Search

The production of HbS - an abnormal hemoglobin (Hb) - in sickle cell disease (SCD) results in poorly deformable red blood cells (RBCs) that are prone to microcapillary occlusion, causing tissue ischemia and organ damage. Novel treatments, including gene therapy, may reduce SCD morbidity, but methods to functionally evaluate RBCs remain limited. Previously, we presented the microfluidic impedance red cell assay (MIRCA) for rapid assessment of RBC deformability, employing electrical impedance-based readout to measure RBC occlusion of progressively narrowing micropillar openings. We describe herein the design, development, validation, and clinical utility of the next-generation MIRCA assay, featuring enhanced portability, rapidity, and usability. It incorporates a miniaturized impedance analyzer and features a simplified wash-free operation that yields an occlusion index (OI) within 15 min as a new metric for RBC occlusion. We show a correlation between OI and percent fetal hemoglobin (%HbF), other laboratory biomarkers of RBC hemolysis, and SCD severity. To demonstrate the assay's versatility, we tested RBC samples from treatment-naïve SCD patients in Uganda that yielded OI levels similar to those from hydroxyurea (HU)-treated patients in the U.S., highlighting the role of %HbF in protecting against microcapillary occlusion independent of other pharmacological effects. The MIRCA assay could also identify a subset of HU-treated patients with high occlusion risks, suggesting that they may require treatment adjustments including a second-line therapy to improve their outcomes. This work demonstrates the potential of the MIRCA assay for accelerated evaluation of RBC health, function, and therapeutic effect in an ex vivo model of the microcapillary networks., Competing Interests: Declaration of competing interest U. A. Gurkan and Case Western Reserve University have financial interests in Hemex Health Inc. and BioChip Labs Inc. M. A. Suster, U. A. Gurkan, P. Mohseni, and Case Western Reserve University have financial interests in XaTek Inc. U. A. Gurkan has financial interests in DxNow Inc. P. Mohseni has financial interests in Haima Therapeutics LLC. Financial interests include licensed intellectual property, stock ownership, research funding, employment, consulting fees, and royalties during the conduct of this study. The competing interests of Case Western Reserve University employees are overseen and managed by the Conflict of Interests Committee according to a Conflict-of-Interest Management Plan. S. Oshabaheebwa, C. A. Delianides, A. A. Patwardhan, E. N. Evans, Z. Sekyonda, A. Bode, F. M. Apio, C. K. Mutuluuza, and V. A. Sheehan report no conflict., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neutrophil recruitment to the inflamed endothelium is a multistep process and is of utmost importance in the development of the hallmark vaso-occlusive crisis in sickle cell disease (SCD). However, there lacks a standardized, clinically feasible approach for assessing neutrophil recruitment to the inflamed endothelium for individualized risk stratification and therapeutic response prediction in SCD. Here, we describe a microfluidic device functionalized with E-selectin, a critical endothelial receptor for the neutrophil recruitment process, as a strategy to assess neutrophil binding under physiologic flow in normoxia and clinically relevant hypoxia in SCD. We show that hypoxia significantly enhances neutrophil binding to E-selectin and promotes the formation of neutrophil-platelet aggregates. Moreover, we identified two distinct patient populations: a more severe clinical phenotype with elevated lactate dehydrogenase levels and absolute reticulocyte counts but lowered fetal hemoglobin levels associated with constitutively less neutrophil binding to E-selectin. Mechanistically, we demonstrate that the extent of neutrophil activation correlates with membrane L-selectin shedding, resulting in the loss of ligand interaction sites with E-selectin. We also show that inhibition of E-selectin significantly reduces leukocyte recruitment to activated endothelial cells. Our findings add mechanistic insight into neutrophil-endothelial interactions under hypoxia and provide a clinically feasible means for assessing neutrophil binding to E-selectin using clinical whole blood samples, which can help guide therapeutic decisions for SCD patients., Competing Interests: Declaration of competing interest U. A. G. and Case Western Reserve University have financial interests in Hemex Health Inc. U. A. G. and Case Western Reserve University have financial interests in BioChip Labs Inc. U. A. G. and Case Western Reserve University have financial interests in Xatek Inc. U. A. G. has financial interests in DxNow Inc. Financial interests include licensed intellectual property, stock ownership, research funding, employment, and consulting. Hemex Health Inc. offers point-of-care diagnostics for hemoglobin disorders, anemia, and malaria. BioChip Labs Inc. offers commercial clinical microfluidic biomarker assays for inherited or acquired blood disorders. Xatek Inc. offers point-of-care global assays to evaluate the hemostatic process. DxNow Inc. offers microfluidic and bio-imaging technologies for in vitro fertilization, forensics, and diagnostics. The competing interests of Case Western Reserve University employees are overseen and managed by the Conflict of Interests Committee according to a Conflict-of-Interest Management Plan. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)