Ethylenediaminetetraacetic acid plus citrate‐theophylline‐adenosine‐dipyridamole (EDTA‐CTAD): A novel anticoagulant for the flow cytometric assessment of platelet and neutrophil activation ex vivo in whole blood

Abstract

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant recommended for full blood counts, citrate is recommended for coagulation and platelet studies, and citrate-theophylline-adenosine-dipyridamole (CTAD) inhibits platelet activation. Because the combination of EDTA and CTAD (E/C) is better than EDTA or CTAD alone for measuring platelet parameters on the ADVIA 120 Haematology System, we investigated whether it also offers advantages for the flow cytometric assessment of platelet and/or neutrophil activation and platelet-leucocyte aggregate formation ex vivo. Blood from healthy subjects was collected into E/C or citrate, kept at room temperature or at 4°C, and analysed 0 to 360 min later in the ADVIA 120 and by immunofluorescent flow cytometry. Platelet count, mean platelet volume, number of platelet clumps, mean platelet component, numbers of CD62P ؉ platelets and platelet-leucocyte aggregates, and expression of CD11b on neutrophils changed little over 360 min in blood with E/C kept at 4°C. In contrast, one or more parameter changed when blood was kept with E/C at ambient temperature or with citrate at either temperature. The use of E/C in in vitro and in vivo studies is illustrated. Platelet and neutrophil activation status ex vivo can be reliably assessed if blood is collected into E/C, held at 4°C, and analysed within 6 h. Cytometry Part B (Clin. Cytometry) 52B:30 -40, 2003.

Flow cytometric analysis of microparticle phenotype and their role in thrombin generation

“…Beads 1.1 lm (1 ll) from a manufacturers stock solution diluted 1:1,000 in filtered TS were added to each sample together with Accucount beads (25 ll). Samples were diluted to 1 ml with filtered TS and analysed immediately by flow cytometry as described previously (11)(12)(13).…”

A kinetic model of bone marrow neutrophil production that characterizes late phenotypic maturation

“…Antibody labeling of whole blood, performed within 2 h of sample collection, was a modification of a previously published method (26). Aliquots (5 l) of Hoechst-labeled blood were incubated with 1 l each of CD10-PE and CD16-PE-Cy5 in combination or isotype control monoclonal antibodies (MAbs; saturating concentrations) in 1 mM NaN 3 on ice in the dark for 20 min, diluted to 0.75-1.0 ml with HBSS-BSA-NaN 3, and stored at 4°C in the dark for up to 4 h.…”

Platelet analysis in laboratory hematology

“…One of the reasons is that EDTA requires a certain reaction time, as far as the hydration of the cells is concerned, which is why one should wait for at least 15 min before analyzing a freshly drawn blood sample. For platelet analyses, EDTA plus citrate-theophylline-adenosine-dipyridamole (EDTA-CTAD) has been proposed as a substitute [44].…”