Endocrine Abstracts (2006) 11 P489

Evaluation of a standardized protocol for the collection and storage of adrenal tumor samples – preparation for an European adrenal tumor bank (ENS@T)

I Johnsen1, S Hahner2, M Fassnacht2, J Bertherat3, X Bertagna3, PF Plouin5, M Reincke4, B Allolio2 & F Beuschlein1


1Department of Internal Medicine II, University Hospital Freiburg, Freiburg, Germany; 2Department of Medicine, Endocrine and Diabetes Unit, University of Wuerzburg, Wuerzburg, Germany; 3Département d’Endocrinologie, Institut Cochin, Paris, France; 4Medical Clinic, University Hospital Innenstadt, Ludwig Maximilians University, Munich, Germany; 5Hypertension Unit, Hopital Europeen Georges Pompidou, Paris, France.


Tissue samples from adrenal tumors provide the basis for standard diagnostic procedures such as pathological examination. In addition, these tumor samples have been an invaluable source for the discovery of novel molecular pathways involved in adrenal tumorigenesis. Information on the molecular phenotype are based on DNA mutation analysis and epigenetic changes, RNA and protein expression pattern and sub-cellular localization as well as post transcriptional protein modification. However, storage and tissue handling of surgical adrenal tumor samples has not been optimized or standardized which might affect reproducibility and comparability between different laboratories. To examine different handling and storage procedures with regard to RNA, protein, and DNA quality and subsequent morphological examinations, we subdivided each surgical adrenal tumor sample into six pieces which where either snap frozen or treated with RNAlater (Ambion), after defined storage time at room temperature (up to 60 min). As we could show, DNA and protein recovery as well as integrity of DNA by means of pulse field electrophoresis and long range PCR (MC2-R locus) was not affected by snap freezing or the use of RNAlater after different storage intervals. Moreover, morphology of sectioned tissue samples was comparable between the groups, while overall staining intensity was decreased after RNAlater pre-treatment. In addition, western blotting did not reveal differences in the expression levels of 3ßHSD protein between the groups. However, levels of pERK as an example for a phosphorylated protein was significantly decreased by processing the tissue with RNAlater (snap vs. RNAlater after 15 min storage, 100.0±17.6% vs 57.7±6.4%, P=0.02). In summary, recovery and integrity of DNA and protein is not significantly affected by the investigated handling conditions while protein phosphorylation is dependent on pretreatment. Investigations of subtle differences in the RNA integrity and expression pattern are under way and will further define the optimal handling protocol of a scheduled European adrenal tumor tissue bank.