RBC Manual Count
Prepared by
Yasser El-dahdouh
HEMOCYTOMETER
Hemo: blood
Cyto: cell
Meter: measurement/counter
Thus, it is an instrument used to
count the blood cells.
types of haemocyto metry
Double Neubauer Ruled •
Metallized Counting
Chamber
Petroff-Hausser Counting •
Chamber
types..
C-Chip Disposable •
Hemacytometer
Cellometer™
– The
Disposable
Cell Counting
Chamber
*** Hemacytometers
 a precision-made slide for performing
manual cell counts with the aid of a
microscope.
*** Hemacytometers are used when……
1- automated cell counters and hematology analyzers are unavailable
2- blood cell counts are extremely low
3- to get a cell count for body fluids (spinal fluid, joint
fluid, semen counts, and other bodily fluids)
The most commonly used hemacytometer is the Neubauer chamber.
It includes:
Neubauer’s slide (a
Cover slip (b
RBC pipette (c
WBC pipette (d
NEUBAUER’S SLIDE
** It is the name given to a thick glass
slide. In the centre of the slide, there is an
H- shaped groove. On the two sides of the
central horizontal bar, there are scales
for counting the blood cells.
** The depth of the scales is 1/10mm or
0.1mm.
** Each scale is 3mm
wide and 3mm long.
** The whole scale is
divided into 9 big
squares.
** Each square is 1mm
long and 1mm wide.
Differences Between RBC AND WBC
Pipette.
RBC pipette WBC pipette
1)
It has a red bead It has a white
bead
2)
It has
It has
graduations upto graduations upto
mark 101
mark 11
3)
Size of bulb is
larger
Size of bulb is
smaller
RBC PIPETTE
WBC PIPETTE
DILUTION FACTORS
For CELL counting
Blood is filled till mark 0.5 and fluid is then
filled till mark 101 or 11.
Both are thoroughly mixed.
The numbers
written on the chamber mean that the space between the
chamber and the cover slip is 0.100 mm and
that the smallest square on the grid has an area of 0.0025 mm2.
Clean
**The numbers
written on the chamber mean that the space between the
chamber and the cover slip is 0.100 mm and
that the smallest square on the grid has an area of 0.0025 mm2.
** Clean the Neubauer chamber and the cover slip
with 70% ethanol.
With the microscope, using a 4x objective, identify the
nine main squares of the counting chamber
delimited by three lines each as shown in the following
image:
** Now change to 10x objective and focus one of the 9 main
squares.
**The counting is performed in the
area delimited by three lines.
** Cells that touch the upper and left border are counted (black
color) whilst
cells that touch the right and lower border are not counted (red
color).
The smallest square has an area of 0.0025 mm2 therefore
each main square has an area of 0.04 mm2
(0.0025 x 16 = 0.04).
Erythrocyte Count
• Is the number of erythrocytes per micro litter of
blood.
• Normal ranges:
– Male
– Female
– New born
4.2 – 5.4
3.6 – 5.0
5.5-6.5
10^6/µL
10^6/µL
10^6/µL
• Erythrocyte count increased in case of
polycythemia and decreased in anemia.
Erythrocyte Count
• Principle:
– In order to facilitate RBCs count a specified
volume of blood is diluted with a specified
volume of isotonic fluid.
– Red cell diluting fluid must be:
•
•
•
•
anti-coagulant anti-hemolysis.
anti-aggregation.
anti-Rouleaux.
preserve RBC shape.
Erythrocyte Count
Diluting fluid:
•
One of the following solutions may be used:
1.
Isotonic saline:
– 0.85% sodium chloride (NaCl) in distilled water.
** Dilution with normal Saline:
Maintain the normal disk shape of the RBC
Prevents autoagglutination
2-
Hayam’s solution:
– Sodium Sulphate
– Sodium Chloride
– Mercuric Chloride
– Distilled Water
10 g.
2g.
0.25 g.
100ml
3- Gower’s solution:
Sodium Sulphate
Glacial acetic acid
Distilled water
4- Citrate-formalin solution:
Tri-sodium Citrate
Formalin.
12.5 g.
33.3 ml
100 ml.
•
Note:
– In certain conditions, Hayam’s solution may cause
clumping of RBCs and Rouleaux formations, while
Gower's solution prevents these problems
•
Sample:
– Whole blood using EDTA or heparin as anticoagulant.
Capillary blood may also be used.
•
Equipments:
1. (Pipettes) used one of the following:
•
•
Thoma pipette (RBCs)
Micropipette –20l is the desired volume.
2. Improved Neubauer chamber with the cover slips.
3. Conventional light microscope.
4. Clean gauze.
Procedure:
1. Dilution of the blood:
–
Micropipette (20) 1:201 dilution.
•
Pipette 4.0ml of diluting fluid into a
tube
•
Pipette 20l of will mixed
anticoagulated whole blood to the
tube.
–
Thoma red count pipette.
•
Draw the blood up to exactly the
0.5 mark and dilute to the 101
mark.
•
Mix continuously for 2-3 minutes.
2.
Load the cleaned hematocytometer.
3.
Place the hematocytometer on the
microscope stage, lower the condenser.
Procedure:
4. Focus with x10 objective
lens on the large central
square. This square is
ruled into 25 small squares,
each of which is further
divided into 16 smaller
squares, of the 25 squares,
only the four corner
squares, and one middle
square are used to count
RBCs.
5. Switch to 40 objective lens,
and start counting in the
five designated squares.
• The large center square is
used for RBC and PLT
counts.
• The center square is
divided into 25 smaller
squares, which are each
subdivided into 16
squares.
• Only 5 of the 25 squares
are used to count red
blood cells.
• These 5 are usually the 4
outer squares and the
inner most center square.
• The entire large center
square is used to count
platelets.
Preparation of the slide
Step 1
Place the cover slip
Step 3
Load Step
Step 2
Dilute the sample 1:200
Step 4
Count in the small 5 center squares for
RBC
Calculations:
•
Total RBC Count =
–
•
N x Dilution Factor x Volume Correction Factor
Where:
–
–
•
N = the total number of red cells counted in the counting
chamber.
Dilution 1: 200
•
Dilution Factor = 200
Counted Volume:
–
–
–
•
•
Each counted square has a volume of 0.2 X 0.2 X 0.1 = 0.004
5 squares volume = 5 X 0.004 = 0.02 cumm
Volume correction factor = 1/0.02 = 50
So,
Total RBC count =
–
N X 200 X 50 = N X 10.000
Sources of errors
Falsely high counts:* collection of blood from the area where there is
hemoconcentration.
* improper pipetting of blood as well as the fluid.
* improper mixing.
*Errors in calculation.
Falsely low counts:* Blood dilution with tissue fluid due to edem.
* improper pipetting and dilution.
* errors in calculation.
Discussion
1. In certain conditions, such as polycythemia, the
red blood cell count may be extremely high, which
makes it difficult to obtain an accurate count. In
this instance, make a larger dilution of blood. For
a 1:301 dilution, add 20L of whole blood to 6.0mL
of diluting fluid.
2. For a patient who has severe anemia and in whom
the RBC is low, make a 1:101 dilution by adding
20L of whole blood to 2.0mL of diluting fluid.
Discussion
3. Make certain the pipettes, hemacytometer, and
cover glass are free from dirt, lint, and dried
blood. Ensure that the diluting fluid is free from
blood and other contamination.
4. RBC takes longer to perform than a WBC
because of the larger number of cells. Therefore,
proceed as quickly as possible once the cells
have settled. Drying of the dilution in the counting
chamber causes inaccuracies in the final cell
count.
5. The range of error for a manual RBC is generally
about 10 to 20%