Influence of specimen age and anticoagulant on flow cytometric evaluation of granulocyte oxidative burst generation.

The oxidative burst generation capacity of granulocytes can be reliably detected by a flow cytometric procedure using lysed whole blood, dihydrorhodamine 123 (DHR), and phorbol myristate acetate (PMA). This assay is used to detect chronic granulomatous disease (CGD) and the CGD carrier state. To assess the feasibility of performing this assay in a reference laboratory setting, we investigated the influence of anticoagulant and specimen age on flow cytometric detection of granulocyte oxidative burst generation. Peripheral blood from 20 healthy controls was collected in acid citrate dextrose (ACD), ethylenediaminetetraacetate (EDTA), or sodium heparin (HEP) and held at room temperature. At 4, 24, 48, 72, and 96 h after collection, red cells were lysed, the white cells loaded with DHR, and then activated by PMA. Granulocyte-associated fluorescence, indicative of oxidative burst generation, was assessed by flow cytometry. Blood in any of the three anticoagulants tested gave reliable results (> 90% of granulocytes positive for fluorescence) at 4 h after collection; at 24 h after collection, HEP and ACD specimens performed slightly better than EDTA specimens. At later time points, HEP proved superior to ACD and EDTA for maintaining granulocyte oxidative burst capacity. As a demonstration of the practical utility of the assay, both CGD and the CGD carrier state were accurately detected using heparinized blood specimens analyzed 72 h after collection. These results show that heparinized blood specimens up to 72 h old can be used to reliably assess granulocyte oxidative burst generation.