enumeration of wbcs - tlc & dlc
TRANSCRIPT
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.
Composition of blood
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.
Composition of blood
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
Neubauer’s chamber
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
Neubauer’s chamber
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.
Total Leukocyte Count (TLC)
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.
Total Leukocyte Count (TLC)
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
Total Leukocyte Count (TLC)
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
Total Leukocyte Count (TLC)
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.
Total Leukocyte Count (TLC)
Total Leukocyte Count (TLC)
Total Leukocyte Count (TLC)
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
Differential Leukocyte Count (DLC)
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
Differential Leukocyte Count (DLC)
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.
Differential Leukocyte Count (DLC)
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.
Differential Leukocyte Count (DLC)
Differential Leukocyte Count (DLC)
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
Differential Leukocyte Count (DLC)
Staining of different subsets of WBCs (Wright-Giemsa stain)
Differential Leukocyte Count (DLC)
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