Enumeration of WBC - Differential Count (DC)
Enumeration of WBC - Differential Count (DC)
Enumeration of WBC - Differential Count (DC)
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
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.
Eosinophils 1 – 6% 1 – 6%
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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:
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Chromatin Chromatin Basophils Purple black
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:
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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:
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)
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
Red Blood cells Red Blood cells are lysed & only background stroma remains
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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.
− 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
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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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