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Characterization and quantitation by flow cytometry of membranous microparticles formed during activation of platelet suspensions with ionophore or thrombin.

Abstract：

:The typical data presentation by flow cytometry of platelet suspensions stimulated with calcium ionophore A-23187, thrombin, C5b-9, or other agonists shows a unimodal decrease (or 'left-shift') in forward angle light scatter. Many reports in the literature interpret these findings as indicative of the appearance of small membranous microparticles generated from the platelets as part of the activation response. Investigators may attempt to quantify the microparticles by calculation of the percentage of counts falling below a gate set around the forward angle light scatter distribution of fresh, non-activated platelets. We believe that this approach can lead to erroneous results unless the total particle count in the sample is also determined. The true change in total particle count in a platelet sample is easily estimated on the flow cytometer by adding a known amount of fluorescent beads to the platelet suspension and noting a change in the ratio of bead versus non-bead event counts as a result of the stimulus added. Using this technique under settings considered routine for platelet analysis on a FACScan flow cytometer (Becton-Dickinson), we have found that particle counts increased very little (less than a doubling) in platelet suspensions stimulated with 1-10 microM A-23187 or 0.01-0.5 U/ml thrombin or with sera from patients with diagnosed heparin-induced thrombocytopenia (HIT). Only by employing high sensitivity settings for signal thresholding on orthogonal light scatter, combined with fluorescence gating on high prevalence surface antigens, were we able to detect significant increases (5- or 6-fold) in total particle count in the same experiments. The new events we observed were separated by a decade or more (log scale) from intact platelets on the light scatter plots and fluorescence histograms in a bimodal distribution. We postulate that the unimodal-shifted population of events seen under routine settings after stimulation with ionophore is really degranulated platelets, and only the much smaller new modal subpopulation represents microparticles released as new entities into the platelet suspension. We conclude that without high sensitivity settings for data acquisition, it is most likely incorrect to claim that the left-shifted events on flow cytometry light scatter plots appearing contiguous with the distribution of activated platelets are released microparticles.

journal_name

Platelets

journal_title

Platelets

authors

Bode AP,Hickerson DH

doi

10.1080/09537100050129279

subject

Has Abstract

pub_date

2000-08-01 00:00:00

pages

259-71

issue

eissn

0953-7104

issn

1369-1635

journal_volume

pub_type

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Characterization and quantitation by flow cytometry of membranous microparticles formed during activation of platelet suspensions with ionophore or thrombin.