Development and Evaluation of a Quantitative Fluorescent Lateral Flow Immunoassay for Cystatin-C, a Renal Dysfunction Biomarker.

The diagnosis, prognosis, and control of chronic kidney disease rely on an understanding of the glomerular filtration rate (GFR). The renal clearance of the cystatin-C is closely associated with the GFR. Cystatin-C is a more suitable GFR marker than the commonly used creatinine. General techniques for cystatin-C calculation, such as particle-enhanced turbidimetric and nephelometric assay, are time-consuming and tedious. Here, we propose a rapid, quantitative immunoassay for the detection of cystatin-C. A fluorescence-based lateral-flow kit was developed in a sandwich format by using a monoclonal antibody. A Linear calibration was obtained over the clinical diagnostic range of 0.023-32 µg/mL and the limit of detection (LOD) was 0.023 µg/mL and the limit of quantification (LOQ) was 0.029 µg/mL. Average recoveries from spiked urine samples ranged from 96-100% and the coefficient of variation was less than 4% for both intra and inter-day assays with excellent repeatability. With the comparison with an ELISA kit, the developed kit is highly sensitive, performs well over the detection range, provides repeatable results in a short time, and can easily be used at point-of-care (POC), making it an ideal candidate for rapid testing in early detection, community screening for renal function disorders.

IQVision: An Image-Based Evaluation Tool for Quantitative Lateral Flow Immunoassay Kits.

The development of quantitative lateral flow immunoassay test strips involves a lot of research from kit manufacturers' standpoint. Kit providers need to evaluate multiple parameters, including the location of test regions, sample flow speed, required sample volumes, reaction stability time, etc. A practical visualization tool assisting manufacturers in this process is very much required for the design of more sensitive and reliable quantitative LFIA test strips. In this paper, we present an image-based quantitative evaluation tool determining the practical functionality of fluorescence-labelled LFIA test cartridges. Image processing-based algorithms developed and presented in this paper provide a practical analysis of sample flow rates, reaction stability times of samples under test, and detect any abnormalities in test strips. Evaluation of the algorithm is done with Glycated Hemoglobin (HbA1C) and Vitamin D test cartridges. Practical sample flow progress for HbA1C test cartridges is demonstrated. The reaction stability time of HbA1C test samples is measured to be 12 min, while that of Vitamin D test samples is 24 min. Experimental evaluation of the abnormality detection algorithm is carried out, and sample flow abnormalities are detected with 100% accuracy while membrane irregularities are detected with 96% accuracy.

Assessment of Carotid Arterial Stiffness in Community Settings With ARTSENS®.

OBJECTIVE: We investigate the field feasibility of carotid stiffness measurement using ARTSENS® Touch and report the first community-level data from India. METHOD: In an analytical cross-sectional survey among 1074 adults, we measured specific stiffness index ([Formula: see text]), pressure-strain elastic modulus ([Formula: see text]), arterial compliance (AC), and one-point pulse wave velocity (PWV[Formula: see text]) from the left common carotid artery. Data for established risk factors (waist circumference, blood pressure, plasma glucose, triglycerides, and HDL-C) were also collected. The association of carotid stiffness with age, gender, hypertension/diabetes, smoking, and clustering of risk factors was studied. RESULTS: Measurements were repeatable with a relative difference (RD) between consecutive readings of < 5% for blood pressure and < 15% for [Formula: see text]% of arterial diameter values. The average RDs for [Formula: see text], [Formula: see text], AC, and PWV[Formula: see text], were 20.51%, 22.31%, 25.10%, and 14.13%, respectively. Typical range for stiffness indices among females and males were [Formula: see text]: 8.12 ± 3.59 vs 6.51 ± 2.78, [Formula: see text]: 113.24 ± 56.12 kPa vs 92.33 ± 40.65 kPa, PWV[Formula: see text]: 6.32 ± 1.38 ms-1 vs 5.81 ± 1.16 ms-1, and AC: 0.54 ± 0.36 mm2 kPa-1 vs 0.72 ± 0.38 mm2 kPa-1. Mean [Formula: see text], [Formula: see text], and PWV[Formula: see text] increased (and mean AC decreased) across decades of age; the trend persisted even after excluding hypertensives and subjects with diabetes. The odds ratio of presence of multiple risk factors for [Formula: see text] kPa and/or PWV[Formula: see text] ms-1 was ≥ 2.12 or above in males. In females, it was just above 2.00 for [Formula: see text] kPa and/or PWV[Formula: see text] ms-1 and increased to ≥ 3.33 for [Formula: see text] kPa and ≥ 3.25 for PWV[Formula: see text] ms-1. CONCLUSION: The study demonstrated the feasibility of carotid stiffness measurement in a community setting. A positive association between the risk factors and carotid artery stiffness provides evidence for the device's use in resource-constrained settings. Clinical Impact: The device paves the way for epidemiological and clinical studies that are essential for establishing population-level nomograms for wide-spread use of carotid stiffness in clinical practice and field screening of 'at-risk' subjects.

Gaussian-Mixture Modelling of A-Mode Radiofrequency Scans for the Measurement of Arterial Wall Thickness.

Measurement of arterial wall thickness is an integral component of vascular properties and health assessment. State-of-the-art automated or semi-automated techniques are majorly applicable to B-mode images and are not available for entry-level in-expensive devices. Considering this, we have earlier developed and validated an image-free (A-mode) ultrasound device, ARTSENS® for the evaluation of vascular properties. In this work, we present a novel gaussian-mixture modeling-based method to measure arterial wall thickness from A-mode frames, which is readily deployable to the existing technology. The method's performance was assessed based on systematic simulations and controlled phantom experiments. Simulations revealed that the method could be confidently applied to A-mode frames with above-moderate SNR (>15 dB). When applied to A-mode frames acquired from the flow-phantom setup (SNR > 25 dB), the mean error was limited to (2 ± 1%), and RMSE was 19 µm, on comparison with B-mode measurements. The measured and reference wall thickness strongly agreed with each other (r = 0.88, insignificant mean bias = 7 µm, p = 0.16). The proposed method was capable of performing real-time measurements.

Phantom Assessment of an Image-free Ultrasound Technology for Online Local Pulse Wave Velocity Measurement.

Cardiovascular community has started clinically adopting the assessment of local stiffness, contrary to the traditionally measured carotid-femoral pulse wave velocity (PWV). Though they offer higher reliability, ultrasound methods require advanced hardware and processing methods to perform real-time measurement of local PWV. This work presents a system and method to perform online PWV measurement in an automated manner. It is a fast image-free ultrasound technology that meets the methodological requirements necessary to measure small orders of local pulse transit, from which PWV is measured. The measurement accuracy and repeatability were assessed via phantom experiments, where the measured transit time-based PWV (PWVTT) was compared against the theoretically calculated PWV from Bramwell-Hill equation (PWVBH). The beat-to-beat variability in the measured PWVTT was within 3%. PWVTT values strongly correlated (r=0.98) with PWVBH, yielding a negligible bias of -0.01 m/s, mean error of 3%, and RMSE of 0.27 m/s. These pilot study results demonstrated the presented system's reliability in yielding online local PWV measurements.

ARTSENS® Pen-portable easy-to-use device for carotid stiffness measurement: technology validation and clinical-utility assessment.

OBJECTIVE: The conventional medical imaging modalities used for arterial stiffness measurement are non-scalable and unviable for field-level vascular screening. The need for an affordable, easy-to-operate automated non-invasive technologies remains unmet. To address this need, we present a portable image-free ultrasound device-ARTSENS® Pen, that uses a single-element ultrasound transducer for carotid stiffness evaluation. APPROACH: The performance of the device was clinically validated on a cohort of 523 subjects. A clinical-grade B-mode ultrasound imaging system (ALOKA eTracking) was used as the reference. Carotid stiffness measurements were taken using the ARTSENS® Pen in sitting posture emulating field scenarios. MAIN RESULTS: A statistically significant correlation (r > 0.80, p < 0.0001) with a non-significant bias was observed between the measurements obtained from the two devices. The ARTSENS® Pen device could perform highly repeatable measurements (with variation smaller than 10%) on a relatively larger percentage of the population when compared to the ALOKA system. The study results also revealed the sensitivity of ARTSENS® Pen to detect changes in arterial stiffness with age. SIGNIFICANCE: The easy-to-use technology and the automated algorithms of the ARTSENS® Pen make it suitable for cardiovascular risk assessment in resource-constrained settings.

Multi-cartridge Fluorescence Reader for Quantitative Immunoassays.

Lateral flow immunoassays (LFIA) play a significant role in point-of-care (POC) diagnostics, facilitating early diagnosis of medical conditions. With simpler infrastructure requirements, POC diagnostics can be easily adopted in remote areas as well as for end-user level monitoring. As part of the current work, we present a camera based multi-cartridge fluorescence reader. The proposed system can carry out simultaneous analysis of four LFIA test cartridges. It forms a simple, rugged system requiring minimal human intervention and the average time taken for tests is lower as compared to the available LFIA readers. The hardware architecture of the system along with the software algorithms utilized is described in this paper. To validate the system performance, tests were conducted using HbA1C samples. The designed system comprises of four test-slots accommodating four test cartridges in a single go. The correlation of the obtained results with reference sample concentrations was determined and system calibration equations were as well obtained. Repeatability across the slots in terms of % coefficient of variation was calculated and was found to be less than 6%. The obtained results along with challenges in the current system and future modifications are also discussed.

Arterial compliance probe for cuffless evaluation of carotid pulse pressure.

OBJECTIVE: Assessment of local arterial properties has become increasingly important in cardiovascular research as well as in clinical domains. Vascular wall stiffness indices are related to local pulse pressure (ΔP) level, mechanical and geometrical characteristics of the arterial vessel. Non-invasive evaluation of local ΔP from the central arteries (aorta and carotid) is not straightforward in a non-specialist clinical setting. In this work, we present a method and system for real-time and beat-by-beat evaluation of local ΔP from superficial arteries-a non-invasive, cuffless and calibration-free technique. METHODS: The proposed technique uses a bi-modal arterial compliance probe which consisted of two identical magnetic plethysmograph (MPG) sensors located at 23 mm distance apart and a single-element ultrasound transducer. Simultaneously measured local pulse wave velocity (PWV) and arterial dimensions were used in a mathematical model for calibration-free evaluation of local ΔP. The proposed approach was initially verified using an arterial flow phantom, with invasive pressure catheter as the reference device. The developed porotype device was validated on 22 normotensive human subjects (age = 24.5 ± 4 years). Two independent measurements of local ΔP from the carotid artery were made during physically relaxed and post-exercise condition. RESULTS: Phantom-based verification study yielded a correlation coefficient (r) of 0.93 (p < 0.001) for estimated ΔP versus reference brachial ΔP, with a non-significant bias and standard deviation of error equal to 1.11 mmHg and ±1.97 mmHg respectively. The ability of the developed system to acquire high-fidelity waveforms (dual MPG signals and ultrasound echoes from proximal and distal arterial walls) from the carotid artery was demonstrated by the in-vivo validation study. The group average beat-to-beat variation in measured carotid local PWV, arterial diameter parameters-distension and end-diastolic diameter, and local ΔP were 4.2%, 2.6%, 3.3%, and 10.2% respectively in physically relaxed condition. Consistent with the physiological phenomenon, local ΔP measured from the carotid artery of young populations was, on an average, 22 mmHg lower than the reference ΔP obtained from the brachial artery. Like the reference brachial blood pressure (BP) monitor, the developed prototype device reliably captured variations in carotid local ΔP induced by an external intervention. CONCLUSION: This technique could provide a direct measurement of local PWV, arterial dimensions, and a calibration-free estimate of beat-by-beat local ΔP. It can be potentially extended for calibration-free cuffless BP measurement and non-invasive characterization of central arteries with locally estimated biomechanical properties.

An In-Vivo Study on Intra-Day Variations in Vascular Stiffness using ARTSENS Pen.

This work presents investigations on intraday variations in arterial stiffness. For this purpose, an in-vivo study was conducted on five subjects over a duration of five consecutive days. Five stiffness index ($\beta $) measurements were obtained per day for each individual. Our clinically validated ARTSENS device was used to perform fully automated reliable stiffness measurements on the carotid artery. For each measurement, two trials were performed and averaged. These trials were observed to be repeatable with the coefficient of variation $< 0.72$%. For each day, one measurement that was performed immediately after the lunch was subject to intervention due to the consumed food, which significantly $( \mathrm {p}< 0.001)$ deviated from the mean baseline $\beta $ of the day. Such significant deviations were not observed for the rest measurements that were performed in the absence of an intervention. Two subjects who consumed caffeinated beverages during the lunch exhibited an increment in $\beta $ measurement (taken immediately after lunch) as compared to mean baseline $\beta $ of the day. Further, there was no significant (p $=$ 0.97) difference between the mean baseline $\beta $ measured over a day and the mean baseline $\beta $ measured over the entire course of the study. Results obtained from the present study demonstrated that the arterial stiffness does not vary significantly over a short period but varies progressively. However, significant temporary variations in stiffness could be observed due to dietary interventions.

PhoneQuant: A smartphone-based quantitative immunoassay analyser.

There is a vital need for portable and cost-effective point-of-care (PoC) testing technologies that provide reliable and rapid results. Lateral Flow Immunoassays (LFIA) are suitable PoC diagnostic tools with the potential for use in a wide variety of field applications ranging from uses in clinical diagnostics to aiding law enforcement. Quick and reliable diagnosis of non-communicable diseases (NCD) like diabetes is vital especially in developing countries like India where the burden of these diseases is very high and is increasing day by day. In this paper, we have presented the design of smartphone-based fully quantitative LFIA analyser, An automatic image processing algorithm is also described. A repeatability study was done with stable fluorescence reference cartridges. The Coefficient of Variation (CoV) for repeatability study was calculated and it was found to be good (<; 1.5%). The instrument was tested with blood samples to generate a calibration curve for Glycated Haemoglobin (HbA1c) with respect to standard lab instrument. Three different set of settings parameters were used in smartphone camera their calibration curve formed. All three curves were linearly correlated with R2-value greater than 0.985. Finally, the calibration curve was validated through three HbA1c blood sample tests- for each sample, CoV was less than 5%. The PhoneQuant analyser is the portable cost-effective solution to traditional bulky LFIA analysers and it has good potential to be deployed at physician's desk or for in-home PoC testing for quick and reliable diagnosis.

ARTSENS® Mobile: a portable image-free platform for automated evaluation of vascular stiffness.

Vascular stiffness is a key physiological marker significant in screening and early diagnosis of vascular disease. However, state of art techniques for vascular stiffness evaluation are not suited for field deployment. We present ARTSENS® Mobile, a portable, image-free ultrasound technology platform for automated evaluation of carotid artery stiffness, amenable for field deployment. The technology utilises a single element ultrasound transducer operated in pulse echo modality to investigate arterial wall dynamics, assisted by intelligent algorithms for wall identification and motion tracking to completely automate the measurement. The ARTSENS Mobile is built around the NI sbRIO 9651 embedded SOM which runs all algorithms for pulsed transducer excitation, high speed synchronised ultrasound echo capture, real-time signal processing and automated measurement. The system is designed to communicate wirelessly with any smart tablet for data display and wire-free control. The accuracy of automated arterial dimension measurements made by ARTSENS Mobile was verified by phantom studies using a reference ultrasound imaging system. The ability of the system to reliably perform automated measurements was verified by a systematic in-vivo study on 21 volunteers. The intra-operator and inter-operator variability of stiffness index ß, was found to be good with a coefficient of variation (CoV) less than 12% and 16 % respectively. The ARTSENS® Mobile can provide accurate and repeatable measures of arterial stiffness in an easy manner and has strong potential in large scale vascular screening.

Carotid and Jugular Classification in ARTSENS.

Over past few years our group has been working on the development of a low-cost device, ARTSENS, for measurement of local arterial stiffness (AS) of the common carotid artery (CCA). This uses a single element ultrasound transducer to obtain A-mode frames from the CCA. It is designed to be fully automatic in its operation such that, a general medical practitioner can use the device without any prior knowledge of ultrasound modality. Placement of the probe over CCA and identification of echo positions corresponding to its two walls are critical steps in the process of measurement of AS. We had reported an algorithm to locate the CCA walls based on their characteristic motion. Unfortunately, in supine position, the internal jugular vein (IJV) expands in the carotid triangle and pulsates in a manner that confounds the existing algorithm and leads to wrong measurements of the AS. Jugular venous pulse (JVP), on its own right, is a very important physiological signal for diagnosis of morbidities of the right side of the heart and there is a lack of noninvasive methods for its accurate estimation. We integrated an ECG device to the existing hardware of ARTSENS and developed a method based on physiology of the vessels, which now enable us to segregate the CCA pulse (CCP) and the JVP. False identification rate is less than 4%. To retain the capabilities of ARTSENS to operate without ECG, we designed another method where the classification can be achieved without an ECG, albeit errors are a bit higher. These improvements enable ARTSENS to perform automatic measurement of AS even in the supine position and make it a unique and handy tool to perform JVP analysis.

An Imageless Ultrasound Device to Measure Local and Regional Arterial Stiffness.

Arterial stiffness (AS) has been shown to be an important marker for risk assessment of cardiovascular events. Local arterial stiffness (LAS) is conventionally measured by evaluating arterial distensibility at particular arterial sites through ultrasound imaging systems. Regional arterial stiffness (RAS) is generally obtained by evaluating carotid to femoral pulse wave velocity (cfPWV) through tonometric devices. RAS has a better prognostic value than LAS and cfPWV is considered as the gold standard of AS. Over the past few years our group has been developing ARTerial Stiffness Evaluation for Non-Invasive Screening (ARTSENS), an inexpensive and portable device to measure the LAS. It uses a single element ultrasound transducer to obtain A-Mode frames from the desired artery and is fully automated to enable a non-expert to perform measurements. In this work, we report an extension of ARTSENS to enable measurement of cfPWV that now makes it the only fully automatic device that can measure both LAS and RAS. In this paper, we provide a general review of the ARTSENS and compare it with other state-of-the-art AS measurement systems. cfPWV measurement using ARTSENS was cross-validated against SphygmoCor by successive measurements with both devices on 41 human subjects and excellent agreement between both devices was demonstrated (Coefficient of determination and, limits of agreement m/s). The inter-device correlation between ARTSENS and SphygmoCor was found to be better than other similar studies reported in the literature.