Intrinsic indicators for specimen degradation

Abstract

Variable degrees of molecular degradation occur in human surgical specimens before clinical examination and severely affect analytical results. We therefore initiated an investigation to identify protein markers for tissue degradation assessment. We exposed 4 cell lines and 64 surgical/autopsy specimens to defined periods of time at room temperature before procurement (experimental cold ischemic time (CIT)-dependent tissue degradation model). Using two-dimensional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, we performed comparative proteomic analyses on cells at different CIT exposures and identified proteins with CIT-dependent changes. The results were validated by testing clinical specimens with western blot analysis. We identified 26 proteins that underwent dynamic changes (characterized by continuous quantitative changes, isoelectric changes, and/or proteolytic cleavages) in our degradation model. These changes are strongly associated with the length of CIT. We demonstrate these proteins to represent universal tissue degradation indicators (TDIs) in clinical specimens. We also devised and implemented a unique degradation measure by calculating the quantitative ratio between TDIs' intact forms and their respective degradationmodified products. For the first time, we have identified protein TDIs for quantitative measurement of specimen degradation. Implementing these indicators may yield a potentially transformative platform dedicated to quality control in clinical specimen analyses.

Cold Ischemia Score: An mRNA Assay for the Detection of Extended Cold Ischemia in Formalin-Fixed, Paraffin-Embedded Tissue

“…Another potential QC marker for cold ischemia is based on Western Blot analysis of the ratio of intact α-II spectrin and its breakdown products. 23 However, it is not easy to standardize a Western Blot assay, particularly in FFPE tissue where extracting intact proteins of large molecular mass is difficult (intact α-II spectrin is 250 kDa).…”

Increasing the discrimination power of rapid evaporative ionisation mass spectrometry (REIMS) in analytical control tissue quality screening and cell line sample identification

“…A ‘quality measure’ in one context may not be relevant for a different assay or application 22,23 . There have been attempts to measure intrinsic quality indicators, but these can be technically complex and have not been adopted routinely 24 . There is currently no universally accepted method for screening tissue sample quality before use, ideally one that is cheap, rapid, technically simple and relatively non‐destructive or invasive.…”

“…22,23 There have been attempts to measure intrinsic quality indicators, but these can be technically complex and have not been adopted routinely. 24 There is currently no universally accepted method for screening tissue sample quality before use, ideally one that is cheap, rapid, technically simple and relatively nondestructive or invasive. Some tissues are not properly annotated, and therefore the design and interpretation of assays can be difficult.…”

Ischemia in Tumors Induces Early and Sustained Phosphorylation Changes in Stress Kinase Pathways but Does Not Affect Global Protein Levels

“…These studies identified a small number of proteins that changed in abundance (primarily as a result of degradation, presumably via proteolysis) after long (Ͼ 3 h) periods of cold ischemia (19,20). For example, Li et al, using two-dimensional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, identified 26 proteins that changed over 48 h of cold ischemia time, chiefly as a result of degradation (20). The sample collection and analysis approaches used in most of these studies did not permit assessment of changes within the one-hour interval investigated in our study.…”