Ralf Wyrich

A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and Molecular Analyses

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules.

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.

Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer

Molecular diagnostics in personalized medicine increasingly relies on the combination of a variety of analytical technologies to characterize individual diseases and to select patients for targeted therapies. The gold standard for tissue‐based diagnostics is fixation in formalin and embedding in paraffin, which results in excellent preservation of morphology but negatively impacts on a variety of molecular assays. The formalin‐free, non‐cross‐linking PAXgene tissue system preserves morphology in a similar way to formalin, but also preserves biomolecules essentially in a similar way to cryopreservation, which markedly widens the spectrum, sensitivity and accuracy of molecular analytics. In this study, we have developed and tested a protocol for PAXgene‐fixed and paraffin‐embedded tissues for fluorescent in situ hybridization (FISH). The implementation of a 24‐h formalin postfixation step of slides from PAXgene‐fixed and paraffin‐embedded tissues allowed us to use the assays approved for formalin‐fixed and paraffin‐embedded tissues. The equivalence of the methodologies was demonstrated by FISH analysis of HER2 amplification in breast cancer cases. The 24‐h postfixation step of the slides used for FISH can be well integrated in the routine diagnostic workflow and allows the remaining PAXgene‐fixed and paraffin‐embedded tissue to be used for further molecular testing.

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

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 HE 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4071. doi:1538-7445.AM2012-4071

Abstract C98: Evaluation of the PAXgene tissue system: Preservation of morphology and gene expression in human melanoma.

Introduction: PreAnalytiX has recently developed the PAXgene® Tissue System, a formalin-free tissue fixation method for simultaneous preservation of tissue morphology and biomolecules. In this study, PAXgene Tissue was used to stabilize human melanoma specimens. Mirrored samples of melanoma were fixed with either neutral buffered formalin or PAXgene Tissue, or snap frozen in liquid nitrogen (LN2) and were compared for preservation of morphology, antigenicity, RNA and noncoding small RNAs. Materials and Methods: Three cases of melanoma were equally divided into two parts: one part was fixed with PAXgene Tissue reagents, the second part of cases 1 and 2 were fixed with formalin and the second part of case 3 was snap-frozen in LN2. Formalin and PAXgene Tissue fixed samples were processed and embedded in paraffin (resulting in FFPE and PFPE tissue respectively). Tissue sections were stained with HE 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C98.