Assignment Topic

Preparation of Blood Smear,

Thick and Thin Smear,
Wet Preparation and
Buffy Coat Preparation

Group members:
1. Arslan Arshad
2. Muhammad Faisal
3. Muhammad Imran Shahid
4. Muhammad Jalal Serwar Nizami
5. Muteba Arshad
 Blood Smear

• The first and most obvious way to

study the cells of blood is to examine
under the microscope by making blood
smear either thin blood smear or thick
blood smear.
• Blood smear is made from
anticoagulated blood or finger prick on
a microscopic slide and then stained in
such a way to examined blood cells
easily under microscope.
Principle
Smears are prepared by placing a drop of
blood on a clean glass slide and spreading
the drop using another glass slide at an
angle. The slide is then stained and
observed microscopically.

Specimen:
• EDTA specimen
• Smears are made from EDTA
• EDTA blood within 2 to 3 hours
Requirement for Proper Smear
Preparation

Some important requirements are:

• Clean glass slide
• Proper size of blood drop
• Quick, smooth spreading of drop
• Rapid drying of smear
• Proper placement of drop
• Preparation of smear within 3 hours of
collection
Procedure
 Get a finger prick. Discard first two drops
and then take the blood.
 Place the slide on a flat surface.
 Place a 2-3 mm drop of blood about ¼ inch
from the right side of frosted area of the
slide.
 Grasp a second slide(spreader slide) in the
right hand between the thumb and finger.
 Place the spreader slide onto the lower slide
in front of the blood drop, and pull the slide
back until it touches the drop.
 Allow the blood to spread by capillary action
almost to the edges of the lower slide.
Continued…
 Push the spreader slide forward at
approximately 30-40 angle, using a rapid,
even motion.
 Do not press down the spreader slide.
 Perform this step quickly.
 The drop of blood must be spread within
seconds otherwise the cell distribution will be
uneven.
 Label the frosted edge with patient name, ID
and date.
 Allow the blood smear to air dry completely
before staining.
 Do not blow to dry.
Continued…
Slide Method
Proper Smear Preparation

Unstained Giemsa Stained

Distinguishing Good & Bad Smear
Thin and Thick Smear Preparation

Thin Smear:
• Thin blood smear is a drop of blood that is
spread across a large area of slide.
• It is used to confirm if any plasmodium is
present in blood.
• In the edge, the cells should be in a
monolayer, not touching one another while
examining under microscope.
• Used for differential leukocytes count.
• Used for Platelets count.
• Thorough study of morphology of RBC’s.
 Method of Preparing Thin Blood
Smear

Thin smear is prepared by one of three methods:

1. Slide method

2. Cover slip method

3. Spin method
Slide Method
• Slide method is commonly used method for
the preparation of thick smear.
• It uses two slides, one for the smear and the
other serves as spreader.
 Place the spreader slide onto the lower slide
in front of the blood drop, and pull the slide
back until it touches the drop.
 Allow the blood to spread by capillary action
almost to the edges of the lower slide.
 Push the spreader slide forward at
approximately 30-40 angle, using a rapid,
even motion.
Continued…

In step 1 spreader is moved back toward blood drop.

In step 2 spreader is moved in forward direction.
Cover Glass Method

• In this method, cover glass of

(22x22)mm2 area are recommended.
• The cover glass with blood side down
is placed crosswise on another cover
glass so that the corners appear on an
eight pointed star.
• When the blood has spread evenly
between the two surfaces, pull the
cover glasses quickly but firmly apart
on a plane parallel to their surface.
Graphical Representation
Spin Method

• Spin method is done in a cytospin

centrifuge.
• A drop of blood is placed in a center of
glass slide.
• Spin at a high speed in a centrifuge.
• Blood spreads uniformly.
• Dry it and stain it.
Desktop Slide Spinner
Arrangement of Slide and Spinner
Difference Between Wedge Smear & Spin
Smear

Wedge Smear

Spin Smear
 Thick Smear Preparation

• Thick smears consist of lysed/dehemoglobinized

red blood cells.
• The blood elements are more concentrated than
in an equal area of a thin smear.
• Thick smear allows a more efficient detection of
parasites. But they do not permit an optimal
review of parasite morphology.
• Thick smear is often not adequate for species
identification of malarial parasite. If the thick
smear is positive for malaria, the thin smear
should be used for species identification.
Uses of Thick Smear

Thick smears are used in:

• Diagnosis of blood protozoan parasites
• Diagnosis of malaria
• Diagnosis of trypanosomes
• Diagnosis of blood abnormalities e.g.
anemia.
Procedure
• Prepare at least two slides per patient.
• Place a small drop of blood in the center of pre-
cleaned labeled slide.
• Spread it out with a corner of another slide to
cover an area about 4 times of its original area.
• A thick smear of a proper density is one which
if placed over news print, allow you to read the
word.
• Lay the slide flat and allow the smear to dry
thoroughly.
• Do not fix smear with methanol or heat. If
there will be a delay in staining smears, dip the
thick smear briefly in water to hemolyze the
RBCs.
Thick Blood Film Preparation
Thick Blood Film Preparation
Observations
Examine the slide against diffuse light, with
naked eye and observe:
• What is the color of smear?
• Does it appear thick, thin or granular?
• Are there any striations?
• Is it uniformly distributed in the middle, two-
third of slide?
• Is there any hole in the smear?
• Is its head-the starting point straight and
about 1cm from the end?
• Is there a tail?
Continued…
• Are its edge about 2 mm from the
long sides of the slide?

Try to answer all these questions.

Examine the slide under low and high

magnification.

Describe what you see about the various

cells.
Examine Slide with Naked Eye
 Characteristics of Good Smear
Characteristics of a good smear:
• Thick at one end
• Should occupy 2/3 of total slide area
• Should not touch any edge of slide
• It should be tongue shaped
• Has both thick and thick area with
gradual transition
• Does not contain lines and holes

NOTE:
As soon as the drop of blood is placed on the glass slide, the smear
should be made without delay. Any delay result in an abnormal
distribution of white blood cells. Rouleaux of the red blood cells
and platelet clumping may also occur.
Good Blood Smear
Continued…

The thickness of the spread when pulling the

smear is determined by:
• The angle of the spreader slide (the greater
the angle, the thicker and shorter the smear)
• Size of blood drop
• Speed of spreading
• For example, If the hematocrit is increased,
the angle of spreader should be decreased. If
the hematocrit is decreased, the angle of
spreader slide should be increased.
Effects of Angle of Spreader
Causes of Poor Blood Smear

Common causes of poor blood smear:

• Delay in fixing
• Fail to maintain appropriate angle
• Too large or too small drop of blood
• Spreader slide pushed across the slide
in a jerky manner.
• Failure to push the spreader slide
completely across the slide.
• Irregular spread with long tail
• Edge of spreader dirty or chipped.
Continued…
Biological causes
• Lipemia:
Appearance of holes in the smear.
• Rouleaux:
Formation of rouleaux i.e. RBCs will
form stacks resembling coins.
• Cold agglutination:
RBCs will clump together. Warm the
blood at 37 degree Celsius for 5
minutes and then remake the smear.
Precautions
• The slide should be absolutely free
from dust and grease.
• When applying the slide to the blood
drop from a finger picked not touch
the skin with the slide, but only the
periphery of the blood drop. This is to
avoid taking up sweat or other debris.
• The film should be dried by waving it
in the air immediately after spreading
it. Delay can cause not only clumping,
but also crenation and distortion of
red cells.
Wet Mount Preparation
• Wet mount is used for the observation
of living organisms.
• In case of blood, wet mount preparation
is performed for the detection of
Microfilariae in the blood.
Procedure:
• Two or three drops of blood are collected
on a clean glass slide.
• Mixed with two drops of water to lyse
the red cells.
• The preparation is covered with cover
slip and sealed
Continued…
• The preparation is examined under
low-power microscope for the motile
microfilariae, which can be seen
wriggling about.
Buffy Coat Smear
• When the anticoagulated blood is
centrifuged, the three layers are formed
in the centrifuge tube based on the
density
• The bottom layer of RBC’s known as
hematocrit which is normally 45 percent
• The middle layer containing WBC’s and
platelets called the buffy coat layer
which is less than 1 percent of the whole
blood
• The top layer of plasma which is 55
percent
Continued…
 Procedure
• Fill the test tube with anti-coagulated blood.
• Centrifuge the blood at 1000rpm for about 15-25
minutes.
• Remove the supernatant plasma (the fluid above
the buffy coat layer).
• Transfer the buffy coat layer and red cells to the
slide and mix using a pipette. Make a thin film with
the use of a smooth edged spreader.
• Allow a few minutes for the preparation to dry
naturally.
• Once the preparation is dry, fix with ethanol for a
minute or two.
• Stain using Giemsa staining method or another
favorable staining method.
• Examine the preparation under 40X objective.
Diagnostics Significance
• Useful in extracting DNA from the blood of
mammals.
• Helps to detect infection with malaria and
other blood parasites.
• Helps to detect immature or abnormal white
blood cells circulating in the bloodstream.
• Determines cell count in the whole blood.
• Helpful in detecting malarial parasite in
peripheral blood.
• Useful tool in diagnosing filariasis.
• Helpful in diagnosing difficult cases of
visceral Leishmaniasis
Related Video
Thin Smear Preparation

https://youtu.be/R1DU_N6eazg
Related Video
Buffy-Coat Preparation

https://youtu.be/2FJ81vtTfrA
References
• Practical hematology-Islamic university
of Gaza-Health Science Department
• Atlas of clinical hematology,6th edition
by H.Loffler,J. Rastetter, T.Haferlach
• Paniker's text book of medical
parasitology
• A textbook of practical physiology
• District Laboratory Practice in Tropical
Countries Part-1 Monica Chesbrough
• District Laboratory Practice in Tropical
Countries Part-2 Monica Chesbrough
• Manual of Laboratory Medicine