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
Female

4.50 - 6.00 x 106/L (x 1012/L)

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)
Hayems 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

ImprovedNeubauerHemocytometer

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