Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection

ObjectivesTo assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA).Design and methodsIsolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMA-induced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca2+]i) was measured using Fluo-3 AM, a calcium-sensitive dye.ResultsEDTA provided the highest number of isolated neutrophils/mL of blood (1.7 × 106 ± 1.5 × 105) when compared with citrate (0.46 × 106 ± 0.95 × 105) and heparin (0.66 × 106 ± 0.15 × 105). EDTA originated less degree of PMA-induced activation (370 ± 30%) relatively to citrate (830 ± 98%) and heparin (827 ± 77%). [Ca2+]i was lower with EDTA (122 ± 11 nM) when compared with citrate and heparin (150 ± 13 and 230 ± 30 nM).ConclusionThe anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.