Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

A microfluidic device for assessment of E-selectin-mediated neutrophil recruitment to inflamed endothelium and prediction of therapeutic response in sickle cell disease.

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.

Standardized microfluidic assessment of red blood cell-mediated microcapillary occlusion: Association with clinical phenotype and hydroxyurea responsiveness in sickle cell disease.

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.

Microfluidic assessment of red blood cell mediated microvascular occlusion.

Abnormal red blood cell (RBC) deformability contributes to hemolysis, thrombophilia, inflammation, and microvascular occlusion in various circulatory diseases. A quantitative and objective assessment of microvascular occlusion mediated by RBCs with abnormal deformability would provide valuable insights into disease pathogenesis and therapeutic strategies. To that end, we present a new functional microfluidic assay, OcclusionChip, which mimics two key architectural features of the capillary bed in the circulatory system. First, the embedded micropillar arrays within the microchannel form gradient microcapillaries, from 20 µm down to 4 µm, which mimic microcapillary networks. These precisely engineered microcapillaries retain RBCs with impaired deformability, such that stiffer RBCs occlude the wider upstream microcapillaries, while less stiff RBCs occlude the finer downstream microcapillaries. Second, the micropillar arrays are coupled with two side passageways, which mimic the arteriovenous anastomoses that act as shunts in the capillary bed. These side microfluidic anastomoses prevent microchannel blockage, and enable versatility and testing of clinical blood samples at near-physiologic hematocrit levels. Further, we define a new generalizable parameter, Occlusion Index (OI), which is an indicative index of RBC deformability and the associated microcapillary occlusion. We demonstrate the promise of OcclusionChip in diverse pathophysiological scenarios that result in impaired RBC deformability, including mercury toxin, storage lesion, end-stage renal disease, malaria, and sickle cell disease (SCD). Hydroxyurea therapy improves RBC deformability and increases fetal hemoglobin (HbF%) in some, but not all, treated patients with SCD. HbF% greater than 8.6% has been shown to improve clinical outcomes in SCD. We show that OI associates with HbF% in 16 subjects with SCD. Subjects with higher HbF levels (HbF > 8.6%) displayed significantly lower OI (0.88% ± 0.10%, N = 6) compared with those with lower HbF levels (HbF ≤ 8.6%) who displayed greater OI (3.18% ± 0.34%, N = 10, p < 0.001). Moreover, hypoxic OcclusionChip assay revealed a significant correlation between hypoxic OI and subject-specific sickle hemoglobin (HbS) level in SCD. OcclusionChip enables versatile in vitro assessment of microvascular occlusion mediated by RBCs in a wide range of clinical conditions. OI may serve as a new parameter to evaluate the efficacy of treatments improving RBC deformability, including hemoglobin modifying drugs, anti-sickling agents, and genetic therapies.

Leukocyte adhesion to P-selectin and the inhibitory role of Crizanlizumab in sickle cell disease: A standardized microfluidic assessment.

Upregulated expression of P-selectin on activated endothelium and platelets significantly contributes to the initiation and progression of vaso-occlusive crises (VOC), a major cause of morbidity in sickle cell disease (SCD). Crizanlizumab (ADAKVEO®), a humanized monoclonal antibody against P-selectin, primarily inhibits the interaction between leukocytes and P-selectin, and has been shown to decrease the frequency of VOCs in clinical trials. However, the lack of reliable in vitro assays that objectively measure leukocyte adhesion to P-selectin remains a critical barrier to evaluating and improving the therapeutic treatment in SCD. Here, we present a standardized microfluidic BioChip whole blood adhesion assay to assess leukocyte adhesion to P-selectin under physiologic flow conditions. Our results demonstrated heterogeneous adhesion by leukocytes to immobilized P-selectin, and dose-dependent inhibition of this adhesion following pre-exposure to Crizanlizumab. Importantly, treatment with Crizanlizumab following adhesion to P-selectin promoted detachment of rolling, but not of firmly adherent leukocytes. Taken together, our results suggest that the microfluidic BioChip system is a promising in vitro assay with which to screen patients, monitor treatment response, and guide current and emerging anti-adhesive therapies in SCD.

ClotChip: A Microfluidic Dielectric Sensor for Point-of-Care Assessment of Hemostasis.

This paper describes the design, fabrication, and testing of a microfluidic sensor for dielectric spectroscopy of human whole blood during coagulation. The sensor, termed ClotChip, employs a three-dimensional, parallel-plate, capacitive sensing structure with a floating electrode integrated into a microfluidic channel. Interfaced with an impedance analyzer, the ClotChip measures the complex relative dielectric permittivity, ϵr , of human whole blood in the frequency range of 40 Hz to 100 MHz. The temporal variation in the real part of the blood dielectric permittivity at 1 MHz features a time to reach a permittivity peak, , as well as a maximum change in permittivity after the peak, , as two distinct parameters of ClotChip readout. The ClotChip performance was benchmarked against rotational thromboelastometry (ROTEM) to evaluate the clinical utility of its readout parameters in capturing the clotting dynamics arising from coagulation factors and platelet activity. exhibited a very strong positive correlation ( r = 0.99, p < 0.0001) with the ROTEM clotting time parameter, whereas exhibited a strong positive correlation (r = 0.85,  p < 0.001) with the ROTEM maximum clot firmness parameter. This paper demonstrates the ClotChip potential as a point-of-care platform to assess the complete hemostatic process using <10 µL of human whole blood.