Week 4: Hemocytometry • • • • Hemocytometer RBC and WBC Thoma pipet Unopette • Dilution problem • Microscopy Red Cell and White Cell Counts • RBC problem: anemia, polycythemia • WBC.
Download ReportTranscript Week 4: Hemocytometry • • • • Hemocytometer RBC and WBC Thoma pipet Unopette • Dilution problem • Microscopy Red Cell and White Cell Counts • RBC problem: anemia, polycythemia • WBC.
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 • 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 L 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