074 Controlled analysis of preanalytical variables in clinical blood and CSF sample collection, processing and storage: Implications for clinical research

Blood and cerebral spinal fluid (CSF) sample collection, processing, handling and storage protocols are based mainly on accepted practices rather than careful comparative analysis and testing. We set out, therefore, with the support of the NCI and the NINDS, to examine variables intrinsic to each step in the process of obtaining and storing clinical samples, beginning with collection of samples from healthy and diseased subjects in controlled studies. For blood, various tube types were tested including EDTA, heparin, serum and protease inhibitors. For both blood and CSF, various times on bench and temperatures of incubation were compared before and after centrifugation. Markers were found that indicated samples had been left on the bench, such as the S-100 protein which is bound to white cells but falls off into the plasma upon sitting on the bench in the blood tubes prior to centrifugation. The effects of freeze-thaw cycles and long-term storage time in freezer were also examined. Increased changes were correlated with increasing number of freeze-thaw cycles, and specific proteins were found to be more sensitive to these cycles. Samples in serum tubes were found to be most protective to changes induced by these freeze/thaw cycles. Many proteins were sensitive to freezer storage over a 6 month period at −20 and −80°C. This is most likely due to breaking of cells during long term freezer storage. For CSF subjects, seated or recumbent collection and fasted or fed conditions were compared. Sample analysis has been performed by high resolution mass spectrometry, leading to the identification of specific proteins that are affected by the various parameters tested. While different blood collection tubes can be used with reproducible results, there is a marked difference in the protein content obtained from each type, with protease inhibitor tubes offering significant protection from changes to the proteome (of CSF, as well). Freeze thaw cycles affect only a few specific proteins and but changes get pronounced over multiple cycles. CSF samples are robust with respect to subject condition, but are affected by temperature and time of incubation prior to freezing. A multiplexed MRM assay is being assembled for the analysis of stored samples in order to determine sample integrity and utility for use in clinical research. Source of funding: None declared. Conflict of interest: None declared. mroy@caprion.com