Pre-Analytics & Biobanking Christian Viertler, MD Institute of - - PDF document

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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

• C. Viertler, 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

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Pre-Analytical Workflow of Tissues Samples

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Current status of tissue preservation

FFPE Liquid nitrogen, RNAlater Morphology Nucleic acids Drawbacks

Gold standard Gold standard Limited use Limited use

Impaired preservation of biomolecules Crosslinking Impaired morphology preservation Logistics, costs

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Future perspective of tissue preservation

Morphology One tissue specimen

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Biomolecules

Histopathological analyes and molecular studies from the same tissue sample on a high-quality level.

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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

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Comparative Studies

PFPE vs gold standard for morphology FFPE and molecular analyses CRYO

PFPE: PAXgene-Fixation and Paraffin-Embedding. More than 5000 differently processed tissue samples collected within the SPIDIA consortium.

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RNA preservation

Viertler et al, J Mol Diagn. 2012 Representative results for RNA integrity on Agilent Bioanalyzer. Summary of ~800 qRT-PCR reactions, 45 (non-)malignant tissue samples from different organs fixed for 3-120h with PAXgene

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formaldehyde, analyzed in comparison to corresponding cryopreserved reference.

RNA quality control qRT-PCR assay

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Gene expression analysis

PFPE vs FFPE, CRYO ref

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. Viertler et al, J Mol Diagn. 2012

R² PFPE= 0.99 R² FFPE= 0.89

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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.

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• 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

Conclusions

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THANK YOU!

QIAGEN GmbH Uwe Oelmüller Ralf Wyrich PreAnalytiX GmbH Daniel Groelz Erasmus Medical Center Rotterdam Department of Pathology Peter Riegman Marcel Kap Dako Denmark Rosa Winther Technical University of Munich Institute of Pathology Karl-Friedrich Becker Sibylle Gündisch Bilge Reischauer Medical University of Graz Institute of Pathology Kurt Zatloukal Karl Kashofer Peter Regitnig Peter Abuja Viktoria Herbst Monika Moser Daniela Pabst Biobank Graz ZMF CF Molecular Biology

cont@ct: christian.viertler@medunigraz.at

All SPIDIA partners