Introduction.

Chronic granulomatous disease is a defect in phagocytosis due to deficiency of gp91 phox , p22 phox , p47phox, p40 phox , and p67 phox (classic form of the disease). Recently, EROS and p40 phox deficiency were described as responsible for the non-classical form of the disease. The 1,2,3-dihydrorhodamine oxidation technique, with phorbol-12-myristate 13-acetate as a stimulus, is performed to diagnose the classic chronic granulomatous disease. However, oxidation mediated by EROS and p40phox requires stimuli such as zymosan, Escherichia coli, or Staphylococcus aureus.

Objective.

To optimize the 1,2,3-dihydrorhodamine technique using zymosan to assess neutrophil respiratory burst and detect the non-classical chronic granulomatous disease.

Materials and methods.

Blood was obtained from five healthy subjects after the signature of the informed consent. The 1,2,3-dihydrorhodamine technique was performed with phorbol-12-myristate-13-acetate as control and different quantities of opsonized zymosan (150, 100, 50, 20, and 10 µg). We obtained through flow cytometry the mean fluorescence intensity of rhodamine 1,2,3 oxidated in the neutrophil population and calculated the oxidation index. The Kolmogorov-Smirnov test, ANOVA, and Tukey’s post-hoc analysis were used. We considered a p value < 0.05 as statistically significant.

Results.

The phorbol-12-myristate-13-acetate increased the rhodamine 1,2,3 mean fluorescence intensity in healthy subjects. Among the different zymosan conditions tested, we selected 50 µg as the optimal and reproducible amount in all controls according to the statistical analysis and cytometric findings.

Conclusions.

We present the optimization of the 1,2,3-dihydrorhodamine technique using zymosan. We propose its implementation in clinical diagnostic laboratories to expand the diagnosis of chronic granulomatous disease.

        · abstract in Spanish     · text in Spanish     · Spanish ( pdf )