+ Bloodsmear exam 1/12/2014. Goals. Instrument Technologies

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1 In house hematology laboratories: pros and cons of bench-top analyzers and the essentials of a blood film exam. NCSU-CVM Clinical Pathology January 22, 2014 Jaime Tarigo, DVM, PhD, DACVP Goals Understand the main technologies used in bench top hematology instruments Understand the strengths & weaknesses of the most common technology Review essentials of a blood film exam Instrument Technologies Quantitative buffy coat analysis Least info Lowest cost Impedance analysis Flow cytometric analysis Most info Highest cost CBC = Automated analyzer data + Bloodsmear exam 1

2 Quantitative Buffy Coat Analysis (IDEXX QBC VetAutoread) Principle: Sorts cells based on cell density Oversized capillary tube with a special float that expands the buffy coat layers Tube is coated with a dye that binds cellular components Quantitative Buffy Coat Analysis (IDEXX QBC VetAutoread) DeNicola DB. Advances in hematology analyzers. Top Companion Anim Med May;26(2): Review. QBC Results Red blood cell analysis: Calculated 2 Part WBC Differential Estimate Reticulocyte % (poor correlation) 2

3 QBC (IDEXX QBC VetAutoread) Advantages Least expensive technology Performs well in healthy animals Identify increases/decreases in WBCs, RBCs, platelets, and presence of microfilaria and reticulocytes Disadvantages Limited leukogram Cannot characterize patterns (stress/epinephrine) Does not identify bands If cell morphology is altered in disease, will not accurately identify cell categories Impedance Analyzers Examples Heska Hematrue Abaxis VetScan ScilVet ABC Coulter AcT Drew Scientific HemaVet Impedance Analyzers Coulter principle: A simple cell counter and sizer.. Cell Count Cell Size 3

4 Impedance Analysis Whole Blood Red cell/platelet Channel Sample is aspirated & diverted into 2 parts RBC count RBC size (MCV) Platelet count Platelet size (MPV) White cell/ Hemoglobin channel Red cells are lysed Total WBC count WBC differential (3-5part) Hemoglobin Measurement Impedance Analyzer Results Good separation between peaks is needed DeNicola DB. Advances in hematology analyzers. Top Companion Anim Med May;26(2): Review. Impedance Analyzer Results Systems/HemaTrue-Hematology-Analyzer.aspx 4

5 Impedance Analyzers Parameters with potential for Inaccurate Results MCV MCHC Platelets Lymphocyte and monocyte counts Eosinophil and basophil counts Inaccurate Results.. False MCV Severe anemia with WBC count The red cell count includes WBCs Agglutination Large clumps are excluded Small clumps (doublets/triplets) are counted MCV a lot Measured vs. Calculated Values Measured RBC count MCV Platelet count MPV Hemoglobin WBC count WBC differential Calculated Hct MCH MCHC RDW PDW 5

6 Measured vs. Calculated Values Measured RBC count MCV Platelet count MPV Hemoglobin WBC count WBC differential Calculated Hct MCH MCHC RDW PDW Measured vs. Calculated Values Measured RBC count MCV Platelet count MPV Hemoglobin WBC count WBC differential Calculated Hct MCH MCHC RDW PDW Measured vs. Calculated Values Measured RBC count MCV Platelet count MPV Hemoglobin WBC count WBC differential Calculated Hct MCH MCHC RDW PDW 6

7 Potential for Error MCHC A calculated value from calculated values.. MCHC is derived from Hemoglobin and HCT HCT is derived from the MCV and RBC count If MCV or RBC is inaccurate, MCHC will be inaccurate Note: Impedance analyzers measure hemoglobin directly (spectrophotometric measurement) = positive interference can occur Potential for Error- Platelets Pseudothrombocytopenia = # 1 Problem Platelet clumps = Common = False platelet count Occurs more often in cats Quick scan of the feathered edge to look for clumps Less common Cytoplasmic fragments with leukemia, counted as platelets WBCs White cells are counted strictly based on size A 3-5 part differential is provided Total granulocytes Neutrophils, eosinophils and basophils May include band cell (left shift not detected) Monocytes Includes most blast cells May include bands (left shift not detected) Lymphocytes Includes nrbcs Largest Smallest 7

8 Left Shift The only way to identify a left shift is by bloodsmear examination Hemoglobin Hemoglobin Spectrophotometric Measurement Hg is converted to cyanmethemoglobin light transmittance = Hg levels Positive interference: anything that blocks or scatters light! Very high WBC count Unlysed RBCs (agglutination) Heinz bodies Lipemia Oxyglobin Lots of rouleaux (personal observation) Generally good RBC counts Hg Hct (within 10% of spun crit) Total WBC counts Total granulocyte/neutrophil counts** Variable MCV MCHC Lymphocyte and monocyte counts Eosinophil and basophil counts Platelets Inaccurate with clumps Reasonably good in dogs Inaccurate in cats (clumps and size) Impedance Analysis 8

9 Impedance Analysis Easy to use Little hands on time Results in a few minutes Low sample volume ( µl) Provides a graph Cost varies; $6500 used to $15,000 or more new Light Scatter Analysis- Flow Cytometry Principle: Light scattered by a cell can be used to count, size and measure internal components Flow cytometry Analyzers may combine this principle with other methodologies Flow cytometry IDEXX Lasercyte Flow cytometry + peroxidase staining Bayer Advia 120 Flow cytometry + impedance technology Abbott Diagnostics Cell-Dyn, Oxford Scientific Forecyte) Flow cytometry + fluorescent staining + impedance IDEXX ProCyte 9

10 Flow Cytometry IDEXX Lasercyte (Flow only) CELL SIZE IDEXX Procyte (Flow + fluorescent staining) Flow Cytometry- Advia 2120 WBC LUC Monos Size Neutrophils Basophil Method Basos Lymphocytes Eosinophils Lymphs& Monos Neuts & Eosos Peroxidase act. Blasts RBC Platelets RBC Ghosts IMHA Size RBCs Platelets Hemoglobin Automated Analyzers Many advantages A component of the CBC Analyzers are not infallible The bloodsmear exam is an essential component of the CBC Regardless of how advanced the analyzer.. What are some of the critical parameters to assess in ill patients? 10

11 What things do I need to look at a smear for?? Thrombocytopenia Look for platelet clumps Clinical suspicion for sepsis / significant inflammation Identify bands (i.e.) left shift Very high lymphocyte or monocyte count? Identify lymphoma or leukemia Anemia? Screen for: Hemoparasites Agglutination, spherocytes Additional important RBC morphology Schistocytes The Bloodsmear Exam < 5minutes with practice Validate the analzyer data RBC mass (#s, MCV, MCHC) WBC count & differential Platelet count Identify key morphologic features RBC- Features suggestive of underlying cause of anemia WBC- Identify inflammation (bands) Platelet morphology- activated/reactive Bloodsmear Preparation 30-40degree angle Small drop of blood from capillary tube Let the blood wick across the slide before spreading Gentle, fluid swift motion Too slow or too much pressure Leukocytes not evenly distributed, all at the feathered edge Ruptured cells 11

12 Getting the most out of your purple top.. 1.Hematology Analyzer 2. Bloodsmear Microhematocrit tube Microhematocrit Tube PCV is most accurate Rapid ID of large buffy coat, plasma character Bloodsmear Exam- Step 1 LOW power exam (10X) Scan the feathered edge Look for platelet clumps Look for microfilaria Look for atypical large cells Assess distribution of leukocytes Identify monolayer 12

13 Scanning the Feathered Edge (10X) Look for platelet clumps Platelet count is underestimated Bloodsmear Exam- Step 1 Look for platelet clumps If they look atypical, take a closer look Cytauxzoon felis Bloodsmear Exam- Step 2 Monolayer exam Majority of rbcs are not in contact Well spread, best morphologic delineation Assess the series density and morphology Platelets Erythrocytes Leukocytes 13

14 Monolayer Exam- Platelets Clumps on feathered edge? No clumps: #platelets/100xhpf * 20,000 Allows validation of platelet count If platelet clumps are present and there are still platelets in the monolayer then you can guesstimate that you have atleast the # calculated from the monolayer.. Monolayer Exam- Platelets Platelet morphology Small, 2-4um Cytoplasmic fragments, anucleate Species variation Large Activated du/courses/bi108/bi1 08_2005_Groups/10/ webpages/plateletslin k.htm Monolayer Exam- Erythrocytes RBC mass- density normal or decreased? Guesstimate.. PCV (spun crit is most accurate!).. Basic morphology Size (MCV) Color (Polychromasia, Hypochromasia) Atypical morphology of importance in the critically ill Agglutination, Spherocytes Schistocytes Hemoparasites 14

15 Monolayer Exam- Erythrocytes Basic morphology- SIZE Anisocytosis Macrocytosis J. Neel, DVM, DACVP Monolayer Exam- Erythrocytes Basic morphology- COLOR Polychromasia Hypochromasia J. Neel, DVM, DACVP Monolayer Exam- Erythrocytes Morphologic changes- RBC Clumping Agglutination Rouleaux 15

16 Monolayer Exam- Erythrocytes Immune-mediated disease Agglutination Spherocyte Ghost Cells J. Neel, DVM, DACVP Monolayer Exam- Erythrocytes Additional morphologic changes of significance Schistocyte Microangiopathy DIC Severe HWD Splenic torsion Vascular tumor- HSA Monolayer Exam- Erythrocytes Additional morphologic changes of significance Oxidative Damage Heinz body (WG) Phenothiazine Onions/kale Red maple leaves Acetaminophen Phenyl compounds, Propylene glycol (semimoist cat food) Crude oil in birds Ketoacids esp. in cats Severe hemolytic anemia 16

17 Monolayer Exam- Erythrocytes Hemoparasites Tick-borne (protozoal, rickettsial/bacterial) Piroplasms Babesia Cytauxzoon Fleas (bacterial) Chains, Rods,*Rings Mycoplasma Monolayer Exam- Erythrocytes Canine Babesiosis um most of the time um most of the time. Not always so picture perfect 17

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19 Cytauxzoon felis Piroplasms = merozoites Schizonts on feathered edge Looks like B. gibsoni.. Mycoplasma (Formerly Haemobartonella) Extracellular Mycoplasma haemofelis ph/mycoplasma-felis.html Monolayer Exam- Leukocytes Leukocyte Density & Distribution Leukocyte Differential Leukocyte Morphology Important changes consistent with inflammation Toxic change, bands Parasites 19

20 Monolayer Exam- Leukocytes Leukocyte Distribution & Density Clumping of WBCs? Analyzer likely inaccurate All at feathered edge? If no, rough estimate: ~#WBCs/10X obj field * = #cells/ul Monolayer Exam- Leukocytes Leukocyte Differential cell count Neuts Eosos Basos Lymphs Monos Eyeball- ~50-75% mature neuts in health Monolayer Exam- Leukocytes Important morphologic changes Neutrophils Toxic Change Cytoplasmic basophilia Dohle bodies Foamy cytoplasm 20

21 Monolayer Exam- Leukocytes Most important change Bands #s proportional to severity Horse-shoe shaped nucleus Greatest indentation > 1/3 to 1/2 of the thickest region of the nucleus Slight indentations ok Non-filamentous seg vs. band.. Band Not a band Band Monolayer Exam- Leukocytes Bands Splitting hairs.. Immature neutrophils Repeatable? #s? h/modules/heme1/toxic.htm Monolayer Exam- Leukocytes Band vs. Monocyte = Challenging Mono s = problematic Highly variable in appearance Vary in size and nuclear shape SOMETIMES bean shaped nuclei.. Not always distinctly vacuolated ogram/mono.htm 21

22 Monolayer Exam- Leukocytes Infectious organisms Distemper inclusion = only in acute stages Bacteria- rare to find = sepsis Morulae* = rare to find Ehrlichia Erhlichiosis- Reclassification Ehrlichi Anaplasma Neorickettsia E. canis A. phagocytophilum N. sennetsu E. chaffeensis (E. equi) N. risticii E. ewingii A. platys (E. platys) MORPHOLOGICALLY = MORULAE IN DIFFERENT CELLS Granulocytic Ehrlichiosis Monocytic Ehrlichiosis E. ewingii E. canis A. phagocytophilum E. chaffeensis N. risticii Monolayer Exam- Leukocytes E. canis E. ewingii University of Georgia, CVM Noah s Archive Good luck 22

23 Monolayer Exam- Leukocytes Sepsis Actually from tissue- tough to catch in the act in circulation.. Distemper Inclusions pg 110 Monolayer Exam- Leukocytes Leukemia ALL - Acute lymphoblastic - Large, immature CLL - Chronic lymphocytic - Small - normal appearing 23

24 The essential bloodfilm exam Low power scan (10X) Platelet clumps, microfilaria, odd/large cells ID monolayer, distribution of leukocytes Rough diff (neuts ~50-75% of all leukocytes) Monolayer High power (100X) Counts (platelets, WBCs) Morphology changes- platelets, RBCs, WBCs Infectious disease CONCLUSIONS Analyzers are great! But not infallible Pros: Rapid results Accurate values for healthy patients Cons: Cannot identify critical morphologic changes Cannot identify infectious organisms Questions? 24