Elsevier

Translational Research

Volume 166, Issue 6, December 2015, Pages 733-739
Translational Research

Original Article
High correlation between 2 flow cytometry platforms in the microparticles analysis using a new calibrated beads strategy

https://doi.org/10.1016/j.trsl.2015.08.006Get rights and content

Microparticles (MPs) are potential noninvasive biomarkers for diagnosis or prognosis in pathologic conditions. However, the lack of standardization of the preanalytical and analytical methods leads to a wide variability in MPs results. The recently developed Megamix-Plus beads, a new bead-based standardization tool optimized to specific types of flow cytometers, could help circumvent this problem. The aim of the present study was to determine whether the number of total MPs and platelet-derived MPs (PMPs) is similar using 2 different cytometer platforms calibrated with the Megamix-Plus beads. Blood samples from 65 patients with deep venous thrombosis were collected and processed to obtain platelet poor plasma (PPP). The number of total MPs and PMPs in each PPP sample was measured using 2 flow cytometers. Megamix-Plus side scatter channel beads were used to calibrate the LSRFortessa flow cytometer from Becton Dickinson, whereas Megamix-Plus forward scatter channel beads were applied to the Navios flow cytometer from Beckman Coulter. High correlation of total MPs and PMPs values between the flow cytometers was found (r = 0.908, P < 0.01 and r = 0.910, P < 0.001, respectively). However, the absolute numbers of total MPs and PMPs were significantly higher measured with the Navios flow cytometer compared with the LSRFortessa cytometer. Therefore, both platforms are valid for MPs determination in general, although a similar platform with the same calibration tool could be a better choice for multicenter studies.

Introduction

At a Glance Commentary

Sánchez-López V, et al.

Microparticles (MPs) have emerged as promising noninvasive biomarkers for various diseases. Flow cytometry is the most common method to detect MPs, but it is a technical challenge because of the small size of MPs leading to a wide variability in MPs results. A new bead-based standardization tool optimized to specific types of flow cytometers could help circumvent this problem.

We observed high correlations in total MPs and platelet-derived MPs between 2 different cytometers in spite of optimal comparison of absolute MPs numbers remains to be achieved. These results are relevant for further multicenter studies.

Microparticles (MPs) are microvesicles from 0.1 to 1 μm in diameter, which are released from different types of cells during cell activation or apoptotic processes.1, 2 They present cell-specific antigens and cytoplasmic markers and generally express phosphatidylserine on their surface.3, 4 MPs were initially considered to be “cellular dust” without any biological function. They are now, however, of clinical relevance because of their role as vectors and biomarkers for diagnosis or prognosis in vascular damage, blood coagulation, inflammation, angiogenesis, and other pathologic situations.4, 5, 6, 7, 8, 9 Nonetheless, a lack of standardization of the preanalytical and analytical methods has led to a wide variability in the number of MPs detected.10, 11, 12, 13, 14 Flow cytometry is the most common method to detect MPs, which allows the determination of both the number and the cellular origin of MPs based on the antigens presented on their surface. Reliable measurement of MPs by flow cytometry is a technical challenge because of the small size of MPs, which is close to the limit of sensitivity for flow cytometers.15 New sets of fluorescent beads have recently been developed. Megamix-Plus forward scatter channel (FSC) beads (Biocytex, Marseille, France) are designed to obtain the best results using flow cytometers with optimized FSC, such as Beckman Coulter (BC) flow cytometers, whereas Megamix-Plus side scatter channel (SSC) beads (Biocytex, Marseille, France) are more adequate for flow cytometers with optimized SSC, such as Becton Dickinson (BD) flow cytometers. To compare the results between 2 different cytometry platforms (BD and BC flow cytometers), we measured the number of total MPs and platelet-derived MPs (PMPs) in patients with deep venous thrombosis using Megamix-Plus calibration beads.

Section snippets

Blood collection and sample processing

Sixty-five patients diagnosed with deep venous thrombosis were included in the study. The research was carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Written consent was obtained from all subjects according to a protocol approved by the Ethical Committee of the Virgen del Rocio Hospital. Venous blood was taken with a 21-gauge needle and with minimal compression and collected (discarding the first 3 mL) in 0.109-M sodium citrate Vacutainer

Calibration of flow cytometers for MPs detection

The flow cytometers were first calibrated with their corresponding beads before MPs detection. The Megamix-Plus FSC was used to calibrate the BC flow cytometer following the protocol described by the manufacturer: (1) On a fluorescence 1 channel × side scatter (SS) plot, select discriminator on fluorescence 1 channel and distinguish each subpopulation of beads (Fig 1, A); (2) Check forward scatter (FS) resolution: FS resolution is sufficient if the subpopulations 0.3 and 0.5 μm on FS do not

Discussion

We obtained high correlation of total MPs and PMPs values between the flow cytometers, but the absolute numbers of total MPs and PMPs were significantly higher measured with the Navios flow cytometer compared with the LSRFortessa cytometer. Fluorescent beads with different sizes have been widely used to calibrate flow cytometers for MPs identification, although it is still under debate whether polystyrene beads are the best for size calibration because of the potential difference in refractive

Acknowledgments

Conflicts of Interest: Authors do not have any conflicts of interest to declare. All authors have read the Translational Research's policy on disclosure of potential conflicts of interest. All authors have read the journal's authorship agreement, and the article has been reviewed by and approved by all named authors.

This work was supported by research grants from the FEDER funding and Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Instituto de Salud Carlos III,

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