Emerging therapies in sickle cell disease (SCD) aim to restore healthy red blood cell (RBC) function, but they often yield heterogeneous cellular responses. There are no proven techniques to evaluate restored rheological functionality and heterogeneity in these RBCs. We present a biomimetic microcapillary network, high-speed imaging, and computational algorithms to analyze RBC capillary velocity profiles of the entire sample population at single-cell resolution. Using peripheral RBCs from SCD patients and healthy donors, we showed that RBC capillary transit velocity correlated with cell shape, hydrodynamic adaptability, and elongation index. Healthy RBCs exhibited a velocity distribution skewed toward higher values, whereas RBCs from individuals with SCD showed a shift toward lower velocities. SCD samples had a greater fraction of slow RBCs than healthy controls (42.1% ± 12.0% vs. 19.0% ± 4.9%, p < 0.0001). We tested mixtures of healthy and SCD RBCs to simulate heterogeneous therapeutic effects and demonstrated that the assay was sensitive to small fractions of abnormal RBCs. The slow RBC fraction emerged as a potential biomarker associated with SCD disease severity. This fraction significantly increased under hypoxia showing sensitivity to hypoxia-induced sickling. Finally, we assessed in vitro-derived RBCs and observed distinct velocity profiles for nucleated and enucleated cells. Processing methods to enrich enucleated RBCs improved the velocity profile, producing a distribution that was more comparable to that of peripheral RBCs. This platform's ability to assess individual RBCs and generate a velocity profile from a small number of cells makes it well suited for evaluating the rheological properties of in vitro-derived RBCs.

ABSTRACT:In sickle cell disease (SCD), red blood cells (RBCs) are poorly deformable, even under normoxia (NOI). With deoxygenation, deformability of sickle RBCs is further reduced due to polymerization of hemoglobin S (HbS). Rigid, poorly deformable sickle RBCs block microvasculature, causing ischemia, pain, and organ damage. The microfluidic impedance red cell assay (MIRCA) can mechanically measure RBC deformability, providing occlusion index under NOI or hypoxia (HOI) as readouts. We analyzed RBCs from 68 adult and 34 pediatric patients with SCD using the MIRCA. Higher HOI and NOI values were positively associated with markers of inflammation, hemolysis, RBC density, older age, and severe SCD genotypes (homozygous HbSS or HbS β0-thalassemia. Each 1% higher NOI across individuals was associated with a 6.3% higher incidence of acute complications per year. Individuals with chronic complications in the past year had a 3.1% higher median NOI than those without chronic complications. Individuals on chronic transfusion therapy exhibit a subpopulation of poorly deformable RBCs captured by the MIRCA but not by a commercially available device that also measures RBC deformability, the laser assisted optical rotational cell analyzer (LoRRca). In vitro addition of voxelotor or osivelotor to samples from individuals on chronic transfusion therapy improved the deformability of these endogenous RBCs. Longitudinally collected NOI and HOI values in individuals with HbSS were stable, with a median percent point change of 13.3% and 15.7%, respectively. MIRCA can be used in combination with clinical laboratory tests to monitor RBC deformability as a biomarker of clinical status at routine clinic visits and included in clinical trials of disease-modifying agents.

BACKGROUND:Platelet count or function defects can result in hemostatic impairment, leading to life-threatening bleeding complications. While platelet transfusions are used to treat such complications, current clinical practice utilizes only platelet count thresholds to guide transfusion. This is because a single precision diagnostic system that can directly correlate both platelet count and function defects to hemostatic deficits is unavailable. OBJECTIVES:We developed PlateChek, a whole blood-based dielectric coagulometry microsensor that uses a gold electrode surface coated with a platelet-specific agonist, and tested the hypothesis that PlateChek can detect hemostatic impairment due to both platelet count and function defects. METHODS:PlateChek incorporates a gold electrode coated with thrombin receptor-activating peptide (TRAP)-6 to render clotting in a platelet-specific manner. Healthy blood was manipulated in vitro to recapitulate platelet count or function defects and tested in PlateChek to assess platelet-specific readout signatures. Finally, blood samples from patients with thrombocytopenia or platelet aggregation dysfunctions were used to validate PlateChek's ability to detect clinically relevant hemostatic impairment. RESULTS:In PlateChek, clotting kinetics were delayed in samples with platelet counts of < 50 k/μL compared to healthy controls and clot firmness was reduced with platelet counts of < 100 k/μL. PlateChek was sensitive to prolonged clotting time induced by vorapaxar inhibition and reduced clot firmness induced by tirofiban inhibition. PlateChek was sensitive to platelet counts of < 70 k/μL and platelet aggregation dysfunctions in clinical blood samples. CONCLUSION:PlateChek is a novel whole blood-based coagulometer with translational potential that directly correlates both platelet count and function defects to hemostatic impairment.

This work presents an innovative method based on aerosol jet printing (AJP) for surface functionalization of electrodes with bioactive reagents for use in microfluidic sensors. AJP, a high-resolution deposition technique, enables precise and repeatable printing of colloidal suspensions, including biological materials, with excellent edge definition. Specifically, tissue factor (TF) is deposited onto gold electrodes designed for dielectric blood coagulometry and structured into a set number of 4 mm -long lines. This allows systematic and dose-dependent evaluation of the effects of controlled TF deposition on whole blood clotting kinetics. With five printed lines covering ~ 0.58 mm2 of surface area, the resulting clotting kinetics (clotting time of 4. 9 8 ± 0.69 min) closely match those observed with the conventional method of manual mixing of TF (clotting time of 4.81 ± 0.58 min), as assessed by dielectric blood coagulometry measurements. Statistical analysis also confirms the accuracy and reproducibility of AJP-based TF deposition across different fabrication batches, highlighting its utility for dose-dependent control of clotting kinetics. This work underscores the potential of AJP for scalable and automated electrode biofunctionalization in microfluidic sensing applications.

Sickle cell disease (SCD) is characterized by the polymerization of hemoglobin S (HbS) upon deoxygenation, leading to the formation of sickled red blood cells (RBCs) with reduced deformability. Under hypoxic conditions, the impaired RBC behavior significantly contributes to vaso-occlusive events, hemolysis, and end-organ damage. Consequently, RBC deformability serves as a pivotal hemorheological biomarker for evaluating disease severity and therapeutic response. The OcclusionChip, a microfluidic assay, measures RBCs deformability through microcapillary occlusion. However, its current hypoxic assay relies on a complex nitrogen gas setup, rendering it bulky, expensive, and unsuitable for point-of-care diagnostic use. Here, we optimized a chemically induced hypoxia assay using sodium metabisulfite (SMB) within the OcclusionChip platform and validated the hypoxia occlusion index (HOI) as a robust measure of RBC deformability in SCD. Optimal hypoxia conditions were established, replicating nitrogen-induced hypoxia without affecting RBC membrane integrity, reactive oxygen species (ROS) levels, or phosphatidylserine (PS) exposure. Under these conditions, RBCs from individuals with heterozygous (HbAS), HbSC, and HbSS genotypes showed significantly higher HOI compared to healthy controls (HbAA), correlating strongly with clinical biomarkers in SCD. Additionally, the HOI assay effectively assessed the efficacy of therapeutic agents, including hemoglobin-oxygen affinity modifiers (GBT021601, GBT440) and protein kinase R (PKR) activators (PKR-3, FT4202), which significantly reduced OI in SCD RBCs. Notably, combination therapies showed enhanced effectiveness, highlighting the assay's potential for optimizing treatment regimens. This study establishes the chemically induced hypoxia OcclusionChip assay as a reliable and clinically useful tool for evaluating RBC deformability in SCD, with significant potential to improve personalized treatment strategies and thus patient outcomes.

This paper presents a portable, palmtop, battery-operable, dielectric blood coagulometry platform incorporating a single-chip, four-channel, microfluidic sensor. The system incorporates building blocks for generating the sensor excitation signal at 1 MHz, performing multichannel multiplexed impedance measurements, controlled heating of the sensor to a target temperature of 37 °C, and plotting the output data in real time on a 4.3-inch touchscreen display, all within a 17 cm × 9 cm × 5 cm 3D-printed enclosure and weighing ~470 g. The system dissipates ~2 W during active testing and is powered by either a 5-V USB-C input or a 6-Ah, 3.7-V lithium polymer (LiPo) battery, permitting continuous operation for up to 11 hours. When measuring fixed test impedances across all four channels, the system exhibits an excellent agreement with an Agilent 4294A impedance analyzer, with rms errors of ≤0.10% over a capacitance range of 47–330 pF and ≤0.37% over a conductance range of 2.13–10 mS. The system functionality is further evaluated in experiments involving healthy human whole blood samples that are in vitro-treated with heparin and tested in two uncoated and two tissue factor (TF)-coated microchannels of the sensor along with untreated samples. With heparin being an anticoagulant, the system expectedly shows a significant prolongation of coagulation time for heparinized blood samples compared to untreated ones with uncoated microchannels. However, with TF-coated microchannels, there is no significant difference in the coagulation time between untreated and heparinized blood samples due to a strong procoagulant effect of TF. This miniaturized, whole blood-based platform has significant potential for rapid assessment of hemostasis using microliter-range sample volumes at the point-of-need.

This work presents a surface-coating procedure for tissue factor (TF)-based functionalization of gold electrodes of a microfluidic dielectric sensor, termed ClotChip. The procedure, which does not involve any incubation or washing steps, is shown to be highly effective in accelerating the coagulation kinetics through the action of TF in the extrinsic coagulation pathway and in reducing whole blood coagulation time. Specifically, the ClotChip Tpeak readout parameter, which has previously been shown to indicate the coagulation time of a whole blood sample, is measured under different experimental conditions. Across six different blood samples, baseline Tpeak values (i.e., in the absence of any TF) are measured to be 12.03 ± 1.92 min. Manual addition of TF to the blood samples at a concentration of 2 pg/mL reduces Tpeak to 3.23 ± 0.58 min, while TF coating of the ClotChip gold electrodes with the incubation-free and wash-free procedure shortens Tpeak to 4.24 ± 0.44 min. This simplified surface-coating procedure can facilitate multi-reagent biofunctionalization of a multichannel ClotChip microsensor in future work.

Accurate assessment of fibrin clot stability can predict bleeding risk in coagulopathic conditions such as thrombocytopenia and hypofibrinogenemia. Hyperfibrinolysis - a clinical phenotype characterized by an accelerated breakdown of the fibrin clot - makes such assessments challenging by obfuscating the effect of hemostatic components including platelets or fibrinogen on clot stability. In this work, we present a biofunctionalized, microfluidic, label-free, electronic biosensor to elicit unique, specific, and differential responses from the multifactorial processes of blood coagulation and fibrinolysis ex vivo. The microsensor tracks the temporal variation in the normalized real part of the dielectric permittivity of whole blood (<10 μL) at 1 MHz as the sample coagulates within a three-dimensional, parallel-plate, capacitive sensing area. Surface biofunctionalization of the microsensor's electrodes with physisorption of tissue factor (TF) and aprotinin permits real-time assessment of the coagulation and fibrinolytic outcomes. We show that surface coating with TF and manual addition of TF result in a similar degree of acceleration of coagulation kinetics in human whole blood samples. We also show that surface coating with aprotinin and manual addition of aprotinin yield similar results in inhibiting tissue plasminogen activator (tPA)-induced upregulated fibrinolysis in human whole blood samples. Validated through a clinically relevant, complementary assay - rotational thromboelastometry for clot viscoelasticity - we finally establish that a microsensor dual-coated with both TF and aprotinin detects the hemostatic rescue in the tPA-induced hyperfibrinolytic profile of whole blood and the hemostatic dysfunction due to concurrent platelet depletion in the blood sample, thus featuring enhanced ability in evaluating complex, combinatorial coagulopathies.

Background Individuals with sickle cell disease (SCD) have red blood cell functional abnormalities which contribute to acute complications. Hydroxyurea (HU), a fetal hemoglobin (HbF) inducer, is a mainstay therapy that benefits up to 50% of adult individuals with SCD. The patients who experience an increase in HbF levels in response to HU are considered HU responders. Although high HbF is protective on a population level, %HbF does not always correlate with clinical severity on an individual level. Typically, second-line drugs like voxelotor are added to HU after clinical worsening is noted. We hypothesize that worsening RBC function may indicate need for a second-line therapy initiation before clinical decline is apparent. One way to measure RBC deformability is with the MIRCA (Microfluidic Impedance Red Cell Assay) a microfluidics device that mechanically measures the ability of a red blood cell (RBC) to squeeze between pillars. Trapped, poorly deformable cells are measured by an impedance analyzer. The device readouts are an occlusion index (OI) under normoxia (NOI) or with sodium metabisulfite (MBS)-induced hypoxia (HOI). Here we present a clinical validation of MIRCA for use in monitoring the need for second-line agents in individuals with SCD on HU. Methods One hundred and nineteen peripheral blood samples from 66 adults and 35 pediatric individuals (87 HbSS/Sβ0, 14 HbSC/Sβ+/SE) were collected in EDTA under an Emory University IRB approved protocol. Whole blood was run on an ADVIA 2120i hematology analyzer to obtain complete blood counts and percent dense red blood cells (%DRBC). Washed RBCs were suspended to 20% (v/v) in 1X PBS or 1.5% (w/v) MBS in 1X PBS to run on MIRCA. Vaso-occlusive events (VOE), acute chest syndrome (ACS), avascular necrosis (AVN), and SCD complications (retinopathy, nephropathy, priapism, splenic sequestration, chronic pain) were determined by chart review. VOE and ACS in the past year were summed to define an acute events (AE) variable. A composite variable was used for disease complications (DC). The association of MIRCA readouts with laboratory data, AE, AVN, and DC were analyzed with linear mixed model, negative binomial model, or Kruskal-Wallis test using StataNow 18.5 (College Station, TX); a p < 0.05 was considered significant. Age, genotype, and maximum tolerated HU dose (MTD - HU ≥ 35 mg/kg/day or absolute neutrophil count < 4000/µl). were added as covariates while analyzing the laboratory data. Genotype, age, %HbF, %DRBC, absolute reticulocyte count (ARC), and absolute neutrophil count (ANC) were added as covariates while analyzing AE and DC. Results Out of 101 patients, 32 were on HU, and 39 were on HU MTD. Higher NOI was associated with higher DC (p = 0.02) after adjusting for genotypes and DRBC. Age, %HbF, ARC, and ANC were not retained in the model due to their p values >0.05. Higher NOI was associated with higher AE (p = 0.02) in the univariate regression, but the association was not seen when adjusted for HbF and age. HOI was not associated with AE or DC. HOI and NOI were not associated with AVN. NOI and HOI values were 2.64% (p = 0.04) and 10.75% (p = 0.004) higher in adults than in children, respectively. NOI and HOI were lower by 6% (p = 0.002) and 18.2% (p = 0.001) in HbSC, HbSβ+, and HbSE individuals compared to HbSS and HbSβ0 individuals, respectively. NOI and HOI were not associated with sex and ARC. Individuals on HU at MTD had 6.9% lower HOI than those not at MTD (p = 0.04). Higher HOI was associated with higher ANC (p < 0.001), higher DRBC (p = 0.04), and lower Hb (p = 0.049). Higher NOI was associated with higher DRBC (p < 0.001) and lower Hb (p = 0.001). NOI was not associated with MTD and ANC. Discussion Higher NOI values were associated with higher DC after adjusting for covariates, suggesting that rising NOI may be predictive of increased risk of SCD complications. Our cohort included 71 (70.3%) subjects on HU (39 at MTD), indicating that NOI values can be used to monitor patients on HU. NOI may be used to identify subsets of high-risk patients after HU optimization that may benefit from second-line therapies. We plan to use HOI and NOI in combination with traditional clinical laboratory tests to predict the need for additional therapeutic intervention. MIRCA can be used to monitor RBC deformability at routine clinic visits; worsening of deformability and poor laboratory results will trigger initiation of second-line therapies before patients experience clinical decline.

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.

Introduction Sickle cell disease (SCD) is an inherited blood disorder characterized by the production of sickle hemoglobin (HbS). Under hypoxia, HbS polymerizes, causing red blood cells (RBCs) to become sickle-shaped and less deformable. These sickled RBCs can occlude capillaries and contribute to organ damage. We previously developed a microfluidic device to assess RBC-mediated microvascular occlusion under hypoxia using isolated RBCs, which required laborious pre-processing steps to remove white blood cells (WBCs) (Oshabaheebwa et al., 2023). These additional steps increased the assay's complexity, hindering its adoption beyond research laboratories. Here, we present an alternative method to assess RBC-mediated microvascular occlusion under hypoxia using whole blood. We also quantify WBCs' contribution to microcapillary occlusion. This approach significantly reduced the required blood volume, total assay time, number of steps, and overall operation complexity. Methods Microfluidic devices were fabricated using standard photolithography and polydimethylsiloxane (PDMS) micro-molding protocols. The device comprised 6 micropillar arrays with interpillar distances decreasing from 12 μm at the inlet to 3 μm at the outlet, mimicking the capillary network (Oshabaheebwa et al., 2024). Venous blood samples were collected in EDTA tubes from participants with HbAA (n=4) and HbSS (n=12) under an IRB-approved protocol. Whole blood was suspended in a solution containing 6% (v/v) EC-Oxyrase (an oxygen-reducing enzyme), 0.13M sodium lactate, and 1X PBS buffer, then incubated for 30 minutes at 37°C to achieve deoxygenation. The volume of whole blood (range: 4-7 µL) was adjusted to achieve 0.2% hematocrit. Samples were perfused through the microfluidic device at a constant inlet pressure of 200 mbar and the temperature was maintained at 37°C. After 15 minutes of perfusion, images were obtained with an Olympus IX83 microscope, and occluding RBCs and WBCs were counted manually in Adobe Photoshop. The occlusion index (OI) was calculated to represent the percent occlusion of the microcapillary network. The OI obtained from this novel whole blood assay was compared to the previously described method of measuring hypoxic OI from isolated RBCs (Oshabaheebwa et al., 2023). To optimize the incubation time, the rate of deoxygenation by EC-Oxyrase was measured using an oxygen sensor (Ocean Insight, Rochester, NY). Samples containing EC-Oxyrase, or a blank PBS buffer, were added to a conical tube, an oxygen probe was immediately inserted, and the partial pressure of oxygen (PO2) was monitored continuously for 15 minutes. Results Hypoxic OI for HbSS whole blood samples (HOI-WB) was significantly higher than for HbAA (mean ± SD: 14.7% ± 4.79% vs. 1.83% ± 0.79%, P = 0.0011). Occlusions from WBCs alone (OI-WBC) in HbAA samples ranged from 0.23% to 1.65%, with total OI (HOI-WB, WBCs + RBCs) between 1% to 2.6%. In HbSS samples, OI-WBC ranged from 1.31% to 2.62%, while HOI-WB ranged from 8.15% to 21.7%. HOI-WB was strongly correlated (PCC=0.9391, P=0.0054) with Hypoxia OI measured with the previously reported assay that used isolated RBCs. The PO2 in samples containing EC-Oxyrase mixed with either PBS, HbAA, or HbSS diluted whole blood reduced to an average of 0.61 ± 0.2 mmHg within 5 minutes of incubation. Conversely, the PO2 in a blank PBS buffer without EC-Oxyrase remained at an average value of 158 mmHg. Conclusion WBCs made minimal contribution to the microcapillary occlusion thus HOI-WB was mainly driven by RBCs. Moreover, HOI-WB was significantly higher in SCD indicating the assay's sensitivity to HbS-induced RBC impairment. Importantly, HOI-WB strongly correlated with previous OI measurements that used isolated RBCs. While HOI-WB was obtained after 30 minutes of incubation with EC-Oxyrase, samples' full deoxygenation was detected in <5 minutes. Future studies will assess whether similar HOI-WB is observed with 5-minute incubation. This assay utilizes a small blood volume, eliminates need for auxiliary equipment for blood processing, and minimizes the expertise required to run the assay. These improvements in efficiency and simplicity bring us closer to rapid, bedside monitoring of RBC-mediated microcapillary occlusion, and enabling adoption in resource-limited settings. References Oshabaheebwa, S, et al. (2023) Blood 142, 5022 Oshabaheebwa, S, et al. (2024) Biosensors and Bioelectronics 258: 116352

This paper investigates the effect of surface roughness on the kinetics of whole blood coagulation through a comparative study of two different electrode fabrication techniques, screen-printing and sputtering, for a microfluidic dielectric sensor termed ClotChip. Featuring a three-dimensional, parallel-plate, capacitive sensing structure with gold sensing and floating electrodes deposited on polymethyl methacrylate plastic substrates, we have previously shown that ClotChip can measure the temporal variation in the normalized real permittivity of a coagulating whole blood sample (<10 μL) at 1 MHz and provide clinically useful information on the coagulation time of the sample through a time-to-permittivity peak ( Tpeak ) readout parameter. In this work, scanning electron microscope imaging and profilometry measurements reveal that the surface of the sputtered-gold electrode is much smoother than the rough surface of its screen-printed counterpart, having a mean arithmetic average height and root mean square roughness of ∼ 38.0 nm and 47.0 nm versus 5.7 μm and 7.2 μm for the screen-printed electrode. The ClotChip Tpeak parameter has also been measured for both untreated whole blood samples and those modified in vitro with corn trypsin inhibitor, an inhibitor of the activated coagulation factor XII (FXIIa) used to suppress surface-induced coagulation by contact activation. Our results establish that surface-induced, FXIIa-mediated contact activation plays a more prominent role in accelerating the kinetics of whole blood coagulation for rough screen-printed electrodes than for smooth sputtered electrodes.

Introduction: Sickle cell disease (SCD) is a genetic disorder caused by a single mutation in the beta-globin gene, resulting in abnormal sickle hemoglobin (HbS). Under low oxygen tension, HbS polymerizes within red blood cells (RBCs), causing them to sickle, the primary pathological event in SCD. Repeated sickling and unsickling of RBCs yields dehydrated RBCs known as irreversibly sickled cells (ISCs) [1]. The shortened lifespan of sickle RBCs triggers an increase in the production of reticulocytes to sustain stress erythropoiesis. These ISCs, reticulocytes, and other forms of RBCs constitute the subpopulations of sickle RBCs that are key to understanding the disease variability. We previously reported hypercoagulability of SCD blood and reduced clot strength. However, the impact of the different subpopulations of sickle RBCs on the hypercoagulability and dynamic compactness of blood clots has not been examined. Herein, we use a microfluidic dielectric sensor, termed ClotChip®, to investigate the impact of the sickle RBCs subpopulations characterized by enrichment in either reticulocytes, fetal hemoglobin (HbF), or ISC on the clotting kinetics and characteristics. Methods: Venous blood samples from subjects with homozygous HbSS and healthy HbAA were collected in sodium citrate tubes under an IRB-approved protocol. RBCs were isolated from plasma and washed 3× in PBS. Using Percoll-renografin (76%) density gradient fractionation, we isolated three different subpopulations of RBCs in HbAA and HbSS samples that were characterized by enrichment in either reticulocyte (Top layer), HbF (Middle layer), or ISC (Bottom layer) [2]. 1mL of washed RBCs were carefully layered onto 20mL of the gradient mix and ultracentrifuged for 20 min at 35,000g and 5 °C. The subpopulations from each sample were harvested and washed 3× in PBS before reconstituting them back in their plasma at 20% Hematocrit. Samples were mixed with CaCl2 to start coagulation and immediately injected into the ClotChip®. The ClotChip® readout curve, which is defined as the temporal variation of the normalized real part of blood dielectric permittivity at 1 MHz, was obtained using a miniaturized impedance analyzer (MIA) system. Two parameters were extracted from the ClotChip® readout curve, i.e., Tpeak [3], Δεr,max [4]. Data was reported as mean ± standard deviation Results: ClotChip® Tpeak (as a surrogate for clotting time) and Δεr,max (as a surrogate for clot strength) readout parameters for three RBCs subpopulations from HbSS (n=10) and HbAA (n=8) were analyzed. The clotting time of HbSS samples were reduced by ~40% compared to HbAA. No significant difference was observed in the clotting time across all subpopulations for HbAA; (bottom vs. middle layer vs. top layer; p=0.594, p =0.357, p=0.103 respectively). No significant difference was observed in the clotting time across all subpopulations for HbSS (bottom vs. middle layer vs. top layer; p=0.940, p=0.678, p=0.659 respectively). The clot strength of HbSS samples were reduced by ~10% across all subpopulations. There were statistical differences in HbAA (middle vs top layer; p=0.019) subpopulations; however, no differences were observed for all other subpopulations (bottom vs. middle layer; p=0.839 and bottom vs. top layer; p = 0.061). For HbSS, significant differences were observed for clot strength across all subpopulations (bottom vs. middle layer vs. top layer; p=0.024, p=0.001, p=0.000 respectively). p-values were determined using a paired t-test). Conclusion: Our results suggest that RBC heterogeneity impacts clot strength significantly but not clotting time. In SCD, the clot strength is affected by all subpopulations of RBCs; however, reticulocyte-enriched subpopulations improved the clot strength more compared to enriched HbF and ISCs. Our findings also suggest that patient-to-patient variability in the clotting dynamics and characteristics might be due to the heterogeneous distribution of RBCs in SCD. Further investigation is needed to understand the contribution of each subpopulation of RBCs to the clinical outcomes in SCD clotting characteristics. References: 1. Kato, G.J., et al., Nat Rev Dis Primers, 2018. 4: p. 18010. 2. Vettore, L., M.C. De Matteis, and P. Zampini,. Am J Hematol, 1980. 8(3): p. 291-7. 3. Maji, D., et al., J Thromb Haemost, 2018. 16(10): p. 2050-2056. 4. Maji, D., et al., IEEE Trans Biomed Circuits Syst, 2017. 11(6): p. 1459-1469.

Rapid point-of-care (POC) assessment of thrombosis is clinically important in patients who develop significant blood coagulation abnormalities such as noted with sepsis or COVID-19. In this work, we compare the coagulation profiles of whole blood in sepsis and COVID-19 patients using a handheld dielectric coagulometer termed ClotChip ® . ClotChip ® is a three-dimensional, parallel-plate, capacitive sensor integrated into a single-use microfluidic channel with a total volume of <20 µL for sample analysis. The readout curve is defined as the temporal variation in the real part of dielectric permittivity of whole blood at 1 MHz. ClotChip ® is sensitized towards detecting altered coagulation states by adding recombinant thrombomodulin (rTM) or activated protein C (APC). Using a handheld ClotChip ® device, we measure the coagulation status in whole blood samples from hospitalized patients with sepsis and COVID-19 (both regular floor and intensive care unit) and compare it to samples from healthy donors. The coagulation profiles show a difference between COVID-19 and sepsis patients when samples are treated with rTM, as well as a difference between moderate and severe COVID-19 infections when samples are treated with APC. This study demonstrates that ClotChip ® measures a coagulation profile in COVID-19 that is different from that in sepsis, highlighting its future potential as a POC diagnostic/prognostic tool in COVID-19-associated coagulopathy.

Background With the emergence of novel pharmacologic and gene-based therapies, identifying the rheological and biophysical RBC abnormalities of sickle cell disease (SCD) not captured by clinical laboratory techniques is crucial. Ektacytometry (LORRCA), the current standard to assess RBC function, does not reflect mechanical stress that RBCs experience in capillary microvasculature in the body. The device is costly, requires a nitrogen gas tank, and its use is difficult to translate outside of large-scale laboratory settings. In comparison, the Microfluidic Impedance Red Cell Assay (MIRCA) is a low-cost, portable device that mimics capillary microvasculature with micropillar arrays spaced 3-12 µm apart. Thus, MIRCA measures mechanical deformability, which is more physiologic, mimicking RBCs squeezing through microvasculature. An impedance analyzer calculates the MIRCA Occlusion Index (OI), representing the % occlusion of the chip. Here we compare the MIRCA to the LORRCA, assessing the correlations of OI and Elongation Index maximum (EI) to conventional laboratory tests and SCD related complications. Methods Peripheral blood from 53 adult (n = 28) and pediatric (n = 25) individuals (HbSS = 35, HbSB0 = 6, HbSC = 5, HbSB0 = 1, and HbAA controls = 6) were obtained under an Emory University IRB approved protocol. Patients transfused < 90 days prior were excluded. Hospitalizations / emergency department (ED) visits for pain within 12 months of sample collection were determined by chart review. Samples were collected in EDTA, stored at 4°C up to 48 hours, centrifuged at 500g, washed, and resuspended to a 20% hematocrit to run on MIRCA, an oxygen gradient ektacytometer (LORRCA), and an ADVIA hematology analyzer. Polydimethylsiloxane (PDMS) fabricated MIRCA chips were bonded to a standard glass slide with pairs of gold-sputtered electrodes adjacent to each array. Chips were prepared by perfusing ethanol, 1X phosphate-buffered saline (PBS), and 3% bovine-serum albumin (BSA) in 1X PBS via syringe pump and were incubated overnight at 4°C prior to use. Baseline impedance was taken for a 2-minute perfusion of 1X PBS, then OIs were calculated from impedance values 10 minutes after sample introduction normalized to baseline. The data was analyzed using the Mann-Whitney U test, Spearman correlation, and linear regression. OriginPro (Northampton, MA, USA) and Stata 18 (College Station, TX, USA) were used for the analyses and a p < 0.05 was considered significant. Results The correlation coefficients for OI and EI with laboratory tests (Table 1) and to each other (Figure 1) were comparable (p < 0.05). HbSS/SB0 had higher OIs than HbSC/SB+ (median = 13.7% vs 6.8%, p < 0.01) and HbAA (median = 4.6%, p < 0.01). Patients with ≥ 1 vaso-occlusive events (VOE) in the past year had higher OIs and lower EIs compared to those without a VOE (median = 15.3% vs 9.6%, p <0.01; median = 0.4 vs 0.5, p = 0.02, respectively). Patients that received acute medical care (admission or ED visit) had higher OIs and lower EIs compared to those that did not (median = 15.7% vs 9.8%, p < 0.01; median = 0.4 vs 0.5, p = 0.02, respectively). In multiple linear regression, HbSS/SB0 (p < 0.01), adults (p = 0.01), and DRBC (p < 0.01) were associated with OI while HbSS/SB0 (p = 0.01), DRBC (p < 0.01), hemoglobin % (p = 0.04), absolute neutrophil count (p = 0.02), and VOE+ (p = 0.02) were associated with EI. The models explained 55.2% and 66.8% variability of OI and EI, respectively. Lower Akaike's information criterion (AIC, 186 vs -80) and Bayesian information criterion (BIC, 192 vs -71) of the EI model suggest a higher correlation to conventional laboratory values and clinical features compared to OI; OI may possibly be a unique biomarker that both laboratory tests and LORRCA fail to fully characterize. Conclusion MIRCA OI and LORRCA EI normoxic deformability are correlated to each other, and both show statistically comparable correlations to many parameters associated with SCD severity. Both OI and EI are significantly associated with SCD outcomes like VOE and acute care. Due to lower device size, cost, and relative ease-of-use, MIRCA may be more convenient for routine clinical use and for use in low resource settings where LORRCA costs and requirements hamper implementation. As the addition of chemical hypoxia to MIRCA is now underway, future work will examine OI under hypoxia and its associations with SCD-relevant outcomes like pain events, stroke and acute chest syndrome.

Introduction Sickle cell disease (SCD) is caused by a genetic mutation that produces sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, causing red blood cells (RBCs) to become sickle-shaped and poorly deformable. Stiff sickle RBCs contribute to microvascular occlusion and organ damage. Previous studies have developed microfluidic devices for assessing RBC-mediated occlusion under hypoxia, but the complexity of the setup and data acquisition limits their adoption. A rapid, easy-to-use, point-of-care assay for assessing RBC-mediated occlusion could increase throughput and aid in determining outcomes of novel therapies, including gene therapies, for SCD. Here, we induced RBC sickling using a chemical method in a simplified workflow to study hypoxic RBC-mediated microcapillary occlusion for healthy (HbAA), sickle cell trait (HbAS), hemoglobin SC disease (HbSC), and homozygous SCD (HbSS) RBCs. Additionally, a custom miniaturized impedance analyzer (MIA) was developed to provide a rapid and fully electronic measurement of occlusion within 10 minutes of sample perfusion. Methods Microfluidic devices were fabricated using standard photolithography and polydimethylsiloxane (PDMS) micro-molding protocols. Glass slides were coated with gold electrodes by sputter deposition and bound to the PDMS microfluidic devices using oxygen plasma. The microfluidic devices comprised six microcapillary arrays of widths 12, 10, 8, 6, 4, and 3 μm, with each array coupled with a pair of gold electrodes [3]. The MIA included an ADALM2000 software-defined instrument module, a custom-designed front-end interface board, and a Raspberry Pi single-board computing module. The ADALM2000 was used to generate and acquire a 10-kHz measurement signal, while the front-end interface board handled signal amplification and multiplexed signal routing through the microcapillary arrays. A custom graphical user interface on the Raspberry Pi allowed users to set experimental parameters and monitor data in real time. Venous blood samples were collected in EDTA tubes from participants with HbAS (n=9), HbSC (n=9), HbSS (n=13), and HbAA (n=6) under an IRB-approved protocol. To induce hypoxia, washed RBCs were suspended at 20% hematocrit in a 1.5% (w/v) sodium metabisulphite in 1× PBS buffer and incubated for 3 minutes at room temperature. For normoxia, RBCs from the same donors were suspended at 20% hematocrit in PBS and incubated with the same conditions. Samples were then perfused through the microfluidic device at a constant inlet pressure. After 10 minutes of perfusion, the change in electrical impedance across each array was used to calculate an occlusion index (OI), representing the percent occlusion of the capillary network. Results Normoxic OI for HbSS and HbSC was significantly higher than HbAA but there were no statistical differences in HbAA vs. HbAS and HbSC vs. HbSS (Figure 1A). HbAS, HbSC, and HbSS RBCs had significantly higher occlusion under hypoxia than in normoxia (Figure 1A, 1.46 ± 1.67% vs. 8.43 ± 2.78%, P = 0.004 for HbAS, 4.56 ± 2.77% vs. 27.1 ± 17.0%, P = 0.004 for HbSC, and 8.01 ± 6.26% vs. 58.3 ± 26.2%, P = 0.0002 for HbSS). Occlusion of HbAA RBCs remained low in both normoxia and hypoxia (2.70 ± 0.643% vs. 4.72 ± 0.953%, P = 0.0625). Hypoxic OI was significantly different between HbAA vs. HbAS (P = 0.017), HbAS vs. HbSC (P = 0.004), and HbSC vs. HbSS (P = 0.007). Hypoxic OI in HbSC and HbSS was associated with elevated markers of RBC hemolysis - absolute reticulocyte count (ARC, P = 0.0061) and lactate dehydrogenase (LDH, P < 0.0001, Figure 1B). Conclusion In normoxia, HbSC had comparable OI to HbSS likely due to HbC induced efflux of potassium ions and water resulting in cellular dehydration and poor RBC deformability. The hypoxic assay, however, unmasked the greater severity of HbSS. Further, elevated hypoxic OI in HbAS may be linked to higher risk of organ damage in this genotype. Chemically-induced hypoxia combined with electrical impedance-based measurement of RBC-mediated microcapillary occlusion offers a new technique for rapidly assessing RBC health and function in a low-oxygen environment. This approach eliminates the need for expensive, high-resolution microscopes and complex gas-exchange chambers. This may serve as a new standard for assessing the clinical efficacy of new treatments that improve RBC deformability and identifying at-risk patients in otherwise mild SCD genotypes.

Introduction: Sickle cell disease (SCD) is a common genetic red blood cell (RBC) disorder that results from a single substitution of valine for glutamic acid in the β-globin gene, resulting in sickle hemoglobin (HbSS). Under hypoxia, HbS polymerizes and forms fibers within the RBCs, thus making the cells rigid, deformed, and sickle-shaped [1]. Deformed RBCs influence the compactness and dynamics of blood clots [2]. In addition, people with SCD are susceptible to thrombophilia [2,3,4]. Recently, there has been a heightened interest in ameliorating SCD using RBCs pyruvate kinase (PKR) activators, which increase ATP and decrease 2,3 DPG, resulting in increased oxygen affinity and improved RBCs deformability [5]. Voxelotor is also an oxygen affinity-modifying drug, recently FDA-approved. Voxelotor delays deoxy-HbSS formation, preventing in vitro HbSS polymerization and RBC sickling, thus improving RBC deformability [6]. The in vitro impact of PKR activators and Voxelotor on blood clotting in clinical samples from people with SCD has not been examined. Here we use a microfluidic dielectric sensor, termed ClotChip ® [7,8], to investigate the effects of Voxelotor and a PKR activator on the clotting time and clot strength of samples from people with SCD. Methods: Venous blood samples were collected in sodium-citrate from subjects with homozygous HbSS and healthy volunteers (HbAA) under an IRB-approved protocol. ClotChip ® microsensors were fabricated as previously described [7]. Blood samples were centrifuged at 200 g for 10 min. Plasma and buffy coats were removed and stored at 4 °Cfor 6 hours. Isolated, washed RBCs were then re-suspended in PBS at 20% hematocrit and then mixed with 67 mg/mL of Voxelotor (GBT440; Selleckchem) to a final concentration of 600 µM (in DMSO) or a PKR activator (PKR activator 3; MedChem Express) at 10 mM (in DMSO) and incubated at 37 °Cfor 6 hours. For controls, HbSS- or HbAA-containing RBCs at 20% hematocrit were mixed with PBS containing 0.5% v/v DMSO and incubated at 37 °C for 6 hours. After incubation, HbSS+Vox, HbSS+PKR, and control samples were centrifuged at 500 g for 10 min to remove PBS. Each sample's plasma was used to reconstitute RBCs at 20% hematocrit. CaCl 2 was added to the samples to induce coagulation, and the samples were injected into the ClotChip ® microfluidic sensors. An Agilent 4294A impedance analyzer was used to obtain the ClotChip ® readout curve defined as the temporal variation of the normalized real part of blood dielectric permittivity at 1 MHz (see Figure 1A). Based on our previous studies [7,8], the time to reach a permittivity peak (T peak) parameter was taken to indicate the clotting time, whereas the maximum change in permittivity after the peak (ΔƐ r,max) parameter was taken to indicate the clot strength. Data are reported as mean ± standard deviation (SD). Results: We analyzed the ClotChip ® T peak and ΔƐ r,max readout parameters for HbSS (n=11), HbSS+Vox (n=11), HbSS+PKR (n=11), and HbAA control (n=8) samples ( Figure 1B). The clot strength was ~50% higher in HbAA than HbSS (HbAA vs. HbSS: p= 0.001, Mann-Whitney test). However, when the SCD samples were treated with Voxelotor, the clot strength was observed to increase by ~10% (HbSS vs. HbSS+Vox, p=0.01, Paired t-test). Treatment with a PKR activator increased the clot strength by ~30% (HbSS vs. HbSS+PKR, p=0.003, Figure 1B). A significant difference was observed in the clotting time between HbAA vs. HbSS (p=0.001) but treatment with Voxelotor or a PKR activator did not change the clotting time for HbSS (HbSS vs. HbSS+Vox, p=0.43; HbSS vs. HbSS+ PKR, p=0.29) ( Figure 1B). (p values were determined using a non-parametric Mann-Whitney test and paired t-test). Discussion : We found that treatment of HbS-containing RBCs with a PKR activator or voxelotor improves clot strength but not clotting time. PKR activators improved the strength of the clot significantly more than Voxelotor. Patients with SCD had a faster clotting time than healthy participants, which agrees with earlier researchers on hypercoagulability in SCD [4]. These data suggest that HbS polymerization and RBC health affect clot characteristics but not clot kinetics in SCD. Further investigations may be required to relate RBC deformability, which is well-known to impact SCD pathophysiology, to blood clotting and venous thromboembolism.

This article presents a standalone, multichannel, miniaturized impedance analyzer (MIA) system for dielectric blood coagulometry measurements with a microfluidic sensor termed ClotChip. The system incorporates a front-end interface board for 4-channel impedance measurements at an excitation frequency of 1 MHz, an integrated resistive heater formed by a pair of printed-circuit board (PCB) traces to keep the blood sample near a physiologic temperature of 37 °C, a software-defined instrument module for signal generation and data acquisition, and a Raspberry Pi-based embedded computer with 7-inch touchscreen display for signal processing and user interface. When measuring fixed test impedances across all four channels, the MIA system exhibits an excellent agreement with a benchtop impedance analyzer, with rms errors of ≤0.30% over a capacitance range of 47-330 pF and ≤0.35% over a conductance range of 2.13-10 mS. Using in vitro-modified human whole blood samples, the two ClotChip output parameters, namely, the time to reach a permittivity peak (Tpeak) and maximum change in permittivity after the peak (Δϵr,max) are assessed by the MIA system and benchmarked against the corresponding parameters of a rotational thromboelastometry (ROTEM) assay. Tpeak exhibits a very strong positive correlation (r = 0.98, p < 10-6, n = 20) with the ROTEM clotting time (CT) parameter, while Δϵr,max exhibits a very strong positive correlation (r = 0.92, p < 10-6, n = 20) with the ROTEM maximum clot firmness (MCF) parameter. This work shows the potential of the MIA system as a standalone, multichannel, portable platform for comprehensive assessment of hemostasis at the point-of-care/point-of-injury (POC/POI).

Dielectric blood coagulometry is a newly emerging approach for the assessment of the hemostatic potential of blood using the electronic technique of dielectric spectroscopy. Since the aggregation and deformation of red blood cells (RBCs) - two critical processes underlying blood coagulation - play a pivotal role in this approach, it is essential to characterize such measurements at reduced hematocrits for applications that involve anemic blood samples. To that end, we develop a protocol for creating reconstituted whole blood (rWB) samples with diminished hematocrits as low as ~10%. These samples are next evaluated using our microfluidic dielectric coagulometer - termed ClotChip - as well as rotational thromboelastometry (ROTEM), which is the clinical standard for viscoelastic coagulometry. We find that rWB samples with hematocrits as low as~10%still exhibit a characteristic dispersion region at MHz frequencies that is attributed to the interfacial polarization of RBC membranes. Furthermore, we show that the two ClotChip readout parameters indicative of clotting time and clot firmness correlate well with those measured by ROTEM for rWB samples with hematocrits in the range of ~10-40 % . This work illustrates the viability of ClotChip as a dielectric blood coagulometer to assess hemostatic function at hematocrits as low as ~10 % .

This paper presents a standalone, multichannel, miniaturized impedance analyzer (MIA) for dielectric coagulometry measurements on human whole blood in a microfluidic sensor. The system incorporates a front-end interface board for 4-channel impedance measurements at an excitation frequency of 1 MHz, an integrated resistive heater formed by a printed-circuit board (PCB) trace to keep the blood sample at a physiologic temperature of 37°C, an ADALM2000 software-defined instrument module for signal generation and data acquisition, and a Raspberry Pi-based embedded computer with 7-inch touchscreen display for signal processing and user interface. When measuring fixed test impedances, the 4-channel MIA exhibits an excellent agreement to a commercial Agilent 4294A impedance analyzer with rms errors of <0.3% over a capacitance range of 47–330 pF and <0.5% over a conductance range of 2.13–10 mS. Dielectric coagulometry measurements on human whole blood also show a very good agreement between the MIA and the Agilent impedance analyzer.