Guide to the 1-3 Minute Blood Film Microscopic Review: Why and How?
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1 Guide to the 1-3 Minute Blood Film Microscopic Review: Why and How? Dennis B. DeNicola, DVM, PhD, DACVP Chief Veterinary Educator IDEXX Laboratories, Inc. Westbrook, ME USA Adjunct Professor of Veterinary Clinical Pathology Purdue University College of Veterinary Medicine
2 Objectives: Understand the value of a brief blood film microscopic review Validate hematology analyzer performance Add valuable information regarding cellular morphology Understand how to develop standardized logical steps for this brief blood film review Understand that in the majority of the times, less than a one minute review is required and that only a maximum of a three minute review should be required even for the most difficult cases
3 In-house Hematology Analyzers pre1980s 1980s 1990s Manual Quantitative Buffy coat. Impedance Laser ProCyte Dx RBC RBC WBC WBC
4 How frequently do you currently use any of the various cytograms generated by your hematology analyzer? A. Never B. Less than 25% of the time C. Between 25 and 50% of the time D. Between 50 and 75% of the time E. Greater than 75% of the time
5 In-house Hematology Analyzers pre1980s 1980s 1990s Manual Quantitative Buffy coat. Impedance Laser ProCyte Dx RBC RBC WBC WBC
6 What has been gained with hematology analyzer evolution? Greater amount of data sometimes even more than the reference laboratory provides Improved precision and accuracy of results more advanced analyzers perform equal to or better than reference laboratory analyzers Decreased technician time Analyzers have minimal maintenance requirements Operations are generally load and go Microscopic blood film review should still be included Majority of time not performing leukocyte differential Quickly scanning slide for cellular morphology changes
7 Hematology Is a blood film needed? ABSOLUTELY YES!! Blood film examination is needed for all Low-end hematology analyzers IDEXX VetAutoread Hematology Analyzer Impedance-based instruments High-end hematology analyzers LaserCyte, LaserCyte Dx, and ProCyte Dx Hematology Analyzers (in-house) Cell-Dyn (reference laboratory) Advia (reference laboratory) Sysmex (reference laboratory
8 Hematology Why is a blood film needed? Validate numerical data generated RBC mass (RBC count, HCT, HGB) RBC indices (MCV, MCH, MCHC, RDW) WBC count WBC distribution Platelet count Add valuable/critical information that is not generated by the hematology analyzers RBC morphology clues to cause of anemia WBC morphology characterize presence or absence of inflammatory disease Platelet morphology
9 Hematology How long should a blood film review take? 1 3 minutes maximum time on scope What is accomplished? Validate data Provide morphology comments Spending more than 3 minutes results in overinterpretation of potential subtle changes Only prominent changes prove helpful
10 Peripheral Blood Film Preparation degree angle Increased angle with low PCV Decreased angle with high PCV Fluid-controlled motion Results Body Monolayer Feathered edge
11 Patient ID Date Anatomy of the Peripheral Blood Film Feathered Edge
12 Which of the following IS NOT evaluated when examining the feathered edge of a blood film? 1. Clumped platelets 2. Large cells 3. Microfilaria 4. Leukocyte distribution 5. RBC morphology
13 Patient ID Date Anatomy of the Peripheral Blood Film Feathered Edge Clumped platelets Large cells Microfilaria Leukocyte distribution
14 Patient ID Date Anatomy of the Peripheral Blood Film Body Rouleaux formation Agglutination Cell clumping
15 Patient ID Date Anatomy of the Peripheral Blood Film Monolayer Platelet number estimation Leukocyte number estimation Morphologic evaluation Data validation
16 Erythrogram Validate Data
17 Erythrogram Validate Data Measure of RBC mass - severity of anemia
18 Erythrogram - Validate Data Measure of RBC mass - severity of anemia Sample A Sample B
19 Which one of these samples is from a severely anemic dog? 1. Sample A 2. Sample B
20 Erythrogram - Validate Data Measure of RBC mass - severity of anemia Sample A Sample B
21 Erythrogram - Validate Data Measure of RBC mass - severity of anemia Description of RBC population Geo Low MCV, Low MCHC
22 Geo - Erythrogram Geo Normal MCV Normal RBC run dot plot for data validation Geo s MCV Compare to normal
23 Erythrogram - Validate Data Measure of RBC mass - severity of anemia Description of RBC population
24 Erythrogram - Validate Data Measure of RBC mass - severity of anemia Description of RBC population Objective measure of variation in RBC size
25 Erythrogram - Validate Data Measure of RBC mass - severity of anemia Description of RBC population Objective measure of variation in RBC size Objective measure of erythroid response
26 Which of the following is the BEST support for the presence of an increased reticulocyte count? 1. Presence of nucleated red blood cells 2. Presence of large red blood cells 3. Presence of target cells 4. Presence of polychromatophils 5. Presence of pale red blood cells
27 Erythrogram - Validate Data Measure of RBC mass - severity of anemia Description of RBC population Objective measure of variation in RBC size Objective measure of erythroid response Routine Stain New Methylene Blue
28 Erythrogram - Validate Data Measure of RBC mass - severity of anemia Description of RBC population Objective measure of variation in RBC size Objective measure of erythroid response Geo Normal Dot plot review provides rapid confirmation of reticulocyte count
29 Erythrogram Add Morphology Examine morphology of erythrocytes Step 1 Recognize abnormalities Normal
30 Erythrogram Add Morphology Examine morphology of erythrocytes Step 1 Recognize abnormalities Step 2 Learn to identify the abnormalities Normal Acanthocytes Schistocytes Spherocytes
31 Erythrogram Add Morphology Examine morphology of erythrocytes Step 1 Recognize abnormalities Step 2 Learn to identify the abnormalities Step 3 Understand what they mean Normal Acanthocytes Schistocytes Spherocytes
32 Spherocytosis
33 Spherocytosis Immune - Mediated Hemolytic Anemia Macrophage FcR RBC
34 Spherocytosis
35 Erythrogram Add Morphology Examine morphology of erythrocytes Step 1 Recognize abnormalities Step 2 Learn to identify the abnormalities Step 3 Understand what they mean Step 4 Look for RBC inclusions Acanthocytes Schistocytes Spherocytes Normal
36 Miscellaneous Inclusions Mycoplasma (Haemobartonella) Distemper virus inclusions Babesia gibsoni Basophilic stippling
37 Leukogram - Validate Data Validate WBC count
38 Leukogram - Validate WBC Count 3,000 5,000 10,000 25,000 50,000
39 Which of the following is the BEST estimated WBC count based on blood film examination? 1. 3,000 / µl 2. 5,000 / µl 3. 10,000 / µl 4. 25,000 / µl 5. 50,000 / µl
40 Leukogram - Validate WBC Count 3,000 5,000 10,000 25,000 50,000 WBC reported 24,000
41 Leukogram - Validate WBC Count 3,000 5,000 10,000 25,000 50,000
42 Which of the following is the BEST estimated WBC count based on blood film examination? 1. 3,000 / µl 2. 5,000 / µl 3. 10,000 / µl 4. 25,000 / µl 5. 50,000 / µl
43 Leukogram - Validate WBC Count 3,000 5,000 10,000 25,000 50,000 WBC reported 2,000
44 Leukogram - Validate Data Validate WBC count Validate leukocyte distribution
45 Five-Part Differential WBC = x10 3 /µl ( ) NEU = x10 3 /µl ( ) LYM = 4.94 x10 3 /µl ( ) MONO = 1.41 x10 3 /µl ( ) EOS = 0.00 x10 3 /µl ( ) BASO = 0.00 x10 3 /µl ( )
46 Leukogram - Validate Leukocyte Distribution WBC = x10 3 /µl ( ) NEU = x10 3 /µl ( ) LYM = 4.94 x10 3 /µl ( ) MONO = 1.41 x10 3 /µl ( ) EOS = 0.00 x10 3 /µl ( ) BASO = 0.00 x10 3 /µl ( ) Normal WBC DotPlot Eosinophils Core Sample Stream Sheath Flow Channel Leukocyte Distribution 5-part differential Quartz Flow Cell Axial Light Loss Neutrophils Monocytes Low Angle Forward Scatter 2-4 degrees High Angle Forward Scatter 8-12 deg Right Angle Scatter High Numerical Aperture degrees Laser and Lens Sy stem Lymphocytes
47 Leukogram Validate Data Validate WBC count Validate leukocyte distribution Examine WBC morphology Left Shift Immature Neutrophil Forms
48 Blood Film Validate Data Validate WBC count Validate leukocyte distribution Examine WBC morphology Left Shift Immature Neutrophil Forms Neutrophil Toxicity
49 Blood Film Validate Data Validate WBC count Validate leukocyte distribution Examine WBC morphology Abnormal Leukocytes Left Shift Immature Neutrophil Forms Neutrophil Toxicity
50 Belle Bell Normal Immature or toxic neutrophils suspected Monotonous population of large mononuclear cells suspect abnormal cell population
51 Belle Bell Normal Immature or toxic neutrophils suspected
52 Belle Bell Normal Immature or toxic neutrophils suspected Monotonous population of large mononuclear cells suspect abnormal cell population
53 Belle Poikilocytosis Acanthocytes 2+ toxic hyposegmented neutrophil Decreased platelets Two large immature appearing mononuclear cells
54 Thrombogram - Validate Data Validate platelet count
55 Thrombogram - Validate Data Validate platelet count Never accept a low platelet count from any analyzer without confirming with a blood film
56 Platelet Number Evaluation Number of platelets per 100x oil objective field of view Minimum: 8 10 Maximum: Potential semiquantitation 20,000 x number of platelets seen per 100x objective field of view Normal platelet count Thrombocytopenia
57 Thrombogram - Validate Data Validate platelet count Add platelet morphology comments
58 Identification of Enlarged Platelets Platelets larger than normal Potential increased MPV from hematology analyzer In the cat Usually equivocal finding In most other species Indicates marrow response to peripheral demand for platelets Thrombocytopenia not required Inflammation and compensated response by marrow Normal-sized platelets Enlarged platelets
59 Reference Laboratory Hematology Needed? Experience Use the laboratory for complicated cases Potential of a pathology review Use the laboratory as a great teaching resource Quality Assurance Use the laboratory to periodically check your in-house system Realize that different instruments produce different results Realize that aged samples are not the same as fresh samples
60 Questions?
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