Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results.

Kristýna Pekárková,Zbyněk Straňák,Marie Kostelanská,Jakub Soukup,Karel Holada,Jan Širc

doi:10.3390/diagnostics11081320

Kristýna Pekárková, Zbyněk Straňák + Show 4 more

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https://doi.org/10.3390/diagnostics11081320

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Abstract

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

Highlights

There is an ongoing effort toward the standardization of flow cytometry analysis of extracellular vesicles (EVs), resulting in the publication of a joint position paper from the ISEV, ISAC and ISTH [1]
We analyzed frozen aliquots of collected cord blood plasma with the improved cytometer. This method meant that the cytometer had a better sensitivity and resolution, we again, did not see any significant differences in the numbers of detected large EV (lEV) between the studied groups
Using violet nm laser side scatter (VSSC), of the double positive platelet lEVs represented about 1.5% of all events in the lEVs gate, while with FITC + VSSC, the percentage increased to 8.5%

Summary

Introduction

There is an ongoing effort toward the standardization of flow cytometry analysis of extracellular vesicles (EVs), resulting in the publication of a joint position paper from the ISEV, ISAC and ISTH [1]. The MIFlow-Cyt EV establishes rules for EV measurement by flow cytometry, which allow for the reproduction of measured data in the future or by other groups using different cytometers. Efforts are being made to standardize the pre-analytical variables of EV flow cytometry measurement [2,3,4]. Significant variability remained [5,6] This significant variability is caused by the varying sensitivities of cytometers. The conversion of arbitrary units to standard units helps to reduce this variability [7]. These combined efforts have resulted in the design of useful software tools, such as FCMPASS [8], which can assist with the reproducibility of EV measurements

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Results

Conclusion