DIFFERENTIAL LEUCOCYTE
COUNT
(DLC)
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Aim
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To Identify & Count different types of leucocytes in given
blood smear and to express them in %.
Principle
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Blood smear is stained with Leishman’s stain &
each type of WBC is identified until 100
leucocytes are examined, then their percentage
is calculated.
Apparatus
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Compound microscope
Cotton swab
Sterile lancet
4 Glass slides
Leishman’s stain
Staining Rack
Distilled water
Cedar wood oil
LEISHMAN STAIN
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Eosin- it is an acidic dye & stains basic parts such
as, Hb in RBC, granules of eosinophils giving
them a pink colour.
Methylene blue - is a basic dye & stains acidic
parts such as, nuclei and cytoplasm of WBC &
granules of basophils giving a blue violet colour
Methyl alcohol
- It fixes blood smear to glass slide preserving
morphology of the cell
- Should be acetone free because it breaks cell
membrane
PROCEDURE
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Take 4 slides, one is used as a spreader.
Under aseptic precaution, prick finger tip
Discard first drop of blood, because it contains tissue cells
Place drop of blood on slide 1cm from edge.
Place spreader in front of blood
Pull it gently back till it touches blood. blood spreads
along its edge. It can be slightly shaken sideways to
facilitate spread.
Move spreader slowly and steadily at 45°.
Dry it by waving in air & place on the staining rack.
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Peripheral smear
Parts Body
- Head
- Body
- Tail
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Criteria of an ideal smear
It should cover ½ to ¾ of the slide.
Should be thickest at origin & gradually thin out & should
fade away without defined border at end.
Should be tongue shaped.
Should appear pink when viewed with naked eye.
There should be no streaks or gaps in the smear.
It should be single cell thick (of RBC), that is, they should
overlap.
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Pour 8-12 drops of Leishman’s stain on each slide. Leave it
for about 3 mins (fixation time), it fixes the blood smear.
Staining smear: add double the amount of distilled water on
smear with dropper
-Mix stain and water by blowing. A metallic shiny layer
(Greenish scum) forms on top of this mixture
Leave it for 7 mintues (staining time), during which staining
occurs (staining time) due to ionization of dyes
- Then, pour off stain & wash it under tap water till it gets
pinkish tinge
- Water should not fall directly on it
- Wipe backside of the slide with dry cotton and allow it to
dry
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Place a drop of cedar wood oil on blood film.
adjust microscope for oil immersion objective
Plane mirror
Condenser - maximum height
Iris diaphragm–Fully open
Focus under objective and carefully lower it
until it just touches the oil drop.
Counting : use zigzag method i.e. start from one end
of the smear, as it approaches other end, move 2
fields down and in opposite direction.
Count cells throughout the length of the smear,
except the extreme ends of head and tail.
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Granulocytes
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Cell type Size Cytoplasm Granules Nucleus
(µm)
Neutrophils 10-14 Slate- blue in Fine, Violet pink Blue violet
(40-70%) color Do not cover nucleus. 2-6 lobes, connected
by chromatin band.
Eosinophil 10-15 Eosinophilic Large, coarse Blue violet
(1-6%) Light pink-red Brick red to orange 2-3 lobes, often
Granular Do not cover nucleus. bilobed connected
by chromatin band.
Basophil 10-15 Basophilic Large, very coarse Blue violet
(0-1%) Bluish Deep purple Irregular shape,
Granular Completely fill the cell Not seen clearly
and nucleus. because of granules
Agranulocytes
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Cell type Size Cytoplasm Granules Nucleus
(µm)
Monocyte 12-20 Abundant No Pale blue violet
(5-10%) Slate blue Large single
kidney shaped.
Small 7-9 Hardly visible No Deep blue violet
lymphocyte Thin crescent of Single, large, round
(20-40%) clear, light blue
cytoplasm
Large 10-15 Large crescent of No Deep blue violet
lymphocyte clear, light blue Single, large, round
(5-10%)
Blood smear under microscope
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Observation
Draw a large square and divide it into 100 small squares
Enter identified cell by its first letter into small squares. Neutrophil as 'N',
eosinophil as 'E', basophil as B, Monocytes as M, lymphocyte as 'L‘ till the
last small square is filled.
Count each type of cell and express it as percentage.
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Results:
Neutrophils: _____%
Eosinophils: _____%
Basophils: _____%
Lymphocytes: _____%
Monocytes: _____ %
Inference -
Precautions
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Avoid tap water for diluting the stain, as
methylene blue may not stain the cells properly.
Use distilled water
Water should not fall directly on the smear.
Count cells throughout the length of the smear,
except the extreme ends of head and tail.
Questions
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What are normal values of DLC?
Neutrophils: 40-70%
Eosinophils: 1-6%
Basophils: 0-1%
Lymphocytes: 20-45%
Monocytes: 2-10%
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What are uses of blood smear ?
Study of morphology of RBC
Morphological classification of anaemia (Wintrobes)
Abnormal RBC ex Sickling
Normal DLC and abnormal leucocytes eg. leukemias
Sex determination by Barr body in nucleus of neutrophils of
female
Indirect platelet count and their morphology.
Detection of parasites example malaria, microfilaria.
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What is clinical importance of DLC?
It is done to find out if there is an increase or
decrease of a particular type of WBC.
It is important in detecting different types of
infection & allergy .
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What is buffered water? Why it is preferred over
distilled water?
It is phosphate buffer in which the pH is
adjusted at 6.8
At this pH, there is optimal ionization of the
stain particles so that they can penetrate the cells
better.
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What is fixation time & staining time?
Fixation is process that makes the blood film
adheres to the glass slide with preserving
morphology of cells
Fixation time 3 minutes
Staining is a process that stains the nuclei and
cytoplasm of the cells.
It is 7 minutes.
Why tap water should not be used for staining?
Methylene blue may not stain the cells due to
unknown pH & salt content of the tap water.
It may contain impurities that will give false
appearance of the granules.
What are the other stains used ?
Giemsa stain
Field stain
Wrights stain
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Why is cedar wood oil used?
Refractive index of both glass and cedar wood
oil is same (1.5)
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Which part of the blood smear should be avoided
for counting the cells ?
Head and extreme tail should be avoided because
these areas contain few cells which are
commonly distorted.
What is neutrophilia and its causes?
Increases in neutrophil count is called neutrophilia
Physiological conditions: exercise, pregnancy,
stress, parturition, after meal.
Pathological conditions: acute pyogenic infections
e.g., abscess, pneumonia, after hemorrhage, steroid
therapy.
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What is neutropenia and its causes?
Decrease in neutrophil count is called neutropenia
It occurs in conditions like typhoid fever, parasitic
infections like viral infections like measles,
aplastic anemia.
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What is eosinophilia and its causes?
It is increase in eosinophil count.
It occurs in allergic conditions like bronchial
asthma, food allergy, parasitic infections, skin
diseases.
What is eosinopenia and its causes?
It is decrease in eosinophil count
It occurs in ACTH therapy and bone marrow
depression
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What is basophilia and its causes?
It is increase in basophil count
It occurs in tuberculosis, smallpox, chronic myeloid
leukemia.
What is basopenia and its causes?
It is decrease in basophil count
It occur in steroid therapy and bone marrow
depression
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What is lymphocytosis and its causes?
It is increase in lymphocyte count
Physiological conditions: children
Pathological conditions : in chronic infections like
tuberculosis, viral infections, chronic lymphatic leukemia.
What is lymphocytopenia and its causes?
It is decrease in lymphocyte count
It occurs in AIDS and bone marrow depression.
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What is monocytosis and its causes?
It is increase in monocyte count
It seen in malaria, kala azar, infectious
mononucleosis
What is monocytopenia and its causes?
It is decrease in monocyte count.
It is seen in bone marrow depression.
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