A miniaturized wash-free microfluidic assay for electrical impedance-based assessment of red blood cell-mediated microvascular occlusion.

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.

A Multichannel Portable Platform With Embedded Thermal Management for Miniaturized Dielectric Blood Coagulometry.

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).

Assessment of fibrinolytic status in whole blood using a dielectric coagulometry microsensor.

Rapid assessment of the fibrinolytic status in whole blood at the point-of-care/point-of-injury (POC/POI) is clinically important to guide timely management of uncontrolled bleeding in patients suffering from hyperfibrinolysis after a traumatic injury. In this work, we present a three-dimensional, parallel-plate, capacitive sensor - termed ClotChip - that measures the temporal variation in the real part of blood dielectric permittivity at 1 MHz as the sample undergoes coagulation within a microfluidic channel with <10 µL of total volume. The ClotChip sensor features two distinct readout parameters, namely, lysis time (LT) and maximum lysis rate (MLR) that are shown to be sensitive to the fibrinolytic status in whole blood. Specifically, LT identifies the time that it takes from the onset of coagulation for the fibrin clot to mostly dissolve in the blood sample during fibrinolysis, whereas MLR captures the rate of fibrin clot lysis. Our findings are validated through correlative measurements with a rotational thromboelastometry (ROTEM) assay of clot viscoelasticity, qualitative/quantitative assessments of clot stability, and scanning electron microscope imaging of clot ultrastructural changes, all in a tissue plasminogen activator (tPA)-induced fibrinolytic environment. Moreover, we demonstrate the ClotChip sensor ability to detect the hemostatic rescue that occurs when the tPA-induced upregulated fibrinolysis is inhibited by addition of tranexamic acid (TXA) - a potent antifibrinolytic drug. This work demonstrates the potential of ClotChip as a diagnostic platform for rapid POC/POI assessment of fibrinolysis-related hemostatic abnormalities in whole blood to guide therapy.

Microfluidic electrical impedance assessment of red blood cell-mediated microvascular occlusion.

Alterations in the deformability of red blood cells (RBCs), occurring in hemolytic blood disorders such as sickle cell disease (SCD), contribute to vaso-occlusion and disease pathophysiology. There are few functional in vitro assays for standardized assessment of RBC-mediated microvascular occlusion. Here, we present the design, fabrication, and clinical testing of the Microfluidic Impedance Red Cell Assay (MIRCA) with embedded capillary network-based micropillar arrays and integrated electrical impedance measurement electrodes to address this need. The micropillar arrays consist of microcapillaries ranging from 12 µm to 3 µm, with each array paired with two sputtered gold electrodes to measure the impedance change of the array before and after sample perfusion through the microfluidic device. We define RBC occlusion index (ROI) and RBC electrical impedance index (REI), which represent the cumulative percentage occlusion and cumulative percentage impedance change, respectively. We demonstrate the promise of MIRCA in two common red cell disorders, SCD and hereditary spherocytosis. We show that the electrical impedance measurement reflects the microvascular occlusion, where REI significantly correlates with ROI that is obtained via high-resolution microscopy imaging of the microcapillary arrays. Further, we show that RBC-mediated microvascular occlusion, represented by ROI and REI, associates with clinical treatment outcomes and correlates with in vivo hemolytic biomarkers, lactate dehydrogenase (LDH) level and absolute reticulocyte count (ARC) in SCD. Impedance measurement obviates the need for high-resolution imaging, enabling future translation of this technology for widespread access, portable and point-of-care use. Our findings suggest that the presented microfluidic design and the integrated electrical impedance measurement provide a reproducible functional test for standardized assessment of RBC-mediated microvascular occlusion. MIRCA and the newly defined REI may serve as an in vitro therapeutic efficacy benchmark for assessing the clinical outcome of emerging RBC-modifying targeted and curative therapies.

Monitoring DOACs with a Novel Dielectric Microsensor: A Clinical Study.

BACKGROUND: There are acute settings where assessing the anticoagulant effect of direct oral anticoagulants (DOACs) can be useful. Due to variability among routine coagulation tests, there is an unmet need for an assay that detects DOAC effects within minutes in the laboratory or at the point of care. METHODS: We developed a novel dielectric microsensor, termed ClotChip, and previously showed that the time to reach peak permittivity (T peak) is a sensitive parameter of coagulation function. We conducted a prospective, single-center, pilot study to determine its clinical utility at detecting DOAC anticoagulant effects in whole blood. RESULTS: We accrued 154 individuals: 50 healthy volunteers, 49 rivaroxaban patients, 47 apixaban, and 8 dabigatran patients. Blood samples underwent ClotChip measurements and plasma coagulation tests. Control mean T peak was 428 seconds (95% confidence interval [CI]: 401-455 seconds). For rivaroxaban, mean T peak was 592 seconds (95% CI: 550-634 seconds). A receiver operating characteristic curve showed that the area under the curve (AUC) predicting rivaroxaban using T peak was 0.83 (95% CI: 0.75-0.91, p < 0.01). For apixaban, mean T peak was 594 seconds (95% CI: 548-639 seconds); AUC was 0.82 (95% CI: 0.73-0.91, p < 0.01). For dabigatran, mean T peak was 894 seconds (95% CI: 701-1,086 seconds); AUC was 1 (p < 0.01). Specificity for all DOACs was 88%; sensitivity ranged from 72 to 100%. CONCLUSION: This diagnostic study using samples from "real-world" DOAC patients supports that ClotChip exhibits high sensitivity at detecting DOAC anticoagulant effects in a disposable portable platform, using a miniscule amount of whole blood (<10 µL).

A novel, point-of-care, whole-blood assay utilizing dielectric spectroscopy is sensitive to coagulation factor replacement therapy in haemophilia A patients.

BACKGROUND: Reliable monitoring of coagulation factor replacement therapy in patients with severe haemophilia, especially those with inhibitors, is an unmet clinical need. While useful, global assays, eg thromboelastography (TEG), rotational thromboelastometry (ROTEM) and thrombin generation assay (TGA), are cumbersome to use and not widely available. OBJECTIVE: To assess the utility of a novel, point-of-care, dielectric microsensor - ClotChip - to monitor coagulation factor replacement therapy in patients with haemophilia A, with and without inhibitors. METHODS: The ClotChip Tpeak parameter was assessed using whole-blood samples from children with severe haemophilia A, with (n = 6) and without (n = 12) inhibitors, collected pre- and postcoagulation factor replacement therapy. ROTEM, TGA and chromogenic FVIII assays were also performed. Healthy children (n = 50) served as controls. RESULTS: ClotChip Tpeak values exhibited a significant decrease for samples collected postcoagulation factor replacement therapy as compared to baseline (pretherapy) samples in patients with and without inhibitors. A difference in Tpeak values was also noted at baseline among severe haemophilia A patients with inhibitors as compared to those without inhibitors. ClotChip Tpeak parameter exhibited a very strong correlation with clotting time (CT) of ROTEM, endogenous thrombin potential (ETP) and peak thrombin of TGA, and FVIII clotting activity. CONCLUSIONS: ClotChip is sensitive to coagulation factor replacement therapy in patients with severe haemophilia A, with and without inhibitors. ClotChip Tpeak values correlate very well with ROTEM, TGA and FVIII assays, opening up possibilities for its use in personalized coagulation factor replacement therapy in haemophilia.

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.

A PMMA microfluidic dielectric sensor for blood coagulation monitoring at the point-of-care.

This paper describes the design and construct of a fully biocompatible, microfluidic, dielectric sensor targeted at monitoring human whole blood coagulation at the point-of-care (POC). The sensor assembly procedure involves using sputtered electrodes in a microfluidic channel with a physiologically relevant height of 50µm to create a three-dimensional (3D), parallel-plate, capacitive sensing area. The sensor is constructed with biocompatible materials of polymethyl methacrylate (PMMA) for the substrate and titanium nitride (TiN) for the sensing and floating electrodes. The real part of the complex relative dielectric permittivity of human whole blood is measured from 10kHz to 100MHz using an impedance analyzer and under static conditions. The temporal variation in dielectric permittivity at 1MHz for human whole blood undergoing coagulation shows a peak in permittivity at 5 minutes, which closely matches our previously established results. This sensor can pave the way for monitoring blood coagulation under physiologically relevant shear flow rates in the future.

Implantable wireless battery recharging system for bladder pressure chronic monitoring.

This paper presents an implantable wireless battery recharging system design for bladder pressure chronic monitoring. The wireless recharging system consists of an external 15 cm-diameter 6-turn powering coil and a silicone-encapsulated implantable rectangular coil with a dimension of 7 mm × 17 mm × 2.5 mm and 18 turns, which further encloses a 3 mm-diameter and 12 mm-long rechargeable battery, two ferrite rods, an ASIC, and a tuning capacitor. For a constant recharging current of 100 µA, an RF power of 700 µW needs to be coupled into the implantable module through the tuned coils. Analyses and experiments confirm that with the two coils aligned coaxially or with a 6 cm axial offset and a tilting angle of 30°, an external power of 3.5 W or 10 W is required, respectively, at an optimal frequency of 3 MHz to cover a large implant depth of 20 cm.

Monitoring time course of human whole blood coagulation using a microfluidic dielectric sensor with a 3D capacitive structure.

This paper reports on the design, fabrication, and testing of a microfluidic sensor for dielectric spectroscopy (DS) of human whole blood during coagulation. The sensor employs a three-dimensional (3D), parallel-plate, capacitive sensing structure with a floating electrode integrated into a microfluidic channel. Using an impedance analyzer and after a 5-point calibration, the sensor is shown to measure the real part of complex relative dielectric permittivity of human whole blood in a frequency range of 10kHz to 100MHz. The temporal variation of dielectric permittivity at 1MHz for human whole blood from three different healthy donors shows a peak in permittivity at ~ 4 to 5 minutes, which also corresponds to the onset of CaCl2-initiated coagulation of the blood sample verified visually.

A 9 MHz-2.4 GHz Fully Integrated Transceiver IC for a Microfluidic-CMOS Platform Dedicated to Miniaturized Dielectric Spectroscopy.

This paper presents a fully integrated transceiver IC as part of a self-sustained, microfluidic-CMOS platform for miniaturized dielectric spectroscopy (DS) from MHz to GHz. Fabricated in AMS 0.35 µm 2P/4M RF CMOS, the transmitter (TX) part of the IC generates a single-tone sinusoidal signal with frequency tunability in the range of ~ 9 MHz-2.4 GHz to excite a three-dimensional (3D), parallel-plate, capacitive sensor with a floating electrode and 9 µL microfluidic channel for sample delivery. With a material-under-test (MUT) loaded into the sensor, the receiver (RX) part of the IC employs broadband frequency response analysis (bFRA) methodology to measure the amplitude and phase of the RF excitation signal after transmission through the sensor. A one-time, 6-point sensor calibration algorithm then extracts both the real and imaginary parts of the MUT complex permittivity, ϵr, from IC measurements of the sensor transmission characteristics in the voltage domain. The "sensor + IC" is fully capable of differentiating among de-ionized (DI) water, phosphate-buffered saline (PBS), and alcoholic beverages in tests conducted at four excitation frequencies of ∼ 50 MHz , 500 MHz, 1.5 GHz, and 2.4 GHz generated by the TX. Moreover, permittivity readings of PBS by the sensor interfaced with the IC at six excitation frequencies in the range of ~ 50 MHz-2.4 GHz are in excellent agreement (rms error of 1.7% (real) and 7.2% (imaginary)) with those from bulk-solution reference measurements by commercial benchtop equipment. The total power consumption of the IC is with 1.5 V (analog) and 3.3 V (digital) supplies.

Wireless, Ultra-Low-Power Implantable Sensor for Chronic Bladder Pressure Monitoring.

The wireless implantable/intracavity micromanometer (WIMM) system was designed to fulfill the unmet need for a chronic bladder pressure sensing device in urological fields such as urodynamics for diagnosis and neuromodulation for bladder control. Neuromodulation in particular would benefit from a wireless bladder pressure sensor which could provide real-time pressure feedback to an implanted stimulator, resulting in greater bladder capacity while using less power. The WIMM uses custom integrated circuitry, a MEMS transducer, and a wireless antenna to transmit pressure telemetry at a rate of 10 Hz. Aggressive power management techniques yield an average current draw of 9 µA from a 3.6-Volt micro-battery, which minimizes the implant size. Automatic pressure offset cancellation circuits maximize the sensing dynamic range to account for drifting pressure offset due to environmental factors, and a custom telemetry protocol allows transmission with minimum overhead. Wireless operation of the WIMM has demonstrated that the external receiver can receive the telemetry packets, and the low power consumption allows for at least 24 hours of operation with a 4-hour wireless recharge session.