Pre-analytical factors can greatly influence the outcome of molecular analyses in medical diagnostics and research. This also applies to in situ staining techniques such as immunohistochemistry (IHC), where different types of tissue fixation methods lead to different modifications of proteins and thus can affect differently the detection by antibodies. For formalin-fixed paraffin-embedded (FFPE) tissue, antigen retrieval is applied in order to reverse the negative effects of formalin and re-establish immunoreactivity. Most antibodies and protocols used in IHC are optimized for FFPE tissue, but not for paraffin-embedded tissue treated with other fixatives such as non-crosslinking fixatives. We report results from systematic studies on distinct pre-analytical conditions in IHC, immunofluorescence and electron microscopy. Parameters investigated are the impact of crosslinking and non-crosslinking fixatives (comparing formalin and PAXgene Tissue fixation) on whole tissue, subcellular structures and organelles, as well as on ultrastructure. The results generated show that minor changes in antigen retrieval conditions may have a major impact on IHC results and that protocols optimized for crosslinking fixatives may not be used for other fixatives without re-validation. Key antigen retrieval parameters such as buffers with different pH and duration of microwave treatment must be tested systematically for each antibody and fixation protocol.

Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples.

Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples.