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Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow
S Robert1, P Poncelet, R Lacroix
1Unité Mixte de Recherche S 608 (UMR-S 608), Institut National de la Santé et de la Recherche Médicale (INSERM), Université de la Méditerranée, Marseille, France. stephane.robert@pharmacie.univ-mrs.fr
Background:
Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized.
Objectives:
The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size-calibrated fluorescent beads; (ii) to determine intra-instrument and inter-instrument reproducibility; and (iii) to establish PMP values in healthy subjects.
Methods:
Using a blend of size-calibrated fluorescent beads (0.5 and 0.9 mum) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra-instrument and inter-instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet-free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers.
Results:
This calibrated-bead strategy allowed full long-term control of the FCM-based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 microL(-1) (1014-3039 microL(-1)) vs. 656 microL(-1) (407-962 microL(-1))].
Conclusions:
The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.
Insights
Standardizing platelet microparticle (PMP) counts using calibrated beads and flow cytometry (FCM) ensures reproducible results. This method allows for reliable PMP measurement in healthy individuals, aiding vascular risk assessment.
Area of Science:
- Biomedical Engineering
- Clinical Chemistry
- Hematology
Background:
- Platelet microparticles (PMPs) are valuable biomarkers for identifying vascular risk.
- Current PMP counting methods using flow cytometry (FCM) lack standardization, hindering clinical application.
Purpose of the Study:
- To standardize FCM settings for PMP quantification using calibrated fluorescent beads.
- To assess the reproducibility of PMP counts within and between instruments.
- To establish reference PMP values in a healthy population.
Main Methods:
- Utilized size-calibrated fluorescent beads (Megamix) to gate PMPs within a defined size window.
- Employed annexin V and CD41 coexpression for PMP counting in platelet-free plasma.
- Validated reproducibility across multiple instruments and over extended periods.
Main Results:
- The calibrated-bead strategy ensured long-term control and reproducible PMP counts (CV < 10%).
- Optimal FCM settings were transferable between instruments using Megamix as a template.
- Standardized FCM protocol yielded consistent PMP counts across instruments (CV < 12%) and established sex-specific reference values in healthy subjects.
Conclusions:
- A novel strategy for standardizing PMP counts via FCM was developed.
- This standardization facilitates reliable PMP measurement, paving the way for multicenter studies and improved vascular risk assessment.
