Staining and Examination of Blood films
1 By Biruk B 11/16/2022
Learning Objectives
At the end of this chapter, the student will be able to:
Explain the general principle of staining thin blood films in hematology
List commonly used Romanowsky dyes
Perform the technique of staining thin blood films with Romanowsky dyes
Prepare Wright and Leishman stain in the right concentration
Describe the appearance of cells and cell components in Romanowsky-
stained blood films
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Objectives cont’d
Prepare Giemsa and Field stains in the right concentration
Stain thick blood films with Giemsa and Field stain
Explain the principle of thick blood film staining with Giemsa and Field stains
Define panoptic staining
Stain thin blood films with one of the panoptic stains
Identify the advantage of panoptic staining over the simple Romanowsky dyes
List the problems that arise in staining and the possible remedies
Perform quality control for staining
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Outline
Introduction
Principle of staining
Romanowsky Stains
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6.1. Introduction
There is little consistency between laboratories in the precise stain used to
prepare a blood film for microscopic examination, but the multiple stains in use
are based on the Romanowsky stain.
Romanowsky stain was developed by the Russian protozoologist in the late
nineteenth century (1890)
He used a mixture of old methylene blue and eosin to stain the nucleus of a
parasite purple and the cytoplasm blue.
Subsequently, Giemsa modified the stain, combining methylene azure and
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Introduction cont’d
The stain most commonly used in the UK is a combination of Giemsa’s stain
with May-Grünwald stain; it is therefore designated the May-Grünwald–
Giemsa (MGG) stain.
The stain most commonly used in North America is Wright’s stain, which
contains methylene blue
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Principle of Staining
Acidic dyes such as eosin unite with the basic components of the cell
(cytoplasm) and hence the cytopolasm is eosinophilic (acidic).
Conversely, basic stains like methylene blue are combine with the acidic
parts of the cell (nucleic acid and nucleoproteins of the nucleus) and hence
these structures are called basophilic.
Other structures stained by combination of the two are neutrophilic
Romanowsky stains are commonly used in hematology
7 By Biruk B 11/16/2022
Romanowsky Stains
Contain:
eosin Y an acidic anionic dye and
azure B and other thiazine dyes derived from the oxidation or polychroming
of methylene blue are basic cationic dyes
When Romanowsky dye is diluted with buffered water, ionization occurs
Eosin ions are negatively charged and stain the basic components of blood cells
e.g. hemoglobin stains pink –red, and the granules of eosinophils stain orange
red 8
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Romanowsky Stains cont’d
Azure B and other methylene blue derived dyes are positively charged and
stain the acidic component of cells.
Nucleic acids and nucleoprotein, stain various shades of mauve-purple
and violet
Granules of basophils stain dark blue –violet
Cytoplasm of monocytes and lymphocytes stain blue or blue gray.
The staining reaction of Romanowsky stains is pH- dependent, that is why
the stains are diluted in buffered water
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of specific pH. 9
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Romanowsky Stains in Common Use
Wright stain
Peripheral smears
Leishamn
Peripheral smears
Geimsa
For malaria thick films
12 By Biruk B 11/16/2022
1. Wright Stain
Methylene blue is polychromed by heating with sodium carbonate
It is purchased as a solution ready to use or as a powder
Staining properties of Wright’s stain deteriorate rapidly
When the stain absorbs moisture or
If stored at high temperatures or in bright sunlight.
Wright’s stain should also be renewed every 3 months and should be left for 3–5
days before being used
pH of buffered water should be 6.8
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Wright staining procedure
1. Place the air-dried smear film side up on a staining rack (two parallel glass
rods kept 5cm apart).
2. Cover the smear with undiluted stain and leave for 3 minute
Note: The undiluted stain not only acts as a fixative but also partially stains
the smear. This stage is required to obtain the best possible staining results.
3. Add equal the volume of pH 6.8-buffered water (i.e., the same number of
drops as the stain)
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Wright staining procedure cont’d
4. Mix by blowing until a metallic sheen (green ‘scum’)) appears.
5. Allow the diluted stain to act for 3 minutes
6. Wash off the stain with running tap water/wash bottle
Don’t tip off the stain, because this will leave a fine deposit covering the film.
7. Wipe the back of the slide clean and stand it in a draining rack for the smear to
dry (head part down).
8. Examine microscopically
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Wright staining procedure cont’d
As it is methanol based, it doesn’t require a fixation
When an aqueous or diluted stain is used, the air dried smear must first
be fixed by flooding with absolute methanol for 3-5 minutes
If films are left unfixed for a day or more, it will be found that the
background of dried plasma stains pale blue
The blood film should appear neither too pink nor too blue
(check the results microscopically)
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2. Leishman Stain
In its preparation, the methylene blue is polychromed by heating a 1%
solution with 0.5% sodium carbonate at 65oC for 12 hours
A further ripening is allowed to proceed for 10 days before it is mixed
with an equal volume of 0.1% eosin B
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Leishman Staining
Similar to that used in Wright stain except for step 3
i.e. with Leshman stain, dilution is effected with
approximately two volume of distilled water to
one volume of stain
The best guide is the appearance of a metallic
scum
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Appearance of cells and cell components in
Romanowsky-stained blood films
Films stained with either Wright or Leishman stain are pinkish in color when
viewed with the naked eye.
Microscopically,
Red cells – pink with a central pale area
Nuclei of leucocytes – blue to purple (light purple in monocytes)
Eosinophilic granules – red orange each distinctly visible
Basophilic granules – dark blue
Platelets
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– violet granules By Biruk B 20
Appearance of cells and cell components in Romanowsky-
stained blood films:
Cytoplasm
Monocytes – gray blue with fine reddish granules
Neutrophils: light pink with lilac (pale purple) granules
Lymphocytes: varying shades of blue
Malaria parasites – sky blue cytoplasm and red purple chromatin
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[Link] stain
Giemsa Stain
Employs various azure compounds (thionine and its methyl
derivative) with eosin and methylene blue
Is an alcohol-based Romanowsky stain that requires dilution in
pH 7.1-7.2 buffered water
It is excellent in staining malaria parasites in thick films.
Commonly used in combination with Jenner or May – Grünwald stains
constituting “panoptic staining” By Biruk B
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Giemsa stain cont’d
Reagent
Giemsa powder
Buffered water, pH 7.1-7.2 or
Buffered saline water, pH 7.1-7.2
Dilute the Giemsa stain as required
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Giemsa stain cont’d
The two working solutions are:
3% for 30 minute staining
Measure 50 ml of buffered water pH 7.1-7.2
Add 1.5 ml of Giemsa stain and mix gently
The stain can be measured using a dry graduated plastic bulb pipette or a small volume
plastic syringe.
10% solution for 10minute staining
1:10 Giemsa =1 part of stock Giemsa + 9 parts buffered water
Measure 45 ml of buffered water in 50 ml cylinder
Add 5 ml of Giemsa stain to 50 ml mark
Mix gently
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Procedure
[Link] working solution (3% or 10% Gemisa)
2. Place the slides in a staining rod/rack
3. Fix the thin film by carefully dropping methanol onto the thin film only.
4. Let the blood film dry in air on a drying rack or tray.
5. Cover the air-dried smear with working solution as follows:
30 min if using 3% stain solution
10 min if using 10% stain solution
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6. Set the timer to 10 minutes for the staining (if you use 10%gemisa).
7. Gently flush all the stain from the slides by dropping clean water over it.
Wipe the back of each slide clean and place it in draining rack for the
preparation to air dry.
8. Allow the slides to air-dry.
9. Examine microscopically.
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4. Panoptic Stain
Consists of a combination of a Romanowsky stain with another
stain, e.g., Giemsa with Jenner
It improves the staining of cytoplasmic granules and other bodies
like nucleoli of blast cells
Popular methods are:
Jenner-Giemsa and
May–Grunwald-Giemsa.
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4.1. May-Grünwald-Giemsa staining
Air dry the films and fix by immersing in a jar containing methanol for 10-20
seconds
For bone marrow films leave for 20-25 minutes
Transfer the films to a staining jar containing May- Grünwald’s stain freshly
diluted with an equal volume of buffered water and leave for 10-15 minutes
Transfer the slides without washing to a jar containing Giemsa’s stain freshly
diluted with 9 volumes of buffered water pH 6.8.
Allow to stain for 10-15 minutes
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4.2. Jenner-Giemsa staining
Air dry the films
Fix by immersing in a jar containing methanol for 10-20 seconds
Transfer the films to a staining jar containing Jenner’s stain freshly diluted
with 4 volumes of buffered water
Leave for 4 minutes.
Transfer the slides (without washing) to a jar containing Giemsa stain
freshly diluted with 9 volumes of buffered water pH 6.8.
Allow to stain for 7-10 minutes.
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Jenner-Giemsa cont’d
Transfer the slides to a jar containing buffered
water, pH 6.8;
Rapidly wash in 3 or 4 changes of water
Allow to stand undisturbed in water for 2-5 minutes
for differentiation to take place.
Place the slides on vertical end to dry.
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5. Field’s Stain
Is a water based Romanowsky stain composed of two solutions
Field stain A and Field stain B
Was introduced to provide a quick method for staining thick films for malaria
parasites.
Should be buffered to the correct pH
Neither solution requires dilution when staining thick films
Field stain B requires dilution in staining thin solution
More stable compared to Giemsa working stain
Stain well fresh blood films particularly thick films
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Thin film Field’s staining
Required
Field’s stain A
Field’s stain B, diluted 1 in 5
Buffered water (pH 7.1-7.2)
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Thin film Field’s staining cont’d
Place slide on staining rack
Fix with methanol
Cover with 0.5 ml of diluted Field’s stain B
Add immediately an equal volume of Field stain A
Mix using 1 ml graduated plastic bulb pipets
Leave for 1 min
Wash off the stain with clean water
Wipe the back of the slide clean
Allow to air dry
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Thick film Field’s staining
Required
Container of Field’s stain A
Container of Field’s stain B
Two containers of clean water (not buffered)
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Thick film Field’s staining cont’d
Holding the thick side facing downward, dip slide into Field stain A for 5 sec
Drain off excess stain by touching a corner of the slide against the side of the coplin
jar
Wash gently for 5 sec in clean water and drain off excess water
Dip the slide into Field stain B for 3 sec
Drain off excess stain
Wash gently in clean water
Wipe the back of the slide
Allow to air dry By Biruk B 35
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An optimally stained film has the following characteristics:
1. The red blood cells (RBCs) should be pink.
2. Nuclei are dark blue to purple.
3. Cytoplasmic granules of neutrophils are lavender to lilac(light purple).
4. Cytoplasmic granules of basophils are dark blue to black.
5. Cytoplasmic granules of eosinophils are red to orange.
6. The area between the cells should be colorless, clean, and free of
precipitated stain.
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A
F
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6.6. Staining Problems
Excessively Blue Stain
Causes:
Too thick films
Prolonged staining
Inadequate washing
Too high alkalinity of stain or diluent
Appearance of cellular elements on excessively blue stained film:
Erythrocytes – blue green
Nuclear chromatin – deep blue to black
Granules of neutrophils – deeply stained, appear large and prominent
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Staining Problems cont’d
Correction: Excessively Blue Stain
Preparing films with ideal thickness
Reducing staining time (optimize the staining time)
Using less stain and more diluent
Prolonging washing
Adjust pH of buffer or prepare a new batch of stain
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Staining Problems cont’d
Excessively Pink Stain
Causes:
Insufficient staining time
Prolonged washing
Too high acidity of the stain or buffer (exposure of stain or
buffer to acid fumes)
Appearance of cells:
Erythrocytes – bright red or orange
Nuclear chromatin – pale blue
Granules of eosinophils – sparkling brilliant red
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Staining Problems cont’d
Correction: Excessively Pink Stain
Prolonging staining time (optimize staining time)
Reducing washing
Adjust pH of buffer or prepare a new batch of
stain
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Staining Problems
Consider adjusting the pH of stain or buffer if:
White cells look too White cell
blue and red cells granules are
look too barely visible and
grey red cells look too
pale
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Staining Problems cont’d
Precipitate on the Film
Causes:
Unclean slides
Drying during the period of staining
Inadequate washing of slide at the end of the staining period
(excessive rinsing of the stained smear will cause fading of stain)
Use of unfiltered or inadequately filtered stain
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Staining Problems cont’d
Correction: Precipitate on the Film
Use clean slides
Cover the smear with generous amount of the stain and avoid
drying
Wash the slide until thinner parts of the film are pinkish.
Filter stain
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Quality control of staining
When a new batch of stain is prepared, decide the best staining time to use
e.g. stain films made from the same blood at different times: e.g. 5,
7,10,12,15, minutes.
Compare the results with a stained control blood film
By checking the pH of newly prepared buffer water and rechecking it at
weekly intervals
The pH of the buffered water used to dilute the stain must be correct (6.8)
pH is mainly responsible for the staining reactions.
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Technical tips
During preparation of staining solutions, mix the preparation 3-4
times everyday to ensure complete dissolution of the powder
Use magnetic stirrer, if it is available
Ripening of the prepared stain gives good results
Always filter staining solutions before use
Proper rinsing is critical
Water artifact can interfere with smear evaluation
Keep stain jars tightly covered when not in use
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Case Study 1: Analyzing a Smear
A technician prepares to do a set of differential
counts
She notices that the red cells look pale and
washed out and that the white cell granules are
barely visible on every slide
At this point, identification of white cells is difficult
Question:
What is the proper course of action?
1 minute!
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Poorly Stained Smear
Notice that this stain is too pale: it is difficult to tell
any shading detail in the red cells
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Case Study 1 Answer: Analyzing a Smear
Question:
What is the proper course of action?
Answer:
A stain that is too pale indicates stain may be too
acidic due to a buffer problem
Stain a second slide from the patient
Increase the pH, stain, and then assess for
color of granules and red cell color
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Microscopic Examination of Blood Films
Every film should first be inspected at low power (10x) before general
examination is undertaken with the 100x lens
Check for even distribution of cells, staining quality, platelet clumping
It is essential to mount (cover) the film with a cover glass as this permits the
film to be examined with the 10 x and 40 x objectives.
Thus, when the film is completely dry cover it by a rectangular cover glass
permanently with a neutral mountant DPX (Distrene, Plasticiser, Xylene)
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Microscopic Examination of Blood Films cont’d
Survey the film at 10 x magnification to get a general
impression of its quality
Find an area where the red cells are evenly distributed, just
touching but not overlapping, and study their gross
morphology at 40x
Use the 100x objective for studying the fine details of
the cell morphology.
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53
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summary
1. What is the general principle of staining blood films with
Romanowsky dyes?
2. What are the Romanowsky dyes that are commonly used in
staining blood films?
3. Describe the appearance of cells and cell components in
Romanowsky- stained thin blood films
4. What are the staining problems that give rise to
unsatisfactory results?
5. How do you correct these problems?
6. What is panoptic staining? What is the advantage of
panoptic stains over simple Romanowsky dyes?
7. List two dyes that are commonly used in thick blood film
staining?
8. Discuss staining quality control methods 54
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Thank you very much !!
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