Standardization of the Nitroblue Tetrazolium Test: Influence of pH, Dye Concentration and Sample Storage

Abstract

The influence of pH, dye concentration and sample storage on the histochemical nitroblue tetrazolium (NBT) test in 45 patients with acute bacterial infections and 51 healthy individuals has been examined. Even minor deviations in pH and NBT dye concentration markedly influenced the reduction of NBT to formazan by patient and control neutrophils, and the differentiation between results in these groups was optimal at pH 7.2 and a concentration of 0.05% NBT in the final blood-NBT mixture. Storage of heparinized blood samples for more than 2 hours at 21°C and for more than 6 hours at 4°C is not recommended due to overlap between results in patients and controls. The error involved in counting of NBT-positive neutrophils accounted for the major discrepancies in triplicate tests in patients and controls and in tests performed on different days. The reproducibility of the endotoxin stimulated test was inferior to that of the unstimulated test.