Clinical biospecimens are the most commonly used sample-type for research purposes, but are often of poor quality given their susceptibility to uncontrolled and unrecorded preanalytical variables. IBBL’s Biorefinery Department carries out biospecimen research to guide researchers in their sample preparation, selection and processing decisions. Recently, Dr. William Mathieson and his colleagues developed an assay to characterise cold ischemia in tissues, with the aim of providing better insight into the selection of the most appropriate samples for a given downstream research study.
Cold ischemic (CI) time is a preanalytical variable that has been shown to influence gene and protein expression and stability: the longer the CI time, the greater the risk of repercussions on sample quality. Since CI times are not usually recorded upon collection, researchers do not know the extent to which their results will be affected by it.
In order to identify the tissue blocks that have been subjected to long CI times, the IBBL team collaborated with the US National Cancer Institute and the Van Andel Institute to devise a reliable assay based on a gene expression pattern. They extracted RNA from 40 colon, kidney and ovary cancer FFPE blocks with different CI times, then measured the expression levels of 23 preselected genes to identify those that are particularly stable or unstable in response to cold ischemia. The expression of three genes, namely PRKACA, FOS, and EGR1, was found to be upregulated due to CI. Using a gene (POLR2A) whose expression remains unvaried as a baseline, the combination of the three genes gives a Cold Ischemia (CI) Score that distinguishes samples with CI times shorter than 3 hours from those with 12-hours, with 62% sensitivity and 84% specificity.
“The CI Score is easy to validate, practical and accessible to laboratories, enabling researchers to retrospectively qualify legacy collections of FFPE tissue samples with undocumented preanalytics and select the samples that are fit for the purpose of their research”, states Dr. Mathieson.
The detailed results of the study can be found in the full paper here.
The study provides insight into the impact of preanalytical variables linked to sample collection methods, specifically cold ischemia, and presents an assay that helps characterise CI times in tissue samples. However, when it comes to biospecimen processing, the choice of methods used can also affect the sample’s fitness-for-purpose. The final article in this series will focus specifically on the impact of various techniques used to extract nucleic acids from tissue blocks. Stay tuned!
 The time between tissue collection and fixation, during which the tissue is deprived of oxygen and is below body temperature.