High‐Throughput Equine Plasma Profiling Using Laser‐Assisted Rapid Evaporative Ionisation Mass Spectrometry

Remnants of erythrocyte removal from blood constitute therapeutic plasma-a water-based solution containing mainly platelets, extracellular vesicles (EVs), protein and lipid complexes and ions. Plasma-based therapies, including their use to accelerate wound healing in human and animal patients, are increasingly gaining prominence in regenerative medicine; however, methods for the optimal preparation of individual plasma are still under development. Rapid and accurate analysis of plasma composition is essential for the development of therapeutic applications and for ensuring the quality of plasma products before patient administration. This study of equine plasma presents a novel approach that utilises a newly developed automated well-plate sampling system using laser-assisted rapid evaporative ionisation mass spectrometry (LA-REIMS) for rapid plasma profiling under varying sample preparation conditions. This platform provided comprehensive lipidomic and metabolic analyses that gave insights into the molecular composition of plasma within seconds and without extensive sample preparation. The results indicated that specific phosphatidylcholines (PC(34:2), PC(36:2), PC(36:3)) and fatty acids were sensitive to centrifugation parameters, suggesting their potential as indicators of plasma stability and quality. The results were compared with the platelet and EV content of the plasma. The number density (n) of platelets and cellular fragments (P2 particles) was determined by flow cytometry (FCM), whereas EVs were characterised by interferometric light microscopy (ILM). The samples were imaged by scanning electron microscopy (SEM). A clear correlation was observed between n of P2 particles (but not of EVs) and lipid profiles, suggesting platelets' critical role in plasma composition. These findings emphasise the potential of the LA-REIMS platform for high-throughput, rapid plasma analysis, quality control and diagnostics. The system's ability to quickly assess plasma quality highlights its practical use in validating plasma prior to therapeutic applications.