Optical scanner for immunoassays with up-converting phosphorescent labels.

A 2-D optical scanner was developed for the imaging and quantification of up-converting phosphor (UCP) labels in immunoassays. With resolution better than 500 microm, a scan rate of 0.4 mm/s, and a 1-2% coefficient of variation for repeatability, this scanner achieved a detection limit of fewer than 100 UCP particles in an 8.8. x 10(4) microm(2) area and a dynamic range that covered more than three orders of magnitude. Utilizing this scanner, a microfluidic chip immunoassay for the cytokine interferon-gamma (IFN-gamma) was developed: concentrations as low as 3 pM (50 pg/mL) were detected from 100 microL samples with a total assay time of under an hour, including the 8 min readout. For this UCP-based assay, 2-D images of the capture antibody lines were scanned, image processing techniques were employed to extract the UCP emission signals, a response curve that spanned 3-600 pM IFN-gamma was generated, and a five-parameter logistic mathematical model was fitted to the data for determination of unknown IFN-gamma concentrations. Relative to common single-point or 1-D scanning optical measurements, our results suggest that a simple 2-D imaging system can speed assay development, reduce errors, and improve accuracy by characterizing the spatial distribution and uniformity of surface-captured optical labels as a function of assay conditions and device parameters.

Toward a better noninvasive assessment of preejection period: A novel automatic algorithm for B-point detection and correction on thoracic impedance cardiogram.

Impedance cardiography is the most common clinically validated, noninvasive method for determining the timing of the opening of the aortic valve, an important event used for measuring preejection period, which reflects sympathetic beta-adrenergic influences on the heart. Automatic detection of the exact time of the opening of the aortic valve (B point on the impedance cardiogram) has proven to be challenging as its appearance varies between and within individuals and may manifest as a reversal, inflection, or rapid slope change of the thoracic impedance derivative's (dZ/dt) rapid rise. Here, a novel automatic algorithm is proposed for the detection of the B point by finding the main rapid rise of the dZ/dt signal, which is due to blood ejection. Several conditions based on zero crossings, minima, and maxima of the dZ/dt signal and its derivatives are considered to reject any unwanted noise and artifacts and select the true B-point location. The detected B-point locations are then corrected by modeling the B-point time data using forward and reverse autoregressive models. The proposed algorithm is validated against expert-detected B points and is compared with different conventional methods; it significantly outperforms them by at least 54% in mean error, 30% in mean absolute error, and 27% in standard deviation of error. This algorithm can be adopted in ambulatory studies requiring beat-to-beat evaluation of cardiac hemodynamic parameters over extended time periods where expert scoring is not feasible.

Robust neural-network-based classification of premature ventricular contractions using wavelet transform and timing interval features.

Automatic electrocardiogram (ECG) beat classification is essential to timely diagnosis of dangerous heart conditions. Specifically, accurate detection of premature ventricular contractions (PVCs) is imperative to prepare for the possible onset of life-threatening arrhythmias. Although many groups have developed highly accurate algorithms for detecting PVC beats, results have generally been limited to relatively small data sets. Additionally, many of the highest classification accuracies (> 90%) have been achieved in experiments where training and testing sets overlapped significantly. Expanding the overall data set greatly reduces overall accuracy due to significant variation in ECG morphology among different patients. As a result, we believe that morphological information must be coupled with timing information, which is more constant among patients, in order to achieve high classification accuracy for larger data sets. With this approach, we combined wavelet-transformed ECG waves with timing information as our feature set for classification. We used select waveforms of 18 files of the MIT/BIH arrhythmia database, which provides an annotated collection of normal and arrhythmic beats, for training our neural-network classifier. We then tested the classifier on these 18 training files as well as 22 other files from the database. The accuracy was 95.16% over 93,281 beats from all 40 files, and 96.82% over the 22 files outside the training set in differentiating normal, PVC, and other beats.

Sensitivity of cell-based biosensors to environmental variables.

Electrically active living cells cultured on extracellular electrode arrays are utilized to detect biologically active agents. Because cells are highly sensitive to environmental conditions, environmental fluctuations can elicit cellular responses that contribute to the noise in a cell-based biosensor system. Therefore, the characterization and control of environmental factors such as temperature, pH, and osmolarity is critical in such a system. The cell-based biosensor platform described here utilizes the measurement of action potentials from cardiac cells cultured on electrode arrays. A recirculating fluid flow system is presented for use in dose-response experiments that regulates temperature within +/-0.2 degrees C, pH to within +/-0.05 units, and allows no significant change in osmolarity. Using this system, the relationship between the sensor output parameters and environmental variation was quantified. Under typical experimental conditions, beat rate varied approximately 10% per degree change in temperature or per 0.1 unit change in pH. Similar relationships were measured for action potential amplitude, duration, and conduction velocity. For the specific flow system used in this work, the measured environmental sensitivity resulted in an overall beat rate variation of +/-4.7% and an overall amplitude variation of +/-3.3%. The magnitude of the noise due to environmental sensitivity has a large impact on the detection capability of the cell-based system. The significant responses to temperature, pH, and osmolarity have important implications for the use of living cells in detection systems and should be considered in the design and evaluation of such systems.

A closed-loop electrical stimulation system for cardiac cell cultures.

An integrated electrical stimulation and recording system was designed for closed-loop control and analysis of cardiac cultures on planar microelectrode arrays. Stimulated action potentials from HL-1 clonal myocyte cultures were digitized, stimulation artifacts were removed using nulling and filtering methods, and analysis was performed to determine stimulation efficacy in real time. Results of this analysis were used to determine future stimulation waveform parameters such as polarity, amplitude, pulse duration, and rate or pattern. Algorithms were designed utilizing real-time analysis and control to maintain a desired electrophysiological response of the culture, such as an arbitrary capture fraction value. This paper presents the hardware and software design of the stimulus pulse circuitry, artifact extraction, analysis, and control components of the system. Applications of this technology include the study of cardiac cell physiology, improving the speed and accuracy of traditional open-loop stimulation protocols, pharmacological screening, and improving the performance of biosensors based on sensing electrical activity in cardiac cultures.

Noninvasive pulse transit time measurement for arterial stiffness monitoring in microgravity.

The use of a noninvasive hemodynamic monitor to estimate arterial stiffness, by measurement of pulse transit time (PTT), was demonstrated in microgravity. The monitor's utility for space applications was shown by establishing the correlation between ground-based and microgravity-based measurements. The system consists of a scale-based ballistocardiogram (BCG) and a toe-mounted photoplethysmogram (PPG). PTT was measured from the BCG I-wave to the intersecting tangents of the first trough and maximum first derivative of the PPG waveforms of each subject. The system was tested on a recent series of parabolic flights in which the PTT of nine subjects was measured on the ground and in microgravity. An average of 60.2 ms PTT increase from ground to microgravity environments was shown, and was consistent across all test subjects (standard deviation = 32.9 ms). This increase in PTT could be explained by a number of factors associated with microgravity and reported in previous research, including elimination of hydrostatic pressure, reduction of intrathoracic pressure, and reduction of mean arterial pressure induced by vasodilation.

Simultaneous stimulation and recording of cardiac depolarization enabled by high-frequency stimulation.

High-frequency stimulation techniques have been recently proposed for the pacing and control of excitability of cardiac tissues. This paper introduces a system designed specifically for such stimulation, and demonstrates the unique ability to record depolarization events on the same electrode used for stimulation, during the stimulus. Experimental results with HL-1 cardiomyocytes are presented, highlighting key concepts enabled by this system, such as direct strength-duration relationship measurement and beat-to-beat stimulation threshold monitoring following pacing onset or pharmacological modulation.

Standing ballistocardiography measurements in microgravity.

The performance and practicality of a scale-based ballistocardiogram (BCG) system for hemodynamic monitoring of astronauts on extended space missions was demonstrated. The system consists of a modified electronic weighing scale fitted with foot bindings to mechanically couple the subject to the scale. This system was tested on a recent series of parabolic flights in which scale-based and accelerometry-based free-floating BCG of 10 subjects was measured in microgravity. The signal-to-noise ratio (SNR) of the scale-based BCG was, on average, a factor of 2.1 (6.3 dB) higher than the free-floating method, suggesting that the tethered scale approach might be more robust in terms of signal quality. Additionally, this approach enables practical BCG-based hemodynamic monitoring in fractional-g environments, and on small space vehicles such as NASA's upcoming Orion capsule. The scale-based results in microgravity were also compared to ground measurements (1 g), where there was an average 38.7 ms RJ interval reduction from ground to microgravity environments that is consistent across 9 of 10 subjects. This phenomenon is likely due to the transient increase in venous return, and consequent decrease in pre-ejection period, experienced during the microgravity time intervals.

Characterisation of electrophysiological conduction in cardiomyocyte co-cultures using co-occurrence analysis.

Cardiac arrhythmias are disturbances of the electrical conduction pattern in the heart with severe clinical implications. The damage of existing cells or the transplantation of foreign cells may disturb functional conduction pathways and may increase the risk of arrhythmias. Although these conduction disturbances are easily accessible with the human eye, there is no algorithmic method to extract quantitative features that quickly portray the conduction pattern. Here, we show that co-occurrence analysis, a well-established method for feature recognition in texture analysis, provides insightful quantitative information about the uniformity and the homogeneity of an excitation wave. As a first proof-of-principle, we illustrate the potential of co-occurrence analysis by means of conduction patterns of cardiomyocyte-fibroblast co-cultures, generated both in vitro and in silico. To characterise signal propagation in vitro, we perform a conduction analysis of co-cultured murine HL-1 cardiomyocytes and murine 3T3 fibroblasts using microelectrode arrays. To characterise signal propagation in silico, we establish a conduction analysis of co-cultured electrically active, conductive cardiomyocytes and non-conductive fibroblasts using the finite element method. Our results demonstrate that co-occurrence analysis is a powerful tool to create purity-conduction relationships and to quickly quantify conduction patterns in terms of co-occurrence energy and contrast. We anticipate this first preliminary study to be a starting point for more sophisticated analyses of different co-culture systems. In particular, in view of stem cell therapies, we expect co-occurrence analysis to provide valuable quantitative insight into the integration of foreign cells into a functional host system.

A preliminary study investigating the quantification of beat-to-beat in seismocardiogram signals.

Ballistocardiography and seismocardiography are both non-invasive mechanical measurements of the vibrations of the body in response to the heartbeat. The ballistocardiogram (BCG) signal represents the movements of the whole body in response to cardiac ejection of blood into the vasculature; the seismocardiogram (SCG) corresponds to local vibrations of the chest wall associated with sub-audible tissue and blood movement and audio frequency heart-valve closure dynamics. This paper focuses on methods for quantifying "signal consistency"--a quantitative measure of how morphologically similar each heartbeat in a patient's recording is compared to the ensemble average taken over the recording. Before comparing each beat to the average, known physiological sources of inconsistency--such as respiratory amplitude and timing variability--are removed, then the remaining inconsistency is quantified. Previously described methods for BCG signals are expanded to fit the high-frequency (> 20 Hz) components of the SCG. The use of this method in future work could help enable proactive management of heart disease in extra-clinical settings.

Preliminary results from standing ballistocardiography measurements in microgravity.

We report on the feasibility of standing ballistocardiogram (BCG) measurements recorded in a microgravity environment. A clinically-tested BCG monitoring scale was adapted for parabolic flight for the microgravity measurements. Upon completion of this flight campaign, the BCG scale was shown to make measurements in micro-g and one-g environments--which is a first demonstration for a standing BCG system. This screening experiment demonstrated proof-of-concept attributes of the hardware design necessary for future characterization studies with multiple subjects. This scale-based BCG system is proposed as a practical device for hemodynamic monitoring for astronauts in Earth, Lunar, Martian, orbital, and interplanetary environments.

Microfluidic impedance cytometer for platelet analysis.

We present the design and performance characteristics of a platelet analysis platform based on a microfluidic impedance cytometer. Dielectrophoretic focusing is used to centre cells in a fluid stream, which then forms the core of a two-phase flow (dielectric focusing). This flow then passes between electrodes for analysis by differential impedance spectroscopy at multiple frequencies from 280 kHz to 4 MHz. This approach increases the signal-to-noise ratio relative to a single-phase, unfocused stream, while minimising the shear forces to which the cells are subjected. The percentage of activated platelets before and after passage through the chip was measured using flow cytometry, and no significant change was measured. Measuring the in-phase amplitude at a single frequency is sufficient to distinguish platelets from erythrocytes. Using multi-frequency impedance measurements and discriminant analysis, resting platelets can be discriminated from activated platelets. This multifrequency impedance cytometer therefore allows ready determination of the degree of platelet activation in blood samples.

A combined heartbeat detector based on individual BCG and IPG heartbeat detectors.

This paper presents a preliminary approach for heartbeat detection on a weighing scale, using a combined heartbeat detector and an ensemble method. First, two independent sub-detectors are implemented based on the BCG (Ballistocardiogram) and lower-body IPG (Impedance Plethysmogram) signals. Then, the results of these sub-detectors are combined using a higher level decision maker. The BCG, which describes the reaction of the body to cardiac ejection of blood, was measured using the strain gauges in a modified commercial weighing scale. For the lower-body IPG, a small amount of current was injected into the subject through the electrodes under the subject's toes, and the resulting differential voltage across the heels was measured. We tested our method on the first 30 seconds of the BCG and IPG signals collected from 8 subjects. The results show the combination significantly improved over individual detector, with a resulting interval accuracy of 97%.

Spatiotemporally controlled cardiac conduction block using high-frequency electrical stimulation.

BACKGROUND: Methods for the electrical inhibition of cardiac excitation have long been sought to control excitability and conduction, but to date remain largely impractical. High-amplitude alternating current (AC) stimulation has been known to extend cardiac action potentials (APs), and has been recently exploited to terminate reentrant arrhythmias by producing reversible conduction blocks. Yet, low-amplitude currents at similar frequencies have been shown to entrain cardiac tissues by generation of repetitive APs, leading in some cases to ventricular fibrillation and hemodynamic collapse in vivo. Therefore, an inhibition method that does not lead to entrainment - irrespective of the stimulation amplitude (bound to fluctuate in an in vivo setting) - is highly desirable. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of broader amplitude and frequency ranges on the inhibitory effects of extracellular AC stimulation on HL-1 cardiomyocytes cultured on microelectrode arrays, using both sinusoidal and square waveforms. Our results indicate that, at sufficiently high frequencies, cardiac tissue exhibits a binary response to stimulus amplitude with either prolonged APs or no effect, thereby effectively avoiding the risks of entrainment by repetitive firing observed at lower frequencies. We further demonstrate the ability to precisely define reversible local conduction blocks in beating cultures without influencing the propagation activity in non-blocked areas. The conduction blocks were spatiotemporally controlled by electrode geometry and stimuli duration, respectively, and sustainable for long durations (300 s). CONCLUSION/SIGNIFICANCE: Inhibition of cardiac excitation induced by high-frequency AC stimulation exhibits a binary response to amplitude above a threshold frequency, enabling the generation of reversible conduction blocks without the risks of entrainment. This inhibition method could yield novel approaches for arrhythmia modeling in vitro, as well as safer and more efficacious tools for in vivo cardiac mapping and radio-frequency ablation guidance applications.

Extracting respiratory information from seismocardiogram signals acquired on the chest using a miniature accelerometer.

Seismocardiography (SCG) is a non-invasive measurement of the vibrations of the chest caused by the heartbeat. SCG signals can be measured using a miniature accelerometer attached to the chest, and are thus well-suited for unobtrusive and long-term patient monitoring. Additionally, SCG contains information relating to both cardiovascular and respiratory systems. In this work, algorithms were developed for extracting three respiration-dependent features of the SCG signal: intensity modulation, timing interval changes within each heartbeat, and timing interval changes between successive heartbeats. Simultaneously with a reference respiration belt, SCG signals were measured from 20 healthy subjects and a respiration rate was estimated using each of the three SCG features and the reference signal. The agreement between each of the three accelerometer-derived respiration rate measurements was computed with respect to the respiration rate derived from the reference respiration belt. The respiration rate obtained from the intensity modulation in the SCG signal was found to be in closest agreement with the respiration rate obtained from the reference respiration belt: the bias was found to be 0.06 breaths per minute with a 95% confidence interval of -0.99 to 1.11 breaths per minute. The limits of agreement between the respiration rates estimated using SCG (intensity modulation) and the reference were within the clinically relevant ranges given in existing literature, demonstrating that SCG could be used for both cardiovascular and respiratory monitoring. Furthermore, phases of each of the three SCG parameters were investigated at four instances of a respiration cycle-start inspiration, peak inspiration, start expiration, and peak expiration-and during breath hold (apnea). The phases of the three SCG parameters observed during the respiration cycle were congruent with existing literature and physiologically expected trends.

Preliminary results from BCG and ECG measurements in the heart failure clinic.

We report on the preliminary deployment of a bathroom scale-based ballistocardiogram (BCG) system for the in-hospital monitoring of patients with heart failure. These early trials provided valuable insights into the challenges and opportunities for such monitoring. In particular, the need for robust algorithms and adapted BCG metric is suggested. The system was designed to be robust and user-friendly, with dual ballistocardiogram (BCG) and electrocardiogram (ECG) capabilities. The BCG was measured from a modified bathroom scale, while the ECG (used as timing reference) was measured using dry handlebar electrodes. The signal conditioning and digitization circuits were USB-powered, and data acquisition performed using a netbook. Four patients with a NYHA class III at admission were measured daily for the duration of their treatment at Stanford hospital. A measure of BCG quality, in essence a quantitative implementation of the BCG classes originally defined in the 1950s, is proposed as a practical parameter.

High-frequency electrical stimulation of cardiac cells and application to artifact reduction.

A novel modality for the electrical stimulation of cardiac cells is described. The technique is based on HF stimulation-burst of HF (1-25 kHz) biphasic square waves-to depolarize the cells and trigger action potentials (APs). HF stimulation was demonstrated in HL-1 cardiomyocyte cultures using microelectrode arrays, and the underlying mechanisms were investigated using single-cell model simulations. Current thresholds for HF stimulation increased at higher frequencies or shorter burst durations, and were typically higher than thresholds for single biphasic pulses. Nonetheless, owing to the decreasing impedance of metal electrodes with increasing frequencies, HF bursts resulted in reduced electrode voltages (up to four fold). Such lowered potentials might be beneficial in reducing the probability of irreversible electrochemical reactions and tissue damage, especially for long-term stimulation. More significantly, stimulation at frequencies higher than the upper limit of the AP power spectrum allows effective artifact reduction by low-pass filtering. Shaping of the burst envelope provides further reduction of the remaining artifact. This ability to decouple extracellular stimulation and recording in the frequency domain allowed detection of APs during stimulation-something previously not achievable to the best of our knowledge.

Rapid assessment of cardiac contractility on a home bathroom scale.

Analyzing systolic time intervals-specifically the preejection-period (PEP)-is widely accepted as one of the few methods for the noninvasive assessment of cardiac contractility. In this paper, we investigated the ballistocardiogram (BCG) as a way to noninvasively measure myocardial contractility when combined with the ECG. Specifically, we derived a parameter from the BCG and ECG that we hypothesized would be highly correlated to PEP. This is the time delay between the J-wave peak of the BCG and the R-wave of the ECG, which we refer to as the RJ interval. The RJ interval was correlated to PEP (r(2) = 0.86) for 2126 heartbeats across ten subjects, with a y-intercept of 138 ms and slope of 1.05. This suggests that the RJ interval can be reliably used as a noninvasive assessment of cardiac contractility.

Multi-signal electromechanical cardiovascular monitoring on a modified home bathroom scale.

A commercially available bathroom scale was modified to enable unobtrusive and robust cardiovascular monitoring in the home. Handlebar electrodes were interfaced to an ultra-low power, two-electrode electrocardiogram (ECG) acquisition circuit providing consistent and clean heartbeat timing information. In addition, the footpad electrodes were used to detect lower-body electromyogram (EMG) and lower-body impedance plethysmogram (IPG) signals using two parallel circuits. The lower-body EMG signal was used as an indication of excessive motion of the subject on the scale. The lower-body IPG signal is related to blood flow through the legs, and will be investigated further in future studies. Finally, the component of bodyweight that varies with time--the ballistocardiogram (BCG) signal--was amplified from the existing strain gauges built into the scale. A preliminary validation was completed on five healthy subjects of varying sizes. The average signal-to-noise ratio (SNR) values computed over all five subjects for the ECG, IPG, and BCG signals were 17.2, 12.0, and 9.0 dB, respectively.

Automatic detection of motion artifacts in the ballistocardiogram measured on a modified bathroom scale.

Ballistocardiography (BCG) is a non-invasive technique used to measure the ejection force of blood into the aorta which can be used to estimate cardiac output and contractility change. In this work, a noise sensor was embedded in a BCG measurement system to detect excessive motion from standing subjects. For nine healthy subjects, the cross-correlation of the motion signal to the BCG noise--estimated using a simultaneously acquired electrocardiogram and statistics of the BCG signal--was found to be 0.94 and 0.87, during periods of standing still and with induced motion artifacts, respectively. In a separate study, where 35 recordings were taken from seven subjects, a threshold-based algorithm was used to flag motion-corrupted segments of the BCG signal using only the auxiliary motion sensor. Removing these flagged segments enhanced the BCG signal-to-noise ratio (SNR) by an average of 14 dB (P < 0.001). This integrated motion-sensing technique addresses a gap in methods available to identify and remove noise in standing BCG recordings due to movement, in a practical manner that does not require user intervention or obtrusive sensing.