Presentation: Hematology

Topic name: manual RBCs counting method

Group Members
i) M Kashif Aslam

ii) Hafiz Binyameen iii) Anam Rehman

iv) M Muzamil Tayyab v) M Ahsan Raza

 Introduction to RBCs
 Red blood cells, or erythrocytes, are the
most abundant cells in the bloodstream
and contains hemoglobin, the compound
that carries oxygen through the body.
RBCs are synthesized by the bone marrow.
Any disruption of the red blood cells, its
quantity, shape, size, structure or life cycle
can therefore affect the oxygen carrying
capacity of the blood.
 Structure of RBCs:
Color:
RBCs have hemoglobin so these are red in
color.
Shape:
A red blood cell is a biconcave disc. Simply
it is a round ball that is squeezed from two
opposite ends to appear, widest at the sides
and narrowest in the middle.
This unique shape increases surface area
for exchange of gases.
 Structure of RBCs:
Size:
A red blood cell measures about 6 to 8
micrometers in diameter (average = 7.8 um) with
an average thickness of 2 micrometers (2.5 um at
the thickest point and less than 1um at the center).
Flexibility :
RBCs are highly flexible in Nature. As we know
that some capillaries are smaller in diameter than
RBCs size. Due to the flexibility RBCs passes
through these capillaries.
its flexibility allows it to become distorted as it
squeezes through narrow passages and then
restores to its.
 Structure of RBCs:
No nucleus:
They do not have nucleus. Hence, cannot produce any new
proteins.
No mitochondria:
 Mitochondria, powerhouses of cell, use oxygen to produce ATP.
 As they do not have mitochondria, RBC cannot use oxygen to
produce ATP.
 All ATP production occurs by anaerobic method.
Life span:
they have a life span of about 120days.
 RBCs count:
• RBCs count is proceedure in which You know the quantity of red blood cells that how many
You have the RBCs in your blood sample.
symptoms of an abnormal RBCs count
If your RBC count is too high or too low, you could experience symptoms and complications.
These are given below
fatigue
shortness of breath
Weakness
Dizziness
increased heart rate
Joint pain
Pale skin
sleep disturbance
 Purpose of RBCs Count
Purpose:
Main purpose of RBCs count is to check out the different
pathologic conditions like Erthrocytopenia
erythrocytosis
Hematologic disorders.
Anemia.
Blood malignancy
infection diagnosis
 Types of RBCs counting methods:
There are two methods for RBCs counting.
Automated Method:
(Electronic hematology cell counter, e.g.
COULTER)
Manual Method:
(visual using a microscope and counting chamber) which is a
cumbersome and less accurate.
 Manual Methods:

Equipment needed
lancet/needle :
For puncturing the skin and blood vessels to get the Blood.
Cotton swab with alcohol.
To make the puncturing site of skin free from microbes.
Test tubes:
To save the blood sample.
Diluting fluids:
RBCs are around only 5 million/cumm of blood.
 Equipment needed
RBCs diluiting fluids
• Counting this much number is highly impossible.
• Therefore the blood sample is diluted with the help of RBC diluting
fluid.
• It fixes and preserves RBCs.
• It is isotonic to RBCs.
Two types of diluting fluids are used for RBC counting.
1) Hayem’s fluid
2) Dacie’s fluid
 RBCs diluting fluids
Hayem’s fluid
Composition of Hayem’s fluid
a. Sodium chloride -0.5 gm
b. Sodium sulfate -2.5 gm
c. Mercuric chloride -0.25 gm
d. Distilled water – 100 ml.
The sodium sulphate discourages clumping of
the erythrocytes and the mercuric chloride is a
preservative.
 RBCs diluting fluids
Dacie’s fluid
Composition of Dacie’s fluid
i. Trisodium citrate – 3 gm.
ii. Formalin – 1 ml.
iii. Distil water – 99 ml.
This diluting fluid is cheap and commonly
used.
 Equipment needed
RBCs PIPPETS:
The RBCs pipette is used for dilution and
mixing the blood with diluting fluids and is
calibrated to deliver 20ul or 0.02 ml of
blood.

Consist of graduated capillary tube, mixing bulb

with glass bead and aspirating tube

Parts:
stem, bulb, rubber tube
Difference B/W RBC Pipette & WBC Pipette
Sr no. RBC Pipette WBC Pipette

1 Red Bead White Bead

2 It has It has
graduations up to graduations up to
mark 101 mark 11

3 Larger Bulb Smaller Bulb

4 Small Lumen Larger Lumen

 RBC PIPETTE

WBC PIPETTE
 Equipment needed
NEUBAUER’S SLIDE
Thick glass slide with two ruling area on
center
Ruled area are separated by H-shaped
gutter/trough
Beyond the two vertical arm of trough
there are two raised shoulder(ridge)
which support cover glass
lining is coated by shining metal or
rhodium
Each scale is 3mm wide and
3mm long.
Depth of the chamber is
0.1mm.
The whole scale is divided
into 9 big squares.
Each square is 1mm long and
1mm wide.
The four corner
squares are further
divided into
sixteen smaller
squares and are
used for WBC
counting.
Total = 64 small
squares
Central square is divided
into 25 medium sized
square and are separated by
triple line
The medium sized square
are further divided into 16
small square(tiny)
The four corner and central
square are used for platelet
and RBC count.
 Cover Glass
A special cover glasses is used
which has a very smooth,
flattened surface and even
thickness
Different thicknesses are
 0.3mm
 0.4mm(most common)
 0.5mm
Two size are common
 16x22sq.mm
 22x23sq.mm
 procedure
There are 4 Main steps in RBC’S counting procedure.
• Blood collection
• Pipetting
• Charging
• Microscopic Examination.
Blood collection
Steps for safe blood sampling.
Step 1: select equipment , prepare area
Step 2: prepare patient , collect blood sample
Step 3: transfer blood sample
Step 4: waste management
 procedure
Pipetting
Draw the blood directly from
finger or collected sample into
RBC pipette up to 0.5 mark.
Wipe off the tip of pipette to
remove extra blood, if present.
Then immediately draw up the
diluting fluid up to 101 mark.
Now rotate the pipette gently
for 2 – 3 mints so that the
diluting fluid gets mixed
properly. This will give dilution 1:
200.
 procedure
Charging
Place the cover slip in position over the ruled area
of chamber. Once again mix the solution
thoroughly by rotating the pipette.
Discard first 1 – 2 drops of blood from pipette. Now
apply slight pressure on the rubber tube of pipette,
so that the third drop of fluid is in hanging position.
Touch the tip of pipette (hanging drop) against the
edge of cover slip. The angle between pipette and
cover slip is 45°. With this process, chamber gets
filled with the fluid. This is known as charging of
the chamber.
 procedure

Charging
The care should be taken that, no air
bubble is present inside the chamber
and there is no over filling beyond the
ruled area.
Leave the counting chamber as it,
without disturbing for about 3 min.
This will allow the settling down of
RBCs.
 procedure
Microscopic examination:
Place the chamber on stage of microscope. Adjust the light and
ruled area. Make sure that, the distribution of RBC over the
chamber is uniform.
Now count the RBC using 40x in Central Square. The central
square is subdivided into 25 squares.
Out of 25 squares, count four at each corner and one at center.
Each of these five squares is subdivided into 16 small squares.
Thus, RBCs are counted in 16 x 5 = 80 small squares.
Counting Rule
 Formula Calculation (RBCs)
No of RBCs in 80 tiny squares = X
No of RBCs in 1 tiny square = X/80
As we now know that there are 25
smaller squares in central square and
each of these 25 smaller squares
contain further 16 tiny squares hence
the central square contain 400 tiny
squares.
• Area = length x width
• Volume = length x width x depth
• Volume = area x depth
 Formula Calculation (RBCs)
As the area of central square is 1 mm2 and depth
of chamber is 0.1 mm or 1/10 mm.
It is inferred that the area of each tiny square
would be 1/400 mm2.
Then volume of each tiny square =1/400 X 1/10
which can be written as 1/4000mm3.
As the no of RBCs in 1/4000 mm3 =X/80.
So the no of RBCs in 1 mm3 = X/80 / 1/400
= X/80 x 4000
To simplify it
the no of RBCs in 1 mm3 = 50x
 Formula Calculation (RBCs)
Dilution factor= Total Volume / Initial Volume
= 100/0.5
= 200
As blood was diluted 200 times for RBCs
So No of RBCs = dilution factor X 50x
= 200 x 50x
So no of RBCs in 1 mm 3 = 10,000x
https://youtu.be/sDhsyh2wKxc
 Errors
Non-uniform suspensions
Improper filling of chambers
Failure to adopt a convention
for counting cells in contact
with boundary lines or each
other
defective apparatus, non-
calibration etc.
 Precaution
Diluent should be corrected
No overflow
Pipettes used must be clear and dry.
No scratches in the ruled area of chamber.
No air bubbles.
 AUTOMATED METHOD
• Electronic counter is based on the principle
of aperture impendence method .
• Anticoagulated blood is diluted with
particle free diluting fluid such as
physiological saline or phosphate buffer
saline.
• Particles passing through a chamber in
single file scatter the light and convert by a
detector into pulses proportional to size of
the cells which are then counted
electronically.
 Advantages:
Easy and rapid method
Many thousand of cells are counted as compared to fewer cells counted
in manual method.
Disadvantages:
Costly equipment
Calibration error
Altered composition of diluent causes erroneous results.
Giant platelets are counted as RBCs.
High WBC count alters results.
https://youtu.be/iscB51whK9Q
 Reference range:
Adult male – 4.5 to 5.5 million/cumm
Adult female – 3.8 to 5.2 million /cumm
At birth – 6 to 8 million/cumm

Interpretation:
Conditions of increased RBC count:
Dehydration
Polycythemia
Smoking
Genetic causes
Pulmonary disease
New born and infants
Congenital heart diseases.
When you move to a higher altitude, your RBC count may increase for several weeks
because there’s less oxygen in the air.
Certain drugs can also increase your RBC count
gentamicin and methyldopa can increase your RBC count
 Gentamicin is an antibiotic used to treat bacterial infections in the
blood.
 Methyldopa is often used to treat high blood pressure.
Conditions of decreased RBC count:
Anemia
Less production and high destruction of RBCs
Pregnancy
bone marrow failure
erythropoietin deficiency, which is the primary cause of anemia in
patients with chronic kidney disease
Nutritional deficiency, like vitamin B12, iron and folic acid etc.
Certain drugs can also lower your RBC count
chemotherapy drugs
chloramphenicol, which treats
bacterial infections
quinidine, which can treat irregular
heartbeats
hydrations, which are traditionally
used to treat epilepsy and muscle
spasms
 How to increase RBCs cells:
There are two ways to increase RBCs count
1) Legal
maintaining a healthy diet and avoiding vitamin deficiencies
By training athletes at high altitude.
adding iron rich food (such as meat, fish, poultry).
increasing copper in your diet with foods like shellfish, poultry, and nuts
exercising regularly, which requires the body to use up more oxygen
getting more vitamin B12 with foods like eggs, meats, and fortified cereals
2) Illegal
By using Ethropiotin, androgen and their analogues they increases the
RBCs count.
 Reference
ABC_OF_CLINICAL_HAEMATOLOGY_Second_Editi.pdf
Oxford_Handbook_of_Clinical_Haematology.pdf
https://images.app.goo.gl/UNmMXn6xA4u1FD3N6
https://images.app.goo.gl/ymS2wqCBJj8Mtr2QA
https://images.app.goo.gl/2Qd5mX8zkGCARxpVA
https://images.app.goo.gl/cTeReiQS35YGrQ8LA
https://youtu.be/sDhsyh2wKxc
https://youtu.be/iscB51whK9Q