Experiment 3

Experiment 1 Hematology I: Methods of Blood Collection, Processing And Storage of Blood
EXPERIMENT 3
DIFFERENTIAL LEUCOCYTE
COUNT
Structure
3.1 Introduction 3.4 Procedure
Expected Learning Calculation

Outcomes
3.5 Result and Observations
3.2 Principle
3.6 Precautions
3.3 Materials Required
3.7 Terminal Questions
3.1 INTRODUCTION
As the name suggests, differential leucocyte count (DLC) refers to the
counting of relative percentage of each type of white blood cells in the human
blood. A certain number of white blood cells (WBC, leukocytes, or leucocytes)
are present in our blood and serve as part of the body's immune system. DLC
helps to identify abnormal white blood cell population in human blood and Agranular cells can be
monitor relative proportion of different WBCs that may change in case of distinguished from
granulocytes by the absence
particular diseases. Leucocytes are typically divided into granulocytes and
of granules in their cytoplasm.
agranulocytes. Neutrophils, eosinophils and basophils are the granulocytes
while lymphocytes and monocytes are agranulocytes. Neutrophils are the
most abundant leucocytes that constitute 50%-70% of the total leucocytes.
Each type protects the body from foreign pathogens such as viruses, bacteria,
fungi, and parasites in a different way. In this exercise, you will learn and
identify leucocytes in blood smear.
Expected Learning Outcomes

After studying and performing this exercise, you should be able to:
 prepare blood smear;
 stain the leucocytes and identify different types;
 perform count of differential leucocytes; and
 calculate differential white blood cell count. 21
BBCCL-116 Human Physiology: Lab
3.2 PRINCIPLE
This exercise helps us to identify different kinds of leucocytes in the human
blood and estimate the percentage distribution of each type. The differential
blood count is based on the staining of nucleus and cytoplasm of the white
blood cells. Staining of both nucleus as well as cytoplasm enables us to
determine the morphology and other properties of cells.
A blood smear is stained with the Leishman stain and different leucocytes are
counted under microscope.
3.3 MATERIALS REQUIRED

1. Glass slides and cover slip
2. Leishman Stain
3. Blood specimen
4. Distilled water
5. Cedarwood oil
6. Dropper or coplin staining jar
7. Cotton/Filter paper
8. Oil immersion Compound Microscope
3.4 PROCEDURE
A. Preparation of blood smear
Place a drop of blood to a clean glass slide about 1-2 cm away from one end
(Refer Exercise 1.1 capillary blood collection).
Spread the blood drop with the help of another glass slide by placing it at an
angle of 45° on the blood drop and move sidewise. Hold the slide spreader
firmly and move to the other end in a straight line (Fig. 3.1a). Blood smear
should be 3-4cm in length. Allow the blood smear to dry.
B. Fixing and staining blood smear
1. Pour 8-10 drops of Leishman stain solution on the blood smear and
leave it for 2-3 min.
2. Add distilled water over the stained smear with the help of a dropper to
wash the slide and leave the slide for 8-10 min.
3. Shake off extra water and wipe the back of the slide with filter paper.
4. Set the slide it in an upright position for air drying (Fig. 3.2).
22
Experiment 3 Differential Leucoyte Count
Fig. 3.1a Schematic diagram of Blood smear
Fig. 3.1b- unstained and stained and blood smear

C. Examination of the Stained Smear under Oil Immersion
5. Cover the stained smear with the cover slip and place it under low power
of microscope. Choose the good portion of smear for counting and
studying the distribution of patterns of the cells.
6. Add a drop of the cedar wood oil on the blood film. Carefully set the oil-
immersion objective and focus the cells using fine adjustment.
7. Start counting the cells by the shifting of slide laterally and then move
forward and back upward until the total 100 cells are counted.
8. Express the count of different leucocytes as the percentage. 23

3.5 RESULT AND OBSERVATIONS

Look at Fig. 3.2 which showing microscopic view of different types of
leucocytes cells. Fig. 3.3 shows the morphological shape of leucocytes. Table
3.1 summarizes the morphological shape and percentage of leucocytes.
The WBCs can be differentiated from RBCs by the presence of nucleus and
large size. Keep in mind some key factors while identification of WBC
1. Size of the cells
2. Colour and lobes of the nucleus.
3. Presence of the pink or blue; fine or coarse cytoplasmic granules
4. Nuclear/Cytoplasmic ratio.
Fig. 3.2: Microscopic view of Leucocytes in Human Blood.
Fig. 3.3: Identification of different types of leucocytes.

24
Experiment 3 Differential Leucoyte Count
Table 3.1: Structure of different leucocytes
White Blood Cell type Percentage Diameter Morphology

(%) in
Adults
Neutrophils 40-75 10-14μm Multilobednucleus,

pink and
granularcytoplasm
Lymphocytes 20-50 6-9 μm Round nucleus
Monocytes 2-10 12-25 μm Oval or horseshoe-

shaped nucleus
Eosinophils 1-6 10-14 μm Bilobed nucleus,brick

red-pinkish granules
in cytoplasm
Basophils 0.3-1 10-14 μm Bilobed nucleus, deep

blue purple large
cytoplasmic granules
3.6 PRECAUTIONS
1. Prepare a thin and uniform smear without any gaps or streaks.
2. Dry blood smear completely before staining.
3. Pour adequate quantity of stain solution on the smear.
4. Allow sufficient time for fixation of the stain.
5. While washing, ensure that stained slide should not be in direct contact
with the stream of water.
6. Count the cells from one end of the slide to another to avoid repeated
counting.
7. Count different types of WBCs separately.
3.7 TERMINAL QUESTIONS
1. Name the various types of leucocytes.
2. Which stain that used for DLC?
3. Describe how you would distinguish between different types of

leucocytes.
25
Acknowledgement of Figures
Fig. 3.1 b
https://commons.wikimedia.org/wiki/File:Peripheral_blood_smear_-
_stained_and_unstained.jpg
Fig. 3.2 Image source:

https://commons.wikimedia.org/wiki/File:Connective_Tissue_Human_Blood_L
eukocyte_Survey_(39982281920).jpg
Fig. 3.3
Source: Anatomy & Physiology, Connexions Web

site. http://cnx.org/content/col11496/1.6
https://commons.wikimedia.org/wiki/File:1916_Leukocyte_Key.jpg
26

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Experiment 1 Hematology I: Methods of Blood Collection, Processing And Storage of Blood

Expected Learning Calculation

Expected Learning Outcomes

3.3 MATERIALS REQUIRED

6. Dropper or coplin staining jar

8. Oil immersion Compound Microscope

B. Fixing and staining blood smear

Fig. 3.1a Schematic diagram of Blood smear

Fig. 3.1b- unstained and stained and blood smear

8. Express the count of different leucocytes as the percentage. 23

3.5 RESULT AND OBSERVATIONS

1. Size of the cells

2. Colour and lobes of the nucleus.

3. Presence of the pink or blue; fine or coarse cytoplasmic granules

Fig. 3.2: Microscopic view of Leucocytes in Human Blood.

Fig. 3.3: Identification of different types of leucocytes.

Table 3.1: Structure of different leucocytes

White Blood Cell type Percentage Diameter Morphology

Neutrophils 40-75 10-14μm Multilobednucleus,

Lymphocytes 20-50 6-9 μm Round nucleus

Monocytes 2-10 12-25 μm Oval or horseshoe-

Eosinophils 1-6 10-14 μm Bilobed nucleus,brick

Basophils 0.3-1 10-14 μm Bilobed nucleus, deep

2. Dry blood smear completely before staining.

3. Pour adequate quantity of stain solution on the smear.

4. Allow sufficient time for fixation of the stain.

7. Count different types of WBCs separately.

3.7 TERMINAL QUESTIONS

1. Name the various types of leucocytes.

2. Which stain that used for DLC?

3. Describe how you would distinguish between different types of

Fig. 3.2 Image source:

Source: Anatomy & Physiology, Connexions Web

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