The effect of heparin and EDTA on the NBT test

Heparin precipitates nitroblue tetrazolium (NBT) in the absence of protein (plasma). This NBT-heparin precipitate, when presented to neutrophils, results in NBT scores higher than those obtained with NBT in solution. It is postulated that the high NBT scores obtained with increasing heparin concentrations are due to increased NBT precipitation and hence increased phagocytosis. It is proposed that the variability of NBT scores obtained when heparin is used as the anticoagulant is related to the amount of precipitate formed and to inhibition of phagocytosis by excess heparin. EDTA, in the presence of plasma and NBT, forms a precipitate consisting of protein and little, if any, NBT. In the presence of EDTA, NBT scores are lower than those obtained with heparin. EDTA, at concentrations used to prevent coagulation, inhibits phagocytosis and this could explain the low NBT scores. These observations provide a mechanism for entry of NBT dye into neutrophils and may help explain the inconsistent results found in both normal and disease states.