PREPARATION OF BLOOD SMEARS anticoagulants) or from free flowing finger blood by

any of the following three techniques

Smear Preparation o Slide method – most commonly used
- A blood film or a peripheral blood smear is a thin layer o Cover glass method
of blood smeared on a glass microscope and then o Spin method
stained in such a way as to allow the cells to be
Smear Preparation
examined microscopically
- Blood films are usually examined to investigate - Microscopic examination of the shape, size and
hematological problems (disorders of the blood) and, coloration of RBCs is useful for determining the cause
occasionally, to look for parasites within the blood of anemia.
such as malaria and filarial o Disorders such as iron deficiency, sickle cell
- Examination of thin blood films is important in the anemia (moon-shaped RBC), megaloblastic
investigation and management of anemia, infections anemia (enlarged cells) and
and other conditions which produce changes in the microangiopathic hemolytic anemia (involves
appearance of blood cells and differential white cell platelets) result in characteristic
count abnormalities on the blood film
o Macrocytic, microcytic, WBCs - Peripheral blood which is collected by skin puncture is
- A blood film report provide rapidly and at low cost, the most appropriate specimen
useful information about a patient’s condition o If not made from skin puncture, films should
be prepared within 1 hour of blood collection
Peripheral Blood Film
into EDTA
 Types - Adequate mixing is necessary prior to film preparation
o Thin blood film (RIGHT) – needs to be if the blood has been standing for any appreciable
spread to become thin period of time
o Thick blood film (LEFT)
Three Methods of Making Films:

 Two slide or wedge method

 Cover glass method
 Spinner method

- Preparation of blood films on glass slides have the

following advantages:
 Slides are not easily broken
 Easier to label
 When large numbers of films are to be dealt
with, slides will be found much easier to
Three basic
handle
steps to make blood film
Wedge Method (Two-slidemethod )
1. Preparation of blood smear
2. Fixation of blood smear - Simplest and most widely used method
3. Staining of blood smear - Uses two slides, one for the smear and the other
serves as spreader or pusher
Preparation of Thin Blood Film
Procedure:
- Thin peripheral blood film (PBF) can be prepared from
anticoagulated blood (much better if no

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 1. Small drop of blood is placed on the central line or a
slide about 1-2cm from one end. (gitna pero dulo_
Another slide, the spreading slide is placed in front of
the drop at an angle of 30° to the slide and then is
moved back to make contact with the drop of blood.
The drop will spread out quickly along the line of
contact of the spreader with the slide
2. Once the blood has spread completely, the spreader
is moved forward smoothly and with a moderate
speed
o The drop should be of such size that the film
is 3-4cm in length (approximately 3⁄4 of the
length of the slide)
o It is essential that the slide used as a
spreader has a smooth edge and should be
narrower in breadth than the slide on which
the film is prepared so that the edges of the
film can be readily examined
3. It can be prepared in the laboratory by breaking of
2mm from both corners so that its breadth is 4mm
less than the total slide breadth
o If the edges of the spreader are rough, films
with ragged tails will result and gross
quantitative irregularity in the distribution of
cells will be the rule
o The bigger leukocytes (neutrophils and *Smear appearance depend on drop of blood and how it is
monocytes) will accumulate in the margins spread.
and tail while lymphocytes will predominate
in the body of the film Criteria of a Good Smear
4. The ideal thickness of the film such that there is some
 There should be a gradual transition from the thick
overlap of the red cells throughout much of the film’s
area to the thin area (feathery edge)
length and separation and lack of distortion towards
 The smear must cover 2/3 or 3⁄4 of the slide
the tail of the film
 Must have a smooth and an even surface, free from
5. Thickness and length of the film are affected by
ridges, waves or holes
speed of spreading and the angle at which the
 Must have a feathery edge
spreader slide is held
o The faster the film is spread, the shorter it
will be
o The bigger the angle of spreader, the thicker
will be the film
6. Once the slide is dry, the name of the patient and the
date or a reference number is written on the head of
the film using a lead pencil or graphite
o If these are not available, writing can be
done by scratching with the edge of a slide.
Parts of a Thin Blood Smear
A paper label should be affixed to the slide
after staining  Head – portion of blood film near the drop of blood
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  Body – main part of blood film  Holes in film – slide contaminated with fat or grease
 Tail – tapering end of the blood film and air bubbles
 Cellular degenerative changes – delay in fixing,
inadequate fixing time or methanol contaminated with
water
Rejected Blood Smears
Biologic Causes of a Poor Blood Smear

 Cold agglutinin – RBCs clumping together

o To correct, warm the blood at 37°C for 5
minutes and then, remake the smear
 Lipemia (milky appearance) – holes will appear in the
smear
 Rouleaux – RBCs will form into stocks, resembling
coins. There is nothing you can do to correct this.

Requirements to be able to Produce Proper Blood Smears or

Films

 Use of chemically clean slides and cover slips

 Use of not too large nor too small drop of blood
 Work is done quickly before coagulation of blood
 Proper angle and pressure of the spreader

Factors Affecting the Thickness of the Spread

Unacceptable Smears
 Angle of spreader slide (greater angle, thicker and
shorter the smear is)
 Size of the drop of blood
 Speed of spreading

Note: If Hct increased, the angle of the spreader slid should be

decreased. If Hct decreased, the angle of the spreader slide
should be increased

Common Cause of a Poor Blood Smear

 Drop of blood too small or too large

 Spreader slide pushed across the slide in a jerky
manner
 Failure to keep the entire edge of the spreader slide
against the slide while making the smear
 Failure to keep the spreader slide at a 30° angle with
the slide
 Failure to push the spreader slide completely across
the slide
 Irregular spread with ridges and long tail – edges of
spreader dirty or chipped, dusty slide
Cover Glass Method
- Not usually done in the lab
- 22mm x 22mm cover glasses are required
Procedure
1. Touch a clean cover glass to the top of a small drop
of blood without touching the skin and place it blood

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 side down, cross-wise on another cover glass so that
the corners will be an eight-pointed star
o If the drop is not too large and if the cover
glasses are perfectly clean, the blood will
spread out evenly and quickly in a thin layer
between two surfaces
2. Cover glass should be placed film side up on a clean
paper and allowed to dry in the air. After staining, they
are mounted film side down with permount film side
down on glass slides
Spinner Method
- Blood films that combine the advantages of easy
handling of the wedge slide and uniform distribution of
cells of the coverglass preparation may be made with
special types of centrifuges known as spinners
(hemaspinners)
- This is of little practical significance but it does result
in slightly lower monocyte counts in wedge films
- White cells can be easily identified at any spot in the
film
- On a wedge smear, there is a disproportion of
monocytes at the tip of the feather edge, of
neutrophils just in from the feather edge, and of both
at the later edges of the film
- Spinner slide produces a uniform blood film, in which
all cells are separated (monolayer) and randomly
distributed
Procedure
1. Position a clean glass on a platen
2. Place 3-4 drops of blood in the middle of the slide
3. Close instrument, the platen spins at high speed,
excess blood are expelled into a catch basin
4. Resultant slide is completely covered with a thin
monolayer of cells
Note: Modern spinners contain an optical system that has a
sensor that detects if the cells have separated properly, and
prompts the platen to automatically stop spinning
Additional Notes
 Increase angle of spreader slide if one consistently
make thin peripheral blood smears
 Blood film should be narrower in width than the
microscope slide in which it is fixed
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 Lateral edges should be used in counting the cells
 Smears from anticoagulated EDTA blood should be
made within 2 hours of blood collection
 Smears too thin – drop of blood too small
 Smears too thick – drop of blood is too large
 Presence of ridges/waves – uneven pressure in
spreader slide
 Presence of holes in the smear – slides not clean
Common Causes of Too Thick or Too Thin Blood Smears
Too thick smears
 Too large of a drop
 Too fast a spread
 Angle of spreader slide is too high
Too thin smears
 Too small a drop
 Too slow a forward spread
 Too low an angle of the spreader slide
Parts of Right Blood Smear:
 Thick area – Roleaux formation (coin-like stacking of
RBC) is apparent and WBCs are hardly
distinguishable
 Transition period – better distribution of red cells,
WBCs are more distinguishable
Common Examinations that use Blood Smears
 Differential leukocyte count
 Stained red cell examination
 Platelet count (Indirect method)
 Reticulocyte count
 Malarial parasites (blood parasites) examination
 Thorough study of the morphology of blood cells
Factors Affecting the Quality of a Blood Smear
 Size of the blood drop (smears too thin: drop of blood
too small; smears too thick: drop of blood is too large)
 If too large a drop is used, lower the angle of the
spreader to 45 degrees to make a thin smear
 Angle of the spreader slide – the end of the spreader
slide should be brought back into the blood until it
 spreads along the quarters of the edge of the
spreader slide
 Speed at which the smear is made
 Presence of ridges/waves is due to uneven pressure
in spreader slide
 Presence of holes in smear is due to unclean slides

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