Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

Abstract

High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6 h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ≤3 freeze–thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14–17 years of frozen storage (−80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies.

Investigating pre‐analytical requirements for serum and plasma based infrared spectro‐diagnostic

“…Similar results were obtained with plasma samples from the same subjects (Figure S5). These results are consistent with conclusions of proteomics studies observing no significant changes in serum and plasma long‐term storage at −80°C .…”

Plasma Proteome Profiling to detect and avoid sample‐related biases in biomarker studies

“…Especially in case-control studies, any difference in the collection and processing of samples may result in systematic bias. So far, relatively little attention has been paid to this crucial aspect on a proteome-wide scale and these studies mainly investigate preanalytical effects (Rai et al, 2005;Timms et al, 2007;Schrohl et al, 2008;Qundos et al, 2013;Hassis et al, 2015).…”

Analysis of the plasma proteome using iTRAQ and TMT‐based Isobaric labeling

“…Sample handling is a vital consideration in the search for differentially abundant proteins. Hassis et al used iTRAQ to evaluate the effects of pre‐analytical variables on the stability of the plasma proteome . They determined the influence of the time delay (0.5, 6, and 96 h) and temperature (RT & 37°C) before processing, freeze thaw cycles and long term storage (14–17 years).…”

“…Hassis et al used iTRAQ to evaluate the effects of pre-analytical variables on the stability of the plasma proteome. 28 They determined the influence of the time delay (0.5, 6, and 96 h) and temperature (RT & 37°C) before processing, freeze thaw cycles and long term storage (14-17 years). They concluded that the time prior to processing was the most significant variable.…”