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