Routine Staining Research Articles

Efficacy of Transdermal Delivery Nano Ethosomal Gel from Ashitaba Leaves on In-vivo Burn Wound Healing in Albino Rats

This study aimed to evaluate the in-vivo burn wound healing process in albino rats treated using transdermal delivery nano ethosomal gel from ashitaba leaves. Ethosomal vesicles were formulated using soy lecithin, cholesterol, ethanol, water, and ashitaba leaf nanoparticles using the cold method. A total of 25 male rats were randomly divided into 5 groups, i.e., (C-) treated without nanoparticle extract, (C+) treated using 1% Silver Sulfadiazine®, treatment group treated nano ethosomal gel from ashitaba leaves with the respective doses were (T1) 1%, (T2) 2,5%, and (T3) 5%. Therapy was initiated on day 1 or after being induced by a burn wound for 14 days, twice a day. On the 15th day, a termination was carried out to take skin tissue from burn scars, then histopathology preparations were made with routine staining. Microscopic observations with an optical microscope on collagen, polymorphonuclear cell infiltration, angiogenesis, and re-epithelialization. The T3 group that was treated using 5% nano ethosomal gel showed the best burn wound healing, this may be caused by compounds in ashitaba leaf nanoparticles which have antioxidant, anti-inflammatory, and antibacterial effects, thus the use of transdermal delivery therapy of 5% ashitaba leaf nano ethosomal gel was effective for wound treatment burn on rat skin.

The Use of Modified Masson’s Trichrome Stain to Recognize the Stratum Corneum in the Epidermis of the Skin and Measure It

Many types of research have stained the skin to determine its layers, especially the epidermis because it is an important and large body barrier against foreign materials. The most popular method was the routine stain that gives the tissues two colors depending on the acidophilic and basophilic to eosin and hematoxylin when the epidermis stain the trichrome stain the layers appear in two colors: pink color in the cytoplasm of the stratum germanitivium and the stratum spinusium and the stratum corneum, the nuclear stain with violet to gray color, in this paper we attempt to modify the Masson’s trichrome to stain the stratum corneum in a different color to recognize it and measure it by using Bouin’s solution with Carnoy’s solution as fixative material. The result of this modification was the stratum corneum appeared in yellow color and the other layers stayed in the same color as the general trichrome method and compared it with routine stain and measured the dead layer in the three methods. This research concludes indicates that the modified method can identify dead layers of the epidermis and becomes more accurate when measured with modern measurements.

Efficacy of Toluidine Blue Staining in Rectal Biopsy with Patients of Hirschsprung Disease

Background: The diagnosis of Hirschsprung disease (HD) is dependent on the histological study of rectal biopsy for ganglion cells and nerve fibers. Hematoxylin and Eosin (H&E) stain is commonly used. However, diagnosis is not possible with H&E staining every time, because staining has limitations in the diagnosis of immature ganglion cells in neonates and in the submucosal area where the ganglion cells are small in number. Using Toluidine blue (Tb) stain it has become easier to identify ganglion cells.Objective: To find out the efficacy of toluidine blue staining in the diagnosis of Hirschsprung disease.Materials and Methods: A cross-sectional descriptive observational study was carried out with the histological findings of 60 rectal punch biopsies and 60 full-thickness rectal biopsies from the corresponding same patients from March 2018 to February 2020. Ten cases of inadequate punch biopsy were excluded from the study. After paraffin embedding, slides were stained with H&E and Tb stains. Then stained sections were examined for the presence or absence of ganglion cells and hypertrophic nerve fibers in the submucosa and in between muscle layers. Result: Out of 50 punch biopsy cases, 33(66.0%) cases with H&E stain and 35(70.0%) cases with Tb stain showed presence of hypertrophic nerves (HN). In Non-HD cases, with H&E stain 11(22.0%) cases showed somewhat easy to identify ganglion cells. By using Tb stain ganglion cells were easy to identify in 4(8.0%) cases and somewhat easy to identify in 8(16.0%) cases. Out of 60 full-thickness rectal biopsy 38(63.30%) cases showed presence of hypertrophic nerves in both H&E and Tb stains. In Non-HD cases with H&E stain ganglion cells somewhat easy to identify in 8(13.3%) cases; easy to identify in 9(15.0%) cases and very easy to identify in 2(3.3%) cases. By using Tb stain, the identification of ganglion cells were 1(1.7%), 9(15.0%) and 9(15.0%) respectively. Study showed Tb stain was superior to H&E in the identification of ganglion cells and hypertrophic nerves.Conclusion: Toluidine blue stains should be used as the routine stain in adjunct to H&E stain to highlight ganglion cells in suspected Hirschsprung disease.KYAMC Journal Vol. 14, No. 02, July 2023: 69-73.

Classification of Salivary Gland Lesions on Cell Block Preparations with a Panel of Immunohistochemical Markers - a Rapid, Reliable, and Minimally Invasive Diagnostic Modality

<p>Fine-needle aspiration cytology (FNAC) is used as a valuable method for examining suspected salivary gland lesions. It is a simple, costeffective, and minimally invasive procedure with high specificity and sensitivity. Due to the cellular heterogeneity and overlapping architectural features, it can be difficult to distinguish between non-neoplastic processes, benign lesions, and/or malignancies in salivary glands on routine stains. Cell block methods are currently replacing surgical biopsy-based diagnostic methods on the basis of utilizing aspirates from FNAC with a rapid and reliable potential for reaching a conclusive diagnosis. Ancillary investigations including a panel of immunohistochemical markers are frequently applied on cytology specimens in the era of precision diagnostics to offer a specific diagnosis and even prognostic information for optimal patient care.</p>

“Ayoub-Shklar” as a Special Stain for Keratin: A Histopathological Study

Abstract Background: Special stains in histopathological studies are used to identify the structures with different dyes apart from the routine stain hematoxylin and Eosin (H and E). The component which is present in the special stains will have a specific bond and affinity for particular tissue components in the histological specimen. Structures like keratin takes up an eosinophilic stain in routine (H and E) staining. Most of the potentially malignant disorders and carcinomas arise due to the keratinization defect, which makes keratin an important diagnostic tool. There are different stains such as Ayoub-Shklar (A-S), Dane-Herman (D-H), and rapid Papanicolaou (PAP) which is used to identify keratin. In A-S stain, keratins can be stained in magenta-red and orange colors. Aim: we compared A-S special stain and routine stain in terms of staining intensity or quality, the pattern of staining, and specificity for staining keratin. Materials and Methods: Thirty cases from the department archives that included 10 well-differentiated squamous cell carcinoma, 10 verrucous carcinoma, and 10 epithelial dysplasia were taken and each case was stained with both A-S and H and E stain. Results: A-S showed an almost equal distribution of uniform and patchy staining patterns, but H and E showed more patchy staining patterns in the three groups. H and E stain shows good staining quality than A-S. A-S shows almost 90% of satisfactory staining specificity. Conclusion: Special stain like A-S stain can be used to stain keratin in different color, but H and E always remain gold standard stain.

Fungal infections in patients with progressing COVID-19: pathomorphology and description of clinical cases

Invasive pulmonary mycoses are a common complication of severe COVID-19 (COronaVIrus Disease 2019) and are characterized by rapid spread and high mortality. It is especially important to study the epidemiology and pathomorphology of fungal superinfection in order to understand the main vector of the diagnosis and treatment of this complication. The aim of this paper is to consider aspects of the epidemiology, pathomorphologic picture, and clinical manifestations of invasive pulmonary mycoses associated with COVID-19. Clinical cases of fungal superinfections (candidiasis, aspergillosis) associated with COVID-19 in patients receiving appropriate therapy were presented. Results of autopsy examination and light microscopy with routine staining were analyzed. Macroscopic assessment of lesions and histological examination revealed morphological multiorgan changes typical of the combination of fungal (candidiasis, aspergillosis) and viral infection (COVID-19). Conclusion. The presented data of post-mortem examination are important for both science and clinical practice as they form the basis for finding new ways of treating patients with comorbid pathology and developing a prognostic algorithm.

Prognostic Nomogram for Lymph-Node Metastasis in Oral Squamous Cell Carcinoma (OSCC) Using Immunohistochemical Marker D2-40.

Nomograms are proven in "individualized risk prediction" in sarcomas and breast and prostate cancers. Incorporating immunohistochemical markers and histopathological parameters can enhance accuracy of these graphical representations of statistical predictive models concerning metastasis. D2-40, a monoclonal antibody to podoplanin (regulator of motility expressed in malignant epithelial cells), dually predicts metastatic potential of tumour by estimating the motile tumour phenotype and by detecting lymphatic vessels/density, both essential to metastasis in OSCC. Thus, we propose a model that incorporates D2-40 immunostaining of individual tumour cells (ITC) too with other variables (seen in H+E staining) as a predictive nomogram. Sixty cases of OSCC were selected with equal number of cases (n=30) of pN0 and pN+ status. Bryne's grading of invasive front of tumour (ITF) was done on H+E-stained slides followed by D2-40 immunostaining for ITCs at ITF and lymphatic vessels. Multivariate regression analysis was used to generate the nomogram of LNM where the predictive contribution of each covariate, namely depth of invasion, D2-40-stained ITCs, gender, histological grade, and worst pattern of invasion (WPOI), was plotted on a scale of 1-100 points. The nomogram showed that the strongest variable in OSCC was the WPOI in H+E-stained section followed by D2-40-positive ITCs and gender. Our predictive nomogram for LNM in OSCC surprisingly showed that a tumour with lower score of WPOI (islands vs ITC) showed numerous D2-40-positive ITCs, drastically increasing the probability of metastasis. The concept of "individualized risk prediction" can be used to predict lymph node metastasis using a variety of histopathological criteria that can be visualized in routine and immunohistochemical staining in OSCC with the aid of a nomogram.

Histomorphometric analysis of angiogenesis in oral submucous fibrosis and oral squamous cell carcinoma associated with oral submucous fibrosis

Background: Oral submucous fibrosis (OSMF), now worldwide acknowledged as the disease of “Southeast Asia and Indian subcontinent”, has the utmost malignant transformation prevalence amongst oral precancerous disorders. Increased vascularity that is neoangiogenesis has been observed in the superficial connective tissue region of pre-cancerous lesions showing dysplasia. This could be an initial occurrence of the carcinogenesis process. The aim of this study will be to analyse neoangiogenesis by examining immunoexpression of CD105 in OSMF, and OSCC with OSMF cases. Methods: The project will comprise 30 normal oral mucosa samples (group I), 30 clinicopathologically diagnosed cases of OSMF (group II), and on the basis of association of OSMF, 30 surgically operated and histopathologically diagnosed cases of OSCC associated with OSMF (group III). Hematoxylin and eosin stains will be used for routine staining procedures and immunohistochemistry for the expression of CD105. Oral epithelial dysplasia (OED) cases of OSMF will be categorized into two groups, low-risk epithelial dysplasia (LRED) and high-risk epithelial dysplasia (HRED). The micro vessel density (MVD), total microvessel area (TVA), and mean microvessel area (MVA) are within and surrounding the tumor tissue sections immunostained with the CD105 antibody will be determined. ANOVA will be applied for evaluation of the mean scores of MVD, TVA, and MVA of groups II and III. The obtained score will be compared with different parameters of OSCC (TNM stage, lymph node metastasis, and histopathological grades). Conclusions: We postulate the progressively increased vascularity with the disease progression from LRED to HRED and further its transformation to invasive oral squamous cell carcinoma This increased vascularity will be evident by enhanced MVD, TVA and mean MVA, which is expressed by CD105 immunoexpression. This observation will emphasize the significance of neoangiogenesis in cases of OSMF with epithelial dysplasia and its further progression to OSCC.

Morphological Spectrum of Gastritis in Endoscopic Biopsies and Its Association With Helicobacter pylori Infection.

Introduction Helicobacter pylori(H. pylori)infection is the most common cause of gastritis. The consequences of the persistent infection range from acute ulceration to the development of gastric neoplasia. The purpose of the study is to determine the histopathological pattern of gastritis in gastric biopsies and its association with H. pylori. Materials and methods This is a retrospective study of all the gastric biopsies received in the Department of Histopathology, Chughtai Institute of Pathology, Lahore, Pakistan from January 12, 2021 to April 10, 2021. Sections were cut from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Slides were stained with routine, special and immunohistochemical stains. The cases were then blindly reviewed by two pathologists with a special interest in Gastrointestinal Pathology. Microscopic features based on updated Sydney classification were recorded. The collected data were then analyzed by using SPSS version 20(IBM Corp., Armonk, NY). Results A total of 429 gastric biopsies were examined. Out of which, 202 (47.1%) were male and 227 (52.9%) were female with a female-to-male ratio of 1.2:1. Their ages ranged from 12 to 100 years and a peak in the fourth decade of life with a mean age of 43 years (median age 49 years). Among gastritis, activity was seen in 194 (45%) and chronicity in 237 (55%) cases. H. pylori was seen in 174 (40.5%) cases and there was a strong statistically significant association between H. pylori infection and severity of gastritis (p<0.001). Other parameters of Sydney classification, such as atrophic changes, were seen in 144 (33%) cases, and intestinal metaplasia in 10 (2.3%) cases. Conclusion H. pylori organism was the most common cause of gastritis in our environment. The severity of gastritis is directly related to the H. pylori infection. If neutrophils are identified on surface epithelium, then H. pylori should be searched with increased attention on morphology and/or on immunohistochemical stain of H. pylori. Chronic inflammation and H. pylori density can also guide treatment which is necessary to avoid complications.

Rapid, Economic, Acetic acid, Papanicolaou (REAP) staining technique analogous to the routine Papanicolaou staining (PAP) technique

The current research compared classical Papanicolaou stain with REAP (Rapid, Economic, Acetic Acid Papanicolaou) stain to evaluate staining character, range, charge virtue, and sustaining of stain. The PAP Stain is the standard dye in support of shielding cell study for any cell study and is used in various research centers with several reductions. In certainty, PAP Stain has lowered squamous cell carcinoma incidence by 70%, particularly within wealthy provinces with effectively-designed broadcasting systems. But the stain has gone through several changes, moving from the uncreative formal mechanism -&gt; gradual REAP PAP staining in which staining duration level is decreased, it improves the quality, it is cost-effective, easily available, and ethyl alcohol is temporarily superseded with 1% ethanoic acid in advanced nations with guarding programs with careful planning. It has been used to access FNAC (fine needle aspiration cytology). Because of its precise nuclear and nucleoli staining, PAP staining of FNAC and fluid smears is a trustworthy stain. FNAC is a straightforward, speedy, and reliable pre-operative assessment and diagnosis process that has sparked advancements in dyeing technology which utilizes minimal coloring count with a clear cell framework. Around 40 minutes are needed for the standard methodology for traditional PAP Stain. REAP Stain was developed to address this, improving quality while cutting staining time to about one minute. This study's objective was to evaluate the value of the Rapid-Pap stain utilizing quantifiable investigation. The effectiveness of cytoplasmic and nuclear staining, stain preservation, value, and overall course of action for the two techniques—conventional PAP stain and REAP stain—were collated. When compared to traditional Papanicolaou, REAP stain offers a good, quick, and affordable replacement for the screening of cells. The REAP technique also does a superb job for the maintenance of stains.

Sharing of proximal fibers by the anterolateral and lateral collateral ligaments in the human knee: a cadaveric study

Literature is highly inconsistent in describing the proximal attachment of the anterolateral ligament (ALL) and its relationship with the lateral collateral ligament (LCL) in human knees. This observational study aims to investigate that lacuna. The gross dissection was performed in the lower limbs (n = 83) from the donated adult-age (> 18 years) embalmed cadavers from medical institutions in the north and east India. The dissected knee specimens were first examined macroscopically. Further routine and special staining and microscopic examinations were performed. The ALL was absent in approximately 20.4% of the studied knee specimens (17/83). In remaining, the sharing of ALL and LCL proximal fibers was observed as a consistent finding (~ 97%) with rare exceptions. The mean length of the tibial and meniscal limbs of ALL was 1.57 ± 0.8 cm [Range (R) 0.5–4 cm] and 0.73 ± 0.47 cm [Range (R) 0.1–1.6 cm], respectively. In addition, multiple variations in its presentation were observed. We propose that the proximal sharing of LCL-ALL fibers is a dominant feature in the studied population. The sharing of the fibers may impact the biomechanics and injury mechanisms for both ligaments. The possibility of ethnic variations in the ALL morphology should be a concern during reconstruction surgery.

Establishment and Characterization of a TFE3-rearranged Renal Cell Carcinoma Cell Line (FU-UR-2) With the PRCC-TFE3 Fusion Transcript.

Xp11.2-RCC was classified into molecularly defined renal carcinomas and named TFE3-rearranged renal cell carcinoma (TFE3-rRCC) in the 2022 World Health Organization classification of renal tumors. In this study, we established and characterized a TFE3-rRCC cell line from a right-sided renal tumor of a 35-year-old female patient and named it FU-UR-2. FU-UR-2 had been initially diagnosed as a papillary RCC because the patient was 35 years old, a routine immunohistochemical staining for TFE3 was negative, and its morphology was papillary. The G-band analysis revealed an X-chromosome aberration, thus we performed immunohistochemical re-staining for TFE3 and examined the aberration in the TFE3 gene by reverse-transcriptase polymerase chain reaction and fluorescence in situ hybridization. FU-UR-2 was confirmed as a TFE3-rRCC with a PRCC-TFE3 fusion transcript. Cultured FU-UR-2 cells continuously propagated over 90 passages and may provide a new permanent culture model to study pathogenetic mechanisms, investigate biological behavior, and develop new treatments such as molecular-targeting antitumor agents or immunological drugs for TFE3-rRCCs.

The Prognostic Role of Human Papillomavirus and p16 Status in Penile Squamous Cell Carcinoma-A Systematic Review.

PSCC is a rare cancer, with approximately half of all cases related to HPV. While HPV and p16 IHC testing have proven their prognostic value for oropharyngeal cancer, this is not yet established for PSCC. The current level of evidence exploring the relation between PSCC and HPV is moderate, so we conducted a systematic review following PRISMA guidelines to evaluate the prognostic role of HPV and p16 IHC in PSCC clinical outcomes. We searched the PubMed, Embase, and Cochrane databases and identified 34 relevant studies that met our inclusion criteria. Of these, 33 were retrospective cohort studies, and one was a cross-sectional study. Nine studies reported that HPV-positive and p16-positive PSCC had better overall survival (OS) and disease-free survival (DFS). This study highlights the need for a meta-analysis to determine the role of routine HPV status or p16 staining testing as part of the initial diagnosis and staging of PSCC patients worldwide.

Clinical significance of detection of micrometastases in lymph nodes in ovarian cancer

Ovarian cancer of epithelial origin is the most common type of oncological process in this organ and is characterized by a high probability of fatal outcome. It is believed that this is due to insufficiently effective diagnosis of the prevalence and severity of this disease. This review presents data on the development of disease recurrence in a significant number of patients after cytoreductive surgery. According to some authors, the reason for the high risk of recurrence is the insufficient information content of the method of studying histological preparations of regional lymph nodes after staining with hematoxylin and eosin to detect small-sized metastases, including isolated tumor cells and micrometastases. To improve the accuracy of such diagnostics, it is recommended to supplement the study of multilevel sections of lymph nodes using routine staining with the use of immunohistochemistry and other methods using various antibodies to cytokeratins, along with other epithelial markers. The review provides an analysis of the opinions of various researchers on the prognostic significance of the detection of micrometastases in the lymph nodes, which can increase the effectiveness of therapy due to a more accurate assessment of the prevalence of the disease and correction of the tactics of treating patients with various oncological diseases.

Artificial intelligence-based reticulin proportionate area - anovel histological outcome predictor in hepatocellular carcinoma.

Reticulin stain is used routinely in the histological evaluation of hepatocellular carcinoma (HCC). The goal of this study was to assess whether the histological reticulin proportionate area (RPA) in HCCs predicts tumour-related outcomes. We developed and validated a supervised artificial intelligence (AI) model that utilises a cloud-based, deep-learning AI platform (Aiforia Technologies, Helsinki, Finland) to specifically recognise and quantify the reticulin framework in normal livers and HCCs using routine reticulin staining. We applied this reticulin AI model to a cohort of consecutive HCC cases from patients undergoing curative resection between 2005 and 2015. A total of 101 HCC resections were included (median age = 68 years, 64 males, median follow-up time = 49.9 months). AI model RPA reduction of > 50% (compared to normal liver tissue) was predictive of metastasis [hazard ratio (HR) = 3.76, P = 0.004, disease-free survival (DFS, HR = 2.48, P < 0.001) and overall survival (OS), HR = 2.80, P = 0.001]. In a Cox regression model, which included clinical and pathological variables, RPA decrease was an independent predictor of DFS and OS and the only independent predictor of metastasis. Similar results were found in the moderately differentiated HCC subgroup (WHO grade 2), in which reticulin quantitative analysis was an independent predictor of metastasis, DFS and OS. Our data indicate that decreased RPA is a strong predictor of various HCC-related outcomes, including within the moderately differentiated subgroup. Reticulin, therefore, may represent a novel and important prognostic HCC marker, to be further explored and validated.

Digital Hepatic Iron Content: An Artificial Intelligence Model for Spatially Resolved Histologic Iron Quantitative Analysis in Liver Samples

Currently, the precise evaluation of tissue hepatic iron content (HIC) requires laboratory testing using tissue-destructive methods based on colorimetry or spectrophotometry. To maximize the use of routine histologic stains in this context, we developed an artificial intelligence (AI) model for the recognition and spatially resolved measurement of iron in liver samples. Our AI model was developed using a cloud-based, supervised deep learning platform (Aiforia Technologies). Using digitized Pearl Prussian blue iron stain whole slide images representing the full spectrum of changes seen in hepatic iron overload, our training set consisted of 59 cases, and our validation set consisted of 19 cases. The study group consisted of 98 liver samples from 5 different laboratories, for which tissue quantitative analysis using inductively coupled plasma mass spectrometry was available, collected between 2012 and 2022. The correlation between the AI model % iron area and HIC was Rs = 0.93 for needle core biopsy samples (n = 73) and Rs = 0.86 for all samples (n = 98). The digital hepatic iron index (HII) was highly correlated with HII > 1 (area under the curve [AUC] = 0.93) and HII > 1.9 (AUC = 0.94). The percentage area of iron within hepatocytes (vs Kupffer cells and portal tract iron) identified patients with any hereditary hemochromatosis–related mutations (either homozygous or heterozygous) (AUC = 0.65, P = .01) with at least similar accuracy than HIC, HII, and any histologic iron score. The correlation between the Deugnier and Turlin score and the AI model % iron area for all patients was Rs = 0.87 for total score, Rs = 0.82 for hepatocyte iron score, and Rs = 0.84 for Kupffer cell iron score. Iron quantitative analysis using our AI model was highly correlated with both detailed histologic scoring systems and tissue quantitative analysis using inductively coupled plasma mass spectrometry and offers advantages (related to the spatial resolution of iron analysis and the nontissue-destructive nature of the test) over standard quantitative methods.

Rivaling efficacy of special stains in the identification of Barr bodies

Background: Gender determination becomes the first priority in process of identification of a person by a forensic investigator in case of mass disaster, crime investigations, and ethnic studies. Demonstration of nuclear sex plays a vital role in determining the gender of an individual. Barr bodies (BB) are known to arise from the inactivation of the X chromosome in a female somatic cell. They are basophilic structures with varying morphology measuring approximately 0.8 × 1.1 microns in the nucleus during interphase. Various nuclear stains such as Thionine, Papanicolaou, Feulgen, Cresyl-violet, Giemsa, Aceto-orcein, and acridine orange &amp; Routine stains can validate Barr bodies. Aim To evaluate the efficacy of Feulgen, Giemsa, and H&amp;E stains in the demonstration of Barr bodies. Materials And Method Buccal smears were prepared from 25 males &amp; 25 females. These smears were stained by Feulgen, Giemsa &amp; Routine staining methods. The Barr body identification was determined by the presence of a darkly stained condensed area on nucleoplasm. The frequency of Barr body was examined by observing 100 nuclei per smear under a binocular light microscope at 100 magnification. For all BB-positive cases, Barr Body Index(BBI) was calculated and compared for the different stains used. Result Buccal cytological smears stained with Feulgen, Giemsa, and Routine stains. Samples with a presence of Barr bodies ≤2 were recorded as male and those with &gt;2 were recorded as female under microscopic findings. The percentage of Barr bodies in Feulgen-stained slides ranged from 30.2 - 37.9 among females and 0 - 0.1 in males, Giemsa slides ranged 14.2 - 22.2 from among females and 0 - 0.3 in males, while with Routine stained slides ranges recorded were10.2 - 14.9 in females and 0 - 0.5 in males. The sensitivity of Feulgen, Giemsa, and Routine stain for detecting sex accurately was 100% and specificity was 100%, 98%, and 94% respectively. Conclusion Feulgen stain proved to be better than Giemsa and routine stain for visualizing Barr Bodies.

Routine elastin staining improves venous invasion detection in colorectal carcinoma

BackgroundColorectal carcinoma is the second most common cause of cancer-related deaths in North America. Invasion of tumor cells into lymphatic and blood vessels is an imperative step in the metastatic progression of colorectal carcinoma. ObjectivesThis is a before-and-after study conducted by the Department of Pathology and Laboratory Medicine of Mount Sinai Medical Center of Florida to assess the impact on venous invasion (VI) detection by implementing routine elastin staining on all tumor-containing blocks per case, where feasible, in colorectal carcinoma (CRC) resection specimens. MethodsClinicopathological parameters of CRC specimens were collected from January until December 2021 (n = 93) for the pre-implementation cohort and from January until December 2022 (n = 61) for the post-implementation cohort. ResultsVI detection was significantly increased in the post-implementation cohort at a rate of 50.8 % compared to only 18.6 % in the pre-implementation cohort. The majority of VI identified in the pre-implementation cohort was extramural (61.5 %), whereas in the post-implementation cohort it was intramural (41.9 %). On univariate analysis, implementation of routine elastin stain was associated with strikingly increased VI detection rates (OR = 4.5, p-value < 0.001). On multivariate analysis, after adjusting for other clinicopathologic variables, elastin staining retained its independent statistically significant impact on VI detection (OR = 2.6, p-value = 0.034). Of note, there were no significant differences in the pre- and post-implementation cohorts in the frequency of nodal metastases, tumor extent, histologic grade, perineural invasion, T stage or M stage. ConclusionBased on our results and what has been published recently, we confirm an increase in the VI detection rate after implementing routine elastin staining on all tumor-containing blocks in CRC resection specimens.

(098) The Value to Patients of Immunohistochemical Staining of the Vestibule with CD117 and PGP9.5

Abstract Introduction Neuroproliferative vestibulodynia (NPV), associated with severe entrance dyspareunia, is diagnosed by an abnormally high density of nerves and mast cells as established by Bornstein. This cellular pathology cannot be appreciated even on vulvoscopic examination, making diagnosis only clinically suspected after exclusion of all other forms of vestibulodynia. Once NPV is suspected, a recommended treatment is complete vestibulectomy. Surgical specimen are sent to pathology for routine staining with hematoxylin and eosin (H&amp;E) which cannot confirm the suspected diagnosis of NPV. Objective We wished to assess value to the patient of being made aware of pathologic findings in excised vestibular specimen stained by CD117 and PGP9.5. Methods Since 2019, patients suspected of having NPV who underwent complete vestibulectomy had both routine H&amp;E and specially ordered CD117 and PGP9.5 staining of their excised specimen. Patients were asked post-operatively the benefits of this information. Results 56 specimens were studied. Tissue sizes varied from 5-11 cm long x 0.5 – 4 cm wide x 0.1 – 0.7 cm thick. Based on H&amp;E staining, all patients were found to have benign squamous vestibular epithelium with chronic inflammatory infiltrate at the superficial sub-epithelial stroma (Fig 1). The inflammatory infiltrate was composed of mature appearing lymphocytes in 87%, and a mixture of lymphocytes and plasma cells in 13%. Eosinophils were noted in 25% of cases. No ulcers, dysplastic or malignant cells were noted in any specimen. The diagnosis of NPV was confirmed by CD117 and PGP9.5 staining (Figs 2, 3) with excess mast cells, (&amp;gt; 8 cells per high power field x400) noted in all cases. Every patient in this cohort valued the pathologic information regarding the positive CD117 and PGP9.5 staining as final confirmation of their NPV diagnosis. This was particularly meaningful for those patients who had experienced medical gaslighting. Patients consistently used the term “validating”, saying the pathology report provided “tangible evidence” of a “physical problem wrong” when they had been told their “pain didn’t exist”. One woman said, “having that knowledge was priceless. It's one thing to have a belief that I had neuroproliferative vestibulodynia. It's another thing to KNOW. To have that indisputable proof is one of the most gratifying and emotional moments in my healing journey. It shows that there was something wrong. It wasn't all in my head. I didn't need to ‘just relax’.” Conclusions The clinical diagnosis of NPV is based on excluding other forms of vestibulodynia. The pathologic diagnosis of NPV is not based on routine pathology staining but on specialized IHC staining with CD117 and PGP9.5. If a patient is ignored, misdiagnosed, or invalidated by a provider, the patient loses self-confidence and faith in their own abilities. Clinicians need to be aware that not having visible signs when a patient complains of symptoms does not mean the dysfunction does not exist. Providing pathologic evidence of the root cause of entrance dyspareunia plays a significant role in the healing process. Disclosure No

(187) Routine Staining of Excised Vestibular Specimens with Hematoxylin and Eosin and Immunohistochemical Staining with CD117 and PGP9.5: Added Value to Patients

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is associated with increased density of mast cells in the epithelial basement membrane and nerve endings in the sub-epithelial stroma. As cellular pathology cannot be visualized on physical examination, diagnosis is difficult, and often delayed. Patients with entrance dyspareunia are suspected to have NPV only after excluding other forms of vestibulodynia. Surgical specimens from complete vestibulectomy with vaginal advancement flap reconstruction are sent to pathology for routine staining with hematoxylin and eosin (H&amp;E). Since 2019 we have also ordered immunohistochemical staining with CD117 and PGP9. Objective We wished to compare the pathologic findings in excised vestibular specimen examined by routine H&amp;E staining versus by CD117 and PGP9.5, and to assess the significance to the patient of having both pathologic examinations performed. Methods Patients with suspected neuroproliferative vestibulodynia who underwent complete vestibulectomy with vaginal advancement flap reconstruction had both routine H&amp;E staining and CD117/PGP9.5 staining of the excised specimen. Patients were asked post-operatively if and how this information benefitted them. Results Fifty-six patients formed the study group. Based on H&amp;E staining, all patients were diagnosed with benign squamous vestibular epithelium with chronic inflammation. Gross examination showed tissue sizes varying from 5-11 cm long x 0.5 – 4 cm wide x 0.1 – 0.7 cm thick. Microscopic examination showed chronic inflammatory infiltrate at the superficial sub-epithelial stroma (Fig 1). This was composed of mature appearing lymphocytes in 87%, and a mixture of lymphocytes and plasma cells in 13%. Eosinophils were noted in 25% cases. P16 immunohistochemical staining, performed to rule out HPV when appropriate, was negative in all cases. Focal parakeratosis was noted in 7% cases. No ulcers, dysplastic or malignant cells were noted in any specimen. Based on CD117 and PGP9.5 staining, the diagnosis of NPV was established (Figs 2, 3). Positive results consistent with excess mast cells, greater than 8 cells per high power field x400, were noted in 100% of cases. All patients (100%) valued the pathologic information, especially regarding the positive CD117 and PGP9.5 staining as final confirmation of their NPV diagnosis, in particular after experiencing medical gaslighting. They consistently used the term “validating”, providing “tangible evidence” of a “physical problem wrong with me” when they had been told their “pain didn’t exist”. “Having that ‘knowledge’ was priceless. It's one thing to have a belief that I had neuroproliferative vestibulodynia. It's another thing to KNOW. To have that indisputable proof is one of the most gratifying and emotional moments in my healing journey. It shows that there was something wrong. It wasn't all in my head. I didn't need to ‘just relax’.” Conclusions The clinical diagnosis of NPV is based on excluding other forms of vestibulodynia. The pathologic diagnosis of NPV is not based on routine pathology staining but on immunohistochemical staining with CD117 and PGP9.5. If a patient is ignored, misdiagnosed, or invalidated by a provider, the patient loses self-confidence and faith in their own abilities. Providing pathologic evidence of the root cause of entrance dyspareunia plays a significant role in the healing process. Disclosure No