Global Inter-Laboratory Comparison Study to Standardize Measurements of Extracellular Vesicle Concentrations
Edwin van der Pol,
Assistant Professor,
Amsterdam University Medical Centers
Introduction: Concentrations of extracellular vesicles (EVs) in body
fluids are upcoming biomarker for health and disease. The concentration
of cell-type specific EVs can be measured with state-of-the-art flow
cytometers. However, flow cytometers have different detection limits and
therefore measure different EV concentrations in the same sample.
Consequently, clinical research studies reporting EV concentrations lack
reproducibility and are typically single-center studies, which
precludes future clinical implementation. To overcome these problems,
the European Union invested 1.8 million euro into the “METVES II”
consortium, which aimed for developing reference materials and methods
to calibrate flow cytometers. The developed infrastructure was tested in
a global inter-laboratory comparison study including 39 flow cytometers
from 24 laboratories.
Methods: Concentrations of
platelet-derived (CD61-APC) and erythrocyte-derived (CD235a-PE) EVs
were measured in stabilized and pre-labeled human plasma EV test
samples. The flow rates were calibrated using metrologically traceable
silica beads, fluorescence intensities were calibrated using beads with a
known number of fluorescent molecules, and light scattering intensities
were calibrated using polystyrene beads and Mie theory. EV
concentrations were compared between flow cytometers within calibrated
fluorescence and size ranges.
Results:
Preliminary results from 25 flow cytometers show that calibration leads
to reproducible EV concentrations. For the platelet EV concentration,
the coefficient of variation of measured EV concentrations decreased
from 75% without calibration to 25% after calibration.
Conclusions:
This is the first inter-laboratory comparison study demonstrating that
full flow cytometer calibration improves the comparability of EV
concentration measurements between flow cytometers, thereby paving the
road to multi-center clinical research studies on EVs.
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