PB0745 - NETosis in Sickle Cell Anemia

Background: Immune regulatory functions of neutrophils include phagocytosis, generation of reactive oxygen species, degranulation, formation of neutrophil extracellular traps (NETs). Most common cause of morbidity and mortality in sickle cell anemia (SCA) is vascular occlusion. Neuthrophil activation plays an important role in vaso-occlusive events.Detection of NETs formation in these patients may play an important role in determining morbidity and mortality.
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Aims: Study aimed to demonstrate the increased inflammation in SCA by demonstrating the activity of NETosis and to regulate the prophylaxis necessary for life-threatening thrombosis, which may occur at the end of this process.

Methods: Blood samples of patients and healthy persons were obtained. Twenty five patients with SCA and 25 healthy individuals were included in the study. Two mL venous blood samples of patients and healthy individuals, EDTA tube for neutrophil elastase enzyme level received. Blood samples taken into EDTA tube for neutrophil isolation were centrifuged at 4000 rpm for 10 minutes and stored at +4ºC. Neutrophils obtained using MACSxpress® Neutrophil Isolation Kit Human were used for NETs formation. In the NETosis ASSAY Kit stage, cells were suspended with NET assay buffer and incubated, then centrifuged to collect neutrophil elastase enzyme activity.Supernatants were incubated with the addition of NET Assay Neutrophil Elastase Substrate and the film on the plate was opened at 405 nm. Concentration absorbance graph was drawn by utilizing standards. Blood samples from patient and control groups were prepared for appropriate fluorescence and scanning electron microscope images and prepared with appropriate protocols.

Results: Although no statistically significant difference was found, we showed that NETosis formation increases SCA compared to healthy subjects (p=0,329). We also showed that NETosis formation increses with increasing number of painfull crisis in these patients.

Conclusion(s): In this study, it has been shown that NETosis formation increases in patients with SCA compared to healthy individuals.