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

• Published in: American journal of clinical pathology Vol. 152; no. 6; pp. 718 - 724
• Main Authors: Chan, Siaw Li, Yi, Xin, Wysocki, Emily, Bridgman, Rachael, Gutierrez, Jocelyn, Mikrut, Krzysztof, Leung, Edward Ki-Yun, Yeo, Kiang-Teck J, Miller, Jonathan L
• Format: Journal Article
• Language: English
• Published: US Oxford University Press 04.11.2019
• Subjects: Analysis and chemistry; Biochemical assays; Blood; Blood platelets; Chemical properties; Chromatography; Chromatography, Liquid - methods; Humans; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Measurement; Methods; Platelet Function Tests - methods; Platelets; Quality control; Quantitation; Scientific imaging; Secretion; Serotonin; Serotonin - analysis; Serotonin - metabolism; Tandem Mass Spectrometry - methods; United States; Dense granule secretion; LC-MS/MS; Nonradioactive serotonin release assay; Platelet function
• ISSN: 0002-9173; 1943-7722
• DOI: 10.1093/ajcp/aqz094

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.