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The First-in-Man “Si Se Puede” Study for the use of micro-oxygen sensors (MOXYs) to determine dynamic relative oxygen indices in the feet of patients with limb-threatening ischemia during endovascular therapy
Patients with limb-threatening ischemia exhibit uneven patterns of perfusion in the foot, which makes it challenging to determine adequate topographic perfusion by angiography alone. This study assessed the feasibility of reporting dynamic relative oxygen indices and tissue oxygen concentration from multiple locations on the foot during endovascular therapy using a novel micro-oxygen sensor (MOXY; PROFUSA, Inc, South San Francisco, Calif) approach.
A prospective, 28-day, single-arm, observational study was performed in 10 patients who underwent endovascular therapy for limb-threatening ischemia. At least 24 hours before therapy, four microsensors were injected in each patient (one in the arm, three in the treated foot). The optical signal from the microsensors corresponded to tissue oxygen concentration. A custom detector on the surface of the skin was used to continuously and noninvasively measure the signals from the microsensors. The ability to locate and read the signal from each injected microsensor was characterized. Oxygen data from the microsensors were collected throughout the revascularization procedure. The timing of therapy deployment was recorded during the procedure to assess its relationship with the microsensor oxygen data. Oxygen data collection and clinical evaluation were performed immediately postoperatively as well as postoperatively on days 7, 14, 21, and 28.
The study enrolled 10 patients (50% male) with ischemia (30% Rutherford class 4, 70% Rutherford class 5). Patients were a mean age of 70.7 years (range, 46-90 years), and all were Hispanic of varying origin. Microsensors were successfully read 206 of 212 times (97.2%) in all patients during the course of the study. Microsensors were compatible with intraoperative use in the interventional suite and postoperatively in an office setting. In nine of 10 revascularization procedures, at least one of the three MOXYs showed an immediate change in the dynamic relative oxygen index, correlating to deployed therapy. Moreover, there was a statistically significant increase in the concentration of oxygen in the foot in preoperative levels compared with postoperative levels. No adverse events occurred related to the microsensor materials.
This MOXY approach appears to be safe when implanted in patients with limb-threatening ischemia undergoing endovascular recanalization and is effective in reporting local tissue oxygen concentrations over a course of 28 days. Further testing is needed to determine its potential effect on clinical decision making, both acutely on-table and chronically as a surveillance modality, which ultimately can lead to improved healing and limb salvage.
The estimated global prevalence of peripheral artery disease increased 23.5% from 2000 to 2013, rising to an estimated 202 million cases.
There has been a parallel increase in percutaneous lower extremity interventions. In the United States alone, the percentage of percutaneous interventions increased by 296.8% comparing 1997 to 2008, although the number of amputations has only decreased by 4.6%.
The accelerated rate of endovascular therapy adoption and development of new percutaneous technologies have not been met with consensus-based algorithms that allow physicians to engage in evidence-based clinical practice. Most therapeutic guidelines used to date are based solely on arterial lesion anatomy
and fail to include many other important factors such as patient risk factors and frailty, renal function, affected angiosome, extent of the wound, or presence of infection. New classification schemes aim to address some of these concerns and are in the process of being refined and validated.
which at least attempts to follow a more physiologic and topographically logical perspective to the act of revascularization. Unfortunately, apart from a bidimensional angiogram, there exists no validated on-table tool to evaluate local perfusion improvement as a means to assess the end-point of interventions.
The “Si Se Puede” study aims to characterize the safety and feasibility of using a microsensor-based tissue oxygen monitor called MOXY (PROFUSA, Inc, South San Francisco, Calif) to determine the local tissue oxygen concentration in different locations of the foot of patients with severe limb ischemia during and after endovascular revascularization. This report represents the first-in-man evaluation of a potential tool that can provide on-table feedback regarding local optimization of perfusion as well as a means to evaluate long-term perfusion status.
A prospective, single-cohort, phase 1, first-in-man, 28-day, observational safety and feasibility study was reviewed and approved by the CEATE Foundation (Center of Academic Excellence in Endovascular Therapy), a local ethics committee of the San Juan de Dios Hospital (San Jose, Costa Rica). Each patient provided informed consent before any study-related procedure. This was an investigator-initiated study. No funding was provided by PROFUSA, Inc other than the donation of the investigational devices used during the study.
Study candidates who met the following inclusion criteria were recruited: age 18 to 90 years with a history of symptomatic severe lower limb ischemia (Rutherford clinical categories 4-6), planned endovascular revascularization procedure ≤1 week of screening, able to take acetylsalicylic acid and thienopyridine, and a life-expectancy of at least 1 year. Excluded were candidates who were pregnant, premenopausal, had a history of sensitivity to light, keloid formation, dermatitis, known allergy to materials used in the study, or in the investigator's opinion were not suitable for study participation.
After study screening and enrollment, patient demographic and clinical characteristics were collected. At least 1 day before the patient's endovascular procedure, the investigator selected one location on the arm and three locations on the foot of the index lower limb to be treated for microsensor injections. The arm sensor was placed in a region of presumed normal perfusion to serve as a reference. The foot sensors were placed in three locations of interest with consideration of their proximity to an ulcer or wound (if present, ∼5-10 mm away from the ischemic border), angiosome anatomy, and their likelihood of presenting a change in oxygenation as a result of the planned revascularization. Sensors were placed in the subcutaneous tissue, ∼2 to 4 mm below the skin surface, and were injected using sterile technique with a custom-made 18-gauge injector. Sensor injection sites were marked and photographed to facilitate their subsequent localization.
Before the revascularization procedure, each of the four injected microsensors was located using a detector. The portion of the detector in contact with the patient was wrapped in a sterile cover, and once the sensor was located, the detector was affixed to the skin using adhesive tape or elastic wrap, or both, to enable continuous sensor data collection throughout the procedure. After all sensors were located and detectors secured, the nursing staff began sterile preparation for the surgery.
The investigators performed all endovascular revascularization procedures according to the standard of care defined by the hospital and were blinded to all data acquisition from the implanted sensors. During the procedure, local tissue oxygen concentration data were continuously recorded once every 5 to 15 seconds from all accessible microsensors. The time of each therapeutic step, specifically balloon angioplasty (balloon inflation and deflation) and vascular stenting, was recorded. Serial pulse oximetry measurements and intraoperative angiograms were recorded. After the investigator announced the revascularization procedure was complete, oxygen data collection continued for at least 5 minutes before all detectors were removed from the patient. The manner in which the sensors work is discussed below.
After revascularization, data from microsensors were collected weekly until the patient's participation in the study was complete (defined as 28 days after sensor injection). At these weekly measurement sessions, investigators evaluated the skin area of the sensor injection sites. Tissue oxygen concentration was measured from each injected sensor by using a reader to locate the sensor, affixing the reader onto the skin with adhesive tape, and continuously recording sensor data for at least 5 minutes. Upon study completion, the sensors remained in situ.
The investigators and the clinical care team were blinded to all data collected from the microsensors throughout the entire study to avoid any potential treatment bias. The study protocol therefore had no effect on the conduct or the outcome of any subject's revascularization therapy or clinical management.
Tissue oxygen monitor
The tissue oxygen monitor consists of an injectable, oxygen-sensitive microsensor, an injection device, and a detector. The microsensor is composed of a biocompatible hydrogel called poly (2-hydroxyethyl methacrylate; pHEMA) and a near infrared (NIR) oxygen-sensitive palladium-benzoporphyrin molecule (Pd-MABP). The microsensor senses oxygen in the body based on the principle of phosphorescence quenching of metalloporphyrins, a well-established technique with excellent sensitivity and specificity to physiologic oxygen.
Calibration of oxygen-dependent quenching of the phosphorescence of pd-meso-tetra (4-carboxyphenyl) porphine: a phosphor with general application for measuring oxygen concentration in biological systems.
The Pd-MABP molecules are covalently attached to the pHEMA hydrogel, ensuring that the sensing chemistry is retained in the hydrogel structure.
The miniature sensors (0.5 × 0.5 × 5 mm) were designed to remain in the body permanently. They were soft and tissue-like to minimize stress at the material-tissue interface caused by motion and pressure, which can damage or stimulate adjacent immune cells and prolong the inflammatory phase.
To place a microsensor into the tissue, a custom-made 18-gauge injection device was used to advance the microsensor to the desired depth and hold the microsensor in place while the needle was retracted, leaving the microsensor in the tissue of interest.
A detector was used to measure tissue oxygen concentration from a microsensor. Inside the detector there was a light-emitting diode, a temperature sensor, and photodetector circuitry. Each detector was connected to a central controller with a laptop computer for configuring the detector and viewing data. A maximum of four detectors were used at a time in this study. When a detector was placed on the skin over an injected microsensor, a visual indicator provided confirmation of proper sensor-detector alignment. The light-emitting diode of the detector sent pulses of light into the skin to excite the microsensor, which in turn emitted a light signal back through the skin and to the detector.
The detector processed this signal, resulting in a parameter called phosphorescent lifetime decay (τ). Phosphorescent lifetime decay of Pd-MABP correlates directly to the oxygen concentration of its surrounding environment and thus is a robust parameter unaffected by variations in the optical properties of the tissue.
Different methods were used to analyze and present oxygen levels: τ from a sensor was normalized to the τ from a reference sensor to provide a relative oxygen value; calibration algorithms could also be used to convert τ to the oxygen concentration in molarity (μM).
The current detector reports τ as frequently as every 5 seconds and could record from up to 16 sensors in parallel, allowing continuous, multisite, tissue oxygen monitoring. Long-term monitoring is also possible, as has been shown in an animal model with >13 months of use.
Data presentation and analysis
The safety and the technical performance of the tissue oxygen monitor were evaluated in this study. To characterize safety, the number and nature of adverse events (AEs) were captured. AEs were further classified into research material-related, study procedure-related, or unrelated. To evaluate technical performance, the positive sensor detection rate, defined as the number of sensors located and measured successfully divided by the number of sensors injected across patients and across study days, was characterized. To report local relative tissue oxygen level, a dynamic relative oxygen index (DROID) was defined as the ratio of the phosphorescent lifetime decay of the arm and the foot sensor. Perioperative DROIDs were presented as time series data annotated with therapy deployment times to qualitatively assess the relationship between deployed therapy and local tissue oxygen level (Fig 1; Supplementary Fig, online only). The perioperative DROIDs were reviewed by patient and by sensor. Point measurements of the DROID postoperatively were calculated using the average of the latter half of the data collected during a session to allow time for measurements to reach steady state. Descriptive statistics of the postoperative DROIDs are reported across patients and across sensors. Data were analyzed using R 3.0.3 software (The R Foundation for Statistical Computing, http://www.r-project.org/foundation/). General additive model fits were performed using the package mgcv 1.8-3.
Phosphorescent lifetime decay and sensor temperature were converted into estimated oxygen concentration based on lifetime data collected in vitro. These data were fit to the general additive model:
where ln is the natural log, c0 is a constant intercept, c1 is a constant coefficient, e is a normally distributed error with a mean of 0, and tp is a tensor product of smooth bases.
The resulting oxygen concentrations for arm and foot sensors on the day of surgery are reported. Sensor data are reported in oxygen concentration units (μM) for accuracy because conversion to the more familiar oxygen tension units (mm Hg) required knowledge of the exact oxygen solubility coefficient for plasma.
Patterns were explored by graphing autocorrelation, partial autocorrelation, and extended autocorrelation functions of raw data and residuals from fitted models. Independence of model residuals was checked using the Ljung-Box test.
We modeled estimated oxygen concentrations from each sensor as a function of three periods: a baseline period immediately preceding the endovascular intervention (period 1), the period of endovascular intervention (period 2), and the period immediately after the endovascular intervention (period 3). The model matrix for the fit was coded in such a way that the coefficients for period 3 represent (and can be used to test for the significance of) the differences in oxygen levels (concentrations) and slopes (rate of change in oxygen concentration as a function of time) between period 1 and period 3. Generalized least squares
was used to fit the data to represent means and slopes in a similar manner as ordinary least squares regression, while explicitly accounting for the autocorrelation (ie, nonindependence) among sequential measurements common in continuous biological monitoring data.
Inclusion criteria for the data included availability of equally spaced data from the beginning of period 1 to the end of period 3 with no more than one missing data point in a row anywhere in the interval (no large gaps). In addition, we required a minimum of 50 data points for each of periods 1 and 3 to improve model reliability. As a result, all sensor data were excluded from patient 1 (too few data points in period 3), patient 2 (due to a large gap in period 1), and patient 7 (<50 data points available in period 3). Data from the left foot plantar 1 sensor of patient 5 were excluded (due to a gap starting at the end of period 2 extending into period 3).
The present study was a first-in-human experience designed to assess the feasibility of a microsensor based approach to characterize local tissue oxygen levels to aid in the revascularization and care of patients with limb-threatening ischemia. The positive sensor detection rate was powered sufficiently to evaluate if the microsensors could be located and function consistently >90% of the time using a binomial test. This 90% threshold was chosen based on prior experience and failure rate when operating other tissue oxygen-monitoring equipment. Other time series analyses on DROID were done in a preliminary and exploratory fashion; thus, they were not sufficiently powered to draw statistical conclusions at this point.
Patient demographic information is summarized in Table I. A total of 40 microsensors were injected in 10 patients: 10 sensors were placed in the upper arms as reference, 30 were placed in the feet in the anterior tibial (22 [73%], posterior tibial (7 [23%]), and peroneal (1 [4%]) angiosomes.
Of 21 AEs reported in the study, 0 were research material-related, 13 were study procedure-related, and 8 were unrelated, including 3 serious AEs. The 13 procedure-related AEs were classified as mild bruising at the injection site (32.5%) and occurred in six patients. The three serious AEs were associated with complications of the endovascular procedure: closure device failure with need for operative intervention in two patients and one intraprocedural iliac arterial perforation treated with a polytetrafluoroethylene-covered stent.
During the course of the 28-day study, 212 measurement attempts were made from the injected microsensors: 97.2% of sensors were successfully located and measured, exceeding the predetermined per-protocol threshold of 90% (P = .0004). In five of six failed attempts, the microsensors were located and measured successfully at a later time. One of the failed attempts occurred at the patient's last day of participation, and thus, the sensor's functionality could not be reassessed.
Perioperative DROIDs were calculated using data from 10 patients. During these 10 endovascular revascularization procedures, 96 timestamps of therapy deployment were recorded. Fig 1 illustrates two of the 28 perioperative DROID time series collected. Changes in DROID appeared to align temporally with some deployed therapies: a decrease in DROID after balloon inflation (dark-gray sensor, t = 28, 39 minutes), an increase after balloon deflation (dark-gray sensor, t = 32, 44 minutes), and an increase after stent deployment (dark-gray and light-gray sensors, t = 51 minutes) were observed. Differential changes in DROID were also noted across microsensors in this individual: the dark-gray sensor appeared to be responsive to deployed therapies, whereas the light-gray sensor did not indicate a change in DROID until ∼t = 48 minutes later on in the procedure.
Preoperative and postoperative DROIDs were calculated from data obtained in 38 sessions with participants over a course of 4 weeks, yielding 138 data points. Measurements were categorized into preoperative (n = 28), immediately postoperative (n = 28), 1 week (n = 15), 2 weeks (n = 21), 3 weeks (n = 23), and 4 weeks (n = 23) after the revascularization procedure. Descriptive statistics of this data set are presented as a box plot in Fig 2.
In vitro calibration data were used to convert the phosphorescent lifetime decay and temperature values collected from this study into oxygen concentration in μM in an exploratory analysis. Converted data in μM were fitted into a regression model to specifically examine (1) the value changes in oxygen concentration and (2) the slope changes in oxygen concentration before and after the revascularization procedure. Fig 3 illustrates the oxygen concentration value measured in the beginning of the revascularization procedure, before any interventions had been performed (pre) and at the end of the procedure, with the patient still on the table (post). These results were further categorized into arm reference sensors and foot sensors. The median oxygen concentration of the reference arm sensors decreased nominally from 58.1 μM to 55.2 μM (n = 6), and the median oxygen concentration of the foot sensors increased from 10.7 μM to 28.7 μM (n = 16). The increase in median oxygen concentration observed in the foot sensors was statistically significant using a one-tailed Wilcoxon test (P = .0042). Moreover, we observed that the oxygen concentration might not have reached steady state upon procedural completion because the sensor signals trended upwards, indicating continued reoxygenation of the tissue after revascularization. This observation was verified in some cases in which increasing oxygen concentrations were captured in the recovery room. Table II summarizes the arm and foot sensor data in the postintervention period.
Table IISummary of oxygen sensor slope data for arm and foot sensors in the immediate postintervention period
Positive slopes that are significant compared with preintervention slopes (P < .05) are bolded. Most foot sensors (12 of 16) demonstrated a significant increasing oxygen concentration immediately after the intervention. Arm sensors exhibited negligible or decreasing oxygen slopes. Refer to the Results for details on patient exclusions.
Postintervention slope, μM/min
a Positive slopes that are significant compared with preintervention slopes (P < .05) are bolded. Most foot sensors (12 of 16) demonstrated a significant increasing oxygen concentration immediately after the intervention. Arm sensors exhibited negligible or decreasing oxygen slopes. Refer to the Results for details on patient exclusions.
A positive slope indicates oxygen levels were increasing. The regression model showed that 94% of the foot sensors (n = 16) demonstrated a positive slope change, in which 80% of them were considered significant compared with slopes in the preintervention period (P < .05). Arm sensors exhibited negligible or decreasing oxygen slopes.
A major tenet of limb salvage therapy is that one must recognize perfusion deficiencies and be able to grade the severity and monitor their improvement after revascularization. In fact, one of the essential components of the Society for Vascular Surgery Wound, Ischemia, and foot Infection (WIfI) Threatened Limb Classification System is to assess perfusion in all patients with threatened limbs. In modern tertiary vascular practice, where many patients have diabetes and medial calcinosis, with open foot wounds or previous amputations, traditional means of measuring perfusion are problematic and often not applicable or reliable.
The MOXY sensor provides a new method of measuring tissue oxygen concentration without perturbing the tissue after the initial injection. In this first-in-man study, the evaluation of the sensors in the perioperative period demonstrated their safety for use in patients with limb-threatening ischemia. Most device-related AEs were limited to small ecchymotic areas, most likely secondary to the 18-gauge needle inserted to administer the sensor. A simpler and less invasive method of delivery to avoid patient discomfort (although reported to be minimal during the implantation period for this cohort) is a straightforward product development task.
The implantation of the devices was simple and effective, and the sensors showed a very high detection rate of 97.2% during a total of six patient visits (preoperative, postoperative, and at weeks 1, 2, 3, and 4). Although this is a small cohort with a relatively short 28-day follow-up, the ease of sensor localization and robust sensor signal are promising and provide insight into the potential clinical applicability in the acute and chronic phases of care. There exists a straightforward path to detector miniaturization and a form factor conducive to measuring oxygen in the operating room and, eventually, home use, which is critical to technology adoption and a clear potential advantage over other methods, such as fluorescence angiography, for sensing biological oxygen.
Compared with other oxygen-sensing devices in the clinical setting, MOXY offers distinct advantages. First, it directly measures tissue oxygen concentration within the tissue, which differs from completely noninvasive (no injectable sensor) technologies, such as pulse oximetry and near-infrared spectroscopy (NIRS), that measure oxygen saturation in the vasculature (Fig 4). Pulse oximetry measures the oxygen saturation in the arterioles and requires a pulse and is therefore less useful for monitoring tissue oxygen saturation in ischemic tissue. NIRS measurements do not require a pulse but provide estimates of the oxygen available for diffusion into the tissue (StO2). Although studies have been published on the use of NIRS in peripheral arterial disease and ischemic tissues,
The electrode warms the surface of the skin to 44°C to disrupt the outermost skin layer (stratum corneum) so that oxygen can exit the tissue. TcpO2 has been demonstrated to predict the ability of diseased tissue to heal (eg, TcpO2 readings <40 mm Hg correlate with impaired wound healing). Criticisms of the technique include the time and complexities involved with obtaining reliable measurements, interference from edema and inflammation, and the requirement for elevated temperatures at the measurement site causing an artificial measurement environment.
The microsensor technology was proven to be reliable in providing real-time information during the perioperative period. Although the blinded condition of the operators limited a systematic evaluation of sensor response to each interventional step, there was a clear trend upwards in tissue oxygen in the immediate postoperative period. The interpatient baseline oxygen tissue concentration is potentially affected by hypoxemic morbidities as chronic obstructive pulmonary disease or congestive heart failure, as well as intraoperative interventions such as oxygen supplementation.
The DROID relationship was created in an effort to normalize any potential confounding systemic oxygen effects (Fig 2). Although not statistically significant because of the underpowered nature of a small sample, there was a clear trend of improvement, which was fairly acute in nature: the largest change was realized within the postoperative period and stabilized by 1 week. Moreover, the level was fairly stable out to 4 weeks. This observation suggests the potential use of the microsensor as an on-table tool to optimize results. Unpublished data in healthy individuals demonstrated MOXY's ability to sense changes in oxygen levels within seconds, which, if also true for this group of patients, could empower the operator to tailor an intervention to optimal perfusion-goal performance.
The “Si Se Puede” first-in-man study has shown that the MOXY sensor appears to be a safe and effective tool to measure tissue oxygen concentrations in real-time in patients with limb-threatening ischemia during the perioperative period of planned revascularization procedures. The study results show that use of these sensors merits further testing to determine their potential effect during on-table clinical decision making and long-term follow-up, with the ultimate goal of improved wound healing and limb salvage rates.
Conception and design: NW, SG, KH, MMB, LMA
Analysis and interpretation: KA, KH, MMB
Data collection: KA, MMB, LMA, MC, NW
Writing the article: KA, KH, JM, MMB
Critical revision of the article: KA, KH, JM, MMB, NW
Final approval of the article: KA, NW, SG, KH, MMB, JM, MC, LMA
Statistical analysis: KA, KH
Obtained funding: KH, NW
Overall responsibility: MMB
The authors wish to acknowledge Dr Mitch Kostich for his contributions to data and statistical analysis.
Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis.
Calibration of oxygen-dependent quenching of the phosphorescence of pd-meso-tetra (4-carboxyphenyl) porphine: a phosphor with general application for measuring oxygen concentration in biological systems.
Sensor development was supported by W911NF-11-1-0119, W31P4Q-12-C-0205 and R43DK091155, and optical reader development was supported by R43DK093139 and R01EB016414. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Defense Advanced Research Projects Agency or the U.S. Army Research Office.
Author conflict of interest: K.A.Y., N.A.W., S.G., and K.L.H. are employees of PROFUSA, Inc and have shares in the company.
Presented at the Twenty-ninth Annual Meeting of the Western Vascular Society, Coronado, Calif, September 20-23, 2014.
The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest.