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Ex vivo magnetic resonance imaging (MRI) requires chemical fixation to preserve tissue during storage or extended imaging sessions. Although it is commonly understood that fixation may alter tissue volume and shape, the potential confounding effects of fixation and storage on morphometric analyses have not been well characterized. With increasing use of ex vivo MRI for mouse brain phenotying and opportunities for inter-study comparisons, we sought to characterize how changes in fixation and/or storage times affected tissue volume, and how this might impact phenotyping results. Mouse brain samples that had been perfusion fixed, within the skull as per our standard protocol, were immersed in formaldehyde-based fixative for 1 to 5days before being stored in saline or water. Throughout fixation and storage, samples were repeatedly scanned using magnetic resonance imaging, and analyzed for volume expansion or shrinkage. We found that most of the brain continued to shrink post fixation, with the rate of shrinkage dependent on the solution in which the samples were submerged. Maximum changes in volume of 3.5% per day and 3% per month were detected during fixation and storage (in PBS), respectively. Most notably, changes were non-uniform, with some structures shrinking slower, or even expanding, when compared to other structures in the brain. Our results highlight that caution is necessary when interpreting results from experiments with inconsistent fixation and storage protocols, so as not to mistake these changes for phenotypic differences.

Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples.

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

Abstract Fluorinert (perfluorocarbon) represents an inexpensive option for minimizing susceptibility artifacts in ex vivo brain MRI scanning, and provides an alternative to Fomblin. However, its impact on fixed tissue and histological analysis has not been rigorously and quantitatively validated. In this study, we excised tissue blocks from 2 brain regions (frontal pole and cerebellum) of 5 formalin-fixed specimens (2 progressive supranuclear palsy cases, 3 controls). We excised 2 blocks per region per case (20 blocks in total), one of which was subsequently immersed in Fluorinert for a week and then returned to a container with formalin. The other block from each region was kept in formalin for use as control. The tissue blocks were then sectioned and histological analysis was performed on each, including routine stains and immunohistochemistry. Visual inspection of the stained histological sections by an experienced neuropathologist through the microscope did not reveal any discernible differences between any of the samples. Moreover, quantitative analysis based on automated image patch classification showed that the samples were almost indistinguishable for a state-of-the-art classifier based on a deep convolutional neural network. The results showed that Fluorinert has no effect on subsequent histological analysis of the tissue even after a long (1 week) period of immersion, which is sufficient for even the lengthiest scanning protocols.

Immunohistochemistry (IHC) and polymerase chain reaction (PCR) and fragment separation by capillary electrophoresis represent the current clinical laboratory standard for the evaluation of microsatellite instability (MSI) status. The importance of reporting MSI status in colorectal cancer is based on its potential for guiding treatment and as a prognostic indicator. It is also used to identify patients for Lynch syndrome testing. Our aim was to evaluate pre-analytical factors, such as age of formalin-fixed and paraffin-embedded (FFPE) block, neoplastic cell percentage, mucinous component, and DNA integrity, that may influence the accuracy of MSI testing and assess the concordance between three different MSI evaluation approaches. We selected the mucinous colorectal cancer (CRC) histotype for this study as it may possibly represent an intrinsic diagnostic issue due to its low tumor cellularity. Seventy-five cases of mucinous CRC and corresponding normal colon tissue samples were retrospectively selected. MMR proteins were evaluated by IHC. After DNA quality and quantity evaluation, the Idylla™ and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Seventy-three (97.3%) cases were successfully analyzed by the three methodologies. Overall, the Idylla™ platform showed a concordance rate with IHC of 98.0% for microsatellite stable (MSS)/proficient MMR (pMMR) cases and 81.8% for MSI/deficient MMR (dMMR) cases. The TapeStation 4200 system showed a concordance rate with IHC of 96.0% for MSS/pMMR cases and 45.4% for MSI/dMMR cases. The concordance rates of the TapeStation 4200 system with respect to the Idylla™ platform were 98.1% for MSS profile and 57.8% for MSI profile. Discordant cases were analyzed using the Titano MSI kit. Considering pre-analytical factors, no significant variation in concordance rate among IHC analyses and molecular systems was observed by considering the presence of an acellular mucus cut-off >50% of the tumor area, FFPE year preparation, and DNA concentration. Conversely, the Idylla™ platform showed a significant variation in concordance rate with the IHC approach by considering a neoplastic cell percentage >50% (p-value = 0.002), and the TapeStation 4200 system showed a significant variation in concordance rate with the IHC approach by considering a DNA integrity number (DIN) ≥4 as cut-off (p-value = 0.009). Our data pinpoint a central role of the pre-analytical phase in the diagnostic outcome of MSI testing in CRC.

Aims Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. Methods 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. Results Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. Conclusions In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation.

AimsHistopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses.MethodsTotal RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes, 18S and GAPDH.ResultsReference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene.ConclusionsThis study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

Genomic medicine requires the identification of biomarkers and therapeutic targets, which in turn, requires high-quality biospecimens. Achieving high-quality biospecimens requires implementing standard operating procedures to control the variations of preanalytic variables in biobanking. Currently, most biobanks do not control the variations of preanalytic variables when collecting, processing, and storing their biospecimens. However, those variations have been shown to affect the quality of biospecimens and gene expression profiling. To identify evidence-based preanalytic parameters that can be applied and those parameters that need further study. We searched the Biospecimen Research and PubMed databases using defined key words. We retrieved and reviewed 212 articles obtained through those searches. We included 58 articles (27%) according to our inclusion and exclusion criteria for this review. -Preanalytic variables in biobanking can degrade the quality of biospecimens and alter gene expression profiling. Variables that require further study include the effect of surgical manipulation; the effect of warm ischemia; the allowable duration of delayed specimen processing; the optimal type, duration, and temperature of preservation and fixation; and the optimal storage duration of formalin-fixed, paraffin embedded specimens in a fit-for-purpose approach.

High-throughput sequencing efforts in molecular tumour diagnostics detect increasing numbers of novel variants, including variants predicted to affect splicing. In silico prediction tools can reliably predict the effect of variant disrupting canonical splice sites; however, experimental validation is required to confirm aberrant splicing. Here, we present RNA analysis performed for 13 canonical splice site variants predicted or known to result in splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Total nucleic acid was successfully isolated for 10 variants from eight formalin-fixed paraffin-embedded (FFPE) tumour tissues and two B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known variant in the B-cell line, aberrant splicing could only be detected after formalin fixation, which indicated that formalin fixation could possibly inhibit RNA degradation. Aberrant splicing was concluded in three of four predicted splice variants of uncertain significance, supporting their pathogenic effect. With this assay, somatic splice variants can be easily and rapidly analysed, enabling retrospective analysis to support the pathogenicity of variants predicted to result in splicing when only FFPE material is available.