The relationship between red blood cell deformability metrics and perfusion of an artificial microvascular network

Abstract

The ability of red blood cells (RBC) to undergo a wide range of deformations while traversing the microvasculature is crucial for adequate perfusion. Interpretation of RBC deformability measurements performed in vitro in the context of microvascular perfusion has been notoriously difficult. This study compares the measurements of RBC deformability performed using micropore filtration and ektacytometry with the RBC ability to perfuse an artificial microvascular network (AMVN). Human RBCs were collected from healthy consenting volunteers, leukoreduced, washed and exposed to graded concentrations (0-0.08%) of glutaraldehyde (a non-specific protein cross-linker) and diamide (a spectrin-specific protein cross-linker) to impair the deformability of RBCs. Samples comprising cells with two different levels of deformability were created by adding non-deformable RBCs (hardened by exposure to 0.08% glutaraldehyde) to the sample of normal healthy RBCs. Ektacytometry indicated a nearly linear decline in RBC deformability with increasing glutaraldehyde concentration. Micropore filtration showed a significant reduction only for concentrations of glutaraldehyde higher than 0.04%. Neither micropore filtration nor ektacytometry measurements could accurately predict the AMVN perfusion. Treatment with diamide reduced RBC deformability as indicated by ektacytometry, but had no significant effect on either micropore filtration or the AMVN perfusion. Both micropore filtration and ektacytometry showed a linear decline in effective RBC deformability with increasing fraction of non-deformable RBCs in the sample. The corresponding decline in the AMVN perfusion plateaued above 50%, reflecting the innate ability of blood flow in the microvasculature to bypass occluded capillaries. Our results suggest that in vitro measurements of RBC deformability performed using either micropore filtration or ektacytometry may not represent the ability of same RBCs to perfuse microvascular networks. Further development of biomimetic tools for measuring RBC deformability (e.g. the AMVN) could enable a more functionally relevant testing of RBC mechanical properties.

Keywords: Red blood cell deformability; artificial microvascular network; ektacytometry; microfluidics; micropore filtration.

Figure 1

Illustration of the two conventional technologies commonly used to evaluate RBC deformability. (A) In the micro-pore filtration technique, RBCs are passed through 5 μm pores of a polycarbonate filter. The time it takes a pre-set volume of the RBC suspension to pass through the filter (filtration time) is measured to evaluate the ability of RBCs to undergo folding deformations when passing through narrow openings. (B) In an ektacytometer (RheoScan-D), a dilute suspension of RBCs in a highly viscous PVP solution is passed through a stright channel at various shear stresses. A laser beam is passed perpendicularly through the RBC suspension and the change in the diffraction pattern is quantified via the calculation of the elongation index to evaluate RBC deformability.

Figure 2

Illustration of the artificial microvascular network (AMVN) device. (A) In the AMVN device, a 40% hematocrit suspension of RBCs is passed through a network of artificial capillaries under a constant pressure difference between the inlet and the outlet, and the overall flow rate in the venule of the AMVN is quantified. (B) The AMVN perfusion rate evaluates the effective ability of RBCs to undergo a variety of deformations as they pass through the network of artificial capillaries ranging in size from 5 μm to 70 μm. (C) RBCs deform as a result of collisions with other cells and vessel walls at capillary bifurcations. (D) RBCs passing through the narrowest capillaries assume the characteristic bullet-like shape. (E) RBCs deform into the parachute shape in larger capillaries. (F) RBCs experience significant deformations due to the convergence of two streams moving with different velocities. (G) In largest vessels of the AMVN, RBCs undergo shear-induced deformations as well as deformations caused by multiple collisions with other cells in bulk flow. The flow direction is from left to right in all images. Scale bars are 10μm.

Figure 3

A comparison of the normalized deformability indices measured using different methodologies for RBCs incubated with graded concentrations of (A) glutaraldehyde and (B) diamide, and (C) for reconstituted samples containing a mixture of healthy normal cells and varying percentage of non-deformable RBCs hardened by incubation with 0.08% glutaraldehyde. Each data point represents mean ± standard deviation of n = 4 samples (* p<0.05 with respect to every other measurement technique).