HEMATOLOGY

MANUAL CELL COUNT 9. Count all cells in the four corner

squares, start with the one in the upper
- Counting or WBC, RBC, and platelets
left- hand corner. Cells touching the
are basically the same but varies in the
top and left lines should be counted;
dilution, diluting fluid, and area
cells that touch the bottom and right
counted on the hemocytometer.
lines are ignored.
Hemacytometer/ counting chamber

- Two raised surfaces, each with a 3 mm

SOURCES OF ERROR
x 3 mm (total area of 9 mm2) square
counting area or grid separated by an » The hemacytometer and coverslip
H-moat should be cleaned properly before they
- Most common: Levy chamber with are used. Dust and fingerprints may
improved Neubauer ruling cause difficulty in distinguishing the
cells.
» The diluting fluid should be free of
PROCEDURE: contaminants
» If the count is low, a greater area may
1. Clean the hemacytometer and be counted to improve accuracy
coverslip with alcohol and dry » Chamber must be charged properly to
thoroughly with a lint- free tissue. Place ensure an accurate count. Uneven flow
coverslip on. of the diluted blood into the chamber
2. Make 1:20 dilution of well-mixed blood results in an irregular distribution of
into WBC diluting fluid in a small test cells. If the chamber is overfilled or
tube underfilled, the chamber must be
3. Cover the tube and mix by inversion cleaned and recharged.
4. Allow to sit for 10 minutes (ensures » Allow cells to settle for 10 minutes
RBC have lysed) solution will be clear before counting
once lysis has occurred. WBC count » Any nucleated red blood cells (NRBC)
should be performed within 3 hours of present in the sample are not lysed by
dilution the diluting fluid. If five or more NRBC
5. Mix again and fill plain per 100 WBC are observed, WBC count
microhematocrit tube. must be corrected.
6. Charge both sides of hemacytometer » Accuracy of the manual WBC count can
at a 45-degree angle and touching the be assessed by performing a WBC
tip to the coverslip edge where it meets estimate on a Wright-stained
the chamber floor and for 10 minutes peripheral blood film made from the
before counting (do not disturb the same specimen.
coverslip)
7. Horizontal position on the microscope
8. Lower the condenser and focus on the
10x objective lens. Cells should be
distributed evenly in all the squares
 RBC and WBC dilution factor formula:

Dilution factor= Volume of the bulb (constant)/

volume of blood

RBC indices

- Calculated to determine the average volume

and hemoglobin content and concentration of the
RBCs in the sample.
- May be used for initial classification of
anemia

Square/s in hemacytometer Number of squares/ area/

distances
4 corners Subdivided into 16 squares
Center squares Subdivided into 25 squares
Each of the smallest squares 0.2 mm x 0.2 mm (or 0.04
(both 16 and 25 squares) mm2)
Distance between each 0.1 mm
counting surface and the
coverslip
Total volume of one entire 0.9 mm3
counting area on one side of
the hemacytometer
 Red Blood Cell Count White Blood Cell Count
Diluting fluid a. NSS Diluting fluid (weak a. Glacial acetic
(isotonic b. Bethel’s solution acids) acid
solutions) c. Taisson’s solution b. Turk’s
d. Hayem’s solution c. 1% HCl
e. Dacie’s fluid
f. 3.8% Sodium Citrate
g. Gower’s solution
Dilution factor 200 Dilution factor 20
Formula = cells counted x dilution factor/ area counted (mm2) x depth
Normal value Male: 4.20- 6.00 x 10^6/L Normal value 5.0-10.0 x 10^9/L
Female: 3.80-5.00 x 10^6/L

RBC index Definition Formula Unit Reference Terms

interval
Mean cell Average MCV= Hct (%) Femtoliters 80- 100 fL <80-
volume (MCV) volume of the x 10/ RBC (fL) microcytic
* RBC red blood cell (10^12/L) 80-100-
size normocytic
>100-
macrocytic
Mean cell Average MCH= Hgb Picograms 26-32 pg
hemoglobin weight of (g/dL) x 10/ (pg)
hemoglobin in RBC (10^12/L)
a red blood
cell
 Mean cell Average MCHC= Hgb Grams per 32-36 g/dL <32-
hemoglobin concentration (g/dL) x 100/ deciliter (g/dL) hypochromic
concentration of hemoglobin Hct 32-36-
in each normochromic
individual red >36-
blood cells hyperchromic

MCV
Hypochromic RBC contains a control area which is greater than 1/3 the diameter
of the cell
Grading Central pale area is…
1+ >1/3 of the red cell diameter
2+ >2/3 of the red cell diameter
3+ 3 quarter of the red cell volume
4+ Thin rim of hemoglobin is left
Normochromic Central pallor is 1/3 of the red cell diameter
Hyperchromic Decreased or absent central pallor

MCV (fL) MCHC (g/ dL) Red Blood Cell Morphology Found in
<80 <32 Microcytic; hypochromic Iron deficiency anemia, anemia of
inflammation, thalassemia, Hb E disease
and trait, sideroblastic anemia
80- 100 32- 36 Normocytic; normochromic Hemolytic anemia, myelophthisic
anemia, bone marrow failure, chronic
renal disease
>100 32- 36 Macrocytic; normochromic Megaloblastic anemia, chronic liver
disease, bone marrow failure,
myelodysplastic syndrome

Corrected WBC count

** if five or more NRBCs per 100 WBCs are observed on the differential count, the WBC count
must be corrected

Corrected WBC count = uncorrected WBC count x 100/ no. of NRBCs per 100 WBCs + 100

CALCULATIONS:

Total CT = cells counted x DF/ area (mm^2) x depth (0.1)

RBC WBC
Size of bulb Larger Smaller
Color of bead Red White
Size of bore Smaller Larger
Volume of bulb 100 10
 Upper calibration 101 11

Sample problem: RBC ct

1:200 – dilution = cells counted x DF / area x depth (0.1)

336 – cells counted = 336 x 200 / 0.2 mm^2 x 0.1 mm

0.2 mm^2 – area = 336 000 / mm^3 or 336 000/ uL or 3.36 x 10

^6 uL or 3.36 x 10^12/ L

Rule of 3 = Hgb x 3 = Hct