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Describe how to perform a bone marrow differential (simple)
- 500 nucleated cells are counted (NRBCs and WBCs) are counted in successive fields
- Cells without nuclei are not included
What is the appropriate angle for the spreader slide technique, and what happens on either side?
- 30-45 degrees is optimal
- >45 degrees results in a thick slide
- <30 degrees results in a thin slide
What is the normal stain used in a differential or count? What "type" of stain is it? What are the primary ingredients? What is the primary reaction?
- Wright stain
- Romanowsky-type stain
- Includes methylene blue, Eosin B or Y, methyl alcohol, phosphate buffer (pH 6.4-6.7)
- Acid-base affinity induces stain
What is the procedure for a Wright stain?
- Add methylene blue/eosin stain mixture for 3 minutes
- Add phosphate buffer to stain and mix
- Allow both to remain on smear for 5 min
- Rinse slide with water
- Air dry
What aspects of the cells will stain in various colors in a Wright stain? Why?
- Acid substances have an affinity for the basic methylene blue (DNA, RNA)
- Basic substances have an affinity for the acidic eosin and stain red (hemoglobin, various enzymes in cytoplasm)
Describe how to perform an examination of peripheral blood film (in detail)
- Low power scan (10x): evaluate staining quality
- evaluate distribution of cells (clumping? abnormalities?)
- determine optimal examination area
- High power scan (40x): estimate WBC count
- count WBCs in 10 consecutive fields and determine the average per field
- 2-4 = 4,000-7,000 WBC/μL
- 4-6 = 7,000-10,000 WBC/μL
- 6-10 = 10,000-13,000 WBC/μL
- 10-20 = 13,000-18,000 WBC/μL
- Oil immersion: evaluate RBC morphology (anisocytosis, poikilocytosis, hypochromia, polychromasia, RBC inclusions)
- Estimate platelet count
- count platelets in 10 successive fields and determine the average per field
- multiply the average by 20,000 (or 15,000 if automated slide) to determine the #platelets/μL
- normal platelets (130-000-400,000)
- WBC differential count
- Count and ID all WBCs (including immature) in successive fields until 100 have been counted
- Also count (but don't ID) NRBCs
- If >10 NRBCs are found then count needs to be corrected
What are the normal values for a WBC differential?
- N. seg: 50-70%
- N. band: 2-6%
- Eosinophils: 0-4%
- Basophils: 0-2%
- Lymphocytes: 20-44%
- Monocytes: 2-9%