Pulmonary endothelial cell damages caused by neutrophil overactivation could result in acute lung injuries including transfusion-related acute lung injury (TRALI). We previously reported that heme-related molecules derived from hemolysis induced the production of reactive oxygen species from neutrophils. Recently, neutrophil extracellular traps (NETs) have been demonstrated to associate with the onset of TRALI.

Study Design and Methods

In this study, neutrophils' morphologic changes induced by the heme-related molecule hemin were confirmed to be NETs via confocal laser scanning microscopy and electron microscopy (EM). Additionally, concentrations of hemin in red blood cell (RBC) components were measured via enzyme-linked immunosorbent assay and possible contribution of these molecules to the onset of TRALI was discussed.


SYTOX green staining observation via confocal laser scanning microscopy revealed that neutrophil morphology changed rapidly upon addition of hemin. The nuclei began to be enlarged and become segmented after 5 minutes, and NET-like structures were released from neutrophils after 15 minutes. In EM observation, NET-like structures appeared after 10 minutes and the nucleoplasm was partially separated from the nuclear membrane, which were consistent with the features of NET formation. These structures stained positively for both myeloperoxidase and histone H3 antibodies.


Thus, our results suggest that hemin induced NETs in 15 minutes, a quicker reaction than NET induction by phorbol myristate acetate requiring 3 hours. Moreover, since RBC components, especially those with long-term storage, contained sufficient hemin concentration to induce NETs, special attention to hemolysis of stored RBC components is important.