Granulocytes form the first and most prominent line of cellular defense against invading microorganisms. Active recruitment of granulocytes to the sites of infection is fundamentally important for the innate immune system. Phagocytes bind to and ingest microorganisms by a process known as phagocytosis, which typically triggers the production of reactive oxygen species (ROS) and the fusion of cytoplasmic granules with pathogen-containing vacuoles. The process of ROS production is depended on multicomponent enzyme – nicotinamide adenine dinucleotide phosphate (NADP)-oxidase. As a result of further reactions with ROS, a number of toxic compounds that ensure destruction of pathogens in the formed phagosome are produced.

Chronic granulomatous disease (CGD) is a group of five genetic disorders of the phagocyte NADP-oxidase complex generating ROS in response to physiological stimuli such as the phagocytosis of microbes. CGD leads to recurrent life-threatening opportunistic infections and uncontrolled inflammation, often accompanied by granuloma formation. A provisional diagnosis of CGD is made by a DHR assay using flow cytometry or by nitroblue tetrazolium (NBT) using light microscopy. DHR (dihydrorhodamine-1, 2, 3) freely enters the phagocytes and is oxidised intracellularly to rhodamine-1, 2, 3 by diffusible H2O2 after phagocyte stimulation.

In our study we have compared measurement of neutrophils (NADPH)-oxidase complex activity by two methods – NBT-test and DHR assay in in a group of healthy children, children with CGD (three genetically confirmed patients) and the group of children with invasive bacterial infections.

The data shows that both methods – NBT-test and DHR-123 assay – reveal the reduced production of ROS in patients with; however, the test DHR-123 assay is more sensitive and reliable in the diagnosis of this pathology. DHR-123 assay make it possible to clearly distinguish patients with CGD, while NBT-test demonstrate lower production of ROS not only in patients with CGD, but in children with invasive bacterial infections as well. Such factors as antibiotics and immunosuppressants usage, localized infections, tuberculosis, autoimmune diseases that can lead to decreased NBT-results, that make this method unsuitable for differential diagnosis of CGD.

Despite the DHR-123 assay and NBT-test have similar principle – activated granulocytes that produces ROS, visualized with the dyes – that reflect the activity of the NADP oxidase and functional status of granulocytes – the DHR-123 assay shows several more advantages over NBT-test. The DHR-123 assay is faster – it takes 40–50 min to get results, while NBT-test takes at least 60–80 min. Also, the test with DHR-123 is a flow cytometry method that allow to analyze a greater number of cells – at least 10 000 cells, compared to NBT-test, where the analysis takes only 200 cells. One more important aspect in comparison of these two methods is evaluation of results. NBT-test is a light microscopy method, and it requires professional experience and strong skills to visually identify and count the activated cells. Often, due to subjective evaluation, we can get false-negative results. While the results of DHR-123 assay display as histograms or dotplots, allowing objectively evaluate the results.

Thus, the DHR-123 assay have multiple advantages over NBT-test in the laboratory diagnosis of chronic granulomatous disease. DHR-123 assay is a high quality and reliable method and it should be implement in screening laboratory practice for diagnosis of chronic granulomatous disease.

Ключові слова: chronic granulomatous disease, NBT-test, DHR-123 aasay

Повний текст: PDF (Ukr)

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