Haemocytometry by Ravi Kumudesh

Haemocytometry

Ravi KumudeshMSc/BSc/Dip(MLT)

Sri Lanka Society for Medical Laboratory Science

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Hemocytometry

• Hemocytometer• RBC and WBC• Thoma pipet• Unopette• Dilution problem• Microscopy

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Red Cell and White Cell Counts

• RBC problem: anemia, polycythemia• WBC problem: leukocytosis, leukopenia• RBC reference ranges

• Male 4.50 - 6.00 x 106/µL (x 1012/L)• Female 3.70 - 5.20 x 106/µL (x 1012/L)

• WBC reference range• Both 5.0 - 10.0 x 103/µL (x 109/L)

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Diluting Fluid

• For RBC– Physiologic saline (0.8% NaCl)– Hayem’s solution– Reese-Ecker

• For WBC– 3% Acetic acid– 0.1 N HCl

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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

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Improved Neubauer Hemocytometer

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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

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Hemocytometer

Chamber

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Counting Rule

• Do not count cells touching– Bottom line– Right line

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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 (µL)

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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 (µL)

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Unopette

• Self-contained reservoir and 20 µL pipet• Ammonium oxalate diluent• DF = 100• Count leukocytes in all 9 large squares• Special formula to facilitate calculation

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Unopette

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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

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Compound Microscope

• Ocular (adjustable binocular)

• Mechanical stage• Condenser• Objectives (parfocal)

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Care of Microscopes

• Carry with care• Protect long objective lens• Clean and disinfect stage• Wipe oil off OIL• Save lamp life

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Sri Lanka Society for Medical Laboratory Science

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