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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 3.Giemsa 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
11/16/2022 22
 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
1.Prepare 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

11/16/2022 Too alkaline By Biruk B Too acidic 42

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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
11/16/2022 By Biruk B
Thank you very much !!

11/16/2022 By Biruk B 55