SRM Journal of Research in Dental Sciences

: 2022  |  Volume : 13  |  Issue : 2  |  Page : 49--53

Comparative analysis of staining efficacy of Leishman's stain with hematoxylin and eosin stain in the assessment of keratin pearl formation and mitotic figures in different grades of oral squamous cell carcinoma

Tumpuri Srilatha, Jaya Singh, Dinesh Raja, Trupti Jain, Akhilesh Chandra, Rahul Agrawal 
 Unit of Oral Pathology and Microbiology, Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Correspondence Address:
Dr. Rahul Agrawal
Unit of Oral Pathology and Microbiology, Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh


Background: Keratin is an intermediate protein that structures the cytoskeleton of all epithelial cells. Keratin pearl is present in various pathological conditions such as oral squamous cell carcinoma (OSCC). Mitotic figures can be used as a valuable tool in assessing cellular proliferation and act as a prognostic indicator in dysplasia and malignancy of the oral cavity. Aim: The aim of this study was to compare the efficacy of Leishman's stain with hematoxylin and eosin (H and E) stain in assessing keratin pearls and mitotic figures. Materials and Methods: In total, 30 diagnosed cases of OSCC of three different histological grades were taken. In each case, two sections of 4–5-micron thickness were made and stained with routine H and E stain and Leishman's stain. Staining intensity and specificity results were analyzed statistically using the Chi-square test and P = 0.05 was considered to be statistically significant. Results: Both stains can distinguish three different types of OSCC histologically. Leishman's stain demonstrated staining intensity (P = 0.087) and specificity (P = 0.017) comparable to that of routine H and E. Conclusion: Leishman's stain distinctly stained the keratin pearl and mitotic figures with a higher intensity. However, H and E showed a higher specificity.

How to cite this article:
Srilatha T, Singh J, Raja D, Jain T, Chandra A, Agrawal R. Comparative analysis of staining efficacy of Leishman's stain with hematoxylin and eosin stain in the assessment of keratin pearl formation and mitotic figures in different grades of oral squamous cell carcinoma.SRM J Res Dent Sci 2022;13:49-53

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Srilatha T, Singh J, Raja D, Jain T, Chandra A, Agrawal R. Comparative analysis of staining efficacy of Leishman's stain with hematoxylin and eosin stain in the assessment of keratin pearl formation and mitotic figures in different grades of oral squamous cell carcinoma. SRM J Res Dent Sci [serial online] 2022 [cited 2022 Jul 3 ];13:49-53
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Keratinized and nonkeratinized epithelium exists in the oral mucous membrane. The cells of keratinized mucosa undergo continual cell proliferation and differentiation, resulting in a cornified layer of keratin-filled cells.[1] Keratin proteins are intermediate filament proteins that make up the cytoskeleton of all epithelial cells. Both benign and malignant epithelial tumors usually exhibit specific keratin expression linked to the origin cell type. Keratin has been utilized as an immunohistochemistry marker in cancer detection for a long time.[2],[3]

Tissue integrity necessitates the division of cells. Genetic damage is indicated by increased and aberrant mitosis. Premalignancy and malignancy both have this trait. As a result, mitotic figure identification and quantification are integral aspects of the histological grading system used to predict the prognosis of precancerous and cancerous tumors. Over the years, mitotic figure quantification has taken a back seat.[4],[5]

Immunohistochemistry, flow cytometry, autoradiography, and DNA ploidy assays are only a few of the modern techniques that are now being used. All of these approaches, however, are impractical for normal usage due to their high cost and lengthy time requirements.[6],[7]

The hematoxylin and eosin (H and E) stain is a commonly used stain that shows most of the histological structures of tissue and provides enough morphological features for the diagnosis of lesions. In the current study, Leishman's staining method was used as it is possible to visualize the nuclear chromatin pattern and cytoplasmic color contrast more clearly with a single step in one stain solution. The preparation time for the Leishman's stain is also shorter than that of the H and E. In spite of these advantages, the accuracy of histological sections should not be compromised, and hence, the current study was evolved in an attempt to discover as an alternative method to routine H and E for the staining of the tissues. The aim of this study was to compare Leishman's stain to H and E in cases of oral squamous cell carcinoma (OSCC) to develop a simple, cost-effective, and less technique-sensitive method for studying histological characteristics.

 Materials and Methods

Setting and design

The present retrospective study was conducted on histopathologically diagnosed 30 paraffin-embedded tissue blocks of OSCC taken from the unit of oral pathology. The study was approved by the Ethical Committee of the Faculty of Dental Sciences, Banaras Hindu University Varanasi (Dated: October 29, 2021; Ethics Committee No.Dean/2021/EC/2972). These 30 OSCC cases were further divided into three groups based on their histological differentiation as well differentiated, moderately differentiated, and poorly differentiated, and each group contained 10 cases each. Two 4–5-micron thick sections were cut from 30 paraffin-embedded tissue blocks and stained with H and E and Leishman's staining according to usual protocol.

Procedure for hematoxylin and eosin staining

Materials used for the stain solution are Harris hematoxylin, acid alcohol, dehydrating alcohols, and eosin.

After deparaffinizing, the sections were kept in xylene for 10 min, they were rehydrated in various grades of alcohol for 2 min eachStained for 5–10 min with Harris hematoxylin stainFollowing that, the samples were washed in water and differentiated in 1% acid alcohol. The slides were then subjected to bluing for 10–15 min in flowing tap water, followed by 1 min of eosin stainingDehydrated, cleared in xylene, and mounted with D.P.X (Dibutylphthalate Polystyrene Xylene).

Procedure for Leishman's stain

Materials used for the stain solution are powder mixture of methylene blue (a basic stain) and eosin (an acid stain) in methyl alcohol.

Sections were deparaffinized with xylene for 10 min and rehydrated with different grades of alcoholLater, slides were covered with freshly diluted Leishman's stain for 2–3 min. Methanol in the stain acts as a fixative and fixes the preparationLeishman's stain was diluted with twice its volume of distilled water for 10–12 minTissue sections were washed with distilled water or phosphate buffer of pH 7.0, then drained and dehydrated through a series of alcohol, cleared in xylene and mounted with D. P. X.

The staining intensity and specificity were scored by two observers for each case with both the stains based on the study by AI-Maaini and Bryant[8] [Table 1]. The obtained data were analyzed using SPSS version-23 software (Statistical Package for the Social Sciences-23, IBM Corp., Armonk, N.Y., USA) and the level of significance was kept at 5%. Staining intensity and specificity results were compared using the Chi-square test. Kappa values were calculated for intra-observer variations.{Table 1}


In our study, both staining procedures, i.e., H and E and Leishman's stain, showed clear and distinct keratin formation and structural details in histological sections of 30 OSCC. A kappa value of 1.0 was observed indicating a fair agreement between the two observers.

Statistically, a significant difference was not found (P = 0.087) between the staining intensity of both the stains [Table 2] and [Figure 1], [Figure 2]. A noticeable statistically significant difference in staining specificity (P = 0.017*) was observed for H and E stain in the demonstration of structural details and fine cellular details in OSCC [Table 3] and [Figure 3], [Figure 4].{Figure 1}{Figure 2}{Table 2}{Figure 3}{Figure 4}{Table 3}


In the head-and-neck region, OSCC is a malignant epithelial tumor that accounts for 90% of all oral cancers worldwide.[9] OSCC is one of the major causes of death due to complex genetic alterations and transformation of normal cells to malignant cells.[10] A lot of factors affect the prognosis of OSCC, including histological grades.[10] In OSCC, individual cell keratinization, keratin pearl formation and mitotic figures are important features for histological grading.[11]

A variety of malignant and nonmalignant lesions can be diagnosed using cytology. The procedure is simple, painless, and cost-effective.[12] Various stains, including H and E, Romanowsky, and Pap, have been used to stain the cytosmears.[13] Romanowsky stains are widely used to stain blood films and air-dried cytological smears. May–Grunwald–Giemsa (MGG) stain, Wright-Giemsa stain, Leishman's stain, and Diff-Quick are some of the Romanowsky stains that are available. By using metachromasia, these stains can better estimate cell size, nuclear size, and cytoplasm while also identifying ground particles.[14],[15] For air-dried smears in cytology, several laboratories employ MGG. The technique is time-consuming, and the stain tends to precipitate.[16] Leishman's stain is an excellent nuclear stain frequently used in hematology.[17] Belgaumi and Shetty, carried out a study in oral cancer patients and found that Leishman Giemsa cocktail produces equivalent cytoplasmic staining and superior nuclear staining than Pap, and that it could be used in the screening of oral cancer.[16]

In the literature review, several studies apply special staining techniques to paraffin-embedded tissue blocks to study keratin pearl formation and mitosis in oral malignancies and compare the results with routine H and E staining of tissue sections to determine stain intensity and specificity. Although various stains have been used to highlight specific histological features, they do not have standard results.[18]

Bala et al. conducted a study in paraffin-embedded sections of OSCC to compare the special stains such as alcian blue-performic acid–Schiff and rapid Papanicolaou (AB-PAS, R-PAP, and Gram's stain) for keratin with H and E stain and found that all four stains demonstrate keratin, the staining specificity of AB-PAS was found to be the best among all and the staining intensity was comparable to that of H and E.[19] Ramulu et al. conducted a study to compare the efficacy of the modified PAP staining procedure with that of Ayoub–Shklar (A-S) stain and H and E-staining technique, to devise a keratin staining procedure that is simple and effective, and concluded that the efficacy of modified PAP is comparable to that of H and E stain and A-S stain for surface keratin is effectively stained by this stain and therefore efficient for keratin staining.[20] A similar finding was also observed by Rao et al.[21]

In 1955, the application of PAP stain to paraffin-embedded sections of OSCC to detect keratin was described by Johnson and Klein et al. Later, Elzay et al. modified the standard PAP by adding phloxine-B, a red acid dye that stains prekeratin and mature keratin on paraffin-embedded sections in strongly red color.[22],[23]

All of the stains mentioned above are employed as unique stains to see certain tissues and cellular structures, where they bind to cellular components physically or chemically. Even modest foci of epithelial differentiation and minute patches of keratin that are undetectable in standard H and E are highlighted by these stains. All the special stains are focused on the limitations of routine, reliable, simple histological stain H and E in the demonstration of special structures. The detection and demonstration of keratin by all special stains has their own disadvantages like they are time-consuming and are not economical when compared to H and E.[24] To the best of our knowledge, this is the first study that shows Leishman's staining is efficient and reliable, to provide a staining procedure that is less expensive and less time-consuming. Further studies with large sample size are essential to utilize Leishman's stain as an adjuvant to traditional staining procedures.


Both H and E and Leishman's stains provided good overall staining intensity and specificity pattern to demonstrate keratin pearl production and mitotic figures. Leishman's stain distinctly stained the keratin pearl and mitotic figures with a higher intensity. However, H and E showed a higher specificity.

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Conflicts of interest

There are no conflicts of interest.


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