Development of a novel protocol for processing fluorescent microspheres used in quantifying tissue perfusion

Alteration of blood perfusion leads to some of the most common cardiovascular pathologies. Current methods for measuring perfusion use fluorescent polystyrene microspheres (MS) that are systemically injected prior to processing to obtain the absolute number of MS trapped inside the tissue. The current standard method is cost-intensive and carries a high risk of MS loss, leading to underestimation of regional perfusion. This study aimed to develop an improved, cost-efficient protocol for measuring regional perfusion through the processing and direct imaging of fluorescent MS embedded ex vivo. Porcine and control samples treated with MS were chemically digested, filtered through either a polycarbonate (PCTE) or cellulose filter, and fluorescence was measured either through the standard fluorometric method or through the proposed direct imaging method. In the standard fluorometric method, interactions were found between the PCTE filter and porcine samples, leading to dampened signal and the subsequent underestimation of regional perfusion in practice. The proposed direct imaging method with cellulose filters showed improved sensitivity even within low MS levels (limit of detection improved significantly), amplification of sample fluorescence (11-13× when compared to PCTE filters), parity between porcine and control samples, and a reduction in cost providing a significant improvement over the industry standard for fluorescent MS perfusion measurement (28-51 % reduction compared to standard method). The proposed method also removed the need for 2-ethoxy ethyl acetate, a teratogen and plastic softener, and reduced complexity in the workflow.