Blood Basics

Composition of Blood

~45% red blood cells
~55% plasma
<1% leukocytes and thrombocytes

Peripheral Blood Films

Anticoagulated whole blood, venous (EDTA lavender top)
- EDTA (ethylenediaminetetraacetic acid) chelates calcium to prevent activation of coagulation cascade
- Preserves cell components, minimal affects on cell morphology
  - Excess EDTA (underfilled) can cause:
    - Low cell counts & hematocrit
    - Altered staining and morphology
      - Cell shrinkage, crenation, WBC damage
  - Low EDTA (overfilled) can cause clotting and platelet clumps
- Store maximum 4-5 hours at RT before making films or running CBC (12 hours at 4C)
  - Prolonged storage in EDTA can cause:
    - Artifacts (crenated cells and spherocytes)
    - Increased MCV and HCT
    - Decreased cell counts, necrotic cells, vacuolation
Capillary collection before clotting
Making films
- Want a slightly rounded, feathered edge with good monolayer
- Spread with slide at 30-45 degree angle
- Larger WBCs may have tendency to be pushed to edge of smear (especially monocytes and granulocytes)
Slide Staining
- Romanowsky staining (Wright, Wright-Giemsa, etc.)
  - Polychromatic stain
  - Eosin and methylene blue, buffered at pH 6.4-6.8 for staining to occur
    - Eosin: acidic (-), stains basic components red-orange
      - Hemoglobin, eosinophilic granules (protein amine groups)
    - Methylene blue: basic (+), stains acidic components blue
    - Thiazine-eosinate complex: neutral, stains neutral components mix of red and blue
      - Neutrophil granules (lilac coloured)
        Nucleic acids, basophil granules (carboxyllic acids and phosphoric acid groups)
  - If pH is too basic, more things will appear gray/blue. If pH is too acidic, more things will appear red-orange