Lynne Rainen

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

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.

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

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 manufacturer9s 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 (HE 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-142.