Blood Film Preparation and Staining
Value
of Thin blood films
Examination
of thin blood films is important in the investigation and management of
anaemia, infections, and other conditions which produce changes in the
appearance of blood cells and differential white cell count. A blood film report
can provide rapidly and at low cost, useful information about a patient’s
conditsion.
Thick
blood films
The
preparation, staining and reporting of thick blood filmsand colour plates
illustrating malaria parasites, trypanosomes, and microfilariae can be found in
Part 1 of the book.
Making,
fixing and staining blood films
Thin
blood films can be made from free-flowing capillary blood or well mixed EDTA
anticoagulated blood. To prevent EDTA associated blood changes, it is important
to make blood films from EDTA anticoagulated blood with as little delay as
possible, i.e. within 1 hour of collecting the blood.
Technique
of making a thin blood film
1.
Make a blood spreader from a slide which has
ground glass polished sides
2.
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).
3.
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)
Note:
When
the blood is from an anemic patient, increase the angle of spreading and spread
the blood more quickly. When the blood is thick and viscous, reduce the angle
of spreading and spread the blood more slowly.
4.
Wipe clean the end of the spreader.
5.
Immediately air dry the film by waving the slide back and forth.
Protect the dried film from dust and insects.
Note:
When
not using a frosted ended slide, write the patient’s name and number on the dried
blood at the top or along the side of the film using a lead pencil.
6.
When completely dry and within a few minutes of making the blood
film, fix it in absolute methanol
Features
of a well made film
ü Not
too thick, nor too long
ü Free
from lines and holes
ü Has a smooth ‘tail’
Note:
‘Holes’
in a blood film are usually caused by using a slide which is not clean. A
jagged ‘tail’ to a blood film is caused by using a spreader with a chipped end
or end that is not clean. Lines across
A
film is usually due to spreading a film jerkily. Too long a film usually means
that too much blood has been used or anaemic ‘thin’ blood has been spread too
slowly or at too shallow an angle.
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.
Note:
Even
when using a Fleishman or wright staining technique in which the film is first
covered with undiluted stain (which fixes and partially stains), prior fixing
of thin films is recommended because the stain may contain moisture which will
result in poor fixation
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 are shown in picture
Stock
bottles of methanol must be closed tightly after use and the container used to
dispense the methanol must have a cap which can be closed completely.
Note:
When
absolute methanol is not available, absolute ethanol (ethyl alcohol) can be
used but this is more expensive and usually less available than methanol.
Staining
thin blood films
In
district laboratories, thin blood films are usually stained manually using Fleishman,
Giemsa or Wright’s stain. These stains are examples of alcohol containing Romanowsky
stains which stain blood cells differentially.
Romanowsky
stains (Principal)
These
stains contain eosin Y which is an acidic dye and azure B (Derived from the
oxidation, or polychroming, of methylene blue) which is basic dyes. Eosin
stains the basic components of blood cells, e.g. hemoglobin stains pink-red,
and the granules of eosinophil’s stain orange-red. Azure B stains the acidic
components of cells. Nucleic acids and nucleoprotein stain various shades of
mauve-purple and violet, the granules of basophils stain dark blue-violet, and
the cytoplasm of monocytes and lymphocytes stains blue or blue grey. The
staining reactions of Romanowsky stains are pH dependent which is why the
stains are diluted in buffered water of specific pH.
Fleishman
staining technique
Many
of the difficulties in reporting blood films, particularly red cell morphology,
are due to variable staining. It is important therefore for laboratories to use
a reproducible standardized staining technique.
Reagents
1.
Fleishman stain
ü For
daily use, store the stain in an amber container,
ü Make
sure the stain is kept in a cool place (not refrigerated) and never left in direct
sunlight.
ü Bright
light and heat will cause the stain to deteriorate rapidly
2.
pH 6.8 buffered water
Method
1.
Cover the blood film with undiluted stain but do not flood the
slide
.Note:
The
undiluted stain not only acts as a fixative but also partially stains the
smear. This stage is required to obtain the best possible staining results.
2.
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
(time may require adjusting).
3.
Wash off the stain with tap water*. 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 if so, wash the film with pH 6.8
buffered water.
Quality
control
ü When
a new batch of stain is prepared, decide the best staining time to use, e.g.
stain films made from the same blood at different times, e.g. 5, 7, 10, 12, 15
minutes. Compare the results with a stained control blood film.
ü Check
the pH of newly prepared buffered water and re-check it at weekly intervals.
The pH of the buffered water used to dilute the stain must be correct. It is
mainly responsible for the staining reactions.
ü Maintain
consistency in the staining procedure by following exactly a standard operating
procedure (SOP).
Thick
films
Thick
films are required for examination for malarial parasites and certain other
parasites, the red cells being lysed before the film is examined. Parasites are
much more concentrated in a thick film, so that searching for them requires
less time. To make a thick film, several drops of EDTA anticoagulated blood are
placed in the centre of a slide and stirred with a capillary tube or needle of
syringe into a pool of blood of such a thickness that type script or a watch face can be read
through the blood . The blood film is not fixed but after drying, is placed
directly into an aqueous Giemsa stain or Field stain so that lysis of red cells
occurs; this allows the organisms to be seen more clearly.
Giemsa stain
Procedure:
1.
Make dilution
of stain with buffer 1:10 by adding one part of stain and ten part of buffer
2.
Pour this
diluted stain on slide already fixed
3.
Allow stain on
smear for 15 min
4.
Wash off with
water, dry and examine for results microscopically
5.
The time of
staining can be adjusted according to the quality of stain
Water based stains
Fields Stain
Reagents
ü Field stain A:
It contains Methylene blue dissolved
in buffered water
ü Field stain B
It contains Eosin dissolved in
buffered water
Method
1.
Dip the film
for 5-10 second in solution A
2.
Remove from
solution A and immediately rinse with water for few seconds
3.
Dip for 5-10
second in solution B
4.
Rinse
immediately with water, dry and examine for results microscopically
5.
The time of
staining can be adjusted according to
quality of stain
