The Pre-analytical Phase of Pathology Research and Diagnostics De Pre-analytische Fase van Pathologie Onderzoek en Diagnostiek

Variation in pre-analytical procedures can have effects on RNA expression in tissue. Standardization of the pre-analytical workflow is not always an option, therefore it is necessary to know which factors might be of influence and should be noted as meta data in tissuebank databases. Here we describe the discovery and potential use of a RT-qPCR fingerprint of genes sensitive to the ischemic period. The biomarker discovery cohort existed of ten patients undergoing curative operations for metastases in the liver. Sample series taken just after the start and directly after surgery followed by a time series ranging from 30 to 360 minutes enabled RNA expression monitoring during the Warm (370C) and Cold (RT) ischemic time. After gene array analysis 1,146 probe sets were found to react significantly to the surgical phase only. A selection of the identified genes were confirmed by RTqPCR and enabled the design of a pre-analytical gene expression fingerprint, comprised of six genes. This fingerprint was validated using twenty-one liver needle biopsies (no ischemia) and excision samples (warm and cold ischemia), as well as in silico analysis of seven publicly available liver gene array data sets. The samples from these data sets were divided in three sample type groups, which reflect the approximate length of the surgical procedure. Gene expression in vivo can deviate from what is measured in ex vivo tissue samples. The pre-analytical RT-qPCR fingerprint showed low levels of expression before surgery and higher levels after surgery. Needle biopsies showed lower expression levels than samples exposed to surgery. In silico analysis showed that fingerprint expression levels are correlated with surgical technique/gross duration of surgery. Further research is necessary to find a possible linear proportional function between duration of surgery and fingerprint gene expression, to establish an evidence based normalization of gene array data.