Romanowsy stain, PAS and SBB in Hematology

1. TOPIC PRESENTATION
ROMANOWSKY STAIN
Sansar Babu Tiwari, MBBS, PGY I
Department of Pathology
TUTH
29th January
1

2. The colour purple: from royalty to
laboratory
During Roman emperors, wearing purple outside
royal family was considered a crime punishable by
death.
Purple garments were worth more than their weight
in gold.
The red, the blue and the purple:
The development of this purple colour marked the
beginning of a new era in hematology  nucleus of
the malarial plasmodium became visible.

3. Phoenician myth: The Hercules, his dog
and Nymph Tyrus: The tyrian PURPLE
1 murex snail  1
drop of dye
To make a single robe
needs 1.5 g dye
which equals 12,000
mollusks.

4. The chronicles of dye:
The ancient dyes were produced from
natural sources.
• Blue dye from Indigo plant native to
China.
• Red dye brazilin obtained from red
wood. (Brazil is named by its richness
in red-wooded trees)
• Insects genus kermes  crimson.

5. Dye, Stain and Biological stain
• Dyes are colored substances that change the color
of other materials permanently.
• Stains are dyes that have been adapted for
specialized biological or non-biological purposes.
• Biological stains have been adapted to render
microscopic samples more visible.
• Non-biological stains are used for purpose such as
textile coloring.

6. Origin of biological stains.
• In 1684, Vieussens used saffron
dye in his study of brain
vasculature. (spirits of wine
colored with saffron)
• Earliest recorded microscopic
use of biological stain:
Leeuwenhoek: To study muscle
fibre under microscope.

7. Shift to synthetic dye.
• In 1856, Perkin at the young age of 15, noted quinine
(derived from cinchona tree) can also be synthesized in
lab. (Hoffman)
• While in the process he failed, but he found the black
precipitates, which he was about to discard.
• To his surprise, the solution turned purple, tested on
silk fabrics  commercialized as Tyrian purple in 1857,
aniline purple in 1858.
• Finally the dye was known as mauve in French. This
mauve madness swept over Europe.
• He donated much of his earning to charity.

8. Methylene blue and Eosin
• Heinrich Caro, a specialist in
dyeing, in 1876 developed
methylene blue in
collaboration with Baeyer.
• Baeyer had synthesized a red
dye and chose the
appropriate name eosin from
the Greek Eos meaning “flush
of the dawn sky”.

9. Romanowsky-type stains
Methylene blue and its product of oxidation
Toluidine blue or Thionine blue
+
Halogenated fluorescein dye usually Eosin Y
Eosin B, Phloxine, Rose Bengal

10. Methylene Blue
MeMe
MeMe

11. Methylene Blue
MeMe

12. Acidic cellular elements such as nucleoproteins, nucleic acids and primitive
cytoplasmic proteins, react with the basic dyes, methylene blue and its oxidative
products. These elements are basophilic and stain variations of blue.

13. Eosin

14. Basic cellular elements such as hemoglobin molecules and some cytoplasmic
constituents in leukocytes, have an affinity for the acidic dye, eosin. These
elements are acidophilic and stain orange-red

15. Neutrophils: Neutral stain
A neutrophil has neutral staining characteristics and stains blended shades of
purple or pink, representing combinations of acidic and basic molecular groups.
Azure dyes stain the primary or non-specific granules in most myeloid cells red-
purple, hence, the term azurophilic granules.

16. Romanowsky effect
Romanowsky effect is defined as the condition in
which the typical coloration of certain cell
components results from the combined action of a
cationic and anionic dye, and cannot be produced by
either dye acting alone.
Polychromatophilic effect:
The differential staining of the plasmodial nucleus as
well as the appearance of the desired purple color in
other blood components were termed
polychromatophilic effect.

17. History of Romanowsky-type stain
Paul Ehrlich 1880
•Methylene blue and
Acid fuschin.
•Neutral stain stains
granules of
neutrophils named
after this discovery.
•Good research
requires patience,
skill, luck and
money.
Cheslav Ivanovich
Chenzinsky 1888
•Methylene blue and
Eosin
•Noticed daisy head
of P. malariae and
sickled shaped
trophozoite of P.
falciparum.
•Albert Plehn
modified M:E ratio
upto 3:1.
Dmitri
Romanowsky
1890
•Methylene blue and
Eosin (didn’t even
described
polychromasia)
•In 1891 in his
doctoral thesis
described
methylene blue
best as used after
mold started
forming on the
surface.
Ernst
Malachowsky
1891
•Tried acidifying but
failed
•Added Borax to
make the solution
alkaline (July 1890)
•He mentioned
effective staining of
plasmodial nucleus
but dint mention
the purple color.
Unna 1892
•Alkalinization using
carbonated salts
especially
potassium
carbonate facilitates
ripening of
methylene blue
Nocht 1898
•Used Unnas secret
•Later In 1899,
extracted
polychrome
methylene blue
from chloroform.
•He proved the
theory of
polychroming by
Romanowsky and
Malachowsky.
Because the dye mixtures
were dissolved in water,
they precipitated rapidly.
Jenner, 1899
•Used absolute methanol as a
solvent.
•He collected the precipitate
formed after mixture of
methylene blue and eosin and
re-dissolved it in methanol.
•No need of another fixative
and also stained basophil
granules.
3 weeks

18. Basic timeline:

19. Giemsa Stain: An untold secret
Dr. Grubler Co. marketed Giemsa powder as an Azure I
(supposed to contain Azure B, Methylene violet
Bernthsen, and Eosin) which gave gray color to the
leukocyte cytoplasm.
So later modified as Azure II (containing equal volumes of
Azure I and Methylene blue in addition to Eosin) that
gave bluish hue to the leukocyte cytoplasm
Later other companies started preparing Giemsa but
different from the original composition.
In our Institution we use Giemsa from Fischer Scientific.

20. Giemsa Stain Solution: Preparation
Preparation of Stock solution:
• 3.8 gm of Giemsa powder is added to 250 ml of
gently heated Glycerol (approx. 60 C).
• Then 250 ml of methyl alcohol is added.
Preparation of working solution:
• The stock solution is added to the distilled water at
the dilution of 1:4.

21. Giemsa Staining:
• Principle:
• It is specific for the phosphate groups of DNA and
attaches itself to AT rich regions of DNA.
• Uses:
• Blood film stain: PBS and Bone marrow.
• Bacteriology: H. pylori, Yersinia pestis, Chlamydia
trachomatis, Ehrlichiosis, Anaplasmosis (ME GA)
• Virology: Cytomegalovirus
• Parasitology: Plasmodium, Trypanosoma
• Cytogenetics: G-banding in karyotyping.

22. Chlamydia trachomatis

23. Yersinia pestis: Bipolar staining

24. Anaplasmosis and Ehrlichiosis.

25. G-banding in Karyotyping:

26. Giemsa Staining:
• Cytopathological staining and detection of H. pylori:
• Working solution of 1:4 dilution is used and kept for
about 25-30 minutes.
• Washed in tap water and dried.

27. Where lies the place of adding Giemsa when
there is still other Romanowsky stains?
• Other Romanowsky-type stains just gave the effect
principle .
• Giemsa, in addition to this, developed particularly
successful staining composition and procedure.

28. Wright-Giemsa Stain Primary Stain
3 gm Wright’s Powder
+
3 gm Giemsa Powder
Is added to 2.5 liter
acetone free methyl
alcohol.

29. Wright-Giemsa Stain Buffer (pH: 6.8)
2.3 gm Disodium
hydrogen
orthophosphate
+
2.3 gm Potassium
dihydrogen
phosphate
Is added to 500 ml
distilled water.

30. Woronzoff-Dashkoff, K. K. (2002). The Wright-Giemsa stain:
Secrets revealed. Clinics in Laboratory Medicine, 22(1), 15–23.
doi:10.1016/s0272-2712(03)00065-9
Decision to be made is whether to add Giemsa stain to the
Wright stain solution.
The primary effect of the Giemsa stain is to intensify
azurophilic, or reddish purple, hues. The author has found
that the addition of a small amount of Giemsa stain brightens
the primary granules of neutrophils and the dustlike
azurophilic granulation of monocytes. If too much Giemsa
stain is added, however, monocytes begin to resemble
neutrophilic promyelocytes.
The amount of Giemsa stain to be added must be carefully
titrated, and re-evaluated with each new lot of Wright stain.

31. Wright-Giemsa
Staining:
• PBS:
• Smears are placed in rods.
• These smears are dried and Wright Giemsa solution is
poured over the slide.
• Sorensen’s buffer is poured immediately over the slides.
• Kept over the 15-20 minutes and then washed with tap-
water and air-dried.
• Bone Marrow:
• Same as PBS except fixation is done in methanol and kept
for 25-30 minutes.

34. Comparison of Different Staining:
Giemsa (Complex) Leishman MGG Jenner (Simplest)
Azure B Eosin Y replaced
with Eosin B
Didn’t use polychromed methylene blue
3.8 gm powdered
dye
0.5 gm powdered
dye
0.75 gm powdered
dye
1.25 gm powdered
dye
250 ml methanol
250 ml glycerol
250 ml methanol 250 ml methanol 250 ml methanol
Slight heat can be
applied
Warm to 15
degree for 15
minutes
Warm upto 50
degrees and cool

35. SPECIAL STAINS IN HEMATOLOGY

36. Periodic Acid Schiff (PAS) stain:
Periodic Acid:
Oxidizes 1-2 glycol
group to aldehydes
Schiff Reagent:
On exposure aldehydes
give red product

37. Periodic Acid Schiff (PAS) stain:
• Positive with glycogen and other carbohydrates
• Diastase digestion can be seen in all except
glycogen.

38. Periodic Acid Schiff (PAS) stain:
Reagents:
• Fixative: Methanol
• 1% periodic acid.
• Schiff reagent
• Counterstain: Aqueous hematoxylin

39. Periodic Acid Schiff (PAS) stain:
Methods:
Fix in methanol for 15 minutes  Rinse in gently
running water and air dry  Expose to diastase if
required  Flood slides with 1% Periodic Acid
reagent for 10 minutes  Rinse in running tap water
for 10 minutes and air dry  Immerse in Schiff
Reagent for 30 minutes with a lid  Rinse in running
tap water for 10 minutes and air dry.
Counterstain in aqueous hematoxylin for 5-10
minutes.

40. Periodic Acid Schiff (PAS) stain:
Results:
• Cytoplasmic positivity may be diffuse or granular.
• Granulocyte precursors show diffuse weak positivity
with neutrophil showing intense granular positivity
(Internal control)
• Eosinophil granules are negative with diffuse
cytoplasmic positivity.
• Basophils are negative.
• Monocytes variable diffuse positivity.
• Normal erythroid precursors and red cells are negative

41. Periodic Acid Schiff (PAS) stain:
Results:
• Granular positivity with negative background cytoplasm
is found in 10-40% of peripheral lymphocytes.
• Lymphoblast show variable PAS-positive cytoplasmic
granules or blocks on a clear background.
• It can also be useful in AML and MDS to identify
abnormal erythroblasts and dysplastic megakaryocytes
and to demonstrate the cytoplasmic blush that helps to
confirm the diagnosis of APML.

43. Granular PAS positivity in ALL.

44. Sudan Black B stain
SBB is a lipophilic dye that binds irreversibly to an
undefined granule component in granulocytes,
eosinophils and some monocytes.
Reagents:
• Fixative: Vapour from 40% formalin
• Stain: SBB 0.3 gm in 100 ml absolute ethanol
• Phenol buffer
• Working stain solution: Add 40 ml buffer to 60 ml SBB
solution
• Counterstain: MGG or Leishman stain

45. Sudan Black B stain
Method:
• 2 drops formalin in filter paper Put at the bottom of
the Coplin jar  Leave it to vaporize for 15 minutes
with lid covered  Put the slide for 5-10 minutes 
Remove the slide and keep in air for 15 minutes.
• Immerse the slides in working stain solution for 1 hour
with lid on.
• Immediately flood with 70% alcohol in a staining rack
 30 secs  Tip off the alcohol and flood again for 30
secs. (Total 3 times)
• Rinse in gently running tap water and air dry.
• Counterstain with Leishman or MGG stain.

46. Sudan Black B stain
Results:
• The reaction product is black and granular.
• Promyelocytes and myelocytes are the most
strongly staining cells.
• Most primitive myeloblasts are negative, and
metamyelocytes and neutrophils have fewer
positive granules.
• Eosinophil granules stain strongly and have a clear
core.
• 1-2% of ALL show non-granular smudgy positivity
not seen with MPO.