Related Experiment Video
Updated: Apr 12, 2026
Analyzing Platelet Subpopulations by Multi-color Flow Cytometry
Published on: June 10, 2025
Standardized counting of circulating platelet microparticles using currently available flow cytometers and
P Poncelet1, S Robert2, T Bouriche1
1R&T Department, BioCytex, Marseille, France.
Abstract:
Clinical determination of MP counts using flow cytometry has not been fully accepted yet due to the lack of standardization protocols. In the past 5 years, we have proposed two versions of a method with reproducible PMP counts in plasma samples. Both methods use forward scatter (FSC)-based threshold set with reference beads of appropriate sizes; first using 0.5 µm beads and later with 0.3 µm beads. Both systems provide reproducible PMP counts. However, this technique works only with some of currently used commercial flow cytometers. Instruments with limited resolution or generating heterogeneous FSC signals are excluded. Such performances are incompatible with the required interinstrument standardization. Here we show that (i) flow cytometers with sub-optimal FSC capabilities generally have higher SSC resolution and background rejection capacity, and (ii) that the same biological entities, "dim and bright PMP," both can be counted using alternative strategies, either as previously described, based on FSC measurements, or as presented here, based on SSC detection. The critical element in the standardization protocol is the use of different sizes of reference beads. This study was designed to permit simultaneous access to both FSC- and SSC-optimized platforms. A new range of about 0.17-0.6 µm eq. (µm-equivalents) is proposed for an alternative SSC-based MP gate generating the same PMP counts as those obtained in the previously proposed 0.3-1 µm eq. FSC-based MP gate. The two equivalent standardization options reconcile intrinsically different scattering behaviors between SSC- and FCS--triggered instruments and open the opportunity for multicenter studies in the future.
Insights
Standardizing microparticle (MP) counts in plasma requires robust flow cytometry protocols. This study introduces a new side scatter (SSC)-based method using specific bead sizes, offering an alternative to forward scatter (FSC) for reproducible MP quantification across instruments.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Clinical Diagnostics
Background:
- Standardization of microparticle (MP) quantification via flow cytometry is hindered by a lack of universal protocols.
- Previous methods relied on forward scatter (FSC)-based thresholds with 0.5 µm or 0.3 µm beads, limiting instrument compatibility.
- Sub-optimal FSC performance in some flow cytometers necessitates alternative detection strategies.
Purpose of the Study:
- To develop a standardized flow cytometry protocol for reproducible microparticle (MP) quantification in plasma.
- To establish an alternative side scatter (SSC)-based detection method compatible with a wider range of flow cytometers.
- To reconcile FSC- and SSC-based gating strategies for enhanced inter-instrument standardization.
Main Methods:
- Utilized reference beads of varying sizes (0.17–0.6 µm equivalent) for side scatter (SSC) threshold setting.
- Compared SSC-based MP gating with established forward scatter (FSC)-based methods.
- Evaluated MP counts on flow cytometers with varying FSC capabilities and SSC resolution.
Main Results:
- Flow cytometers with lower FSC resolution often exhibit superior SSC resolution and background rejection.
- An SSC-based gating strategy using 0.17–0.6 µm equivalent beads yields comparable MP counts to the FSC-based 0.3–1 µm equivalent method.
- The proposed SSC method provides reproducible PMP counts, independent of FSC signal quality.
Conclusions:
- A novel SSC-based standardization protocol enables accurate MP quantification across diverse flow cytometer platforms.
- This dual approach (FSC and SSC) addresses instrument variability and facilitates multicenter studies.
- The use of precisely sized reference beads is critical for achieving standardization in MP counting.
