The major goal of the HUPO Plasma Proteome Project (HUPO PPP) is comprehensive characterization of the plasma proteome. The first phase of this project serves to

* Originally published in Proteomics 2005, 13, 3262-3277

establish the advantages and disadvantages of the various technologies, and allows for an initial assessment of the different sample types that can be used. The technologies for analysis include MS-based methods, (e.g., MALDI-TOF, SELDI-TOF, MS/MS, and FT-ICR-MS), 2-DE, antibody microarrays, and other methodologies.

The Specimen Collection and Handling Committee (SCHC) was created in order to evaluate a number ofpre-analytical variables that can potentially impact the outcome of results, but are not related to inherent sample (e.g., patient or donor) differences. These include the choices of sample type and collection system after collection, and handling issues which include stabilization, processing, storage, and potential effects of additives. We focused on these issues in the PPP pilot phase. Other issues such as patient status, venipuncture, phlebotomy technique and collection devices were not addressed, as a centralized collection procedure and strictly controlled conditions were necessary for such a large scale collection of a limited number of sample types.

The HUPO PPP elected to use limited pooled specimens in order to reduce the number of variables that could potentially confound the analysis and comparison of methods. While inter-individual variation is also important, this finer discrimination between donors will be reserved for later phase studies. After careful consideration of the objectives, a rigorous, standardized specimen collection procedure was established and the collection of HUPO reference specimens was initiated [1, 2, and Section 2]. The decision was made to collect and pool sufficient quantities from two individuals, derived from each ethnic group studied.

Another important question was that of comparing serum and plasma specimens with regard to the human proteome. Historically, serum samples dominate archives, however they were amassed based on requirements of conventional assays and not necessarily because they represent the most appropriate sample for protein analytics. For serum samples, clotting only by glass/silica-based activation was used, to eliminate any variability from the addition of other unwanted ex vivo effects, such as protease activation. On the other hand, the acquisition of plasma requires the use of anticoagulants. Plasma specimens were derived using the three most common anticoagulants, namely potassium-EDTA, lithium-heparin, and sodium-citrate. Depending on analytical objectives and/or target peptides or proteins, use of either plasma or serum may impact both method and results.

The HUPO PPP specimens were collected from three ethnic groups: Caucasian American, Asian American and African American. In addition, the National Institute of Biological Standards and Control (NIBSC, UK) provided a lyophilized plasma specimen, which was compared with the frozen specimens. The use of protease inhibitors was also discussed. The committee developed protocols that were comprehensive in nature, applicable to most methods or techniques and practical for use in a clinical laboratory setting, where the criteria of specificity, sensitivity, quality, reproducibility, and consistency are of critical importance.

After specimen collection, the impact of processing time and storage temperature on sample integrity was investigated. The effect of freeze-thaw cycles was also deemed important and was discussed. We sought to better define many of these issues and conditions related to blood collection and handling. What follows are the collective observations from PPP participants, related to each of these parameters.

These results and suggestions come from this first comprehensive effort designed to address the many aspects of sample selection and multiple issues that may result in pre-analytical variation. Clearly, these observations indicate that a great deal more work must be done to define a true "best practices" approach to plasma or serum samples. Through this manuscript, we hope to emphasize that a better appreciation of these issues will increase awareness and understanding of their consequences and sensitize researchers to the confounding artifacts that can result from non-ideal conditions. Sound experimental design, an educated choice of specimen type, and careful sample collection and handling procedures can have a profound impact on results, especially in inter-laboratory or multi-facility studies. The experimental observations and the resulting recommendations are described herein.

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