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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.)

Oxygen (O 2 ) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O 2 based on pH, where a lower pH results in a lower Hb-O 2 affinity and higher Hb-O 2 dissociation. Hb-O 2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O 2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O 2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O 2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O 2 dissociation can be used to screen for and detect Hb-O 2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O 2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O 2 affinity.

Objective: Effective management of diabetes largely benefits from early diagnosis followed by intensive long-term regulation of blood glucose. The levels of glycohemoglobin (HbA1 and HbA1c) have been used as standard biomarkers to assess long-term blood glucose concentrations for diabetes diagnosis and management. Gold standard laboratory methods for HbA1 and HbA1c testing are often costly and not widely available. Moreover, currently available point-of-care (POC) immunoassay-based glycohemoglobin tests may produce inaccurate test results for patients with co-existing diseases such as hemoglobin disorders and anemia. Here, we report a POC platform, HemeChip-GHb, for quantitative HbA1 detection leveraging paper-based affinity electrophoresis., Methods: We describe the design and development of the HemeChip-GHb test. Feasibility and accuracy of the HemeChip-GHb system were demonstrated by testing blood samples collected from healthy donors, patients with prediabetes, and patients with diabetes., Results: HbA1 levels measured with HemeChip-GHb show 0.96 correlation to the levels reported from the clinical standard HPLC tests, and with a bias of -0.72% based on Bland-Altman analysis. 99.6% of the HbA1 levels for paired HemeChip-GHb and HPLC fell within A and B zones of no difference in clinical outcome based on error grid analysis., Conclusion: Using HemeChip-GHb we achieved accurate diabetes status detection with sensitivity and specificity of 100%., Significance: We presented a novel POC paper-based affinity electrophoresis platform that has the potential for accurately diagnosing diabetes, and addressing an unmet need for accurate and affordable diagnostics in resource-challenged environments.

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.)

Hemoglobin (Hb) disorders affect nearly 7% of the world's population. Globally, around 400,000 babies are born annually with sickle cell disease (SCD), primarily in sub-Saharan Africa where morbidity and mortality rates are high. Screening, early diagnosis, and monitoring are not widely accessible due to technical challenges and cost. We hypothesized that multispectral imaging will allow sensitive hemoglobin variant identification in existing affordable paper-based Hb electrophoresis. To test this hypothesis, we developed the first integrated point-of-care multispectral Hb variant test: Gazelle-Multispectral. Here, we evaluated the accuracy of Gazelle-Multispectral for Hb variant newborn screening in 265 newborns with known hemoglobin variants including hemoglobin A (Hb A), hemoglobin F (Hb F), hemoglobin S (Hb S) and hemoglobin C (Hb C). Gazelle-Multispectral detected levels of Hb A, Hb F, Hb S, and Hb C/E/A 2 , demonstrated high correlations with the results reported by laboratory gold standard high performance liquid chromatography (HPLC) at Pearson Correlation Coefficient = 0.97, 0.97, 0.93, and 0.95. Gazelle-Multispectral demonstrated accuracy of 96.8% in subjects of 0-3 days, and 96.9% in newborns. The ability to obtain accurate results on newborn samples suggest that Gazelle-Multispectral can be suitable for large-scale newborn screening and for diagnosis of SCD in low resource settings., Competing Interests: The authors declare the following conflict of interests: RA, QZ, UAG, and Case Western Reserve University have financial interests in Hemex Health Inc. UAG and Case Western Reserve University have financial interests in BioChip Labs Inc. UAG and Case Western Reserve University have financial interests in Xatek Inc. UAG 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 disorder., (© 2022 The Author(s).)

Red blood cell (RBC) deformability is a valuable hemorheological biomarker that can be used to assess the clinical status and response to therapy of individuals with sickle cell disease (SCD). RBC deformability has been measured by ektacytometry for decades, which uses shear or osmolar stress. However, ektacytometry is a population based measurement that does not detect small-fractions of abnormal RBCs. A single cell-based, functional RBC deformability assay would complement ektacytometry and provide additional information. Here, we tested the relative merits of the OcclusionChip, which measures RBC deformability by microcapillary occlusion, and ektacytometry. We tested samples containing glutaraldehyde-stiffened RBCs for up to 1% volume fraction; ektacytometry detected no significant change in Elongation Index (EI), while the OcclusionChip showed significant differences in Occlusion Index (OI). OcclusionChip detected a significant increase in OI in RBCs from an individual with sickle cell trait (SCT) and from a subject with SCD who received allogeneic hematopoietic stem cell transplant (HSCT), as the sample was taken from normoxic (pO2:159 mmHg) to physiologic hypoxic (pO2:45 mmHg) conditions. Oxygen gradient ektacytometry detected no difference in EI for SCT or HSCT. These results suggest that the single cell-based OcclusionChip enables detection of sickle hemoglobin (HbS)-related RBC abnormalities in SCT and SCD, particularly when the HbS level is low. We conclude that the OcclusionChip is complementary to the population based ektacytometry assays, and providing additional sensitivity and capacity to detect modest abnormalities in red cell function or small populations of abnormal red cells., Competing Interests: YM and UAG are inventors of the OcclusionChip Technology and a patent application has been filed by Case Western Reserve University. EK, CF, UAG and Case Western Reserve University have financial interests in BioChip Labs Inc., which offers commercial clinical microfluidic biomarker assays for inherited or acquired blood disorders and is currently commercializing the OcclusionChip technology. 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 remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Man, An, Monchamp, Sekyonda, Kucukal, Federici, Wulftange, Goreke, Bode, Sheehan and Gurkan.)