(260b) High-Throughput Measurement of the Human RED Blood Cell Shear Modulus Distribution As a Function of PO2

One of the fundamental questions around the human circulatory system is to determine how blood flow is regulated so that blood can be distributed to the right place when and where it is needed. For example, neural activity has to be accompanied by an increase in local blood flow to satisfy increased glucose and oxygen demand–the partnership between neural activity and local blood flow is termed functional hyperemia. In landmark work, J. Wan and his colleagues[1] very recently showed that cerebral functional hyperemia is initiated in the capillaries with RBCs acting as oxygen sensors to regulate cerebral blood flow. In this case, the interactions between deoxygenated hemoglobin (deoxyHb) and band 3 protein in the RBC membrane are the molecular switch that responds to local PO₂ changes and controls RBC deformability and capillary blood cell velocity. We believe that this phenomena, whose study is in its nascent stages, may directly bear on COVID19 and the development of this and other diseases. This follows directly, since it is now known that SARS-COV-2 attacks hemoglobin directly and hypoxia is a generic symptom of COVID19[2]. Indirectly, decreased flexibility of RBCs has already been noted as a biomarker for sepsis, with the cell membrane altered by the attacking micro-organisms. Note in this context, that sepsis creates a coagulopathy involving bleeding, while SARS-COV-2 creates a coagulopathy associated with thrombosis—the latter of which is currently a subject of intense study[3].

RBC structure and deformability are largely governed by the membrane shear modulus. State-of-the-art methods to measure the shear modulus of RBCs are not high-throughput and, microfluidic platforms for high-throughput measurements of RBC mechanical properties have not yet enabled measurement of the shear modulus. These limitations challenge the development of diagnostic devices based on RBC shear modulus biomarkers. In this talk, we will review the development of our high-throughput microfluidic platform[4], coupled with high-fidelity simulations to address this significant gap in technology. In contrast with existing technologies, this approach allows us to measure the shear modulus of individual RBCs and generate shear modulus distributions (for a given individual or multiple individuals) including measurements of thousands of cells in a few seconds of experimental data acquisition. We demonstrate that our platform provides mean values of the modulus that are in quantitative agreement with other low throughput measurement techniques in the literature (e.g optical tweezers), but that the distribution for each donor follows a broad lognormal distribution.

We will then discuss the modification of our platform to make similar measurements of an individual’s shear modulus distribution but at controlled PO₂ saturation levels in the blood. Thus, our microfluidic chip is modified to include gas channels that run along the blood flow channel. The PO₂ titration in the gas channels is accurately controlled such that for each individual blood sample measurements of the shear modulus are made at the equilibrium PO₂ of the gas channel. The result yields accurate and independent measures of the correlation of RBC shear modulus with PO₂ present in healthy individuals for the first time.

[1] Zhou et al., Oxygen tension–mediated erythrocyte membrane interactions regulate cerebral capillary hyperemia, Sci. Adv. 2019; 5 : eaaw4466 29 May 2019; [2] Cavezzi et al., COVID-19: hemoglobin, iron, and hypoxia beyond inflammation. A narrative review; Clin Pract. 2020 May 19; 10(2): 1271.doi: 10.4081/cp.2020.1271; [3] Connors, J. And J. Levy, Blood, 135(23) pp.2033-2040 [4] Saadat et al., High-throughput measurement of an individual’s red blood cell shear modulus distribution, Lab on a Chip , 20, pp. 2927-2936 (2020).