1. A peripheral blood film (PBF) is a thin layer of blood smeared on a microscope slide that is stained to allow examination of blood cells. 2. An ideal blood smear is uniformly thick, occupies the central portion of the slide, and tapers from thick at the head to thin at the tail. 3. Preparation involves using a spreader slide at a 40-45 degree angle to pull blood into a feathered tongue shape from a drop of blood on the slide.

1. Preparation and staining of
Peripheral blood Film
Dr Kamla Choudhary
Assistant Professor
Physiology

2. Definition
“A blood smear is a blood test that gives
information about the number and shape of
blood cells.”
(Textbook of medical physiology- Guyton & Hall)
Alternative Names
Peripheral blood smear (PBF).

3. REAGENTS, EQUIPMENTS
• Reagents-Leishman's stain & distil water/
buffer water
• 4-5 Glass slides, 3 x 1 inch with smooth edge
• Pricking apparatus (needle or Lancet 22 G)
• Compound Microscope

5. Aim of blood smear
•Blood films are usually examined to
investigate hematological problems (disorders of the blood)
and, occasionally, to look for parasites within the blood such
as malaria and filaria.
•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 condition.

6. Three basic steps to make blood film:
1.Preparation of blood smear.
2.Fixation of blood smear.
3.Staining of blood smear.
The peripheral blood film (PBF) is of two types:
1. Thin blood film
2. Thick blood film
This is prepared for detecting blood
parasites such as malaria and microfilaria.

7. A-PREPARATION OF BLOOD SMEAR
Blood films can be made from anticoagulated, or finger-
prick blood
Blood Smear- ***is a thin layer of blood on glass slide.
Blood film- ***
A blood film or peripheral blood film is a thin layer of blood smeared
on a microscope slide and then stained in such a way to allow the
various blood cells to be examined microscopically.
Example of
properly prepared
thick and thin film
blood smears

8. PROCEDURE
1. Take 3 or 4 clean glass slides, the surface of which should be even
and smooth and place it on your work-table,.
2. Prick your finger and allow a medium-sized drop of blood to form on the
finger-tip Or use blood sample as shown in picture
3. Lift a slide from the table, holding it along its long edges. Then touch its
centre, about 1 cm from the narrow end on right side, to the blood drop.

9. 4. Place the slide on a flat surface.
5. With your right hand, place the smooth clean edge of a second
(spreader) slide on the specimen slide, just in front of the blood
drop.
6. Hold the spreader slide at a 40°or 45° angle, and draw it back
against the drop of blood.(Control thickness of the smear by
changing the angle of spreader slide)
PROCEDURE conti……
7. Pull the spreader back gently so that it touches the front of
the blood drop Hold it there, (or move it a little from side to
side), allow the blood to spread almost to the edges of the slide.

10. 8. Push the spreader forward with one light, smooth, and fluid motion
9. A thin film of blood in the shape of tongue or a bullet with a
feathered edge will remain on the slide.
10. Allow the blood film to air-dry completely before staining. (Do not
blow to dry. The moisture from breath will cause RBC artifact.)

12. STEPS FOR BLOOD FILM

13. Parts of a Thin Blood Film
A peripheral blood film consists of 3 parts :
1. Head i.e. the portion of blood film near the drop of
blood.
2. Body i.e. the main part of the blood film.
3. Tail i.e. the tapering end of the blood film.

14. POINTS TO BE NOTED
• A good blood film preparation will be thick at the drop end and
thin at the opposite end.
• As soon as the drop of blood is placed on the glass slide, the
smear should be made without delay. Any delay results in an
abnormal distribution of the white blood cells, with many of the
large white cells accumulating at the thin edge of the smear.
• The blood smear should occupy the central portion of the
slide and should not touch the edges.
• The thickness of the spread when pulling the smear is
determined by the
 1) angle of the spreader slide (the greater the angle, the
thicker and shorter the smear),
 2) size of the blood drop and
 3) speed of spreading.

15. Characteristics /Characteristics /features of an ideal blood Smearfeatures of an ideal blood Smear
1. The blood film should occupy the middle two-thirds (about 5 cm) of
the slide, with a clear margin of about 2 mm on either side.
2. It should be tongue-shaped, i.e. broad at the head (starting point),
and taper towards the other end, but without any ‘tails’.
3. It should be translucent, uniformly thick throughout, with no vacant
areas, striations (longitudinal or transverse), or ‘granular’ areas.
4. It should be neither very thick nor very thin (this can be learned only
with practice). A thin film looks faintly pink against a white surface.

16. Examples of unacceptable smears

17. Examples of unacceptable smears
A: Blood film with jagged tail made from a spreader with achipped end.
B: Film which is too thick
C: Film which is too long, too wide, uneven thickness and made on a
greasy slide.
D: A well-made blood film.

18. Common causes of a poor blood smear:
– Drop of blood too large or too small.
– 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 40 angle with the
slide.
– Failure to push the spreader slide completely across the
slide.

19. B-B- BLOOD SMEARBLOOD SMEAR
FIXATIONFIXATION
&&
C-C-STAININGSTAINING

20. B-Fixing the blood Smear
Fixation is the process that makes the blood smear and its cells
adhere to the glass slide. It also preserves the shape and chemistry
of blood cells as near living cells as possible.
• To preserve the morphology of the cells, films must be
fixed as soon as possible after they have dried.
• It is important to prevent contact with water before fixation
is complete.
• To fix the films, pour 8-12 drops of Leishman's stain on
slide, just enough to cover the smear. Leave it for 2-3
minutes.
This 2-3 minutes duration of time period is known as
fixation time******

21. C-Staining of blood smear
Staining is the process that stains (colors) the nuclei and cytoplasm of
the cells. Both these purposes are achieved by the Leishman’s stain.

22. Various stains for peripheral blood film:
 Romanowsky stains are universally employed for staining of
blood films. All Romanowsky combinations have two essential
ingredients i.e. methylene blue and eosin or azure.
• Methylene blue is the basic dye and has affinity for acidic
component of the cell (i.e. nucleus) and eosin/azure is the
acidic dye and has affinity for basic component of cell (i.e.
cytoplasm).
• Most Romanowsky stains are prepared in methyl alcohol so
that they combine fixation and staining.

23. Various stains included under Romanowsky family
are as under:*****
1. Leishman stain
2. Giemsa stain
3. Wright stain
4. Field stain
5. Jenner stain
6. JSB stain

24. Leishman's stain: belongs to Romanowsky
group of stain
LEISHMAN STAIN
It contains a compound dye—eosin and methylene-blue dissolved in
acetone-free methyl alcohol.
i. Eosin - It is an acidic dye (negatively charged) and stains basic (positive)
gives an orange-red color to hemoglobin and eosinophil granules.
ii. Methylene-blue It is a basic dye (positively charged) and stains
acidic (negatively charged) granules in the cytoplasm, nuclei of leukocytes,
especially the granules of basophils, a blue-violet color and weakly to
granules of neutrophils .

25.  iii. Acetone-free and water-free absolute methyl alcohol. The methyl
alcohol is a fixative and must be free from acetone and water. It
serves two functions:*********
 a. It fixes the blood smear to the glass slide. The alcohol
precipitates the plasma proteins, which then act as a ‘glue’ which
attaches (fixes) the blood cells to the slide so that they are not
washed away during staining.
 b. The alcohol preserves the morphology and chemical status of
the cells.
 The alcohol must be free from acetone because acetone being a
very strong lipid solvent, will, if present, cause crenation, shrinkage,
or even destruction of cell membranes. This will make the
identification of the cells difficult. (If acetone is present, the stain
deteriorates quickly).
 The alcohol must be free from water since the latter may result in
rouleaux formation and even hemolysis. The water may even wash
away the blood film from the slide.

26. Staining the film
• At the end of 2 -3 min. add an equal amount of buffer
water (pH 6.8 ) or double the no. of drops of distilled water
and Mix the stain and buffer slowly by gently blowing air
intermittently.
• Leave the mixture on the slide for 10-15 min. (Staining
Time)*****
• Pour off the stain and wash the slide with tap water
gently.
• Stand slide on end, and let dry in air.

27. Assessment of Stained Blood Smears
• Take an assessment of all the blood films. Examine with
naked eye first, and then under low and high
magnifications.
• Choose the best stained films for cell counting.
• Make sure that you can identify all the cells with certainty.
• Ensure that you are examining the blood smear side of the
slide. Hold the slide in bright light and tilt it this way and
that to see if there are any reflections. The clean side
shows reflections, while the side which has the blood
smear appears dull and does not show any reflections.
(You may also scratch the margin of the smear with a pin).
27

28. Features of a Well-stained Blood Smear
Naked eye appearance.
•The smear appears translucent and bluish-pink when seen against a
white surface, its thickness being uniform throughout. (It is assumed
that an ideal blood film has been stained).
Under low magnifications (100 x).
• The red cells appear as dots, uniformly spread-out in a single
layer. The WBCs cannot be differentiated. There should be no
stain precipitate present on smear.
Under high magnification (450 x or 400 x).
•The red cells are stained dull orange-pink and show a central pallor
(due to biconcavity) which, if wide, may give the appearance of
rings. The WBCs, with their nuclei deep blue-violet, lie unevenly
here and there among the red cells. The platelets occur in small
groups.
28

29. 29
Naked eye
appearance
Under low power of compound
microscope
Under high power of compound
microscope

30. understainedunderstained suitablesuitable overstainedoverstained

31. Causes & correction
• Understained smear/ Too Acid Stain:Understained smear/ Too Acid Stain:
1.1. insufficient staining timeinsufficient staining time
2.2. prolonged buffering or washingprolonged buffering or washing
3.3. old stainold stain
• Correction:Correction:
1)1) lengthen staining timelengthen staining time
2)2) check stain and buffer pHcheck stain and buffer pH
3)3) shorten buffering or wash timeshorten buffering or wash time

32. • Overstained/ Too Alkaline Stain:Overstained/ Too Alkaline Stain:
1.1. thick blood smearthick blood smear
2.2. prolonged stainingprolonged staining
3.3. insufficient washinginsufficient washing
4.4. alkaline pH of stain componentsalkaline pH of stain components
• Correction :Correction :
1)1)check pHcheck pH
2)2)shorten stain timeshorten stain time
3)3)prolong buffering timeprolong buffering time

37. The thickness of the spread when pulling the smear is
determined by :
1. The angle of the spreader slide. (the greater the angle, the
thicker and shorter the smear).
2. Size of the blood drop.
3. Speed of spreading.
Notes:
1. If the haematocrit increased, the angel of the spreader slide
should be decreased.
2. If the haematocrit decreased, the angel of the spreader
slide should be increased.