1

COLLECTION OF BLOOD

PRICK METHOD

2
 COLLECTION OF BLOOD
METHODS:
Blood can be collected by using two methods for Hematological studies

1. prick method
2. vein puncture method

PRICK METHOD:
Blood may be obtained by pricking the finger or ear lobe for adults, great toe for infants
for pricking lancet of 3mm depth was used. The area selected for pricking should have not had
any edema (or) congestion. The area selected should be free from dirt, oil and grease. The area
should be wiped with %spirit (or) alcohol. The first drop is wiped off because the blood contains
tissue fluids. Then the blood can be collected for Hematological test. After collecting the
required amount of blood, clean cotton is pressed on the pricked area.

A separate lancet should be used for each patient because the use of same lancet for
another patient may lead to infection n such as AIDS, Hepatitis B etc.

3
VEIN PUNCTURE METHOD

4
VEIN PUNCTURE METHOD:
Puncturing of the vein to obtain larger quantity of blood by using syringe and needle is
known as vein puncture. Anticubital vein in the forearm is selected for puncture.

The upper part of the arm is constricted by twisting a tourniquet of rubber taping
around it, or placing a blood pressure cuff and inflating it to about 40mm Hg. The patients
forearm is rested. On, cleaned with 90% spirit with cotton. Sterilized 22 gauge needle is filter
into the syringe and inserted at an angle of about 30^c into the anticubital vein. The piston is
withdrawn to such the blood. After obtaining the required amount of blood, the tourniquet is
removed first, the cotton is kept on the needle, then the needle and syringe was withdrawn
back by pressing the cotton and the patient was asked to fold the elbow.

5
ESTIMATION OF HAEMOGLOBIN

SAHLIS’ METHOD

6
 ESTIMATION OF HAEMOGLOBIN
AIM:
To estimate the amount of Hemoglobin present in the given sample.

METHODS:
Sahli’s method (or) Acid Haematin method.

PRINCIPLE:
Hemoglobin is converted into acid haematin by the addition of N/10 (or) 0.1 N HCl and
the resulting brown colour is compared with the colour comparator.

REQUIREMENTS:
Haemometer set, blood lancet, spirit soaked cotton, blood, N/10 HCl and distilled water.

PROCEDURE:
Take N/10 HCl till the lowest ‘2’ mark of the graduated tube. Then add 0.02 ml of blood
and mix well and then kept for 10 minutes. Distilled water is added drop to the graduated tube
to make the colour suitable with the comparator. When the colour of the solution is the
graduated tube matches with the comparator and the reading in the graduated tube is noted.

RESULT:
The given blood sample contains 10.3 gms/dl.

NORMAL RANGE:
1. Men - 13 - 16 Gms % (average 14.5 Gms %)
2. Women - 12 - 15 Gms % (average 13.5 Gms %)
3. At Birth - 17 - 20 gms %
4. 1 to 2 years - 11 - 12 gms %

7
8
CLINICAL SIGNIFICANCE:
Increased amount of Hemoglobin.

 Haemoconcentration
 Congenital heart disease

Decreased amount of Hemoglobin.

 Haemodilution
 After hemorrhage

9
ESTIMATION OF HAEMOGLOBIN

10
 ESTIMATION OF HAEMOGLOBIN
AIM:
To estimate the amount of Hemoglobin present in the given sample.

METHOD:
Cyarmethaemoglobin method.

REQUIREMENTS:
Colorimeter, sahli’s pipette, 5 ml pipette, test tube, lancet, spirit soaked cotton (or) 70%
alcohol and Drabkin’s solution.

PRINCIPLE:
Hemoglobin is converted into methaemoglobin in the presence of potassium ferric
cyanide. Methaemoglobin is converted into cyanomethaemoglobin in the presence of
potassium cyanide.

TEST PROCEDURE:
Take 5 ml of Drabkin’s solution in a clean test tube add 0.02 ml (or) 20 µl of patients
whole blood. Mix well and wait for 5 minutes. Take reading of test solution using green filter by
adjusting the optical density to zero with Drabkin’s solution.

STANDARD CYANMETHAEMOGLOBIN:
Standard is provided in the kit and having a concentration of 5.0 mg/dl. This readymade
standard solution is read directly on a colorimeter using green filter.

RESULT:
The given blood sample contains 12.04 g/dl of hemoglobin.

NORMAL VALUE:

1. Men - 13 -16 gms % (average 14.5 gms %)

2. Women - 12 - 15 Gms % (average 13.5 Gms %)
3. At Birth - 17 - 20 gms %
4. 1-2 years – 11 - 12 gms %

11
CALCULATION:
𝑜𝑝𝑡𝑖𝑐𝑎𝑙 𝑑𝑒 𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑡𝑒𝑠𝑡
= 𝑜𝑝𝑡𝑖𝑐𝑎𝑙 × 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟.
𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

0.29
= 0.36 × 0.06 × 251

=0.80 × 0.06 × 251

=12.04 g/dl.

12
CLINICAL SIGNIFICANCE:
Increased amount of hemoglobin.

 Haemoconcentration
 Congenital heart disease

Decreased amount of Hemoglobin.

 Haemodilution
 After Hemorrhage.

13
HAEMOCYTOMETER

14
 HAEMOCYTOMETER
IMPROVED NEUBAUER COUNTING CHAMBER:
The counting chamber is used to count various blood cells, spermatozoa, total count of
RBC, WBC, Platelet and eosinophil. It is also called Haemocytometer.

The total area of counting chamber is 3×3 mm, 9 square mm. It is divided into 9
squares. The corner squares are divided into 16 smaller squares. The central squares area is
divided into 25 squares. Each of these squares is further divided into 16 small squares (i.e.)
25*16= 400 smaller squares.

 4 corner squares – Total WBC count (Low power)

 5 central small squares – Total RBC count (High power)
 5 central small squares – platelet count (High power)
 9 squares are used – Absolute eosinophil count ( low power)
 9 squares are also used for cavity fluids, csf, sperm count under high power

COVERSLIP OR COVERGLASS:
The cover glass has very smooth surface and even thickness. Area of cover glass is 22*23
mm.

Thickness of cover slip is 0.3 mm, 0.4 mm and 0.5 mm. 0.4 mm thickness is commonly
used for all total count.

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TOTAL RED BLOOD CELL COUNT

DIRECT METHOD

16
 TOTAL RED BLOOD CELL COUNT
AIM:
To find out the number of RBC’s present in the given blood sample.

METHOD:
Direct method

REQUIREMENTS:
Haemocytometer set, RBC pipette, RBC diluting fluid, Blood lancet, spirit soaked cotton (or) 70
% alcohol and microscope.

PRINCIPLE:

FORMAL CITRATE SOLUTION:

Trisodium citrate – acts as anticoagulant.
Formalin – preserves cell morphology.

TEST PROCEDURE:
Withdraw the patient’s blood upto 0.5 mark using RBC pipette. Immediately aspirate
RBC diluting fluids upto 101 mark, mix well and wait for 5 minutes. This gives 1/200 dilution.
After discarding the stem fluids, charge the mixer into the chamber. By holding the tip of the
RBC pipette as an angle of 45°C. then the chamber is kept under moist temperature for 5 – 10
minutes.

OBSERVATION:
RBC’s were observed and counted in central heavy ruled squared area of four outer
corner and one centre square under 45* objectives of microscope.

RESULT:
The given blood sample contains 5.5 millions cells/ cu mm.

17
CALCULATION:
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 × 𝐷𝑒𝑝𝑡 ℎ 𝑓𝑎𝑐𝑡𝑜𝑟
= 𝑇𝑜𝑡𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐴𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

550×200×10
= 1/5

=5.5 millions cells/ cu mm.

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NORMAL VALUE:
4.5 – 6.5 million cells/cu.mm.

CLINICAL SIGNIFICANCE:
Increased number of RBC’s (Erythrocytosis)

 Haemoconcentration
 Chronic heart disease

Decreased number of RBC’s (Erythrocytopenia)

 Haemodilution
 Anaemia

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TOTAL RED BLOOD CELL COUNT

BULK DILUTION METHOD

20
 TOTAL RED BLOOD CELL COUNT

AIM:
To find out the number of RBC’s present in the given blood sample.

METHOD:
Bulk dilution method

REQUIREMENTS:
Haemocytometer set, RBC diluting fluid, blood, lancet, spirit soaked cotton (or) 70%
alcohol, test tube, sahlis pipette, 5 ml pipette and microscope.

PRINCIPLE:

FORMAL CITRATE SOLUTION:

Trisodium citrate – act as anticoagulant

Formalin – preserves the cells morphology.

PROCEDURE:
3.98 ml of RBC diluting fluid and 0.02 ml (or) 20 µl of whole blood is mixed well and kept
for 2 -3 minutes. Again mixed well then charged into the counting chamber which was covered
with coverslip. Then the charged chamber is kept under moist temperature for 5 -10 minutes.

OBSERVATION:
RBC’s were observed and counted in central heavy ruled square area of four outer
corner squares and one center square under 45x objective of microscope.

RESULT:
The given blood sample contains_5.86 million cells/ cu mm.

NORMAL VALUE:
4.5 – 6.5 million cells/ cu.mm.

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FORMULA:
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ×𝐷𝑒𝑝𝑡 ℎ 𝑓𝑎𝑐𝑡𝑜𝑟
= .
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

586×200×10
= 1/5

=1172000/0.2

=5860000

=5.86 million cells/ cu mm.

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CLINICAL SIGNIFICANCE:
Increased number of RBC’s (Erythrocytosis)

 Haemoconcentration
 Chronic heart disease.

Decreased number of RBC’s (Erythrocytopenia)

 Haemodilution
 Anaemia.

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TOTAL LEUCOCYTE COUNT

DIRECT METHOD

24
 TOTAL LEUCOCYTE COUNT
AIM:
To find out the number of WBC’s present in the given blood sample.

METHOD:
Direct method.

REQUIREMENT:
Haemocytometer set, WBC pipette, WBC diluting fluid, blood, lancet, spirit soaked
cotton (or) 70% alcohol and microscope.

PRINCIPLE:

TRUCK’S FLUID:
Glacial acetic acid – lyses RBC’s and platelets

Violet – stains the WBC’s

PROCEDURE:
Withdraw rhea patient’s blood up to 0.5 mark using WBC pipette and immediately
aspirate WBC diluting fluid up to 101 mark. Mix well and kept for 5 minutes, this gives 1/20
dilution.

After discarding the stem fluid, charge the chamber which has been covered with cover
glass be holding the tip of the WBC pipette at an angle of 45°C.

OBSERVATION:
WBC’s were observed and counted is four corner squares under 10× objective of
microscope.

RESULT:
The given blood sample contains 7600 cells/cu mm.

NORMAL RANGE:
4500 – 10,000 cells/cu.mm.

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FORMULA:
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ×𝐷𝑒𝑝𝑡 ℎ 𝑓𝑎𝑐𝑡𝑜𝑟
= 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

152×20×10
= 4

=7600 cells/cu mm.

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CLINICAL SIGNIFICANCE:
Increased number of WBC’s (Leucocytosis)

 Muscular exercise
 Leukemia

Decreased number of WBC’s (Leucocytopenia)

 Bacterial and viral infection

 Anaemia

27
TOTAL LEUCOCYTE COUNT

BULK DILUTION METHOD

28
 TOTAL LEUCOCYTE COUNT
AIM:
To find out the number of WBC’s present in the given blood sample.

METHOD:
Bulk dilution method.

REQUIREMENT:
Haemocytometer set, sahlis pipette, 1 ml pipette, test tube, WBC diluting fluid, blood,
lancet, spirit soaked cotton (or) 70% alcohol and microscope.

PRINCIPLE:

TRUCK’S FLUID:
Glacial acetic acid – lyses RBC and platelets

Violet – stains the WBC’s

PROCEDURE:
0.38 ml of diluting fluid and 0.02 ml of whole blood is mixed well and kept for 2 – 3
minutes. Again mixed well then charged into the counting chamber which was covered with
cover glass then the 4 corner squares are counted under low power.

OBSERVATION:
WBC’s were observed and counted is four corner squares under 10× objective of
microscope.

RESULT:
The given blood sample contains 7400 cells /cu mm.

NORMAL RANGE:
4500 – 10,000 cells/cu.mm.

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FORMULA:
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ×𝐷𝑒𝑝𝑡 ℎ 𝑓𝑎𝑐𝑡𝑜𝑟
= 𝑇𝑜𝑡𝑎 𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

148×20×10
= 4

=29600/4

=7400 cells/cu mm.

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CLINICAL SIGNIFICANCE:
Increased number of WBC’s (Leucocytosis)

 Muscular exercise
 Leukemia

Decreased number of WBC’s (Leucocytopenia)

 Bacterial and viral infection

 Anaemia

31
TOTAL PLATELET COUNT

BULK DILUTION METHOD

32
 TOTAL PLATELET COUNT
AIM:
To find out the number of platelets present in the given blood sample.

METHOD:
Bulk dilution method.

REQUIREMENT:
Haemocytometer set, RBC pipette, platelet diluting fluid, blood, lancet, spirit soaked
cotton (or) 70% alcohol and microscope, test tube, sahlis pipette.

PRINCIPLE:

DACIE’S FORMAL CITRATE SOLUTION:

Tri sodium citrate – act as anticoagulant

Formalin – preserves cell morphology

Brilliant cresyl blue.

PROCEDURE:
1.98 ml of diluting fluid and 0.02 ml (or) 20 ml of blood was added to a test tube. Mixed
well and kept for 2 – 3 minutes. Again mixed well and charged into a counting chamber covered
with coverslip. The charged chamber was kept under moist temperature for 5 – 10 minutes.

OBSERVATION:
Platelets were observed and counted in center heavy ruled square area of four outer
corners and one center square under 45× objective of microscope.

RESULT:
The given blood sample contains 140000 cells/ cu mm.

NORMAL RANGE:
1.0 – 3.0 lakhs cells/cu.mm.

33
FORMULA:
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ×𝐷𝑒𝑝𝑡 ℎ 𝑓𝑎𝑐𝑡𝑜𝑟
= 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

140×100×10
= 1

=140000 Cells / cu mm.

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CLINICAL SIGNIFICANCE:
Increased number of platelets (thrombocytosis)

 Allergic conditions
 Hemorrhage

Decreased number of platelets (thrombocytopenia)

 Scarlet fever
 Acute infection.

35
TOTAL PLATELET COUNT

DIRECT METHOD

36
 TOTAL PLATELET COUNT
AIM:
To find out the number of platelets present in the given sample of blood.

METHOD:
Direct method.

REQUIREMENT:
Haemocytometer set, RBC pipette, platelet diluting fluid, blood, lancet, spirit soaked
cotton (or) 70% alcohol and microscope.

PRINCIPLE:

DACIE’S FORMAL CITRATE SOLUTION:

Tri sodium citrate – act as anticoagulant

Formalin – preserves cell morphology

Brilliant cresyl blue.

PROCEDURE:
With draw the patient blood up to 0.5 marks in the RBC pipette. Immediately aspirate
platelet diluting fluid up to 101 marks. Mixed well and kept for 5 minutes. This gives 1/200
dilution. After discarding the stem fluid and charged into the counting chamber which was
covered with coverslip by the holding the tip of the RBC pipette at angle of 45°C. Then charged
chamber was kept under moist temperature for 15 minutes.

OBSERVATION:
Platelets were observed and counted in center heavy ruled square area of four outer
corners square and one center square under 45× objective of microscope.

RESULT:
The given blood sample contains 160000 cells/ cu mm.

37
FORMULA:
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ×𝐷𝑒𝑝𝑡 ℎ 𝑓𝑎𝑐𝑡𝑜𝑟
= 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

160×100×10
= 1

=1.60000 cells/ cu mm.

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NORMAL RANGE:
1.0 – 3.0 lakhs cells/cu.mm.

CLINICAL SIGNIFICANCE:
Increased number of platelets (Thrombocytosis)

 Allergic conditions
 Hemorrhage

Decreased number of platelets (Thrombocytopenia)

 Scarlet fever
 Acute infection.

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ABSOLUTE EOSINOPHIL COUNT

DIRECT METHOD

40
 ABSOLUTE EOSINOPHIL COUNT
AIM:
To find out the number of eosinophils present in the given blood sample.

METHOD:
Direct method.

REQUIREMENTS:
Haemocytometer set, WBC pipette, Eosinophil diluting fluid, blood, lancet, spirit soaked
cotton (or) 70 alcohol and microscope.

PRINCIPLE:

HINGLE MAN’S SOLUTION:

Yellow eosin stains the eosinophilic granules

Phenol – lyses RBC’S, platelets and other WBC’S

Formalin – preserves cells morphology

PROCEDURE:

Withdraw the patient’s blood up to 0.5 marks in the WBC pipette. Immediately aspirate
eosinophil diluting fluid up to 11 marks. Mixed well and waited for 5 minutes. This gives 1/20
dilution.

After discarding the stem fluid charge the chamber which has been covered with
coverslip by holding the tip of the WBC pipette at an angle of 45°C. Then the charged chamber
is kept under moist temperature for 5 minutes.

OBSERVATION:

Eosinophils were observed and counted at all the 9 squares under 10x objective of
microscope.

41
FORMULA:
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ×𝐷𝑒𝑝𝑡 ℎ 𝑓𝑎𝑐𝑡𝑜𝑟
= 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

= 120 cells/ cu mm.

42
RESULT:
The given blood sample contains 120 cells/cu mm.

NORMAL RANGE:
40 – 440 cells/cu.mm.

CLINICAL SIGNIFICANCE:
Increased number of Eosinophils (Eosinophilia)

 Asthma
 Parasitic infection.

43
ABSOLUTE EOSINOPHIL COUNT

BULK DILUTION METHOD

44
 ABSOLUTE EOSINOPHIL COUNT
AIM:
To find out the number of eosinophils present in the given blood sample.

METHOD
Bulk dilution method.

REQUIREMENTS:
Haemocytometer set, sahlis pipette, 1ml pipette, test tube, Eosinophil diluting fluid,
blood, lancet, spirit soaked cotton (or) 70 alcohol and microscope.

PRINCIPLE:

HINGLE MAN’S SOLUTION:

Yellow eosin stains the eosinophilic granules

Phenol – lyses RBC’S, platelets and other WBC’S

Formalin – preserves cells morphology

PROCEDURE:

0.36 ml of diluting fluid and 0.04 ml of whole blood was added to a test tube. Mixed well
and kept for 2 – 3 minutes. Again mixed well and then charged into a counting chamber which
was covered by a cover slip.

OBSERVATION:

Eosinophils were observed and counted at all the 9 squares under 10x objective of
microscope.

RESULT:
The given blood sample contains 100 cells /cu mm.
45
FORMULA:
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ×𝐷𝑒𝑝𝑡 ℎ 𝑓𝑎𝑐𝑡𝑜𝑟
= 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑟𝑒𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

= 100 cells/ cu mm.

46
NORMAL RANGE:
40 – 440 cells/cu.mm.

CLINICAL SIGNIFICANCE:
Increased number of Eosinophils (Eosinophilia)

 Asthma
 Parasitic infection.

47
SMEAR MAKING AND STAINING

48
 SMEAR MAKING AND STAINING
AIM:
To find out the blood components (RBC’S, WBC’S and Platelets) and blood parasites in a
given patient’s blood sample.

REQUIREMENTS:
Slides, spreader, Pasteur pipette, blood, lancet, spirit soaked cotton (or) 70% alcohol,
Leishman’s stain, buffer water (PH – 6.8) and microscope.

PRINCIPLE:

LEISHMAN’S STAIN:
Methylene blue – Basic dye

Eosin – acidic dye

Methanol – acts as fixative.

The basic components of the cells are stained by acidic dye. So, it is called acidophilic;

The acidic components of the cells are stained by basic dye. So, it is called basophilic.

The neutral components of the cells are stained by both of the dyes.

PROCEDURE:

SMEAR MAKING:
Take a clean grease fuce glass slide, place a small drop of blood near the edge of the
slide place the spreader slide at an angle of 30° - 35° then pull back of the spreader until it
touches the drop of blood. Let the blood run along the edge of the spreader forward to the end
of the slide with a smooth movement dry the blood smear at room temperature.

IDENTIFICATION MARKING:
By using lead pencil (or) ball pen write the patients ID number on the slide.

49
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STAINING:
A well dried smear is taken and kept on staining rods then poured the leishman stain
and kept for 2 minutes for fixation of cells. Then dilute it with the double the amount of buffer
water and kept for 10 minutes and then washed with tap water and dried. The dried slide is
observed under low power objective of microscope and then turned to oil immersion objective
and the cells are counted on the area where the RBC is not touching each other. In this area
WBC’s are clear morphology. Total WBC’s have a clear morphology in are counted and number
of each type of white cells are expressed in percentage.

51
DIFFERENTIAL LEUKOCYTE COUNT

52
 DIFFERENTIAL LEUKOCYTE COUNT
AIM:
To find out the percentage of Neutrophil, Eosinophil, Basophil, Lymphocyte, Monocyte
in the given blood smear.

METHOD:
BREADTH wise method.

REQUIREMENTS:
Stained slides, microscope, oil, Differential cell counter.

PRINCIPLE:

LEISHMAN’S STAIN:
Methylene blue – Basic dye

Eosin – acidic dye

Methanol – acts as fixative

The basic components of the cells are stained by acidic dye. So, it is called acidophilic.

The acidic components of the cells are stained by basic dye. So, it is called basophilic.

The neutral components of the cells are stained by both the dyes.

PROCEDURE:
A smear was made dried and examined under low power objective of microscope and
then turned to oil immersion objective cells are counted on the area where the RBC’s are not
touching each other. WBC’s also having a clear morphology in this area. WBC’s are counted and
number of each type of white cells is expressed in percentage.

NEUTROPHIL:
Size 10 – 12 µ, contains 3 lobes cytoplasm contains pink colour granules.

BASOPHIL:
53
Size 8 – 10 µ, contains kidney shaped nucleus, purplish black colour granules.

54
LYMPHOCYTES:

LARGE LYMPHOCYTES:
Size 12 – 15 µ and even smaller, cytoplasm of T- lymphocytes may contain non- specific
granules.

SMALL LYMPHOCYTES:
Size 7 – 10 µ in size.

MONOCYTE:
Large cell has a size of 16 – 22 µ, irregular shape.

EOSINOPHIL:
Size 10 – 12 µ contains two lobed nucleus, cytoplasm contains thick coarse granules
which are orange colour.

OBSERVATION:
Count 100 WBC’s in the smear under 100x objective of microscope.

NORMAL VALUE:

 Neutrophil – 40 – 75%
 Eosinophil – 01 – 06%
 Basophil – 00 – 01%
 Lymphocyte - 20 – 45%
 Monocyte - 02 – 10%

RESULT:

 Neutrophil – 55 %
 Eosinophil – 05%
 Basophil – 00%
 Lymphocyte – 35%
 Monocyte - 05%

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CLINICAL SIGNIFICANCE:

Increased number of Neutrophil (Neutrophilia)

 Acute infection
 Metabolic disorder

Decreased number of Neutrophil (Neutropenia)

 Viral infection
 A plastic anemia

Increased number of Eosinophil (Eosinophilia)

 Blood parasitism
 Scarlet fever

Increased number of Basophil (Basophilia)

 Small pox
 Polycythemia vera
Increased number of Monocyte (Monocytosis)
 Tuberculosis
 Malaria

Increased number of Lymphocyte (Lymphocytosis)

 Infectious mononucleosis
 Malnutrition

57
BLOOD PICTURE

58
 BLOOD PICTURE

AIM:

To find out the morphological characteristics of RBC’s, WBC’s and abnormalities in the
given blood sample.

METHOD:

Breadth wise method

REQUIREMENTS:

Stained slides, microscope, oil and Differential cell counter.

PRINCIPLE:

LEISHMAN’S STAIN:

Methylene blue – Basic dye

Eosin – Acidic dye

Methanol – acts as fixative

The basic components of the cells are stained by acidic dye. So, it is called acidophilic.

The acidic components of the cells are stained by basic dye. So, it is called basophilic.

The neutral components of the cells are stained by both the dyes.

PROCEDURE:

By examining the blood film it is possible to estimate the number of each cellular
element and to study the morphology of cells and to identify blood parasites. It is also used to
study the response of the body to various diseases and the progress of treatment.

Distribution and staining of the cells and microfilaria

59
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UNDER HIGH POWER:

Estimate the number of white cells.

UNDER OIL IMMERSION:

RBC’S SIZE:

RBC’s size is measured with the help of micrometer.

NORMOCYTES:

Normal RBC’s having a size of about 7.2 micron.

MICROCYTES:

Less than 6 micron in size of RBC’s.

MACROCYTES:

More than 8 micron in size of RBC’s.

ANISOCYTES:

RBC’s having various sizes (or) different sized RBC’s.

SHAPE:

SPHEROCYTES:

RBC’s which are not in biconcave shape and does not possess central pallor area and
have small surface area.

TARGET CELLS:

These RBC’s have a centrally stained area.

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SICKLE CELL:

RBC’s are in the shape of sickle or crescent.

OVALOCYTES:

RBC’s are oval in shape.

ELLIPTOCYTES:

More oval than ovalocytes.

ACANTHOCYTES:

These types of RBC’s have irregular shape and have thorn like projection on its outer
surface.

BURR CELLS/ ECHINOCYTES:

These RBC have blunt projections which are numerous in number and every spaced
around the circumference.

CRESENT BODIES:

Ruptured RBC’s having the shape of quatermoon.

CRENATED CELL:

RBC’s having irregular outer edges.

POIKILOCYTOSIS:

RBC’s having various shapes.

COLOUR:

HYPERCHROMIA:

Fully haemoglobinized cell.

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HYPOCHROMIA:

Decrease in the hemoglobin concentration of RBC.

ANISOCHROMIA:

Both hyper and hypochromia cells are present.

RBC INCLUSIONS:

BASOPHILIC STIPPLING: (RNA)

RBC’s having numerous purple coloured granules that are dispersed throughout the
cytoplasm.

HOWELL – JOLLY BODIES: (DNA)

Small round cytoplasmic red cell inclusion. These bodies are DNA that stain dark purple
within the red blood cell.

SIDEROCYTES:

These RBC’s containing non hemoglobin iron granules that stains purple.

CABOT RING:

These RBC’s contain reddish – blue threadlike ring or has figure 8 configuration.

These are remnants of the nuclear membrane or microtubules.

ABNORMALITIES OF WBC’S:

TOXIC GRANULATION:

Neutrophil contain prominent granules. It is due to severe bacterial infection.

FOOD VACULATION:

Occurrence of vacuoles in the cytoplasm.

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HYPER SEGMENTATION:

More than 5 lobes are present in Neutrophil.

HYPO SEGMENTATION:

Failure of normal lobe development in Neutrophil.

AUER RODS/ AUER BODIES:

Auer rods (cytoplasmic inclusion) are needle shaped structures found in the cytoplasm
of myeloid cells and indicate acute myeloid leukemia.

DOHLE BODIES:

They are small round (or) oval blue cytoplasmic inclusion that is remnants of rough
endoplasmic recticulum.

SMUDGE CELLS/ BASKET CELLS:

They are degenerated lymphocyte and are seen in chronic lymphocytic leukemia.

PLATELETS:

8 – 25 cells/ field in 10 field – adequate

0 – 5 cells/ field in 10 field – inadequate.

67
RECTICULOCYTE COUNT

68
 RECTICULOCYTE COUNT

AIM:

To find out the number of Recticulocytes present in the given blood sample.

METHOD:

 Wet preparation
 Dry preparation

REQUIREMENTS:

Slide, spreader, Pasteur pipette, test tube, Recticulocyte diluting fluid, blood lancet,
spirit soaked cotton (or) 70% alcohol and microscope.

PRINCIPLE:

The recticulocyte count is based on the property of ribosomal RNA to react with isotonic
solution of supravital stain which stains living material. For the detection of ribosomal
RNA in Recticulocytes they should be fixed. Blood is mixed with the stain and incubated.
The RNA in the cell gets precipitated as dark blue network or Recticulum.

PROCEDURE:

DRY METHOD:

In a dry test tube 2 – 3 drops of blood and then 2 – 3 drops of supra vital stain mixed
well and then incubate at 37°C for 20 minutes. Then make a thin smear in a glass slide using
spreader. Dry the smear and view under oil immersion objective of microscope 1000 RBCs are
counted and number of Recticulocytes among them are noted.

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WET METHOD:

As in dry method, the blood and stain solution is mixed and incubated. From this a drop
of solution is placed in a glass slide and covered with a coverslip the number of Recticulocytes
among 1000 RBCs is noted.

OBSERVATION:

Recticulocytes were observed and counted among 1000 RBCs under 100x objective of
microscope.

RESULT:

The percentage of Recticulocytes present in the given blood sample 0.7 %.

NORMAL VALUE:

Adult – 0.5 – 2.0 %

Infants – 2.6 %

CLINICAL SIGNIFICANCE:

Increased number of Recticulocytes (recticulocytosis)

Decreased number of Recticulocytes (recticulocytopenia)

71
ERYTHROCYTE SEDIMENTATION RATE

WINTROBES’ METHOD

72
 ERYTHROCYTE SEDIMENTATION RATE

AIM:

To determine the Erythrocyte sedimentation rate of the given blood sample.

METHOD:

Wintrobe’s method.

REQUIREMENTS:

Wintrobe’s tube, capillary Pasteur pipette, ESR stand and blood sample.

PRINCIPLE:

When anti coagulated blood is allowed to stand undisturbed for a period of time. The
erythrocytes tend to sink to the bottom. Two layers are formed the upper plasma layer and the
lower RBC’s. The rate at which the erythrocyte falls is known as erythrocyte sedimentation rate.

PROCEDURE:

Well mixed anticoagulated blood is taken and filled into the wintrobe’s tube with the
help of capillary Pasteur pipette. The tube is labeled and placed in the exact vertical position on
the stand.

OBSERVATION:

Read the plasma level after 30 minutes and 60 minutes and the result is expressed in
mm.

RESULT:

The erythrocyte sedimentation rate of the given blood sample 17 mm.

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NORMAL RANGE:

 Men - 0 – 9 mm
 Women - 0 – 20 mm

CLINICAL SIGNIFICANCE:

Increase in erythrocyte sedimentation rate

 Chronic infections
 Inflammatory diseases.

75
ERYTHROCYTE SEDIMENTATION RATE

WESTERGREN’S METHOD

76
 ERYTHROCYTE SEDIMENTATION RATE

AIM:

To determine the Erythrocyte sedimentation rate of the given blood sample.

METHOD:

Westergren’s method.

REQUIREMENTS:

Westergren’s pipette, ESR stand, 1 ml pipette, saline, test tube, blood sample and 3.8%
Trisodium citrate.

PRINCIPLE:

When anticoagulated blood is allowed to stand undisturbed for a period of time. The
erythrocytes tend to sink to the bottom. Two layers are formed the upper plasma layer and the
lower RBC’s. The rate at which the erythrocyte falls is known as erythrocyte sedimentation rate.

PROCEDURE:

WITHOUT ANTICOAGULATED BLOOD:

0.4 ml of Trisodium citrate and 1.6 ml of blood was taken in a test tube and mixed well.

ANTICOAGULATED BLOOD:

0.4 ml of saline and 1.6 ml of blood was taken in test tube and mixed well then the
mixed solution was sucked into the westergren’s pipette up to ‘0’ mark with the help of rubber
bulb. Then fix the Westergren’s pipette in ESR stand in vertical position.

OBSERVATION:

For one hour, 20 minutes once read the result plasma level and the result is expressed
in mm.

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RESULT:

The erythrocyte sedimentation rate of the given blood sample 15 mm.

NORMAL RANGE:

 Men - 0 – 15 mm
 Women - 0 – 20 mm

CLINICAL SIGNIFICANCE:

Increase in erythrocyte sedimentation rate

 Chronic infections
 Inflammatory diseases.

79
PACKED CELL VOLUME

80
 PACKED CELL VOLUME

AIM:

To determine the value of packed cell volume of the given blood sample.

METHOD:

Wintrobe’s method.

REQUIREMENTS:

Wintrobe’s tube, capillary Pasteur pipette, blood sample and centrifuge.

PRINCIPLE:

When anticoagulated blood in hematocrit tube is centrifuged at high speed, the RBC’s
settle at the bottom of the tube leaving the plasma at the top. PVC is defined as the volume
occupied by erythrocytes in a given volume of blood and is usually expressed as a percentage of
the volume of the whole blood sample.

PROCEDURE:

Well mixed anticoagulated blood is filled in the wintrobe’s tube with the help of
capillary Pasteur pipette without any air bubbles. Then the label the tube and place it in
centrifuge and balance the opposite side and centrifuged for 30 minutes at 3000 rpm. After
centrifugation three layers are formed. The bottom layer consists of erythrocytes and the
middle layer is a thin layer consists of white blood cells and platelets known as Buffy coat. The
upper layer consists of plasma.

OBSERVATION:

Note the level of RBC’s in the Wintrobe’s tube and it is expressed in percentage (%).

RESULT:

The packed cell volume of the given blood sample is 50 %.

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NORMAL RANGE:

 Men - 40 – 50 %
 Women - 36 – 47 %

CLINICAL SIGNIFICANCE:

Increase in packed cell volume – Polycythemia

 Haemoconcentration
 Congenital heart disease.

Decrease in packed cell volume – Anemia

 Haemodilution
 After Hemorrhage.

83
BLEEDING TIME

DUKE’S METHOD

84
 BLEEDING TIME
AIM:

To determine the bleeding time of the patient.

METHOD:

Duke’s method

REQUIREMENT:
Lancet, filter paper, stops watch, spirit and cotton.

PRINCIPLE:
A 1mm deep incision is made on the earlobe (or) patients’ finger. The length of time
required for bleeding to cease is recorded.

PROCEDURE:
Cleaned the ear lobe (or) finger with spirit cotton allow to dry and puncture the ear lobe
or finger by using lancet. Start the stop watch. The blood should flow freely without squeezing
the earlobe or finger 15 (or) 30 seconds. Collect the drops of blood in a filter paper. Do not
touch the skin (or) wound. After reading the step for every 15 – 30 seconds, when bleeding
seems to stop. Stop the stopwatch and note the time.

OBSERVATION:
When no blood stain was seen on the filter paper after a gentle touch. Stop the stop
watch.

RESULT:

The bleeding time of the patient by duke method 1 min 30 sec.

NORMAL RANGE:

1 – 5 minutes.

85
BLEEDING TIME

IVY METHOD

86
 BLEEDING TIME

AIM:
To determine the bleeding time of the patient.

METHOD:
Ivy method.

PRINCIPLE:
A standard incision was made on the skin of the patient and the length of the time
required for bleeding to stop is recorded.

REQUIREMENTS:

Sphygmomanometer, lancet, spirit, cotton and filter paper.

PROCEDURE:

Place the sphygmomanometer cuff on the patients’ arm above the elbow and inflate to
40 mm Hg and hold choose an area finger wide below the bend in the elbow on the volar
surface and clean the area with alcohol, stretch the skin laterally between the thumb and fore
finger and hold in a tout position.

Take a disposable lancet with point about 3 mm in length. Hold the patient and quickly
make two or three skin punctures in the cleansed area of the patients’ fore arm. The wound
should be deep in depth and 2 – 3 mm wide. A stop watch is started at the time of the first skin
puncture was made gently touch the drop of blood which comes over the wound for every 30
seconds with a filter paper. Do not rub the sheet.

OBSERVATION:

The bleeding time is reported as the time when no blood stain is seen on the filter paper
after gentle touch and stop the stopwatch.

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RESULT:

The bleeding time of the patient by Ivy method 1 min.

NORMAL RANGE:

2 – 6 minutes.

CLINICAL SIGNIFICANCE:

Prolonged bleeding time occurs in

 Thrombocytopenia
 Von willebrand disease
 Factor VIII deficiency.

89
CLOTTING TIME

90
 CLOTTING TIME

AIM:

To determine the doing time of the patient.

METHOD:

Capillary method.

REQUIREMENTS:

Lancet, capillary pipette, spirit, cotton and stopwatch.

PRINCIPLE:

Blood is collected in a capillary pipette by a finger prick and the stopwatch is started.
The formation of fibrin string is noted by breaking the capillary pipette at regular intervals.

PROCEDURE:

Clean the finger tip with spirit and cotton allow to dry. A deep incision with a sterile
lancet was made and the stopwatch was started. First drop of blood was wiped and the blood
was collected in a capillary pipette. After that 1 cm capillary pipette was broken for every 30
seconds.

OBSERVATION:
When the fibrin string appears, stop the stopwatch and note the time.

RESULT:
The clotting time of the patient 5 min .

NORMAL RANGE:
4 – 9 minutes.

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CLINICAL SIGNIFICANCE:

Prolonged clotting time indicates

 Mild to moderate deficiency of plasma factor

 Severe clotting disorder

93
 CLOTTING TIME

MODIFIED LEE AND WHITE METHOD

94
 CLOTTING TIME

AIM:

To determine the clotting time of the patient.

METHOD:
Modified lee and white method (Double syringe method).

REQUIREMENTS:
Sphygmomanometer, test tube, 2 syringes, needle, water bath, spirit, cotton.

PRINCIPLE:
Venous blood is collected in a clean and dry test tube without anticoagulant required for
the clotting of the blood.

PROCEDURE:
This method was performed using 2 syringes. So it is called two syringe techniques. First
1 ml of blood was drawn into the first syringe, and then the syringe was detached from the
needle without disturbing the position of the point of the needle within the lumen of the vein.
A second syringe is promptly attached and blood is drawn into it. 1 ml of blood was taken in
two test tubes and the tubes were compulsory kept in water bath at 37°C.

OBSERVATION:
Observe for the clotting of blood in the test tube and stop the stopwatch.

RESULT:
The clotting time of the given blood sample 6 min 30 sec.

NORMAL RANGE:
5 – 12 minutes.

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CLINICAL SIGNIFICANCE:

Prolonged clotting time indicates

 Mild to moderate deficiency of plasma factor

 Severe clotting disorder

97
PROTHROMBIN TIME

QUICKS’ METHOD

98
 PROTHROMBIN TIME

AIM:

To determine the prothrombin time of the given blood sample.

METHOD:
Quicks’ method.

REQUIREMENTS:
Water bath, stop watch, test tube, brain thromboplastin,0.15 g/dl and calcium chloride.

PRINCIPLE:

Thrombokinase preparation (thromboplastin preparation of human or rabbit brain)

containing calcium ions is added to citrated plasma. In the presence of factor VII stage 2 of
coagulation mechanism is triggered and the clotting time is recorded after the addition of
thrombokinase in the presence of calcium ions. Since factors XII, XI, VIII and platelets are
bypassed. The test depends upon the activity of factor VI, V, X and I. deficiency of any of these
factors may cause prolonged time for clot formation in this test.

PROCEDURE:

0.1 ml of thromboplastin and 0.1 ml by calcium chloride into a test tube & mixed well
then kept in waterbath at 37°C for 1 minute then 0.1 ml of the test plasma which was brought
to 37°C in a waterbath. The prewar med plasma is added to the test tube containing
thromboplastin calcium mixture then the stopwatch is started. After few seconds of gentle
mixing of the tube, held in waterbath the tube is lifted up and tipped back and forth for a clot
then stopwatch is stopped and the time is noted.

RESULT:

The prothrombin time of the given sample was 16 seconds.

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NORMAL RANGE:

15 – 20 seconds.

CLINICAL SIGNIFICANCE:

Prolonged prothrombin time indicate deficiency of factor II, X, XII, VII.

101