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
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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
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
20031990s1980spre1980s
Manual Quantitative
Buffy coat.
Impedance Laser
2010+
In-house Hematology Analyzers
RBC
WBC
RBC
WBC
ProCyteDx
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
20031990s1980spre1980s
Manual Quantitative
Buffy coat.
Impedance Laser
2010+
In-house Hematology Analyzers
RBC
WBC
RBC
WBC
ProCyteDx
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
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
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
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 over-interpretation of potential subtle changes
•Only prominent changes prove helpful
Peripheral Blood Film Preparation
• 30–45 degree angle
• Increased angle with low PCV
• Decreased angle with high PCV
• Fluid-controlled motion
•Results
• Body
• Monolayer
• Feathered edge
Anatomy of the Peripheral Blood Film
Pati
en
t ID
Date
Feathered Edge
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
Anatomy of the Peripheral Blood Film
Pati
en
t ID
Date
Feathered Edge
• Clumped platelets
• Large cells
• Microfilaria
• Leukocyte distribution
Anatomy of the Peripheral Blood Film
Pati
en
t ID
Date
Body
• Rouleaux formation
• Agglutination
• Cell clumping
Anatomy of the Peripheral Blood Film
Monolayer
• Platelet number estimation
• Leukocyte number estimation
• Morphologic evaluation
• Data validation
Pati
en
t ID
Date
Erythrogram – Validate Data
Erythrogram – Validate Data
Measure of RBC mass -severity of anemia
Measure of RBC mass -severity of anemia
Erythrogram - Validate Data
Sample A Sample B
Which one of these samples is from a severely anemic dog?
1. Sample A
2. Sample B
Measure of RBC mass -severity of anemia
Erythrogram - Validate Data
Sample A Sample B
Erythrogram - Validate Data
Geo
Measure of RBC mass -severity of anemia
Description of RBC population
Low MCV, Low MCHC
Geo - Erythrogram
NormalNormal
MCV
Geo’s
MCV
Geo RBC run dot
plot for data
validation
Compare to
normal
Erythrogram - Validate Data
Measure of RBC mass -severity of anemia
Description of RBC population
Erythrogram - Validate Data
Measure of RBC mass -severity of anemia
Description of RBC population
Objective measure of variation in RBC size
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
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
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
Erythrogram - Validate Data
Geo
Measure of RBC mass -severity of anemia
Description of RBC population
Objective measure of variation in RBC size
Objective measure of erythroid response
Normal
Dot plot review provides rapid confirmation of reticulocyte count
Erythrogram – Add Morphology
Examine morphology of erythrocytes
• Step 1 – Recognize abnormalitiesNormal
Erythrogram – Add Morphology
Examine morphology of erythrocytes
• Step 1 – Recognize abnormalities
• Step 2 – Learn to identify the abnormalities
Normal
AcanthocytesSchistocytes
Spherocytes
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
AcanthocytesSchistocytes
Spherocytes
Spherocytosis
Immune - Mediated Hemolytic Anemia
FcRMacrophage
RBC
Spherocytosis
Spherocytosis
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
Normal
AcanthocytesSchistocytes
Spherocytes
Miscellaneous Inclusions
Mycoplasma
(Haemobartonella)
Basophilic stippling
Distemper virus
inclusions
Babesia gibsoni
Leukogram - Validate Data
• Validate WBC count
Leukogram - Validate WBC Count
3,000 5,000 10,000 25,000 50,000
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
Leukogram - Validate WBC Count
3,000 5,000 10,000 25,000 50,000
WBC reported
24,000
Leukogram - Validate WBC Count
3,000 5,000 10,000 25,000 50,000
Which of the following is the BEST estimated WBC count based on blood film examination?