ERYTHROCYTE COUNT
AIM: To count the total number of erythrocytes in one cubic mm (or1microliter) of
blood.
PRINCIPLE: For ease of counting, we are diluting the given blood sample (1 in 200)
with a diluting fluid, since our blood has huge number of RBCs per cubic mm of blood.
This diluted fluid is then introduced into the counting chamber of known dimensions.
The erythrocytes in a definite smaller volume (1/50 [Link]) are counted. To get the
actual number per cubic mm of undiluted blood, the counted number is multiplied by
the dilution factor (200) and the volume factor (1/50).
REQUISITES:
a) All the equipments required for Universal Safety Precaution.
b) Student Microscope
c) Improved Neubauer chamber
d) Cover slip
e) Whole blood (anticoagulated blood)
f) RBC (diluting) pipette
g) Absorbent cotton
h) Syringe 10 mL
i) Diluting fluid: A number of solutions have been recommended.
Hayem's Solution, used in this lab, has the following composition:
Sodium chloride : 0.5gm% (maintains osmolarity)
Sodium sulphate : 2.5gm% (preserves RBC without clumping)
Mercuric chloride : 0.25gm% (preservative for the solution)
Distilled water to make up to 100 mL
PROCEDURE:
1. Follow the universal safety precaution protocol.
2. Keep the microscope ready.
3. Place the Neubauer chamber in the given tile. Ensure that Improved Neubauer
chamber and the cover slip are dry and lint free. Place the cover-slip to cover
both platforms of the counting chamber.
4. Mix the given whole blood following figure of eight pattern.
5. Ensure that RBC pipette is clean and dry. (Look for the free movement of the
red bead in the bulb)
6. Attach the syringe to the saline tube of the RBC pipette.
7. Draw the blood up to 0.5 mark on the RBC pipette by gently pulling the piston
of the syringe.
8. Wipe the excess blood in the sides of the pipette using the absorbent cotton
from bulb to the tip. Avoid touching the tip bore.
9. Dilute the blood by drawing the diluting fluid up to 101 mark on the RBC
pipette. Ensure that tip of the pipette is always immersed in the diluting fluid to
avoid air bubbles.
10. Wipe the excess diluting fluid on the sides of the pipette using the absorbent
cotton from bulb to the tip. Avoid touching the tip bore.
11. Mix the contents in the bulb by holding the pipette horizontally and rolling it
with your fingers for few seconds.
12. Since the fluid in the stem of the pipette has not mixed with the blood, discard
the first two drops of the fluid from the pipette using syringe.
13. Place the tip of the pipette at the junction between the coverslip and the
Neubauer chamber.
14. Carefully control the flow of the diluted fluid either by pressing the saline tube
or by using syringe and allow the solution to gradually seep under the coverslip
into the platform. (The fluid will be drawn into the chamber underneath the
coverslip by capillary action.) Avoid overcharging and undercharging. If the
chamber gets flooded or contains air bubbles, clean the counting chamber, wipe
it dry and charge it again.
15. Allow 2-3 minutes for the cells to settle down, so that they are all in the same
plane. If you wait for longer than 3 minutes, drying will begin and the cells will
be disturbed by currents of evaporation. The chamber should not be exposed to
direct sunlight or draughts of air.
16. While transferring the counting chamber to the microscope stage, see that it is
kept absolutely horizontal, because tilting the chamber will cause dragging of
the cells to one side and lead to wrong counts.
17. First examine the whole of the platform of the counting chamber under low
power to assess the uniformity of the distribution of the cells. (If the distribution
is not uniform, or if there are any air bubbles, clean the chamber and charge it
again.)
18. Viewing under low power, bring the RBC counting area into the field. The
usual practice is to count the RBC in the 5 medium-sized squares shown below.
19. Switch to high power and bring the top left corner medium-sized square (of
1/25 [Link] area) in the RBC area for counting.
20. Count the cells in each of the 16 small square divisions of this square. As you
count, you should keep your left hand on the fine adjustment screw of the
microscope and continuously rotate it back and forth to pick cells in various
"layers".
RBC COUNTING CHAMBERS
21. While counting, only the cells contained within the square and those cells
touching or lying on the lines of any two adjacent sides (bottom and left or top
and right) are included in the count. By doing so, all the cells in a square will
have been counted, none being omitted and none being counted twice.
22. Enter the count in the grid in your counting paper.
23. Identify the next medium size square to be counted and in this way proceed to
count all the five selected squares in high power.
24. Add up the total number of cells in each medium-sized square. If the total of
one medium sized square is found to differ from another by more than 20, the
distribution of cells is unsatisfactory and the count has to be abandoned. In that
case, washout the counting chamber, charge it again and make a fresh count.
25. If the distribution is satisfactory, add up the total count in all the five and enter
in the space provided.
REPORT:
CALCULATION:
Area of 5 medium sized RBC squares 1/25 x 5 = 1/5 [Link].
Depth under the cover slip (height of diluted blood) = 1/10 mm.
Volume of 5 medium sized squares = 1/5 x 1/10 = 1/50 [Link].
Number of cells in 1/50 [Link] volume of diluted blood = n RBCs
Therefore, 1 [Link] of diluted blood contains = n x 50/1 RBCs.
The dilution factor is 1:200. (0.5 parts of blood in 100 parts of Hayem’s fluid)
Therefore, 1 [Link] of undiluted blood contains = n x 50/1 x 200/1.
= n x 10000 RBCs
Counting multiple grids and taking the average reduces the inherent technical and
field errors of the method. Counting of only one grid has to be done for your exams.
PRECAUTIONS:
1. Speed is essential to complete counting before fluid starts to dry up.
2. The tip of the pipette should dip in the blood. Otherwise, air will also enter along
with blood.
Technical errors:
a) Improper pipetting
b) Failure to wipe away the blood on the outside of the pipette tip before dilution
c) Improper mixing
d) Uneven distribution in the chamber.
Significance of RBC count: This is relatively an inaccurate procedure as in any
manual method. Manual counts are not done in most labs these days, since Hb
estimation and PCV will provide similar information. However, RBC counts are
required for calculating red cell indices, and for this purpose, automated cell counters
only can provide reliable counts.
QUESTIONS:
1. Are WBCs seen in this count?
2. What are the other RBC diluting fluids?
3. Why are we counting only the specific 5 medium sized squares?
4. What is the normal range of erythrocyte count?
5. What are the physiological variations in RBC count?
6. Explain the terms "anaemia" and "polycythaemia" and classify in brief their various
types.
7. Describe the terms "Physiological anaemia of pregnancy" and "relative
polycythaemia".
8. How will you alter the technique of erythrocyte counting in?
a. Severe anaemia
b. Polycythaemia
9. List the factors which would cause uneven distribution of cells in the
counting chamber?
10. What are the other uses of the RBC pipette?
11. Which anticoagulant is more suitable for this experiment?
12. What are the other methods available for counting RBCs?