Romanowsky staining or Romanowsky–Giemsa staining, is a prototypical staining technique, widely used in hematology and cytopathology. They are used to differentiate cells for microscopic examination in air dried cytological smears or pathological specimens, especially blood and bone marrow films, and to detect parasites such as malaria within the blood. Romanowsky stains is a neutral dye containing both acid and basic dyes in combination. It contains both azure B (electron acceptor) and eosin Y (electron donor). The value of Romanowsky staining lies in its ability to produce a wide range of hues, allowing cellular components to be easily differentiated. This phenomenon is referred to as the Romanowsky effect, or more generally as metachromasia. These stains allow better estimation of cell size, nuclear size, cell cytoplasm and identify ground substances by metachromasia.

1. LEISHMAN-GIEMSA COCKTAIL
Is it an Effective Stain for Air Dried Cytology Smears ?
DR. ISHITA SINGHAL
MDS SECOND YEAR

3. INDEX
INTRODUCTION
AIM
MATERIAL REQUIRED
RESULTS
DISCUSSION
LIMITATION
CONCLUSION

4. INTRODUCTION
◄ FNAC is a preliminary diagnostic procedure for
wide spectrum of non-neoplastic and neoplastic
lesions.
◄ It is safe, faster, easy to perform and cost
effective procedure.
◄ It has high grade specificity and sensitivity.
◄ Example: H & E, Romanowsky and Pap have
been used for staining the FNAC smears.

5. ROMANOWSKY STAINS
◄ Romanowsky staining or Romanowsky–Giemsa staining, is a prototypical staining
technique, widely used in hematology and cytopathology.
◄ They are used to differentiate cells for microscopic examination in air dried cytological
smears or pathological specimens, especially blood and bone marrow films, and to
detect parasites such as malaria within the blood.
◄ Romanowsky stains is a neutral dye containing both acid and basic dyes in
combination. It contains both azure B (electron acceptor) and eosin Y (electron donor).
◄ The value of Romanowsky staining lies in its ability to produce a wide range of hues,
allowing cellular components to be easily differentiated. This phenomenon is referred
to as the Romanowsky effect, or more generally as metachromasia.
◄ These stains allow better estimation of cell size, nuclear size, cell cytoplasm and
identify ground substances by metachromasia.
https://en.wikipedia.org/wiki/Romanowsky_stain

6. The staining technique is named after
The Russian physician Dmitri Leonidovich Romanowsky (1861–1921)
https://en.wikipedia.org/wiki/Romanowsky_stain

7. ROMANOWSKY
STAINS
GIEMSA STAIN
[GS]
WRIGHT'S
AND WRIGHT-
GIEMSA
STAINS [WGS]
MAY
GRUNWALD
GIEMSA
[MGG]
LEISHMAN
STAIN [LS]
LEISHMAN
GIEMSA
COCKTAIL
[LGC]
https://en.wikipedia.org/wiki/Romanowsky_stain

8. AIM
◄ The study was done to evaluate the quality of
staining of LGC in air dried cytology smears and
compare the quality of staining of LGC with
MGG.

9. MATERIAL REQUIRED
◄ Location: Tertiary care centre
◄ The number of cases studied: 100
◄ Duration: 2 months
◄ It included all the patients who were sent to
cytology department for FNAC.
◄ Smears were stained with MGG (HI media, India)
and LGC - Combination of Leishman stain
(Chromo span, India) and Giemsa stain (Nice,
India) respectively.

10. PREPARATION OF STAINS
MGG LGC
• Dilute Giemsa Stain 1:20 with deionized
water. For bluer coloration, water
buffered at pH 7.2 may be used in place
of deionized water.
• The unit volume of Giemsa was mixed
with equal amount of distilled water to
prepare Giemsa working solution (1:1
dilution)
• An equal volume of Leishman’s stain
was filtered and mixed with an equal
volume of the above Giemsa working
solution (1:1)

11. PROCEDURE
MGG LGC
• Slides were stained in May-Grunwald
stain diluted 1:5 with pH 6.8 buffer for 5
mins.
• Rinsed in buffer.
• Stained in Giemsa's stain diluted 1:10
with pH 6.8 buffer for 15 mins.
• The slides were washed in buffer, dried,
cleared and mounted.
• The air dried smears were flooded with
LG cocktail and left for 1 minute.
• An equal volume of buffer/distilled water
was added and left for 6 minutes.
• The slides were washed in tap water,
dried, cleared and mounted.

12. ◄Both the slides of all the cases were labelled and numbering was
done continuously to prevent bias. These blinded slides were
analyzed by the 2 pathologists.
The slides were viewed and compared by giving scores as
Score 1= Satisfactory, Score 2= Good, Score 3= Excellent.
Based on 5 parameters i.e., Overall Staining, Clarity Of Staining,
Cytoplasmic Staining, Nuclear Staining & Background Material
Staining.
◄The maximum score for a single case was calculated taking into
account all parameters and it was 15.
The overall maximum possible score in the study was calculated by
multiplying the number of cases by 15 for each of the 2 stains. QI of
the stain was obtained by ratio of actual score obtained/maximum
score possible.

13. RESULTS
◄ Out of the 100 cases:
1. 53 were Males
2. 47 were Females
◄ All the 200 slides from 100 cases were viewed
and included lesions like:
1. Lymph Node Lesions (38)
2. Thyroid (30)
3. Breast (18)
4. Subcutaneous Tissue (7)
5. Salivary Gland (3)
6. Others (4)

14. S.NO. PARAMETERS MGG LGC
1
OVERALL STAINING
SATISFACTORY 23×1=23 (No. of cases × score) 10×1=10
GOOD 67×2=134 45×2=90
EXCELLENT 10×3=30 45×3=135
SCORE 187 235
2
CLARITY OF STAINING
SATISFACTORY 31×1=31 10×1=10
GOOD 59×2=118 34×2=68
EXCELLENT 10×3=30 56×3=168
SCORE 179 246
3
CYTOPLASMIC STAINING
SATISFACTORY 43×1=43 10×1=10
GOOD 46×2=92 30×2=60
EXCELLENT 11×3=33 60×3=180
SCORE 168 250
4
NUCLEAR STAINING
SATISFACTORY 36×1=36 10×1=10
GOOD 53×2=106 40×2=80
EXCELLENT 11×3=33 50×3=150
SCORE 175 240
5
BACKGROUND MATERIAL STAINING
SATISFACTORY 37×1=37 10×1=10
GOOD 47×2=94 50×2=100
EXCELLENT 16×3=48 40×3=120
SCORE 179 230
ACTUAL SCORE OBTAINED/MAXIMUM POSSIBLE SCORE 888/1500 1201/1500
QUALITY INDEX 0.59 0.80

21. The differentiation between myoepithelial
and ductal epithelial cells in the breast.
The background material like colloid in
thyroid and intercellular secretions in
salivary gland acini.
The cytoplasmic and nuclear features of
the lymphoid cells along with
lymphoglandular bodies.
The nuclear features of malignant cells like
nuclear chromatin and nucleolus.
LGC was better than MGG
as it differentiated from
the background over
staining which could be
confused for background
material, and helped to
arrive at the diagnosis.

22. DISCUSSION
◄ FNAC is minimally invasive and the preferred
technique for the diagnosis of wide spectrum of
benign and malignant lesions.
◄ There are many factors which affect the
interpretation of the FNAC smears.
◄ Among which the method of sampling and
quality of staining are important.
◄ Sampling method is mainly dependent on the
experience of pathologist and quality of staining
depends on the type of stain and method
followed for staining.

23. LEISHMAN STAIN GIEMSA STAIN
• Leishman stain is a good nuclear stain. • Giemsa stain is a good cytoplasmic stain.
• Leishman stain is used widely for staining the
peripheral smears, intraoperatively for imprint
cytology in ovarian neoplasms, and in fluid
cytology for cell count and cell type.
• Giemsa stain is used for staining the peripheral
smears, bone marrow specimens, Plasmodium
species that cause malaria and some other
spirochete and protozoan blood parasites.
• When used alone it stains the nucleus and
extracellular ground substance intensely but
under-stains the individual cells, 3-dimensional
clusters and cytoplasmic granules.
• It provides lighter staining of the cell nucleus and
cytoplasmic granules.
Hence, combination of these two stains forms the LG cocktail which is relatively a new staining
technique which provides good nuclear morphology, crisp nuclear and cytoplasmic contrast,
staining of cytoplasmic granules and metachromasia to the background material.
In any malignant case the diagnosis depends on the nuclear features. The nuclear features like
variation and enlargement of the nucleus are exaggerated in air dried smears. If the background
stain is too intense the visualization of the nuclear features in cell clusters is prevented. In such cases
LGC is a good staining option.
◄ Romanowsky stains are stains with differential staining capabilities of a
combination of dyes. The major problem with these stains is instability.

24. PREVIOUS STUDIES PRESENT STUDY
Study by Garbyal RS et al.
• Cytoplasmic staining was found to be good with LGC
and excellent with MGG.
• Nuclear staining, morphology and background staining
were excellent with LGC.
• It was observed that LGC stain was better than MGG for
cytoplasmic staining.
Gajendra S et al.
• Peripheral blood and bone marrow smear examination
showed better nuclear and cytoplasmic staining using
LGC as compared to Leishman stain or Giemsa stain
when used alone.
• It also helped in morphological diagnosis and sub
classification of mainly leukaemia cases.
• In anaemia cases, polychromasia and RBC inclusions
were better demonstrated.
• Ring forms of the plasmodium also showed better
staining with LGC.
• The overall staining and clarity of staining was good
with LGC when compared to MGG stain.
Belgaumi UI et al.
• LGC provided comparable cytoplasmic staining, better
nuclear staining, morphology and background staining
to PAP in exfoliative cytology.
• It also has potential application in the screening of oral
cancer.
• LGC stain had same features when compared to MGG
stain. Cytoplasmic granules were stained better in LGC.

25. COMPONENTS
PAPANICOLAOU
STAIN
LEISHMAN STAIN GIEMSA STAIN
LEISHMAN
GIEMSA COCKTAIL
NUCLEI
Blue color due to
hematoxylin
Deep blue to blue-
violet
Blue color Deep blue color
CYTOPLASM Light blue Pink Light blue
KERATINIZED CELLS Orange/pink - - -
SUPERFICIAL CELLS Orange/pink - - -
INTERMEDIATE AND
PARABASAL CELLS
Green/blue - - -
METAPLASTIC CELLS Green and pink - - -
GROUND
SUBSTANCE
- - -
Moderate
metachromasia
Sidhu SK, Ramalingam K, Goyal S, Poonia M, Rajawat GS, Sharma N. Comparing the efficacy of leishman–giemsa cocktail stain, giemsa stain, and Papanicolaou stain in potentially malignant
oral lesions: A study on 540 cytological samples. Journal of cytology. 2018 Apr; 35(2):105.

26. MAY GRUNWALD GIEMSA PAPANICOLAOU STAIN LEISHMAN GIEMSA COCKTAIL
Chromatin pattern cannot be studied Excellent for chromatin staining Chromatin pattern can be studied
Metachromatic stain Not a metachromatic stain Metachromatic stain
Not a transparent stain Transparent stain Transparent stain
Good for demonstration of extracellular
substance
Not good for background or mucin
Good for demonstration of extracellular
substance
Procedure Time: 45 minutes Procedure Time: 45 minutes Procedure Time: 10 minutes
Expensive Expensive Cheap
Laborious multistep procedure requiring
many solutions
Multistep procedure requiring large
volumes of alcohol
One step procedure requiring 2 solutions
Needs prior fixation with methanol
Needs prior fixation with alcohol that
causes cell shrinkage by removing
intracellular water and gives a sharp
nuclear detail
No fixation required
Working solution is to be prepared every
day, so can stain only few slides
Rapid PAP kit can be used for staining
many slides
Working solution is not to be prepared
every day, so can stain many slides
Has tendency to precipitate due to which
there is high background staining.
Staining intensity decreases when material
dries.
It provides metachromasia to the
background material.
Dey P. Routine Staining in Cytology Laboratory. In Basic and Advanced Laboratory Techniques in Histopathology and Cytology 2018 (pp. 133-138). Springer, Singapore, and Belgaumi UI, Shetty
P. Leishman Giemsa cocktail as a new, potentially useful cytological technique comparable to Papanicolaou staining for oral cancer diagnosis. Journal of Cytology/Indian Academy of Cytologists.
2013 Jan; 30(1):18.

27. Smear showing differential staining of
cytoplasm (×400)
PAP
Smear showing inadequate cellular
details (×400)
MGG
Smear showing clear cellular details
with an enlarged nucleus and crisp,
granular chromatin (×400)
LGC
Belgaumi UI, Shetty P. Leishman Giemsa cocktail as a new, potentially useful cytological technique comparable to Papanicolaou staining for oral cancer diagnosis. Journal of Cytology/Indian
Academy of Cytologists. 2013 Jan; 30(1):18.

28. Garbyal RS, Agarwal N, Kumar P. Leishman-Giemsa cocktail. Acta cytologica. 2006; 50(4):403-6.

29. Intracytoplasmic mucin in
adenocarcinoma
Intracytoplasmic pink granules of
medullary carcinoma of the thyroid.
Extracellular mucin in
adenocarcinoma.
Garbyal RS, Agarwal N, Kumar P. Leishman-Giemsa cocktail. Acta cytologica. 2006; 50(4):403-6.

30. Metgud R, Naik S, Patel S. Spritzer: For diagnostic cytopathology. Journal of cancer research and therapeutics. 2017 Oct 1; 13(6):964.
Differential staining of cytoplasm in
healthy controls
PAP
Clear cellular details with a prominent
nucleus and crisp, granular chromatin
in healthy controls
LGC
Inadequate cellular details in healthy
controls
FEULGEN STAIN

31. Gajendra S, Jha B, Goel S, Sahni T, Sharma R, Shariq M, Jaiswal S, Sachdev R. Leishman and Giemsa stain: a new reliable staining technique for blood/bone marrow smears. International
journal of laboratory hematology. 2015 Dec; 37(6):774-82.
LGC

32. Gajendra S, Jha B, Goel S, Sahni T, Sharma R, Shariq M, Jaiswal S, Sachdev R. Leishman and Giemsa stain: a new reliable staining technique for blood/bone marrow smears. International
journal of laboratory hematology. 2015 Dec; 37(6):774-82.
LS

33. Gajendra S, Jha B, Goel S, Sahni T, Sharma R, Shariq M, Jaiswal S, Sachdev R. Leishman and Giemsa stain: a new reliable staining technique for blood/bone marrow smears. International
journal of laboratory hematology. 2015 Dec; 37(6):774-82.
GS

34. LIMITATIONS
◄ The limitation of this study is the sample size
and duration.
◄ The other stains like H & E and PAP stain were
not taken for comparing the cytological
parameters.

35. CONCLUSION
◄ In this study comparing the LGC and MGG
staining of air dried FNA smears, LGC stain was
found to have good overall staining
characteristic with a QI of 0.8 as compared to
0.59 of MGG stain.
◄ Hence, this cocktail can be used for staining
routinely of air dried smears to provide good
staining quality which adds overall efficacy of
the report generated.
◄ It also proves to be economical by saving time
and manpower.
◄ Can be used in situations with heavy outpatient
load, screening intra—operative smears, and
community screening programs.

36. REFERENCES
◄ https://en.wikipedia.org/wiki/Romanowsky_stain
◄ Culling CF, Allison RT, Barr WT. Cellular pathology technique. Elsevier; 2014
May 19.
◄ Dey P. Routine Staining in Cytology Laboratory. In Basic and Advanced
Laboratory Techniques in Histopathology and Cytology 2018 (pp. 133-138).
Springer, Singapore.
◄ Belgaumi UI, Shetty P. Leishman Giemsa cocktail as a new, potentially useful
cytological technique comparable to Papanicolaou staining for oral cancer
diagnosis. Journal of Cytology/Indian Academy of Cytologists. 2013 Jan;
30(1):18.
◄ Garbyal RS, Agarwal N, Kumar P. Leishman-Giemsa cocktail. Acta cytologica.
2006; 50(4):403-6.
◄ Metgud R, Naik S, Patel S. Spritzer: For diagnostic cytopathology. Journal of
cancer research and therapeutics. 2017 Oct 1; 13(6):964.
◄ Gajendra S, Jha B, Goel S, Sahni T, Sharma R, Shariq M, Jaiswal S, Sachdev R.
Leishman and Giemsa stain: a new reliable staining technique for blood/bone
marrow smears. International journal of laboratory hematology. 2015 Dec;
37(6):774-82.
◄ Sidhu SK, Ramalingam K, Goyal S, Poonia M, Rajawat GS, Sharma N.
Comparing the efficacy of leishman–giemsa cocktail stain, giemsa stain, and
Papanicolaou stain in potentially malignant oral lesions: A study on 540
cytological samples. Journal of cytology. 2018 Apr; 35(2):105.