Indian Journal of Dental Research

ORIGINAL RESEARCH
Year
: 2007  |  Volume : 18  |  Issue : 3  |  Page : 101--105

Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma


Madhuri R Ankle, Alka D Kale, Seema Charantimath, Seema Charantimath 
 Department of Oral Pathology and Microbiology, Institute of Dental Sciences, K.L.E.S's Academy of Higher Education and Research, Nehru Nagar, Belgaum - 590 010, India

Correspondence Address:
Madhuri R Ankle
Department of Oral Pathology and Microbiology, Institute of Dental Sciences, K.L.E.SSQs Academy of Higher Education and Research, Nehru Nagar, Belgaum - 590 010
India

Abstract

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. Aim of the study: 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. Materials and Methods: Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Man-Whitney U test. Results: A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections ( p = 0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.( p = 0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet ( p = 0.4429) or the H and E-staining techniques ( p = 0.2717). Conclusion: One per cent crystal violet provides a definite advantage over the H and E-stained sections in selectively staining the mitotic figures.



How to cite this article:
Ankle MR, Kale AD, Charantimath S, Charantimath S. Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma.Indian J Dent Res 2007;18:101-105


How to cite this URL:
Ankle MR, Kale AD, Charantimath S, Charantimath S. Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma. Indian J Dent Res [serial online] 2007 [cited 2020 Jul 7 ];18:101-105
Available from: http://www.ijdr.in/text.asp?2007/18/3/101/33784


Full Text

Mitosis is a process wherein a mother cell divides exactly into two identical daughter cells. The various phases of mitosis are prophase, metaphase, anaphase and telophase, some of which are seen in tissue sections. Defects of mitosis results in various nuclear abnormalities, namely, micronuclei, binucleation, broken egg appearance, pyknotic nuclei and increased number of and / or abnormal mitotic figures [Figure 1]. [1] These abnormal mitotic figures are commonly seen in oral epithelial dysplasia and squamous cell carcinoma. Increased numbers of and / or abnormal mitotic figures are important criteria that carry increased weightage in the grading of dysplasias. [2]

The distinction between a pyknotic nucleus, an apoptotic cell and a mitotic cell in a routinely stained tissue section may pose a problem. Errors in identifying a mitotic cell can thus weaken the reliability of histological grading due to the loose use of morphologic criteria. Combination of stains and modification of the existing histochemical techniques can overcome these problems. A literature search revealed numerous selective stains like crystal violet, toluidine blue and giemsa which highlight chromatin patterns. These stains have been used in brain tissue, uterus and breast carcinoma. [3],[4],[5]

Crystal violet is a basic dye which has a high affinity for the highly acidic chromatin of mitotic cells. Mitotic cells are stained magenta and stand out distinctly against a light blue background of resting cells. Therefore, this study was done with an aim to compare i) the staining of mitotic cells in H and E with that of crystal violet, ii) the number of mitotic figures in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in the crystal violet-stained sections with those in the H and E-stained sections.

 Materials and Methods



Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were retrieved from the archives of the Department. Two sections of 3 microns each were cut from the blocks. One section was stained with Haematoxylin and Eosin and the other section was stained with 1% crystal violet. Mitotic figures were counted using an oculometer grid in 15 grid fields under a magnification of x400 in a stepladder fashion [Figure 2]. The oculometer grid had 10 x 10 squares. But, the periphery of the square grid was restricted by the limited, circular field of the eyepiece. So, only the central, 4 columns and 10 rows (4 x 10 = 40 squares) were considered for the purpose. Hence, a grid field in this study consisted of 40 squares.

Criteria to identify the mitotic cells:

The criteria given by Van Deist et al. [6] were used to assign a structure as a mitotic figure in this study:

The nuclear membrane must be absent indicating that cells have passed the prophase.Clear, hairy extensions of nuclear material (condensed chromosomes) must be present-either clotted (beginning metaphase), in a plane (metaphase / anaphase) or in separate clots (telophase)Two parallel, clearly separate chromosome clots to be counted as if they are separate.

These are the criteria, which help to distinguish between various phases of mitosis from other commonly seen nuclear changes like pyknotic nuclei, apoptosis, karyorrhexis [Figure 3].

Mitotic count was expressed as the mitotic count per grid field and the mitotic count per square millimeter.

The mitotic count per grid field was calculated as, mitotic count / grid field

[INLINE:1]

The mitotic count per square milimeter was calculated as follows:

a) Area of 1 grid field = 0.0128 mm 2

b) Mitotic count per square milimeter =

[INLINE:2]

Collected data was statistically analysed using the Mann-Whitney U test at a confidence interval of 95%. All the data analysis was done using the Statistical Package for Social Sciences (SPSS) [Version 10].

 Results



No mitotic cells were seen either in the crystal violet-stained or in the H and E-stained sections in ten cases of normal oral mucosa. In epithelial dysplasia, the mean mitotic count /15 grid fields was 2.8667 (SD = standard deviation = 2.7997) in the H and E-stained sections. The mean mitotic count in crystal violet-stained sections was 6.2667 (SD = 4.9924).

In the carcinoma group, a mean mitotic count of 5.00/15 grid fields was obtained in the H and E-stained sections (SD = 5.4903) while a mean mitotic count of 7.9333 /15 grid fields was obtained in the crystal violet-stained sections (SD = 6.3524) [Table 1]. A significantly increased mitotic count was observed in crystal violet-stained sections of oral epithelial dysplasia and oral squamous cell carcinoma [Table 2],[Table 3] [(u = 2.1361; p = 0.0327) and (u = 2.0117; p = 0.0443) respectively] when compared with H and E-stained counterparts. No significant results were obtained when comparison of mitotic counts was made between the dysplasia and the carcinoma groups [Table 4],[Table 5].

 Discussion



Cell division is required to maintain tissue integrity. Increased and abnormal mitoses indicate genetic damage. This is an important feature in precancer and cancer. Thus identification and quantitation of mitotic cells forms an indivisible part of the histological grading systems used for prognostication of precancerous and cancerous lesions. The quantitation of mitotic figures has been on the backseat over the decades. Newer prognosticators like immunohistochemistry, flow cytometry, autoradiography, DNA ploidy measurements are now on the forefront. However, cost and time factors make them less feasible. A properly standardized histochemical stain and precise use of morphologic criteria for identification of a mitotic cell can overcome these problems.

The aim of this study was to use a simple, cost-effective technique to study the mitotic cells using 1% crystal violet as a selective stain and compare it with H and E staining in ten cases of normal oral mucosa and 15 cases each of oral epithelial dysplasia and squamous cell carcinoma. In this study, no mitotic cell could be identified either with H and E or with crystal violet stains in normal oral mucosa. The proliferative compartment in normal oral mucosa is confined to the basal and parabasal areas and the cells cycle very slowly. [7] Leary [8] said that mitotic figures are rare and are typically found in [3] Use of 1% crystal violet also eliminates the erroneous inclusion of pyknosis, apoptosis and karyorrhexis as mitotic figures thereby eliminating false positive results, which provides easy discrimination. The dysplasia group in this study included moderate to severe dysplasia cases whereas well to moderately differentiated carcinomas were considered in the carcinoma group.Comparison of the mitotic counts between the dysplasia and the carcinoma groups in either the crystal violet-stained sections or the H and E-stained sections showed no significant variations [Table 4],[Table 5]. Severe dysplasia frequently coexists with in situ carcinoma or in invasive carcinoma. [9] The boundaries between severe dysplasia, in situ carcinoma and early invasive carcinoma are indistinct. This could probably be the reason for the minimal difference in mitotic counts obtained in this study between the dysplasia and carcinoma groups. This can also be attributed to variation in the metabolic activity, stromal quantity, tumour cell heterogeneity and varying cellularity in a tumour tissue. [10] The proliferative compartment is the major target of carcinogens in the process of carcinogenesis, which is why many studies emphasize on the proliferative markers.

A literature search revealed no study wherein mitotic counts were compared between dysplasia and carcinoma by the use of a selective histochemical stain. Various methods that are being used to study proliferation include autoradiographic determination of thymidine labelling for proliferation. This is a time-consuming procedure requiring at least ten days. S phase fraction based on DNA content analysis measured by cytometry is another method, which is inaccurate in aneuploid tumours. Immunohistochemical expression of various proliferative markers such as Ki67, epidermal growth factor receptor (EGFR) and proliferating cell nuclear antigen (PCNA) is increased in dysplasia and carcinomas, more so at the invasive front of the carcinoma. [11] Although prognostically significant, the reaction pattern of immunohistochemical markers is similar in certain benign and reactive lesions. [2]

Therefore, considering all these drawbacks, although notoriously considered unreliable, mitotic cell counting is the easiest, cheapest and fastest way of assessing proliferation. It can be reproducible when precisely standardized staining techniques and identification criteria are strictly followed. Thus, the present study recommends the use of 1% crystal violet as a selective stain for mitotic figures.

 Conclusion



Crystal violet (1%) is a step ahead of the standard H and E staining in the staining of mitotic figures. How far a single tissue section can be representative of an entire tumour mass is a matter of debate. Thus, it is recommended that larger samples of the study groups and serial sections should be ideally studied to determine the reliability of mitotic figure counting as a reliable parameter. Finally, to validate the usefulness of crystal violet as a stain for evaluating proliferation / mitosis in tissues, a comparison of the mitotic counts obtained with 1% crystal violet should be carried out with an immunohistochemical proliferative marker.

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