Differences Between Thick Blood Smear and Thin

Blood Smear
Last updated: August 10, 2022 by Sagar Aryal

S.N
Thick Blood Smear Thin Blood Smear
.

1. Thick blood smears are most useful for Thin blood smears helps to discover which
detecting the presence of parasites. species of parasite is causing the infection.

2. A thick blood smear is a drop of blood A thin blood smear is a drop of blood that is
on a glass slide. spread across a large area of the slide.

The blood films must be laked before or The purpose is to allow malarial parasites
3. during staining to rupture all the RBC so to be seen within the RBC and to assess the
that only WBC, platelets and parasites size of the infected RBCs compared to
are visualized. uninfected RBCs

Thick smears allow a more efficient

4. Less sensitive than a thick film especially
detection of parasites (increased
where there is a low parasitemia.
sensitivity 11 times than thin smear).

5. It is not fixed in methanol. It is fixed in methanol.

 S.N
Thick Blood Smear Thin Blood Smear
.

Thin smears allow the examiner to identify

6. Thick smears are mainly used to detect malaria species, quantify parasitemia, and
infection and to estimate parasitemia. recognize parasite forms like schizonts and
gametocytes.

Thick Blood smear

Thick blood film samples a relatively large volume of blood, thus allowing more
efficient detection of parasites (increased sensitivity).

Thick smears consist of a thick layer of dehemoglobinized (lysed) red blood cells
(RBCs), which provides a better opportunity to detect parasitic forms against a
more transparent background. However, they do not permit an optimal review of
parasite morphology.

Making Thick Blood Smear

1. Using the corner of a clean slide, spread the drop of blood in a circle the size of
a dime (diameter 1-2 cm).
 Do not make the smear too thick or it will fall off the slide. (you should be able
to read newsprint through it.)
2. Allow the smear to dry thoroughly. Insufficiently dried smears (and/or smears
that are too thick) can detach from the slides during staining. You can accelerate
the drying by using a fan or hairdryer.
 #Do not fix thick smears with methanol or heat.
3. If there will be a delay in staining smears, dip the thick smear briefly in water to
hemolyse the RBCs.

Quality Control
Visually, the smear should appear as a round to oval smear of blood about 2 cm in
diameter. It should be of such thickness that newsprint can barely be seen through
the wet or dry smear.

Limitation of Thick Smear

 Making a species identification of malarial parasites may be impossible, even for
experienced technicians.
 A thin film should always be examined if a definitive identification based on
morphology is required.
 Smears must be prepared from anticoagulated blood within one hour after
venipuncture. The morphology of parasitic forms and the erythrocytes become
atypical after that time from the direct action of the anticoagulant.
Blood smears should be stained as soon as possible after they are prepared.
Storage of unstained slides for a few days in the hot and humid atmosphere without
staining will result in auto-fixation, and the thick film will be useless for
microscopy.

Thin Blood Smear

Prepared smear

Thin smears consist of blood spread in a layer such that the thickness decreases
progressively toward a monolayer. It allows optimal assessment of the morphology
of any parasitic forms that may be present. Thin blood film is prepared similarly to
the differential white cell count.

Making Thin Blood Smear

1. Bring a clean spreader slide, held at a 45° angle, toward the drop of blood on the
specimen slide.
2. Wait until the blood spreads along the entire width of the spreader slide.
3. While holding the spreader slide at the same angle, push it forward rapidly and
smoothly.
4. Wait until the thin films are completely dry before staining.
5. Fix the thin film with methanol (100% or absolute) for 15-30 seconds and let
it dry completely before staining.
 Note: fixation time for ethanol is 20 minutes

Limitation of a thin smear

 Parasitic forms may be missed in light infections. In such instances, a thick film
must be examined.
 Smears must be prepared from anticoagulated blood within 1 hour after
venipuncture. The morphology of parasitic forms and the RBC become atypical
after that time from the direct action of the anticoagulant.
Giemsa Staining of Thick and Think Blood Smear
P.
falciparum trophozoite stage in thick (right) and thin (left) smear.

Giemsa stain is the most reliable method for staining thick and thin blood films.
Giemsa solution is composed of eosin and methylene blue (azure). The cytoplasm
appears blue (stained by methylene blue), and the nucleus appears red (stained by
eosin).

Counts the number of slides to be stained. Each slide requires approximately 3

mL of stain. If you have 16 slides to stain, you can prepare 50 mL Giemsa
working solution. Two commonly used working solutions are;

1. 10% Giemsa stain working solution: It is used in hospital/diagnostic

laboratories for a quick diagnosis. It is slightly costly, as more stain is consumed.
2. 3% working solution: This cost-effective slow method is mostly used to stain
slides for teaching or epidemiological purposes.
Check the preparation of Giemsa working solution in this blog post.

1. Fix the thin film by carefully dropping methanol onto the thin film using
a Pasteur pipette.
Alternatively, you can dip the thin film for 2 seconds into a small container or
beaker containing methanol. Avoid contact between the thick film and methanol.
Methanol vapors quickly fix the thick film, thus interfering in its hemolysis.

2. Let the blood film dry in the air or on a drying rack or tray.
Allow methanol-fixed thin smear to dry by placing the slides on a flat surface. It
will approximately take 2 minutes to completely air dry the specimen. Never dry
slide in a vertical position with the thin film down, as this may lead to fixing of
the thick film by methanol vapor.

3. Place the slides individually on the staining rack with blood films facing up.
 Ensure that these slides are not touching each other.

4. Gently pour the stain onto the top of the slides until the blood films are
covered.
Each slide will require approximately 3 mL of stain. Avoid pouring the stain
directly onto thick films.

5. Set the timer for the staining. Leave the stain on the slides for 45-60 min (if
you are using 3% Giemsa working solution) or 8-10 min (while using 10%
Giemsa solution).
Optimum staining duration should be determined previously by testing the batch
of stock staining solution used (i.e. your internal quality control will dictate the
exact duration requirement for good staining).

6. Gently flush all the stains from the slides by dropping buffered water (pH
7.2) over them.
Buffered water should be poured onto the slides from the thin film end to avoid
undue disturbance and to wash off the thick films. The surface becomes covered
with a metallic green scum during the staining process. You should avoid getting
it onto blood films during rinsing, as it can impair examination.

7. When the stain has been washed away, remove each slide individually and
allow air drying.
Ensure that thick films are not scraped against the edge of the rack.

8. Discard any remaining Giemsa working solution.

Microscopic examination

Examining the thick film

 Examining
the thick blood films

1. Place the Giemsa-stained blood film to be examined on the microscope stage,

with the label to the left. Position the thick film in line with the 10X objective
lens.
2. Switch on the microscope, adjust the light source optimally and find the focus by
looking through the ocular and the 10x objective.
3. Scan the blood film for parasites and blood elements. Select part of the film
that is well stained and has evenly distributed white blood cells.
4. Place a small drop of immersion oil on the thick film. To avoid cross-
contamination, ensure that the immersion oil applicator never touches the
slide. Do not allow the 40x objective to touch the oil.
5. Switch the 100x oil immersion objective over the selected portion of the
thick film. Use the fine focus adjustment to see the image. Raise the mechanical
stage to avoid damaging the slide.
6. Using the fine adjustment, focus on the cell elements, and confirm that the film
is acceptable for routine examination; 15-20 white blood cells per thick film
field will give a satisfactory film thickness. Films with fewer white blood cells
per field will require more extensive examination.
7. Examine the slide systematically. Start at the top left of the film (marked with a
vertical green arrow) and begin at the periphery of the field, then move
horizontally to the right, field by field.
8. When the other end of the film is reached, move the slide slightly downwards,
then to the left, field by field, and so forth. For efficient examination,
continuously focus and refocus with the fine adjustment throughout the
examination of each field.
9. Examine the thick film under the oil immersion objective, field by field,
horizontally or vertically. Use the fine adjustment to focus.
10.A minimum of 100 high-power fields must be examined before a thick film
can be declared as having “no malaria parasites seen.” If possible, the whole
thick film should be scanned.
11.If parasites are found, scan additional 100 fields to increase the chance of
identifying mixed infections.
12.Identify all species and stages observed, and record them.

Examining Thin Films

The thin blood film should always be examined to identify parasite species or
mixed infections after examining the thick film. Unlike the thick film, the thin film
allows visualization of parasite and red cell morphology. Perform an examination
at the feathery end or edge of the thin film.
 Examining the
thin blood films

1. Place a drop of immersion oil on the feathered edge of the thin film.
2. Move from the 10x lens to the 100x oil immersion lens.
3. Examine the feathery end of the edge of the thin film where red cells lay side by
side, and there is minimal overlap. Follow the pattern of movement shown in
Fig. 2. Move along the edge of the film, then move the slide outwards by one
field, inwards, returning in a lateral movement, and so on.
4. Continue examining the thin film until the presence and species of malaria
parasites have been confirmed. Identify and record all species and stages
observed in the malaria microscopy blood register.
.

Thick and Thin Blood Smears for Malaria—Overview

Doctors use thick and thin blood smears to determine whether you have malaria. If
one test is negative and no parasites are found, you will have repeated blood
smears every 8 hours for a couple of days to confirm that there is no malaria
infection.

Blood smears are taken most often from a finger prick. Thick and thin blood
smears will let doctors know the percentage of red blood cells that are infected
(parasite density) and what type of parasites are present.

 A thick blood smear is a drop of blood on a glass slide. Thick blood smears
are most useful for detecting the presence of parasites, because they examine
a larger sample of blood. (Often there are few parasites in the blood at the
time the test is done.)
  A thin blood smear is a drop of blood that is spread across a large area of the
slide. Thin blood smears helps doctors discover what species of malaria is
causing the infection.


Why It Is Done

Introduction
A well-prepared blood smear is important to produce good results on
analysis after doing a Giemsa stain, in identifying blood cells or/and
demonstrating the presence of parasites in a sample. Below, we discuss the
procedures for preparing both thin and thick smear for Giemsa
staining technique, Importance, and applications of blood smears, in detail.
Blood smears are mostly done for Differential Leukocyte count (DLC)i.e it
quantifies the white blood cells and specifies the morphologies of each
leukocyte. Normally, peripheral blood is used to prepare smears and
depending on the function of the smear, two types of smear can be
prepared.
a. Thin blood smear – for demonstration and differentiation of leukocytes.
b. Thick blood smear – for diagnosis of blood protozoan parasites and
blood abnormalities eg anemiae.
 Image Source: Haematology in a NutShell, Microbiology Info, and
DOI: 10.5336/caserep.2015-47850
NOTE: Dry smears are the best for staining, so ensure your smear is
completely dry before applying a staining technique.
Blood smears are simple procedures to perform aiming at demonstrating
and acquiring information on blood cells, qualitatively and quantitatively.
Quantitative importance enables the numbering of blood cells while the
qualitative function is to demonstrate and identify the cell morphologies,
including types of leukocytes, erythrocytes, monocytes, and platelets.
Blood smears have also been used in detecting hematological disorders i.e
by observing the morphologies and quantifying the cell numbers.
Objectives
1. To prepare a thin blood smear
2. To prepare a thick blood smear
Requirements
 Sterile syringe & needle
 EDTA vials
 Tourniquet
 70% isopropyl alcohol
 Sterile Lancet
 Microscopic glass slides
Thick Blood Smear Preparation
Specimen: Venous blood sample
Principle
Thick blood smears require larger volumes of blood than the thin blood
smears. this allows them to be used for the detection of blood parasites in
the blood samples. A thick blood smear is made by spreading a large blood
drop in a small area of about 1 cm which provides a better opportunity to
detect various parasitic forms against a more transparent background.
Procedure
 Collect blood sample by venipuncture and put in a clean test tube
 Using a capillary tube collects blood from the tube and put two large
drops at the center of a sterile microscopic slide.
 Holding the slide between your thumb and index finger, gently shake the
slide to spread the blood about 10mm in diameter.
 Air-dry the smear for 20-30 minutes till its completely dry then apply the
appropriate Romanowski stain.
Thin Blood Smear Preparation
Specimen: Peripheral Blood sample
Principle
The Thin Blood smear is prepared by making a drop of well-mixed venous
blood, 2mm in diameter at the center of a sterilized microscopic glass slide.
Some borders are left around the smear for easy counting and
differentiating of the cells. A second glass slide is used as a spreader,
streaking the blood into a thin film across the glass slide. This preparation is
allowed to dry and then fixed with an appropriate Romanowski stain,
depending on your objective.
Procedure
 Using a sterile pricking needle, make a prick on the index finger
 apply some pressure on the finger and put two drops of blood at the
edge, leaving a margin on a sterile Microscopic slide.
 Place the edge of the sterile microscopic slide over the drops of blood, at
an angle of 30-450, and make two streaks rapidly but smoothly forward
from the blood sample and spread it. This will leave a thin film of blood
resembling a tongue-shape.
 Allow the slide to air dry and stain with an appropriate staining
technique.
Applications of blood smears
 For classification of blood disorders including types of anemia, bleeding
disorders
 To characterize blood-related disorders such as leukemias
 To detect immune-mediated inflammatory disorders and infections
 To detect protozoal parasites: Plasmodium falciparum, Mycoplasma spp
(Mycoplasma haemofelis and Mycoplasms haemominutus and Bacteria
such as Bartonella spp.
Advantages
 It is a rapid simple technique which requires basic equipment
 It can be performed with very small volumes of blood.
Disadvantages
 Use clean slides to avoid the formation of grease spots (holes in the
smear).
 Rapid air drying of smear to preserve cell morphologies
 Regular use of the technique to produce useful blood smears

What is the principle of Giemsa stain?

The stain is also used for the demonstration of some microorganisms.
PRINCIPLE: The “neutral” dyes combining the basic dye methylene blue and the
acid dye eosin, give a wide color range when staining. The pH of the staining
solution is critical and ideally should be adjusted for different fixatives.