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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
Hemocytometer

RBC and WBC

Thoma pipet

Unopette

Dilution problem

Microscopy

Week 4: Hemocytometry
red cell and white cell counts
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
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

Thoma Pipet
hemocytometer chamber
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
counting rule
Counting Rule
  • Do not count cells touching
    • Bottom line
    • Right line
rbc example
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
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
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 wbc example
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
Compound Microscope
  • Ocular (adjustable binocular)
  • Mechanical stage
  • Condenser
  • Objectives (parfocal)
care of microscopes
Care of Microscopes
  • Carry with care
  • Protect long objective lens
  • Clean and disinfect stage
  • Wipe oil off OIL
  • Save lamp life