – monocyte origin Erythroleukemia – red cell origin Reactive Lymphocytes Present in small numbers. Associated with strong immune response. Commonly seen in infectious disease. Canine distemper Atypical Circulating Cells Genetic Abnormalities Pelger-Huët Decreased lobulation of granulocyte nuclei Not associated with disease Chediak-higashi Syndrome Lysosomal storage diseases 22 An introduction to Cytology Cytology A useful tool in veterinary medicine. Ease of collection Inexpensive Quick Relatively low-risk to patient with possibility for return of valuable information. Cytology Disadvantages No architecture to evaluate. Histology usually required as a follow-up in neoplastic diseases. Requires patience to obtain good samples. Cytology: the formula Assess cellularity. Assess degree of hemodilution. Note number of intact cells. Characterize the primary cell populations. Evaluate background. Search for etiologic agents if indicated. Types of Cytological Preparations Imprints Of surface lesions. Of excised tissue. Scrapings Fine needle aspirates of mass lesions Aspirates of body cavity fluids 23 Sample Preparation Handling of air-dried slides. Slides should not be exposed to formalin. Causes poor staining and loss of cellular detail. Slides should not be placed in a refrigerator. Causes water condensation and cell lysis. Sample Preparation Body cavity fluids. Pleural fluid, abdominal fluid, synovial fluid, pericardial fluid. Always submit in an EDTA tube (LTT). Only exception is CSF (RTT). If culture is needed – split the sample and submit portion for culture in clot tube (RTT). Sample Submissions Fine needle aspirates direct preparations. Air-dried slides – do not fix. To prestain or not to pres