SCD-Biochip: A Functional Assay for Red Cell Adhesion in Sickle Cell Disease

Vaso-occlusion, a hallmark of sickle cell disease (SCD), is caused by abnormal adhesion of blood cells to the endothelium. Analyses of RBC-endothelial interactions are technically challenging, which has limited the widespread use of these evaluations clinically or as a research tool. Here, we present a microfluidic device (SCD-Biochip) that allows serial quantitative and qualitative examination of RBC adhesion and deformability to endothelium associated protein-immobilized microchannels (fibronectin, FN, and laminin, LN), in a closed preprocessing-free system (Fig. 1A). 15 µL of surplus blood, after clinical lab tests had been completed, was taken from subjects with HbSS (transfused, non-transfused, and with high or low levels of HbF), HbSC, HbSBeta-thalassemia and from control subjects (HbAA). Samples were injected, without processing, on to FN- and LN-immobilized microchannels. This was followed by wash steps at or above the physiological flow velocities of post-capillary venules (0-10 mm/s). Adhered RBCs were then quantified. We observed a statistically significant difference in RBC adherence per unit area to FN (Fig. 1B), after 0.8 mm/s flow velocity (low wash), amongst RBCs from subjects with HbSS (90±84.0, n=26), with compound heterozygous HbSC or Sβthalassemia (21±6.8, n=4), or with normal hemoglobin (HbAA, 4±3, n=10, Mann-Whitney, p<0.05). Strikingly, this test was sensitive to physiological changes that were not well-reflected in routine lab tests (e.g., adherence to FN did not differ between samples from transfused or non-transfused HbSS patients (77±62, n=6, compared with 88.1±82.6, n=17, P=0.75, respectively), despite routine scheduled transfusions of HbAA-containing RBCs in the former. Samples from HbSS subjects with a low HbF were more likely to be highly adherent to FN than were samples from subjects with a high HbF (5/7 with <8% HbF compared with 1/10 ≥8% HbF, p=0.035). Increased adherence of HbSS- (707±904.8, n=7), compared with HbAA-containing RBCs (9±6, n=7), to LN at low wash was also seen. Of interest, a single sample from an HbSS subject with acute pain showed an adherence to FN and LN of 250 and 2176, respectively. Qualitatively, HbSS-containing RBCs showed decreased adherence to FN at increased flow rates; adhered RBCs decreased from 90±84 (n=25) at low flow wash to 69±52 (n=13) at 3.3 mm/s (intermediate flow) and 36±48.2 (n=11) at 41.7 mm/s (high flow). Analyses revealed deformable HbAA- and HbSS-containing RBCs, but only HbSS-containing samples also had a significant population of non-deformable RBCs. We analyzed single cell deformation and detachment in the presence of physiological fluid flow. Deformable HbAA- and HbSS-containing RBCs detached at relatively low flow rates (6.11±0.48 n=3, and 9.3±1.44 n=6, respectively), compared to non-deformable HbSS-containing RBCs (40±6.97 n=5). Deformable HbAA- or HbSS-containing RBCs displayed a single adhesion pivot point, compared to non-deformable HbSS-containing RBCs, which showed multiple adhesion sites. At highest flow (41.7mm/s), 75±17% (n=9) of adhered RBCs were non-deformable on FN. Of note, flow evaluations revealed two characteristic RBC profiles in HbSS subjects: either one in which adherent non-deformable cells predominated at all flow rates or one in which less adherent deformable cells comprised >50% of the RBC population. The SCD-Biochip evaluates, simply and with small blood sample volumes, complex biophysical properties of adhesion and deformability, which reflect clinical phenotypes, including hemoglobin composition. This adaptable technology may give important biophysical insights into disease pathophysiology when widely applied in SCD. Figure 1. A functional assay for RBC adhesion in SCD (A) The SCD-Biochip allows serial examination of RBC adhesion and deformability to endothelium-associated proteins and provides closed, preprocessing-free, and direct analysis of blood. (B) Identification of different phenotypes in SCD-Biochip based on adhesion of RBCs (per unit area of 32 mm2) to fibronectin immobilized microchannels. Figure 1. A functional assay for RBC adhesion in SCD (A) The SCD-Biochip allows serial examination of RBC adhesion and deformability to endothelium-associated proteins and provides closed, preprocessing-free, and direct analysis of blood. (B) Identification of different phenotypes in SCD-Biochip based on adhesion of RBCs (per unit area of 32 mm2) to fibronectin immobilized microchannels. Disclosures No relevant conflicts of interest to declare.