Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.

Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.

The quality of human tissue specimens can have a significant impact on analytical data sets for biomarker research. The aim of this study was to characterize fluctuations of protein and phosphoprotein levels in human tissue samples during the preanalytical phase. Eleven intestine and 17 liver specimens were surgically resected, aliquoted, and either snap-frozen or fixed in formalin immediately or exposed to different ischemic conditions before preservation. Protein levels in the resultant samples were investigated by reverse phase protein array, Western blot analysis, and liquid chromatography-tandem mass spectrometry. Our data revealed that the degree of sensitivity of proteins and phosphoproteins to delayed preservation varied between different patients and tissue types. For example, up-regulation of phospho-p42/44 MAPK in intestine samples was seen in some patients but not in others. General trends toward up- or down-regulation of most proteins were not evident due to pronounced interpatient variability but signal intensities of only a few proteins, such as cytokeratin 18, were altered from baseline in postresection samples. In contrast, glyceraldehyde 3-phosphate dehydrogenase was found to be stable during periods of cold ischemia. Our study represents a proper approach for studying potential protein fluctuations in tissue specimens for future biomarker development programs.