Formalin-free Fixation Research Articles

Interstitial deletion of the Apc locus in β-catenin-overexpressing cells is a signature of radiation-induced intestinal tumors in C3B6F1 ApcMin/+ mice†.

Recent studies have identified interstitial deletions in the cancer genome as a radiation-related mutational signature, although most of them do not fall on cancer driver genes. Pioneering studies in the field have indicated the presence of loss of heterozygosity (LOH) spanning Apc in a subset of sporadic and radiation-induced intestinal tumors of ApcMin/+ mice, albeit with a substantial subset in which LOH was not detected; whether copy number losses accompany such LOH has also been unclear. Herein, we analyzed intestinal tumors of C3B6F1 ApcMin/+ mice that were either left untreated or irradiated with 2Gy of γ-rays. We observed intratumor mosaicism with respect to the nuclear/cytoplasmic accumulation of immunohistochemically detectable β-catenin, which is a hallmark of Apc+ allele loss. An immunoguided laser microdissection approach enabled the detection of LOH involving the Apc+ allele in β-catenin-overexpressing cells; in contrast, the LOH was not observed in the non-overexpressing cells. With this improvement, LOH involving Apc+ was detected in all 22 tumors analyzed, in contrast to what has been reported previously. The use of a formalin-free fixative facilitated the LOH and microarray-based DNA copy number analyses, enabling the classification of the aberrations as nondisjunction/mitotic recombination type or interstitial deletion type. Of note, the latter was observed only in radiation-induced tumors (nonirradiated, 0 of 8; irradiated, 11 of 14). Thus, an analysis considering intratumor heterogeneity identifies interstitial deletion involving the Apc+ allele as a causative radiation-related event in intestinal tumors of ApcMin/+ mice, providing an accurate approach for attributing individual tumors to radiation exposure.

Formalin-free fixation and xylene-free tissue processing preserves cell-hydrogel interactions for histological evaluation of 3D calcium alginate tissue engineered constructs

Histological evaluation of tissue-engineered products, including hydrogels for cellular encapsulation, is a critical and invaluable tool for assessing the product across multiple stages of its lifecycle from manufacture to implantation. However, many tissue-engineered products are comprised of polymers and hydrogels which are not optimized for use with conventional methods of tissue fixation and histological processing. Routine histology utilizes a combination of chemical fixatives, such as formaldehyde, and solvents such as xylene which have been optimized for use with native biological tissues due to their high protein and lipid content. Previous work has highlighted the challenges associated with processing hydrogels for routine histology due to their high water content and lack of diverse chemical moieties amenable for tissue fixation with traditional fixatives. Thus, hydrogel-based tissue engineering products are prone to histological artifacts during their validation which can lead to challenges in correctly interpreting results. In addition, chemicals used in conventional histological approaches are associated with significant health and environmental concerns due to their toxicity and there is thus an urgent need to identify suitable replacements. Here we use a multifactorial design of experiments approach to identify processing parameters capable of preserving cell-biomaterial interactions in a prototypical hydrogel system: ionically crosslinked calcium alginate. We identify a formalin free fixative which better retains cell-biomaterial interactions and calcium alginate hydrogel integrity as compared to the state-of-the-art formalin-based approaches. In addition, we demonstrate that this approach is compatible with a diversity of manufacturing techniques used to fabricate calcium alginate-based scaffolds for tissue engineering and cell therapy, including histological evaluation of cellular encapsulation in 3D tubes and thin tissue engineering scaffolds (∼50 μm). Furthermore, we show that formalin-free fixation can be used to retain cell-biomaterial interactions and hydrogel architecture in hybrid alginate-gelatin based scaffolds for use with histology and scanning electron microscopy. Taken together, these findings are a significant step forward towards improving histological evaluation of ionically crosslinked calcium alginate hydrogels and help make their validation less toxic, thus more environmentally friendly and sustainable.

A new substitute for formalin: Application to embalming cadavers.

The development of formalin-free fixatives is an urgent issue in gross anatomy because of the health hazard and the tissue-hardening actions of formalin. We recently identified the fixative, antimicrobial, and preservative effects of N-vinyl-2-pyrrolidone (NVP), a precursor of the water-soluble macromolecular polymer polyvinylpyrrolidone, in animal experiments. The aim of the present study is to investigate whether NVP solution can be used as an alternative to formalin in human cadaveric dissection. Twelve donated cadavers were infused with NVP via the femoral and common carotid arteries using a peristaltic pump. Experienced teaching staff members in our department dissected the cadavers and examined their macroanatomical properties. The NVP-embalmed corpses showed no sign of decomposition or fungal growth. The bodies remained soft and flexible. Notably, the shoulder, elbow, wrist, phalangeal, hip, knee, cervical spine, and temporomandibular joints were highly mobile, almost equivalent to those of living individuals. The range of motion of most joints was greater in the NVP-fixed than formalin-fixed cadavers. Under the dermis, the subcutaneous fat was markedly reduced and the connective tissues were transparent, so the ligaments, cutaneous nerves, and veins were easily discernible. The abdominal wall and the visceral organs remained pliable and elastic, resembling those of fresh cadavers. The lungs, liver, and gastrointestinal tract were moveable in the thoracic and abdominal cavities and were readily isolated. NVP can be used successfully as a fixative and preservative solution for human cadavers; furthermore, NVP-embalmed bodies could be valuable for learning clinical skills and for training, and for developing innovative medical devices. Clin. Anat. 31:90-98, 2018. © 2017 Wiley Periodicals, Inc.

Non-formalin fixative versus formalin-fixed tissue: A comparison of histology and RNA quality

Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin.Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RT PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition.In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RT PCR regardless of amplicon length.

Abstract 4071: PAXgene Tissue: A new tissue fixation technology for simultaneous preservation of morphology and nucleic acids

Abstract Introduction: PAXgene Tissue (PAXgene) is a new, formalin-free fixation technology developed for simultaneous preservation of morphology and biomolecules in tissue samples. PAXgene fixed, paraffin embedded (PFPE) tissue is suitable for conventional histochemical and immunohistochemical staining as well as for extraction of high quality nucleic acids. Material and Methods: Cases of human lung, breast and colorectal cancer were divided and fixed in formalin, PAXgene, or snap frozen in liquid nitrogen (LN2). PAXgene fixed, stabilized specimens were stored for up to four days at room temperature prior to processing. Formalin fixed, paraffin embedded (FFPE) and PFPE tissue morphologies were compared using H&E stain. RNA, miRNA and DNA were isolated from FFPE, PFPE and LN2 tissue. RNA expression was analyzed in 96-well RT-qPCR arrays with pre-designed assays, and miRNA expression profiling was performed with single Sybr-green or primer/probe based assays. DNA was analyzed by agarose gel electrophoresis, long-range, and multiplex PCR. Results: Morphology of PFPE samples was similar to or indistinguishable from FFPE tissue. A high correlation of both RNA and miRNA gene expression results was observed between PFPE and LN2 samples. For all PCR assays, Ct values were within +/− 2 Ct. In contrast, poor correlation was observed between RNA from FFPE compared to RNA from LN2 with differences of up to 10 Cts. Correlation of FFPE to LN2 was higher in the case of miRNA but was still not as high as that of miRNA from PFPE. High molecular weight DNA which performed well in PCR could be isolated from LN2 and PFPE tissue, but DNA from FFPE failed in long-range PCR and showed biased or no amplification in multiplexed PCR. Conclusion: The PAXgene Tissue fixation technology preserves morphology and nucleic acids in tissue samples. It enables morphological and multimodal research biomarker testing with one sample. PAXgene Tissue is for research use only. It is not intended for diagnostic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4071. doi:1538-7445.AM2012-4071

Comparison of Formalin-Free Tissue Fixatives: A Proteomic Study Testing Their Application for Routine Pathology and Research

Formalin-fixed, paraffin-embedded tissue is the routine processing method for diagnostics practiced in pathology departments worldwide. To determine the potential value of non-cross-linking, formalin-free tissue fixation for diagnostics in pathology and proteomic investigations. We tested 3 commercially available, formalin-free tissue fixatives-FineFIX, RCL2, and HOPE-in lung cancer specimens from 10 patients. The fixatives were evaluated for their effects on tissue morphology, protein recovery, and immunoreactivity for a selected panel of proteins differently expressed in lung cancer, using immunohistochemistry and Western blotting. Tumor-cell analysis with hematoxylin-eosin worked equally well for all tested fixatives when compared with the standard formalin-fixed, paraffin-embedded procedure. Movat pentachrome stains showed comparable results for the different matrices and cellular proteins analyzed. The RCL2 (P = .01) and HOPE fixatives (P = .03) improved protein recovery when compared with formalin-fixed, paraffin-embedded or frozen tissues. Our data clearly show that the fixatives evaluated influenced immunoreactivity to matched, formalin-fixed, paraffin-embedded lung cancer tissue. In particular, membrane-bound proteins, such as epidermal growth factor receptor EGFR, can be detected more efficiently by immunohistochemistry and Western blotting. We have demonstrated that formalin-free fixatives have the potential in routine pathology and research to replace formalin in histomorphology and protein preservation.

Evaluation of formalin-free tissue fixation for RNA and microRNA studies

FineFix, RCL-2 and HOPE, three formalin-free fixatives, were compared to the common used formalin fixed tissue samples of lung cancer and were evaluated for their effects on quality, quantity and integrity of RNA and microRNA. Two commercially available RNA extraction Kits (RNeasy FFPE by Qiagen and RecoverAll™ Nucleic Acid Isolation by Ambion) were tested and optimized in order to determine an extraction protocol for RNA as well as miRNA independent of the fixative. Two selected miRNAs were quantified via TaqMan MicroRNA assays. The optimized RNA extraction protocol for Qiagen's Kit leads to similar results for RNA quality and integrity for all fixatives. Highest RNA yield was obtained for formalin and the highest average miRNA ratio was found for FineFix. RNA fragments smaller than 500 bases were detected in FineFix, formalin and RCL2 fixed tissues; HOPE was the only fixative showing long fragments in one third of the samples. Our findings demonstrate that formalin-free fixatives are in general not superior for RNA studies. With our optimized RNA extraction protocol, there is no difficulty in extracting great amounts of RNA with high quality. According to the quality obtained, quantitative real-time PCR analysis can be performed without any negative impact. Similar results can be achieved for the tested fixatives and therefore no fixative seems to represent a new “gold-standard” for tissue fixation.

Abstract LB-142: Preservation of gene expression profile and histomorphology in human breast tumor tissue with the new PAXgene® tissue system

Abstract Introduction: PreAnalytiX has developed a system for preservation of histomorphology and nucleic acids in paraffin embedded tissue samples. The system is comprised of a collection container for formalin-free fixation and stabilization of tissue specimens and purification kits for isolation of DNA, RNA or microRNA (miRNA) from PAXgene Tissue fixed, paraffin embedded (PFPE) tissue samples. In this case study, a tissue specimen of human infiltrating ductal carcinoma (HIDC) of the breast was divided into three parts after resection and fixed either in neutral buffered formalin (NBF) or the PAXgene Tissue Container, or snap frozen in liquid nitrogen (LN2). The three, different preparations of the tumor sample were compared for preservation of histomorphology, RNA integrity, and gene expression profile. Materials and Methods: The part of the tumor specimen treated with PAXgene Tissue reagents (PFPE) was fixed for two hours and stored for 20 days at 4°C before processing and paraffin embedding according to manufacturer's instructions. The NBF fixed sample (FFPE) was fixed for eight hours, processed, and embedded in paraffin. PFPE and FFPE samples were stained with hematoxylin and eosin (H&E) or immunohistochemical stains for HER2 and estrogen receptor α (ERα). RNA was purified from sections of PFPE, FFPE and from frozen tissue using the PAXgene Tissue RNA Kit (PFPE), the RNeasy® FFPE Kit (FFPE) and the RNeasy® Mini Kit (LN2). RNA was analyzed on the Agilent Bioanalyzer for purity and integrity. Expression levels for 92 genes associated with cancer and 4 housekeeping genes were quantified in real-time RT-PCR using the Applied Biosystems TaqMan® Array Human Molecular Mechanisms of Cancer 96-Well Plate. Results: H&E stained sections of PFPE were similar to, or indistinguishable from, FFPE tissue. However, the differentiation of both chromatin structure and nuclear substructure were preserved better in PFPE than in FFPE tissue. Immunohistochemical staining of HER2 and ER gave comparable staining intensities in PFPE and FFPE samples. RNA from frozen and PFPE samples were of high integrity with average RIN values of 9.5 and 5.6 respectively, compared to RIN value of 2.2 from FFPE. Gene expression analysis of 96 genes in real time RT-PCR showed a high correlation of CT (threshold cycle) values for the samples from frozen and PFPE tissue (r2=0.98), and a poor correlation between frozen and FFPE (r2=0.64). Conclusion: The PAXgene Tissue System preserves morphology and gene expression profile in paraffin embedded breast cancer tissue samples. Histomorphology is preserved similarly to that seen in FFPE tissue, while the gene expression profile shows a high correlation to snap frozen tissue. For research use only, not for use in diagnostic procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-142.

Morphological quality and nucleic acid preservation in cytopathology

Background:Fixation is a chemical or physical procedure to prevent the degradation of proteins and tissue morphology. To optimise molecular analysis of archival tissues, it is essential that fixation preserves morphology...

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system

BackgroundThe potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer – estrogen receptor and HER2 – at the RNA and protein levels.MethodsParallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR.ResultsThe immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens.ConclusionThe formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

Theory and practice of combining coagulant fixation and microwave histoprocessing

The German, F. Blum, introduced formalin as a fixative in 1893. Formalin rapidly became popular for hardening and preserving gross human and animal specimens. As a result, microscopy for diagnostic pathology by combining paraffin embedding and formalin fixation was developed. Alcohol-based fixatives have coagulation of proteins as their main preservative effect. Because there is no cross-linking, immunostaining is not compromised, and DNA and RNA is not damaged. Ethyl alcohol was used by Dutch scientists of the 18th century, but was replaced by the cheaper formalin. Addition of low molecular weight polyethylene glycol (PEG) optimized the coagulant fixative, Kryofix. The polyethylene glycol prevents excessive hardening and enhances the speed of coagulation of proteins. Kryofix was used on a large scale for skin biopsies in Leiden between 1987 and 2001. DNA preservation by the formulated coagulant fixative, BoonFix, is related to the concentration of ethyl alcohol, PEG and acetic acid. BoonFix has been used since 2004 in Leiden for over 40,000 diagnostic skin biopsies and more than 100,000 cervical samples. A literature review and three decades of experience with coagulant, formalin-free fixatives in pathology suggest that when health authorities realize that formalin invalidates expensive tests, it might eventually be eliminated legislatively from diagnostic pathology. Finally, coagulant fixation is optimal for microwave histoprocessing where ethyl alcohol is followed by isopropanol.

A Novel Fixative Improves Opportunities of Nucleic Acids and Proteomic Analysis in Human Archive??s Tissues

All tissues from biopsy or surgery origin are fixed and paraffin embedded as a routine procedure in the hospital departments of pathology. The traditional method of tissue preservation is the fixation in formalin, followed by paraffin embedding. In this way tissue's integrity is ensured also for future analyses, because there is no further chemical degradation of nucleic acids and proteins in tissues embedded in paraffin. After few sections for the histopathological examination the tissues are stored for decades in the hospital archives. Even if formalin fixation compromises the quality and integrity of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA and RNA from these archive's tissues, even of autopsy origin. Protein analysis is on the contrary completely blocked, due to the fact that formalin fixation creates covalent links between proteins and the only way to study protein expression is immunohistochemistry. In this study we present our results concerning the use of a new formalin free fixative, the FineFIX. After extraction of nucleic acids, PCR and RT-PCR analyses were performed in DNA and RNA respectively. For DNA analysis it was possible to obtain amplicons of 2400 bps, while in formalin-fixed samples the maximum length achieved was less than 400 bps. RT-PCR analysis show that it was possible to study RNA fragments of 600 bps from FineFIX fixed tissues, against a maximum length of about 150 bps achieved by formalin-fixed tissues. These tissues were analyzed also by Western Blot analysis, showing that the proteins obtained from FineFIX treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues. Protein extracts from FineFix treated tissues were also compared with fresh tissues'ones by two dimensional electrophoresis, demonstrating that the protein pattern were well comparable for number and distribution of the spots.

Mast Cell Heterogeneity in the Small Intestine of Normal, Gnotobiotic and Parasitized Pigs

A formalin fixative and a formalin-free fixative were used to study mast cells in the small intestine of conventional, gnotobiotic and parasitized pigs. Many more mast cells were identified after basic lead acetate fixation ('mucosal mast cells', MMC) than after routine formalin fixation ('connective tissue mast cells'). The MMC were preferentially localized in the lamina propria. There were no differences between conventional and gnotobiotic pigs. However, in parasitized animals, the number of mast cells was several times higher, mainly because there were more MMC. The heterogeneity of intestinal mast cells in the pig indicates that this might be an interesting model for functional studies on mast cell subsets.