Other preanalytical variables and control considerations

Proteomic investigations involving blood products (serum/plasma) are especially complicated for a variety of reasons. Not only is the plasma proteome very complex, there are a wide range of other variables that can affect the source, acquisition and treatment of the blood sample itself, and many of these variables could lead to alteration of the detected proteome. These effects may range in severity from subtle or not detectable, to those having a great impact. The variables fall into distinct categories, as shown in Tab. 2. An appreciation of these conditions and their effects, and the diligent tracking of these variables, may prove to be essential for reproducibility of results.

For the HUPO PPP effort, BD Diagnostics prepared reference specimens under strictly controlled conditions and these were subsequently made available to all laboratories [1, 2, and Section 2], thus eliminating many of the potential variations. Additional studies will be necessary to evaluate all of these variables in Tab. 2, and the severity of their impact upon the plasma proteome. Such studies can be conducted in the future with national or regional proteomics organizations. Until a more conclusive body of data concerning these variables can be generated, pre-analytical effects on individual studies may only be discovered in retrospect. Thus, meticulous tracking ofpre-analytical variables is critical for analysis, evaluation and ensuring reproducibility of results.

With regards to the storage and handling of samples, there are a number of issues that are worthy of mention. It is critical to accurately document the temperature storage history of each individual archived serum or plasma sample, in addition to establishing realistic and well defined sample processing times. Conventional wisdom, in the absence of other data, suggests that samples should be aliquoted and stored in liquid nitrogen as soon as possible after collection. However, this approach will probably be impractical for most sample collection efforts. A common practical compromise is to process and aliquot serum and plasma samples within 1 to 2 h of collection followed by flash freezing and storage at — 80°C in a freezer where temperature fluctuations are minimized. However, potentially degrading enzymatic processes may be damaging, even during this 1-2 h timeframe (as demonstrated by the SELDI data presented above). Further, the number of freeze-thaw cycles should be minimized, ideally using sample without refreez-ing, but certainly to no more than once, with an absolute limit of two. Such procedures, and accompanying rigorous documentation oftemperatures and times, will help to maintain the integrity of samples, which is paramount in any proteomic analysis. Researchers will need to be aware of developing data on such variables, to improve the quality of plasma proteome research in general.

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