CHAPTER 5

Preparation of Blood Smear

Objectives

After completion of this chapter, the student will be

able to:
 State the importance of a properly made blood smear
 Prepare thin blood films on slides and cover glasses
 Identify the desirable qualities of a good thin blood film
 Prepare thick blood films
 Describe the effects of the various situation that create
unacceptable blood smears
 Prepare plasma and serum
 Describe the difference between plasma and serum
 Apply quality control methods
Outline

 Preparation of Thin film

 Preparation of thick blood smears
 Preparation of other types of smears
 INTRODUCTION
i. peripheral blood smear
 Examining peripheral blood
smears is an important
procedure performed in the The validity or reliability
Hematology laboratory. of the information
 It is useful in: obtained from blood film
 Providing diagnostic information evaluation depends
(anemia, infections, and other heavily on well-made
conditions which produce changes and well- stained films!!
in the appearance of blood cells )
 Guiding the selection and
monitoring of therapy
 Indicating adverse effects of
treatment
 Differential white cell count.
Introduction cont’d

 There are two kinds of blood films

 Thin blood film
 Thick blood film
 Thin or thick 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, but not later than 3 hours.
5.1 Preparation of Thin Blood Films

 A thin blood film is a drop of blood that has been

systematically spread/smeared on a slide and which has
more or less a monolayer of cells
 Blood samples for smear preparation can be obtained
from:
 a free-flowing capillary blood or
 well mixed EDTA anticoagulated blood
 It can be prepared using:
 glass slides (Wedge method) or
 cover glasses method
 The most common method is the wedge or push slide
smear technique
5.1.1. Wedge method (Two-slide method)

 Compared to the cover glass method, preparation of

blood films on glass slides have the following
advantages
 Slides are not easily broken
 Slides are easier to label
 When large numbers of films are to be dealt with,
slides will be much easier to handle
Materials Needed

 Clean glass microscope slides

 Well-mixed EDTA blood sample
 Device to make a drop of blood
 Marking pen or pencil
 Gloves
 Waste and sharps disposal containers
 Making a spreader slide
 Make a blood spreader from a slide which has ground
glass polished sides as follows:
 Only good quality slides will have ground glass polished
sides. Such sides are essential to make blood
spreaders.
 Examine each end of the slide and select the end
which is completely smooth, i.e. no chips in the glass.
If one end of the slide is frosted, use the non-frosted
end (ensure it is smooth).
 Using a glass marker and glass cutter, etch across a
corner of the slide as shown in Fig.
 Holding the slide between a piece of cloth, break off
the corner and discard safely the broken off piece of
glass.
 Procedure for preparation of thin blood film

1.Use a glass slide that is free of dust, grease and debris

Note: It is essential to ensure slides are washed free from
traces of detergent and the surface of the slide is
completely clean and not greasy.

2. Place a small drop of well mixed EDTA blood (about 2-3

mm), 1.0 cm from the end of the glass slide, using
either a plain capillary tube or other type of blood
dropping device
 when the blood is from an anemic patient larger drop
of blood should be used
 If capillary blood to be used, follow SOP for proper
collection of capillary blood
Procedure cont’d
 Ifusing an anticoagulated blood sample, be sure the
specimen is well-mixed

 The drop should be placed (opposite from the end that

is frosted, if that type of slide is provided) and in the
middle of the slide
3. the spreading slide is placed in front of the drop of blood
at an angle of about 30o -40 o to the slide and then is
moved back to make contact with the drop
Cont..
4. The drop will spread out quickly along the line of contact
of the spreader with the slide
5. The spreader is advanced with a smooth steady motion
so that a thin film of blood is spread over the slide
6. Allow the smear to air-dry
 Do not blow on the smears as this can disrupt cellular
morphology and cause the formation of unwanted
artifacts, like target cells
 Also do not use heat for drying
7. Label the name of the patient and date or reference
number on the head of the film using a lead pencil, a
diamond marker or a graphite
Technique for Making a Wedge Smear
 Thickness and length of the film
are affected by:
 speed of spreading
 the angle at which the
spreader slide is held
 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.
Technical tips
 Mix EDTA anticoagulated blood properly before making
the smear
 Use capillary tubes to deliver the original drop of blood –
they are easier to control
 The angle of push may need to be adjusted depending
on the viscosity and volum of the blood
 Slides must be clean, not damaged, with no chips or
cracks
 Smooth and bullet shaped appearance of film indicates
a good smear
 The faster the film is spread the thicker and shorter it will
be
 The bigger the angle of spreading the thicker will be the
film
Technical tips cont’d

Caution!
During smear preparation,
after applying the small drop
of blood, if the drop sits for
longer than 3-5 seconds
before spreading, clumping
of platelets and white cells,
and rouleaux formation of
the RBCs occurs!
Acceptable Smears are:
 Smooth, homogeneous and have no lines,
vacuoles or holes, jerks, streaks, or ridges
 Note: ‘Holes’ in a blood film are usually caused
by using a slide, which is not clean (greasy)
 Lines across a film are usually due to spreading
a film jerkily

 Availability of sufficient examination area (working

area) i.e., the length of the film should be 1/2 to 3/4
of the length of the slide
Acceptable Smears Are:
 smooth at the edge
 A jagged ‘tail’ to a blood film is caused by
using a spreader with a chipped end or end
that is not clean.
 Free of visible clotting
 Not too thick and not too thin
 Gradual transition to thickness from the thick to
thin areas terminating in smooth ‘tail’
Characteristics of an
Acceptable Smear

Smooth edge
‘Feathered’

Origin of
blood drop
Smooth, homogeneous;
no clots, holes, bubbles

½ to ¾ length
of slide
Card no- 2453/09

Date – 12/09/09
Pt name- Almaz

Wright’s stained
blood smear
Sources of error In Making a Smear

 Size of blood drop

 Too much blood makes Caution!
the blood film thicker
 Wrong angle
 Too low and the smear will
be too long
 Too high and the smear
will be too short
 Too much pressure on the
push slide
Sources of error cont’d

 Dirty slides
 Delay in spreading blood drop
 Failure to completely spread blood drop
 Stopping abruptly before completely spreading the blood
drop
 Pushing the spreader slide too quickly or too
slowly(speed)
 Too quick- thicker and shorter film
 Too slow- longer and thinner film
Examples of Peripheral Blood Smears

Figure. Blood films made on slides. A: A well-made film. B: An irregular patchy film on a dusty
slide. C: A film that is too thick and made with jerky movement. D: A film that has been spread
with inconsistent pressure and using an irregularly edged spreader, resulting in long tails. E: A film
made on a very greasy slide.
Fixing thin blood films

 Fixing helps to preserve the morphology of the cells and

attach (stick) the cells on the slide
 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.
 Satisfactory fixing of thin blood films requires the use of
water-free absolute methanol.
 When absolute methanol is not available, absolute
ethanol (ethyl alcohol) can be used but this is more
expensive and usually less available than methanol.
Advantage of thin blood film
 Important in confirmation of the plasmodium species
 Enable us to see the different features of RBC as a
result of being parasitized by malaria parasites and /or
other causes (RBC morphology)
 Greatly assists in the diagnosis of mixed infections.
 Makes possible the calculation of percentage (%) of
parasitized RBC so important in follow-up of malaria Rx.
 Other??
 Summary of thin blood film (two slide method) preparation

Figure: Collecting drop of blood Figure: position the spreader in front of the blood

Figure: drow the spreader back until it touch the blood Figure: allow the blood to flow along the edge of the spreader

Figure: push the spreader to the end of the slide with a smooth Figure:(A) Correctly and (B) incorrectly prepared
movement blood film
5.1.2 The Cover Glass Method

Material: 22mm  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
(when using capillary blood)
and place it down
Figure: blood film preparation using cover
2. Cross- wise on another cover glass method

glass so that the corners will

appear as an eight-pointed
star.
Cont..

- 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 the two surfaces.
3. Separate the two cover slips by pulling them in opposite
direction
4. Cover glasses should be placed film side up on a clean
paper and allowed to dry in the air
5. After they are stained they are mounted with DPX
mountant film side down on glass slides
Advantage of cover slip method

 WBC and Platelets are more evenly distributed

 More of the prepared film can be examined
 decrease (low) sampling error
 Used for Bone Marrow aspiration smear
Advantage of Slide method

 Slides are not easily broken

 Slides are easier to label and stain
 When large numbers of films are to be dealt with, slides
will be found much easier to handle.
 Easier to learn performing the technique
 Unlike the cover slip method, RBCs are well distributed
5.2. Preparation of thick blood film

 Thick blood smears are widely used in the diagnosis of

blood parasites particularly malaria.
 It gives a higher percentage of positive diagnosis in
much less time since it has ten times the thickness of
normal smears.
 Five minutes spent in examining a thick blood film is
equivalent to one hour spent in traversing the whole
length of a thin blood film.
Procedure

 Place a small drop of blood on a clean slide

 spread it with an applicator stick or the corner of another
slide until small prints are just visible through the blood
smear
 The prepared smear corresponds to a circle of
approximately 2cm diameter.
Fig. Preparing a thick blood film Fig. Labelling the slide
5.3. Other types of smears

Other types of smear preparation include:

 Automated spun smear
 Buffy coat smear

 Buffy – coat smear

 Is a smear prepared from buffy coat layer (the layer
that contains WBC and Platelets)
 Has a great value especially in leucopenia
 Also important in the diagnosis of amastigotes stages
of visceral leishmaniasis
Review Questions/Summary
1. What is a thin blood film?
2. List two types of methods for making a thin film
3. Which technique of blood film preparation is commonly
employed and how is the method of preparation?
4. What are the desirable qualities of a thin blood film?
5. What are the possible effects of using a blood sample
that has been standing at room temperature for some
time on blood cell morphology?
6. What is the effect of delaying in spreading after applying
the small drop of blood?
7. What is a buffy coat smear and its application?
8. List at least five common sources of error in blood
smear preparation