Walking a thin line between fixation and epitope binding - characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue.

Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde's cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.

Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens.

We present a rigorous validation strategy to evaluate the performance of Ultivue multiplex immunofluorescence panels. We have quantified the accuracy and precision of four different multiplex panels (three human and one mouse) in tumor specimens with varying levels of T cell density. Our results show that Ultivue panels are typically accurate wherein the relative difference in cell proportion between a multiplex image and a 1-plex image is less than 20% for a given biomarker. Ultivue panels exhibited relatively high intra-run precision (CV ≤ 25%) and relatively low inter-run precision (CV >> 25%) which can be remedied by using local intensity thresholding to gate biomarker positivity. We also evaluated the reproducibility of cell-cell distance estimates measured from multiplex images which show high intra- and inter-run precision. We introduce a new metric, multiplex labeling efficiency, which can be used to benchmark the overall fidelity of the multiplex data across multiple batch runs. Taken together our results provide a comprehensive characterization of Ultivue panels and offer practical guidelines for analyzing multiplex images.

An algorithm-based technique for counting mitochondria in cells using immunohistochemical staining of formalin-fixed and paraffin-embedded sections.

PURPOSE: Visualizing mitochondria in cancer cells from human pathological specimens may improve our understanding of cancer biology. However, using immunohistochemistry to evaluate mitochondria remains difficult because almost all cells contain mitochondria and the number of mitochondria per cell may have important effects on mitochondrial function. Herein, we established an objective system (Mito-score) for evaluating mitochondria using machine-based processing of hue, saturation, and value color spaces. METHODS: The Mito-score was defined as the number of COX4 (mitochondrial inner membrane) immunohistochemistry-positive pixels divided by the number of nuclei per cell. The system was validated using four lung cancer cell lines, normal tissues, and lung cancer tissues (199 cases). RESULTS: The Mito-score correlated with MitoTracker, a fluorescent dye used to selectively label and visualize mitochondria within cells under a microscope (R2 = 0.68) and with the number of mitochondria counted using electron microscopy (R2 = 0.79). Histologically, the Mito-score of small cell carcinoma (57.25) was significantly lower than that of adenocarcinoma (147.5, p < 0.0001), squamous cell carcinoma (120.6, p = 0.0004), and large cell neuroendocrine carcinoma (111.8, p = 0.002). CONCLUSION: The Mito-score method enables the analysis of the mitochondrial status of human formalin-fixed paraffin-embedded specimens and may provide insights into the metabolic status of cancer.

[Conventional paraffin embedding technique and double-embedding technique for minute oral biopsies and delicate pulp tissue - a comparative study]. / Traditsionnyi metod zalivki v parafin i metod dvoinoi zalivki dlya malen'kikh bioptatov polosti rta i myagkikh tkanei pul'py ­ sravnitel'noe issledovanie.

BACKGROUND: Among oral biopsies, small incisional tissues, have to be preserved all through the processing and embedding to ensure optimal visualization of all the mucosal layers without compromise. Optimal tissue orientation is the most critical step in tissue processing for demonstration of definitive morphology in the sections, which is often more challenging in cases of minute/small or thinner sections using routine paraffin techniques to evaluate accurate diagnosis. Some modification is needed to handle these samples to get a better result. Double embedding technique with some modification has been widely used for small/ thin/ multiple biopsies and gives excellent results in many other fields like general pathology and biotechnology. The double embedding technique though produced excellent and significant results in mucosal biopsies yet, it is of minimal interest among oral pathologists. To best of our knowledge, this is the first study to use double embedding technique for pulp tissues. OBJECTIVE: The present study was aimed to evaluate and compare the ease of embedding and sectioning sections using Agar-Paraffin double embedding technique for small oral mucosal biopsies and thin pulp tissues. MATERIAL AND METHODS: A total of 40 oral tissue samples categorized into two groups were taken for the present study. Group I included 20 small oral mucosal biopsy samples of size ranging from 0.2 to 0.5 cm and Group II included 20 pulp tissues obtained from freshly extracted non carious tooth. 10 blocks were prepared by routine paraffin method and 10 blocks were prepared by modified double embedding method for each group. Scores were given by comparing all the criteria with that of the routine paraffin technique. Chi-square test was used for statistical analysis. RESULTS: The average ease score for the Agar-Paraffin double embedded small/minute biopsies showed better scores than the pulp tissue with that of the routine technique. However, no statistically significant difference was seen among embedding and sectioning sections between the two groups. CONCLUSION: Modified double embedding method is simple and reliable alternative technique that helps in better orientation, processing and sectioning especially for oral small or thin biopsies and delicate pulp tissues.

Case report: Cutaneous anthrax diagnosed using mNGS of a formalin-fixed paraffin-embedded tissue sample.

The metagenomic next-generation sequencing (mNGS) method is preferred for genotyping useful for the identification of organisms, illumination of metabolic pathways, and determination of microbiota. It can accurately obtain all the nucleic acid information in the test sample. Anthrax is one of the most important zoonotic diseases, infecting mainly herbivores and occasionally humans. The disease has four typical clinical forms, cutaneous, gastrointestinal, inhalation, and injection, all of which may result in sepsis or meningitis, with cutaneous being the most common form. Here, we report a case of cutaneous anthrax diagnosed by mNGS in a butcher. Histopathology of a skin biopsy revealed PAS-positive bacilli. Formalin-fixed paraffin-embedded (FFPE) tissue sample was confirmed the diagnosis of anthrax by mNGS. He was cured with intravenous penicillin. To our knowledge, this is the first case of cutaneous anthrax diagnosed by mNGS using FFPE tissue. mNGS is useful for identifying pathogens that are difficult to diagnose with conventional methods, and FFPE samples are simple to manage. Compared with traditional bacterial culture, which is difficult to cultivate and takes a long time, mNGS can quickly and accurately help us diagnose anthrax, so that anthrax can be controlled in a timely manner and prevent the outbreak of epidemic events.

Novel method for classification of prion diseases by detecting PrPres signal patterns from formalin-fixed paraffin-embedded samples.

Prion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Sträussler-Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8-9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 µg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

Heat 'n Beat: A Universal High-Throughput End-to-End Proteomics Sample Processing Platform in under an Hour.

Proteomic analysis by mass spectrometry of small (≤2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a nearly universal, rapid, and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centered on a one-tube homogenization and digestion method called Heat 'n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided into separate challenges. The initial sample washing and final peptide cleanup steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin embedded (FFPE). Third, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenization treatment, and reduction, alkylation, and proteolysis were optimized into a single step. The refinements produced near doubled peptide yield when compared to our earlier method ABLE delivered a consistently high digestion efficiency of 85-90%, reported by ProteinPilot, and required only 38 min for core processing in a single tube, with the total processing time being 53-63 min. The robustness of HnB was demonstrated on six organ types, a cell line, and a cancer biopsy. Its suitability for high-throughput applications was demonstrated on a set of 1171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 h, producing highly consistent peptide yield and quality for over 3513 MS runs.

Isolation of total DNA from Placenta Samples, both Fresh and following Formalin and Paraffin Treatment.

The isolation of DNA from placental tissue suspected of infection is an important tool for identifying microorganisms such as bacteria, fungi, and viruses associated with complications during and after pregnancy. While experts primarily process placental tissue, the preservation methods employed pose challenges to extracting complete DNA. Therefore, selecting the appropriate protocol is paramount to achieving greater efficiency in obtaining genetic material.

Virtual formalin-fixed and paraffin-embedded staining of fresh brain tissue via stimulated Raman CycleGAN model.

Intraoperative histology is essential for surgical guidance and decision-making. However, frozen-sectioned hematoxylin and eosin (H&E) staining suffers from degraded accuracy, whereas the gold-standard formalin-fixed and paraffin-embedded (FFPE) H&E is too lengthy for intraoperative use. Stimulated Raman scattering (SRS) microscopy has shown rapid histology of brain tissue with lipid/protein contrast but is challenging to yield images identical to nucleic acid-/protein-based FFPE stains interpretable to pathologists. Here, we report the development of a semi-supervised stimulated Raman CycleGAN model to convert fresh-tissue SRS images to H&E stains using unpaired training data. Within 3 minutes, stimulated Raman virtual histology (SRVH) results that matched perfectly with true H&E could be generated. A blind validation indicated that board-certified neuropathologists are able to differentiate histologic subtypes of human glioma on SRVH but hardly on conventional SRS images. SRVH may provide intraoperative diagnosis superior to frozen H&E in both speed and accuracy, extendable to other types of solid tumors.

Enhancing Specific Fluorescence In Situ Hybridization with Quantum Dots for Single-Molecule RNA Imaging in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

Comparative Analyses of Targeted Myeloid Cancer Next-Generation Sequencing Panel in Fresh Blood, Bone Marrow and FFPE Material.

Next-generation sequencing is a vital tool for personalized diagnostics and therapies in cancer. Despite numerous advantages, the method depends on multiple parameters regarding the sample material, e.g., sample fixation. A panel's ability to ensure balanced pre-amplification of the regions of interest is challenging, especially in targeted sequencing approaches, but of significant importance to its applicability across hematological malignancies and solid tumors. This study comparatively evaluated the technical performance of the commercially available OncomineTM Myeloid Panel in fresh and Formalin-fixed paraffin-embedded (FFPE) material by using an Ion Torrent™ Personal Genome Machine™ System and Ion GeneStudio S5 System platform. In total, 114 samples were analyzed, including 55 fresh materials and 59 FFPE samples. Samples were sequenced with a minimum of one million reads. Amplicons with coverage below 400 reads were classified as underperforming. In fresh material, 49/526 amplicons were identified as performing insufficiently, corresponding with 18 genes. Using FFPE material, 103/526 amplicons underperformed. Independent of input material, regions in 27 genes, including ASXL1, BCOR and BRAF, did not match quality parameters. Subsequently, exemplary mutations were extracted from the Catalogue of Somatic Mutations in Cancer database. This technical evaluation of the OncomineTM Myeloid Panel identified amplicons that do not achieve adequate coverage levels and which need to be considered when interpreting sequencing.

Supported Immunohistochemical Staining of Mucins.

Immunohistochemistry (IHC) staining is the most common method to detect the distribution and localization of biomarkers in different parts of a tissue. Antibodies for tandem repeat peptide of mucins are very popular, but antibodies for glycosylation or others are also used. IHC for mucin is the same protocol as IHC for others. This description includes IHC according to ABC method for processed formalin-fixed paraffin-embedded (FFPE) tissues. Protocol of in situ hybridization is also shown.

Two complete 1918 influenza A/H1N1 pandemic virus genomes characterized by next-generation sequencing using RNA isolated from formalin-fixed, paraffin-embedded autopsy lung tissue samples along with evidence of secondary bacterial co-infection.

The 1918 influenza pandemic was the most devastating respiratory pandemic in modern human history, with 50-100 million deaths worldwide. Here, we characterized the complete genomes of influenza A virus (IAV) from two fatal cases during the fall wave of 1918 influenza A (H1N1) pandemic in the United States, one from Walter Reed Army Hospital in Washington, DC, and the other from Camp Jackson, SC. The two complete IAV genomes were obtained by combining Illumina deep sequencing data from both total RNA and influenza viral genome-enriched libraries along with Sanger sequencing data from PCR across the sequencing gaps. This study confirms the previously reported 1918 IAV genomes and increases the total number of available complete or near-complete influenza viral genomes of the 1918 pandemic from four to six. Sequence comparisons among them confirm that the genomes of the 1918 pandemic virus were highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases. Interestingly, in the Washington, DC, case, evidence is presented of the first reported Rhodococcus-influenza virus co-infection. IMPORTANCE: This study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of Rhodococcus-influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history.

Multiplex Immunofluorescence Staining Protocol for the Dual Imaging of Hypoxia-Inducible Factors 1 and 2 on Formalin-Fixed Paraffin-Embedded Samples.

Hypoxia is a common condition in rapidly proliferating tumors and occurs when oxygen delivery to the tissue is scarce. It is a prevalent feature in ~90% of solid tumors. The family of HIF (hypoxia-inducible factor) proteins-HIF1α and HIF2α-are the main transcription factors that regulate the response to hypoxia. These transcription factors regulate numerous downstream gene targets that promote the aggressiveness of tumors and therefore have been linked to worse prognosis in patients. This makes them a potential biomarker to be tested in the clinical setting to predict patient outcomes. However, HIFs have been notoriously challenging to immunolabel, in part due to their fast turnover under normal oxygen conditions. In this work, we developed a multiplexed immunofluorescence (mIF) staining protocol for the simultaneous detection of HIF1α and HIF2α in the same formalin-fixed paraffin-embedded (FFPE) tissue section.

A reliable transcriptomic risk-score applicable to formalin-fixed paraffin-embedded biopsies improves outcome prediction in localized prostate cancer.

BACKGROUND: Clinical manifestation of prostate cancer (PCa) is highly variable. Aggressive tumors require radical treatment while clinically non-significant ones may be suitable for active surveillance. We previously developed the prognostic ProstaTrend RNA signature based on transcriptome-wide microarray and RNA-sequencing (RNA-Seq) analyses, primarily of prostatectomy specimens. An RNA-Seq study of formalin-fixed paraffin-embedded (FFPE) tumor biopsies has now allowed us to use this test as a basis for the development of a novel test that is applicable to FFPE biopsies as a tool for early routine PCa diagnostics. METHODS: All patients of the FFPE biopsy cohort were treated by radical prostatectomy and median follow-up for biochemical recurrence (BCR) was 9 years. Based on the transcriptome data of 176 FFPE biopsies, we filtered ProstaTrend for genes susceptible to FFPE-associated degradation via regression analysis. ProstaTrend was additionally restricted to genes with concordant prognostic effects in the RNA-Seq TCGA prostate adenocarcinoma (PRAD) cohort to ensure robust and broad applicability. The prognostic relevance of the refined Transcriptomic Risk Score (TRS) was analyzed by Kaplan-Meier curves and Cox-regression models in our FFPE-biopsy cohort and 9 other public datasets from PCa patients with BCR as primary endpoint. In addition, we developed a prostate single-cell atlas of 41 PCa patients from 5 publicly available studies to analyze gene expression of ProstaTrend genes in different cell compartments. RESULTS: Validation of the TRS using the original ProstaTrend signature in the cohort of FFPE biopsies revealed a relevant impact of FFPE-associated degradation on gene expression and consequently no significant association with prognosis (Cox-regression, p-value > 0.05) in FFPE tissue. However, the TRS based on the new version of the ProstaTrend-ffpe signature, which included 204 genes (of originally 1396 genes), was significantly associated with BCR in the FFPE biopsy cohort (Cox-regression p-value < 0.001) and retained prognostic relevance when adjusted for Gleason Grade Groups. We confirmed a significant association with BCR in 9 independent cohorts including 1109 patients. Comparison of the prognostic performance of the TRS with 17 other prognostically relevant PCa panels revealed that ProstaTrend-ffpe was among the best-ranked panels. We generated a PCa cell atlas to associate ProstaTrend genes with cell lineages or cell types. Tumor-specific luminal cells have a significantly higher TRS than normal luminal cells in all analyzed datasets. In addition, TRS of epithelial and luminal cells was correlated with increased Gleason score in 3 studies. CONCLUSIONS: We developed a prognostic gene-expression signature for PCa that can be applied to FFPE biopsies and may be suitable to support clinical decision-making.

High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022.

High pathogenicity avian influenza viruses (HPAIVs) H5Nx of clade 2.3.4.4b have been circulating increasingly in both wild and domestic birds in recent years. In turn, this has led to an increase in the number of spillover events affecting mammals. In November 2022, an HPAIV H5N1 caused an outbreak in a zoological park in the south of France, resulting in the death of a Tibetan black bear (Ursus thibetanus) and several captive and wild bird species. We detected the virus in various tissues of the bear and a wild black-headed gull (Chroicocephalus ridibundus) found dead in its enclosure using histopathology, two different in situ detection techniques, and next-generation sequencing, all performed on formalin-fixed paraffin-embedded tissues. Phylogenetic analysis performed on the hemagglutinin gene segment showed that bear and gull strains shared 99.998% genetic identity, making the bird strain the closest related strain. We detected the PB2 E627K mutation in minute quantities in the gull, whereas it predominated in the bear, which suggests that this mammalian adaptation marker was selected during the bear infection. Our results provide the first molecular and histopathological characterization of an H5N1 virus infection in this bear species. IMPORTANCE: Avian influenza viruses are able to cross the species barrier between birds and mammals because of their high genetic diversity and mutation rate. Using formalin-fixed paraffin-embedded tissues, we were able to investigate a Tibetan black bear's infection by a high pathogenicity H5N1 avian influenza virus at the molecular, phylogenetic, and histological levels. Our results highlight the importance of virological surveillance programs in mammals and the importance of raising awareness among veterinarians and zookeepers of the clinical presentations associated with H5Nx virus infection in mammals.

Using formalin fixed paraffin embedded tissue to characterize the microbiota in p16-positive and p16-negative tongue squamous cell carcinoma: a pilot study.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is the most common oral cavity cancer, and p16 immunohistochemistry is an exact and available tool in the prognostic and predictive characterization of squamous cell cancers in the head and neck. Microorganisms have a close relationship with the development of TSCC. However, the association between oral bacteria and p16 status has not been well defined in the case of TSCC. Compared with traditional clinical microbial collection methods, formalin-fixed paraffin-embedded (FFPE) tissue samples have several advantages. METHODS: To compare the microbiota compositions between p16-positive and p16-negative patients with TSCC, we performed a small pilot study of microbiological studies of TSCC by paraffin tissue. DNA from FFPE tissue blocks were extracted and microbiomes were profiled by sequencing the 16 S-rRNA-encoding gene (V1-V2/V3-V4/V4 regions). Alterations in the functional potential of the microbiome were predicted using PICRUSt, Tax4Fun, and BugBase. RESULTS: A total of 60 patients with TSCC were enrolled in the study, however, some challenges associated with DNA damage in FFPE tissues existed, and only 27 (15 p16-positive and 12 p16-negative) passed DNA quality control. Nevertheless, we have tentatively found some meaningful results. The p16 status is associated with microbiota diversity, which is significantly increased in p16-positive patients compared with p16-negative patients. Desulfobacteria, Limnochordia, Phycisphaerae, Anaerolineae, Saccharimonadia and Kapabacteria had higher abundances among participants with p16-positive. Moreover, functional prediction revealed that the increase of these bacteria may enhance viral carcinogenesis in p16-positive TSCC. CONCLUSIONS: Bacterial profiles showed a significant difference between p16-positive TSCC and p16-negative TSCC. These findings may provide insights into the relationship between p16 status and the microbial taxa in TSCC, and these bacteria may provide new clues for developing therapeutic targets for TSCC.

Value of original and modified pathological scoring systems for prognostic prediction in paraffin-embedded donor kidney core biopsy.

BACKGROUND: No study has validated, compared and adapted scoring systems for prognosis prediction based on donor kidney core biopsy (CB), with less glomeruli than wedge biopsy. METHODS: A total of 185 donor kidney CB specimens were reviewed using seven scoring systems. The association between the total score, item scores, score-based grading, and allograft prognosis was investigated. In specimens with less than ten glomeruli (88/185, 47.6%), scoring systems were modified by adjusting weights of the item scores. RESULTS: The Maryland aggregate pathology index (MAPI) score-based grading and periglomerular fibrosis (PGF) associated with delayed graft function (DGF) (Grade: OR = 1.59, p < 0.001; PGF: OR = 1.06, p = 0.006). Total score, score-based grading and chronic lesion score in scoring systems associated with one-year and 3-year eGFR after transplantation. Total-score-based models had similar predictive capacities for eGFR in all scoring systems, except MAPI and Ugarte. Score of glomerulosclerosis (GS), interstitial fibrosis (IF), tubular atrophy (TA), and arteriolar hyalinosis (AH) had good eGFR predictive capacities. In specimens with less than ten glomeruli, modified scoring systems had better eGFR predictive capacities than original scoring systems. CONCLUSIONS: Scoring systems could predict allograft prognosis in paraffin-embedded CB with ten more glomeruli. A simple and pragmatic scoring system should include GS, IF, TA and AH, with weights assigned based on predictive capacity for prognosis. Replacing GS scores with tubulointerstitial scores could significantly improve the predictive capacity of eGFR. The conclusion should be further validated in frozen section.