title: 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 degrees 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 degrees 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 degrees C, and analysed within 6 h.
authors.affiliation: Department of Haematology, The Royal London Hospital, Whitechapel, London, United Kingdom. marion.macey@royalhos-tr.nthames.nhs.uk
authors.forename: Marion
authors.initials: M
authors.lastname: Macey
authors.forename: Desmond
authors.initials: D
authors.lastname: McCarthy
authors.forename: Urooj
authors.initials: U
authors.lastname: Azam
authors.forename: Timothy
authors.initials: T
authors.lastname: Milne
authors.forename: Peter
authors.initials: P
authors.lastname: Golledge
authors.forename: Adrian
authors.initials: A
authors.lastname: Newland
mesh_terms: D000241:Adenosine; D000328:Adult; D000925:Anticoagulants; D001792:Blood Platelets / Q000737:chemistry / Q000166:cytology; D039481:CD11b Antigen / Q000032:analysis; D002951:Citrates; D004176:Dipyridamole; D004492:Edetic Acid; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D019041:L-Selectin / Q000032:analysis; D017493:Leukocyte Common Antigens / Q000032:analysis; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis; D008875:Middle Aged; D018375:Neutrophil Activation; D009504:Neutrophils / Q000737:chemistry / Q000166:cytology; D019007:P-Selectin / Q000032:analysis; D010316:Particle Size; D015539:Platelet Activation; D013806:Theophylline
chemical_list: D000925:Anticoagulants; D039481:CD11b Antigen; D002951:Citrates; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D019041:L-Selectin; D004176:Dipyridamole; D004492:Edetic Acid; D013806:Theophylline; D017493:Leukocyte Common Antigens; D000241:Adenosine