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An investigation of plasma collection, stabilization, and storage procedures for proteomic analysis of clinical samples

Abstract

In order to evaluate the critical components of the process necessary to preserve clinical plasma samples collected at research sites for proteomic analysis, various collection and preservation protocols with controlled experimentation were evaluated. The presence of a protease inhibitor cocktail (PIC) included in the blood draw tube would stabilize the plasma proteins was hypothesized. To test this hypothesis, four plasma samples from each of 14 volunteers were collected. Samples were treated following a standard protocol that included PIC or were subjected to various processing treatments that included time, temperature, different anticoagulants, and the absence of PIC. Large format two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis and enzyme immunoassay (EIA) were used to detect differences between the treatment groups. A novel 2D-PAGE quality scoring method was developed to determine global differences in the treatment groups, wherein a rating scale questionnaire was used to quantify the quality of each 2D-PAGE gel. The data generated from EIAs, classical 2D-PAGE image analysis and 2D-PAGE quality scoring, each generated similar results. Inclusion of protease inhibitor cocktail in the sample tubes, provided stable and reliable human plasma samples that yielded reproducible results by proteomic analysis. When PIC was included, samples retained stability under less stringent processing, such that refrigeration for several hours before processing or one freeze-thaw cycle had little detrimental effect. We demonstrated that samples without PIC, from either heparin or ethylenediaminetetraacetic acid (EDTA) plasma tubes, gave results that varied significantly from the control samples. Also, even with PIC present in blood tubes, we found it was important to quickly decant the separated plasma from the cellular components found in the blood tubes following centrifugation, as prolonged exposure again yielded different results from the standard procedure.

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Correspondence to Jeffrey D. Hulmes.

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Key Words

  • Proteomics
  • plasma collection
  • twodimensional gel electrophoresis
  • sample stability
  • protease inhibitors
  • 2D-PAGE evaluation