IMEX   05356
INSTITUTO DE MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Flow Cytometry Mepacrine Uptake/ Release Combined with CD63 Assay in Diagnosis of Patients with Platelet Disorders
Autor/es:
ASENSIO M; AGAZZONI M; ALBERTO MF; ROMERO ML; BERMEJO E; SANCHEZ LUCEROS, ANALIA
Lugar:
Melbourne
Reunión:
Congreso; XXVII Congress of the International Society on Thrombosis and Haemostasis (ISTH); 2019
Institución organizadora:
International Society on Thrombosis and Haemostasis
Resumen:
Background : Aggregometry with ATP release determination by bioluminescentassay (lumi- aggregometry), is an important tool to study platelet functional defects despite having some limitations (sample volume, thrombocytopenia, etc.). Flow cytometry (FC) can solve some of them as it has been demonstrated in diagnosis of storage pool disease (SPD) with FC mepacrine uptake/release assay (Mep u/r) and CD63 expression (CD63e).Aims : To evaluate some technical aspects of FC Mep u/r and CD63e and the contribution along with lumi- aggregometry in functional characterization of different thrombocitytopathies.Methods : We evaluate a normal group (NG, n: 37) and six patients (P1- 6); P1 a syndromic SPD, P2 an acquired SPD; P3 presenting a Cal- DAG- GEFI mutation (RASGRP, 2p. GLy305Asp); P4- P5 two brothers with Leucocyte Adhesion Deficiency and P6 having a defect on GPR´s activation pathway on lumi- aggregometry assay.Lumi- aggregometry was performed using standard agonists (ADP, ADR, arachidonic acid, collagen, and ristocetin). FC Mep u/r and CD63e was carry out as Cai et al. 2016, with a few modifications. Trap6 as platelet activator was used in several concentrations (20- 60 uM). Platelet count dependency was tested with platelet rich plasma from NG (n:6) having 50- 100- 300. 10 9 platelets/L.Results : Patient ' s results are shown on table 1. P1 and P2 had similar results in ATP release and Mep u/r but differences between CD63e suggest absence of dense granules and ?empty sack? syndrome respectively. Ca 2+ downstream integrin IIbIIIa activation does not affect Mep u/r and CD63e showing conserved ATP release mechanism on FC where elements are evaluated independently (P3- 5). P6 had moderate decrease response in both ATP release and Mep u/r. FC techniques are shown to be independent of the platelet count(Table 2).Conclusions : The combination of lumy- aggregometry and FC techniques allows a deeper knowledge of various platelets disorders. FC assays shows to be independent of platelet count so it could be used on thrombocytopenia.