ROMANOWSKY STAINS

Preparation of peripheral blood smear

The purpose of examining peripheral blood smear include assessing various elements of
peripheral blood cells such as erythrocytes, leukocytes and platelets and look for parasites such
as malaria, trypanosoma, microfilaria and others 1. Smear preparations that are well made and
daubed are an absolute requirement for getting good examination results.

Basic of Rowanowsky stains

The basis of Romanowsky staining is the use of two different dyes, namely Azur B
(Trimetiltionim) which is alkaline and eosin y (tetrabromoflurescein) which is acidic. Azur B will
color cell components that are acidic such as chromatin, DNA, and RNA. Whereas eosin y will
color basic cell components such as eosinophil granules and hemoglobin. The eosin y bond on
the aggregated Azur B can produce a purple color, and this condition is known as the
Romanowsky Giemsa effect. This effect occurs very significantly in DNA but not in RNA giving
rise to a colored nucleus with a cytoplasm that coloring is blue.2

The best examination material is fresh blood from the capillaries or veins, which are
blotted on the slide. In certain circumstances EDTA blood can also be used. 3

Types Romanowsky stains

1. Wrights Stain4

Wright’s stain is a polychromatic stain consisting of a mixture of Eosin and Methylene blue.
As the Wright stain is methanol based, it doesn’t require a fixation step prior to staining.
However, fixation helps to reduce water artefact that can occur on humid days or with aged
stain.

Methanol fixes the cells to the slide. Eosin Y is an acidic anionic dye and methylene blue is
basic cationic dye. When diluted in buffered water, ionization occurs. Eosin stains the basic
components such as hemoglobin and eosinophilic granules an orange to pink color. Methylene
blue stains acidic cellular components such as nucleic acid and basophilic granules in varying
shades of blue. The neutral components of the cells are stained by both components of the dye,
producing variable colors.
2. Leishman Stain5

Leishman's stain is applied in conventional staining techniques to uniformly stain

chromosomes. These techniques leave centromers constricted, thus enabling the
measurement of chromosome length, centromeric position, and arm ratio. Slides can be
easily destained and banded by most banding procedures. Orceinstained chromosomes
cannot be destained.
Weigh out 0.2 g of the powdered dye, and transfer it to a conical flask of 200–
250 ml capacity. Add 100 ml of methanol and warm the mixture to 50°C for 15 min,
occasionally shaking it. Allow the flask to cool and filter. It is then ready for use, but it
will improve on standing6.

3. May Grunwald Stain

May-Grunwald-Giemsa staining method is used for morphological inspection and

differential counting of blood cells. May-Grünwald staining combines the effect of acidic
eosin and alkaline methylene blue. Giemsa staining makes effect of azure. This staining
stains all cellular components. The pH is a very important factor in staining, so any
change will lead to wrong staining reaction. The limits of the most suitable pH are
between 6.5 and 6.8.7
Weigh out 0.3 g of the powdered dye and transfer to a conical flask of 200–250
ml capacity. Add 100 ml of methanol and warm the mixture to 50°C. Allow the flask to
cool to c 20°C and shake several times during the day. After letting it stand for 24 hours,
filter the solution. It is then ready for use, no “ripening” being required. 8

4. Giemsa Stain

Giemsa stain is a type of Romanowsky stain, named after Gustav Giemsa, a

German chemist who created a dye solution. It was primarily designed for the
demonstration of malarial parasites in blood smears, but it is also employed in histology
for routine examination of blood smear.
Giemsa stain is a differential stain and contains a mixture of Azure, Methylene
blue, and Eosin dye. It is specific for the phosphate groups of DNA and attaches itself to
where there are high amounts of adenine-thymine bonding. Azure and eosin are acidic
dye which variably stains the basic components of the cells like the cytoplasm, granules
etc.
Methylene blue acts as the basic dye, which stains the acidic components,
especially the nucleus of the cell. Methanol act as a fixative as well as the cellular stain. 9
 Automated Staining10

Automatic staining machines are available that enable large batches of slides to be
handled. They may be either stand-alone staining machines or a part of a large automated
blood counting instrument. In many instances, the instrument spreads, fixes, and stains blood
films. Some automated instruments incorporating staining can only be programmed to prepare
and stain a single film per sample. Others can prepare and stain multiple films from a single
blood sample; this is useful in a teaching programme with a large number of students.

Some systems apply staining solutions to slides lying horizontally (flat-bed staining),
whereas others either immerse a slide or slides in a bath of staining solution (“dip-and-dunk”
technique) or spray stain onto slides in a cytocentrifuge. Problems include increased
background staining, inadequate staining of neutrophil granules, degranulation of basophils,
and blue or green rather than pink staining of erythrocytes. These problems are usually related
to the specific stains and staining protocols used rather than to the type of instrument,
although flat-bed stainers are more likely to cause problems with stain deposit. However, as a
rule, staining is satisfactory provided that reliable stains are used and there is careful control of
the cycle time and other variables. 11 Flat-bed stainers may not stain an entire film (e.g., a bone
marrow film) if the film exceeds the standard length.

Perls Staining

Prussian blue (Perls’) reaction is a method for staining non-haem iron in normoblasts
(siderocytes), macrophages (haemosiderin), and other cells containing particulate iron. The
granules are formed of a water-insoluble complex of ferric iron, lipid, protein and carbohydrate.
The method allows assessment of both the amount of iron in reticulo-endothelial stores and
availability of iron to developing erythroblasts. Principle of perls staining is The granules
(containing ferric iron) react with pottassium ferrocyanide [K4Fe(CN)6] to form a blue compound
ferriferrocynanide), Prussian blue reaction.12
Fixation of perls staining is Avoid the use of acid fixatives. Chromates will also interfare
with the preservation of iron.

Procedures:

1. Deparaffinize and bring the sections to water.

2. Treat the sections with freshly prepared acid ferrocyanide solution for 10-30
minutes.
3. Wash well in distilled water.
4. Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red.
5. Wash rapidly in distilled water.
6. Dehydrate, clear and mount.
REFERENCES

1. Baker JR (1958) Principles of Biological Microtechnique . Methuen, London. p. 272.

2. Lillie RD (1944) Factors infl uencing the Romanowsky staining of blood fi lms and the
role of methylene violet. J. Lab. Clin. Med. 29: 1181–1197.
3. Horobin, R. W., & Walter, K. J. (1987). Understanding Romanowsky staining.
Histochemistry, 86(3), 331–336.
4. Marshall, P. N., Bentley, S. A., & Lewis, S. M. (1978). Staining properties and stability of a
standardised Romanowsky stain. Journal of Clinical Pathology, 31(3), 280–282.
5. Sathpathi S, Mohanty AK, Satpathi P, Mishra SK, Behera PK, Patel G, Dondorp AM.
Comparing Leishman and Giemsa staining for the assessment of peripheral blood smear
preparations in a malaria-endemic region in India. Malar J. 2014 Dec 30;13:512.
6. Bain, B. J., & Mitchell Lewis, S. (2006). Preparation and staining methods for blood and
bone marrow films. Dacie and Lewis Practical Haematology, 59.
7. Rijal, N. (2019, July 13). Giemsa Stain: Principle, Procedure and Results.
https://microbeonline.com/giemsa-stain-principle-procedure-and-results/
8. Bain, B. J., & Mitchell Lewis, S. (2006). Preparation and staining methods for blood and
bone marrow films. Dacie and Lewis Practical Haematology, 77.
9. Wittekind DH 1983 On the nature of RomanowskyGiemsa staining and its significance
for cytochemistry and histochemistry: an overall view. Histochemical Journal 15:1029–
1047.
10. Favuzzi, John A., et al. "Method and apparatus for automated pre-treatment and
processing of biological samples." U.S. Patent No. 7,850,912. 14 Dec. 2010.
11. Bain, B. J., & Mitchell Lewis, S. (2006). Preparation and staining methods for blood and
bone marrow films. Dacie and Lewis Practical Haematology, 70.
12. Anderson, J. (2017, May 13). An Introduction to Routine and Special Staining.
https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-routine-and-
special-staining/