Formalin Fixation Time Research Articles

Forensic STR and SNP Genotyping of Formalin-Fixed Skeleton Samples With Illumina's ForenSeq System.

Formalin fixatives are widely used in forensics to preserve tissues. However, extracting high-quality genomic DNA from formalin-fixed samples is challenging. Traditional short tandem repeat (STR) analysis using capillary electrophoresis (CE) for forensic DNA typing frequently results in failure. Massively parallel sequencing (MPS) can handle many samples and thousands of genetic markers, usually single-nucleotide polymorphisms (SNPs) and STRs, in a single test. Thus, it is useful for assessing highly deteriorated forensic evidence. Few studies have examined the effectiveness of STRs and SNPs genotyping of formalin-fixed skeletons using MPS. In this study, 55 skeletal samples from 5 individuals that were treated under different formalin fixation times (5-75 days) were examined and sequenced using the ForenSeq DNA Signature Prep Kit on the Illumina MiSeq FGX platform. The results showed that as the duration of formalin fixation increased, the detection rates of STRs and SNPs gradually decreased. After 75 days of fixation, the average detection rates for STRs and SNPs were 4% and 10%, respectively. The cumulative discrimination power (CDP) of individual identification SNPs (iiSNPs) was >0.9999 on the 45th day. However, the CDP of STRs was 0.9930 on the 22nd day. Low detection rates were observed for six STRs (D1S1656, PentaE, D22S1045, PentaD, DX8378 and DX10103) and five SNPs (rs2920816, rs354439, rs1736442, rs338882 and rs1031825). In conclusion, DNA extracted from formalin-fixed skeletons decomposes rapidly over time, and MPS technology can be a useful tool for detecting forensic genetic markers in such samples.

Proteomic profiling of FFPE specimens: Discovery of HNRNPA2/B1 and STT3B as biomarkers for determining formalin fixation durations

Recent advancements in proteomics technologies using formalin-fixed paraffin-embedded (FFPE) samples have significantly advanced biomarker discovery. Yet, the effects of varying sample preparation protocols on proteomic analyses remain poorly understood. We analyzed mouse liver FFPE samples that varied in fixatives, fixation duration, and storage temperature using LC/MS. We found that variations in fixation duration significantly affected the abundance of specific proteins, showing that HNRNPA2/B1 demonstrated a significant decrease in abundance in samples fixed for long periods, whereas STT3B exhibited a significant increase in abundance in samples fixed for long durations. These findings were supported by immunohistochemical analysis across liver, spleen, and lung tissues, demonstrating a significant decrease in the nuclear staining of HNRNPA2/B1 in long-duration acid formalin(AF)-fixed FFPE samples, and an increase in cytoplasmic staining of STT3B in long-duration neutral buffered formalin-fixed liver and lung tissues and granular staining in all long-duration AF-fixed FFPE tissue types. Similar trends were observed in the long-duration fixed HeLa cells. These results demonstrate that fixation duration critically affects the proteomic integrity of FFPE samples, emphasizing the urgent need for standardized fixation protocols to ensure consistent and reliable proteomic data. SignificanceThe quality of FFPE samples is primarily influenced by the fixation and storage conditions. However, previous studies have mainly focused on their impact on nucleic acids and the extent to which different fixation conditions affect changes in proteins has not been evaluated. In addition, to our knowledge, proteomic research focusing on differences in formalin fixation conditions has not yet been conducted. Here, we analyzed FFPE samples with different formalin fixation and storage conditions using LC/MS and evaluated the impact of different fixation conditions on protein variations. Our study unequivocally established formalin fixation duration as a critical determinant of protein variation in FFPE specimens and successfully identified HNRNPA2/B1 and STT3B as potential biomarkers for predicting formalin fixation duration for the first time. The study findings open new avenues for quality assessment in biomedical research and diagnostics.

The impact of formalin fixation in the elemental content of tissues: Parametrization up to 48 h

Formalin fixation is a crucial step in the preparation of tissue samples for anatomic pathology studies, however, research showed that prolonged formalin fixation, can alter the elemental composition of tissues with implications for pathology and biomedical research. In this study we aim to assess and parametrize the influence of short-term formalin fixation in tissues.Six sets of human colon tissue samples were exposed to different formalin fixation times, up until 48 h. The elemental content throughout time was compared to the elemental content of the snap-frozen sample of the same tissue that was not exposed to formalin using Energy Dispersive X-ray Fluorescence.Results showed a clear decrease of Cl and K concentration in the tissues solution reaching a plateau between 1 h and 3 h of fixation. Also, there is an uptake of P in the tissue, likely due to the buffered formalin solution, within the first 15 min of fixation. This behaviour was concomitant with an increase of Na determined using Particle Induced gamma-ray emission analysis (PIGE) and Elastic Backscattering Spectroscopy (EBS). The dynamic changes determined in the studied elements demonstrate that a better understanding of the fixation-related mechanisms is in demand as well as optimization of the fixation protocols implemented in anatomic pathology laboratories.

Effects of Extended Fixation on Advanced Gastric Cancer HER2 Status Assessment Using IHC and FISH.

In gastric cancer, accurate determination of human epidermal growth factor receptor type 2 (HER2) status is crucial for treatment decision-making. However, the optimal formalin fixation time of gastric cancer specimens for HER2 status determination remains unestablished. Here, we investigated real-world data on formalin overfixation and its effect on HER2 status determination in gastric cancer. We comprehensively analyzed HER2 testing results in 228 gastric cancer specimens, including those subjected to formalin overfixation. Subsequently, we divided 52 resected specimens of advanced gastric cancer into three groups and studied the effects of short-term (6-72 h) and long-term (1 and 2 weeks) fixation on HER2 status determination using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). A total of 21.5% (49/228) of the specimens were HER2-positive, whereas 78.5% (179/228) were negative. Among the HER2-negative specimens, no biopsies were overfixed, whereas 12.5% (9/72) of the surgical resection specimens were overfixed. The HER2 status of the 6-72-h group was 82.7% and 76.9% identical to that of the 1- and 2-week groups, when determined using IHC, and 73.1% and 36.5%, when determined using FISH, respectively. However, HER2 determination was not feasible in 26.9% and 63.5% of the specimens in the 1- and 2-week groups, respectively. Formalin overfixation may hinder the determination of HER2 status and should be avoided in gastric cancer sample preparation.

Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study.

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity.

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.

The risk of weekend biopsy: Impact of specimen source and fixation status on HER2 assessment in the treatment of advanced gastric cancer (The HER_WEEKEND study).

Accurate evaluation of human epidermal growth factor receptor type 2 (HER2) expression is crucial for determining chemotherapy regimens in gastric cancer. However, formalin fixation status has been identified as an important factor affecting HER2 assessment reliability. This retrospective cohort study aimed to investigate the correlation between sample collection day (weekday vs. weekend) and source (biopsy vs. surgical specimens) in assessing HER2 expression in patients with unresectable advanced/recurrent gastric cancer. Data were collected from gastric cancer patients who received chemotherapy at a single public hospital in Japan from 2008 to 2021. The analysis included 177 patients (109 men, 68 women) with a median age of 68.0 (21-88) years, and the primary outcome was the HER2 positivity rate. The overall HER2 positivity rate was 18.1%, with higher rates on weekdays (20.0%) compared to weekends (12.8%). Biopsies had higher positivity rates on weekdays (23.9%) but lower rates on weekends (11.1%) than surgical specimens. Significant differences were observed in formalin fixation times between weekdays and weekends for both biopsies and surgical samples. The study findings suggest that longer formalin fixation times on weekends may lead to underestimating HER2 expression, particularly in biopsies. Therefore, it is crucial to be cautious of excessive formalin fixation when collecting samples, especially during weekend biopsies.

Evaluation of the influence of the formalin fixation time on the elemental content of tissues measured with X-ray fluorescence

One of the most widely used methods for tissue preservation and fixative during transportation is the conservation in formalin during variable amounts of time. In this study, we have evaluated the influence of formalin fixation time in the elemental composition of human tissue samples using Energy Dispersive X-Ray Fluorescence (EDXRF).Ten sets of human tissue samples (from colon, ileum, stomach, and spleen) were exposed to different formalin fixation times, between 2 and 24 days, and for each tissue, the elemental content throughout time was compared to the elemental content of the snap-frozen sample of the same tissue that was not exposed to formalin. Additionally, in order to further evaluate the transference of elements, the formalin solution was also analysed using EDXRF.Our results showed a clear decrease of Cl and K in the tissues, transferred to the formalin solution. Conversely, there is an uptake of P in the tissue, likely due to the buffered formalin solution. The consistent alterations seen in the studied elements across all the ten different tissues allow us to hypothesize that in the future, there will be different thresholds for their use as diagnostic tools in unfixed (intraoperative exams) as well as formalin fixed paraffin embedded tissues.

Deep oncopanel sequencing reveals within block position-dependent quality degradation in FFPE processed samples

BackgroundClinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized diploid cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified.ResultsTissue sections that fail more frequently show low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces are more likely to show FFPE-specific errors, akin to “edge effects” seen in histology, while the inner samples display no quality degradation related to fixation time.ConclusionsTo assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.

The effect of prolonged formalin fixation on the expression of proteins in human brain tissues

Formalin-fixed, paraffin-embedded (FFPE) tissues have been widely used in researches. Proteins and nucleic acids in prolonged FFPE tissues display different degrees of degradation. We investigated the effect of prolonged formalin fixation on protein expression in human brain tissues. Twenty-eight middle prefrontal front cortex tissue blocks from human brains prefixed in formalin were obtained from a brain bank. The tissue blocks were divided into two groups, the control group and the prolonged fixation group. Quantitative immunocytochemistry was used to analyse the biological markers of Fox-3, Rbfox3 (NeuN), glial fibrillary acidic protein (GFAP), ionized calcium binding adapter molecule-1 (IBA-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nissl staining showed that positive signaling of Nissl body was significantly decreased by 16.6% in the prolonged fixation group. In addition, the staining intensity of Nissl body was negatively correlated with fixation time. The level of NeuN immunoreactivity (ir) was significantly reduced by 19.31% in the prolonged fixation group. Moreover, there was a significant negative correlation between NeuN-ir and fixation time. There were no significant changes in GFAP-ir, IBA-1-ir and GAPDH-ir between control group and the prolonged fixation group. Prolonged formalin-fixed tissues showed time- and molecule-dependent protein changes, which may be potential confounders in the clinic and researches. Our study suggested short formalin fixation time is recommended when using PPFE brain tissues.

Breast cancer pathology services in sub-Saharan Africa: a survey within population-based cancer registries

BackgroundPathologists face major challenges in breast cancer diagnostics in sub-Saharan Africa (SSA). The major problems identified as impairing the quality of pathology reports are shortcomings of equipment, organization and insufficiently qualified personnel. In addition, in the context of breast cancer, immunohistochemistry (IHC) needs to be available for the evaluation of biomarkers. In the study presented, we aim to describe the current state of breast cancer pathology in order to highlight the unmet needs.MethodsWe obtained information on breast cancer pathology services within population-based cancer registries in SSA. A survey of 20 participating pathology centres was carried out. These centres represent large, rather well-equipped pathologies. The data obtained were related to the known population and breast cancer incidence of the registry areas.ResultsThe responding pathologists served populations of between 30,000 and 1.8 million and the centres surveyed dealt with 10–386 breast cancer cases per year. Time to fixation and formalin fixation time varied from overnight to more than 72 h. Only five centres processed core needle biopsies as a daily routine. Technical problems were common, with 14 centres reporting temporary power outages and 18 centres claiming to own faulty equipment with no access to technical support. Only half of the centres carried out IHC in their own laboratory. For three centres, IHC was only accessible outside of the country and one centre could not obtain any IHC results. A tumour board was established in 13 centres.ConclusionsWe conclude that breast cancer pathology services ensuring state-of-the-art therapy are only available in a small fraction of centres in SSA. To overcome these limitations, many of the centres require larger numbers of experienced pathologists and technical staff. Furthermore, equipment maintenance, standardization of processing guidelines and establishment of an IHC service are needed to comply with international standards of breast cancer pathology.

Using postmortem formalin fixed paraffin-embedded tissues for molecular testing of sudden cardiac death: A cautionary tale of utility and limitations

For archived cases of previously young healthy individuals where cause of sudden death remains undetermined, formalin fixed paraffin-embedded tissues (FFPE) samples are often the only biological resource available for molecular testing. We aim to ascertain the validity of postmortem molecular analysis of 95 cardiac genes using the FFPE samples routinely processed in the offices of medical examiners - typical fixation time in formalin ranges from days to months. The study was conducted in the College of American Pathologists accredited Molecular Genetics Laboratory within the City of New York Office of Chief Medical Examiner. Twelve cases, with FFPE samples and corresponding non-formalin fixed samples (RNAlater-preserved tissues or bloodstain card), were chosen for testing results comparison. The methods of extracting DNA from FFPE samples using Covaris, Qiagen, and Promega products showed comparable results. The quality of the extracted DNA, the target-enriched DNA libraries of 95 cardiac genes using HaloPlex Target Enrichment system by Agilent Technologies, and sequencing results using Illumina Miseq instrument were evaluated. Compared to the sequencing results of the nonfixed samples, the FFPE samples were categorized into three groups: 1) Group 1 samples fixed in formalin 2–6 days, had greater than 55 % sequencing regions ≥30x and 94%–100% variant concordance. 2) Group 2 samples fixed in formalin for 8 days, showed intra-sample sequencing variations: the surface tissues showed 25%–27% extra variants (false positive) and 8.1%–9.7% missing variants (false negative), whereas the repeated core tissues showed reduced extra variants to 1.6 % and the false negative error was unchanged. 3) Group 3 samples fixed in formalin 29–136 days, had 2–55 % sequencing regions ≥30x, up to 52.2 % missed variants and up to 6.3 % extra variants. All reportable variants (pathogenic, likely pathogenic or variant of uncertain significance) identified in the nonfixed samples were also identified in FFPE, albeit three variants had low confidence variant calling. In summary, our study showed that postmortem molecular diagnostic testing using FFPE samples routinely processed by the medical examiners should be cautioned, as they are replete with false positive and negative results, particularly when sample fixation time is longer than 8 days. Saving non-formalin fixed samples for high fidelity molecular analysis is strongly encouraged.

MS3-1 IMPLEMENTATION OF GENE PANEL TESTING USING NEXT-GENERATION SEQUENCING

The advance of next-generation sequencers (NGS) has dramatically improved the performance of genomic analysis of clinical samples in cancer precision medicine. The practical use of gene panel testing for clinical applications has begun in Japan. At present, “OncomineTM Dx Target Test” is listed as a companion diagnostic system using NGS, and “FoundationOne CDx Cancer Genomic Profile” and “OncoGuide™ NCC Oncopanel System” are listed as gene panel testing under insurance coverage. Formalin-fixed paraffin-embedded specimen have been routinely used for molecular diagnosis testing, therefore quality control such as formalin fixation time and tumor contents is important to ensure validity of diagnostic results. In this presentation, the issue to obtain evaluable results of gene panel testing using formalin-fixed paraffin-embedded specimen will be discussed.Due to evolution of detection technologies, we can detect gene mutation with high sensitivity. Detection of gene mutation in circulating tumor DNA is feasible approach for diagnostic testing in cancer treatment. Liquid biopsy has been approved as a companion diagnostic testing to detect EGFR mutations in NSCLC. Examples of the clinical utility of plasma testing in cancer treatment will be presented.

MS3-2 TREATMENT STRATEGY BASED ON THE RESULTS OF GENE PANELS

The advance of next-generation sequencers (NGS) has dramatically improved the performance of genomic analysis of clinical samples in cancer precision medicine. The practical use of gene panel testing for clinical applications has begun in Japan. At present, “OncomineTM Dx Target Test” is listed as a companion diagnostic system using NGS, and “FoundationOne CDx Cancer Genomic Profile” and “OncoGuide™ NCC Oncopanel System” are listed as gene panel testing under insurance coverage. Formalin-fixed paraffin-embedded specimen have been routinely used for molecular diagnosis testing, therefore quality control such as formalin fixation time and tumor contents is important to ensure validity of diagnostic results. In this presentation, the issue to obtain evaluable results of gene panel testing using formalin-fixed paraffin-embedded specimen will be discussed.Due to evolution of detection technologies, we can detect gene mutation with high sensitivity. Detection of gene mutation in circulating tumor DNA is feasible approach for diagnostic testing in cancer treatment. Liquid biopsy has been approved as a companion diagnostic testing to detect EGFR mutations in NSCLC. Examples of the clinical utility of plasma testing in cancer treatment will be presented.

Accelerated Tissue Processing With Minimal Formalin Fixation Time for 9-Gauge Vacuum-Assisted Breast Biopsy Specimens.

ObjectivesVacuum-assisted biopsy (VAB) of the breast seems unsuitable for rapid processing due to large size. We tested microwave-based acceleration.MethodsAs a proof-of-principle study, 9-gauge VAB specimens were taken from eight mastectomy specimens. Forty-two biopsy specimens were processed. Quality of H&E was evaluated in 84 slides, and estrogen receptor (ER), progesterone receptor (PR), E-cadherin, and human epidermal growth factor receptor 2 (HER2) stains were evaluated in six slides. Preoperative biopsy specimens were used as a control.ResultsDiagnostic quality of H&E slides was good in 87%, reasonable in 12%, and low in 1%. Quality of E-cadherin was good in 75% and reasonable in 25%. Quality of ER was good in 83% and reasonable in 17%. PR and both HER2 immunohistochemistry and fluorescence in situ hybridization were good in all slides. Quality of experimental slides was similar to control slides.ConclusionsNine-gauge VAB specimens can be processed within 4 hours. Slides are suitable for all routine pathologic stains. This enables a same-day diagnosis.

Abstract 3539: Impact of formalin time on targeted NGS performance in FFPE tissue

Abstract Targeted next generation sequencing (NGS) panel testing is now a standard of care for management of advanced stage cancer. At present, most targeted NGS oncology testing occurs with formalin fixed paraffin embedded (FFPE) tissue. This is due to the established utility and prevalence of FFPE in routine histologic classification, grading, staging, and ancillary testing. In addition, FFPE materials are an optimally suited medium for determining adequacy of the specimen prior to downstream NGS targeted panel testing. For these reasons, as well as a growing trend of minimally invasive procedures, clinical specimens are increasingly prioritized for FFPE to meet the bulk of standard oncology care. Currently, formalin fixation time guidelines do exist for specific immunohistochemical and in-situ hybridization oncology tests. However, specific FFPE processing guidelines have yet to be established that would optimize targeted NGS panel testing performance. In this study, our aim was to develop contrived FFPE materials using the FDA-led Sequencing Quality Control Phase II (SEQC2) consortium working group 2 (WG2) cell line materials. The overarching goal was to use these FFPE materials to identify an upper limit of formalin time associated with minimal impact on somatic variant calling performance in targeted NGS oncology panels. The SEQC2 - WG2 normal lymphoblast cell line was selected as a well-characterized test material to undergo systematic alteration in formalin fixation time (1, 2, 6 and 24 hours) prior to “routine” tissue processing and embedding (FFPE). These FFPE cell-block materials, derived from a single sample source (with both extracted FFPE DNA as well as shaved sections), were distributed to multiple labs running various targeted NGS amplicon and hybridization oncology panels. The expected outcome of these studies is to provide general guidance to the community on how formalin time in FFPE specimens impacts targeted NGS oncology panel testing performance. In addition, we hope to identify genomic regions (internal control regions) that serve as markers for the full range of formalin time FFPE-effect. This information will assist in improving the lower limit of detection for variant calling (<5% variant allele frequency). Citation Format: Thomas M. Blomquist, Erin Crawford, James Willey, Joshua Xu, Onco-panel Working Group of the Sequencing Quality Control Phase 2 (SEQC2) Consortium. Impact of formalin time on targeted NGS performance in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3539.

The effect of prolonged formalin fixation on the staining characteristics of archival human brain tissue.

Neurodegenerative disorders include wide range of conditions, which affect millions of people worldwide. Unfortunately, they are incurable and irreversibly progressive. Immunohistochemical staining of paraffin-fixed tissues for both diagnostic and research purposes are widely used. However, large amount of brain tissues are fixed but little is known about whether they are suitable for retrospective studies. The study aimed at investigating the effects of prolonged formalin fixation time on immunohistochemical expression of some common neurodegenerative markers in archival brain specimens. Twenty brain specimens were obtained from hu- man cadavers in the Anatomy Department of King Abdulaziz University that were prefixed in 10% formalin. They were divided into two equal groups according to time of fixation: group 1 - less than 1 year, group 2 - up to 20 years. Histological examination of white and grey matter was done using haematoxylin and eosin, luxol fast blue (LFB) for myelin staining, Con- go red for amyloid plaques, CD 68 for microglial cells, tenascin-C (large ex- tracellular matrix glycoprotein) and caspase 3 antibody for apoptotic cells. For both groups, corpus callosum sections displayed myelination with LFB staining. The distribution of CD 68 positive microglial cells was evi- dent in frontal and temporal grey matter, but not in corpus callosum sections. Strongly positive masses were seen in Congo red-stained frontal and temporal sections. Anti-caspase 3 immunostaining revealed positively stained neurons. Histological and immunohistochemical techniques yielded repro- ducible staining results when applied to human brain tissue stored in formalin for long periods; so they can be used in well preserved biobank material which are the most targeting research areas in neuropathology.

Association Between Formalin Fixation Time and Programmed Cell Death Ligand 1 Expression in Patients With Non-Small Cell Lung Cancer.

The expression of programmed cell death ligand 1 (PD-L1) determined by immunohistochemistry (IHC) may be associated with tissue formalin fixation time in non-small cell lung cancer (NSCLC) samples. We investigated the association between the PD-L1 expression and formalin fixation time, and clarified the optimal duration of fixation for accurate PD-L1 evaluation. We collected 55 tumor specimens from resected NSCLC patients. The samples were halved and immediately fixed in 10% buffered formalin for 12-24 h (normal fixation), or 96-120 h (prolonged fixation). Each specimen was stained using two assay systems (22C3 and SP263) for PD-L1. The mean PD-L1 tumor proportion score was not significantly different between normal and prolonged fixation groups for either 22C3 or SP263 (normal fixation: 18.8%; prolonged fixation: 16.3%, p=0.277; normal fixation: 16.2%; prolonged fixation: 17.6%, p=0.560, respectively). Formalin fixation duration for up to 120 h does not affect PD-L1 IHC expression. PD-L1 tumor proportion score of tumor specimens can be evaluated by IHC even if these have been fixed in formalin outside the recommended duration in clinical practice.

Factors affecting KRAS mutation detection in colorectal cancer tissue

BackgroundWith the recent development of molecular tests for various biomarkers, it has become even more important to prepare adequate tissue samples. However, little is known about how the effect of cold ischemia time or formalin fixation time can affect KRAS mutation detection in colorectal cancer. MethodsThis study included the results of KRAS mutation tests for colorectal cancer in 401 specimens. We investigated clinicopathologic factors that may affect DNA quality of formalin-fixed, paraffin-embedded (FFPE) tissue including specimen type, cold ischemia time, and formalin fixation time and assessed the detection rate of the KRAS mutation in samples with varying DNA quality. ResultsSample DNA quality for KRAS mutation test was better in biopsy specimens, which showed markedly shorter cold ischemia time and shorter formalin fixation time compared to resection specimens. A cold ischemia time of one hour or less was associated with better sample DNA quality. But the formalin fixation time was not a significant factor when it fell within the range performed in routine pathology diagnosis. When prolonged formalin fixation was tested, we confirmed that the specimen DNA quality gradually got worse from one month to three months. ConclusionsThe biopsy specimens showed better sample DNA quality for KRAS mutation test compared to resection specimens. In a routine diagnostic pathology setting, the cold ischemia time was an important factor affecting DNA quality and the formalin fixation had a wide time range for optimal DNA quality.

Assessment of HER2 status in breast cancer biopsies is not affected by accelerated tissue processing.

To establish whether core needle biopsy (CNB) specimens processed with an accelerated processing method with short fixation time can be used to determine accurately the human epidermal growth factor receptor 2 (HER2) status of breast cancer. A consecutive case-series from two high-volume breast clinics was created. We compared routine HER2 immunohistochemistry (IHC) assessment between accelerated processing CNB specimens and routinely processed postoperative excision specimens. Additional amplification-based testing was performed in cases with equivocal results. The formalin fixation time was less than 2 h and between 6 and 72 h, respectively. Fluorescence in-situ hybridisation and multiplex ligation-dependent probe amplification were used for amplification testing. One hundred and forty-four cases were included, 15 of which were HER2-positive on the routinely processed excision specimens. On the CNB specimens, 44 were equivocal on IHC and required an amplification-based test. Correlation between the CNB specimens and the corresponding excision specimens was high for final HER2 status, with an accuracy of 97% and a kappa of 0.85. HER2 status can be determined reliably on CNB specimens with accelerated processing time using standard clinical testing methods. Using this accelerated technology the minimum 6 h of formalin fixation, which current guidelines consider necessary, can be decreased safely. This allows for a complete and expedited histology-based diagnosis of breast lesions in the setting of a one-stop-shop, same-day breast clinic.