Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume

Siaw Li Chan; Xin Yi; Emily Wysocki; Rachael Bridgman; Jocelyn Gutierrez; Krzysztof Mikrut; Edward Ki Yun Leung; Kiang Teck J. Yeo; Jonathan L. Miller

doi:10.1093/ajcp/aqz094

Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume

Siaw Li Chan, Xin Yi, Emily Wysocki, Rachael Bridgman, Jocelyn Gutierrez, Krzysztof Mikrut, Edward Ki Yun Leung, Kiang Teck J. Yeo, Jonathan L. Miller

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

Objectives: Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods: Citrated whole blood (200 μL) was incubated with deuterated serotonin (d₄5-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d₄5-HT by micro-LC-MS/MS. Results: This study demonstrated a broad linear range of 50 to 2,000 pg/mL d₄5-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Conclusions: Quantification of d₄5-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

Original language	English (US)
Pages (from-to)	718-724
Number of pages	7
Journal	American Journal of Clinical Pathology
Volume	152
Issue number	6
DOIs	https://doi.org/10.1093/ajcp/aqz094
State	Published - Nov 4 2019

Keywords

Dense granule secretion
LC-MS/MS
Nonradioactive serotonin release assay
Platelet function

ASJC Scopus subject areas

Pathology and Forensic Medicine

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10.1093/ajcp/aqz094

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Chan, S. L., Yi, X., Wysocki, E., Bridgman, R., Gutierrez, J., Mikrut, K., Leung, E. K. Y., Yeo, K. T. J., & Miller, J. L. (2019). Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume. American Journal of Clinical Pathology, 152(6), 718-724. https://doi.org/10.1093/ajcp/aqz094

Chan, SL, Yi, X, Wysocki, E, Bridgman, R, Gutierrez, J, Mikrut, K, Leung, EKY, Yeo, KTJ & Miller, JL 2019, 'Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume', American Journal of Clinical Pathology, vol. 152, no. 6, pp. 718-724. https://doi.org/10.1093/ajcp/aqz094

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title = "Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume",

abstract = "Objectives: Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods: Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC-MS/MS. Results: This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Conclusions: Quantification of d45-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.",

keywords = "Dense granule secretion, LC-MS/MS, Nonradioactive serotonin release assay, Platelet function",

author = "Chan, {Siaw Li} and Xin Yi and Emily Wysocki and Rachael Bridgman and Jocelyn Gutierrez and Krzysztof Mikrut and Leung, {Edward Ki Yun} and Yeo, {Kiang Teck J.} and Miller, {Jonathan L.}",

note = "Funding Information: Acknowledgment: We thank SCIEX for providing equipment support for this project. This work was supported by the Department of Pathology, University of Chicago. Publisher Copyright: {\textcopyright} 2019 American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.",

year = "2019",

month = nov,

day = "4",

doi = "10.1093/ajcp/aqz094",

language = "English (US)",

volume = "152",

pages = "718--724",

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AU - Chan, Siaw Li

AU - Yi, Xin

AU - Wysocki, Emily

AU - Bridgman, Rachael

AU - Gutierrez, Jocelyn

AU - Mikrut, Krzysztof

AU - Leung, Edward Ki Yun

AU - Yeo, Kiang Teck J.

AU - Miller, Jonathan L.

N1 - Funding Information: Acknowledgment: We thank SCIEX for providing equipment support for this project. This work was supported by the Department of Pathology, University of Chicago. Publisher Copyright: © 2019 American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.

PY - 2019/11/4

Y1 - 2019/11/4

N2 - Objectives: Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods: Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC-MS/MS. Results: This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Conclusions: Quantification of d45-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

AB - Objectives: Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods: Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC-MS/MS. Results: This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Conclusions: Quantification of d45-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

KW - Dense granule secretion

KW - LC-MS/MS

KW - Nonradioactive serotonin release assay

KW - Platelet function

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Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume

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