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PERIPHERAL BLOOD FILM EVALUATION WHAT LIES BENEATH? งานประชุมวชิ าการ คณะสัตวแพทยศาสตร์ มหาวิทยาลัยเชียงใหม่ 2563 Multi Systemic Disease Nawin Manachai (DVM., MSc., PhD.) Small Animal Clinic Department of Companion Animal and Wildlife Clinic Faculty of Veterinary Medicine Chiang Mai University • คำถำม ? ในช่วง 6 เดอื นทผี่ ่ำนมำท่ำนดู blood smear บ่อยแค่ไหน ? 1. อย่ำงน้อย 1 ครงั้ ตอ่ สปั ดำห ์ 2. อย่ำงน้อย 1 ครงั้ ตอ่ เดอื น 3. อย่ำงน้อย 1 ครงั้ ตอ่ 3 เดือน 4. อย่ำงน้อย 1 ครงั้ ตอ่ 6 เดือน 5.ไม่เคยดูเลย Peripheral blood smear (PBS) Screening Diagnosis • Hematological disorders • simply -anemia • safe -leukopenia -thrombocytopenia -unexplained cytosis -malignancies • Non-hematological disorders (hematologic manifestations in Early management Monitoring systemic disease) • Peripheral blood smear (PBS) Iron deficiency IMHA Megaloblastic anemia ITP Myelophthisis blood picture MAHA blood picture Hematologic malignancy Blood parasite infection What is included in a complete blood count (CBC) ? Scatter plot data Analyzer data Blood film microscopic review Provided by automated analyzers Provided by automated analyzers 5 Peripheral blood smear (PBS) 1. EDTA-blood 2. Glass slide 3. Coverslip 4. Fixative agent 5. Staining • Wright’s stain • Diff-quick 6. Light microscope 7. You Standard area… stacked RBCs on standard area Advantage zone of morphology Verify automate analyzer results Identify critical diagnostic features that analyzers cannot evaluate Identify morphologic abnormalities can be present even in patients with quantitatively normal results for all Peripheral blood film (smear) feathered edge hematologic parameters Make blood smears soon after collection to reduce the risk of artifacts Make a good quality smear 10X Always start from LOW POWER 10X 1. RBC distribution • degree of anemia • rouleaux formation • autoagglutination 2. WBC estimated number • 10-15 cell/LPF approximate to normal 3. Small number of suspected cells and large haemoparasite • nRBCs, blast • evaluate for the presence of microfilaria 4. Identify platelet clumps • might artifactually decrease platelet numbers Always start from LOW POWER 10X presence of microfilaria at feathered edge presence of platelet clump at feathered edge 100X Let’s proceed to … High power field with oil immersion lens x100 • Perfect in all 3 series 1. Assess to RBC 2. Assess to WBC 3. Assess to platelet 4. Assess to others Look at… RED CELLS • Size and staining • Anisopoikilocytosis • Bone marrow response • Red cell distribution • Others (inclusions) 1. Size and staining ICSH : The International Council for Standardization in Haematology • Hypochromic microcytic -grading +1, +2, +3 -mild, moderate, marked • Normochromic normocytic Small lymphocyte • Macrocytic • According to red cell size (MCV) … Macrocytic • MCV >77 fL (>45, cat) 1 Megaloblastic anemia Normocytic -Vit B12 deficiency • MCV 60-77 fL (40-45, cat) Non-regenerative anemia -Folate deficiemcy Microcytic 1.Anemia of inflammation 2. Reticulocytosis • MCV < 60 fL (<40, cat) 2. CKD 3. FeLV, MDS, 3. Sepsis • Yong animal Aplastic anemia 4. Hemoparasites • 4. Liver disease Iron deficiency anemia I(D) 5. Metabolic defects 5. Drugs • Anemia of inflammation 6, Hypothyroidism (severe) 7. Androgen deprivation 6. False due to agglutinate • Lead toxicity 8.Bone marrow disease RBC • Chronic blood loss 9. Hemolytic anemia • PSS 10. Blood loss • According to Red cell staining (MCH and MCHC)… • Iron deficiency anemia -increased central pallor • Microcytic hypochromic -decreased Hb -secondary to low-grade, Hypochromic RBC chronic blood loss. from the GI tract (ulcers, neoplasia, parasites) non-regenerative anemia CKD BM diseases Normohromic RBC AI Endocrinopathies • Indicates a regenerative BM response to anemia • Blood loss Polychromasia RBC • Hemolysis • Reflect to aggregate reticulocytes 14 • Hypochromic microcytic anemia in dog • Iron deficiency (ID) • Chronic blood loss Peripheral Blood Film… Dog 3 yo PCV = 30% Hb = 10 g/dL MCV = 57 fL Chronic diarrhea with bloody content for several weeks VET MED CMU Variation in RBC size on a blood smear, normally correlate to Red Cell 2. Anisocytosis Distribution Width (RDW) measure by automates - Normal finding in cat - Frequently found with poikilocytosis - Assess to others morphologic changes A) Normal distribution of RDW (11-14% in dog) (14-20% in cat) B) Increase distribution of RDW - Anisocytosis VET MED CMU • Aniso-poikilocytosis Important to assess RBC morphology…. (HPF100X) • To progress understanding of the type of anemia present and its possible causes • Poikilocytes may be removed premature from circulation • Some abnormalities have specific significance relating to a particular • Disease while others may be non-specific • Spleen and liver an important role Aniso-poikilocytosis 6.Codocyte/Leptocyte (target cell) 1.Schistocyte (fragmented cell) 7.Stomatocyte 2.Dacrocyte (tear drop cell) BM diseases: myelofibrosis 8.Spherotocyte 3.Acanthocyte (spur cell) - Lack of central pallor Micro-spherocyte 4.Echinocyte (bur cell) 9. Keratocyte (bite cell) (Basket/blister cell) (oxidative injuries) 5.Eliptotocyte (ovalocyte) • Schistocytes.. (fragmented RBCs) • Microangiopathic hemolytic anemia (MAHA) • MAHA blood picture (1-2 / HPF) Red cell fragmentation (fragmented RBC) caused by mechanical injury to RBC in vasculature (shearing force) Common in dogs with DIC, vasculitis, hemangiosarcoma, TTP, HUS • MAHA blood picture Peripheral blood film Feline, 1 yo M anorexia, depress -Acanthocytes +3 and presence of skin -Schistocytes +2 and mucosal hemorrhage Coagulogram PE: Pale mm Thrombocytopenia Petechial and PT and aPTT Ecchymosis prolongation Hemorrhage Underlying disease? Presentation Title • Acanthocytes (spur cells)... -No central pallor -Irregular spine Pathologic: Abnormal lipid composition of RBC membrane Increase free cholesterol in plasma e.g. liver failure, hepatic lipidosis in cat Severe liver disease : Spur cell anemia Fragile RBC Fragmentation injury: DIC ; co-finding with schistocyte or keratocyte (MAHA blood picture) • Echinocyte (crenation, burr cell)… Presence of central pallor Regular spicule Change in membrane lipid or pH or oxidative injury Type I Type III Normal or artifacts: Drying artifacts in smear preparation, prolonged storage in EDTA Pathologic: Electrolyte abnormality, uremia, rattlesnake envenomation glomerulonephritis • Echinocyte and Acanthocyte Peripheral Blood Film… Dog 12 yo, male PCV = 32% Hb = 10 g/dL MCV = 60 fL Abdominal effusion ALT = 400 U/L H ALP = 362 U/L H Albumin = 2 g/dL L Liver US : multiple hyperechoic mass VET MED CMU • Echinocytosis from hematotoxin suspected snake venom Parameter Day 1 Day2 Hct (%) 54 27 Hb (g/dl) 19 9.7 Plt (cell/ul) 82,000 36,000 WBC (cell/ul) 15,100 49,650 VET MED CMU Band - 5,165 VET MED CMU (cell/ul) Creat.(mg/dl) 1.6 2.83 Identify morphologic abnormalities can be present in patients with quantitatively normal results for all hematologic parameters • Stomatocyte… (mouth-like) • Pathologic: anemia (non-specific): Hereditary stomatocytosis in Alaskan Malamute cirrhosis, obstructive liver disease • Leptocyte… (target cells) Increase ratio of surface area to volume Decreased ratio of Hb (relative or absolute) • Fold in the membrane a central bar of Hb Artifacts: Drying artifacts in smear preparation and excessive EDTA • Pathologic (dog): anemia (non-specific) Hepatic diseases cirrhosis esp. cholestatic disease Post-splenectomy Renal disease (nephrotic syndrome), Regenerative anemia Anemia of inflammation • Leptocyte… (target cells) : Chlolestatic disease Peripheral Blood Film… Dog 8 yo, male PCV = 28% Hb = 9.1 g/dL MCV = 58 fL Abdominal enlargement ALT = 350 U/L H ALP = 1,240 U/L H Cholesterol= 250 mg/dL H Lipemia serum VET MED CMU 27 • Spherocytes (micro-spherocyte)... • No central pallor • Smaller than RBCs Remove a portion of a damaged RBCs cell membrane Result from partial phagocytic removal of antibody-coated Suggestive of immune-mediated hemolytic anemia (IMHA) (only spherocyte membrane Spherocytes are prematurely removed from found) or RBC fragmentation (a few spherocyte + schistocyte, acanthocyte etc.) circulation by splenic macrophages Only observed in dogs due to prominent central pallor because of their reduced ability to deform • Spherocytosis... with other IMHA blood picture -Spherocyte -nRBC -Polychromasia -Autoagglutination VET MED CMU VET MED CMU • Keratocytes (helmet cells)….. Red cell fragmentation caused by a ruptured vesicle or oxidative injury Often result from oxidative damage to RBC membrane DIC, hepatic lipidosis in cats • Blood loss 3. Bone marrow response… • RBC destruction (hemolysis) • Myelophthisis (BM disease) Minor evidence of regeneration of BM response Nucleated red blood cells (nRBC) Increased RBC size (MCV) - macrocytosis Polychromasia Increased Howell-Jolly bodies (with anisocytosis) -nuclear remnant -splenic diseases or splenectomized dogs -small number in healthy cats Aggregate reticulocyte (NMB) 3. Bone marrow response… Nucleated red blood cells (nRBC) Polychromasia VET MED CMU VET MED CMU CLL with hemolytic anemia in cat with FeLV • Don’t be confuse!! between small lymphocyte and nucleated red cell VET MED CMU VET MED CMU Small lymphocyte Nucleated Red Cell Small lymphocyte nRBC 4. Red cell distribution… Rouleaux formation • Rouleaux formation • Normal in healthy horses and cats -stacks formation of RBCs • Excessive rouleaux formation indicates hyperglobulinemia and /or hyperfibrinogenemia -Inflammatory disease: FIP, Reactive lymphadenopathy -Neoplasia: B-cell neoplasm : MM, CLL • Autoagglutination
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