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Introduction: Exfoliative cytology in age estimation is a simple, painless, less invasive collection of exfoliative cells from epithelial layers, used as a diagnostic aid for age estimation. The oral cavity is an ideal site for exfoliative epithelial cells with a physiological turnover of cells, turnover decreases as the age increases show age variation with cellular morphological changes. Age estimation is one of the important factors to identify an individual and also helps to know the chronological age of a person. Aim: To analyze and estimate the age from buccal smear and comparing the average cellular size under Image morphometric analysis. Materials and Methods: Buccal mucosal smears are taken using a wooden spatula in gentle motion of scraping and smeared on a clean glass slide and fixed in 95% ethanol immediately after smearing a minimum of around 15 minutes and stained with Haematoxylin and eosin stain. After staining, the cells were observed by microscope and measured by a paint tool. Pearson correlation analysis was done using SPSS software. Results: The cell and nuclear size difference values observed using a Pearson correlation coefficient were statistically significant with p value

OBJECTIVE It remains a challenge to predict individual risk for recurrence after primary treatment of ductal carcinoma in situ (DCIS). While DCIS is contained within the duct, prior studies have pointed to the importance of stromal collagen in cancer progression. Using hematoxylin and eosin (H&E) stain-based images, we studied the collagen structure surrounding DCIS in a case-control cohort of women to determine whether stromal collagen could provide additional risk information. METHODS We present a quantitative histology image analysis pipeline (termed “histomics”) that consists of collagen segmentation, quantitative feature extraction and statistical analysis to predict recurrence. A cohort of 73 patients with DCIS (33 cases with recurrence matched with 40 controls) were retrospectively analyzed. H&E images were obtained from pathologic slides and regions-of-interest (ROI) of the collagen in the stroma adjacent to DCIS was automatically segmented using an in-house trained deep learning algorithm. A total of 123 histomics features (intensity, statistical and textural) were then extracted from the segmented ROI for each patient. Kruskal-Wallis test and receiver-operating characteristic methodology was used to assess performance of the features to differentiate patients with and without recurrence. RESULTS We found substantial variations in recorded H&E signal between samples, and the intensity and statistical features did not reach statistical significance. However, twelve percent of the textural features had an AUC between 0.62 to 0.61 with p-value between 0.06 to 0.08. Two textural histomics features in particular were significantly associated with DCIS patient recurrence; inverse variance (AUC: 0.64, p-value: 0.046) feature based on grey-level cooccurrences matrix (GLCM) and complexity (AUC 0.65, p-value: 0.025) feature based on neighborhood grey tone difference matrix (NGTDM). CONCLUSION Textural stromal features extracted from H&E image-based histomics indicate association of some features with tumor progression in DCIS. H&E image based histomics features can be a promising quantitative tool to develop predictive signatures of recurrence in patients with DCIS. We are processing additional patients through our quantitative image analysis pipeline to determine the significance of histomics features compared to conventional clinical features for the prediction of DCIS recurrence. Citation Format: Taman Upadhaya, Ronald Balassanian, Olivier Morin, Catherine Park. Prediction of breast ductal carcinoma in situ recurrence using histomics analysis of stromal features from hematoxylin and eosin stain-based images [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-43.

OBJECTIVE. A clinical challenge of ductal carcinoma in situ (DCIS) is to accurately predict individual risk for recurrence. Prior studies have highlighted the importance of stromal collagen surrounding breast ducts in breast cancer aggression. We studied the collagen structure surrounding DCIS in a matched case-control cohort of women with DCIS using second-harmonic generation (SHG) and hematoxylin and eosin (H&E) images to determine whether stromal collagen could provide additional risk information. METHODS. A cohort of 122 patients with DCIS (61 cases with recurrence matched with 61 controls) were retrospectively analyzed. Whole-side SHG and H&E images were obtained and regions-of-interest (ROI) of stromal collagen adjacent to DCIS was automatically segmented using an in-house trained deep learning algorithm. A quantitative histology image analysis pipeline (termed “histomics”) was exploited to extract a total of 123 histomics features (intensity, statistical and textural) from the segmented stromal region from both SHG and H&E ROI images. Clinical features previously shown to associate with DCIS recurrence including tumor size, margins and treatment were also collected. Conditional logistic regression model with lasso penalty was used to assess the performance of these features to stratify patients with and without recurrence. RESULTS. We found the combination of SHG, H&E and clinical features reached the highest concordance of 0.93 and the combination of only SHG and H&E features reached a concordance of 0.91. In contrast, clinical features alone attained a concordance index of 0.86. Model comparison by the Akaike information criterion (AIC​) shows that the higher concordance of the combination of SHG, H&E and clinical features may indicate better discrimination, whereas the higher concordance of combination of only SHG and H&E features was merely the result of including the larger number of features. Various textural features were found to be statistically significant (p Citation Format: Taman Upadhaya, Mary-Kate Hayward, Mi-Ok Kim, Ronald Balassanian, Valerie Weaver, Olivier Morin, Catherine Park. Prediction of breast ductal carcinoma in situ recurrence using histomics analysis of stromal collagen from second-harmonic generation and hematoxylin and eosin stain-based images [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-16.

Accuracy in morphological diagnosis is the cornerstone of anatomic pathology. Proficiency with the microscope offers values to the health care system that cannot be overestimated. However, that skill is only possible if high-quality histological substrates are available for assessment, particularly focusing on hematoxylin and eosin (H&E)-stained slides. This brief review considers the several steps that are necessary to control in the preparation of high-quality H&E sections, including those dealing with fixation, embedding, microtomy, histochemical staining, and coverslipping. A table for the troubleshooting of problem slides is also included.

Background: Immunohistochemical analyses of anterior cruciate ligament (ACL) allografts following remnant-preserving ACL reconstructions using Achilles tendon allografts have provided evidence for the presence of neural elements. In this study, we aimed to examine the expression of neural elements and quantify the presence of neural cells in ACL remnants and Achilles allografts using nerve growth factor (NGF) therapy after remnant-preserving ACL reconstruction. Methods: Experiments were conducted on 5 pairs of rats (approximately 8 weeks old and weighting 320 g at the time of surgery). Longitudinally-split Achilles tendons from the paired rats were freshly frozen and later defrosted with warm saline and allografted onto the right ACL of the other, which was partially detached at the femoral attachment site. A sham operation was conducted on the left knee to be used as Control. NGF was injected in both the knee joints 1 week after surgery. The presence of neural cells in the ACL of the sham-operated knee, allografted Achilles tendon, and ACL remnant was examined 6 weeks post surgery using H and E and immunohistochemical staining. Results: H and E staining did not reveal neural cells in any of the three groups. However, immunohistochemical analysis showed the presence of nestin-positive neural elements in normal ACL as well as ACL remnants. Additionally, neural elements were examined in 7 of the 8 (87.5%) allograft tissues. Quantitative analysis showed no difference in the number and area of nuclei among the three groups. However, the number and area of neural cells in Achilles allograft were significantly lower than in the other two groups (p=0.000 and p=0.001, respectively). Conclusion: Our observations indicate that ACL remnants promote new ingrowth and persistence of neural cells. We suggest that the ingrowth of neural elements could support the persistence and new ingrowth of mechanoreceptors, thereby enhancing the functional stability of knee joints. Moreover, the expression of neural cells in Achilles allograft was lower than that of normal ACL or ACL remnants in the quantitative evaluation, thereby confirming the essential role of ACL remnants in knee joint functionalization. Key terms: anterior cruciate ligament, remnant preservation, immunohistochemistry, nerve growth factor

Melanomas represent the malignant transformation of melanocytes, cells found primarily in the skin to protect epithelium and underlying connective tissues from harmful effects of ultraviolet light. Melanomas vary greatly in morphology and may continue to produce melanin markedly, mildly, or not at all. Performing and evaluating hematoxylin and eosin stains as well as immunohistochemical stains on pigmented melanomas has been a long-standing challenge due to the obscuring pigment. Protocols for removing melanin to reveal cellular morphology have been used successfully for years, but coupling these protocols with stains for immunohistochemistry represents an added challenge. In this study, the investigators evaluated results of various melanin bleaching protocols on tissue morphology, completeness of melanin removal, and immunohistochemistry staining quality. It was found that 1% formamide in 3% H2O2 under bright light without heating outperformed other tested protocols.

Histological analysis is a fundamental and principal method used in biological research and even for disease diagnosis. The result shows the status of cells and tissues in organs and enables us to infer the condition of the whole body. The tissue staining method known as hematoxylin and eosin staining (HE) is one of the most general methods of investigating the status of cells and tissues. Hematoxylin stains the nucleus violet and eosin stains cytosol pink. HE staining shows the unique morphologies of tissues and cells. However, after being stained with HE, tissues are very difficult to use in another histological analysis because hematoxylin is hard to remove from the sections due to its stain stability. Therefore, serial sections of the tissue are used to obtain more information through another staining, including immunohistochemistry. The adjacent tissue section is not the same as the HE-stained section, however, so the results from the adjacent sections can cause confusion or ambiguity. The present study showed that our decolorization solution can decolor the hematoxylin or iron hematoxylin stain from stained structures, including the nucleus, and the decolored section could be stained again in another staining, including immunohistochemistry. This decolorization method is very valuable, in that it can determine the accurate distribution of substances and features in cells and tissues, and thus it can improve the robustness of the resulting data.

Background: Keratins are the most abundant proteins and are characteristic findings in many epithelial pathologies, making it a diagnostically important marker, both histopathologically and immunohistochemically. Since, immunohistochemistry is an expensive diagnostic tool, special stains to detect the degree of keratinization could serve as a faster and economic option. The aim of the present study was to compare the efficacy of special stains for keratin with standard hematoxylin and eosin stain (H and E). Objectives include: (i) To subject the diagnosed cases of keratin disorders to the selected special stains: Ayoub-shklar method, Dane-Herman method, Alcian blue –periodic acid Schiff ’s (PAS), rapid papanicolaou (PAP) and Gram’s stain. (ii) To compare the staining specificity and staining intensity of special stains with respect to routine hematoxylin and eosin (H and E) stain. (iii) To compare the efficacy of special stains to routine H and E stain in identification of the type of keratin present in the selected cases. Materials and Methods: A total of 80 cases of known pathology for keratin were retrieved from the department archive, which included 10 each of normal gingiva, hyperkeratosis, squamous papilloma, verrucous hyperplasia, verrucous carcinoma, well-differentiated squamous cell carcinoma, orthokeratinized odontogenic cyst and keratocystic odontogenic tumors. Six sections of 4 µ each from the paraffin blocks were made, stained with H and E and the special stains and these were evaluated by 2 pathologists based on the modified scoring criteria from Rahma Al-Maaini and Philip Bryant 2008. Results: The results were tabulated using Chi square and kappa statistics. The statistical values for identification of the type of keratinization was insignificant showing that ortho and parakeratinized epithelia could be correctly identified by both H and E as well as all the special stains. Furthermore, all the special stains showed a positive result and statistical significance (P < 0.001) with respect to the staining of keratin. Conclusion: To conclude, though the special stains distinctly stained the keratin with a higher intensity, H and E proves to be overall better stain with respect to specificity

Background. Gastrointestinal cytomegalovirus (CMV) infection is a serious complication in immunocompromised patients; clinicians often expect expedited results for biopsy specimens. Our goal is to determine the accuracy of identification of CMV on hematoxylin and eosin (H&E) stain. Methods and Results. A total of 361 biopsy specimens from 273 patients with suspicion for CMV infection were retrieved. CMV was detected by immunohistochemistry (IHC) in 37 specimens acquired from 33 individual patients (average age = 54 years). Among the CMV-positive patients, 29 (88%) were reported to be immunosuppressed. Colon was the most common affected location. Of 37 CMV-positive specimens by IHC, 28 were positive by H&E (76%), 6 were negative (16%), and 3 were suspicious (8%). Of the 29 positive specimens on H&E, 28 were confirmed by IHC (97%) and 1 was indeterminate (3%). The sensitivity and specificity of H&E were 84% and 94%, respectively; the positive predictive value was 97%, and the negative predictive value was 93% ( P < .00001). Conclusion. Our results show that a preliminary diagnosis of CMV infection, based on H&E stains, can be reported with high specificity and low risk for false-positive results. Suspicious cases should be deferred pending the result of IHC stains.

Objective: The nature of calcifications in fibro-osseous lesions is difficult to differentiate under hematoxylin and eosin (H and E) stain and could be misleading. Special stains could be used. Modified Gallego's stain is a differential stain for hard tissues, which has been discussed recently in the literature. Methods: Retrospective study was done from June to December 2015 to differentiate various types of mineralized tissues in ossifying fibroma (OF), cemento-OF (COF), and cementifying fibroma (CF), using modified Gallego's stain and its correlation with H and E stain. Control group comprised of decalcified section of bone, tooth, and odontoma, stained with modified Gallego's stain. Study group comprised of 30 lesions (10 OF, 10 COF, and 10 CF) stained with both modified Gallego's stain and H&E stain. This study did not have any numerical data; therefore, no appropriate statistical test could be performed. Hence, cross tabulation of the categorical data was used followed by descriptive statistical analyses. Results were presented on continuous measurements using mean ± standard deviation, and results on categorical measurements were presented in number (%). Results: Modified Gallego's staining showed that, out of 10 cases of OF, 9 cases were interpreted as OF; one case of juvenile psammomatoid OF was interpreted as juvenile psammomatoid COF. Out of 10 cases of COF, 4 cases were interpreted as OF. Out of 10 cases of CF, 2 cases were interpreted as COF and 3 cases as OF. Conclusions: Fibro-osseous lesions are difficult to diagnose using H and E staining alone. Modified Gallego's stain could be a best adjunct.

The parasitic dinoflagellate Amoebophrya infects broad range of marine organisms. Particularly, Amoebophrya infections in planktonic dinoflagellates can prevent or delay the formation of algal blooms, and recycle undergrazed planktonic dinoflagellates back to the microbial loop by disrupting host cells. Its ecological significance was gradually recognized along with the discovery of its enormous molecular diversity in oceanic and coastal ecosystems. Thus, we developed a reliable, easily accessible and less time-consuming assay, to detect and assess Amoebophrya infections in planktonic dinoflagellates. The modified hematoxylin and eosin staining assay provided reliable diagnosis of Amoebophrya infection by identifying the characteristic "beehive" of the multinucleate trophonts. After staining, the typical multinucleate "beehive" is evidently distinguishable from the compact nuclei of uninfected host cells. The modified hematoxylin and eosin (H & E) staining assay is easy to use, that can be routinely performed within 3h (up to 20 samples/batch) using general laboratory equipment, supplies and chemical reagents. The produced slides with agar-embedded dinoflagellate cells can be stored for several months or even years in a dry place without noticeable loss in quality of staining. With suitable calculation, the modified H & E assay can be applied to assess the prevalence of Amoebophrya infection in planktonic dinoflagellates. This efficient and powerful assay will facilitate the investigation on the ecological roles of Amoebophryidae in coastal and oceanic ecosystem.