Enumeration of WBCs - TLC & DLC

M.Sc. Zoology Semester I Genetics

Enumeration of WBCs - TLC & DLC

Praveen DeepakAssistant Professor

Department of Zoology

S. S. College, Jehanabad

Cytology

Introduction

Study of blood is related to haematology. Haematology is, simply, defined as the

science or study of blood, blood-forming organs and related disease.

The tests related to the blood, blood cells and blood chemistry is known as

haematological profile or complete blood profile (CBC) that includes haemoglobin

estimation (Hb), RBC, WBC, Platelets (PLT), Total leukocytes (TLC), Lymphocytes,

Monocytes, Eosinophiles, Basophils, etc ., in per micro-litre volume of blood.

Haemoglobin is estimated as gram per decilitre (gm/dl).

Blood is specilalized body fluid and it is considered as connective tissue due to

presence of matrix (plasma acts as matrix).

Basically, it is composed of 90% of water and rest 10% is plasma proteins and various

blood cells, like red blood cells (RBCs), white blood cells (WBCs), and platelets (PLT).

WBCs are of many types, e.g. lymphocyte, monocytes, neutrophils, basophils and

eosinophils.

Composition of blood

When blood is centrifuged in centrifuge machine, distinct bands can be observed.


Plasma

It is a pale yellow sticky liquid which is about 55% of the blood’s volume.

It is composed of 92% water, 8% dissolved proteins, and rest glucose, amino acid, CO2, hormones, antibodies

It carries .dissolved materials such as glucose, amino acids, minerals, vitamins, salts, CO2, urea, hormones, and heat.

After the removal of clotting factor, rest of plasma is known as serum, which is straw colour liquid.

Red Blood Cells

These are tiny biconcave disc-shaped anucleated cells.

They also do not contain any organelles including mitochondria.

These cells are rich in haemoglobin, an iron containing biomolecule that can bind oxygen and is responsible for blood’s red colour.

Thus, they function as principal mean of delivering oxygen (O2) to the body tissue via blood flow through the circulatory system.


White Blood Cells

They are also called and leukocytes which are colourless.

Unlike to RBCS, they possess a nucleus and other subcellular organelles.

Their main function is to defense against pathogens.

Thus, some of them act as phagocytes, called as neutrophils, macrophages,

dendritic cells, etc., some secretes antibodies and other active biomolecules,

called lymphocytes.

Platelets

They are also called as thrombocytes.

They also lack nucleus and other subcellular organelles.

They are tiny fragments of large bone marrow cells.

They carry specialized blood clotting factors, which are released upon injury.

Formation of blood cells

Complete Blood Count (CBC)

https://www.ncbi.nlm.nih.gov/books/NBK2263/table/ch1.T1/

Types of white blood cells

Normal count

Men: 5,000 and 10,000; Women: 4,500 and 11,000; and Children: 5,000 and 10,000 per μl

Percentage population of different WBC types

Name of leukocyte Male (% of total

leukocytes)

Female (% of total leukocytes)

Neutrophils 40 – 75% 30 – 75%

Lymphocytes 20 – 50% 20 – 50%

Monocytes 2 – 10% 2 – 10%

Eosinophils 1 – 6% 1 – 6%

Basophils 0.3 – 1% 0.3 – 1%

Physiological condition associated with

disbalanced WBC population

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

Blood Cell Counting

Haemocytometer

A haemocytometer is a device developed for

counting of blood cells. It was invented by

Louis-Charles Malassez..

It includes:

A Neubauer’s chamber

Cover slips

RBC pipette

WBC pipette

Neubauer’s chamber

Neubauer’s slide


It is a thick glass slide.

In the center of the slide, there is an H – shaped groove.

On the two sides of the central horizontal bar, there

are scales for counting the blood cells

The depth of the scales is 1

10𝑚𝑚 or 0.1mm.

Each scale is 3mm wide and 3mm long, i.e. 9mm2.

The whole scale is divided into 9 large squares.

Each large square is 1mm long and 1mm wide, i.e.

1mm2.

The central large squares are further divided in 3

directions; 0.25 x 0.25 mm (0.0625mm2), 0.25 x

0.20mm (0.05mm2) and 0.20 x 0.20mm (0.04mm2).

Neubauer’s slide


The central square is further subdivided into 0.05 x

0.05mm (0.0025mm2) squares.

The raised edges hold the coverslip 0.1mm off the grid

giving each square a defined volume.

Neubauer’s slide

Thoma pipette

RBC Pipette

WBC Pipette

Blood Cell Count

For cell counting

Blood is filled till mark 0.5 or 1.0 and fluid is then filled till mark 101 or 11

according to the need.

Both are thoroughly mixed.

Dilution factors

Total Leukocyte Count (TLC)

Total leukocyte count or TLC is a laboratory procedure to quantify the number of total number of leukocyte in the blood sample.

It is the process similar to that of enumeration of red blood cells (RBC), and is also performed by Hemocytometer chamber or Neubauer’s slide .

It helps us to find out whether a person has got any infection, if yes then what type of infection.

The TLC analysis makes us able to differentiate between acute and chronic infection.

It helps us to find out certain types of cancer in a patient.

It helps the physician to follow the patient with chemotherapy.

It helps in analyzing the effect of drugs.


Principle

A cationic or basic dye of Romanowsky stain (methylene blue or its oxidation

products such as azure B) binds to anionic sites, i.e, nucleic acid and gives

blue-grey colour. It stains basophilic granules of basophils intensely and

faintly to that neutrophils.

An anionic or acidic dye of Romanowsky stain such as eosin Y or eosin B,

binds to cationic sites on proteins and gives an orange-red colour. It stains

cytoplasm and eosinophilic granules to orange – red in colour.


Reagents

Diluting fluid or dilution buffer – PBS can be used. If PBS is not utilized,

Türk's solution, is used which dilutes the sample and stain the cells as ewell

70% ethyl alcohol for cleaning microscope lens

Türk's solution: It contains gentian violet or crystal violet (methyl violet 10B or

hexamethyl pararosaniline chloride) which stains the nuclei into deep purple colour

allowing easy identification and counting. Additionally, acetic acid in the solution lyses the

RBCs. Composition of Turk’s solution is following:

Glacial acetic acid 1ml

Gentian violet 2ml (1% aqueous solution)

Distilled water 100 ml


Equipments

Pipettes – Thoma peipette (RBCs) or tube or micropipette.

Improved Neubauer chamber with the cover slips.

Light microscope.

Clean gauze

Sample

Whole blood using EDTA or heparin as anticoagulant. However, capillary

blood may also be used


Procedure

Clean the hemocytometer and its cover slip with 70% ethyl alcohol and then dry with a dry wipe.

Draw blood in a clean dry WBC pipette up to the mark 0.5 with all possible accuracy.

Draw the diluting fluid up to mark 11 (dilution 1 in 10). For tube method, take 0.02 ml blood and mix it with diluting fluid 0.38 ml of which is taken in a small tube. Mix the blood with diluting fluid (1:20 dilution).

Mix the contents of pipette for minutes.

Dispel the first 4 drops of the contents.

Load the Neubauers chamber with the pipette contents by holding the pipette at angle of 54 degree carefully.

Allow 2 minute for setting of cells.

Count and calculate the total leukocyte in the sample.


Calculation

The total area of each the corner large squares is 1mm2

Since the depth is Τ1 10 𝑚𝑚.

Therefore, the volume of each corner large square is 1 𝑚𝑚2 × Τ1 10𝑚𝑚 = Τ1 10𝑚𝑚3.

Therefore, the volume of the four corner large squares is

Τ1 10× 4 = Τ4 10𝑚𝑚3

The number of WBCs in 1mm3 …………………………………………..….. 𝑋

The number of WBCs in Τ4 10 ………………………………………..…….. 𝑌

Therefore,

𝑋 =𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑊𝐵𝐶𝑠 𝑖𝑛 𝑎𝑙𝑙 𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑒𝑟𝑠 ×1

𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎𝑙𝑙 𝑐𝑜𝑟𝑛𝑒𝑟 𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑎𝑟𝑒𝑠=

𝑌 ×1

Τ4 10= 𝑌

1

Τ4 10= 𝑌 ×

10

4= 𝑌 × 2.5

Since, dilution is 20.

Therefore number of WBC in 1mm3, i.e. 𝑋 = 𝑌 × 2.5 × 20 = 𝑌 × 50

Therefore,

𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑊𝐵𝐶 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 50

Differential Leukocyte Count (DLC)

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

Micropipette

Glass slides with cover slips

Microscope

Clean gauge or cotton

Sample

Whole blood using EDTA or heparin as anticoagulant. However, capillary blood may also be used.

Reagents

Stains – Commonly used satins are Leishman’s stain, Wright stain, Giemsa stain, and Filed stain. However, only one stain is used while doing differential count. Generally, Wright’s stain is used.

70% Ethanol

Distilled water


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 is added to the

mixture.


Procedure

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.


Staining with Wright’s stain

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:

Cytoplasm

Sky blue

Eosinophils:

cytoplasm

Blue Platelets Violet to purple

granules


Staining of different subsets of WBCs (Wright-Giemsa stain)


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

Interpretation of results

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.

Sources of errors

Presence of microclots in the sample.

Inadequate mixing of contents.

Improper filling of the chamber.

Improper dilution.

Mistake in the calculation.

Further reading

Bain B.J., Bates I., Laffan M.A. 2016. Dacie and Lewis Practical Haematolog. Elsevier

Health Sciences, Philadelphia, USA

Kale R.R. & Kale S.R. 2002. Haematology, Practical Human Anatomy and Physiology.

Eight Edition. 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

https://www.labtestsguide.com/differential-leukocyte-count-dlc-test-procedure

Enumeration of WBCs - TLC & DLC

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