Pre-Analytics & Biobanking Christian Viertler, MD Institute of Pathology Medical University of Graz EScoP, Basic Molecular Biology for Pathologists Graz, March 31 – April 4, 2014 Outline • Critical variables within the pre-analytical workflow of tissue samples • Current status and future perspective of tissue preservation • RNA quality control and gene expression analysis from (fixed and paraffin-embedded) tissue samples • SPIDIA comparative studies PFPE, FFPE and cryopreserved samples • Preservation of morphology, antigenicity, nucleic acids • Conclusions C. Viertler, 2014 1
Pre-Analytical Workflow of Tissues Samples C. Viertler, 2014 Current status of tissue preservation FFPE Liquid nitrogen, RNAlater Morphology Gold standard Limited use Nucleic acids Limited use Gold standard Impaired preservation of biomolecules Impaired morphology preservation Drawbacks Crosslinking Logistics, costs C. Viertler, 2014 2
Future perspective of tissue preservation One tissue specimen Morphology Biomolecules + Histopathological analyes and molecular studies from the same tissue sample on a high-quality level. C. Viertler, 2014 Gene Expression Analysis Fixation and storage effects Fixation and storage introduces major gene-to-gene variations in qRT-PCR. Viertler, Kashofer et al; PLoS ONE 2013 C. Viertler, 2014 3
Comparative Studies PFPE vs gold standard for morphology FFPE and molecular analyses CRYO PFPE : P AXgene- F ixation and P araffin- E mbedding. More than 5000 differently processed tissue samples collected within the SPIDIA consortium. C. Viertler, 2014 RNA preservation RNA quality control qRT-PCR assay Summary of ~800 qRT-PCR reactions, 45 (non-)malignant Representative results for RNA integrity on Agilent Bioanalyzer . tissue samples from different organs fixed for 3-120h with PAXgene or formaldehyde, analyzed in comparison to corresponding cryopreserved reference. Viertler et al, J Mol Diagn. 2012 C. Viertler, 2014 4
Gene expression analysis PFPE vs FFPE, CRYO ref R² PFPE= 0.99 R² FFPE= 0.89 Viertler et al, J Mol Diagn. 2012 Gene expression analysis of 92 cancer pathway-associated and 4 endogenous control genes (18S, GAPDH, GUSB, HPRT1) by qRT-PCR on predefined TaqMan array “Human Molecular Mechanisms of Cancer” plate. C. Viertler, 2014 DNA integrity, long-range& multiplex PCR Viertler et al, J Mol Diagn. 2012 FFPE, PFPE, and snap-frozen (CRYO) samples of 5 human colorectal cancer cases. C. Viertler, 2014 5
Conclusions � The quality and reliability of tissue-based molecular analyses can only be defined in the context of pre-analytical procedures � Pre-analytical variables e.g. ischemia, preservation method, fixation time and storage conditions impact molecular analyses � Established methods for RNA QC are well suited for frozen tissue but less reliable for (formalin-fixed and) paraffin-embedded tissues • Additional QC recommended depending on the level of standardisation of the pre-analytical workflow � Documentation of the pre-analytical sample history is crucial for biobanking � New alternatives to routine formalin fixation should be considered for: • comprehensive tissue diagnostics, clinical trials, biomedical research • biomarker discovery programmes in a routine clinical setting • molecular studies whenever a collection of snap frozen samples is not possible • direct correlation of disease phenotypes with alterations of biomolecules C. Viertler, 2014 THANK YOU! QIAGEN GmbH Uwe Oelmüller Ralf Wyrich PreAnalytiX GmbH Daniel Groelz Dako Denmark Medical University of Graz Rosa Winther Institute of Pathology Kurt Zatloukal Karl Kashofer Erasmus Medical Center Rotterdam Peter Regitnig Department of Pathology Peter Abuja Peter Riegman Viktoria Herbst Marcel Kap Monika Moser Daniela Pabst Technical University of Munich Institute of Pathology Biobank Graz Karl-Friedrich Becker ZMF CF Molecular Biology Sibylle Gündisch Bilge Reischauer All SPIDIA partners cont@ct: christian.viertler@medunigraz.at C. Viertler, 2014 6
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