Comparison of neutrophil respiratory oxidative burst activity between flow cytometry using dihydrorhodamine (DHR) 123 and conventional nitroblue tetrazolium test (NBT)

Background: Neutrophil plays a defense role against bacteria and fungi specifically by the neutrophil phagoburst activity. The nitroblue tetrazolium (NBT) test is the gold standard method to measure this but the flow cytometry assays also have been developed. The aim of the study was to evaluate the performance of flow cytometry using Dihydrorhodamine (DHR) 123 and to compare with the conventional NBT test. Methods: This study was carried out to determine the phagoburst activity at Universiti Kebangsaan Malaysia Medical Centre on 26 normal healthy donors and 5 chronic myeloid leukemia (CML) patients using both methods. Results: In flow cytometry, higher fluorescence intensity was observed in Phorbol myristate acetate (PMA) stimulated neutrophils. Comparing the mean fluorescent intensity (MFI) between CML and healthy donor showed CML patients have significant lower MFI (312.15 vs 738.22; p 0.05) indicating poorer respiratory burst in CML patients. The PMA stimulated positive cells were higher in flow cytometry than the NBT (98.06% vs 87.5%). Although there was poor correlation between two methods, a good agreement between flow cytometry assay and NBT test results was observed in terms of positive and negative results in simulated and unstimulated cells. Moreover, in flow cytometry, 95% of stimulated cells suggest the higher sensitivity. All samples with negative NBT showed negative DHR, reflecting high degree of agreement between these two methods and eliminating possible false negative result in flow cytometry. Conclusion: This study showed that flow cytometry assay has superior technical advantages compared to NBT test as it is easy to perform, quick, more sensitive and requires only a small amount of blood sample.

Automated summary

This study compared the performance of flow cytometry using Dihydrorhodamine (DHR) 123 with the conventional nitroblue tetrazolium (NBT) test in evaluating neutrophil phagoburst activity. The results showed that flow cytometry had higher fluorescence intensity and better sensitivity compared to the NBT test. There was good agreement between the two methods in terms of positive and negative results, with flow cytometry showing a higher proportion of stimulated cells. Overall, flow cytometry was found to be an easy, quick, and sensitive method that required a small blood sample.