hematology

1. CHAPTER 6
Staining and Examination of Blood films

2. Acknowledgements
 Addisa Ababa University
 Jimma University
 Hawassa University
 Haramaya University
 University of Gondar
 American Society for Clinical Pathology
 Center for Disease Control and Prevention-Ethiopia

3. Learning Objectives
At the end of this chapter, the students will be able to:
 Explain the general principle of staining 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

4. 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 staining with Giemsa and Field
stains
 Define panoptic staining
 Stain thin blood films with 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

5. Outline
 Introduction
 Principle of staining
 Romanowsky Stains
 Panoptic stains

6. 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
 Ehrlich was the first to use aniline dyes at first in
sequence and latter as a premixed acidic – basic stains
(neutral dyes).
 Jenner (1880) found that the precipitate formed when
eosin and methylene blue are mixed could be dissolved
in methyl alcohol to form a useful stain combining
certain properties of both parent dye stuffs.

7. Introduction cont’d
 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 malarial parasite purple and the
cytoplasm blue.
 Subsequently, Giemsa modified the stain, combining
methylene azure and eosin.
 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.

8. Introduction cont’d
 The stain most commonly used in North America is
Wright’s stain, which contains methylene blue
 Romanowsky found that when old (ripened and therefore
"polychromed") methylene blue solution is mixed with
eosin and the precipitate dissolved in methyl alcohol, a
stain results that has a wider range than Jenner’s stain
staining cell nuclei and platelet granules (which Jenner’s
mixture failed to stain).

9. 6.2. 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
attracted to and 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

10. 6.3. 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

11. 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 of specific pH.

12. 6.3.1 Romanowsky Stains in Common
Use
 Modern Romanowsky stains in common use are:
 Wright stain
 Leishman stain
 Giemsa stain
 Wright and Leishman are basically similar to
Romanowsky’s original method, the difference
being the method of polychroming the methylene
blue.

13. 6.3.1.1. 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

14. 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 1
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.
 The methyl alcohol fixes the smear.
 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

15. Wright staining procedure cont’d
3. Add equal the volume of pH 6.8-buffered water (i.e., the
same number of drops as the stain)
4. Mix by blowing until a metallic sheen appears.
5. Allow the diluted stain to act for 3-5 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. The blood film should appear neither too pink nor too
blue (check the results microscopically)

16. Wright staining procedure cont’d
 Note:
 Diluting the stain in buffered water brings about full
staining of the blood cells
 The exact staining time to use should be decided
when a new batch of stain is prepared
 It should be checked also at the beginning of each
week.
Each Lab should
determine the
optimal staining
time!!!

17. 6.3.1.2. 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

18. Leishman Staining
 similar to that used in Wright stain except for step 3
 i.e. with Leishman 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

19. 6.3.2. 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 – violet granules

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
 Note: if the stain fails, it is the size of the granules that will
mark the cell

21. 6.3.1.3.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”

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
 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.

23. Giemsa stain cont’d
 10% solution for 10 minute staining
 Measure 45 ml of buffered water in 50 ml cylinder
 Add 5 ml of Giemsa stain to 50 ml mark
 Mix gently
 Place the slides in a staining rod/rack
 Do not fix the films before staining.
 Cover the air-dried smear with a 1:10 diluted Giemsa
using buffered distilled water at pH 7.2 (recommended for
malaria parasites in order to stain schuffner’s granules) as
a diluent
 1:10 Giemsa =1 part of stock Giemsa + 9 parts
buffered water

24. Giemsa stain cont’d
 stain the slides as follows:
 30 min if using 3% stain solution
 10 min if using 10% stain solution
 Wash the stain from the slide gently
using clean water (not necessarily
distilled water or buffered water)
 Wipe the back of each slide clean and
place it in draining rack for the
preparation to air dry.
Do NOT fix thick
films before
Giemsa staining

25. 6.4. 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.

26. 6.4.1. 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

27. May-Grünwald-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

28. 6.4.2. 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.

29. 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.

30. 6.5. Field’s Stain
 Is a water based Romanowsky stain composed of two
solutions
 Field stain A
 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

31. 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)

32. 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

33. 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)

34. 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

35. Indications of the different
stains for use
 Wright stain
 Peripheral smears
 Leishamn
 Peripheral smears
 Geimsa
 For malaria thick films

36. 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

37. Staining Problems cont’d
 Correction:
 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

38. 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

39. Staining Problems cont’d
Excessively Pink Stain
 Correction:
 Prolonging staining time (optimize staining time)
 Reducing washing
 Adjust pH of buffer or prepare a new batch of stain

40. Staining Problems
White cells look too
blue and red cells
look too
grey
White cell
granules are
barely visible and
red cells look too
pale
Consider adjusting the pH of stain or buffer if:
Too alkaline Too acidic

41. 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

42. Staining Problems cont’d
 Correction:
 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
 N.B It is possible to re-stain the slide after washing with
methanol
 but this should be only done when it is not possible to
make a new smear
 If re-staining is to be done: flood smear with methanol,
flood with tap water as many times as possible, restain

43. 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.

44. 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

45. 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!

46. Poorly Stained Smear
 Notice that this stain is too pale: it is difficult to tell any
shading detail in the red cells

47. Case Study 1 Answer:
Analyzing a Smear
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
Question:
What is the proper course of action?

48. 6.7. 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)

49. 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
 At the same time, scan the film to get an impression of
the quantitative distribution of while blood cells

50. Microscopic Examination of
Blood Films cont’d
 identify any unusual or abnormal cells,
 estimate the relative proportion of platelets and note the
presence of abnormally large platelets.
 Use the 100x objective for studying the fine details of the
cell morphology.

51. Review Questions/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