Preparation of Blood Smear

Biruk Bayleyegn (BSc, MSc)

January 2022

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 Objectives
 After completion of this chapter, the students will be able to:
 State the importance of a properly made blood smear

 Prepare thin and thick blood films

 Identify the desirable qualities of a good thin blood film

 Describe the effects of the various situation that create unacceptable blood smear
 Apply quality control methods
 Prepare plasma and serum
 Describe the difference between plasma and serum

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 Introduction
 Examining peripheral blood smears is an important procedure performed in the
Hematology laboratory.
 Peripheral blood smear evaluation is the capstone of CBC
• It is useful in:
– WBC and Platelet estimation
– Estimation of relative proportion of different types of WBCs
– To assess morphology of blood cells
– Providing diagnostic information
– Guiding the selection and monitoring of therapy
– Indicating adverse effects of treatment
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 Peripheral blood smear ….
Clinical significance of blood films
– It is important in the investigation and management of:
• Anemia
• Infections
• Other conditions which produce changes in the appearance of blood cells and
differential white cell count
• RBC inclusion bodies
 The validity or reliability of
blood film result is depends on
 Well-made and well- stained
films
 Skilled and talented lab
personnel
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• There are two kinds of blood films:

– Thin blood film

– Thick blood film

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 Difference between thin and thick smear

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 1. Preparation of Thin Blood Films
• A thin blood film is a drop of blood that has been systematically
spread/smeared on a slide.
Thin smear has a monolayer of cells
Sample:
– A free-flowing capillary blood or
– Well mixed EDTA anticoagulated blood
• Should be done within 2 to 3 hours of collection
• >5hours: Echinocytes, spherocytes, vacuolated cells
• Prevent platelet clumping, used to prepare multiple slides
• May cause platelet satellitism and agglutination (Use Sodium
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Citrate) 7
Method of thin blood smear:
– Glass slides (Wedge method) or
– Cover glasses method
– Automated spun smear

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 Wedge method (Two-slide method)
• Also called push slide smear technique

Materials Needed
– Clean microscope slides
– Capillary/Well-mixed EDTA blood sample
– Device to make a drop of blood
– Marking pen or pencil
– Gloves
– Waste and sharps disposal containers
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 Procedure

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

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
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
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 Cont..
4. The drop will spread out quickly along the line of contact of the spreader with
the slide
– As the drop of blood spreads, be careful not to let it spread to both edges of
the spreader
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 is written on the
head of the film using a lead pencil, a diamond marker or a graphite
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 Steps For Blood Film

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 tail body head

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 Factors affecting Wedge Smear
 Thickness and length of the film are affected by:
1. Speed of spreading
2. Angle at which the spreader slide is held
3. Blood volume
 When the blood is from 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.

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 Sources of error in making Thin smear
• Size of blood drop
– Too large blood: thicker and longer blood film
– Too small blood: short or thin blood film
• Wrong angle
– Too low and the smear will be too long
– Too high and the smear will be too short
• Pushing the spreader slide too quickly or too slowly
• Too much pressure on the push slide
• Dirty slides
• Delay in spreading blood drop
• Failure to completely spread blood drop
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 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 i.e., the length of the film should be 1/2 to
3/4 of the length of the slide.

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 Morphologic changes due to area of smear
• Thin area- Spherocytes which are really "spheroidocytes" or flattened red cells.
True spherocytes will be found in other (Good) areas of smear.
• Thick area - Rouleaux, which is normal in such areas. Confirm by examining
thin areas.
• If true rouleaux, two-three RBC's will stick together in a "stack of coins"
fashion..

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 The Cover Glass Method

Material: 22mm  22mm cover glasses are required

Procedure:
1. Place a small drop of blood on a clean cover glass

2. Gently place a second cover glass over it so that the two cover
glasses form a sixteen-sided figure. As soon as the two cover
glasses come together, the blood begins to spread.

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 Cont..
3. Just before the spreading is complete, 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

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 Advantage of cover slip method

• WBC and Platelets are more evenly distributed

• More of the prepared film can be examined

– decrease sampling error
• Used for Bone Marrow aspiration smear

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 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

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 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.

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 Cont’d
Thick smear procedure

1. Place a small drop of blood on a clean slide

2. spread it with an applicator stick or the corner of another slide until small
prints are just visible through the blood smear

3. The prepared smear corresponds to a circle of approximately 2cm

diameter.

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 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 malaria, amastigotes of visceral
leishmaniasis
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 Preparation of Plasma and Serum
• Plasma is the fluid part of anticoagulated blood
• Serum is the fluid part of clotted blood, does not contain fibrinogen

• Plasma preparation: Straw Yellow

– Collect blood using a vacutainer tube containing an appropriate anticoagulant
• If syringe method is used, dispense the collected blood carefully in a test
tube, containing required anticoagulant
– Mix the blood by inverting the test tube, Do not shake

– Centrifuge the blood at 300R for 10 minutes.

– Separate the upper clear fluid (plasma) using long neck pasture pipette and
dispense it in a clean test tube
• Avoid mixing of RBCs with the fluid part
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 Serum preparation
• Collect blood using plane vacutainer tube (tube with no anticoagulant)
– If syringe method is employed for sample collection, collect blood and dispense in dry leak proof glass
tube
– Avoid plastic tubes because blood does not clot well in plastic container
• Allow the blood to completely clot and retract at room temperature
– This might take more than 30 minutes
• After complete clotting centrifuge the clotted blood

• Collect the upper clear fluid (serum) using long neck pasture pipette and dispense it in clean
test tube
– Serum can be stored for several days if kept refrigerated
– For long term storage, store in deep freezers
Note: Fridge temperatures should be monitored and documented

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 Types of Specimens

Serum
Plasma • Allow blood to clot for 20 - 30
• Centrifuge whole blood, minutes
separate plasma from cells • Centrifuge 10 – 15 minutes,
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separate cells from serum 34
 Sources of error in plasma and serum preparation
• Centrifugation time and speed

• Not waiting until blood clots in serum preparation

• Use of wet or unclean test tubes (contaminated with detergent) for

centrifugation

• Improper separation of cells and plasma/serum

• Wringing of the clot with applicator stick to enhance clotting

• Any factors that cause hemolysis during specimen collection affect

the quality of plasma/serum
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 Review Questions/Summary
1. What is a thin blood film?

2. List 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

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drop of blood? 36
 Thank you!!

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 Next : Staining

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