Every year, improprieties and inconsistencies in pre-analytical handling and storage of blood plasma/serum (P/S) specimens generate unacceptably large numbers of costly false leads in biomedical research. Experts in the field are calling for this problem to be immediately stopped or at least minimized. The issue is particularly important in the scope of NIH-sponsored research: A recent study found that out of 455 NCI-sponsored extramural grants that relied on biospecimens, 63% employed pre-existing biospecimens?107 of which involved pre-existing P/S. Surprisingly, no criteria for establishing pre-existing sample integrity have been set and no quality thresholds exist that must be met before NCI (taxpayer) funds are spent on studies involving pre-existing P/S. Exposure to the thawed state (which includes temporary storage at -20 C) represents one of the most common pre-analytical variables (PAVs) encountered by P/S. Considering the entire life of a research specimen, no other PAV is more difficult to control and track?and, as demonstrated in the Research Strategy, even the best standard operating procedures (SOPs) in the most respected hands cannot guarantee that all specimens are handled ideally (i.e., in accord with the SOP). Our preliminary data argue that objective, molecular-level evidence based on measurements of molecular damage?above and beyond sole reliance on SOPs?is crucial to unambiguously establishing sample integrity. To date, however, no gold standard marker of P/S integrity yet exists. The goal of this project is to validate a simple, inexpensive, rapid test requiring 10 L of P/S that provides a representative assessment of the oxidative damage that P/S proteins have incurred due to exposure to the thawed state. The test is based on the fact that the relative abundance of S-cysteinylated (oxidized) albumin (S-Cys-Alb) increases substantially over time (but to a maximum value) when P/S is handled/stored above its freezing point of -30 C. Thus by measuring S-Cys-Alb before and after an intentional incubation period that causes S-Cys-Alb to hit its maximum value, the difference between these values, ?S- Cys-Alb, is then readily interpreted as inversely proportional to the degree of ex vivo oxidation that occurred prior to the first measurement of S-Cys-Alb. Thus, for example, a ?S-Cys-Alb value of zero would indicate a badly mistreated sample. Herein we will validate ?S-Cys-Alb as a marker of P/S integrity via four Specific Aims:
Specific Aim 1 : Experimentally validate the predicted range of ?S-Cys-Alb that can be expected from freshly collected cancer patient plasma and serum samples.
Specific Aim 2 : Systematically map out how ?S- Cys-Alb behaves under ?realistic? mistreatment conditions and link known, unstable cancer markers to ?S-Cys- Alb.
Specific Aim 3 : Conduct a blind challenge to quantify the ability of ?S-Cys-Alb to identify biospecimen mistreatment within cancer patient plasma and serum samples.
And Specific Aim 4 : Use ?S-Cys-Alb to quantify the integrity of plasma samples collected from a representative large cancer study in which samples were collected at multiple sites under a single SOP and eventually transferred to a single repository.
Exposure of blood plasma/serum (P/S) biospecimens to temperatures above their freezing point of -30 C can dramatically impact clinical research results?a phenomenon that has been shown to produce an inordinate number of false leads in biomedical research. Well over 100 NIH-sponsored extramural grants employ pre- existing P/S?yet, surprisingly, no criteria for establishing pre-existing sample integrity have been set and no quality thresholds exist that must be met before NIH/NCI (taxpayer) funds are spent on studies involving pre- existing P/S. This project will validate a promising candidate marker of P/S integrity that is based on inexorable protein oxidation events that occur ex vivo when a sample is exposed, even temporarily, to temperatures above -30 C.
