Despite the development of microfluidics, experimental challenges are considerable for achieving a quantitative study of phase separation, i.e., the non-proportional distribution of Red Blood Cells (RBCs) and suspending fluid, in microfluidic bifurcations with channels smaller than 20 μm. Yet, a basic understanding of phase separation in such small vessels is needed for understanding the coupling between microvascular network architecture and dynamics at larger scale. Here, we present the experimental methodologies and measurement techniques developed for that purpose for RBC concentrations (tube hematocrits) ranging between 2% and 20%. The maximal RBC velocity profile is directly measured by a temporal cross-correlation technique which enables to capture the RBC slip velocity at walls with high resolution, highlighting two different regimes (flat and more blunted ones) as a function of RBC confinement. The tube hematocrit is independently measured by a photometric technique. The RBC and suspending fluid flow rates are then deduced assuming the velocity profile of a Newtonian fluid with no slip at walls for the latter. The accuracy of this combination of techniques is demonstrated by comparison with reference measurements and verification of RBC and suspending fluid mass conservation at individual bifurcations. The present methodologies are much more accurate, with less than 15% relative errors, than the ones used in previous in vivo experiments. Their potential for studying steady state phase separation is demonstrated, highlighting an unexpected decrease of phase separation with increasing hematocrit in symmetrical, but not asymmetrical, bifurcations and providing new reference data in regimes where in vitro results were previously lacking.
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