HEMATOLOGY 1 Laboratory

HEMA311| Prelims L5: RBC Counting

Guzman, Agnes Almodal

RBC Counting Procedure

● The red cell count is the number of red cells in 1
[Link] (cubic millimeter) 1. Draw blood up to 0.5 mark using the RBC pipette.
● The RBC count is one of the tests that are used for
the diagnosis of anemia and polycythemia.

If you have difficulty taking it directly from the lavender tube, you
can place a drop on the coverslip or on the slides and then
aspirate from there.
2. Wipe the outside walls (body) of the pipette with a
clean gauze.

RBC pipette

The RBC pipette has three calibrations at 0.5, 1, and

101. Unlike the WBC pipette, its bulb is rounder and
contains a red bead, which helps in mixing since this is a
manual method. The standard dilution factor for RBC
counting is 200, which is calculated as 100 ÷ 0.5 This is to avoid excess blood from getting into the diluting fluid
when aspirating. Never touch the tip because it will reduce your
0.5, if you do so, repeat from the beginning.
Materials and Equipment 3. Dip the pipette into diluting fluid, then aspirate the
diluting fluid into the pipette slowly until the mixture
● Anticoagulated blood reaches the 101 mark.
● RBC Pipette
● Diluting Fluid (Hayem’s Solution)
● Tally counter
● Counting Chamber
● Special Cover Slip (thicker for viewing the grid)
● Gauze pad
● Microscope
● Test tube

There should be no bubbles and gaps

4. Gently rotate the pipette to mix the diluting fluid
and blood

5. Mix for 3-5 minutes. Until it becomes a

homogeneous solution
Transcribed by: Dimarucut, Stephanie R. Our Lady of Fatima University
 6. Discard the first 3-4 drops of the diluted sample

Use the index finger to control not the thumb. This is done
because the RBCs in your stem/body are more on diluting fluid,
so if you don't dispense and then go straight to the
hemocytometer, no RBCs will be seen.
7. Prepare the counting chamber

How to Count RBC Using the Neubauer Ruling

In the Neubauer counting chamber, the four large corner squares (A, B, C,
and D) are used for WBC counting. The distance from A to B is 3 mm,
Put the coverslip in the middle
and from B to C is another 3 mm, giving a total ruled area of 9 mm². Each
8. Charge both sides of the hemocytometer with a large WBC square measures 1 mm × 1 mm and is subdivided into 16
drop of diluted sample and allow to stand for few small squares (4 × 4). Thus, the four WBC squares (A, B, C, D) contain a
minutes total of 64 small squares.

The central square (also called the secondary square) is used for RBC
and platelet counting. This central square is divided into 25 smaller
squares (5 × 5). Each of these small squares (tertiary squares) is further
subdivided into 16 tiny squares. Therefore, when counting RBCs, we use
5 tertiary squares (the four corners and the center), which gives a total of
80 tiny squares.

When performing the count, RBCs are read in the upper left, upper right,
lower right, lower left, and center tertiary square.

For measurements:

It should not be too much or it should not end up in the mold(?) ● One small square in the WBC counting area = 0.25 mm².
or the canal. Then wait for 5 minutes for the RBC to settle. ● One tiny square in the tertiary area = 0.05 mm².
9. While keeping the hemacytometer in a horizontal
position, place it on the microscope stage.
10. Using High Power , count the red cells in the 5 “R”
squares of the central secondary square.
Microscopically:
● one large square is made up of nine 1-mm
x 1-mm squares
● each of WBC squares is divided further to
16 squares
● the center square for RBC is subdivided Low power view
into 25 smaller squares 11. Calculate the number of RBC per liter of each side
● The distance between each counting of the hemocytometer.
surface and coverslip (depth) is 0.10 mm SUBTOPIC 1
● The total volume is 9 cu mm
Calculations
● Since the cells in 1/5 [Link] are counted, the
dilution is 1:200, and the depth of the counting
chamber is 1/10 mm

Transcribed by: Dimarucut, Stephanie R. Our Lady of Fatima University

 ● RBC count = # of RBC counted (on the 5 squares)
x area correction factor x dilution correction factor x
depth correction factor
- DIlution factor = 100/amount of blood
aspirated
● RBC count = # of RBC count in millions/[Link] x
.000001 = total no. of RBC counted x 1012/L
Ex: 5,400,000 x .000001= 5.4 x 1012/L

Area used in the 1/5 [Link]

actual cell count

Area correction factor 5

Depth of the counting 0.1 or 1/10 mm

chamber (constant)

Depth correction factor 10

Dilution 1:200 or 1/200

Dilution correction factor 200

RBC count = no. of cells counted x 5 x 10 x
200 or no. of cells counted x 10,000
VARIATION IN TECHNIQUES
Dilution varies if:
● Polycythemia or Erythremia
- Blood is drawn to 0.3 mark of the RBC
pipette and diluent up to 101, 1:333
(100/0.3 = 333)
● Anemia
- Blood up to 1 mark and the diluent to 101,
the dilution is 1:100 (100/1 = 100)
REFERENCE VALUES
Normal Values:
● Female: 3.6 –5.6 x 1012/L
● Male: 4.2 –6.0 x 1012/L
● At Birth: 5.0 –6.5 x 1012/L
PHYSIOLOGIC VARIATION
● Increased count in dehydration
● Increased count in exercise
● Newborn children have higher counts than adults
● Women have lower counts than male
● Individuals living at higher altitudes have higher
counts.
- the oxygen is thinner (partial pressure is low) the
tendency is to grasp more oxygen
● Increase erythrocyte count in polycythemia
- Can use JAK2 (Janus Kinase 2) gene mutation test
(best test)
● Increase in pulmonary tuberculosis and pulmonary RULES IN COUNTING
fibrosis. 1. Observe the boundary line of the square
● Increase in acute poisoning a. Double line – The outer line is the
● Decrease in anemia and after hemorrhages. boundary of the square
b. Triple Line – The middle line is the
boundary of the square

Transcribed by: Dimarucut, Stephanie R. Our Lady of Fatima University

 2. Count the cells touching halfway in and halfway out
of the upper and left boundary lines
3. Never count cells touching halfway in and halfway
out of the lower and right boundary lines of the
square
4. Never count cells drying preparation
5. Never count cells in underfilled or overfilled
chamber

6. Do not count cells touching: (to avoid double

counting)
a. Bottom Line
b. Right Line

7. Counting 2 sides in L shape (i.e count the cells

settle on the Top and left sides (colored sides) and
exclude the cells on the right and the bottom sides)
8. Counting is Zigzag in all squares
Blue cells is counted
gray cells is not counted

Transcribed by: Dimarucut, Stephanie R. Our Lady of Fatima University