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Thromb Haemost 2018; 118(09): 1612-1624
DOI: 10.1055/s-0038-1668544
New Technologies, Diagnostic Tools and Drugs
Georg Thieme Verlag KG Stuttgart · New York

Analytical Considerations in Nanoscale Flow Cytometry of Extracellular Vesicles to Achieve Data Linearity

Janice Gomes   *
1   Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Ontario, Canada
2   Kidney Clinical Research Unit, London Health Sciences Centre, London, Ontario, Canada
,
Fabrice Lucien   *
1   Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Ontario, Canada
3   Department of Urology, Mayo Clinic, Rochester, Minnesota, United States
4   Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario, Canada
,
Tyler T. Cooper
5   Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
,
Yohan Kim
1   Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Ontario, Canada
3   Department of Urology, Mayo Clinic, Rochester, Minnesota, United States
4   Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario, Canada
,
Karla C. Williams
3   Department of Urology, Mayo Clinic, Rochester, Minnesota, United States
4   Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario, Canada
,
XinYang Liao
4   Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario, Canada
,
Lauren Kaufman
6   Department of Chemistry, University of Western Ontario, London, Ontario, Canada
,
Francois Lagugné-Labarthet
6   Department of Chemistry, University of Western Ontario, London, Ontario, Canada
,
Oliver Kenyon
7   Apogee Flow Systems Inc., Hertfordshire, United Kingdom
,
Justin Boysen
8   Division of Hematology, Mayo Clinic, Rochester, Minnesota, United States
,
Neil E. Kay
8   Division of Hematology, Mayo Clinic, Rochester, Minnesota, United States
,
Christopher W. McIntyre
1   Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Ontario, Canada
2   Kidney Clinical Research Unit, London Health Sciences Centre, London, Ontario, Canada
,
Hon S. Leong
1   Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Ontario, Canada
3   Department of Urology, Mayo Clinic, Rochester, Minnesota, United States
4   Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario, Canada
› Author Affiliations
Funding H.S.L. is supported by a Movember/Prostate Cancer Canada Rising Star Award (#RS2012–008 and #RS2016–56) and received operating grant support by Prostate Cancer Fight Foundation and Ontario Institute for Cancer Research (SPS 0613–03). Y.K. is supported by an Ontario Graduate Scholarship Award. K.C.W. is funded by a CIHR Postdoctoral Fellowship (140880). L.K. and F.L.L. are supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada.
Further Information

Publication History

25 April 2018

11 July 2018

Publication Date:
15 August 2018 (online)

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Abstract

Background Platelet microparticles (PMPs) and their abundance in the blood are a prognostic biomarker in thrombotic disorders and cancer. Nanoscale flow cytometry (nFC) is ideal for high-throughput analysis of PMPs but these clinical assays have not been developed previously.

Objective This article demonstrates that nFC is a suitable technology to enumerate PMPs present in plasma samples in a clinical setting.

Materials and Methods nFC was performed using the Apogee A50-Micro instrument. Instrument settings and acquisition parameters were developed with the use of fluorescent beads and plasma samples. Sample preparation and handling was also optimized.

Results nFC allows for linear detection of particles between approximately 200 and 1,000 nm based on calibration beads and was dependent on dilution factor and flow rate. Linearity in event analysis as samples became more diluted was lost when events approximately 100 nm were gated while linearity was maintained despite dilution of sample in events larger than 200 nm in diameter. Higher flow rates lead to an under-estimation of events analysed per microlitre of analyte and this was more pronounced when plasma samples were not diluted more than 1/20×.

Conclusion nFC offers multi-parametric analysis of PMPs when optimal calibration of acquisition and sample processing settings is performed. Analysis of plasmas from metastatic prostate cancer patients and leukaemia patients revealed that PMP levels were larger than 100 nm and were equally abundant in patients that responded to or failed androgen deprivation therapy or between patients representing different stages of leukaemia.

Keywords

cancer - microparticles - flow cytometry

Authors' Contributions

J.G. and F.L. performed all experiments, designed all experiments and wrote the manuscript. Y.K., K.C.W., T.L., L.K. and F.L.L. performed experiments. C.M. and H.S.L. wrote the manuscript. N.E.K. and J.B. provided plasma samples and reviewed and edited the manuscript.


* Janice Gomes and Fabrice Lucien equally contributed to the study.


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