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
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Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied.

Author(s): van Ierssel SH, Van Craenenbroeck EM, Conraads VM, Van Tendeloo VF, Vrints CJ, Jorens PG, Hoymans VY

Publication: Thromb Res, 2010, Vol. 125, Page 332-9

PubMed ID: 20117824 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of high speed centrifugation and storage on the phenotypic characterization of endothelial microparticles (EMP) in platelet poor plasma (PPP).

Conclusion of Paper

The addition of a third centrifugation at 10,000 g decreased the abundance of CD31+ CD42b- EMP compared to specimens only centrifuged twice at 1550 g. The abundance of CD144+ EMP was significantly reduced by storage of PPP for 7 h at 4 degrees C compared to initial levels. Storage of PPP at -80 degrees C led to significant increases in CD31+ CD42b- EMP and CD62E+ EMP and decreases in CD144+ EMP, but CD144+ CD105+ EMP levels were not affected by storage of PPP at -80 degrees C. There was little variability in EMP counts between repeat measurements, but the variability in counts increased with increasing storage.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of high speed centrifugation and storage on the phenotypic characterization of EMP in PPP. Peripheral blood was collected in acid-citrate dextrose tubes, and the first 3 mL was discarded. Specimens were centrifuged twice at 1550 x g, for 20 min, without brakes to obtain PPP before some specimens were subjected to a third centrifugation step at 10000 x g.

    Summary of Findings:

    The addition of a third centrifugation at 10,000 g decreased the abundance of CD31+ CD42b- EMP compared to specimens only centrifuged twice at 1550 g (p=0.004), but the abundance of all other EMP was unaffected. The abundance of CD144+ EMP was significantly reduced by storage of PPP for 7 h at 4 degrees C compared to initial levels (p=0.012), but no other counts were significantly affected. Storage of PPP at -80 degrees C for 7 days led to a significant increase in CD31+ CD42b- EMP (p=0.004) and a decrease in CD144+ EMP (p=0.019). Storing PPP at -80 degrees C for 28 days resulted in a significant increase in CD62E+ EMP (p=0.004), but the levels of CD144+ CD105+ EMP were unaffected by storage of PPP at -80 degrees C. There was little variability in EMP counts between repeat measurements, but the variability in EMP counts increased with increasing storage.

    Biospecimens
    Preservative Types
    • None (Fresh)
    • Other Preservative
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    Cell count/volume Flow cytometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Storage duration 0 h
    7 h
    7 days
    28 days
    Storage Storage temperature 4 degrees C
    - 80 degrees C
    Biospecimen Preservation Type of fixation/preservation Frozen
    None (fresh)
    Refrigeration
    Biospecimen Aliquots and Components Centrifugation Multiple speeds compared
    Different number of centrifugation steps compared
    View more

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