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Formalin fixation and paraffin embedding is a timeless, cost-efficient, and widely adopted method of preserving human tissue biospecimens that has resulted in a substantial reservoir of formalin-fixed, paraffin-embedded blocks that represent both the pathology and preanalytical handling of the biospecimen. This reservoir of specimens is increasingly being used for DNA, RNA, and proteomic analyses. To evaluate the impact of preanalytical factors associated with the formalin fixation and paraffin embedding process on downstream morphological and molecular endpoints. We surveyed the existing literature using the National Cancer Institute's Biospecimen Research Database for published reports investigating the potential influence of preanalytical factors associated with the formalin fixation and paraffin embedding process on DNA, RNA, protein, and morphological endpoints. Based on the literature evidence, the molecular, proteomic, and morphological endpoints can be altered in formalin-fixed, paraffin-embedded specimens by suboptimal processing conditions. While the direction and magnitude of effects associated with a given preanalytical factor were dependent on the analyte (DNA, RNA, protein, and morphology) and analytical platform, acceptable conditions are highlighted, and a summary of conditions that could preclude analysis is provided.

Context .—While formalin fixation and paraffin embedding has become a universal mechanism of tissue preservation and a gold standard for immunohistochemistry, fixation and processing variables that may confound assay effectiveness have received little attention from the scientific community. Objective .—To identify discrete steps in specimen fixation and processing that may impact immunostaining, assess the magnitude of reported effects in the literature, and highlight preanalytical variables that require further investigation. Data Sources .—Thirty-nine primary research articles that investigated immunohistochemical effects of 1 or more preanalytical variables were identified by our literature survey. Thresholds identified in the literature were then compared with published immunohistochemistry guidelines for formalin-fixed, paraffin-embedded specimens. Conclusions .—Of the 62 preanalytical variables identified, 27 were examined in published research. Meta-analysis revealed 15 preanalytical variables that were capable of impacting immunohistochemistry (including fixation delay; fixative type; time in fixative; reagents and conditions of dehydration, clearing, and paraffin impregnation; and conditions of slide drying and storage) and 12 variables with no reported influence (including the type of processor used; the number and position of specimens during dehydration, clearing, and paraffin impregnation; and the duration of paraffin block storage). Variables with antigen-dependent or inconsistent effects were highlighted. Comparison of literature-supported thresholds with published recommendations revealed (1) strong agreement among preanalytical variables for optimal immunostaining, (2) discrepancies among thresholds for adequate immunostaining, and (3) the continued need for rigorous research and comprehensive guidelines on specimen fixation, processing, and storage.

The National Cancer Institute conducted the Biospecimen Pre-analytical Variables (BPV) study to determine the effects of formalin fixation and delay to fixation (DTF) on the analysis of nucleic acids. By performing whole transcriptome sequencing and small RNA profiling on matched snap-frozen and FFPE specimens exposed to different delays to fixation, this study aimed to determine acceptable delays to fixation and proper workflow for accurate and reliable Next-Generation Sequencing (NGS) analysis of FFPE specimens. In comparison to snap-freezing, formalin fixation changed the relative proportions of intronic/exonic/untranslated RNA captured by RNA-seq for most genes. The effects of DTF on NGS analysis were negligible. In 80% of specimens, a subset of RNAs was found to differ between snap-frozen and FFPE specimens in a consistent manner across tissue groups; this subset was unaffected in the remaining 20% of specimens. In contrast, miRNA expression was generally stable across various formalin fixation protocols, but displayed increased variability following a 12 h delay to fixation.

Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown. To elucidate molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program. The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls. DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point-specific effects of DTF and TIF. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV (representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay. DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.

Evaluation of how genetic mutations or variability can directly affect phenotypic outcomes, the development of disease, or determination of a tailored treatment protocol is fundamental to advancing personalized medicine. To understand how a genotype affects gene expression and specific phenotypic traits, as well as the correlative and causative associations between such, the Genotype-Tissue Expression (GTEx) Project was initiated The GTEx collection of biospecimens and associated clinical data links extensive clinical data with genotype and gene expression data to provide a wealth of data and resources to study the underlying genetics of normal physiology. These data will help inform personalized medicine through the identification of normal variation that does not contribute to disease. Additionally, these data can lead to insights into how gene variation affects pharmacodynamics and individualized responses to therapy.

The National Institutes of Health Genotype-Tissue Expression (GTEx) project was developed to elucidate how genetic variation influences gene expression in multiple normal tissues procured from postmortem donors. To provide critical insight into a biospecimen's suitability for subsequent analysis, each biospecimen underwent quality assessment measures that included evaluation for underlying disease and potential effects introduced by preanalytic factors. Electronic images of each tissue collected from nearly 1000 postmortem donors were evaluated by board-certified pathologists for the extent of autolysis, tissue purity, and the type and abundance of any extraneous tissue. Tissue-specific differences in the severity of autolysis and RNA integrity were evaluated, as were potential relationships between these markers and the duration of postmortem interval and rapidity of death. Tissue-specific challenges in the procurement and preservation of the nearly 30 000 tissue specimens collected during the GTEx project are summarized. Differences in the degree of autolysis and RNA integrity number were observed among the 40 tissue types evaluated, and tissue-specific susceptibilities to the duration of postmortem interval and rapidity of death were observed. Ninety-five percent of tissues were of sufficient quality to support RNA sequencing analysis. Biospecimens, annotated whole slide images, de-identified clinical data, and genomic data generated for GTEx represent a high-quality and comprehensive resource for the scientific community that has contributed to its use in approximately 1695 articles. Biospecimens and data collected under the GTEx project are available via the GTEx portal and authorized access to the Database of Genotypes and Phenotypes; procedures and whole slide images are available from the National Cancer Institute.

The National Institutes of Health (NIH) Genotype-Tissue Expression (GTEx) project was designed to evaluate how genetic variation and epigenetic effects influence gene expression in normal tissue. To ensure that the grossly normal-appearing tissues collected were free from disease, each specimen underwent histologic evaluation. In total, nearly 30 000 tissue aliquots collected from almost 1000 postmortem donors underwent histologic review by project pathologists, and detailed observations of any abnormalities or lesions present were recorded. Despite sampling of normal-appearing tissue, in-depth review revealed incidental findings among GTEx samples that included neoplastic, autoimmune, and genetic conditions; the incidence of some of these conditions among GTEx donors differed from those previously reported for other populations. A number of age-related abnormalities observed during histologic review of tissue specimens are also described. Histologic findings from the GTEx project may serve to improve populational awareness of several conditions and present a unique opportunity for others to explore age- and sex-influenced conditions. Resources from the study, including histologic image and sequencing data, are publicly available for research.

* Context.--The National Institutes of Health Genotype-Tissue Expression (GTEx) project was developed to elucidate how genetic variation influences gene expression in multiple normal tissues procured from postmortem donors. Objective.--To provide critical insight into a biospecimen's suitability for subsequent analysis, each biospecimen underwent quality assessment measures that included evaluation for underlying disease and potential effects introduced by preanalytic factors. Design.--Electronic images of each tissue collected from nearly 1000 postmortem donors were evaluated by board-certified pathologists for the extent of autolysis, tissue purity, and the type and abundance of any extraneous tissue. Tissue-specific differences in the severity of autolysis and RNA integrity were evaluated, as were potential relationships between these markers and the duration of postmortem interval and rapidity of death. Results.--Tissue-specific challenges in the procurement and preservation of the nearly 30 000 tissue specimens collected during the GTEx project are summarized. Differences in the degree of autolysis and RNA integrity number were observed among the 40 tissue types evaluated, and tissue-specific susceptibilities to the duration of postmortem interval and rapidity of death were observed. Conclusions.--Ninety-five percent of tissues were of sufficient quality to support RNA sequencing analysis. Biospecimens, annotated whole slide images, de-identified clinical data, and genomic data generated for GTEx represent a high-quality and comprehensive resource for the scientific community that has contributed to its use in approximately 1695 articles. Biospecimens and data collected under the GTEx project are available via the GTEx portal and authorized access to the Database of Genotypes and Phenotypes; procedures and whole slide images are available from the National Cancer Institute. (Arch Pathol Lab Med. 2025;149:217-232; doi: 10.5858/arpa.2023-0467-OA)

The active debate about the return of incidental or secondary findings in research has primarily focused on return to research participants, or in some cases, family members. Particular attention has been paid to return of genomic findings. Yet, research may generate other types of findings that warrant consideration for return, including findings related to the pathology of donated biospecimens. In the case of deceased biospecimen donors who are also organ and/or tissue transplant donors, pathology incidental findings may be relevant not to family members, but to potential organ or tissue transplant recipients. This paper will describe the ethical implications of pathology incidental findings in the Genotype-Tissue Expression (GTEx) project, the process for developing a consensus approach as to if/when such findings should be returned, possible implications for other research projects collecting postmortem tissues and how the scenario encountered in GTEx fits into the larger return of results/incidental findings debate.

(Arch Pathol Lab Med. 2025;149:233-241; doi: 10.5858/ arpa.2023-0468-OA) The Genotype-Tissue Expression (GTEx) project, a National Institutes of Health (NIH) Common Fund study, was designed to evaluate how genetic variation and epigenetic effects influence gene expression in normal tissue and to gain insight into tissue-specific gene expression and regulation.1'2 GTEx tissue collection was coordinated by the National Cancer Institute (NCI) Biorepositories and Biospecimen Research Branch using a specialized infra-structure developed in accordance with the NCI Best Practices for Biospecimen Resources.3,4 Tissue collection, when completed, resulted in more than 25 000 tissue samples from up to 40 grossly nondiseased tissues from nearly 1000 postmortem or organ donors. The GTEx project has (1) established a molecular resource database with aggregated genotypic and gene expression data (www.gtexportal.org), (2) shared the full data set, including raw DNA/RNA sequences and the available clinical data for each donor, to the database of genotypes and phenotypes (dbGaP; dbGaP accession No. phs000424; https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/ study.cgi?study_id=phs000424), (3) created a view-only tissue image library of micrographs (biospecimens.cancer.gov/gtex-biobank/histology_viewer.asp) as well as micrographs compatible with image analysis that are available upon request (email: ncibbrb@nih.gov), and (4) established an associated tissue bank (biospecimens.cancer.gov/gtexbiobank and gtex-portal.org/home/biobank) for the scientific community to pur-sue additional studies. Uninten-tional inclusion of missampled or diseased tissue could increase variability and potentially confound genetic analysis. [...]a multiphase approach was applied: tissue that appeared grossly normal upon collection was preferentially sampled; each tissue sample collected underwent histologie review by a project pathologist to confirm the sample was rep-resentative of the target tissue, was of suitable quality and was absent of disease; and sequencing data were used to ver-ify recorded donor sex and the absence of chromosomal anomalies and genetic conditions. Briefly, research authorization was obtained from a member of each donor's family.3'6'7 In the present study, any of the following resulted in donor exclusion: an antemortem transfusion within 48 hours of death, a history of metastatic malignancy, expo-sure to HrV/AIDS or the hepatitis C virus within the 5 years pre-ceding death, repeated positive reactive screening tests for HIV-1 or HIV-2 antibodies, active sepsis, or multiple documented or con-firmed pathologie conditions.