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Single cells have unique biophysical signatures that can rapidly change during various disease states. For instance, cellular density is an inherent property differing between cell types. Characterizing changes in fundamental density properties down to the single-cell level can reveal sub-populations in pathological states. Here, we have developed a microfluidic, magnetic levitation-based assay (MagDense) that detects minute density differences of individual red blood cells (RBCs) down to 0.0001 g mL−1 resolution. This assay fractionates RBCs based on their density profiles in a non-ionic paramagnetic medium flowing in a capillary microchannel placed between magnets with same poles facing each other. Based on precisely measured levitation height and density of individual RBCs at their specific equilibrium state, we demonstrated that MagDense can accurately analyze the density of sickle hemoglobin (HbS)-containing RBCs and normal hemoglobin (HbA)-containing RBCs. In addition, the precise density and cell size measurements at the single cell level showed three different sub-populations of RBCs in blood samples from individuals with homozygous sickle cell disease receiving blood transfusions; where less dense, HbA-containing RBCs levitated higher, while the denser, HbS-containing RBCs levitated lower. We compared the mean RBC densities of sickle cell disease subjects with healthy controls and found distinctly separated bands of RBC density for each group denoting the likely range of cell densities seen in the blood samples. The high resolution of our method enabled measurement of deviation from the mean RBC density. Moreover, we introduced a new term as a measure of density dispersion, "RBC levitational density width, RLDW". Mean RBC density in sickle cell disease associated with hemoglobin from complete blood count (p = 0.032, linear regression) and RLDW associated with absolute reticulocyte count (ARC) and RBC distribution width (RDW) from complete blood count (p = 0.002 for ARC and p = 003 for RDW, linear regression). Our magnetic levitation-based assay enables rapid, accurate, density-based imaging, profiling and label-free monitoring of single RBCs. Our approach can be broadly applicable to investigate blood cell disorders and the effects of emerging pharmacological and curative therapies in patient outcomes. [ABSTRACT FROM AUTHOR]

Anemia affects over 25% of the world's population with the heaviest burden borne by women and children. Genetic hemoglobin (Hb) variants, such as sickle cell disease, are among the major causes of anemia. Anemia and Hb variant are pathologically interrelated and have an overlapping geographical distribution. We present the first point-of-care (POC) platform to perform both anemia detection and Hb variant identification, using a single paper-based electrophoresis test. Feasibility of this new integrated diagnostic approach is demonstrated via testing individuals with anemia and/or sickle cell disease. Hemoglobin level determination is performed by an artificial neural network (ANN) based machine learning algorithm, which achieves a mean absolute error of 0.55 g dL−1 and a bias of −0.10 g dL−1 against the gold standard (95% limits of agreement: 1.5 g dL−1) from Bland–Altman analysis on the test set. Resultant anemia detection is achieved with 100% sensitivity and 92.3% specificity. With the same tests, subjects with sickle cell disease were identified with 100% sensitivity and specificity. Overall, the presented platform enabled, for the first time, integrated anemia detection and hemoglobin variant identification using a single point-of-care test. [ABSTRACT FROM AUTHOR]

Objectives: We present a standardized in vitro microfluidic assay and Occlusion Index (OI) for the assessment of red blood cell (RBC)–mediated microcapillary occlusion and its clinical associations in sickle cell disease (SCD). Methods: Red blood cell mediated microcapillary occlusion represented by OI and its clinical associations were assessed for seven subjects with hemoglobin‐SC disease (HbSC), 18 subjects with homozygous SCD (HbSS), and five control individuals (HbAA). Results: We identified two sub‐populations with HbSS based on the OI distribution. HbSS subjects with relatively higher OIs had significantly lower hemoglobin levels, lower fetal hemoglobin (HbF) levels, and lower mean corpuscular volume (MCV), but significantly higher serum lactate dehydrogenase levels and absolute reticulocyte counts, compared to subjects with HbSS and lower OIs. HbSS subjects who had relatively higher OIs were more likely to have had a concomitant diagnosis of intrapulmonary shunting (IPS). Further, lower OI associated with hydroxyurea (HU) responsiveness in subjects with HbSS, as evidenced by significantly elevated HbF levels and MCV. Conclusions: We demonstrated that RBC‐mediated microcapillary occlusion and OI associated with subject clinical phenotype and HU responsiveness in SCD. The presented standardized microfluidic assay may be useful for evaluating clinical phenotype and assessing therapeutic outcomes in SCD, including emerging targeted and curative treatments that aim to improve RBC deformability and microcirculatory health. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • An E-selectin functionalized microfluidic device partially mimicking the inflamed endothelium is presented. • In sickle cell disease (SCD), neutrophil binding to E-selectin is mediated by L-selectin and inversely correlates with hemolysis. • Neutrophil binding to E-selectin can be used to stratify clinical phenotypes and predict therapeutic response in SCD. [ABSTRACT FROM AUTHOR]