HEMOCYTOMETRY

HEMOCYTOMETR
Y
Kimberly A. Bugayong, RMT

Kimberly A. Bugayong, RMT

MATERIALS:
◦ THOMA PIPETTE:
◦ -small pipette with short stem with graduations
◦ -bulb near the top
◦ -Markings at the top of the bulb:
◦ -101: red cell pipette
◦ -11: white cell pipette
◦ SUCKING TUBE:
◦ -facilitates aspiration of the sample and diluting
◦ fluid
MATERIALS:
◦ HEMOCYTOMETER:
◦ -Levy hemocytometer with improved
Neubauer ruling.
◦ -total area: 9mm^2, divided into 9 large
squares (1mm x 1mm)
◦ -two chambers are separated by a shallow H-
shaped moat in the middle.
◦ -corner squares: WBC - divided into 16
smaller squares (0.25mm)
◦ -central square: RBC – divided into 25 smaller
squares (0.04mm^2) for the smallest squares in
the center
-Distance between coverslip and counting chamber:
0.1mm
-Total volume of one counting area = 0.9mm^3
MATERIALS:
◦ HEMOCYTOMETER:
◦ -Fuch’s – Rosenthal Hemocytometer
◦ -2 counting chambers
◦ -each counting chamber:
◦ *total area of 16mm^2 divided into 16 large
squares (1mm x 1mm), *depth = 0.2mm
◦ -Each large square is further divided into 16 smaller
squares (0.25mm x 0.25mm)
MATERIALS:
◦ HEMOCYTOMETER:
◦ -Spiers – Levy
◦ -4 counting chambers
◦ each counting chamber:
◦ *total area = 10mm^2 divided into 10 large
squares arranged horizontally each measuring 1mm
x 1mm,
◦ *depth = 0.2mm
◦ each large square: divided into 16 smaller squares
(0.25 x 0.25)
PRINCIPLE:
◦ -Dilution of blood
◦ -Counting cells from a sample of diluted blood
◦ -Counts are expressed as cubic millimeters (mm3) because of the linear dimensions of
the hemocytometer. (traditional unit of reporting)

◦ International Committee for Standardization in Hematology has recommended the

liter (L) as the unit of volume.
◦ -cell counts are therefore expressed as the number of cells or formed elements per
liter of blood
DILUTION of BLOOD:
◦ DILUTING FLUIDS:
◦ RBC = isotonic, prevent coagulation of red cells
◦ WBC = weak acid solutions used to lyse red cells to facilitate white cell counts
CHARGING of the HEMOCTOMETER:
1. Discard the first few drops of fluid in the pipette.
2. Secure the coverslip on top of the hemocytometer, making sure it will not be disturbed during charging
3. Carefully fill the counting chambers with the sample as seen in Figure A, then allow to settle for 5 minutes.
MANUAL CELL COUNT
MANUAL CELL COUNT
POISSON’s LAW of DISTRIBUTION:
◦ -Cells settle in a random manner.
◦ -Even though cell counts in the different squares are different, they must not
go beyond the acceptable difference:
>/= 15 (WBC), >/=20 (RBC)
◦ Otherwise, the count is considered invalid = recharge the hemocytometer
again.
CALCULATIONS and REPORTING:
◦ General formula for cell counts:

Total cell count = # of cells counted x Dilution Factor

Area in mm^2 x Depth
CALCULATIONS and REPORTING:
◦ Alternatively,
Total cell count = # of cells counted x DF x Volume Correction Factor
Wherein:
DF = volume of fluid in bulb
volume of sample
VCF = Desired volume to be counted (1)
Actual volume counted
Actual number counted = number of squares x volume per square
SOURCES of ERROR
RED BLOOD CELL COUNT

◦ WHO: Erythrocyte number concentration

◦ Not recommended
◦ Poor reproducibility and accuracy
◦ Usually done on bodily fluids to which red cell count is requested
RED BLOOD CELL COUNT
◦ Procedure:
1. Using RBC thoma pipettes, draw blood to the 0.5 mark
-no air bubbles
-wipe excess blood on the outside of the pipette
2. Draw diluting fluid to the 101 mark
-no air bubbles
RED BLOOD CELL COUNT
◦ Procedure:
3. Cover the tip of the pipette with finger, remove suction device, shake
4. Discard a few drops
-First few drops are primarily diluting fluid
-Hence, deduction of 1 unit in the computation for dilution factor
5. On a clean hemocytometer with coverslip, charge
-Allow to settle for 3 minutes: moist chamber
6. Under 10x magnification, locate the central square
-shift to 40x: count red cells in ruled areas
7. Repeat on the next chamber
RED BLOOD CELL COUNT
◦ Sample computation:
◦ Total cell count
◦ 525 x 200 x 50
◦ 5,250,000 cells/mm3
◦ 5.25 x 10^6cells/mm^3 or
cells/uL cells/uL

Total cell count = # of cells x DF x VCF

DF = 101-1 = 200 VCF = 1 =50 Ref:
0.5 5.(0.005mm3) M: 4.20-6.0x10 6/uL
F: 3.80-5.20x10 6/uL
WHITE BLOOD CELL COUNT

◦ Same procedure as RBC count but using WBC thoma pipette

-Draw blood to the 0.5 mark
-Draw diluting fluid to 11 mark
-Dilution: 1:20
-Count on the 4 large WBC squares
WHITE BLOOD CELL COUNT
◦ Sample Computation Total cell count
◦ 134 x 20 x 2.5
◦ =6700 cells/mm3
◦ =6.7 x 10 3 cells/mm3 or cells/uL
◦ =6.70 x 10 9/L

Total cell count = # cells x DF x VCF Ref values:

DF = 11-1 =20 VCF = 1 =2.5 Adults: 3.6-10.6 x 10 3/uL
0.5 4(0.1mm3) (x10 9/L)
WHITE BLOOD CELL COUNT
CORRECTED WBC COUNT:
-Nucleated red cells are not lysed by the diluting fluid
-nRBCs are counted as WBCs (cannot be differentiated in a hemocytometer)

**IF there are more than 5 nRBCs for every 100WBCs in a PBS during a diff count,
a corrected WBC count is recommended:

CORRECTED WBC CT = Uncorrected WBC count x 100

Number of nRBCs per 100 WBC + 100
WHITE BLOOD CELL COUNT
CORNER COUNT
SQUARE
I
II
III
IV
TOTAL
WHITE BLOOD CELL COUNT
CORNER COUNT
SQUARE
I
II
III
IV
TOTAL