Impact of Human Blood Specimen Collection, Processing and Storage on Protein Integrity and Implications for Use in Clinical Research.
Subcontractor: Caprion Proteomics
Principal Investigator: Daniel Chelsky, Ph.D.
Dr. Chelsky and his team systematically investigated the impact of preanalytical variables on protein integrity in human plasma and serum. The variables that were investigated using a mass spectrometry approach include collection tube type, incubation time and temperature before and after centrifugation, storage time and temperature and incremental freeze/thaw cycles. In collaboration with Palo Alto Hospital, their team obtained blood from normal healthy and breast/prostrate cancer subjects. Sample collection and processing were controlled using a bench top computer program that alerted the operator in real time to initiate each process step, as well as by a series of standard operating procedures. Data demonstrated that incubation of blood resulted in more changes in the proteome as compared to serum or plasma. Serum and plasma samples incubated at room temperature (~ 20 °C) for up to 24 hours were relatively stable. Longer incubation times induced significant changes to a subset of the proteome. Some proteins showed changes upon freezer storage for 6 months, with smaller changes occurring after 18 months. Overall, there were more changes at -20 °C than at -80 °C. Increasing number of freeze/thaw cycles also affected specific proteins and the P100 and serum tubes with mechanical separators were found to be most protective against freeze/thaw cycles. These results provided extensive and independently verified information on the effect of pre-analytical variables on blood samples and pointed to clear evidence-based guidelines for their handling. Dr. Chelsky’s group designed a multiplexed multiple reaction monitoring (MRM) assay, targeting less stable proteins that could be used to evaluate sample integrity and establish utility of stored samples in clinical research.
A manuscript titled “Impact of Human Blood Specimen Collection, Processing and Storage on Protein Integrity and Implications for Use in Clinical Research ” has been prepared.