Increased perimeter red cell concentration in filter paper bloodspot samples is consistent with constant-load size exclusion chromatography occurring during application

Abstract

Background Filter paper bloodspot samples exhibit increased red cell concentration near the bloodspot perimeter compared to the interior. We examined whether simulation of size exclusion chromatography separating cell and liquid components during bloodspot formation was consistent with the observed red cell distribution. Methods Whole blood was defined as a mixture of 60% liquid and 40% solids (hematocrit). Bloodspot formation was simulated by step-wise center additions of 1 μl blood up to a total volume of 50 μl. Partitioning of the liquid component of the liquid volume into space not available to the solid component was calculated using a partitioning coefficient K = V i / V m, where V i is the immobile (stationary) liquid volume and V m is the mobile liquid volume. After each volume addition step, relative red cell concentration was calculated as the solids volume fraction of total volume as a function of radial distance from the center. Results Simulation for K = 0.3 resulted in final red cell distribution that was quantitatively consistent with bloodspot data. Conclusions Increased perimeter red cell concentration in bloodspots is consistent with partitioning of the liquid fraction into mobile and immobile components during bloodspot formation according to the principles of constant-load size exclusion chromatography.