NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Method validation for preparing serum and plasma samples from human blood for downstream proteomic, metabolomic, and circulating nucleic acid-based applications.

Author(s): Ammerlaan W, Trezzi JP, Lescuyer P, Mathay C, Hiller K, Betsou F

Publication: Biopreserv Biobank, 2014, Vol. 12, Page 269-80

PubMed ID: 25075813 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of centrifugation parameters and temperature of blood storage on microparticle counts, hemoglobin, circulating DNA yield and metabolite and protein levels in serum and plasma.

Conclusion of Paper

A single 10 min centrifugation at 2000 x g for serum or a 20 min centrifugation at 2000 x g for plasma with medium braking was determined to be optimal for microparticle counts, and DNA and hemoglobin yields. Centrifugation at 2-8˚C instead of room temperature led to lower microparticle counts and circulating DNA yield from serum and lower microparticle counts and hemoglobin content from plasma. When blood specimens were stored at room temperature instead of 4˚C before centrifugation, microparticle counts were higher in 1 of 2 serum specimens and 3 of 3 plasma specimens. Further, 1.6% of detected proteins in serum and 4.4% of detected proteins in plasma were affected by storage at room temperature as opposed to 4˚C.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of centrifugation speed, duration, temperature and braking on microparticle counts, circulating DNA and hemoglobin yield, and metabolite profiles in serum and plasma. Blood specimens from a single healthy donor were collected into CAT (serum) and K2EDTA (plasma) Vacutainer tubes. Serum tubes were stored upright for 45 min at room temperature before centrifugation, and plasma was centrifuged immediately. Centrifugation was performed at 1000, 2000 or 4000 x g for 10 or 20 min (plasma only) with soft, medium or hard braking. The effect of a second centrifugation of plasma was also investigated. Nucleic acids were extracted using the QIAamp circulating nucleic acid kit.

    Summary of Findings:

    Microparticle counts in serum declined non-significantly with increasing centrifugation speed. Thrombocyte counts in plasma declined with increasing centrifugation speed but were too high in plasma centrifuged once. Microparticle counts were comparable when plasma was centrifuged at 2000 x g followed by a second spin at 1000 x g or 2000 x g, but they were significantly lower when the second spin was at 4000 x g (p=0.004). Further, microparticle counts were comparable in plasma spun twice for 10 min at 2000 x g and plasma spun once for 20 min at 2000 x g. A single 10 min centrifugation at 2000 x g for serum or a 20 min centrifugation at 2000 x g for plasma resulted in acceptable microparticle counts and DNA and hemoglobin yields. Centrifugation at 2-8˚C instead of room temperature did not alter hemolysis but led to lower microparticle counts (p=0.028) and circulating DNA yield (p=0.014) from serum and lower microparticle counts (p=0.017) and hemoglobin content (p=0.020) from plasma. Centrifugation temperature also affected 11 of 297 metabolites in serum and 6 of 304 metabolites in plasma. Braking speed significantly affected microparticle counts and circulating DNA content in serum and microparticle counts and hemoglobin yields in plasma, with the best result obtained with medium braking.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Fluorometry
    Protein Spectrophotometry
    Small molecule GC-MS
    Protein LC-MS or LC-MS/MS
    Protein GC-MS
    Cell count/volume Hematology/ auto analyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
    Multiple durations compared
    Multiple speeds compared
    Multiple temperatures compared
    Multiple brake speeds compared
    Biospecimen Aliquots and Components Blood and blood products Plasma
    Serum
  2. Study Purpose

    The purpose of this study was to determine the effects of delayed centrifugation on microparticle counts, hemoglobin and circulating DNA yields and metabolite and protein levels in serum and plasma. 2 serum and 3 plasma specimens were obtained from healthy donors using CAT and K2EDTA Vacutainer tubes, respectively. Serum tubes were stored upright for 45 min or 2 h at room temperature or 4˚C before centrifugation, and plasma was centrifuged immediately or after 2 h. Nucleic acids were extracted using the QIAamp circulating nucleic acid kit.

    Summary of Findings:

    When blood was stored for 2 h at room temperature instead of 4˚C before centrifugation, the microparticle counts in serum were significantly higher for 1 of the 2 patients (p=0.006) and hemoglobin was significantly higher for the other (p=0.019). However, only an average of 1.6% (4.8% for one patient and 0% for the other) of detected proteins in serum were affected by storage of blood at room temperature as opposed to 4˚C for 2 h prior to centrifugation. Storage of blood at room temperature instead of 4˚C for 45 min prior to centrifugation led to increased microparticle counts in plasma from all three patients (p<0.001, p=0.003 and p<0.001) and reduced hemoglobin in the plasma from 1 patient (p=0.042). 4.4% of detected proteins in plasma were affected by storage of blood at room temperature as opposed to 4˚C for 45 min, with 1.5%, 4.8% and 0% of proteins affected for individual patients.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    DNA Fluorometry
    Cell count/volume Hematology/ auto analyzer
    Small molecule GC-MS
    Protein LC-MS or LC-MS/MS
    Protein Spectrophotometry
    Protein GC-MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Aliquots and Components Blood and blood products Plasma
    Serum
    Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
    Storage Storage temperature 4˚C
    Room temperature
    Storage Storage conditions 0 min
    45 min
    2 h

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