Rapid two-temperature formalin fixation.
Ontology highlight
ABSTRACT:
SUBMITTER: Chafin D
PROVIDER: S-EPMC3548901 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues.
| S-EPMC3115967 | biostudies-literature
Project description:INTRODUCTION. Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. RESULTS. The cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues.
2011-09-05 | GSE27175 | GEO
Project description:INTRODUCTION. Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. RESULTS. The cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues. Thirty samples from human cancers processed in different ways before RNA extraction: (i) Fresh-Freezing (FF), (ii) Standard Fixation with formalin (SF), (iii) Cold-Fixation (CF), a new formalin fixation procedure preserving nucleic acids. The first six samples compare CF with SF and FF in one colorectal cancer (CRC) specimen and one breast cancer (BRCa) specimen. The subsequent 24 samples compare only CF with FF in various cancer specimens. Expression data are neither normalized nor backound subtracted to preserve the original signal distribution for each sample, which is essential to compare expression profiles obtained after different tissue preservation methods.
2011-09-04 | E-GEOD-27175 | biostudies-arrayexpress
Project description:PURPOSE:Chemical fixatives such as formalin form cross-links between proteins and affect the relaxation times and diffusion properties of tissue. These fixation-induced changes likely also affect myelin density measurements produced by quantitative magnetization transfer and myelin water imaging. In this work, we evaluate these myelin-sensitive MRI methods for fixation-induced biases. METHODS:We perform quantitative magnetization transfer, myelin water imaging, and deuterium oxide-exchanged zero TE imaging on unfixed human spinal cord tissue at 9.4 Tesla and repeat these measurements after 1 day and 31 days of formalin fixation. RESULTS:The quantitative magnetization-transfer bound pool fraction increased by 30.7% ± 21.1% after 1 day of fixation and by 42.6% ± 33.9% after 31 days of fixation. Myelin water fraction increased by 39.7% ± 15.5% and 37.0% ± 15.9% at these same time points, and mean T2 of the myelin water pool nearly doubled. Reference-normalized deuterium oxide-exchanged zero TE signal intensity increased by 8.17% ± 6.03% after 31 days of fixation but did not change significantly after 1 day of fixation. After fixation, specimen cross-sectional area decreased by approximately 5%; after correction for shrinkage, changes in deuterium oxide-exchanged zero TE intensity were nearly eliminated. CONCLUSION:Bound pool fraction and myelin water fraction are significantly increased by formalin fixation, whereas deuterium oxide-exchanged zero TE intensity is minimally affected. Changes in quantitative magnetization transfer and myelin water imaging may be due in part to delamination and formation of vacuoles in the myelin sheath. Deuterium oxide-exchanged signal intensity may be altered by fixation-induced changes in myelin lipid solid-state 1 H T1 . We urge caution in the comparison of these measurements across subjects or specimens in different states, especially unfixed versus fixed tissue.
| S-EPMC6626568 | biostudies-literature
Project description: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.
| S-EPMC6502812 | biostudies-literature
Project description:Sequencing technologies now provide unprecedented access to genomic information in archival formalin-fixed paraffin-embedded (FFPE) tissue samples. However, little is known about artifacts induced during formalin fixation, which could bias results. Here we evaluated global changes in RNA-sequencing profiles between matched frozen and FFPE samples. RNA-sequencing was performed on liver samples collected from mice treated with a reference chemical (phenobarbital) or vehicle control for 7 days. Each sample was divided into four parts: (1) fresh-frozen, (2) direct-fixed in formalin for 18 h, (3) frozen then formalin-fixed, and (4) frozen then ethanol-fixed and paraffin-embedded (n = 6/group/condition). Direct fixation resulted in 2,946 differentially expressed genes (DEGs) vs. fresh-frozen, 98% of which were down-regulated. Freezing prior to formalin fixation had ≥ 95% fewer DEGs vs. direct fixation, indicating that most formalin-derived transcriptional effects in the liver occurred during fixation. This finding was supported by retrospective studies of paired frozen and FFPE samples, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription initiation pathways with direct fixation. Notably, direct formalin fixation in the parent study did not significantly impact response profiles resulting from chemical exposure. These results advance our understanding of FFPE samples as a resource for genomic research.
| S-EPMC7468282 | biostudies-literature
Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. Previously, we identified a consistent shift in global RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was from fixing fresh tissue in formalin. To test this idea, RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs), 98% of which were down-regulated. Freezing prior to fixation resulted in ≥95% fewer DEGs vs. FR, indicating that most formalin-derived transcriptional effects occurred with fixation. This was supported by follow-up studies, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with formalin fixation. Notably, formalin fixation in the parent study did not significantly impact chemical response profiles, which were consistent with CAR/PXR activation and 600 ppm phenobarbital exposure. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples.
2020-09-08 | GSE148174 | GEO
Project description:In our study we systematically compared the alternative fixatives acidified formal alcohol (AFA), PAXgene®, HOPE®, and combinations of AFA or formalin with ultrasound treatment to standard (buffered) formalin fixation. We examined general morphology and detectability of protein structures by immunohistochemistry of the membrane receptors epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), and phosphorylated human epidermal growth factor receptor 2 (phospho-HER2). In order to allow for stringent comparability of different fixation techniques, we used matched mouse xenograft tumor samples from three different human cancer cell lines (colon, ovarian, and non-small cell lung cancer), either fixed conventionally with formalin or an alternative fixative. Tissue morphology after fixation with AFA and PAXgene® was comparable to formalin-fixed paraffin-embedded tissue (FFPET) morphology. Ultrasound fixations resulted in slightly inferior morphology and HOPE® fixation preserved morphology only poorly compared to FFPET in this system. None of the tested alternative fixatives enabled immunohistochemical detectability of all three targets in the same manner as FFPET. Pronounced staining was possible for EGFR and IGF-1R with all alternative fixatives but HOPE®, and phospho-HER2 staining was only noteworthy with formalin-ultrasound-fixed tissue. Therefore, the use of alternative fixatives comes with the need for careful validation of obtained IHC results individually for each target.
| S-EPMC3432218 | biostudies-literature
Project description:Immunohistochemistry (IHC) is routinely used in diagnostic pathology to detect infectious agents, to immunophenotype neoplastic cells, and to prognosticate neoplastic diseases. Formalin fixation is considered a limiting factor for IHC because formalin can cross-link antigens and mask epitopes. Prolonged formalin fixation is presumed to result in decreased antigen detection; however, this effect has only been evaluated with a few antibodies. The goal of this study was to evaluate the effect of prolonged formalin fixation on the immunohistochemical detection of 61 different antigens. Approximately 5-mm-thick tissue slices were fixed in 10% neutral-buffered formalin. Tissue slices were removed from formalin, processed, and paraffin-embedded at 1-day, 3-day, and then at approximately 1-week intervals. IHC was performed on all sections in tandem after all tissues were processed. Immunoreactions were evaluated by three pathologists according to a four-tier grading system. Immunoreactivity of cytokeratin 7, high-molecular-weight cytokeratin, and laminin was diminished by prolonged formalin fixation. However, immunohistochemical reactivity remained moderate to strong with up to 7 weeks of fixation for all other antibodies. These results suggest that prolonged formalin fixation has minimal effects on antigen detection for most commonly used antibodies. These results further validate the use of IHC in diagnostic pathology.
| S-EPMC2713075 | biostudies-other
Project description:Imaging mass spectrometry is becoming a key technology for the investigation of the molecular content of biological tissue sections in direct correlation with the underlying histology. Much of our work has been done with fresh-frozen tissue sections that has undergone minimal protein degradation between the time a tissue biopsy is sampled and the time it is snap-frozen so that no preserving or fixing agents need to be added to the frozen biopsy. However, in many sampling environments, immediate flash freezing may not be possible and so we have explored the use of ethanol-preserved, paraffin-embedded tissue specimens for proteomic analyses. Solvent-only preserved tissue specimens provide long-term preservation at room temperature, generation of high quality histological sections and little if any chemical alteration of the proteins. Using mouse organs, several key steps involved in the tissue dehydration process have been investigated to assess the potential of such preserved specimens for profiling and imaging mass spectrometry investigations.
| S-EPMC2927987 | biostudies-literature