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Data, Dots and Cells: How to get the most out of your CBC Dennis B. DeNicola, DVM, PhD, DACVP Chief Veterinary Educator IDEXX Laboratories, Inc. Adjunct Professor of Veterinary Clinical Pathology Purdue University College of Veterinary Medicine Objectives Understand when hematologic evaluations should be considered Understand how some of the graphics from different hematology analyzers are produced Understand how to use analyzer graphics to complement to CBC data and blood film microscopic evaluation to: Help validate the data generated Add valuable information not provided by the analyzer Understand how to use dot plots to recognize abnormal blood samples What is included in a CBC Data – report with numbers Dots – graphic representation of how analyzer performed Cells – rapid (less than 1-3 minutes) microscopic evaluation of the blood film What is included in a CBC Data – report with numbers Dots – graphic representation of how analyzer performed Cells – rapid (less than 1-3 minutes) microscopic evaluation of the blood film CBC – Data Evaluation RBC mass RBC morphology Objective measure of BM response WBC mass WBC relative distribution WBC absolute distribution PLT mass PLT morphology What is included in a CBC Data – report with numbers Dots – graphic representation of how analyzer performed Cells – rapid (less than 1-3 minutes) microscopic evaluation of the blood film CBC: Cells • Erythrocytes • Confirm count: clumping/agglutination Normal • Confirm reticulocyte count with scan • Examine morphology of erythrocytes Acanthocytes Schistocytes Spherocytes CBC: Cells • Validate WBC count • Validate leukocyte distribution • Examine WBC morphology “Left Shift”—Immature Neutrophil Forms Neutrophil Toxicity Abnormal Leukocytes CBC: Cells • Confirm count Normal platelet count • Inspect for clumping • Characterize morphology Platelet clumping at feathered edge Thrombocytopenia What is included in a CBC Data – report with numbers Dots – graphic representation of how analyzer performed Cells – rapid (less than 1-3 minutes) microscopic evaluation of the blood film In-house Hematology Analyzers pre1980s Manual 1980s Quantitative Buffy coat. 1990s 2003 2010+ Impedance Laser ProCyte Dx RBC RBC WBC WBC How often do you use the graphic presentation provided by your analyzers? 1. 2. 3. 4. 5. Never < 25% 25 – 50% 50 – 75% > 75% Quantitative Buffy Coat Analysis Which situation related to the IDEXX VetAutoread BEST fits your experience? 1. Currently use a VetAutoread 2. Have used a VetAutoread in the past 3. Have heard of the VetAutoread but have never used one 4. Have never heard of the VetAutoread 43% 32% 24% 0% 1. 2. 3. 4. Centrifuged Anticoagulated Blood Platelets Agranulocytes Lymphocytes Monocytes Granulocytes Eosinophils Basophils Neutrophils Immature erythrocytes Nucleated RBCs Quantitative Buffy Coat Analysis Quantitative Buffy Coat Analysis Quantitative Buffy Coat Analysis DNA RNA EOS Plasma RETIC Top of the float Change Bottom of the float PLT L/M GRANS RBCs Quantitative Buffy Coat Analysis Dog – Acute Anemia HCT = 28.8 % Regenerative or non-regenerative? Dog with acute anemia – Regenerative or Non-regenerative? 1. 2. 3. 4. Regenerative Non-regenerative WAG regenerative WAG non-regenerative Dog – Acute Anemia Dog with acute anemia – Cause? 1. 2. 3. 4. Acute blood loss Chronic blood loss Hemolytic Hemolytic – oxidant injury 5. Hemolytic – immunemediated 6. Not able to identify Dog – Acute Anemia Dog with acute anemia – Cause? 1. 2. 3. 4. Acute blood loss Chronic blood loss Hemolytic Hemolytic – oxidant injury 5. Hemolytic – immunemediated 6. Not able to identify Dog – Acute Anemia - Spherocytosis Dog – Leukocytosis and Mild Anemia Dog with leukocytosis and anemia – Interpretation of leukogram? 1. Inflammation 2. Glucocorticoid influence (“stress”) 3. Epinephrine influence (“excitement”) 4. Inflammation plus “stress” 5. Inflammation plus “excitement” 6. Cannot characterize Dog – Leukocytosis and Mild Anemia Dog – Leukocytosis and Mild Anemia Common Leukogram Patterns Moderate Glucocorticoids Epinephrine Inflammation (“Stress”) (“Excitement”) - - N- Band Neutrophil - N N Lymphocyte - N- Monocyte N - N- N Eosinophil N Basophil N- N N Leukocyte Type Mature Neutrophil Impedance Counting Analyzers Which situation related to the use of an impedance analyzer BEST fits your experience? 1. Currently use an impedance analyzer 2. Have used an impedance analyser in the past Impedance Analyzers Improved precision and accuracy of cell counts Ability to produce accurate MCV values True hemoglobin measurement Allows MCHC calculation The Coulter Impedance Method Vacuum Change in resistance is proportional to cell volume Aperture Current Pathway Internal Electrode External Electrode Suspension of Cells External Housing (Aperture Bath) Aperture Aperture Housing Electrically conductive diluent Detail of Aperture Impedance technology Heska Difficult to accurately count monocytes even with normal samples Abaxis Impedance technology Impedance technology Heska Abaxis Platelet and RBC Volume Histograms Fixed Thresholds Floating Thresholds Impedance technology Problematic samples when platelets and red blood cells are similar in size Many feline samples present with this problem and typically have an overestimation of platelet numbers Feline large platelets Impedance Analyzers Improved precision and accuracy of cell counts Ability to produce accurate MCV values True hemoglobin measurement Partial differential counting Allows MCHC calculation Typically a three part differential Granulocytes, Lymphocytes and Monocytes Potential problems Nucleated red blood cells Platelet clumping and size Inaccurate counts, misidentified as RBC Flow Cytometry Analyzers LaserCyte LaserCyte Dx Which situation related to the IDEXX LaserCyte or LaserCyte Dx BEST fits your experience? 1. Currently use LaserCyte 2. Have used LaserCyte in the past 3. Have heard of the LaserCyte but have never used one 4. Have never heard of the LaserCyte LaserCyte and LaserCyte Dx LaserCyte LaserCyte Dx Schematic LaserCyte Technology Core Sample Stream Sheath Flow Channel Quartz Flow Cell Axial Light Loss High Angle Forward Scatter 8-12 deg Low Angle Forward Scatter 2-4 degrees Right Angle Scatter High Numerical Aperture 50-130 degrees Laser and Lens Sy stem Data Collection Report Light absorption / cell size Sadie Shaeffer – 11yr, F Shep Mix (07-09-08) Doublets Reticulocytes Erythrocytes Platelets Granularity / complexity Sadie Shaeffer – 11yr, F Shep Mix (07-09-08) Light absorption / cell size Eosinophils Neutrophils Monocytes Lymphocytes nRBCs Granularity / complexity Light absorption / cell size Rufisee - Feline Granularity / complexity Nadia – 7 yr old, Female, Mixed Breed dog Clinical presentation Not acting normal Slightly decreased appetite Slightly decreased exercise tolerance Physical examination Slightly depressed Suspected mildly enlarged spleen Confirmed with radiographs Nadia – 7 yr old, Female, Mixed Breed dog Nadia – 7 yr old, Female, Mixed Breed dog What is the BEST interpretation for this Erythrogram? 1. Normal 2. Resolving anemia 3. BM compensation for blood loss 4. BM compensation for hemolytic disease 5. Analyzer failure – no true reticulocytosis Nadia – 7 yr old, Female, Mixed Breed dog Reticulocytes Nadia – 7 yr old, Female, Mixed Breed dog Nadia – 7 yr old, Female, Mixed Breed dog Which of the following is the most significant RBC morphology abnormality? 1. Normal – no abnormality present 2. Stomatocytosis 3. Spherocytosis 4. Anisocytosis Nadia – 7 yr old, Female, Mixed Breed dog Nadia – 7 yr old, Female, Mixed Breed dog Confident in platelet count based on dot plot presentation Nadia – 7 yr old, Female, Mixed Breed dog Valid leukocyte differential?? Has the LaserCyte provided a valid leukocyte differential? 1. Yes 2. No 3. I don’t know Nadia – 7 yr old, Female, Mixed Breed dog Confident in leukocyte differential based on dot plot presentation Interpretation?? What is the BEST interpretation of the Leukogram? 1. 2. 3. 4. 5. Normal Inflammation “Stress” “Excitement” Inflammation plus “stress” 6. Inflammation plus “excitement” 7. Cannot characterise Nadia – 7 yr old, Female, Mixed Breed dog Nadia – 7 yr old, Female, Mixed Breed dog Nadia – 7 yr old, Female, Mixed Breed dog Nadia – 7 yr old, Female, Mixed Breed dog Case outcome Compensated immune-mediated hemolytic disease Associated inflammation Hemolytic crisis the day following this CBC Responded to treatment Poco – 3 yr old, Female, Norfolk Terrier Clinical presentation Mild weight loss Decreased appetite Mild depression Physical examination Depressed Painful abdomen on palpation Enlarged liver and spleen Poco – 3 yr old, Female, Norfolk Terrier Poco – 3 yr old, Female, Norfolk Terrier Is the anemia regenerative or nonregenerative? 1. Regenerative 2. Non-regenerative Poco – 3 yr old, Female, Norfolk Terrier With this marked regenerative anemia, what is the likely mechanism? 1. Blood loss 2. Hemolytic disease Poco – 3 yr old, Female, Norfolk Terrier What is the cause for the hemolytic anemia? 1. Immune-mediated 2. Oxidant injury 3. Mechanical / fragmentation 4. Underlying metabolic disease 5. Cannot determine at this time Poco – 3 yr old, Female, Norfolk Terrier Poco – 3 yr old, Female, Norfolk Terrier Which of the following is the primary RBC morphologic abnormality? 1. 2. 3. 4. 5. Poikilocyte Acanthocyte Spherocyte Schistocyte Dacryocyte Poco – 3 yr old, Female, Norfolk Terrier Poco – 3 yr old, Female, Norfolk Terrier Case outcome Aspirates of liver and spleen Malignant lymphoma, large cell type, high grade Owner elected euthanasia because of extend of disease Advanced Flow Cytometry Analyzers ProCyte Dx Which situation related to the IDEXX ProCyte Dx BEST fits your experience? 1. Currently use ProCyte Dx 2. Have used ProCyte Dx in the past 3. Have heard of the ProCyte Dx but have never used one 4. Have never heard of the ProCyte Dx Advanced Technology Laminar flow impedance Better alignment of cells being analyzed Allows fast, precise and accurate cell counts Advanced Technology • Laser Flow Cytometry • Advanced cellular interrogation • Optical fluorescence • Reticulocyte count • Feline platelet count • Additional specificity for leukocyte identification Size (Forward Scatter) CBC: RBC-PLT Dot Plot Red blood cells Reticulocytes Platelets RNA content (Fluorescence) Reticulocytes The ProCyte Dx analyzer uses a fluorescent stain to more precisely identify reticulocytes. RETIC = 202 K/µL Size Size RETIC = 19 K/µL Canine Fluorescence Canine Fluorescence Canine • Laminar flow impedance Size Platelet counting • Accurate counting and sizing for most species Fluorescence Feline • Accurate platelet counting for cats Size • Optical fluorescence Fluorescence “Sable” – 10 yr, Fs, Mixed breed dog “Not feeling well” for about a week Not eating well Slight depression Physical examination Good body condition and hair coat Pink mucous membranes Tender abdomen on palpation Sable – 10 yr, Fs, Mixed breed dog Sable – 10 yr, Fs, Mixed breed dog 10 - 110 Normal RBC mass Normal RBC indices True Reticulocytosis?? Is the reticulocyte count valid based on the dot plot evaluation? 1. Yes 2. No 3. I don’t know “Sable” – 10 yr, Fs, Mixed breed dog 10 - 110 Normal RBC mass Normal Normal RBC indices Mild-to-moderate reticulocytosis Clear evidence of reticulocytosis “Sable” – 10 yr, Fs, Mixed breed dog Which of the following is the most significant RBC morphology abnormality? 1. Normal – no abnormality present 2. Acanthocytosis 3. Spherocytosis 4. Anisocytosis “Sable” – 10 yr, Fs, Mixed breed dog Case Outcome Diagnostic imaging of abdomen Radiographs revealed enlarged spleen and liver Ultrasound suggested blood-filled cavities • Suspect splenic hemangiosarcoma • Suspect hepatic metastases Owner elected surgical exploration Large splenic mass with evidence of hepatic metastases Owner elected euthanasia at surgery Final diagnosis - hemangiosarcoma RNA content (Fluorescence) CBC: WBC Dot Plot Granularity (Side Scatter) Onyx: 6-year-old, Mn, Cocker spaniel Several days of weakness, decreased exercise tolerance and decreased appetite No significant previous clinical problems Current on all vaccinations and parasite controls Good general body condition Slightly pale mucous membranes “Onyx” – 6 yr, Mn, Cocker spaniel “Onyx” – 6 yr, Mn, Cocker spaniel What is an RDW?? Moderately severe anemia Macrocytic What does the mildly increased RDW indicate? 1. Polychromasia 2. Anisocytosis 3. Mixture of large and normal sized red blood cells 4. Mixture of small and normal sized red blood cells 5. Mixture of small, normal and large sized red blood cells Red Cell Indices Red Cell Distribution Width (RDW) coefficient of variation of the red cell volume distribution index of variation in size of erythrocyte population (anisocytosis) RDW = 16.2% (14.7 – 17.9) # of RBCs RDW = 23.6% (14.7 – 17.9) Size of RBCs “Onyx” – 6 yr, Mn, Cocker spaniel Moderately severe anemia Macrocytic Anisocytosis Probable large and normal Which of the following is the best interpretation of the reticulocyte count? 1. Within normal limits 2. Mild increase – probable blood loss regenerative 3. Moderate to marked increase – probable hemolytic regenerative 4. Increased - regenerative “Onyx” – 6 yr, Mn, Cocker spaniel Moderately severe anemia Macrocytic Anisocytosis Probable large and normal Regenerative – either blood loss or mild/early hemolytic disease “Onyx” – 6 yr, Mn, Cocker spaniel Which of the following is the most significant RBC morphology abnormality? 1. Normal – no abnormality present 2. Acanthocytosis 3. Spherocytosis 4. Anisocytosis “Onyx” – 6 yr, Mn, Cocker spaniel Onyx: 6-year-old, Mn, Cocker spaniel Moderate leukocytosis Neutrophilia Eosinopenia Normal or abnormal dot plot? Is Onyx’s WBC dot plot normal or abnormal? 1. Normal 2. Abnormal Onyx: 6-year-old, Mn, Cocker spaniel * * * WBC abnormal distribution Band neutrophils suspected Onyx: 6-year-old, Mn, Cocker spaniel Onyx: 6-year-old, Mn, Cocker spaniel Onyx: 6-year-old, Mn, Cocker spaniel Case outcome Immune-mediated hemolytic anemia Unidentified cause Inflammation Responded well to treatment Laboratory values returned to baseline within 2 weeks Blizzard 8 year old, intact male, Eng Springer Spaniel Clinical presentation Sudden decreased exercise tolerance Difficult breathing Clinical assessment Fever, depressed Auscultation – fluid in thoracic cavity Consolidated lungs Blizzard Blizzard Blizzard Blizzard Blizzard Case outcome Pyothorax Pulmonary abscess Slow and steady recovery Abby – 6yr, Female, Labrador Retriever Original presentation Normal behavior but presented with recurring nose bleeds (without associated trauma) Physical examination Good body condition Palpable slightly enlarged spleen Laboratory findings Marked thrombocytopenia (< 30 K/µL) SNAP 4Dx negative Being treated for immune-mediate thrombocytopenia of unidentified etiology Abby – 6yr, Female, Labrador Retriever Abby – 6yr, Female, Labrador Retriever Moderate anemia Regenerative - Reticulocytosis Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Moderate polychromasia Poikilocytosis Anisocytosis Large immature appearing lymphocytes Abby – 6yr, Female, Labrador Retriever Decreased PLT density Lymphoglandular body Decreased RBC density Schistocytes Burr cells Acanthocytes Howell-Jolly bodies Anisocytosis Platelet Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Schistocytes Burr cells Acanthocytes Howell-Jolly bodies Anisocytosis Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Moderate polychromasia Anisocytosis Abby – 6yr, Female, Labrador Retriever Moderate leukocytosis Automated differential Monocytosis / Eosinopenia Dot Plot evaluation Abnormal distribution Qualified results Mandates blood film review Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Moderate polychromasia Large immature appearing lymphocytes Reactive lymphocyte (center bottom) Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Howell-Jolly body Large immature appearing lymphocyte Mildly toxic neutrophil Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Howell-Jolly body Polychromasia Large immature appearing lymphocyte Mildly toxic hyposegmented neutrophil Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Howell-Jolly body Anisocytosis Large immature appearing lymphocyte Reactive lymphocyte (top left) Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Howell-Jolly body Polychromasia Lymphoglandular body Large immature appearing lymphocytes Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Polychromasia Large immature appearing lymphocytes Abby – 6yr, Female, Labrador Retriever Decreased PLT density Decreased RBC density Polychromasia Large immature appearing lymphocyte Platelet Lymphoglandular body Abby – 6yr, Female, Labrador Retriever “Wooley” – 8 yr, Mn, Mixed breed Acute onset severe depression and abdominal pain Dehydrated Moderate fever Tender abdomen on palpation Suspected fluid in abdomen “Wooley” – 8 yr, Mn, Mixed breed Platelet Abnormal Distribution “Wooley” – 8 yr, Mn, Mixed breed No significant abnormalities “Wooley” – 8 yr, Mn, Mixed breed Platelet Abnormal Distribution Severe thrombocytopenia What would be the next logical diagnostic procedure? 1. Bone marrow aspirate for cytology 2. Bone marrow core biopsy for histopathology 3. Platelet factor 3 measurement 4. Coombs test 5. Blood film review Platelet Count — Validate Data Platelets Confirm count Never accept a low platelet count from any analyzer without confirming with a blood film Platelet Number Evaluation Number of platelets per 100x oil objective field of view Normal platelet count Minimum: 8 – 10 Maximum: 35 – 40 Potential semiquantitation 20,000 x number of platelets seen per 100x objective field of view Thrombocytopenia “Wooley” – 8 yr, Mn, Mixed breed “Wooley” – 8 yr, Mn, Mixed breed What is the significance of large platelets in the peripheral blood of a dog? 1. No significance – common finding 2. Indicates increased rate of bone marrow production 3. Indicates toxicity to bone marrow – damaged megakaryocytes 4. Indicates dysplastic bone marrow production – common with preleukemia Identification Of Enlarged Platelets Platelets larger than normal Potential increased MPV from hematology analyzer In the cat Normal sized platelets Usually equivocal finding In most other species Indicates marrow response to peripheral demand for platelets Thrombocytopenia not required Inflammation and compensated response by marrow Enlarged platelets “Wooley” – 8 yr, Mn, Mixed breed Case Outcome Pancreatitis Wooley responded well to treatment and was released to owner Questions?
