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Week 4: Hemocytometry

Hemocytometer RBC and WBC Thoma pipet Unopette. Dilution problem Microscopy. Week 4: Hemocytometry. Red Cell and White Cell Counts. RBC problem: anemia, polycythemia WBC problem: leukocytosis, leukopenia RBC reference ranges Male 4.50 - 6.00 x 10 6 /  L (x 10 12 /L)

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Week 4: Hemocytometry

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  1. Hemocytometer RBC and WBC Thoma pipet Unopette Dilution problem Microscopy Week 4: Hemocytometry

  2. Red Cell and White Cell Counts • RBC problem: anemia, polycythemia • WBC problem: leukocytosis, leukopenia • RBC reference ranges • Male 4.50 - 6.00 x 106/L (x 1012/L) • Female 3.70 - 5.20 x 106/L (x 1012/L) • WBC reference range • Both 5.0 - 10.0 x 103/L (x 109/L)

  3. Diluting Fluid • For RBC • Physiologic saline (0.8% NaCl) • Hayem’s solution • Reese-Ecker • For WBC • 3% Acetic acid • 0.1 N HCl

  4. RBC Aspirate to 0.5 or 1.0 Dilute to 101 DF = 200 or 100 WBC Aspirate to 0.5 or 1.0 Dilute to 11 DF = 20 or 10 Discard volume of one (1) in the stem Thoma Pipet

  5. Improved Neubauer Hemocytometer

  6. Hemocytometer Chamber • Depth = 0.1 mm • Grids • RBC use 5 small squares in the center large square • WBC use 4 corner large squares • Unopette for WBC use all 9 large squares

  7. Hemocytometer Chamber

  8. Counting Rule • Do not count cells touching • Bottom line • Right line

  9. RBC Example… • Aspirate blood to 0.5, then dilute to 101 • DF = 200 • Counted 500 erythrocytes in 5 small squares RBC = 500 rbc x 25 Sm Sq x DF (200) 5 Sm Sq 0.1 mm3 = 5.00 x 106/mm3 (L)

  10. WBC Example… • Aspirate blood to 0.5, then dilute to 11 • DF = 20 • Counted 160 leukocytes in 4 large squares WBC = 160 wbc x 1 Lg Sq x DF (20) 4 Lg Sq 0.1 mm3 = 8.0 x 103/mm3 (L)

  11. Unopette • Self-contained reservoir and 20 mL pipet • Ammonium oxalate diluent • DF = 100 • Count leukocytes in all 9 large squares • Special formula to facilitate calculation

  12. Unopette

  13. Unopette WBC Example… • Counted 70 leukocytes in 9 large squares • Add 10% of count • Multiply by DF WBC = (70 wbc + 7 wbc) x DF (100) mm3 = 7.7 x 103/mm3 = 7.7 x 109/L

  14. Compound Microscope • Ocular (adjustable binocular) • Mechanical stage • Condenser • Objectives (parfocal)

  15. Care of Microscopes • Carry with care • Protect long objective lens • Clean and disinfect stage • Wipe oil off OIL • Save lamp life

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