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Background Morphometry and quantitative image analysis have shown considerable potential for diagnostic applications because they eliminate subjectivity and increase the measurement reproducibility. In our study, we investigated the use of morphometric quantitative image analysis as a tool to aid diagnosis of lesion tissues stained with seven staining techniques. Material and methods Thirteen already established lesions were examined in this study. Tissues were sectioned at 5 μm thickness using the rotary microtome and prepared for staining according to standard methods before staining them with seven different histochemical dyes: Haematoxylin and Eosin (H&E), Masson's Trichrome (MT), Periodic Acid Schiff (PAS), Phosphotungstic Acid Haematoxylin (PTAH), Southgate Mucincamine (SGM), Alcian Blue (AB) and Verhoeff Van Gieson (VVG). Photomicrographs were analyzed using imageJ for percentage area and intensity measurement. Results The average % area and intensity measurement based on the seven staining techniques used in this study were 47.88% and 122.23 points (pts). The lesions, adenomyosis in MT (80%), squamous cell carcinoma in PAS (80%) and fibroma in PTAH (77%) stained above the average % area. The squamous cell carcinoma stained with MT was the most intense tissue (55 pts), followed by Brenner tumour in PTAH (61 pts) and squamous cell carcinoma in PAS (69 pts). There were significant correlations between the % area of tissues covered by PAS and H&E ( r = 0.599, p = 0.030), and PAS and MT ( r = 0.572, p = 0.041) as well as in the intensity of tissues in PAS and H&E (r=0.615, p=0.025), VVG and H&E ( r = 0.707, p = 0.007), and VVG and PTAH ( r = 0.577, p = 0.039). Conclusion MT staining technique produced the best results in the % area of tissues covered and the intensity measurements, and therefore was recommended for routine use alongside with H&E in diagnostic histopathology.

Acquired perforating dermatosis is a group of disease characterized by transepidermal elimination of altered dermal constituents of unknown pathogenesis. The giant variant was first described in 2006, as an emerging entity with seven reported cases to date. Here is an 83-year-old male presented with a 4-year history of gradually enlarging soft tisssue mass with ulcerartions at the left knee joint. Imaging revealed an extra-articular, single, heterogeneous, multinodular mass, suspicious for a soft tissue sarcoma. Wide local excision of the mass showed fleshy, hemorrhagic nodules communicating with epidermal ulcers. Microscopy showed cystic spaces straddling dermis and subcutis, containing eosinophilic, amorphous, granular material extruding through epidermal craters, surrounded by exuberant myofibroblastic proliferation. Trichrome and van-Gieson stains confirmed that the extruded material is collagen and the histology was compatible with the giant variant of acquired perforating collagenosis. Awareness of histological appearance prevents misdiagnosis and overtreatment of this entity, masquerading as a sarcoma clinically.

Introduction Tissues submitted for histopathological diagnoses are routinely fixed in formaldehyde fixative which facilitates tissue storage for the extended period of time for further investigation or research purpose. The literature has reliable data relating to immunohistochemistry and molecular studies done using tissue fixed in formalin for longer duration and in paraffin-embedded tissue blocks. We have attempted to evaluate staining adequacy of archival specimens using hematoxylin and eosin (H&E) and trichrome stains. Methodology We compared staining adequacy in tissues stored in formalin for a minimum period of 5 years labeled as long term fixed tissues (LFT) and corresponding 5-year-old paraffin-embedded tissue blocks of same cases available in the archives designated as old tissue blocks (OTB), along with freshly fixed tissues (FFT) used as controls. Fifty-one tissue sections in each study group were stained with H&E, Mallory's trichrome and Van Giesons' and microscopic evaluation was carried out using Research Microscope B × 51. Results Staining adequacy of the tissue sections was found to be superior in FFT, followed by OTB and LFT using all three stains. Staining for H&E, was found to be superior as compared to trichrome stains. Conclusion Trichrome stains were poorly demonstrated in LFT indicating the possible alteration in protein structure and tissue architecture in LFT whereas H&E, stained LFT specimens showed relatively good staining character. Hence, OTB proved to be better as compared to LFT in terms of staining quality and hence archiving tissue in paraffin embedded tissue blocks serve as valuable bio-bank and source for prospective studies.

Background: Solid tumors such as oral squamous cell carcinoma (OSCC) are composed of malignant epithelial cells and the stroma in which these cells are dispersed. As the tumor progresses, the extracellular matrix undergoes dramatic morphological and architectural changes. Special stains make analysis easy and less erroneous by highlighting the area of interest and can be used to study these changes. Aim: The aim of the study was to analyze morphological changes in collagen fibers in various histological grades of OSCC using Masson's trichrome (MT) and Picrosirius red (PSR). Study Design: The study comprised 74 tissue samples, divided into two groups: Group I consisted of 63 cases of histologically proven OSCC (39 cases of well-differentiated squamous cell carcinoma [WDSCC], 17 moderately differentiated squamous cell carcinoma [MDSCC] and 7 poorly differentiated squamous cell carcinoma [PDSCC]) and Group II consisted of 11 cases of normal mucosa as controls. Materials and Methods: Sections were stained with hematoxylin and eosin, MT and PSR and observed under light and polarizing microscope, respectively. Statistical Analysis: ANOVA, Tukey's honestly significant difference post hoc multiple comparison test, Chi-square test and paired t-test were used for the statistical analysis. Results: As the grade of OSCC progressed, collagen fibers became thin, loosely packed and haphazard. The mean area fraction also decreased. They exhibited orange–red hue and strong birefringence in WDSCC, yellowish-orange hue and strong birefringence in MDSCC and greenish-yellow hue and weak birefringence in PDSCC. Conclusion: Initially, there is a reorganization of the collagen fibers in an attempt to prevent the invasion of tumor cells, but as cancer progresses, the stromal change enhances movement of the tumor cells within it, leading to metastasis.

Previously we found carbon tetrachloride (CCl4) induced cirrhosis associated cardiac hypertrophy and apoptosis. The purpose of this study is to determine whether further CCl4 treatment would induce cardiac cell fibrosis. The cardiac tissues were analyzed by H&E. histological staining, Trichrome Masson staining and Western blotting. The results showed that the CCl4-treated-only group exhibits more trichrome staining, meaning that more fibrosis is present. Moreover, CCl4 could further induce cardiac-fibrosis via TGF-β-p-Smad2/3-CTGF pathway. However, our data showed that the CCl4- indcued cardiac abnormalities were attenuated by Ocimum gratissimum extract (OGE) and silymarin cotreatments. In conclusion, our results indicated that the OGE and silymarin may be a potential traditional herb for the protection of cardiac tissues from the CCl4 induced cirrhosis associated cardiac fibrosis through modulating the TGF-β signaling pathway.

Background: Identification and differentiation of stromal hard tissue components is a challenging task. Numerous methods of demonstrating these components have been worked upon in the past. Although some of the methods have been successful, there are many drawbacks of employing them. The need of the hour, therefore is to develop and use a simple, rapid and cost-effective method of identifying stromal hard tissues as they may signify an important change in the diagnosis of the pathology. Our aim is therefore to observe the usability of tetrachromic VOF stain over Hematoxylin and Eosin and Masson's Trichrome in routinely encountered head and neck pathologies. Materials and Method: Routine cases such as Central and peripheral ossifying fibromas, osteomas, giant cell granulomas, osteomyelitis and malignancies like osteosarcomas were retrieved from the department archives and 3 sections from each block were prepared to stain with H and E, Masson's trichrome and modified tetrachromic VOF stains respectively using standard staining protocol. Results: Tetrachromic VOF takes an upper hand in stromal hard tissue differentiation irrespective of the pathology. Conclusion: Modified tetrachromic VOF is simple, cost-effective method and can be employed for diagnosis of cases with hard tissue differentiation within the stroma on routine basis.

Naturally derived bone substitute materials have to undergo controlled purification processes to be suitable for clinical application. The aim of the present article is to analyze the structure and composition of a new commercially available bovine-derived bone substitute material, SmartBone®, for the presence and localization of organic and inorganic constituents. Standardized conventional methods were performed for the histological analysis using hematoxylin and eosin, Azan, Masson-Goldner trichrome, as well as specific tartrate-resistant acid phosphatase staining, and were evaluated by light microscopy. The results showed a calcified trabecular structure with preserved lamellar substructure. In addition, organic structures were found within the osteocyte lacunae, the haversian canals, and the inter-trabecular region. The presence of organic remnants within the bone block raises the question on what extent including organic material could affect the regeneration process and application safety in clinical settings and whether including specific organic material such as collagen could be beneficial for bone regeneration. Thereby, further preclinical and clinical studies are necessary to evaluate the bone block examined here and to determine whether a clinician should accept the organic remnants within bone blocks.

Background: Oral sub mucous fibrosis (OSMF) is chronic insidious diseases of the oral cavity, pharynx and larynx with unknown etiology. It is characterized by mucosal rigidity. The proposed etiological factor for causing OSMF areca nut. Areca nut and arecoline cause fibroblast proliferation and collagen production. Aim and Objectives: Correlate the clinical and histopathological grading using Masson's trichrome and Van Gieson's stain in OSMF. Materials and Methods: Thirty patients with clinically proven cases of OSMF and grouped according to their mouth opening. Incisional biopsy was done. The sections were stained with hematoxylin and eosin for histopathological conformation. Selected cases were sectioned and stained with Masson's trichrome and Van Gieson special stain and graded histopathologically. Data was analyzed using SPSS 20.0 version. Pearson's correlation coefficient and Chi-square test were done to find out the association between variables. Student's t-test was used to find clinical and histopathological correlation. Results: The mean age of OSMF patients was 20–40 years and when compared with gender males were affected more than females. The stains between Masson's trichrome with Van Gieson's stain showed significant correlation, but when compared between clinical and histopathological grading there was no statistically significant. Conclusion: Clinical staging and histopathological grading in OSMF there is no correlation when using special stains such as Masson's trichrome and Van Gieson's stain.

Background and aim Odontogenic tumors are tumors that arise from the odontogenic epithelium and/or odontogenic ectomesenchyme. Ameloblastoma (AB) and keratocystic odontogenic tumor (KCOT) are common epithelial odontogenic tumors that have an aggressive and invasive character. Tumor microenvironment has an important role in tumor behavior and growth as it provides nutrients, oxygen through angiogenesis and myofibroblasts (MFs). This work was aimed to study the stromal architecture such as collagen organization, microvessel density, and MFs' number in ABs and KCOTs and determine the stromal architecture influence on their aggressiveness. Material and methods Forty-eight specimens were used as follow, 24 ABs, 20 KCOTs, two ameloblastic carcinomas, and two tissues of dental follicle (as a normal control). Specimens were examined by Masson's trichrome special stain as well as anti-CD34 and anti-human alpha smooth muscle actin antibodies for immunohistochemical staining. Results Statistical significant differences between ABs and KCOTs in microvessel density as well as MF number were obviously noticed. Conclusion Stromal architecture of epithelial odontogenic lesions might be regarded not only a structural support, but it may have a role in the pathogenesis of these lesions.

Background: Benign vascular lesions such as vascular malformation and hemangioma at times pose difficulty in diagnosis both for clinicians and pathologists. Vascular malformations are difficult to treat while hemangiomas resolve spontaneously in most instances. There are instances when vascular malformations, especially arteriovenous malformations (AVMs) have been misdiagnosed as hemangiomas and vice-versa. Clinical and radiological correlation with histopathological confirmation of these anomalies is important for the management of these lesionsAim: The aim was to study the histological characteristics of hemangiomas and vascular malformations and to study the utility of histochemical stains in their diagnosis. Materials and Methods: We retrospectively studied fifty cases retrieved from the records of Department of Pathology which were diagnosed as hemangioma (n=32) and vascular malformation (n=18) on Hematoxylin and Eosin (H and E) stain over a period of 18 months. The cases were analyzed based on findings of histochemical stains such as Verhoeff-van Gieson (VVG), Masson's trichrome (MT), and toluidine blue. Results: After reviewing all the cases with the use of histochemical stains, two of the three cases originally diagnosed as hemangioma turned out to be AVM and one to be venous malformation. An increased number of intra-lesional nerves were found in 16 of 19 cases of AVM and in both cases of venous and lymphatic malformation. Hemangiomas did not show increase in nerve bundles. Mast cells were found to be increased in proliferating hemangiomas and pyogenic granulomas as compared to AVMs. Conclusion: Hemangiomas and vascular malformations should be clearly differentiated to reduce the risk of treatment failure and recurrence. With the use of histochemical stains such as VVG, MT and toluidine blue, the diagnostic difficulty can be reduced and definitive diagnosis is possible.

Introduction: Oral submucous fibrosis (OSMF) is graded according to various histological factors which include the epithelial changes and the connective tissue changes. These features though could be identified in routine hematoxylin and eosin (H and E) staining; they could be better appreciated in special stains. This pilot study is an attempt to identify a single special stain that can act as an adjunct to H and E stain to help grade this potentially malignant disease. Aims and Objectives: To assess if special stains can improvise on differentiating the various histological changes seen in OSMF and to accordingly grade OSMF cases. Materials and Methods: Formalin-fixed paraffin-embedded tissue sections of OSMF-10 cases of each grade (n = 30). Three special stains: Van-Gieson, Mallory's trichrome and Masson trichrome. Statistical Analysis: The results obtained were tabulated and statistically analyzed using Chi-square test. Observations and Results: The thickness and degree of keratinization were best detected in Mallory's stain (100%) and were statistically significant; the subepithelial changes were better detected using special stains, especially Mallory's stain (100%). The changes in collagen fibers were better visualized in all three special stains but were not statistically significant. The changes in blood vessels were better detected in Van-Gieson's and Mallory's stain; the obtained results were statistically significant. The degree of fibrosis between muscle bundles could be detected in all the three special stains, but when compared the results were not statistically significant. The questionable areas of muscle degeneration, especially in deeper connective tissue were better detected in Mallory's (43%) and Masson's stain (43%) as compared to Van-Gieson stain (14%) and the results obtained were statistically significant. The inflammatory cells and dysplastic features are better visualized in routine H and E stains. Conclusion: Pathogenesis of OSMF is related to fibro-elastic and muscle degenerative changes in the connective tissue followed by secondary changes in epithelium. Routine H and E, stains all the connective tissue components in various shades of pink, use of special stains bestows contrast between different components of connective tissue, thus improvising grading of OSMF. Mallory's stain can be used as a single adjunct to H and E stain as both pattern of keratinization in the epithelium and changes in the superficial and deeper connective tissue could be ascertained.

Thunbergia laurifolia Lindl (TL) has been traditionally used as an antidote, anti-inflammatory, and anti-drug addiction. This study investigated the burn wound healing activity of TL leaf extract (TLL) from supercritical CO 2 extraction in rats. The extract was prepared to 2.5%, 5%, and 10% gel (TLL gel). Rats were induced to second-degree burn wounds. They were randomly divided into six groups (six rats/group), which five groups were topically applied gel base, 1% silver sulfadiazine gel, 2.5%, 5%, and 10% TLL gel, respectively, for 14 days. Six untreated burn rats were used as the control group. The rats in each group were evaluated for wound healing rate, histological parameters, and wound collagen content. Rats treated with 10% TLL gel had a higher wound healing rate than rats in the control and untreated groups. An increase in collagen content, which indicates good regeneration of wound skin, was observed in the TLL treated rats from a pathological study by Masson′s trichrome and collagen content assay. The results from this study suggest that T. laurifolia leaf extract obtained by supercritical CO 2 extraction promotes the recovery of wound skin by shortening the inflammation phase, increasing collagen content, and stimulating fibroblasts proliferation and migration in wound healing.

Context: Angiogenesis, the process that leads to the formation of new blood vessels, continues to be a topic of major scientific interest. There is an increasing hope that new discoveries will lead to newer therapies that target angiogenesis as a reliable option for disease therapy. Aims: The objective of this study was to assess the role of vascularity, correlation of morphometric aspects of vascularity, investigate its usefulness in the histopathological classification and prognosis in normal buccal mucosa (NBM), leukoplakia and squamous cell carcinoma (SCC) of buccal mucosa. Materials and Methods: The study sample consisted of 15 cases of NBM, 30 cases of leukoplakia, and 30 cases of SCC of buccal mucosa. The 75 archival samples were stained by hemotoxylin and eosin (H and E) and Masson's trichrome (MT). The stained sections were analyzed using image analysis software. Statistical analysis used : Statistical Package for Social Sciences (SPSS) 12.0 statistical software. Results : The combined mean vessel density (MVD) of all the cases in H and E was 0.1112 and for MT it was 0.2150. The difference of MVD between H and E and MT was statistically significant. The mean MVD in SCC (0.3455) for MT was higher than NBM (0.1314) and leukoplakia (0.1263). The mean MVD increased from stage III (0.3563) to IV (0.5312). It also increased from NBM (0.1314) to hyperkeratosis (0.1505) and decreased from grade I (0.3556) to II (0.2795) of oral SCC (OSCC). Conclusions: MVD can be used as an adjunct with other diagnostic modalities. Further studies are needed to standardize baseline levels for different sites and age groups.

We report a rare case of keratoconus with granular dystrophy with a follow-up of two decades, documenting the sequential presentation of two diseases confirmed by histology and genetic studies. A 13-year-old boy was diagnosed in 1988 with keratoconus in both eyes (BE) based on slit-lamp biomicroscopy findings of corneal ectasia in BE accompanied by Fleischer′s ring, Vogt′s striae, a small, old, healed hydrops. The left eye (LE) had central corneal thinning and scar in the central area involving the mid and posterior stroma secondary to healed hydrops. Penetrating keratoplasty (PKP) was advised. The boy was lost to follow-up till 1991 and presented with white, dot-like opacities in the central cornea in the RE only, suggestive of granular corneal dystrophy. Similar findings of white, dot-like opacities were noted in the LE in 1995 and the patient subsequently underwent PKP in BE. Histopathology of corneal buttons confirmed the presence of patchy, crystal-like orange deposits, which stained bright red with Masson′s trichrome. Mutational analysis of the TGFBI gene in patient′s DNA revealed a heterozygous mutation corresponding to a change in Arg555Trp in the keratoepithelin protein. Granular dystrophy recurred after 8 years in the RE.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:24:48Z No. of bitstreams: 0 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:24:48Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:24:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-01 Made available in DSpace on 2014-05-27T11:24:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-01 Background: Actinic cheilitis (AC) is a premalignant condition intimately related to exposure of the lips to sun rays. Aim: The objective of this study was to evaluate the elastic and collagen fibers in the lamina propria of AC. The degree of epithelial atypia was correlated with the quantity of elastic and collagen fibers. Materials and Methods: Fifty-one cases were investigated. One slide was stained with hematoxylin-eosin for the evaluation of atypia, the second was stained with Weigert′s resorcin-fuchsin for the assessment of elastic fibers, and the third slide was stained with Mallory′s trichrome for the analysis of collagen fibers. Results: Ordinal logistic regression analysis revealed a significant correlation between the presence of atypia and collagen fibers (P