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

Flow cytometric analysis of circulating platelet-monocyte aggregates in whole blood: methodological considerations.

Author(s): Harding SA, Din JN, Sarma J, Jessop A, Weatherall M, Fox KA, Newby DE

Publication: Thromb Haemost, 2007, Vol. 98, Page 451-6

PubMed ID: 17721630 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of type of anticoagulant, collection method, processing delays, hemolysis, and refrigerated storage of fixed specimens on platelet-monocyte aggregate evaluation by flow cytometry.

Conclusion of Paper

Significantly different percentages of platelet-monocyte aggregates were observed between blood specimens incubated for 5 minutes in the different anticoagulants. The highest percentage of aggregates was measured in lithium heparin blood, followed by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK), then citrated blood, and finally EDTA blood. Specimens collected via intravenous catheter rather than venipuncture had significantly higher baseline levels of platelet-monocyte aggregation, and platelet-monocyte aggregation increased with sequential collection via intravenous catheter over time, but it did not increase with multiple venipunctures. Room temperature processing delays between 10 and 60 minutes caused platelet-monocyte aggregate percentages to increase for both PPACK and citrated blood (other anticoagulants not tested). Erythrocyte lysis by the addition of FACS lyse solution had no effect on platelet-monocyte aggregation. Storing fixed specimens at 4 degrees C for up to 24 h before analysis had no effects on the percentage of platelet-monocyte aggregates measured.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of type of anticoagulant, collection method, processing delays, hemolysis, and refrigerated storage of fixed specimens on platelet-monocyte aggregate evaluation by flow cytometry. All experiments were performed with blood from 8 healthy volunteers.

    Summary of Findings:

    Significantly different percentages of platelet-monocyte aggregates were observed between blood specimens incubated for 5 minutes in the different anticoagulants. The highest percentage of aggregates was measured in lithium heparin blood, followed by PPACK, then citrated blood, and finally EDTA blood. Specimens collected via intravenous catheter rather than venipuncture had significantly higher baseline levels of platelet-monocyte aggregation, and platelet-monocyte aggregation increased with sequential collection by intravenous catheter over time, but it did not increase with multiple venipunctures. Room temperature processing delays between 10 and 60 minutes caused platelet-monocyte aggregate percentages to increase for both PPACK and citrated blood (other anticoagulants not tested). Erythrocyte lysis by the addition of FACS lyse solution had no effect on platelet-monocyte aggregation. Storing fixed specimens at 4 degrees C for up to 24 h before analysis had no effects on the percentage of platelet-monocyte aggregates measured.

    Biospecimens
    Preservative Types
    • None (Fresh)
    • Other Preservative
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    Cell count/volume Flow cytometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Acquisition Anticoagulant Lithium heparin
    EDTA
    PPACK
    Sodium citrate
    Biospecimen Acquisition Method of fluid acquisition IV catheter
    Venipuncture
    Biospecimen Acquisition Time of biospecimen collection Baseline
    30 min
    50 min
    85 min
    Biospecimen Aliquots and Components Aliquot sequential collection 1st collection
    2nd collection
    3rd collection
    4th collection
    Storage Time at room temperature 0 min
    10 min
    20 min
    30 min
    60 min
    Biospecimen Aliquots and Components Hemolysis Chemically-induced
    Not induced
    Storage Storage duration 0 h
    4 h
    8 h
    12 h
    24 h
    Biospecimen Preservation Type of fixation/preservation None (fresh)
    Refrigeration

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