1. Pictures/Diagrams Copyright © University of Nebraska
CLS416 - Clinical Hematology Day 2
Blood Smear Examination, Reticulocyte Counts
Pictures/Diagrams Copyright © University of Nebraska

2. Manual Cell Counting Dilution, diluting fluid & counting area varies
Hemocytometer has two counting chambers
Chamber dimensions: 3mm x 3mm x 0.1mm

3. Objectives
Refer to lecture objectives 1-12 on page 34

4. Peripheral Blood Smear Exam
The blood smear is a wonderful tool used to verify instrument ‘numbers’
CBC parameters should agree with findings observed on the blood smear
The CBC & Differential is the most frequently requested analysis
Differentials may be automated or manual
Microscopic exam of a Wright’s stained smear:
Manual WBC differential
WBC estimates
Platelet estimates

5. No band # or cell morphology
CBC & Automated Differential
‘Normal’ reference range for age and sex
WBC Types
No band # or cell morphology

6. Significant variations from normal are noted on report
CBC & Manual Differential
Sickle cell anemia
Significant variations from normal are noted on report

7. Peripheral Blood Smear Exam
Macroscopic exam of smear
Microscopic exam of smear
Low power (10x)
Scan to access overall smear quality
Detect big, ‘scary’ cells
High dry power (40x)
WBC estimates
Oil immersion (100x)
WBC differentials, cell morphology, platelet estimates
Patient Name/#
Fibrin
Fibrin
10x
10x
100x

8. Peripheral Blood Smear Exam
WBC estimates are done to verify the validity of automated WBC counts
40x, high dry power = HPF
Count WBCs in 10 HPFs where red cells slightly overlap
Include fields with no cells in estimate
Count broken or smudge cells
Take average # per HPF x 2,000
The WBC estimate is never ‘reported’
WBC estimate = 6,000/cmm
40x

9. Peripheral Blood Smear Exam
Manual differentials are done if significant patient abnormalities exist and/or automated differential results are ‘flagged’
Requires well-made/stained smear
Oil immersion (OIFs) and high light
Rainbow area where red cells
just touch
Rainbow area
Edge, too thin
Too thick
Stain precipitate
Under-stained

10. Smear Exam - Differentials
Differential includes:
WBC differential that classifies WBC types
Slide is scanned, counting and
identifying 100 consecutive WBCs
Reported in % (and absolute #)
Cell morphology
WBCs, RBCs and platelets are evaluated
Significant variations from normal cell appearance are noted or quantitated on report  give clues to cause of a disorder
Platelet estimate
Used to verify the accuracy of automated platelet counts

11. WBC Differentials Reference percentage ranges vary with age
^Seg Neutrophils and Lymphs are the most numerous WBCs in the blood of normal adults ^ ^ *
*Due to low #, count Eos & Basos using same counter key…keep track of # of each *
Significance:
Deviations from normal reference ranges may indicate disease  provide clues to diagnosis
No immature cells should be present
May see smudge, broken or necrotic (dying) cells
Critical values: Blasts cells

12. Identify the cells at 1, 2, and 3 Match each cell with the correct WBC listed
Segmented neutrophil
Eosinophil
Basophil 2 1 3 1 3 2
KEY Question 2: A Basophil; B Segmented neutrophil; C Eosinophil

13. Identify the cells at 1, 2, and 3 Match each cell with the correct WBC listed
Large lymphocyte
Monocyte
Small lymphocyte 2 2 1 1
KEY Question 3: A Small lymphocyte; B Monocyte; C Large lymphocyte 3

14. Identify the cells at 1 and 2 Match each cell with the correct WBC listed
Monocyte
Band neutrophil 1 1
KEY Question 4: A Band neutrophil; B Monocyte 2

15. Identify the cells at 1 through 7 Match each cell with the correct WBC listed2 4 3 5 6 7 1
Segmented neutrophil
Band neutrophil
Eosinophil
Basophil
Small lymphocyte
Large lymphocyte
Monocyte 4 7 2 6
KEY Question 6: A Segmented neutrophil; B Basophil; C Monocyte; D Band neutrophil; E Large lymphocyte; F Small lymphocyte; G Eosinophil 5 3

16. Necrotic neutrophil w pyknotic nucleus
Other Differential Findings
Necrotic neutrophil w pyknotic nucleus
Smudge cell
Band Neutrophil
Neutrophil
Lymph
Broken eos
Broken neutrophil
Eosinophil

17. Which cells are normal blood findings?
Identify the cells at 1, 2, and 3 Match each cell with the correct cell listed 2
Nucleated red cell
Blast cells
Small lymphocyte 2 3 1 1 3
Which cells are normal blood findings?

18. Identify the cell shown
A ‘heart’ cell

19. Platelet Estimates
PLT estimates are done to verify the validity of automated PLT counts
Oil immersion (100x) and high light
Count platelets in 10 OIFs where red cells just touch
Include fields with no cells in estimate
Find the average # per OIF
The platelet estimate is never ‘reported’
100x
Platelet estimate = 120,000 uL

20. Have blood sample redrawn Perform manual platelet count
Platelet Counting Errors
Schistocytes
Platelet Clumping
Action??
Action??
Have blood sample redrawn
Perform manual platelet count

21. Automated Cell Counting
Two main principles
Coulter electronic impedance principle
Optical light scatter principle

22. Auto Instrument Examples

23. Checks done to prevent error
The most fundamental responsibility of a laboratory is to ensure quality test results
Assure specimen integrity to prevent pre-analytic errors
Monitor reagents/instruments with maintenance & QC samples to prevent analytic errors
Checks done to prevent erroneous results:
Critical values are confirmed, called and/or redrawn
‘Flagged’ parameters are verified by smear review or manual method…e.g., estimates
Patient results are compared to previous results
Inconsistent data is verified…instrument values should correlate with findings observed on the blood smear

24. Example of Hemodilution:
Line 04:45 from ICU patient…critically low Hgb & Hct results are obtained
Redraw to confirm was requested

25. Correlation of CBC & Blood Smear

26. Reticulocyte Counts
Measures the rate of RBC production by the bone marrow
Retics appear as polychromasia on a Wright’s stained blood smear…a retic count is done for number
Automated or manual to confirm auto results
Supravital ‘living cell’ stain  New methylene blue
Precipitates RNA into filaments or granules
Supravital Stain
Wright’s stained smear
NRBC
Polychromasia
Supravital stained smear
Reticulocytes

27. Reticulocyte Counts Two “dots” or more is a retic
Count 500 red cells, separating mature RBCs vs retics
1000 total red cells are counted, # of retics/500 RBCs must agree + 2 retics on two different slides
Another slide must be counted if results do not agree
Reported in relative number (%), to nearest tenth and absolute #, to nearest thousand
Absolute # is more reliable than %
Reticulocytes
Supravital stain
Supravital stain
Artifact

28. Reticulocyte Counts Sources of error for manual retic counts
Improper blood to stain ratio or inadequate mixing
Improper counting
Reference range
Adult % ,000/cmm Absolute#
Newborn %
Significance
↑ absolute Retic count….Reticulocytosis
Increased RBC production
Will observe increased amount of polychromatophilic red cells on Wright’s stained blood smear
↓ absolute Retic count….Reticulocytopenia
Decreased RBC production