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

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 Mann-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.

Role of potassium hydroxide mount in rapid diagnosis of fungal corneal ulcers.

The aim of the study was to establish the importance of KOH mount in rapid diagnosis of fungal corneal ulcers. Corneal scrapings were collected from 50 patients with corneal ulcers where clinical features suggested fungal aetiology and ulcers showed no signs of healing on initial treatment. Corneal scrapings were subjected to wet mount with 10% KOH, Gram's stain, and culture on SDA media. Wet mount showed fungal elements in 34 cases (68%) and were classified according to morphology. Gram's stain of corneal scrapings did not provide much help. Fungal culture was positive in 9 (26.4%) out of 34 cases. Specific antifungal treatment was instituted in all cases that showed positive wet mount without waiting for culture reports and on follow-up all these 34 patients showed improvement. Thus wet mount with 10% KOH can be relied upon as the singlemost important screening tool for rapid diagnosis of fungal corneal ulcer and treatment should be dispensed on its basis.

Early diagnosis of mycotic keratitis: predictive value of potassium hydroxide preparation.

Potassium hydroxide (KOH) preparation is an underutilized modality in the diagnosis of mycotic keratitis. We have earlier shown its utility in the diagnosis of Nocardia and Acanthamoeba keratitis. The aim of this study was (i) to evaluate the sensitivity, specificity and predictive value of KOH preparation, and (ii) to compare its efficacy with other methods of corneal scraping examination, for the diagnosis of mycotic keratitis. The study was conducted in two phases. In phase I, randomized corneal scrapings were examined by KOH, Gram's stain, and lactophenol cotton blue (LPCB) in 91 infectious keratitis subjects. In phase II, 53 corneal scrapings were stained with KOH and calcofluor white (CFW), and viewed with bright field (KOH) and fluorescence (CFW) microscopy. The KOH and CFW readings were recorded by an observer masked to the clinical findings and culture results. Nineteen scrapings were examined by two masked observers. In 22 culture positive fungal keratitis patients in phase I, the sensitivity of KOH, Gram's stain, and LPCB methods was 100%, 86.4%, and 77.3%, respectively. In phase II, the specificities of KOH and CFW were identical (83.8%), while the sensitivities were 81.2% and 93.7%, respectively (p = 0.59), in 16 culture positive mycotic keratitis patients. There was no significant difference between the negative and positive predictive values of KOH and CFW. Furthermore, no significant interobserver variability was found in the specificity and sensitivity. The KOH method compares well with other microscopy methods in the diagnosis of keratomycosis and has a definite place in the armamentarium of diagnostic techniques.

Corneal vital staining with gentian violet.

We have used 0.5% gentian violet solution as a corneal vital stain in 112 patients with variable degrees of corneal involvements and in 40 normal eyes as control. Gentian violet stained the epithelial defects and degenerated epithelial cells of cornea. The stain persisted 3-5 minutes and disappeared by 8-10 minutes. There was no cross infection from dye use. The dye did not hamper usual process of repair of corneal lesions. It is found highly feasible, with a sensitivity about 100% and specificity about 95%, with some minor side effects. Thus it could be an excellent method in community ophthalmology for early diagnosis of corneal affections, thereby commencing prompt and appropriate treatment in early stage.