S. S.

College, Jehanabad
Department: Zoology
Class: M.Sc. Semester I
Subject: Zoology
Topic: Enumeration of WBC - Differential Count (DC)
Mode of teaching: Google classroom & Zoom
Date & Time: 16.07.2021 & 03:00 Zoom
Teacher: Praveen Deepak, Assistant Professor, Department
of Zoology, S. S. College, Jehanabad

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ENUMERATION OF WBC – DIFFERENTIAL COUNT (DC)
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White blood cell or leukocytes are heterogeneous group of nucleated cells that are important
component of blood like Red Blood Cells or RBCs. However, leukocytes are devoid of
hemoglobin, but have prominent subcellular organelle. They are also found in circulation in
circulation for at least a period of their life. Their normal concentration in blood varies between
4500 and 11,000 per microliter. They play an important role in defense against the foreign
invasion or non-self through phagocytosis and immune response. It is capable of motility and
largely involved in ingesting foreign materials and cellular debris by acting as scavenger in the
tissue. They are larger than the RBCs in size and also differ in the shape; white blood cells
(WBCs) are rounded, amoeboid and irregular in shape while red blood cells (RBCs) are
biconcave discoid. The size of WBCs is about 15µm while that of RBCs is 7.5µm.
As leukocytes are heterogeneous group of blood cells, they are classified into various groups
depending on their size and cytoplasmic as well as nuclear characteristics. They exist in two
forms, such as, granulocytes and agranulocytes. Granulocytes have granular appearance due to
the presence of specific granules in their cytoplasm, e.g., eosinophils, basophils and neutrophils,
whereas agranulocytes have smooth appearance due to the absence of such granules in the
cytoplasm, e.g., lymphocytes and monocytes (see figures below to differentiate the cells). These
cells are variedly distributed in terms of numbers as shown in table.

Table: Percentage of different cell types of white blood cells

Name of leukocyte Male (% of total leukocytes) Female (% of total leukocytes)

Neutrophils 40 – 75% 30 – 75%

Praveen Deepak, Department of Zoology, S. S. College, Jehanabad

Lymphocytes 20 – 50% 20 – 50%

Monocytes 2 – 10% 2 – 10%

Eosinophils 1 – 6% 1 – 6%

Basophils 0.3 – 1% 0.3 – 1%

Genesis of white blood cells (WBCs)

White blood cells develop from the hematopoietic stem cells (HSCs) in the bone marrow. The
HSCs differentiate into two intermediate precursors namely myeloid stem cells and lymphoid
stem cells. Lymphoid stem cell leads the formation of lymphoblast cells which later on
differentiate into large granular natural killer cells or NK cells and small lymphocytes. Myeloid
lymphoid cells are differentiated into four types of intermediate cells namely megakaryoblast,
proerythroblast, myeloblast and monoblast. Megakaryoblast differentiate into platelets with
megakaryocyte is a, intermediate stage, proerythroblasts are differentiated into erythrocytes with
reticulocyte as an intermediate stage, myeloblasts are differentiated into eosinophils, basophils,
and neutrophils, while monoblasts are differentiated into monocytes.

Differential count of WBCs

The process of evaluation of relative percentage of each type of wile blood cell is known as
differential blood count or differential count of WBC (DC). It also helps to reveal abnormal

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white blood cell populations (e.g., blasts, immature granulocytes, and circulating lymphoma cells
in the peripheral blood.
Aim of the differential count
Aim of the blood differential leukocyte count (DLC) is to make a blood smear and to count the
different types of leukocytes present in a stained blood smear and express their relative value in
percentage.
Principle
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. For differential count, generally the combination of polychrome
methylene blue and eosin stains are used because of their selective staining properties; methylene
blue stains nucleus while eosin stains cytoplasm. Staining is followed by quantitating the
different types of cells
Equipment
1. Micropipette
2. Glass slides with cover slips
3. Microscope
4. Clean gauge or cotton
Sample
1. Generally blood sample is collected in EDTA (Ethylene-Diamine-Tetraacetic acid).
2. Freshly prepared peripheral blood smear can also be used.
Reagents and solution
1. Well mixed whole or anticoagulant blood
2. Stains – Commonly used satins are Leishman’s stain, Wright stain, Giemsa stain, and
Filed stain. Out of these stains, only one stain needs to be used while doing differential
count (DLC) test.
3. 70% Ethanol
4. Distilled water
Preparation of stains
Leishman’s stain: It is constituted by mixing 0.150gm of Leishman stain in 100 ml of
absolute methanol. Leishman’s staining results in following colour formation:

Nuclear staining Staining of granules

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 Chromatin Chromatin Basophils Purple black

Nucleoli Nucleoli Eosinophils Red orange

Cytoplasm staining Neutrophils Purple

Erythrocytes Erythrocytes Platelets Purple

Reticulocytes Reticulocytes

Lymphocytes Lymphocytes

Monocytes Monocytes

Neutrophils Neutrophils

Basophils Basophils

Wright’s stain: It is a histologic stain that facilitates the differentiation of blood cell types. It
is classically mixture of eosin (red) and methylene blue dyes. It is used primarily to stain
peripheral blood smears, urine samples, and bone marrow aspirates and examined under light
microscope. It is prepared by mixing 1.0gm of Wright’s stain powder in 400ml absolute
methanol. Thereafter 100ml phosphate buffered saline (Potassium dihydrogen phosphate
0.663gm and disodium hydrogen phosphate 0.256gm added in 100ml distilled water; 0.15M,
pH 6.5/6.8) is added to the mixture. Staining with Writght’s stain results in following colour
formation:

WBC types Colour WBC types Colour

Erythrocytes Yellowish – red Eosinophils: Granules Red or Orange – red

Neutrophils: Nucleus Dark purple Basophils: Nucleus Purple to dark blule

Neutrophils: Cytoplasm Pale – pink Basophils: Granules Very dark purple

Neutrophils: Granules Reddish – lilac Lymphocytes: Nuclei Dark purple

Eosinophils: Nulclei Blue Lymphocytes: Sky blue

Cytoplasm

Eosinophils: cytoplasm Blue Platelets Violet to purple

granules

Giemsa stain: It is a type of Romanowsky stain (neutral stains composed of a mixture of

oxidized methylene blue (azure) dyes and Eosin Y). It was named after Gustav Giemsa, a
German chemist who created a dye solution for the demonstration of malarial parasites in
blood smears, but now it is applied in routine examination of blood smear because it is

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 specific for the phosphate groups of DNA and attaches itself to where there are high amounts
of adenine-thymine bonding. It is prepared by adding 7.6gm of Giemsa powder to the 500ml
of glycerol and finally 500ml of absolute methanol is added to the mixture. For thin blood
smears, 1:20 dilution and for thick blood smear, 1:50 dilution of this preparation is used.
Staining with Giemsa stain results in following colour formation:

Blood cell types Colour Blood cell types Colour

Red Blood cells Mauve – pink Lymphocytes Nuclei: Dark blue

Cytoplasm: Light blue

Neutrophils Nuclei: Reddish purple Monocytes Nuclei: Purple

Cytoplasm: Pink Cytoplasm: Pink
Eosinophils
Nuclei: Purple Platelets Violet to purple colour
granules
Cytoplasm: Faintly pink
Granules: Red to orange
Basophils
Nuclei: Purple
Granules: Blue coarse

Field stain: It is also a type Romanowsky stain developed in order to discover malarial
parasites in thick blood smears and now used for histological staining of blood smears. It
enables worker for rapid processing of the specimens. It contains methylene blue (basic dyes)
and eosin (acidic dyes). It is prepared in two parts; Field’s stain A and Field’s stain B.

Field’s Stain A (Methylene Blue Solution) Field’s Stain B (Eosin Blue Solution)

Methylene blue 1.300gm Eosin 1.300gm

Potassium dihydrogen phosphate 6.250gm Potassium dihydrogen 6.250gm

phosphate

Disodium hydrogen phosphate 5.000gm Disodium hydrogen phosphate 5.000gm

Distilled water 550.000ml Distilled water 500.000ml

It can also be prepared by adding 1.3gm methylene blue in 550ml phosphate buffer saline
(PBS) for Field Stain A and 1.3g, eosin in 500ml PBS for Field stain B. Filed stain A has a
dark violet coloured solution, while Field stain B has a orange coloured solution. Staining is
performed by flooding or dipping slide in Field’s stain A for 2 – 3 seconds and in Field’s
stains B for 2 – 3 seconds after washing which leads to following colour formation:

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 Cell types Colour Cell types Colour

Neutrophils granules Lilac Leukocytes Nuclei Purple

Eosinophils granules Orange Leukocytes cytoplasm Pale blue

Nuclei
Blue Malarial parasite Deep red chromatin &
pale blue cytoplasm

Red Blood cells Red Blood cells are lysed & only background stroma remains

Preparation of slides and staining

− Collect drops of blood on the end side of a glass slide.
− Spread the blood drop with another glass slide by placing it at an angle of 45 degree and
move sidewise.
− Hold the spreader firmly and move it on the previous slide to the other end in a straight
line with same force and pressure.
− Allow the glass slide to dry after formation of the smear.
− Air dry the slide or use any fixative to fix the smear.
− Stain the slides with a stain stated above.

Observation and counting

− Put the slides on the stage of light microscope and focus appropriately.
− Identify different types of WBC under medium magnification on the basic nuclear
morphology and granules.
− Count all cells without differentiating cell types present on the slide.
− Move downwards and in chain like manner till 100 cells are observed.

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 − Count the different types of WBC one by one observed on the equivalent area of the slide
and calculate percentage out of total leukocytes.

Significance of differential count

− It gives the relative percentage of each type of white blood cell.

− It helps in analyzing for any abnormal white blood cell population, e.g. blasts, immature
granulocytes and circulating lymphoid cell, etc., that may be a sign of potential health
issue.
− It helps us diagnosing an infection or inflammation in the body.
− It helps in detecting any disorder of the immune system.
Interpretation of test results
− An increased percentage of neutrophils in your blood can mean that you have:
• Neutrophilia, a white blood cell disorder that can be caused by an infection, steroids,
smoking, or rigorous exercise
• an acute infection, especially a bacterial infection

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 • acute stress, tissue injury due to trauma
• pregnancy
• inflammation, such as inflammatory bowel disease or rheumatoid arthritis
• chronic leukemia

Condition associated with increased or decreased number of WBC types

Type of WBC Increased number Decreased number

Neutrophils Neutrophilia Neutropenia

Lymphocytes Lymphocytosis Lymphocytopenia

Monocytes Monocytosis Monocytopenia

Eosinophils Eosinophilia Eosinopenia

Basophils Basophilia Basopenia

− A decreased percentage of neutrophils in your blood can indicate:

• Neutropenia, a white blood cell disorder that can be caused by a lack of neutrophil
production in the bone marrow
• aplastic anemia, a decrease in the number of blood cells produced by your bone
marrow
• a severe or widespread bacterial or viral infection
• recent chemotherapy or radiation therapy treatments
− An increased percentage of lymphocytes in your blood may be due to:
• lymphoma, a white blood cell cancer that starts in your lymph nodes
• a chronic bacterial infection
• hepatitis
• multiple myeloma, a cancer of the cells in your bone marrow
• a viral infection, such as mononucleosis, mumps, or measles
• lymphocytic leukemia
− A decreased percentage of lymphocytes in your blood can be a result of:
• bone marrow damage due to chemotherapy or radiation treatments
• HIV, tuberculosis, or hepatitis infection
• leukemia
• a severe infection, such as sepsis
• an autoimmune disorder, such as lupus or rheumatoid arthritis
− A heightened percentage of monocytes in your blood can be caused by:
• chronic inflammatory disease, such as inflammatory bowel disease

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 • a parasitic or viral infection
• a bacterial infection in your heart
• a collagen vascular disease, such as lupus, vasculitis, or rheumatoid arthritis
• certain types of leukemia
− An increased percentage of eosinophils in your blood can indicate:
• eosinophilia, which can be caused by allergic disorders, parasites, tumors, or
gastrointestinal (GI) disorders
• an allergic reaction
• skin inflammation, such as eczema or dermatitis
• a parasitic infection
• an inflammatory disorder, such as inflammatory bowel disease or celiac disease
• certain cancers
− An increased percentage of basophils in your blood might be caused by:
• a serious food allergy
• inflammation
• leukemia

Precautions
− Always wear protective gloves/protective clothing/eye protection/face protection before
handling the dilution fluid.
− Follow good microbiological lab practices while handling specimens and culture.
− Standard precautions as per established guidelines should be followed while handling
clinical specimens.
Bibliography
− Bain B.J., Bates I., Laffan M.A. 2016. Dacie and Lewis Practical Haematology. Elsevier
Health Sciences, Philadelphia, USA
− Kale R.R. & Kale S.R. 2002. Haematology, Practical Human Anatomy and Physiology.
Eight Editions. Nirali Prakashan, Pune, India
− Singh T. 2017. Text and Practical Haematology for MBBS. Arya Publications, New
Delhi, India
− https://www.labtestsguide.com/differential-leukocyte-count-dlc-test-procedure
− https://www.healthline.com/health/blood-differential

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