Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Impact of Formalin- and Cryofixation on Raman Spectra of Human Tissues and Strategies for Tumor Bank Inclusion.

Reliable training of Raman spectra-based tumor classifiers relies on a substantial sample pool. This study explores the impact of cryofixation (CF) and formalin fixation (FF) on Raman spectra using samples from surgery sites and a tumor bank. A robotic Raman spectrometer scans samples prior to the neuropathological analysis. CF samples showed no significant spectral deviations, appearance, or disappearance of peaks, but an intensity reduction during freezing and subsequent recovery during the thawing process. In contrast, FF induces sustained spectral alterations depending on molecular composition, albeit with good signal-to-noise ratio preservation. These observations are also reflected in the varying dual-class classifier performance, initially trained on native, unfixed samples: The Matthews correlation coefficient is 81.0% for CF and 58.6% for FF meningioma and dura mater. Training on spectral differences between original FF and pure formalin spectra substantially improves FF samples' classifier performance (74.2%). CF is suitable for training global multiclass classifiers due to its consistent spectrum shape despite intensity reduction. FF introduces changes in peak relationships while preserving the signal-to-noise ratio, making it more suitable for dual-class classification, such as distinguishing between healthy and malignant tissues. Pure formalin spectrum subtraction represents a possible method for mathematical elimination of the FF influence. These findings enable retrospective analysis of processed samples, enhancing pathological work and expanding machine learning techniques.

Strand Orientation Bias Detector to determine the probability of FFPE sequencing artifacts.

Formalin-fixed paraffin-embedded tissue, the most common tissue specimen stored in clinical practice, presents challenges in the analysis due to formalin-induced artifacts. Here, we present Strand Orientation Bias Detector (SOBDetector), a flexible computational platform compatible with all the common somatic SNV-calling pipelines, designed to assess the probability whether a given detected mutation is an artifact. The underlying predictor mechanism is based on the posterior distribution of a Bayesian logistic regression model trained on The Cancer Genome Atlas whole exomes. SOBDetector is a freely available cross-platform program, implemented in Java 1.8.

Developmental and regional dependence of macromolecular proton fraction and fractional anisotropy in fixed brain tissue.

An important advantage of imaging fixed tissue is a gain in signal-to-noise ratio and in resolution due to unlimited scan time. However, the fidelity of quantitative MRI parameters in fixed brain tissue, particularly in developmental settings, requires validation. Macromolecular proton fraction (MPF) and fractional anisotropy (FA) indices are quantitative markers of myelination and axonal integrity relevant to preclinical and clinical research. The goal of this study was to assert the correspondence of MR-derived markers of brain development MPF and FA between in vivo and fixed tissue measures. MPF and FA were compared in several white and gray matter structures of the normal mouse brain at 2, 4, and 12 weeks of age. At each developmental stage, in vivo imaging was performed, followed by paraformaldehyde fixation and a second imaging session. MPF maps were acquired from three source images (magnetization transfer weighted, proton density weighted, and T1 weighted), and FA was obtained from diffusion tensor imaging. The MPF and FA values, measured in the cortex, striatum, and major fiber tracts, were compared before and after fixation using Bland-Altman plots, regression analysis, and analysis of variance. MPF values of the fixed tissue were consistently greater than those from in vivo measurements. Importantly, this bias varied significantly with brain region and the developmental stage of the tissue. At the same time, FA values were preserved after fixation, across tissue types and developmental stages. The results of this study suggest that MPF and FA in fixed brain tissue can be used as a proxy for in vivo measurements, but additional considerations should be made to correct for the bias in MPF.

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.

Effect of formalin fixation on measured concentrations of deposited gadolinium in human tissue: an autopsy study.

BACKGROUND: Generally, studies of gadolinium (Gd) deposition in humans measure concentration by analyzing formalin fixed postmortem tissue. However, the effect of formalin fixation on measured Gd concentration has not been well investigated. PURPOSE: To evaluate the effect of fixation by comparing Gd concentration in fresh versus formalin-fixed postmortem human tissues. MATERIAL AND METHODS: Fresh samples of bone and skin were collected from autopsy cases with previous exposure to Gd-based contrast agents (GBCAs). The type of GBCA administered, dose, and estimated glomerular filtration rate were recorded. Each tissue sample was cut into three aliquots. Paired samples were stored fresh frozen while the remaining two were stored in 10% neutral buffered formalin for one and three months, respectively. Gd concentration was measured using ICP-MS. RESULTS: Of 18 autopsy cases studied, 12 were exposed to only macrocyclic GBCA, one to only linear agents, and five received both macrocyclic and linear agents. On average, Gd concentration for bone decreased 30.7% after one month of fixation (P = 0.043) compared to non-fixed values. There was minimal, if any, change in concentration between one and three months (average decrease 1.5%; P = 0.89). The findings were numerically similar for skin tissue with an average decrease of 36.9% after one month (P = 0.11) and 6.0% (P = 0.73) between one and three months. CONCLUSION: Formalin fixation appears to decrease Gd concentration in bone and skin by approximately 30%-40% on average. The largest decrease occurs within the first 30 days of fixation followed by a considerably smaller decrease at 60 days.

Changes in magnetic resonance imaging relaxation time on postmortem magnetic resonance imaging of formalin-fixed human normal heart tissue.

BACKGROUND: Postmortem magnetic resonance imaging (MRI) has been used to investigate the cause of death, but due to time constraints, it is not widely applied to the heart. Therefore, MRI analysis of the heart after formalin fixation was previously performed. However, the changes in MRI signal values based on the fixation time of formalin were not investigated. The objective was to investigate changes over time in the T1- and T2-values of MRI signals in normal areas of hearts removed during autopsy, hearts subsequently fixed in formalin, and heart specimens sliced for the preparation of pathological specimens. METHODS: The study subjects were 21 autopsy cases in our hospital between May 26, 2019 and February 16, 2020 whose hearts were removed and scanned by MRI. The male:female ratio was 14:7, and their ages at death ranged from 9 to 92 years (mean age 65.0 ± 19.7 years). Postmortem (PM)-MRI was conducted with a 0.3-Tesla (0.3-T) scanner containing a permanent magnet. A 4-channel QD head coil was used as the receiver coil. Scans were performed immediately after removal, post-formalin fixation, and after slicing; 7 cases were scanned at all three time points. RESULTS: The T1- and T2-values were calculated from the MRI signals of each sample organ at each scanning stage. Specimens were sliced from removed organs after formalin fixation, and the changes in T1- and T2-values over time were graphed to obtain an approximate curve. The median T1-values at each measurement time point tended to decrease from immediately after removal. The T2-values showed the same tendency to decrease, but this tendency was more pronounced for the T1-values. CONCLUSION: MRI signal changes in images of heart specimens were investigated. Formalin fixation shortened both T1- and T2-values over time, and approximation formulae were derived to show these decreases over time. The shortening of T1- and T2-values can be understood as commensurate with the reduction in the water content (water molecules) of the formalin-fixed heart.

Effect of a formalin-based fixation method on bone mineral content in human ex-vivo specimens.

OBJECTIVE: Histopathology of formalin-fixated human ex-vivo specimens may be used as reference standard for evaluation of diagnostic index tests like CBCT or MRI. The aim was to estimate changes in bone mineral content (BMC) over time in human ex-vivo bone specimens fixated in a formalin-based solution for 24 h followed by storage in an alcohol-based medium for six months, assessed by dual-energy X-ray absorptiometry (DXA). METHODOLOGY: Bone specimens (n = 19) from human ex-vivo mandibles donated for science were included. BMC was measured by DXA before fixation (D0), after 24 h of immersion fixation in a formalin-based solution (D1), and hereafter every 30 days (M1-M6) during storage in a 30% ethanol-based storage medium for 6 months. Changes in BMC from D0 to D1 and from D0 to M6 were calculated and mean change in BMC estimated. RESULTS: Mean change in BMC from D0 to D1 was -0.73% (95% CI -1.75%; 0.29%), and from D0 to M6 -1.19% (95% CI -2.14%; -0.23%). CONCLUSIONS: No changes in BMC of ex-vivo human bone specimens were found after 24 h formalin-based immersion fixation. After six months storage in an ethanol-based medium, BMC mean loss of 1% was detected. In this range, changes in BMC are not clinically relevant.

Rapid tissue processing using a temperature-controlled collection device to preserve tumor biomarkers.

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold-hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold-hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process.

Formalin tissue fixation biases myelin-sensitive MRI.

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.

Optimization of RNA extraction protocol for long-term archived formalin-fixed paraffin-embedded tissues of horses.

A suitable RNA extraction protocol was established to gain high quality RNA from formalin-fixed paraffin-embedded tissues to perform reliable molecular assays either applicable for using FFPE tissue archives or tissues with harsh formalin-fixation. Eighteen FFPE samples from the central nervous system of horses, stored up to 11 years, were used as archive cases. To test the influence of the fixation period, brain, liver, kidney, and skeletal muscle tissue fragments from another horse, were treated either with water or tris-acetate-EDTA buffer after fixation under different timepoints with 10% unbuffered formalin. Two deparaffinization methods and three proteinase K-based lysis step were tested and translated into three protocols. After detailed statistical analysis it was determined that a longer period and increase in volume of proteinase K incubation provide higher yields and purity of RNA (P < 0.01) of archived samples. Alongside, amplification of equid-housekeeping gene up to 298 bp was successful with the protocol adaptations. For different formalin-fixation timepoints, it was demonstrated that the right choice for treatment and formalin-fixation period is organ-related (P ≤ 0.05). Essentially, little alterations to pre-existing extraction protocols unwound the RNA of up to 11-year-old samples, enabling the use of FFPE tissue archives or e.g. harshly fixed material needed in infection research under high biosafety levels for a variety of molecular analysis.

Effects of concentration and vendor specific composition of formalin on postmortem MRI of the human brain.

PURPOSE: Formalin fixation prevents tissue autolysis by crosslinking proteins and changes tissue microstructure and MRI signal characteristics. Previous studies showed high variations in MR relaxation time constants of formalin fixed brain tissue, which has been attributed to the use of different formalin concentrations. Our investigations confirmed the influence of formalin concentration on relaxation times and unexpectedly revealed an influence of vendor specific formalin composition, which has not been investigated so far. METHODS: We systematically analyzed relaxation times of human brain tissue fixed with 4% and 10% formalin compared with unfixed condition at 3 Tesla MRI. Furthermore, we assessed relaxation times of nine formalin solutions from different vendors and performed comparisons of their magnetic susceptibility by SQUID (superconducting quantum interference device) magnetometry. RESULTS: Tissue relaxation times decreased approximately twice as fast using 10% than in 4% formalin fixation. The vendor specific composition of the formalin solutions and concentration dependent paramagnetic effects showed a substantial contribution to differences in relaxation times of formalin. CONCLUSION: Our study demonstrates that differences of the formalin composition have substantial effects on MRI signal characteristics after fixation, which can explain the divergence of reported relaxation times beyond the effect of differences in formalin concentration. Magn Reson Med 79:1111-1115, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Proteomic distinction of renal oncocytomas and chromophobe renal cell carcinomas.

BACKGROUND: Renal oncocytomas (ROs) are benign epithelial tumors of the kidney whereas chromophobe renal cell carcinoma (chRCCs) are malignant renal tumors. The latter constitute 5-7% of renal neoplasias. ROs and chRCCs show pronounced molecular and histological similarities, which renders their differentiation demanding. We aimed for the differential proteome profiling of ROs and early-stage chRCCs in order to better understand distinguishing protein patterns. METHODS: We employed formalin-fixed, paraffin-embedded samples (six RO cases, six chRCC cases) together with isotopic triplex dimethylation and a pooled reference standard to enable cohort-wide quantitative comparison. For lysosomal-associated membrane protein 1 (LAMP1) and integrin alpha-V (ITGAV) we performed corroborative immunohistochemistry (IHC) in an extended cohort of 42 RO cases and 31 chRCC cases. RESULTS: At 1% false discovery rate, we identified > 3900 proteins, of which > 2400 proteins were consistently quantified in at least four RO and four chRCC cases. The proteomic expression profiling discriminated ROs and chRCCs and highlighted established features such as accumulation of mitochondrial proteins in ROs together with emphasizing the accumulation of endo-lysosomal proteins in chRCCs. In line with the proteomic data, IHC showed enrichment of LAMP1 in chRCC and of ITGAV in RO. CONCLUSION: We present one of the first differential proteome profiling studies on ROs and chRCCs and highlight differential abundance of LAMP1 and ITGAV in these renal tumors.

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

The effect of formalin fixation on the size of pelvic sidewall lymph nodes.

PURPOSE: Although several studies have demonstrated that the size of harvested lymph nodes can be a prognostic predictor in colorectal cancer patients, some considered the size of freshly harvested nodes and others assessed the size after formalin fixation. Because the size change of lymph nodes during fixation has not been fully investigated, we conducted the present study comparing the size of lateral lymph nodes that were surgically harvested from rectal cancer patients, before and after formalin fixation. METHODS: A total of 19 consecutive patients diagnosed with rectal adenocarcinoma who underwent total mesorectal excision and dissection of lateral pelvic sidewall lymph nodes were prospectively enrolled. The largest diameters of lymph nodes were measured immediately after manual harvest and after formalin fixation. The ratio of post-fixation size to pre-fixation size and the size difference between pre- and post-fixation were assessed for each lymph node. RESULTS: The average ratio (± standard deviation) of post-fixation size to pre-fixation size was 0.88 ± 0.40, with median value of 0.8. The size of the lymph nodes decreased by an average of 1.04 mm after fixation, and the median size change after fixation was a 1-mm decrease. CONCLUSIONS: Although measuring lymph node size after formalin fixation can be a viable alternative to measuring the size of fresh lymph nodes before fixation, a 10 to 20% shrinkage or 1-mm size reduction should be considered when interpreting the examination findings.

The myth: in vivo degradation of polypropylene-based meshes.

INTRODUCTION AND HYPOTHESIS: Polypropylene is a base polymer used in biomaterial applications, including sutures and mesh products, for the treatment of pelvic organ prolapse, stress urinary incontinence, and hernia repairs. Previous studies have dismissed the value of formulation additives employed in polypropylene, and the importance and necessity of an effective mesh explant cleaning protocol when characterizing explanted devices. However, both are critical to understanding the alleged degradation of polypropylene-based meshes. METHODS: An effective, nondestructive, hydrolytic cleaning process, supplemented with light microscopy (LM), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) data, was used to evaluate 78 explanted Prolene meshes (with duration of implantation ranging from 0.4 to 11.7 years). RESULTS: The cleaning process exposed clean, unoxidized, nondegraded Prolene fibers with smooth surfaces and with no visible evidence of gradient-type or ductile damage. LM showed identical translucent and sometimes clear, cracked/flaking material on both blue and clear fibers, instead of clear cracked/flaking material on the clear fibers and blue cracked/flaking material on the blue fibers. FTIR confirmed progressive protein removal and loss of protein absorption intensity after each cleaning step. CONCLUSIONS: Our effective cleaning of explanted Prolene meshes and subsequent analyses showed that they did not degrade in vivo, confirming the in vivo stability of properly formulated polypropylene. Instead, the cracked layer that some researchers have identified as degraded Prolene is an adsorbed protein-formaldehyde coating, resulting from the well-established formalin-protein fixation process, which occurs immediately upon placing an explant in formalin.

Shrinkage in oral squamous cell carcinoma: An analysis of tumor and margin measurements in vivo, post-resection, and post-formalin fixation.

PURPOSE: To quantify changes in tumor size and tumor-free margins following surgical resection and formalin fixation of oral cavity squamous cell carcinoma. MATERIALS AND METHODS: Nineteen patients were studied via cohort design. Between May and December 2011, measurements of tumor size and tumor-free margin were made in patients with squamous cell carcinoma of the oral cavity. Mucosal reference points were marked with sutures, representing tumor diameter and two separate resection margins. Measurements were recorded immediately before resection, after resection, and following fixation in formalin. RESULTS: The overall mean shrinkage in tumor size was 10.7% (95% CI 3.4-18.0, p=0.006). When comparing mean tumor measurements, most of the tumor size decrease (6.4%, 95% CI 0.4-12.4, p=0.039) occurred between pre- and post-excision measurements. To a lesser extent, tumor size decreased following formalin fixation. Comparison of tumor-free margin measurements revealed a pre-excision to post-fixation mean decrease of 11.3% (95% CI 2.9-19.6%, p=0.011), with a statistically significant decrease of 14.9% (95% CI 8.5-21.3%, p<0.001) occurring between pre- and post-excision, and no significant decrease from post-excision to post-formalin fixation. CONCLUSION: Mucosal dimensions of both tumor and tumor-free margins in oral cavity squamous cell carcinoma specimens decrease between surgical resection and pathologic analysis. Most of this decrease occurs prior to fixation, especially for margins, and may be due to intrinsic tissue properties rather than formalin effects.

Effects of formalin fixation and temperature on MR relaxation times in the human brain.

Post-mortem MRI of the brain is increasingly applied in neuroscience for a better understanding of the contrast mechanisms of disease induced tissue changes. However, the influence of chemical processes caused by formalin fixation and differences in temperature may hamper the comparability with results from in vivo MRI. In this study we investigated how formalin fixation and temperature affect T1, T2 and T2* relaxation times of brain tissue. Fixation effects were examined with respect to changes in water content and crosslinking. Relaxometry was performed in brain slices from five deceased subjects at different temperatures. All measurements were repeated after 190 days of formaldehyde immersion. The water content of unfixed and fixed tissue was determined using the wet-to-dry ratio following drying. Protein weight was determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fixation caused a strong decrease of all relaxation times, the strongest effect being seen on T1, with a reduction of up to 76%. The temperature coefficient of T1 was lower in the fixed than unfixed tissue, which was in contrast to T2, where an increase of the temperature coefficient was observed following fixation. The reduction of the water content after fixation was in the range of 1-6% and thus not sufficient to explain the changes in relaxation time. Results from SDS-PAGE indicated a strong increase of the protein size above 260 kDa in all brain structures examined. Our results suggest that crosslinking induced changes of the macromolecular matrix are responsible for T1 shortening and a decreased temperature dependency. The relaxation times provided in this work should allow optimization of post-mortem MRI protocols for the brain.

Skin punch biopsy sectioning: before or after tissue processing?

BACKGROUND: We intended to study whether there is a meaningful difference in microscopic examination between dividing a biopsy section into two equal parts before tissue processing (first method) or after (second method). METHODS: A total of 400 cases were included in the study. Punch biopsies (PB) were cut into two pieces using the first method in 200 cases and just before paraffin embedding in another 200 cases using the second method. We microscopically evaluated the epidermal mesh view, the presence of a cross-cut hair follicle and bow shape because of epidermal angling, the presence of two pieces on the slide and if there was a difference of >2 mm between the parts, and the number of new sections and new slides. RESULTS: Cross-cut hair follicle (p = 0.018), epidermal mesh view (p = 0.036), difference of >2 mm between the parts (p = 0.008), the number of new sections (p < 0.001) and new slides (p < 0.001) were considerably higher when the first method was used compared with the second method. The presence of two pieces was less (p < 0.001) when using the first method. CONCLUSIONS: We noted a meaningful difference in the quality of microscopic evaluation between the first and second methods. Better sections were obtained with the second method. In addition, the decrease in the number of new slides will reduce workload, archival work and cost.

A reference image-based method for optimization of clinical immunohistochemistry.

AIMS: Cold ischaemic and formalin fixation time (CIT and FFT) are considered to be crucial parameters for intralaboratory variation in immunohistochemistry (IHC). Here we describe a new method to optimize IHC, by using control tissue blocks with known pre-analytical history and comparing the IHC outcome with digitized reference slides. METHODS AND RESULTS: Tissue specimens (two per tissue type) were divided into eight samples, which were subjected to different CIT and FFT. Immunohistochemistry was performed with 34 routinely used antibodies, following standard operating procedures. Relative staining intensity of four sections per slide was scored. Of the antibodies studied, seven were influenced by CIT, 13 by FFT and five by both parameters. IHC protocols were adapted until most sections on the slide showed the same intensity. Changing the antibody dilution for 10 protocols and the antigen retrieval method for six protocols improved the consistency of the IHC staining. Nine protocols could not be optimized. The optimized staining results were compared to reference slides and were found to be of adequate quality. CONCLUSIONS: It was possible to optimize most IHC protocols by adapting the analytical, rather than the pre-analytical, phase. If global references can be established, this method could decrease interlaboratory variation, preceding standardization of the pre-analytical workflow.