CHAPTER 5

Preparation of Blood Smear, Blood

Plasma and Serum
 Objectives
After completion of this chapter, the students 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

 Preparation of plasma and serum

 Introduction
I. peripheral blood smear
 Examining peripheral blood smears is
an important procedure performed in
The validity or reliability of
the Hematology laboratory. the information obtained
 It is useful in: from blood film evaluation
depends heavily on well-
 Providing diagnostic information made and well- stained
films!!
 Providing additional data

 Guiding the selection and

monitoring of therapy
 Indicating adverse effects of

treatment
 Introduction cont’d

 There are two kinds of blood films

Thin blood film

Thick blood film

Cont’d

II. Clinical significance of blood films:

It is important in the investigation and management of:

 anemia,

infections,

and other conditions which produce changes in the

appearance of blood cells and differential white cell count.

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
 Capillary/Well-mixed EDTA blood sample
 Device to make a drop of blood
 Marking pen or pencil
 Gloves
 Waste and sharps disposal containers
Procedure
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 in
diameter ), 1.0 cm from the end of the glass slide, using either a
plain capillary tube or other type of blood dropping device
1. when the blood is from an anemic patient larger drop of blood
should be used
2. If capillary blood to be used, follow SOP for proper collection
of capillary blood
Procedure cont’d
If using 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 oC 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
 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
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 for blood film preparation
 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 of the blood

 Slides must be clean, not damaged, with no chips or cracks

 A feather edge or bullet shaped appearance 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 i.e., the length of the film

should be 1/2 to 3/4 of the length of the slide

Acceptable Smears Are:

 Straight at the feathered 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

Wright’s stained
blood smear
Sources of error In Making a Smear
 Size of blood drop
Caution!
• Too much blood makes the blood film

thicker
 Wrong angle

• Too low and the smear will be too

long
• Too high and the smear will be too

short
 Speed

 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

Examples of Peripheral Blood Smears
 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

2. Cross- wise on another cover 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 (lower) 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.

 Cont’d

 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.
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 malaria, amastigotes of

visceral leishmaniasis
5.4. Preparation of Plasma and Serum
 Plasma is the fluid part of anticoagulated blood

 Serum is the fluid part of clotted blood

Serum does not contain fibrinogen

Plasma preparation:
Collect the needed volume of blood (5 ml) using a vacutainer

tube containing an appropriate anticoagulant

If syringe method is used for blood sample collection,

dispense the collected blood carefully in a test tube, which

contain the required anticoagulant
Procedure Cont..
 Mix the blood by inverting the test tube

8-10 times
Do not shake

 Centrifuge the blood at 300g 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
Serum preparation
 Collect about 5 ml of blood using plane vacutainer tube (a tube

with no anticoagulant)
 If syringe method is employed for sample collection, collect 5 ml

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
 Cont..
 After complete clotting and retraction centrifuge the clotted blood

at 300g for 10 minutes

 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

Types of Specimens

 Plasma

Plasma contains fibrinogen

Centrifuge whole blood,

separate plasma from cells

Types of Specimens
 Serum

Allow blood to clot for 20 - 30

minutes
Glass tube

Plastic 15- 20 minutes

Centrifuge 10 – 15 minutes,

separate cells from serum

Serum does not contain

fibrinogen
Chemistry Testing
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

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 drop of blood?
7. What is the difference between serum and plasma?
8. What is a buffy coat smear and its application?
9. List at least five common sources of error in blood smear
preparation