Peripheral Blood Smear Examination

(Blood film)

Dr: AMER WAHAN

 Peripheral Blood Smear Examination

outline
 Introduction
 Stains for Blood Smear
 Preparation of the Peripheral Blood Smear
 Fixation of the Smear
 Staining of the Smear
Examination of a Peripheral Blood Smear
 Red Blood Cells
 White Blood Cells
 Platelets
 Hemoparasites

INTRODUCTION
Peripheral smear (peripheral blood film) is the most important, valuable and
frequently asked investigation in hematology laboratory. It provides the following
information:
• Red cell morphology: Morphological features of RBCs are important for diagnosis
of anemias and other hematologic disorders.
• WBC disorders and differential leukocyte count (DLC): Quantitative and
qualitative changes in WBCs help in diagnosis of both hematologic and non-
hematologic disorders.
• Platelet number and morphology: These features are useful in the diagnosis of
bleeding disorders.
• Cross check the CBC parameters: It helps in cross-checking the complete blood
count (CBC) parameters derived from automated cell counters.
• Detection of blood parasites (Hemoparasites).

STAINS FOR BLOOD SMEAR

• The blood cells contain cellular structures which vary in their reaction (pH), some
are acidic and others being basic.
• The aniline dyes used in staining blood smears are of two general classes: basic dyes
such as methylene blue and acidic dyes such as eosin.
• All stains which are made of combinations of acidic and basic dyes are called
Romanowsky stains. The differences between the various Romanowsky stains are
mainly in the proportion of the reagents and in their preparation.

Romanowsky Stains
A mixture of methylene blue (basic stain) and eosin (acidic stain) was first prepared
by Romanowsky in 1891 and employed on his work on malarial parasite. The action
of these stains depends on compounds formed by the interaction of methylene blue

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and eosin. Methylene blue on oxidation produces colored compounds called azures
that have the ability to combine with eosin. Oxidation is achieved during
maturation/chemical treatment of the stain. The azures are responsible for different
shades of staining in the smears.
• Nuclei and structures in the blood which are stained by the basic dyes are called
basophilic.
• Structures that take up only acidic dyes are called acidophilic or eosinophilic.
Romanowsky group includes the following stains:
• Leishman stain which contains Leishman powder and acetone free methyl alcohol
(methanol).
 Preparation of stain: Dissolve 1 gm of Leishman powder in 500 ml of acetone free
methyl alcohol in a conical flask. Warm it at 500 C for 15 minutes. Keep it for
ripening by exposing to sunlight for 2 days. Acetone free methyl alcohol acts as
a fixative. Since acetone destroys the cells, methyl alcohol should be free from
acetone.
• Giemsa stain contains Giemsa stain powder, glycerol and acetone free methyl
alcohol.
 Preparation of stain: Dissolve 1 gm of Giemsa stain powder in 66 ml of glycerol
at 60° C for 2 hours. After cooling add 66 ml of acetone free methyl alcohol and
mix it. Keep it at 37° C for one week for ripening.
• Wright stain
• Jenner stain
• Jenner-Giemsa stain

PREPARATION OF THE PERIPHERAL BLOOD SMEAR (FIGS 1. A TO D)

• A drop of blood is placed on a clean grease free glass slide 1 cm away from one end.
• Take a spreader (either another slide or a narrower piece of slide with smooth edge)
and placeits smooth edge over the drop of blood so as to spread the blood along its
edge.
• Place the slide at about 30° to 40° angle and make the smear with a smooth (not
jerky) forward movement. The resulting ideal smear should be about 2.5 to 3.5 cm
in length.
• Allow it to dry at room temperature and label.

Features of a Well-made Peripheral Smear

• Length: Smear should be 2/3 to 3/4 of the length of the slide.
• Shape: The smear should be either tongue or finger-shaped (smooth without any
irregularities or holes).

Sources of Error
• Presence of grease on the slide causes blank oval areas in the smear.
• If spreader edge is not smooth, the tail of the smear shows ragged edges and most
of the neutrophils accumulate in the tail end. The rest of the smear shows paucity of

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 neutrophils, a process termed as tailing of the smear. This results in erroneous
differential counts.

Figs 1A to D: Different steps in the preparation of peripheral blood smear and appearance of a well-
made smear

FIXATION OF THE SMEAR

• The blood smears should be fixed within 4 hours for good staining.
• Blood films are ‘fixed’ by acetone-free methyl alcohol which is present in most of
the commonly used stains, namely Leishman or Wright or Giemsa stain.
• It can also be fixed by dipping the slide 8 to 10 times in a coplin jar with methyl
alcohol.

STAINING OF THE SMEAR

Staining of the smears is carried out using Romanowsky stains which stain red cells,
white cells, platelets and parasites.

Procedure of Staining with Leishman Stain

• Keep the peripheral smear on the staining rack.
• Pour the undiluted stain on the slide with a dropper so that stain covers the entire
upper surface of slide.
• Wait for 2 minutes for the methyl alcohol to fix the smear. Then pour double the
amount of buffer water on the slide. Gently mix using a dropper.
• Wait for another 8-10 minutes (time depends on the batch of stain) for staining.
Look for greenish metallic scum to appear.

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• Displace the stain by pouring running water from one side of the slide so that the
stain scumis drained off the slide and no more stain is left on the slide.
Precaution: Do not discard the stain from slide by just tilting it, since it will result
in stain precipitates over the smear.
• Remove the slide from staining rack and keep it in slanting position to dry it up.
• Examine the slide under microscope.

Sources of Error
• Buffered pH of 6.8 is important for good quality of staining.
 Acidic pH red cells appear pink and WBCs takes up very light stain. 
 Basic pH red cells appear blue and WBCs appear bluish-black.
• Staining errors:
 If smear is overstained, wash the smears with undiluted Leishman stain or
methanol for few seconds.
 If smear is under stained, restrain by adding both Leishman stain and buffer for
required period depending on the extent of under staining.

Procedure of Staining with Giemsa Stain

• Peripheral smear prepared is fixed by dipping 8 to 10 times in a coplin jar containing
methanol.
• Allow the slide to dry.
• Pour the diluted stain (1 in 10) and wait for 20-30 minutes.
Note: The amount of dilution and time for staining varies with each batch of stain.
• Wash the slide (as in staining by Leishman) and dry it.

EXAMINATION OF A PERIPHERAL BLOOD SMEAR

A well prepared peripheral smear usually has three parts namely head, body and tail.
The best area of the smear to study the morphology of cells is the body, where the cells
are well separated out and touch one another without overlapping. Apply a thin layer
of oil over the smear which makes the appearance of cells very clear.
First look under low power 10x, followed by high power 40x and oil immersion.
Observe the following:
• The quality of the film and uniformity of staining.
• Red blood cells: Select an area (best between tail and body of the smear) suitable
for examination of red cells in the thin area of the smear where the red cells are
evenly placed and just touch each other. Look for number, distribution and staining
of the red cells, degree of rouleaux formation if present.
• White blood cells: Number and distribution of white cells
• Platelets: Number and distribution
• Hemoparasites (such as malaria, microfilaria)
• Any abnormal cells.

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RED BLOOD CELLS
Red cells are examined for (1) size (2) shape (3) color (4) inclusions and (5) other
abnormalities.
1. Size: In the peripheral smear, size of the red blood cells is compared with that of a
small lymphocyte which has size of about 7-8 μm. The red cells are called microcytic
if smaller than small lymphocyte and macrocytic if larger than 9 μm. Appearance of
size variation and conditions responsible for these variations are shown in Fig.2.

Fig.2: Variation in size of red blood cells and associated conditions

2. Shape: Variation in red cell shape and conditions responsible for these variations
are shown in Figs 3 A and B.

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Fig.3A

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 Fig. 3B
Figs 3A and B: Variation in shape of red blood cells and associated conditions

3. Color: Variations in red cell color are depicted in Fig..4.

• Sometimes red cells appear to be hyperchromic without a central area of pallor.
This is not actually due to an increase in the concentration of hemoglobin in the
cell, but due to change in cell shape and increase in the thickness of the cell
membrane in macrocytes.

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 Fig. 4: Variation in color of red blood cells

4. Inclusions in the red cells: Inclusions in red blood cells and associated conditions
are presented in Fig. 5.
5. Other abnormalities: Example, rouleaux formation (Fig.6).

WHITE BLOOD CELLS

White cells (leukocytes) are examined for the following features:
• Number: Note whether the number is decreased, normal or increased.
Assessment of approximate total white cell count: Observe at least 10 high
power fields in the thick portion of the smear where red blood cells overlap.
Estimate the approximate number of WBCs/high power filed. Each WBC/high
power field is approximately equivalent to 2,000 WBC/cu mm.
• Abnormal or immature forms
• Predominant cell type: This is determined by performing the differential white cell
count. At least 100 white cells should be counted. Normal range of differential
leukocyte count is shown in Table 2

Table 2: Normal range of different leukocyte count (DLC)

Type of white blood cells Normal range

Granulocytes
Neutrophil (polymorphonuclear leukocyte) 40-70% (2.0-7.0 × 109/L)

Eosinophil 1-6% (0.02-0.5 × 109/L)

Basophil 0-1% (0.02-0.1 × 109/L)
Lymphocytes 20-40% (1.0-3.0 × 109/L)

Monocyte 2-10% (0.2-1.0 × 109/L)

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Fig 5. Inclusions in red blood cells and associated conditions

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 Fig 6: Rouleaux formation and associated conditions

PLATELETS
They are examined for the following features under oil immersion.
A single platelet under oil immersion is equivalent to 15,000 to 20,000 platelets/cu mm.
• Number: Normal/ increased/decreased
• Form: Note the size and shape.
• Aggregation: Smears made from finger-prick show aggregation of platelets or
platelet clumps.

HEMOPARASITES
These include (1) malarial parasites, (2) microfilaria, (3) trypanosomes, (4) Leishmania
Donovani and (5) babesiosis
1. Malaria: Malarial parasites can be demonstrated in the peripheral smear; most
common are plasmodium vivax and falciparum.
Thick Film for Malarial Parasite: Thick film method is preferred for mass surveys,
quick diagnosis and when malarial parasites are likely to be few. It is advisable to
make both thin and thick films on the same slide.
Thick blood films can be stained by Field’s rapid method or Jaswant-Singh-
Bhattacherji (JSB) stain.

2. Filariasis: Microfilaria can also be demonstrated in peripheral blood (Fig.7B).

3. Trypanosomes: These are motile flagellate protozoa.
4. Leishmania Donovani: It causes kala-azar, amastigote forms known as LD bodies
can be found in the reticuloendothelial cells of the bone marrow, spleen and buffy
coat preparations of peripheral blood. LD bodies are small, round, 2 to 4 µm in
diameter with a nucleus and a rod shaped kinetoplast.
5. Babesiosis: It is a malaria like parasitic disease caused by babesia.

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 A

Figs 7A and B: Plasmodium vivax ring (A upper), schizont (A left lower) and falciparum gametocyte (A
right lower) and microfilaria (B)

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Reporting of Peripheral Smear:
Format for peripheral smear reporting is shown in Table 3.

Table.3: Format for reporting peripheral blood smear

1. RBCs: Comment on the following features

 Size: Normocytic (normal), microcytic or macrocytic (predominant
feature to be mentioned)
 Chromasia Normochromic (normal), hypochromic, absence of
central pallor
 Anisocytosis : Present (slight /marked) or absent
 Poikilocytosis: Present (slight /marked) or absent
 Other abnormalities: Spherocytosis, sickle cells, target cells
 Polychromatophilia: Present or absent
 Nucleated red cells: Normoblasts- per 100 leukocytes
2. WBCs:
 Comment on total WBC count as normal/increased /decreased
 Differential Count (on 100 cells) Type of WBC (percentage)
o Neutrophils
o Eosinophils
o Basophils
o Lymphocytes
o Monocytes
 Immature leukocytes: Specify different cells
3. Platelets: Approximate judgment on adequate/increased /reduced
4. Hemoparasites

SUMMARY

• Peripheral blood smear examination is the most important and valuable

investigation which is done in a well-prepared and well stained smear. Smears are
stained by Romanowsky stains, most commonly Leishman and Giemsa stains.
• Examination of peripheral blood smear in a systematic manner gives a lot of
information in both hematological and non-hematological diseases.
• In the RBC series, examine size, shape and color. In the WBC series look for number
and Distribution of white cells. Examine platelets for number and distribution.
Search for any hemoparasites (e.g. malaria, microfilaria) or abnormal cells.

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