Perfusion fixation methods for preclinical biodistribution studies: A comparative assessment using automated image processing.

Studying the biodistribution of novel therapeutics and biomaterialsin vivorequires effective and consistent perfusion and fixation of major organs. Standard methods for removing red blood cells (RBCs) and fixing tissue often involve transcardial perfusion, such as brain-targeted perfusion (via the left ventricle) or lung-targeted perfusion (via the right ventricle). Using autofluorescence measurements and a bespoke ImageJ macro to quantify RBC content from histology, we compared the efficacy and consistency of three whole-body perfusion techniques. We show that lung-targeted perfusion evacuates more blood from the lung vasculature than brain-targeted perfusion (20 ± 54% fewer RBCs), and that our novel approach of 'dual-targeted' perfusion (via the right and left ventricles sequentially) had even higher efficacy (30 ± 6% fewer RBCs). Furthermore, by combining aspects of brain- and lung-targeted methods, dual-targeted perfusion achieved the highest consistency in autofluorescence emissions from major organs (64% and 65% lower variance than brain- and lung-targeted perfusion respectively). Since RBC content and autofluorescence can be confounding factors in biodistribution studies using fluorescent probes, our findings and proposed novel approach offer insight into perfusion fixation techniques for pre-clinical studies.