ENUMERATION OF RED BLOOD CELLS
• AIM
• PRINCIPLE
• APPARATUS
• PROCEDURE
• OBSERVATION AND RESULTS
• STEPS OF CALCULATIONS
• NORMAL VALUE
• VARIATIONS
• AIM-
To find out the number of RBCs per cmm of blood
• PRINCIPLE-
The number of RBCs in a known volume of diluted blood is
counted. From this, the number of RBCs in 1cmm of
undiluted blood is calculated.
• APPARATUS-
- Microscope
- Neubauer’s counting chamber
- RBC diluting pipette
- RBC dilution fluid [ Hayem’s fluid]
- cover glass
- spirit, cotton, sterile lancet
• Haemocytometer/ Neubauer’s counting chamber-
. It’s a solid heavy glass slide.
. Divided into 3 platforms by 2 deep gutters.
.Central platform is 1/10 mm lower than side 2 platform,
so that when placed a cover glass can rests on the
platforms on either side, giving a gap for central platform.
.Divided into 2 parts by transverse gutter. This each half
contains large square, called as counting areas to count
cells.
. Each counting area has a large square of 3mm, and again
Into 9 primary squares of 1mm.
.The 4 corner primary squares are used for counting WBCs,
which again divided into 16 tertiary squares[1/4 mm],
thus total 64 smallest squares are used for counting WBCs.
.The central primary square 1mm is divided into 25
secondary squares[1/5mm side], by triple lines, which again
divided into 16 tertiary squares[1/20mm]. For counting
RBCs the central and four secondary squares are used, so
total 80 tertiary squares are used.
9 Primary
Squares
1mm
WBC WBC
¼ mm
16
Secondary
Squares
each
WBC WBC
RBC
25
Secondary
Squares
RBC RBC
1/5 mm
RBC
RBC RBC
16
Tertiary
1/5 mm Squares
each
1/20 mm
• So total no: of tertiary squares = 16 x 5 = 80
RBC RBC
RBC
RBC RBC
• So depth of the chamber = 1/10 mm
• So depth of the chamber = 1/10 mm
RBC pipette-
.It has a capillary stem of uniform bore with conical tip and
bulb above the stem.
.The capacity of the bulb is 100 times that of stem.
.The stem is divided into 10 equal parts and two markings
0.5 and 1 below the stem and 101 just above the bulb.
.The bulb contains a red bead----
- helps in mixing the blood with the diluting fluid
- Easy identification of the pipette
.The other end of the pipette is fitted with a soft and light
rubber tubing and a mouth piece[ red colour]
RBC diluting fluid[ Hayem’s fluid] – each 100 mL contains
1. NaCl- 0.5 gm– to maintain isotonicity of fluid
2. Na2SO4- 2.5gm- to prevent rouleaux formation
3. Mercuric Perchloride- 0.25gm- acts as a preservative,
antifungal, antibacterial.
4. Distilled H2O- to make up 100ml solution
PROCEDURE-
. Clean the chamber and cover glass, keep on plain surface,
and place the cover slip on the chamber.
. With all aseptic precaution take a prick.
. Suck blood into the RBC pipette up to mark 0.5----
- not to less or more
- avoid air bubbles
.Immediately suck in the diluting up to the mark 101.
.Hold the pipette horizontally and roll it between the palms,
for thorough mixing.
• CHARGING OF THE CHAMBER-
Discard the first few drops of fluid in the stem…..
Because the fluid in the stem does not take part in the
mixing and contain only the diluting fluid. Mixing takes place
only in the bulb.
.Then hold the pipette at an angle of 45 degree, touch the
edge of the cover glass.
.Due to the capillary action the fluid runs into the capillary
space between the slide and cover glass and fills it………
- avoid overcharging and undercharging
- avoid air bubbles
.Wait for 3-5mins to settle down the cells, the cells should
not be in motion while counting is being done.
.Adjustment of microscope to focus RBC----
.Focus chamber under low power[10X], then under high
power[40X].
.Focus the RBC square, count RBCs in 4 corner and one
central square.
.While counting the cells, count the cells in the square, left
and lower border line and omit the cells on upper and right
border.[ Rule of L] to avoid counting the cell twice.
Lower & Left deserve a
Like
1/20
Steps of Calculation mm
1. Side of 1 tertiary square = 1/20 mm
2. Area of 1 tertiary square = 1/400 mm2
3. Depth of the chamber = 1/10 mm
4. Therefore, volume of 1 tertiary square = 1/4000 mm3
5. Total no: of tertiary squares counted = 16 x 5 = 80
6. Therefore, volume of 80 tertiary squares = 80 x 1/4000
= 1/50 mm3
7. Let N be the number of RBCs in 1/50 mm3 of diluted blood
8. Then the number of RBCs in 1 mm3 diluted blood = 50 x N
9. Dilution factor = 0.5 in 100 or 1 in 200
10. Therefore total no: RBCs in 1 mm3 undiluted blood
= 50 x 200 x N = 10000 N
7 6 3 2 5 6 4 2
5 4 4 6 5 4 3 6
68 66
3 4 2 1 5 4 2 1
4 6 7 4 3 6 6 4
7 5 3 6
5 6 3 2
3 4 2 1
4 6 6 2 7 5 3 5
4 6 7 4
5 5 4 6 5 4 4 6
3 6 2 1 68 3 5 4 1
4 6 4 4 68 73 4 6 7 4
• N1 + N2 + N3 + N4 + N5 =
• 68 + 66 + 68 + 68 + 73 = 343
• RBC count = 343 X 10,000 = 3430000
• = 3.43 millions/cmm
• Normal values-
- Males- 4.5-6.5 millions/cmm [5.4millions/cmm]
- Females – 3.5-5.5 millions/cmm[4.8millions/cmm]
• Variations in the count-
• Physiological-
• Increase Decrease
. Males .Females
.Infants
.high temperature
.Low O2 tension .Malnutrition
.After exercise . pregnancy
• Pathological
• Increase Decrease
.Polycythemia .Anaemia