Objective:

Dihydrorhodamine (DHR) 123 measurement by flow cytometry is widely used to detect neutrophil phagocytosis and oxidative burst activity. Our study aimed to evaluate the performance characteristics of DHR 123 assay results of neutrophils isolated by two different techniques and stimulated with two bacterial strains according to the validation principles.

Material and Methods:

The oxidative burst index of neutrophils was measured by flow cytometry using healthy human venous blood samples. Granulocytes were separated by two different density separation methods, Ficoll and dextran sedimentation, and stimulated with two bacterial strains (ATCC 25923 Staphylococcus aureus subsp. aureus Rosenbach and ATCC 25913 methicillin-resistant S. aureus). Flow cytometric measurements were performed at five different time points (0, 10, 20, 30, 60 min). Statistical analysis was performed using GraphPad Prism version 8 software (GraphPad Software, Boston, USA) to assess linearity, precision, and sensitivity and to compare methods, bacterial strains, and incubation times.

Results:

Our study showed that isolation by the dextran method was more suitable due to low limits of detection and quantification. Both ATCC strains were suitable for use, but ATCC 25923 may be preferred because the dextran isolation method with strain ATCC 25923 had the lowest limit of detection and quantification. Our data also showed that measurement at 0 and 30 min was appropriate.

Conclusion:

Our study contributes to the standardization of functional methods for neutrophil oxidative burst analysis.

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