High-Resolution Genotyping of Formalin-Fixed Tissue Accurately Estimates Polygenic Risk Scores in Human Diseases.

Formalin-fixed paraffin-embedded (FFPE) tissues stored in biobanks and pathology archives are a vast but underutilized source for molecular studies on different diseases. Beyond being the "gold standard" for preservation of diagnostic human tissues, FFPE samples retain similar genetic information as matching blood samples, which could make FFPE samples an ideal resource for genomic analysis. However, research on this resource has been hindered by the perception that DNA extracted from FFPE samples is of poor quality. Here, we show that germline disease-predisposing variants and polygenic risk scores (PRS) can be identified from FFPE normal tissue (FFPE-NT) DNA with high accuracy. We optimized the performance of FFPE-NT DNA on a genome-wide array containing 657,675 variants. Via a series of testing and validation phases, we established a protocol for FFPE-NT genotyping with results comparable with blood genotyping. The median call rate of FFPE-NT samples in the validation phase was 99.85% (range 98.26%-99.94%) and median concordance with matching blood samples was 99.79% (range 98.85%-99.9%). We also demonstrated that a rare pathogenic PALB2 genetic variant predisposing to cancer can be correctly identified in FFPE-NT samples. We further imputed the FFPE-NT genotype data and calculated the FFPE-NT genome-wide PRS in 3 diseases and 4 disease risk variables. In all cases, FFPE-NT and matching blood PRS were highly concordant (all Pearson's r > 0.95). The ability to precisely genotype FFPE-NT on a genome-wide array enables translational genomics applications of archived FFPE-NT samples with the possibility to link to corresponding phenotypes and longitudinal health data.

Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples and in normal urine.

We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.

Histochemical and biochemical analysis of collagen content in formalin-fixed, paraffin embedded colonic samples.

Collagen is the most abundant structural protein and extracellular matrix component in mammals. In the colon, collagen fibres reside in all the major sublayers; namely, the mucosa, submucosa, muscularis externa and the serosa. Methods to quantify collagen content in formalin-fixed, paraffin-embedded (FFPE) stained sections are required and image analysis offers a technique by which the spatial distribution and localisation of collagen fibres can be easily measured. This laboratory protocol was developed from established techniques using FFPE colon. Human colonic samples embedded transversally in paraffin wax were serially sectioned and stained with either Masson's trichrome (MT) or Picrosirius red (PSR). Quantitation estimation of collagen content in each sublayer was performed via ImageJ processing. Hydroxyproline content was quantified using a rapid and sensitive assay in sectioned tissue. Either MT or PSR staining followed by morphometric image analysis via ImageJ provided equally appreciable quantitative results. Moreso, analysis of hydroxyproline content in our samples indicate that this protocol could be useful in retrospective studies for FFPE samples. This laboratory protocol provides a systematic and reproducible method that can be utilized to accurately assess collagen content in individual sublayers of the colonic wall as well as detection of overall hydroxyproline content in FFPE specimens.

Colorectal Cancer Archaeome: A Metagenomic Exploration, Tunisia.

Colorectal cancer (CRC) is a serious public health problem known to have a multifactorial etiology. The association between gut microbiota and CRC has been widely studied; however, the link between archaea and CRC has not been sufficiently studied. To investigate the involvement of archaea in colorectal carcinogenesis, we performed a metagenomic analysis of 68 formalin-embedded paraffin fixed tissues from tumoral (n = 33) and healthy mucosa (n = 35) collected from 35 CRC Tunisian patients. We used two DNA extraction methods: Generead DNA FFPE kit (Qiagen, Germantown, MD, USA) and Chelex. We then sequenced the samples using Illumina Miseq. Interestingly, DNA extraction exclusively using Chelex generated enough DNA for sequencing of all samples. After data filtering and processing, we reported the presence of archaeal sequences, which represented 0.33% of all the reads generated. In terms of abundance, we highlighted a depletion in methanogens and an enrichment in Halobacteria in the tumor tissues, while the correlation analysis revealed a significant association between the Halobacteria and the tumor mucosa (p < 0.05). We reported a strong correlation between Natrialba magadii, Sulfolobus acidocaldarius, and tumor tissues, and a weak correlation between Methanococcus voltae and healthy adjacent mucosa. Here, we demonstrated the feasibility of archaeome analysis from formol fixed paraffin-embedded (FFPE) tissues using simple protocols ranging from sampling to data analysis, and reported a significant association between Halobacteria and tumor tissues in Tunisian patients with CRC. The importance of our study is that it represents the first metagenomic analysis of Tunisian CRC patients' gut microbiome, which consists of sequencing DNA extracted from paired tumor-adjacent FFPE tissues collected from CRC patients. The detection of archaeal sequences in our samples confirms the feasibility of carrying out an archaeome analysis from FFPE tissues using a simple DNA extraction protocol. Our analysis revealed the enrichment of Halobacteria, especially Natrialba magadii, in tumor mucosa compared to the normal mucosa in CRC Tunisian patients. Other species were also associated with CRC, including Sulfolobus acidocaldarius and Methanococcus voltae, which is a methanogenic archaea; both species were found to be correlated with adjacent healthy tissues.

Quantitative analysis of lncRNA in formalin-fixed paraffin-embedded tissues of oral squamous cell carcinoma.

The study evaluated expression profiles of few regulatory lncRNAs in oral squamous cell carcinoma and normal mucosa adjacent to oral cancer using paired fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues stored at a different duration of time (1-5 years) using real-time quantitative PCR. The quantity and quality of total RNA isolated from FFPE tissues was less compared with that of fresh frozen tissues, which resulted in a noncorrelation of quantification cycle values. Following normalization, the expression of lncRNAs in the paired tissues did not differ significantly. The differential expression of the lncRNAs in the study was consistent with The Cancer Genome Atlas head and neck squamous cell carcinoma database. The study findings demonstrate the possibility of performing accurate quantitative analysis of lncRNAs using short amplicons and standardized real-time quantitative PCR assays in oral squamous cell carcinoma FFPE samples.

Analysis of Copy Number Variation of DNA Repair/Damage Response Genes in Tumor Tissues.

Cells experience increased genome instability through the course of disease development including cancer initiation and progression. Point mutations, insertion/deletions, translocations, and amplifications of both coding and noncoding regions all contribute to cancer phenotypes. Copy number variation (CNV), i.e., changes of the number of copies of nuclear DNA, occurs in the genome of even normal somatic cells. Studies to understand the effects of CNV on tumor development, especially aspects concerning tumor aggressiveness and the influence on outcomes of therapeutic modalities, have been reignited by the breakthrough technologies of the molecular genomics. This section discusses the significance of analyzing CNVs that cause simultaneous increase/decrease of clusters of genes, using the expression profile of XRCC1 with its neighbor genes LIG1, PNKP, and POLD1 as an example. Methods for CNV assay at the individual gene level on formalin-fixed, paraffin-embedded (FFPE) tissues using the NanoString nCounter technology will then be described.

In Situ Proximity Ligation Assay to Visualize Protein-Protein Interactions in Tumor Specimens.

Protein-protein interactions (PPI) are the basis of various biological phenomena, such as intracellular signal transduction, gene transcription, and metabolism. PPI are also considered to be involved in the pathogenesis and development of various diseases, including cancer. PPI phenomenon and their functions have been elucidated by gene transfection and molecular detection technologies. On the other hand, in histopathological analysis, although immunohistochemical analyses provide information pertaining to protein expression and their localization in pathophysiological tissues, it has been difficult to visualize the PPI of these proteins. An in situ proximity ligation assay (PLA) was developed as a microscopic visualization technique for PPI in formalin-fixed, paraffin-embedded (FFPE) tissues as well as in cultured cells and frozen tissues. PLA using histopathological specimens enables cohort studies of PPI, which can clarify the significance of PPI in pathology. We have previously shown the dimerization pattern of estrogen receptors and significance of HER2-binding proteins using breast cancer FFPE tissues. In this chapter, we describe a methodology for the visualization of PPI using PLA in pathological specimens.

Genotyping of dengue virus from infected tissue samples embedded in paraffin.

Dengue has become one of the vector-borne diseases that affect humans worldwide. In Latin American countries, Colombia is historically one of the most affected by epidemics of this flavivirus. The underreporting of signs and symptoms of probable cases of dengue, the lack of characterization of the serotypes of the infection, and the few detailed studies of postmortem necropsies of patients are among other conditions that have delayed progress in the knowledge of the pathogenesis of the disease. This study presents the results of fragment sequencing assays on paraffin-embedded tissue samples from fatal DENV cases during the 2010 epidemic in Colombia. We found that the predominant serotype was DENV-2, with the Asian/American genotype of lineages 1 and 2. This work is one of the few reports of the circulating genotypes of dengue during the 2010 epidemic in Colombia, one of the most lethal dates in the country's history.

Fungal Integrated Histomolecular Diagnosis Using Targeted Next-Generation Sequencing on Formalin-Fixed Paraffin-Embedded Tissues.

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Biomarker Analysis of Formalin-Fixed Paraffin-Embedded Clinical Tissues Using Proteomics.

The relatively recent developments in mass spectrometry (MS) have provided novel opportunities for this technology to impact modern medicine. One of those opportunities is in biomarker discovery and diagnostics. Key developments in sample preparation have enabled a greater range of clinical samples to be characterized at a deeper level using MS. While most of these developments have focused on blood, tissues have also been an important resource. Fresh tissues, however, are difficult to obtain for research purposes and require significant resources for long-term storage. There are millions of archived formalin-fixed paraffin-embedded (FFPE) tissues within pathology departments worldwide representing every possible tissue type including tumors that are rare or very small. Owing to the chemical technique used to preserve FFPE tissues, they were considered intractable to many newer proteomics techniques and primarily only useful for immunohistochemistry. In the past couple of decades, however, researchers have been able to develop methods to extract proteins from FFPE tissues in a form making them analyzable using state-of-the-art technologies such as MS and protein arrays. This review will discuss the history of these developments and provide examples of how they are currently being used to identify biomarkers and diagnose diseases such as cancer.

DIGE Saturation Labeling for Scarce Amounts of Protein from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue.

In this chapter, we describe the utility of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) as a proteomics platform for the global detection of expressed proteins in formalin-fixed paraffin-embedded (FFPE) tissues and its use for biomarker discovery/identification of proteins that may contribute to cancer development and progression. Formalin fixation and paraffin embedding of tissue is the standard processing methodology practiced in pathology laboratories worldwide, resulting in a highly stable form of tissue that is easily stored due to its inherent stability at room temperature. Consequently, FFPE tissues represent an attractive reservoir of clinical material for conducting retrospective protein biomarker analysis. A limitation for proteomics research in this type of clinical sample is the amount of viable protein that can be obtained from fixed tissues. Tissue biopsies are precious samples that can generally be acquired in very small amounts due to the invasive nature of the sample collection, mainly during surgery or biopsy. Subsequently, the amount of extracted protein can be, in many cases, very limited. The saturation 2D-DIGE technology has emerged as a useful method for protein analysis where only scarce amounts of protein are available. This approach can be adapted successfully to label low-level protein isolated from FFPE tissue.

Relative Quantification of Proteins in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue Using Multiplexed Mass Spectrometry Assays.

The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery.

Evaluation and Comparison of Genomic DNA Extraction Methods and PCR Optimization on Archival Formalin-Fixed and Paraffin-Embedded Tissues of Oral Squamous Cell Carcinoma.

Recovery and amplification of nucleic acids from archived formalin-fixed tissue samples is the most developing field in retrospective genetic studies. We compared different deparaffinization methods and DNA isolation techniques, and intergroup comparisons were performed to evaluate the effectiveness of different storing methods for archival OSCC samples based on obtained mean DNA quantity, quality, and PCR amplification of the P53 gene. The study comprised 75 archival histologically diagnosed OSCC samples which were divided into Group I: Formalin-fixed paraffin-embedded tissue blocks and Group II: Long-term formalin-fixed tissue. A comparison of different deparaffinization methods showed that xylene deparaffinization is an efficient method to obtain suitable DNA. Comparing different DNA isolation techniques illustrated that the conventional phenol-chloroform method gives better integrity to DNA in contrast with the kit method. Comparison between FFPET and long-term FFT samples demonstrated that samples fixed in formalin overnight and embedded in wax yield better quality and quantity DNA in comparison with long-term samples fixed in formalin. To obtain suitable integrity of DNA, tissue samples should be stored by fixing in formalin overnight followed by preparation of paraffin tissue blocks, deparaffinization by xylene, and subjecting them to the conventional phenol-chloroform DNA isolation protocol.

Moving translational mass spectrometry imaging towards transparent and reproducible data analyses: a case study of an urothelial cancer cohort analyzed in the Galaxy framework.

BACKGROUND: Mass spectrometry imaging (MSI) derives spatial molecular distribution maps directly from clinical tissue specimens and thus bears great potential for assisting pathologists with diagnostic decisions or personalized treatments. Unfortunately, progress in translational MSI is often hindered by insufficient quality control and lack of reproducible data analysis. Raw data and analysis scripts are rarely publicly shared. Here, we demonstrate the application of the Galaxy MSI tool set for the reproducible analysis of a urothelial carcinoma dataset. METHODS: Tryptic peptides were imaged in a cohort of 39 formalin-fixed, paraffin-embedded human urothelial cancer tissue cores with a MALDI-TOF/TOF device. The complete data analysis was performed in a fully transparent and reproducible manner on the European Galaxy Server. Annotations of tumor and stroma were performed by a pathologist and transferred to the MSI data to allow for supervised classifications of tumor vs. stroma tissue areas as well as for muscle-infiltrating and non-muscle infiltrating urothelial carcinomas. For putative peptide identifications, m/z features were matched to the MSiMass list. RESULTS: Rigorous quality control in combination with careful pre-processing enabled reduction of m/z shifts and intensity batch effects. High classification accuracy was found for both, tumor vs. stroma and muscle-infiltrating vs. non-muscle infiltrating urothelial tumors. Some of the most discriminative m/z features for each condition could be assigned a putative identity: stromal tissue was characterized by collagen peptides and tumor tissue by histone peptides. Immunohistochemistry confirmed an increased histone H2A abundance in the tumor compared to the stroma tissues. The muscle-infiltration status was distinguished via MSI by peptides from intermediate filaments such as cytokeratin 7 in non-muscle infiltrating carcinomas and vimentin in muscle-infiltrating urothelial carcinomas, which was confirmed by immunohistochemistry. To make the study fully reproducible and to advocate the criteria of FAIR (findability, accessibility, interoperability, and reusability) research data, we share the raw data, spectra annotations as well as all Galaxy histories and workflows. Data are available via ProteomeXchange with identifier PXD026459 and Galaxy results via https://github.com/foellmelanie/Bladder_MSI_Manuscript_Galaxy_links . CONCLUSION: Here, we show that translational MSI data analysis in a fully transparent and reproducible manner is possible and we would like to encourage the community to join our efforts.

A One-Stop Protocol to Assess Myocardial Fibrosis in Frozen and Paraffin Sections.

Masson's Trichrome Staining (MTS) is a useful tool for analyzing fibrosis in a plethora of disease pathologies by differential staining of tissue components. It is used to identify collagen fibers in different tissues like heart, lung, skin, and muscles. Especially in cardiac fibrosis, MTS stains the collagen fibers (blue color), which helps in the distinction of scar area versus the healthy area (red color). However, there are several challenges to stain both paraffin-embedded sections and frozen (cryosections) using a single protocol. Therefore, the goal of this study was to develop a simple short protocol to assess cardiac fibrosis in both paraffin-embedded and cryo heart sections. MTS uses three different stains, i.e., Weigert's Iron Hematoxylin, Biebrich scarlet-acid fuchsin, and aniline blue to detect nuclei, cytoplasm, and collagen, respectively. In this study, we developed a simple short protocol that can be adapted by any lab to easily assess cardiac fibrosis in paraffin and frozen heart sections. Furthermore, we have addressed the challenges that are commonly faced during the immunostaining process and troubleshooting techniques. Overall, we have successfully developed a simple one-step protocol to assess myocardial fibrosis in paraffin-embedded and frozen cryosections.

An optimized protocol for total RNA isolation from archived formalin-fixed paraffin-embedded tissues to identify the long non-coding RNA in oral squamous cell carcinomas.

Approximately 93% of the human genome is translated into RNAs, of which only 2% code for proteins and the rest 98% are noncoding RNAs. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs of > 200 nucleotides length that are emerging as novel players in the field of cancer diagnostics or prognostics. Recently, lncRNAs are known to be associated with oral squamous cell carcinomas (OSCC). The demonstration of stable lncRNA has been a challenge in formalin-fixed paraffin-embedded tissues (FFPE). The survivability and expression level of lncRNA in FFPE tissues compared with fresh tissues is not well documented in the literature. Hence, we designed the current pilot study with the main aim to optimize modified TRI (Total RNA isolation) reagent RNA isolation protocol to identify the lncRNA expression in archived FFPE tissues of OSCC in comparison to the standard RNA isolation kit method. The findings of our study demonstrated that the RNA quantity and quality were comparatively better with the optimized TRI reagent modified protocol than the standard RNA isolation kit method. Furthermore, ct (cycle threshold) values after reverse-transcription and qRT-PCR (Quantitative Real time PCR) were comparable and almost equal in both the methods for normal mucosa (control) and OSCC samples.

Proteome profiling of formalin-fixed, paraffin-embedded lung adenocarcinoma tissues using a tandem mass tag-based quantitative proteomics approach.

Over the past few decades, increasing efforts have been made to improve the understanding of, and treatment options for, lung adenocarcinoma (LUAD). However, considering the heterogeneity of LUAD, precise proteomics-based characterization at the molecular level is an urgent clinical requirement for effective treatment. Formalin-fixed, paraffin-embedded (FFPE) tissue is a good option as the working tool for proteomics studies. The present study aimed to obtain a global protein profile using LUAD FFPE tissue samples. Using a quantitative proteomics approach, the study revealed that 360 proteins were significantly more highly expressed in LUAD than in adjacent nontumor lung tissues. Also, 19 differentially expressed membrane proteins were found to be primarily responsible for immune processes. Epidermal growth factor (EGF)-like domain and laminin EGF domain showed markedly different expression levels between cancer tissues and tumor-adjacent normal tissues. Furthermore, Gene Ontology functional enrichment analysis showed that significantly upregulated proteins were associated with the endoplasmic reticulum lumen, protein disulfide isomerase activity, vitamin binding, cell cycle G1/S phase transition, to name but a few. Also, numerous kinases and post-translational modification enzymes were significantly upregulated across all eight LUAD samples compared with paracarcinoma tissues. Proteomics analysis revealed that AAA domain containing 3A (ATAD3a), a member of the ATPase family, was highly expressed in LUAD tissues, which was supported by immunohistochemical analysis. Furthermore, the study confirmed that ATAD3a enhanced the cisplatin sensitivity of LUAD cells. Collectively, the findings of the present study provide new potential candidate targets in patients with LUAD, and may aid auxiliary LUAD diagnosis and surveillance in a noninvasive manner.

Performances of single tube nested polymerase chain reaction and GeneXpert ultra on Formalin fixed paraffin embedded tissues in the diagnosis of tuberculous spondylodiscitis.

INTRODUCTION: Tuberculous Spondylodiscitis is the most common form of musculoskeletal tuberculosis. Molecular techniques on fresh tissues are proved to improve the diagnosis of tuberculous spondylodiscitis and to allow a rapid diagnosis to initiate the treatment and prevent neurological complications. OBJECTIVES: The objective of the present study was to assess the diagnostic performances of single tube nested PCR and GeneXpert ultra in the diagnosis of tuberculous spondylodiscitis on formalin fixed paraffin embedded tissues. METHODS: This study included 63 tuberculous spondylodiscitis cases collected from June 2014 to January 2020 and corresponding to 27 definite tuberculous spondylodiscitis with positive microbiology, and 36 probable tuberculous spondylodiscitis, with histopathological, clinical and radiological findings consistent with tuberculous spondylodiscitis but with negative microbiology. The sensitivity, specificity, positive predictive value and negative predictive value of nested PCR and GeneXpert ultra were determined with reference to microbiology. RESULTS: Nested PCR was positive in 47 (75%) cases: 26/27 definite tuberculous spondylodiscitis and 21/36 probable tuberculous spondylodiscitis. GeneXpert ultra was positive in only 6 (10%) cases corresponding to definite tuberculous spondylodiscitis. The sensitivity, specificity, positive predictive value and negative predictive value of nested PCR on formalin fixed paraffin embedded tissues were 96%, 100%, 100%, 83% respectively. For GeneXpert ultra, these rates were 22%, 100%, 100% and 25% respectively. CONCLUSION: Nested PCR and GeneXpert ultra on formalin fixed paraffin embedded tissues are useful tools for the diagnosis of tuberculous spondylodiscitis, especially for cases where microbiological investigations were not carried out. Both techniques have excellent specificity but single tube nested PCR is more sensitive. Key Points • Molecular techniques are routinely performed on fresh tissues • GeneXpert and nested PCR on formalin fixed paraffin embedded tissues are reliable for the diagnosis of tuberculous spondylodiscitis • Nested PCR is more sensitive than Genexpert for diagnosing tuberculous spondylodiscitis.

Creation of Formalin-Fixed, Paraffin-Embedded 3D Lung Cancer Cellular Spheroids for the Optimization of Immunohistochemistry Staining Procedures.

In an era of precision medicine important treatment decisions are dictated by expression of clinically informative tumor protein biomarkers. These biomarkers can be detected by immunohistochemistry (IHC) performed in tumor tissue sections obtained from biopsies or resections. Like all experimental procedures, IHC needs optimization for several of its steps. However, the investigator must avoid optimizing the IHC procedure using valuable human biopsy samples which may be difficult to obtain. Ideally, valuable biopsy samples should only be subjected to IHC once the IHC protocol has been optimized. In this chapter, we describe a procedure for IHC optimization using tri-dimensional (3D) cellular spheroids created from cultured cells. In this approach, cultured cells are pelleted into 3D spheroids, which are then processed just like a tissue sample, namely, fixed, embedded, sectioned, mounted on slides, and stained with IHC just like a human tissue sample. These 3D cellular spheroids have a tissue-like architecture and cellularity resembling a tumor section, and both cellular and antigen structure are preserved. This method is therefore acceptable for IHC optimization before proceeding to the IHC staining of human tumor samples.

Performance of the MeltPro MTB Assays in the Diagnosis of Drug-Resistant Tuberculosis Using Formalin-Fixed, Paraffin-Embedded Tissues.

OBJECTIVES: The MeltPro MTB assays for detection of resistance to antituberculosis (TB) drugs perform well in genotypic drug susceptibility testing (DST) of clinical samples, but their effectiveness with formalin-fixed, paraffin-embedded (FFPE) tissues is unknown. METHODS: FFPE tissues were obtained from 334 patients with TB. Susceptibility to rifampicin (RIF), isoniazid (INH), and fluoroquinolones was examined using the MeltPro MTB assays, with Xpert MTB/RIF (Xpert) and/or phenotypic DST (pDST) results as references. Samples with discordant results were analyzed by multiplex polymerase chain reaction-targeted amplicon sequencing (MTA-seq). RESULTS: With pDST as the reference, the MeltPro MTB assays sensitivity for RIF, INH, levofloxacin (LVX), and moxifloxacin (MXF) was 95.00%, 96.00%, 100%, and 100%, respectively, and the specificity was 95.15%, 95.92%, 94.69%, and 89.92%, respectively. Concordance was 99.08% between the MeltPro MTB and Xpert (κ = 0.956) for RIF and 95.12% (κ = 0.834), 95.93% (κ = 0.880), 95.12% (κ = 0.744), and 90.24% (κ = 0.367) between the MeltPro MTB and pDST for RIF, INH, LVX, and MXF, respectively. MTA-seq confirmed the discordancy between the MeltPro MTB and pDST for 26 (89.66%) of 29 samples. CONCLUSIONS: The MeltPro MTB assays rapidly and efficiently predict Mycobacterium tuberculosis resistance to the main first- and second-line anti-TB drugs in FFPE tissues.