Week 4: Hemocytometry Hemocytometer RBC and WBC Thoma pipet Unopette Dilution problem Microscopy
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)
Diluting Fluid For RBC For WBC Physiologic saline (0.8% NaCl) Hayem’s solution Reese-Ecker For WBC 3% Acetic acid 0.1 N HCl
Thoma Pipet 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
Improved Neubauer Hemocytometer
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
Hemocytometer Chamber
Counting Rule Do not count cells touching Bottom line Right line
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)
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)
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
Unopette
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
Compound Microscope Ocular (adjustable binocular) Mechanical stage Condenser Objectives (parfocal)
Care of Microscopes Carry with care Protect long objective lens Clean and disinfect stage Wipe oil off OIL Save lamp life