Flow cytometry detection of serotonin content and release in resting and activated platelets.
Early detection of platelet activation is important for the diagnosis and follow-up of several pathological conditions that primarily or secondarily involve platelets in their pathogenesis. The golden standard assay to detect thrombocyte activation is represented by the release of serotonin, classically performed by demanding methodologies, such as high-performance liquid chromatography, 14C-labelling and enzyme-linked immunosorbent assay (ELISA). We developed a non-radioactive method, based on individual cells, for the detection of serotonin content in activated and resting platelets by flow cytometry. The assay was standardized on cells activated by Ca2+ ionophore or by sera from patients with heparin-induced thrombocytopenia (HIT). Cells were identified by CD41a surface staining and their serotonin content measured by specific antiserotonin intracytoplasmic staining, while their activation was independently shown by annexin V binding. Cellular degranulation was detected by flow cytometry in all the cases that were also positive by standard ELISA. Moreover, multiparametric flow cytometry analysis revealed that, although virtually all activated cells bind annexin V, serotonin was released only by the platelet subset that downmodulates surface CD41a.
Gobbi
Giuliana
G
Department of Anatomy, Pharmacology and Forensic Medicine, Human Anatomy Section, University of Parma, Ospedale Maggiore, Via Gramsci. 14, I-43100 Parma, Italy.
Mirandola
Prisco
P
Tazzari
Pier Luigi
PL
Ricci
Francesca
F
Caimi
Luigi
L
Cacchioli
Antonio
A
Papa
Stefano
S
Conte
Roberto
R
Vitale
Marco
M
D001792:Blood Platelets / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015539:Platelet Activation; D012701:Serotonin / Q000378:metabolism*
D012701:Serotonin