The various romanowsky stains explained

1. MBARARA UNIVERSITY OF SCIENCE &
TECHNOLOGY
DEPARTMENT OF MEDICAL LABORATORY
SCIENCE
MLS 2112: HAEMATOLOGY & BLOOD
TRANSFUSION
TOPIC: ROMANOWSKY STAINS
BY
Tumukunde Benjamin

2. Objectives
By the end of the topic, we should be able to;-
• Define Romanowsky stains,
• State the principle of Romanowsky stains,
• Know the types of Romanowsky stains,
• Know how different Romanowsky stains are
prepared,
• Know how different Romanowsky stains are
used,
• Know QC procedures in the use of
Romanowsky stains.

3. Introduction
• Romanowsky Stains are named after a man called
Dmitri Leonovich Romanowsky who invented it
in 1891
• Defined as; stains that are used in haematology
and cytological studies to differentiate cells in
microscopic examinations of blood and bone
marrow samples.
• They are also applied to detect the presence of
haemoparasites such as malaria, trypanosomes,
leishmania and others parasites.

4. Introduction cont’d
• Romanowsky stains are neutral in a way that
they are composed of oxidized methylene
blue (azure) dyes and Eosin Y.
• The azures are purplish- blue and are basic
while the eosin is pinkish red and is acidic.
• They stain the cell components differently
depending on their PH.
• This ability to produce a wide range of hues
(colours) allowing cellular components to be
easily differentiated is called Romanowsky
effect/ metachromasia

5. Principle
• Romanowsky stains work on the principle that
“the acidic components of the cell are stained
by the basic dye (azure) forming a blue-purple
colour while the basic components of the cell
are stained by the acidic dye (eosin Y) forming
a pin- red colouration.”

6. Staining characteristics
Azure B (Methylene blue) staining
Eosin Y staining

7. Types of Romanowsky stains
• Field stains (A and B)
• Giemsa stain
• May-Grünwald
• Leishman stain
• Wright's

8. Uses of Romanowsky stains
• Staining of blood and bone marrow specimens
for cytopathological findings. Eg Leukemias
• Detection of malaria and other
haemoparasites. Eg Trypanosomes.

9. Preparation of Romanowsky stains
1. Field stains
• Are water based stains.
• Field stain A is basic containing methylene blue derivatives
and
• Field stain B is acidic containing eosin Y
Preparation of Field stain A from commercially available
Powder.
• Heat distilled water up to 60 °C
• Measure 600 ml heated distilled water in a conical flask
• Add 5 grams of commercially available Field stain A powder
• Mix the powder until it dissolves completely
• Filter the solution
• Label with date of preparation.

10. Field stains cont’d
Preparation of Field stain B from
commercially available Powder.
• Heat distilled water up to 60°C
• Measure 600 ml heated distilled water in a
conical flask
• Add 4.8 grams of commercially available Field
stain B powder
• Mix the powder until it dissolves completely
• Filter the solution
• Label with date of preparation.

11. 2. Giemsa preparation
Giemsa stock solution
• Dissolve 7.6 g of Giemsa powder with 500 ml of methanol
• Heat the solution up to 60°C
• Add 500 ml of glycerin
• Filter the solution
• Label the container with preparation date
• Store the solution for 1 – 2 months before use in a cool dark place
Note: Ensure that the glass ware is dry while weighing Giemsa powder
because any contact with water will spoil the remaining powder
Giemsa working solution (10%)
• Measure 10 ml of Giemsa stock solution in a measuring cylinder
• Add 10 ml of methanol
• Add 80 ml of distilled water
• Mix thoroughly
• The reagent is ready for use

12. 3. May-Grunwald preparation
Stock solution
• Weigh 0.3 g of May Grunwald dye
• Dissolve it in 100 mL absolute methanol
• Warm the mixture to 50°C in a water bath for a few hours
and allow it to cool to room temperature.
• Gently mix on an automatic rotator for 24 hours.
• Filter the mixture and stain is ready for use.
Working reagent (15%)
• Measure 30 ml of May-Grunwald solution
• Add 20 ml of buffered water
• Add 150 ml of distilled water and mix. The stain is ready
for use
N.B: Some Labs use 10%

13. 4. Wright’s stain preparation
• Weigh 1.0 g of Wright’s powder
• Mix with 400 ml of water free methanol
• Label the container with preparation date

14. 5. Leishman stain preparation
Leishman stock solution
• Weigh 0.15g of Leishman stain powder and grind it in a glass
• Put the powder into a bottle through a funnel and gently add
20 ml of methanol through the same funnel to ensure that all
the dry stain is washed into the bottle
• Shake the mixture in a circular motion for 2-3 minutes
• Add the remaining amount of methanol to the mixture
through the same funnel up to 100ml mark
• Cap the bottle and shake for more 2 minutes
• Label the bottle and keep the mixture for a week. There after,
the stain is ready for use.

15. Leishman stain preparation cont’d
Leishman working reagent
• Measure 50 ml of Leishman stock solution into
a measuring cylinder
• Add 75 ml of phosphate buffer (PH btn 6.4 –
7.0)
• Add distilled water up to 250 ml mark (175 ml)
• Mix and let the solution stand for 10 minutes
before use

16. Staining procedure for Leishman
A. Flooding method
• Place the slide on the staining rack and flood it with methanol
for 1 minute
• Flood the slide with 20 drops of Leishman stain solution and
allow to stand for 1 minute. Do not rinse
• Apply 30 drops of buffer diluent
• Mix gently by rocking the slide and allow to stand for 3
minutes
• Pour off the mixture and rinse with distilled water for 10 – 15
minutes
• Air dry the smear and examine microscopically.
B. Dipping method
• For the same steps with reagents in the coplin jars

17. Staining procedure for Wright’s stain
• Cover the air dried smear with un diluted stain
for 2- 3 minutes. This partially fixes the smear.
• Add equal amount of buffered water until a green
scum appears on top.
• Leave it stand for 5 minutes.
• Without disturbing the slide, flood with distilled
water and wash until the thinner part of the
smear turns pinkish red
• Allow the smear to air dry and examine
microscopically

18. Staining procedure for May-Grunwald stain
It is together used with Giemsa stain
May-Grunwald Giemsa staining
• Fix the smear with methanol of 2 minutes
• Cover the smear with May- Grunwald stain (10%/
15%) for 5 minutes and tip off the excess
• Cover the smear with 50% Giemsa stain for 15
minutes.
• Rinse the smear with distilled water and leave it
to air dry.
• Examine microscopically

19. Giemsa staining procedure
A. Thick smear
• Prepare the stain using 1:50 dilution ratio (1 ml of the stain +
49 ml of buffered water)
• Dip the smear in diluted Giemsa stain for 30 sec- 1 minute
• Rinse the smear in distilled water 3- 5 times
• Allow the smear to air dry.
B. Thin smear
• Prepare the stain using 1:20 dilution ratio (2 ml of the stain +
38 ml of buffered water)
• Dip the smear in diluted Giemsa stain for 20 - 30 minutes
• Rinse the smear in distilled water 2- 3 times
• Allow the smear to air dry.

20. Staining procedure for Field stains
A. Thick smear
• Dip an air dried smear in Field stain A for 4 seconds
• Rinse with tap water
• Dip the smear in Field stain B for 3 seconds
• Rinse with tap water
• Clean the back of the slide and leave to air dry.
B. Thin smear
• Fix the air dried smear with methanol for 1 minute
• Dry in the air.
• Dip fixed smear to Field Stain B (Red Stain) for 5 to 6 seconds.
• Wash in running tap water.
• Dip smear into Field Stain A (Blue Stain) for 10 to 30 seconds.
• Wash in running tap water.
• Leave to air dry

21. Quality control procedures for preparing
& use of Romanowsky stains
• Use of SOPs while preparing and using the stains
• Appropriate weighing of powder stains while
preparing stock solutions
• Use of water free methanol while preparing the
methanol based stains
• Ensure that all solute particles of the powder are
dissolved
• Filtering of the stains before use
• Use of smears from normal and abnormal samples to
control stains before use
• For stored samples, bring them to room temperature
before processing them

22. All the best in your endeavours