Differential WBC Count - White bood cell count

1. Dr. S. MEENATCHISUNDARAM
ASSOCIATE PROFESSOR
DEPARTMENT OF MICROBIOLOGY
SNMV COLLEGE OF ARTS AND SCIENCE
COIMBATORE
https://orcid.org/0000-0002-8691-449X
95496
https://scholar.google.com/citations?user=IkdZ5XsAAAAJ&hl=en
DIFFERENTIAL WHITE CELL COUNT

2. INTRODUCTION
A differential white cell count provides information on the
different white cells present in the circulating blood, i.e.
neutrophils, lymphocytes, monocytes, eosinophils, basophils
(rarely seen).
Providing the total WBC count is known, the absolute number
of each white cell type, i.e. number of each cell per litre of blood,
can be calculated and an assessment made of whether the
number of a particular cell type is increased or decreased
DIFFERENTIAL WHITE CELL COUNT

3.  Blood must be collected with care and adequate safety precautions to ensure test results are
reliable, contamination of the sample is avoided and infection from blood transmissible
pathogens is prevented.
 Protective gloves should be worn when collecting and handling blood samples.
 Lancets, needles, and syringes must be sterile, and dry, and blood collecting materials must be
discarded safely to avoid injury from needles and lancets.
CAPILLARY BLOOD
 Capillary blood is mainly used when the patient is an infant or young child and the volume of
blood required is small, e.g. to measure haemoglobin, perform a WBC count, and to make thick
and thin blood films.
 Thick blood films for malaria parasites are best made from capillary blood (anticoagulated blood
is more easily washed from slides during staining).
Capillary blood can be collected from
 The ‘ring’ finger of a child or adult
 The heel of an infant up to one year old
DIFFERENTIAL WHITE CELL COUNT

4. TECHNIQUE FOR COLLECTING CAPILLARY BLOOD
 Cleanse the puncture area with 70% ethanol.
 Allow the area to dry.
 Using a sterile pricker or lancet, make a rapid puncture,
sufficiently deep to allow the free flow of blood.
 Wipe away the first drop of blood with a dry piece of cotton
wool and use the next few drops for the test.
 Do not squeeze too hard because this will result in an unreliable
test result.
 When sufficient blood has been collected, press a piece of dry
cotton wool over the puncture area until bleeding stops.
COLLECTION OF CAPILLARY
BLOOD FROM A FINGER
COLLECTION OF BLOOD FROM
THE HEEL OF AN INFANT
DIFFERENTIAL WHITE CELL COUNT

5. Place a drop of blood on the end of a clean dry
slide. Avoid making the drop too large (if too
large, use a drop from the excess blood to
make the film)
Using a clean smooth edged spreader, draw
the spreader back to touch the drop of blood
and allow the blood to extend along the edge
of the spreader. Holding the spreader at an
angle of about 30, spread the drop of blood to
make a film about 40–50 mm in length (two
thirds of the slide)
DIFFERENTIAL WHITE CELL COUNT

6. Wipe clean the end of the spreader.
Immediately air dry the film by waving the
slide back and forth. Protect the dried film
from dust and insects
When completely dry and within a few
minutes of making the blood film, fix it in
absolute methanol
Features of a well made film
● Not too thick, nor too long
● Free from lines and holes
● Has a smooth ‘tail’
DIFFERENTIAL WHITE CELL COUNT

7. METHANOL FIXING THIN BLOOD FILMS
When completely dry, fix a blood film with absolute methanol (methyl alcohol). Place the film on a
staining rack and add 1–2 drops of moisture-free methyl alcohol and allow it to dry on the film.
Alternatively, the blood film can be immersed in a container of absolute methanol for about 2 minutes,
but this is a more expensive method of fixing and also in tropical humid climates there is a greater risk
of the alcohol absorbing water from the atmosphere which will result in poor fixing of blood cells.
Water-free methanol
Satisfactory fixing of thin blood films requires the use of water-free absolute methanol. Poorly fixed
cells due to methanol containing water
When absolute methanol is not available, absolute ethanol (ethyl alcohol) can be used but this is more
expensive and usually less available than methanol
DIFFERENTIAL WHITE CELL COUNT

8. LEISHMAN STAINING TECHNIQUE
Method
Cover the blood film (preferably methanol prefixed) with undiluted stain but do not flood
the slide. If using a dropper bottle count the number of drops required to cover the film.
Add twice the volume of pH 6.8 buffered water (i.e. twice the number of drops as stain).
The diluted stain should not overflow. Ensure the water is well mixed with the stain by
blowing on the diluted stain or mixing the stain and water using a plastic bulb pipette.
Allow to stain for 10 minutes
Wash off the stain with tap water* (filtered if not clean). Do not tip off the stain, because
this will leave a fine deposit covering the film. Wipe the back of the slide clean and stand it
in a draining rack for the smear to dry. The blood film should appear neither too pink nor
too blue
Tap water: If the tap water is highly acidic, resulting in too pink a blood film or highly
alkaline, resulting in too blue a blood film, try using boiled cooled water or filtered rain
water. If neither of these are suitable, wash the film with pH 6.8 buffered water.
DIFFERENTIAL WHITE CELL COUNT

9. STAINING RESULTS
Red cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pink-red
Nucleus of cells . . . . . . . . . . . . . . . . . . . Purple-violet
Cytoplasm
Neutrophils, eosinophils . . . . . . . . . . . . . . Pale pink
Large lymphocytes . . . . . . . . . . . . . . . . . . Clear blue
Small lymphocytes . . . . . . . . . . . . Darker clear blue
Monocytes . . . . . . . . . . . . . . . . . . . . . . . . . Grey-blue
Granules
Eosinophils . . . . . . . . . . . . . . . . . . . . . . . Orange-red
Neutrophils . . . . . . . . . . . . . . . . . . . . . Mauve-purple
Toxic granules. . . . . . . . . . . . . . . . . . . . . . Dark violet
Basophils. . . . . . . . . . . . . . . . . . . . . . Dark blue-violet
Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . Purple-blue
Inclusions
Malaria pigment (in monocytes) . . . . . . Brown-black
Howell-Jolly body. . . . . . . . . . . . . . . . . . Purple-violet
Auer body (in myeloblast) . . . . . . . . . . . . Purple-red
DIFFERENTIAL WHITE CELL COUNT

10.  Thin blood film is prepared and stained.
 Allow the stained film to dry completely before examining it.
 Keep the prepared glass slide under low power objective lens
 Then identify different types of WBC under medium power
 Draw a table with 10 boxes both on horizontal and vertical axis
on the observation book
 Fix the slide on the platform and choose a area towards the
corner
 Note the different types of WBC found on the table in an
abbreviated
 Move downward and in a chin like manner till 100 cells are
observed
 After counting 100 cells prepare the report
DIFFERENTIAL WHITE CELL COUNT

13. REPORT
Neutrophils - 61%
Lymphocytes - 29%
Eosinophils - 5%
Monocytes - 5%
Basophils - 0
TOTAL - 100
DIFFERENTIAL WHITE CELL COUNT

14. INTRODUCTION
Calculate the absolute number of each white cell type by multiplying the number of each cell
counted (expressed as a decimal fraction) by the total WBC count.
Example
If: Percentage of neutrophils counted = 80%
i.e. 0.80 when expressed as a decimal fraction.
If total WBC count = 8.6 x 109/L
Absolute neutrophil count is 0.80 x 8.6 = 6.9x 109/L
DIFFERENTIAL WHITE CELL COUNT