Hypoxia-enhanced adhesion of red blood cells in microscale flow

Abstract

Objectives: The advancement of microfluidic technology has facilitated the simulation of physiological conditions of the microcirculation, such as oxygen tension, fluid flow, and shear stress in these devices. Here, we present a micro-gas exchanger integrated with microfluidics to study RBC adhesion under hypoxic flow conditions mimicking postcapillary venules.

Methods: We simulated a range of physiological conditions and explored RBC adhesion to endothelial or subendothelial components (FN or LN). Blood samples were injected into microchannels at normoxic or hypoxic physiological flow conditions. Quantitative evaluation of RBC adhesion was performed on 35 subjects with homozygous SCD.

Results: Significant heterogeneity in RBC adherence response to hypoxia was seen among SCD patients. RBCs from a HEA population showed a significantly greater increase in adhesion compared to RBCs from a HNA population, for both FN and LN.

Conclusions: The approach presented here enabled the control of oxygen tension in blood during microscale flow and the quantification of RBC adhesion in a cost-efficient and patient-specific manner. We identified a unique patient population in which RBCs showed enhanced adhesion in hypoxia in vitro. Clinical correlates suggest a more severe clinical phenotype in this subgroup.

Keywords: erythrocyte; lab on a chip; laminin; microfluidics; oxygen; sickle cell disease.

Figure 1. Single-use micro-gas exchanger integrated with microchannels for oxygen tension control of cell microenvironment

(A) Blood deoxygenated in the micro-gas exchanger during flow and reached the functionalized microchannels, where the flow conditions were physiologically relevant. The system was applied to SCD blood to observe sickling and adhesion behavior of RBCs. (B) The microfluidic system, composed of three parallel microchannels of 50 µm height, outlet tubing, and micro-gas exchanger inlet tubing, as shown. Inset shows adhered sickle RBC on endothelium associated protein (fibronectin, FN, and laminin, LN) functionalized surface. Scale bar indicates 20 µm of length. (C) The micro-gas exchanger, comprised of two concentric tubing; an inner gas-permeable tubing and an outer gas-impermeable tubing, as shown. Controlled gas flow takes place between the inner and outer tubing; blood flows inside the inner tubing. Deoxygenation of blood occurred due to gas diffusion (5% CO₂ and 95% N₂) through inner gas-permeable tubing wall. The micro-gas exchanger is single-use, allowing easy adaptation to biological and point-of-care microfluidic systems.

Figure 2. Computational modeling of flow and gas equilibrium in the micro-gas exchanger

(A) Schematic for axisymmetric cross section of the micro-gas exchanger shows gas concentration and fluid flow boundary conditions. Gas diffusion rate depends on the flow rate, gas concentration, and tubing length. Micro-gas exchanger length is specifically designed to allow CO₂ and O₂ equilibrium before blood reaches to the microchannel. (B & C) CO₂ and O₂ concentration (%) shift in blood flowing within the micro-exchanger tubing. Contour plots (i-iii) on the axisymmetric cross section of blood and permeable tubing in micro-gas exchanger show (B) CO₂ and (C) O₂ concentration in blood at different lengths of the tubing. CO₂ concentration of blood starts to increase as blood flows through the permeable tubing and reaches equilibrium at 5% in 250 mm. O₂ concentration of blood reaches 7.5% in 250 mm through the micro-gas exchanger tubing, which corresponds to results obtained in the validation experiments (see Fig. S1 for additional details).

Figure 3. Deoxygenation of adhered RBCs in blood samples from SCD (Hb SS) and normal (Hb AA) subjects

(A) Shown are single adhered HbSS containing RBCs, with (hypoxic) and without (normoxic) deoxygenation, from patients with SCD, under different flow conditions. Under hypoxic conditions, the aspect ratio of sickle RBCs decreased and displayed decreased deformability, defined by changes in aspect ratio in response to fluid flow shear stress application, in comparison with normoxic conditions. (B) Shown are single HbAA containing RBCs, from patients without SCD. Overall, these RBCs showed a preserved morphology and deformability characteristic under hypoxic conditions, with greater deformability than that seen in HbSS-containing RBCs ^,. The scale bars indicate 5 µm of length.

Figure 4. Heterogeneity in RBC adhesion in response to hypoxic microscale blood flow

(A) Shown are blood samples from different SCD patients, with heterogeneous responses to hypoxia in terms of increase in number of adhered RBCs. Among the analyzed patient blood samples, a subpopulation of patients (HNA) showed a negligible increase in the number of adhered RBCs to FN and LN. Samples from the remaining patients (HEA) displayed a dramatic increase in number of adhered RBCs to FN and LN in response to hypoxia. Colored dots indicate adhered RBCs. (B) Analyzed patient blood samples showed significantly greater increase in number of adhered RBCs to LN compared to FN in response to hypoxia. (N=35) (C) Patients clustered in two subpopulations, HEA and HNA, based on increase in number of adhered RBCs to FN (N=17) and LN (N=26) in response to hypoxic conditions. The HEA patient subpopulation showed significantly greater increase in the number of adhered RBCs with hypoxia compared to HNA patient subpopulation (p<0.05, See Fig. S2). The horizontal lines between individual groups represent a statistically significant difference based on a one-way ANOVA test (P<0.05). Data point cross bars represent the mean. ‘N’ represents the number of subjects.

Figure 5. Association of hypoxic RBC adhesion with clinical phenotypes in SCD

Patients with HEA RBCs (N=8), compared with HNA RBCs (N=15), showed (A) significantly higher fetal hemoglobin (Hb F) levels (p=0.0079), (B) significantly higher reticulocyte counts (p<0.0001), and (C) significantly higher LDH levels (p=0.0102). The horizontal lines between individual groups represent a statistically significant difference based on a one-way ANOVA test (p<0.05). Data point cross bars represent the mean. ‘N’ represents the number of subjects.

Figure 6. The effect of BCAM/Lu blocking on RBC adhesion in normoxic and hypoxic conditions for HEA and HNA patient populations

Shown is mean percent change (+/− standard deviation) in the number of adhered RBCs under hypoxic conditions compared to normoxic conditions in patients with HEA RBCs (N=4) and in patients with HNA RBCs (N=6), following exposure to BCAM/Lu blocking antibodies. The horizontal lines between individual groups represent a statistically significant difference based on a one-way ANOVA test (p<0.05). ‘N’ represents the number of subjects.