Comparative Analysis of Cardiac Puncture and Perfusate Blood Collection for Murine Extracellular Vesicle Isolation.

Reliable characterization of circulating extracellular vesicles (EVs) in rodents may be significantly influenced by how blood is collected, yet systematic comparisons of commonly used sampling methods remain limited. Here, we directly evaluate the effects of cardiac puncture and perfusate blood collection on EV yield and surface-marker profiles in naïve mice, as well as in mice subjected to neurotrauma using a contusion spinal cord injury (SCI) model. Using matched isolation procedures and MACSPlex immunophenotyping, we analyzed newly generated cardiac puncture plasma alongside previously published perfusate-derived datasets, with both cohorts matched for age, sex, weight, injury severity, and post-injury timepoint. Cardiac puncture produced substantially higher particle concentrations due to access to undiluted blood, whereas perfusate samples exhibited modest increases in select markers, such as CD9, consistent with method-associated influences on platelet-derived vesicles. Despite these quantitative differences, both approaches yielded broadly similar EV phenotypes, and SCI-associated marker patterns remained stable across sampling methods. The consistency between cardiac puncture and perfusate datasets validates the robustness of our earlier perfusate-based findings and demonstrates that key biological signatures are preserved regardless of collection technique. These results provide practical guidance for optimizing murine EV studies and underscore the importance of methodological transparency and standardization in preclinical EV research.