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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