1. 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
  2. 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
  3. 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
  4. 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
  5. 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)
  6. 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 
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  7. 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%
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