Analysis of neutrophil distribution in cultured whole blood clots, an ex vivo thrombus model

Abstract

Neutrophils, a type of white blood cell, play a crucial role in immune function and wound repair. In humans, there is evidence that neutrophils aid blood coagulation through interactions with the injured endothelium and fibrin. Myeloperoxidase (MPO) can be used as a marker for neutrophil infiltration into tissues because it is one of the most abundant proteins in neutrophil cells. In the present study, three cultured human blood clot samples were immunostained for MPO expression using goat anti-MPO primary antibody and donkey anti-goat green fluorescent secondary antibody. Nuclei were stained with a blue fluorescent DNA-binding dye called Hoescht 33342. MPO staining patterns were compared across samples, with a focus at the top of the blood clot. Analysis using fluorescence microscopy revealed co-localization of cell nuclei and MPO proteins in all three samples as well as proximity of multiple stained MPO proteins to cell nuclei. Based on the results of this study, it can be concluded that neutrophils, as indicated by MPO staining, are present and actively involved in the clotting process in human blood clots. These findings support the existing evidence that neutrophils survive and have a functional role in blood coagulation and provide specific insights into their localization within the clot structure. In the future, double immunostaining could be conducted with multiple neutrophil markers to differentiate between types of neutrophils to better understand the role of neutrophils encapsulated in a thrombus.