Soft Gripper Robotics

RoSE: A Robotic Soft Esophagus for Endoprosthetic Stent Testing Dipankar Bhattacharya1,2 , Sherine Jesna V. A.1,2, Leo K. Cheng2,3,4, Weiliang Xu1,2,4 Project page: https://bhattner143.github.io/rose-stent.github.io/

Abstract

Soft robotic systems are well suited for the developing devices for biomedical applications. A bio-mimicking robotic soft esophagus (RoSE) is developed as an in vitro testing device of endoprosthetic stents for dysphagia management. Endoprosthetic stent placement is an immediate and cost-effective therapy for dysphagia caused by malignant esophageal strictures from esophageal cancer. However, later stage complications, like stent migration could weaken the swallow efficacy in the esophagus. The stent radial force (RF) on the esophageal wall is pivotal in avoiding stent migration. Due to limited randomized controlled trials in patients, the stent design and stenting guidelines are still unconstructive. To address the knowledge deficit, we have investigated the capabilities of the RoSE by implanting two stents (stent A and B) of different radial stiffness characteristics, to measure the stent RF and its effect on the stent migration. Also, endoscopic manometry on the RoSE under peristalsis has been performed to study the impact of stenting and stent dysfunctionality on the intra-bolus pressure signatures (IBPSs) in the RoSE, and further its effects on the swallowing efficacy. Each implanted stent in the RoSE underwent a set of experiments with various test variables (peristalsis velocity and wavelength, and bolus concentrations). In this paper, the conducted tests are representatives to show the application of RoSE to perform a wide-ranging assessment of the stent behavior. The usability of RoSE has been discussed by comparing the results of stent A and B, for various combinations of the test variables mentioned above. The results have demonstrated that the stiffer stent B has a higher RF, whereas, stent A maintained its RF at a low profile due to its lesser stiffness. The results have also implicated that a high RF is necessary to minimize the stent migration under prolonged peristaltic contractions in the RoSE. For the manometry experiments, stent A slightly increased the IBPS, but the stiffer stent B significantly decreased the IBPS, especially for the higher concentration boluses. It was found that if a stiffer stent buckles, it can reduce the swallow efficacy, and cause recurrent dysphagia. Therefore, RoSE is an innovative soft robotic platform capable of testing various endoprosthetic stents, thereby offering a solution to many existing clinical challenges in the area of stent testing.

Keywords: stent migration; radial force; soft robotics; robotic esophagus; biomimetics

Ntroduction

Swallowing is a complex but orderly physiological process transporting saliva or food from the mouth to the stomach. The swallowing physiology and anatomy are elucidated with the critical insights from the in vivo studies.1 Any esophageal impairment compromises the efficiency of 4 Medical Technologies Centre of Research Excellence, Auckland 1010, New Zealand.

This is the author accepted manuscript (AAM). The Version of Record is available at: Citation: Bhattacharya D, Ali SJV, Cheng LK, Xu W. RoSE: A Robotic Soft Esophagus for Endoprosthetic Stent Testing. Soft Robotics. 2021;8(4).

swallowing, is known as dysphagia.2 Severe pathologies, for instance, benign esophageal strictures from various injuries, esophageal cancer, and esophageal perforations, distressing lumen patency explicitly leading to dysphagia. Unaccompanied by comprehensive care, a vicious cycle materializes as malnutrition and dehydration aggravating the dysphagia itself, subsequently increasing morbidity and even mortality.3 The standard clinical practices involve evaluation of the etiology of the swallowing deficit using perception studies, and in vivo analysis, followed by intervention strategies for nutritional support.4 Depending upon the severity, the method of maintaining nutrition, varies from oral dietary supplements, texture modified foods, implanting nasogastric feeding tubes, surgical correction, endoscopic dilation

Of The Sphincters, And Esophageal Stenting.2, 5

The textured food modification is observed to maintain nutrition in the dysphagia and to improve swallowing safety and efficacy.2, 5 In altered food swallowing, videofluoroscopy and manometry are considered as gold standards to study the temporal-spatial aspects of bolus geometry and intrabolus pressure, respectively. In an esophageal peristaltic transport of food bolus, the manometry recordings can be distributed into two intraluminal pressure segments. In the first segment, within the bolus fluid, the recorded pressure is solely due to the bolus hydrodynamic pressure, which is also known as the intrabolus pressure signature (IBPS). At the tip of the bolus tail, the pressure undergoes a transition from IBPS to esophageal direct contact pressure with the manometry catheter, which can be regarded as the second segment. The maximum IBPS occurs at the bolus tail tip, and after which, no bolus fluid exists, leading to direct contact pressure. Since pressure cannot be transmitted axially in the absence of bolus fluid; thus, these two segments are independent of each other.6 Videofluoroscopy, and manometry techniques are used hand in hand to determine the maximum IBPS. The shortcomings of these techniques are X-ray radiation exposure and catheterization (inserting a manometry catheter) that accumulate to the efforts of swallowing and the general health of

Subjected Individuals.7

Malignant and benign esophageal strictures from esophageal cancer, can be addressed with the endoprosthetic stent placement, commonly known as esophageal stenting.8 The earlier use of uncovered stents for palliation was limited and contributed to in situ erosion, occlusion, and fistulation. However, with the advent of fully coated removable self-expandable plastic stents (SEPSs), self-expandable metallic stents (SEMSs), and biodegradable stents, remarked new applications with changeable success.9 These stents designate a novel, alternative, immediate, and cost-effective therapy for managing adequate oral nutrition during dysphagia. A silicone covered SEMS is a tubular braided mesh of interwoven helical springs made of corrosion- resistant materials, like nitinol, stainless steel, and polymers.10 These stents are proven to be efficient endoprosthetic management for both malignant and benign esophageal strictures, as they could hold open the esophagus and hence, relieve the impediments of compromised lumen patency in such cases (Fig. 1). Furthermore, the insertion procedure of a SEMS is less traumatic because of its flexibility, and it can be readily compressed into a smaller delivery system.10-12.

However, this method of palliative treatment does have its inadequacies, and one of the significant shortcomings is stent migration, caused due to the interactions of an implanted stent with the continuous peristaltic contractile forces of the esophageal wall.13 The stent deployment has reported about 30-50% long term success rates, with migration being the main associated complication in most of the failure cases.14, 15 The efforts to mitigate pain and migration, to improve stent removability and flexibility, and to ensure stent patency have revolutionized the SEMS designs. The evolution of the stent design has made stenting a more effective treatment in both benign and malignant esophageal diseases. However, in the event of esophageal stent implantation, manometry and videofluoroscopy on the patients are less preferred for a compelling study on bolus geometry, IBPS, and stent inadequacies (such as stent migration), because the techniques are not very comforting for the patients, and there are major ethical concerns associated with such kind of studies. Despite recent technological innovations in this field, evidence to show which stent design is better than the other is confined to a few randomized controlled trials in patients with malignant esophageal strictures.16 The SEMSs have been found considerably similar to the esophageal prostheses.17According to the current US FDA review guidance for esophageal and tracheal prostheses devices bench testing of esophageal prostheses is needed to establish substantial equivalence.18 The review guidance has recommended six different stent testing protocols. They are as follows: 1) Compression force testing: It measures the force required by the stents for compression. 2) Expansion force testing: It measures the force exerted by the stents during expansion. 3) Corrosion testing: It tests the compatibility of the stent materials with the corrosive environment in the esophagus. 4) Tensile strength tests: If a stent includes a bonded or welded part, then this test needs to be performed. 5) Deployment testing: It validates the accuracy and repeatability of the stent delivery system. 6) Dimensional testing: It verifies the dimensional reproducibility of the stents after deployment. The first four tests are related to stent performance after its deployment, and the last two tests are relevant in terms of the insertion procedure. The compression and expansion force testing of the stent includes stent radial force (RF) measurement, which is crucial in designing a stent for maintaining the in situ lumen patency, which is, unfortunately, still an unknown parameter due to the poorly understood association of the RF and clinical outcomes (Fig. 1A).11, 12 Since the adequate data on migration issues, critical complications, and morbidity in stent deployment, as well as extraction, are lacking, the stenting acting on the esophageal wall is the opening RF applied by the stent per unit contact area of the stent (𝜋𝑑𝑒𝑙𝑒). The internal pressure accounts for the stress in the esophageal wall, known as the circumferential (hoop) stress (𝜎𝜃,Fig. 1B).19 RF assures proper fixation of the stent in the esophagus, and an essential factor in limiting stent migration.

Fig. 1. Cross-sectional view and free body diagram of an esophagus with an implanted stent. (A) Schematic of isometric view showing the cross-section of an esophagus with an implanted stent. The purple blue arrows represent the radial force (RF) applied by the stent. (B) Free body diagram of the esophagus region, where the stent resides. The hoop stress, 𝜎𝜃 is given by 𝑝𝑑𝑒/2𝑡𝑒, where 𝑝 is the applied pressure, and equal to RF/𝜋𝑑𝑒𝑙𝑒.19 While a considerable amount of literature is available on novel stent designs, little attention has been paid on comparing the differences in RF and its clinical impacts among the available stents in the market. An investigation in such a direction could potentially play a pivotal role in dealing with the migration and symptoms of pain. The mathematical modeling of the stent interaction with the vessel wall, provided in various studies, could be useful for predicting the mechanical properties of the stent. Still, the studies fail to embrace the esophageal peristalsis complexity and precisely replicate the association between the esophageal wall and the stent.

The analytical model of the stent based on the theory of virtual work by Jedwab and Clerc 20 is useful for predicting the mechanical properties of a stent (such as the RF), but it model fails to provide any implication of stent migration with its mechanical properties. Hirdes et al.11 evaluated RF of several commercially available stents, but the author could not offer clear evidence to show that high stent RF can prevent stent migration. Likewise, numerous numerical studies have focussed solely on explaining the mechanical deformation of the stent inside the host tissue.21, 22 Very few numerical models have examined the interaction between the stent and the esophagus under peristalsis.15, 23 Garbey et al.15 analyzed stent migration in a numerical esophageal model with stent flare design, stent length, diameter, and radial stiffness but not directly with stent RF. Mozafari et al.23 identified flared stent ends, and a higher frictional coefficient of the stent can have a considerable impact on mitigating stent migration. The study was done with an FEA built esophagus, under axial pulling (peristalsis was not considered). In Soft robotics in vitro models has proved to be a complementary and supplemental approach to mathematical models, and clinical studies to investigate the human physiology and to validate medical procedures.24 The excellent adaptability of soft robots to their environment has been explored in the recent decades, leading to the development of next-generation soft materials and soft actuators, soft stretchable electronics, and control, and processing.25-28 Soft robotics also widens the opportunities for developing devices in the field of biomedical applications, such as therapeutic devices,29 rehabilitation devices, and prostheses,30 devices for training and biomechanical studies.31 With Kobayashi et al.32 pioneering the robotic swallowing simulator works, the studies in silico and in vitro model of esophageal phase have provided reasonable mechanistic insights for many interesting archetypes observed in the in vivo investigations of the human swallow physiology.33 Hence a bio-mimicking robotic soft esophagus (RoSE) is developed as an esophagus simulator with extended biomedical applications.34 In the medical domain, testing bolus formulation and transport are hindered by the inter-person swallow and the inter-swallow variability in human test subjects.35 Variations in muscle actuation strength and peristalsis wave speed greatly influence the manometry captured IBPS in the subjects; thus, testing and measuring the swallow efficacy in a man is qualitative. In the mathematical field, the modeled boluses do not reflect the complicated behavior of bolus materials such as multi-phase flow and non-Newtonian property.36 Besides, it is challenging to model the shear fields generated by peristaltic actuation and their associated time-shear dependent behavior. The soft-bodied robot, RoSE (Fig. 2) can physically mimic the human swallowing action, by generating peristaltic waves (Fig. 2), to transport the food bolus along the conduit. Unlike other in vivo methods, RoSE offers a more steady swallowing behavior and does not hold the risk associated with testing actual human subjects.34 RoSE bridges the gap between clinical and mathematical modeling fields, and it is proposed to deform materials to achieve clinically significant rheometry more faithfully. Besides, RoSE could vary the bolus parameters and the peristaltic parameters (such as wave speed and wavefront length) independently, which is crucial to comprehend the effect of these variations as a fit to a broader bolus formulations were tested on the RoSE with manometry and videofluoroscopy. The manometric pressure profiles achieved are comparable to human swallowing behavior.37 Since RoSE can physically mimic the human swallowing behavior; thus, instead of actual patients, RoSE can be used to conduct the study on the various stent designs and their inadequacies before implanting them in patients with malignant and benign esophageal strictures. Besides, to evaluate swallow efficacy, RoSE can also be used to study the effect of stenting on IBPS of different texture modified foods for dysphagia patients.

This study aims to investigate the following capabilities of RoSE, 1) to perform experiments on two stents having different radial stiffness characteristics for evaluating their respective RFs, 2) to study the effect of the RFs on the migration of the stents, 3) to learn the impact of the reduction of RoSE conduit diameter caused by stent deployment on IBPS, and 4) to analyze the effect of stent dysfunctionality on bolus transport, and hence, swallow efficacy.

The RoSE stent testing protocols provides a novel in vitro platform to perform a wide-ranging assessment of the stent behavior. Besides, the protocols are capable of generating spatio- temporal peristaltic waves of different characteristics in the RoSE conduit. In this study, commercially designed stents are subsequently deployed in the RoSE and analyzed to validate the above-vested capabilities of the Mechatronics device. Along with the circumferential loading of the stent, studies on the effect of stent implantation on bolus transport, under peristaltic contractile forces are done by controlling the RoSE conduit deformation.

The analysis of the bolus pressure signatures with stenting in a soft-robotic in-vitro platform, like the RoSE, has extended our knowledge of swallow efficacy after stent implantation during dysphagia. Besides, this study has also done endoscopic manometry in the presence of stents, and it has also considered esophageal peristalsis for stent-related measurements. To the best of our knowledge, studies correlating the effect of stent implantation and dysfunctionality on swallow efficacy with parameters (such as IBPS and IBPS gradient) that can be measured, still does not exist. Little is known about the IBPS and IBPS gradient due to the implantation of stents and their dysfunctionality. Much of the research up to now are clinical studies.

Investigating stent RF has been a continuing concern within the clinicians and the scientific community. It has been challenging to fully predict the clinical outcome of a stent based on its RF and bolus pressure signature data.11 However, the presented results show that RoSE can provide a platform to assess various stent behaviors, that can help the researchers and the endoscopists to elucidate the patency of the stents in the occurrence of unfavorable events, during and after the stent implantation. Also, the results comparing the efficiency of the stents could aid the endoscopists in the selection of a suitable stent for an individual patient, which is otherwise guided solely by their experience and availability of the stents. The results will also aid in the optimization of future stent designs to improve the behavior of the stent during deployment and to mitigate migration.

Rose Actuator, Design And Materials

RoSE actuator has 12 layers (L1, L2, ..., L12) of regular and repeating pneumatic hollow chambers arranged horizontally along the axis of the device, with a whorl width of 10 mm each (Fig. 2). Each layer has four chambers arranged axis symmetrically, and have whorl boundaries of 5 mm thickness with theoretically identical properties (Fig. When pressurized with air, the four chambers expand and closes the cross-sectional area of the food passage and push off the housing structure to cause the radially occlusive peristaltic motion, mimicking circular muscle activation. Though the actuation in RoSE is distributed, by reducing the actuator chamber width, whorl boundary and conduit thickness, continuous peristaltic actuation is achieved (Fig.

2). The actuator design specifications are based on the FEA analysis conducted by Chen et al.38 Table 1 provides the quantitative and qualitative characteristics of RoSE, which are required for performing stent RF and migration, and stent implanted RoSE swallow efficacy testing.

Fig. 2. Side and top sectional view of robotic soft esophagus (RoSE). The left and right drawings reflect the longitudinal and axis-symmetric arrangement of the chambers, respectively. Table 1: Quantitative and qualitative characteristics of RoSE

Kpa

Pneumatic pressure required to achieve medical wave

Overlapping-Sequential

RoSE is fabricated using custom-designed mold and housings, which are rapid prototyped via fused deposition of acrylonitrile butadiene styrene (ABS) plastic (Fig. 3A). An RTV silicone rubber material (Ecoflex 0030, Smooth-on, USA) is chosen to build the actuator (Fig. 3B). The silicone rubber was chosen for its low 100 % modulus and pour viscosity, high tear resistance, and surplus elongation at break (900 %), which is needed during actuator deformation. In addition, RoSE design is based on the idea of actuating many layers in a subsequent overlapping manner, which mimics the esophagus in recruiting muscles in a rostro-caudal way, to achieve peristaltic transport smoothly. The multi-casting fabrication procedure of the RoSE is shown in Fig. 3A to F.

Fig. 3. Multi-casting fabrication procedure of RoSE. (A) The actuator mold and housing are rapidly prototyped by the printing method from ABS plastic (Elite Printer, Dimension, USA), and the initial assembly is built. (B) In vertical orientation, the conduit and the chambers are constructed by casting silicone rubber (Ecoflex 00-30, Smooth-On, USA). (C) After the silicone cured, the chamber place holders and the plastic rod is removed. (D) In horizontal orientation, four outer castings of silicone are made to seal the chambers from the sides. (E) Four outer casings are added to confine the chamber deformation within the conduit. (F) Pneumatic tubes and tees are connected to provide air pressure to the whorl of chambers (G) Final assembly of RoSE.

Rose Firmware Protocol

Custom firmware modules, written in Python 3.7, are developed on Raspberry Pi 3B+ to assert the robot, with independent, continuously variable, pressure input (Fig. 4). The RoSE firmware module is divided into two significant sub-modules: A) Symmetric actuation protocol, for simultaneous inflation and deflation of the RoSE conduit, to continuously record the radial force during contraction and expansion of stents. B) Peristaltic actuation protocol, for generating spatio-temporal waves in the RoSE conduit, to learn the stent migration and effect of stenting on the IBPS (Fig. The user can select an appropriate testing protocol module by following the prompts on the IDLE (Python’s integrated development and learning environment) shell window. Each testing protocol (sub-module) selects a CSV file where 8-bit digital values corresponding to a pressure range are stored for L1, L2, ..., L12 layers of RoSE.

Fig. 4. Flow chart illustrating system description of RoSE. RoSE firmware protocol is capable of generating two different actuation trajectories (symmetric and peristaltic actuation) based on the input commands of the user. The firmware protocol for symmetric and peristaltic actuation, developed on Raspberry Pi 3B+, is enclosed within the red and green dashed lines, respectively. The blocks outside the dashed lines represent the RoSE hardware, which consists of interfacing ADC and DAC board, assembly of pneumatic valves, and sensors such as TOF and valve in-built pressure sensor.

Pneumatic pressure to RoSE is regulated in open-loop by an electro-pneumatic interface which consists of a series of 12 pressure regulating proportional valves (ITV-0030-3BS, SMC, Noblesville, IN, USA), and an interfacing ADC (MAX11605, Maxim Integrated, San Jose, CA, U.S.) and DAC (AD8802, Analog Devices, Norwood, MA, USA) board, interfaced with the Pi (Fig. 5). The Pi communicates over SPI and I2C with the DAC and ADC, respectively, to perform assertion and feedback measurement. The DAC converts 8-bit digital values, stored in the CSV files, to voltage levels, which controls the valves. The ADCs are then used to receive various sensor outputs such as in-built valve pressure sensor and force sensing potentiometer (FSP01CE, Ohmite, active area – 10 x 13 mm2). The valve pressure sensors are used to record the chamber pressure-time trajectory (Plot A and B, Fig. 4).

Fig. 5. Schematic and image of the electro-pneumatic interface controlling RoSE. An assembly of 12 proportional pneumatic valves, sourced by compressed air supply, are implemented to control the pressurization of 12 layers of RoSE. The valves assembly is controlled by an custom-built interfacing ADC and DAC board, connected to a Raspberry Pi 3B+. Firmware protocols are developed on the Pi with Python (v3.7) to actuate RoSE with independent, continuously variable pressures.

In symmetric actuation, layers L1 to L12 of the RoSE were actuated, with the same pressure input, from 11 KPa to 71.5 KPa in 1 KPa increments. In a CSV file, 8-bit digital values corresponding to the pressure range is stored (File A, Fig. 4). Each value was held for 5 s to check the repeatability of the sensors (data was collected at 1-second interval) and to allow the chambers to reach their settling deformation.

For manometry, and migration experiments, three adjacent RoSE layers are sinusoidally actuated with multi-level pressures to achieve the spatial-temporal aspects of peristalsis waves. In the peristaltic actuation protocol, the displacement of the conduit can take the shape of a sinusoid with complete occlusion at the point where the minima of the sinusoid occur. By displacing the sinusoid from L1 to L12 in small increments, continuous peristalsis wave in RoSE was achieved. The sinusoid can be represented by (1).

(1)

where 𝐻(𝑥,𝑡) is the time-dependent conduit radius (mm),  is the minimum conduit radius (mm),  is peak-to-peak peristalsis wave amplitude (mm), c is peristalsis wave velocity (mmps), /2 is sinusoidal wavefront length (mm), x is conduit axial displacement (mm), and t is time (s) .

From the IDLE shell window, the user can define the peristalsis wave speed (c) and wavefront length (/2). Each combination of the wave speed and wavefront length selects a specific CSV file (File B, Fig. 4) that stores a set of 8-bit digital values to impose a time-variant pressure trajectory between adjacent RoSE layers (Plot B, Fig. 4), so that RoSE conduit can displace in a peristaltic manner. For complete occlusion, an operating pressure range of 11 KPa to 71.5 KPa was used. The actuation and the continuity of peristalsis pattern are verified using medically inspired techniques such as videofluoroscopy,37 articulography,34 and image

Stent Configuration

Fifteen commercial, covered SEMSs having distinct structures, cover materials, and cover material patterns, and similar dimensions were initially tested for stent migration in RoSE. Based on the maximum and minimum recorded migration, candidate stents: stent A and B were selected for further measurement and analysis of different stent-related parameters (such as stent RF, radial stiffness and migration), and bolus pressure signatures in RoSE with and without the stents.

The commercial stent A and B with different radial stiffnesses, under test, are cylindrical with flares at both ends, covered in silicone, and braided from a single thread of highly elastic nitinol wire of 0.10 mm diameter (Fig. 6). When fully expanded, the main body of the stents measured 110 mm in length and 23 mm in diameter. The flare ends are 10 mm in length and 5 mm wider in diameter than the body.

Fig. 6. Silicone covered self-expandable metallic stents (SEMSs). (A, and B) Stent A and B have a mean radial stiffness of 1.55  0.24, 3.13  0.53 Nmm-1 respectively. Although both the stent has the same nitinol braided wire mesh configuration, the silicone coating thickness and pattern are different. Stent A has no pattern on its silicone cover, while stent B has much thicker silicone cover with a pattern on it.

Food Bolus

Synthetic boluses of starch-thickened water (Altrix Rapid Thickener, Douglas Nutrition Ltd, New Zealand), have been used as a clinically significant substitute to masticated boluses throughout experimentation. Boluses were formulated on a concentration basis (72, 108, 144 gL−1) by thorough mixing, after which they were left to stand for an hour to stabilize. The settling time allowed the starch granules to take up water and achieve a stable structure which directly affects the rheological properties of the boluses. These boluses formulations covered the range from syrup thin to pudding thick based on the product specification as per International Dysphagia Diet Standardization Initiative (IDDSI) specification.5 All the formulations of the starch-thickened water have exhibited shear thinning behavior. Due to this non-Newtonian effect, the viscosity will be depended on the flow characteristics such as peristalsis wave velocity. In this study, the three used bolus concentrations mixes are labeled as bolus I, bolus II, and bolus III, respectively (Table 2).

Table 2. Characteristics of the starch-thickened food boluses.

(Gl-1)

The protocol of stent RF and migration measurement To ensure prolonged functionality of the esophageal stent in the face of the repetitive peristaltic contractile forces associated with the peristalsis, a high RF and fatigue strength, corrosion resistance, and deformability are required. After the deployment of the stent in RoSE conduit (Fig. 7 A, to C), the stent apply an opening radial pressure to the conduit wall, resulting in a hoop (circumferential) stress in the wall, increasing its diameter.19 A similar phenomenon occurs in a stent implanted esophagus (Fig. 1). The pressure is associated with the stent RF on the RoSE conduit wall as it tries to expand back to its initial diameter. The pressure can be defined as the stent opening RF acting on the conduit wall per unit cross-sectional area of the RoSE conduit (Fig. 7C) whereas, the hoop force (HF) is the hoop stress multiplied by the cross- sectional area of the RoSE conduit wall, where the stent resides (footprint of the stent).

Similarly, radial pressure on the stent imposed by the RoSE contraction, generates RF and HF on the stent wall (Fig. 7 D to F). Fig. 7. Experimental setup to measure the radial force (RF) exerted by a stent on RoSE conduit. (A) Schematic of the experimental setup used for measuring the stent RF. Firstly, the testing protocol comprised of implanting a force-sensing potentiometer (FSP), followed by the subsequent deployment of the stents. Secondly, all the RoSE layers (L1, L2, ..., L12) were inflated and deflated cyclically with the same air pressure to generate cylindrical contractions, and expansions respectively. (B) Endoscopic image showing the cross-sectional view of the RoSE conduit during contraction, captured from the distal end of the RoSE. (C) Schematic showing the position of the stent and the FSP inside the RoSE conduit. The purple arrows represents the opening force applied by the stent. (D, and E) Schematic showing the side and top view of the stent-FSP implanted RoSE under contraction (blue arrows are representing the loading on the stent). (F) Free body diagram of the stent to represent RF and HF.

The RF can be classified into: 1) Radial resistance force (RRF): RRF can be defined as the stent resistance to the loading when the RoSE conduit (esophagus) is contracting (RRF is the RF shown in Fig. 7 D to F). 2) Chronic outward force (COF): When the RoSE conduit (esophagus) is expanding, the force exerted by the stent to regain its original diameter during its unloading (Fig. 7C) is known as COF. RRF and COF are forces associated with the loading and unloading of the stents, respectively.

By using a force-sensing potentiometer (FSP01CE, Ohmite, active area – 10 x 13 mm2) sensor, the RF exerted by the stent on the RoSE conduit wall was measured (Fig. 7). After lubricating the conduit of the RoSE with artificial saliva (Aquae Dry Mouth Spray, Hamilton), the stent under the test was deployed from the distal side of the RoSE by using an intruder sheath and a retrieval thread fastened to the proximal end of the stent. While holding the stent with a rat tooth forceps from the distal side of the RoSE, and by withdrawing the intruder sheath gradually from the proximal end of the RoSE, the stent was released slowly in the conduit. Owing to the stent stored strain energy, it self-expanded and exerted an RF on the conduit wall, and hence on the FSP sensor, which readily hooped around the stent (Fig. 7E, Movie S1). To achieve uniform axial displacement during compression, both the stent ends were allowed to move freely.

To ensure a consistent reference configuration, the stent center was positioned to coincide with the distal end of FSP active region having a width of 𝑊𝑓 (Fig. 7C). The 3 mm smaller diameter of the RoSE conduit from the stent body caused transverse loading of the stent which deformed the stent both axially and radially. If 𝑙𝑠 and 𝑑𝑠 are the new deformed length and diameter of the stent respectively, and if 𝐹𝑓 is the force recorded by the FSP, then stent radial pressure (𝑃𝑠) can

(1)

By considering uniform diameter throughout the stent, the lateral surface area of the stent is given by, 𝐴𝑠= 𝜋𝑑𝑠𝑙𝑠. Hence, the stent RF (𝐹𝑠) can be written as:

(2)

Eq. (2) is also applicable for stent loading (contraction) and de-loading (expansion) under symmetric actuation of RoSE (Fig. 7D). The stent radial stiffness (𝑘) can be defined as how much diameter of the stent is reduced by the application of the force exerted by the RoSE conduit. The stiffness signifies the effectiveness of the stent in resisting diameter change during RoSE contraction and expansion. By differentiating (2) with respect to 𝑑𝑠 and taking its

(4)

For measuring the elongation strain of the stent during the pressurization cycle, a time of flight (TOF) sensor was mounted vertically at the distal side of the RoSE. By using a nylon thread through a pulley, a vertically displacing paperboard marker was connected to the distal end of the stent. The TOF sensor was used to record the displacement of the marker (stent elongation) in the vertical direction (Fig. A1 A of Supplementary Materials). Finally, the elongation strain was calculated by the change in the marker displacement (stent length) per unit stent’s initial length.

The RF stent testing protocol included symmetric actuation of the RoSE layers from L1 to L12, with the same pressure simultaneously (Fig. A1 B of Supplementary Materials, Movie S1). Within the RoSE conduit, stent A and B were expanded to a diameter of 19.7 mm and 18.7 mm and then compressed to a diameter of 11.7 mm and 7.2 mm respectively.

For stent migration testing, instead of symmetric actuation, RoSE was actuated with peristaltic actuation which includes spatio-temporal sinusoidal peristalsis waves of different characteristics (Table 3, Fig. A1 C of Supplementary Materials). The migration studies elucidated the contributions of stent RF, bolus concentrations, and peristaltic wave trajectories of RoSE towards the stent migration in terms of both displacement and direction. To ensure repeatability, all the RF and migration experiments were conducted five times.

Table 3. Manipulated parameters to cover a range of swallowing scenarios.

, 30, 40 Mms-1 6

Wavefront lengths 40, 50, 60 mm

Rose Manometry Protocol

In RoSE manometry protocol, RoSE was implanted with a stent, and a manometric motility catheter (P3315205CC152, Sandhill Scientific, USA) positioned akin to the clinical in vivo observations (Fig. 8, A and B).6 The pressure signatures, intrabolus and intraluminal, associated with each swallow was captured using the manometric motility catheter and data acquisition system (S98-200C, Sandhill Scientific, USA). The catheter is a long flexible tube with a 4 mm diameter, which consists of 5 pressure sensors evenly spaced at 50 mm. The swallow investigation was carried out by aligning one of the sensors to the layer L4 of the RoSE (Fig.

8A). Due to the limited visibility inside the conduit, the configuration was achieved according to the dimension of the RoSE and the catheter. RoSE was kept in a supine position to evade the effect of gravity on the transport of the food boluses. Feeding pipes were connected at both ends of the RoSE conduit, and by using a funnel, bolus was fed to the RoSE through the left pipe.

Fig. 8. RoSE manometry test setup to measure bolus swallow pressure signatures. (A, and B) Schematic, and image of the manometry test setup to statistically analyze the mean maximum intrabolus pressure (IBPS), and the IBPS gradient as a function of bolus concentration, peristalsis wave velocity, and stent placement (before and after).

The peristaltic actuation described in RoSE firmware protocol generates peristaltic waves of various wavefront lengths and velocities (Table 3). The wave generates a contractile force that squeezes and pushes the bolus from left to right of the RoSE conduit, with or without the presence of a stent (Fig. 8). The manometry protocol has been implemented to inspect the macro bolus behavior under peristalsis (Movie S2). During the bolus transport, the pressure signature recorded by the catheter pressure sensor in the presence of the bolus, near the bolus tail, is known as IBPS whereas, the intraluminal pressure signature (ILPS) is the overall contact pressure (IBPS and RoSE-catheter contact pressure) recorded along the catheter axis. The peak ILPS describes the normal contact pressure exerted by the RoSE lumen on the manometry catheter. Due to the non-Newtonian nature of the bolus fluid behavior and different peristaltic deformation characteristics owing to various wave velocities and wavefront lengths, the IBPS gradient in the region of bolus tail is an essential indicator of RoSE swallow efficacy.

The RoSE manometry protocol is designed to study the impacts of stent stiffnesses, bolus viscosities, peristaltic wave velocities, and wavefront lengths on the IBPSs (Table 3) and its gradient. The manipulated parameters (Table 3) are designed to cover a range of different

Authors:

Peder EZ Larson 1, 2,* , Jenna ML Bernard1, James A Bankson 3, Nikolaj Bøgh 4, Robert A Bok1, Albert P. Chen 5, Charles H Cunningham 6,7, Jeremy Gordon1, Jan-Bernd Hövener 8, Christoffer Laustsen 4, Dirk Mayer 9,10, Mary A McLean11 12, Franz Schilling13, James Slater1, Jean-Luc Vanderheyden5, 14, Cornelius von Morze 15, Daniel B Vigneron1, 2, Duan Xu1, 2, and the HP 13C

94143, Usa.

Denmark. 5 GE Healthcare, Menlo Park, California, USA. 6 Physical Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada.

8 Section Biomedical Imaging, Molecular Imaging North Competence Center (MOIN CC), Medicine, Baltimore, MD, USA. Cambridge, United Kingdom.

14Jlvmi Consulting Llc, Dousman, Wi, Usa

#See Acknowledgements for a list of all HP 13C MRI Consensus Group Members This work was supported by the ISMRM Hyperpolarized Media MR Study Group, the ISMRM Hyperpolarization Methods & Equipment Study Group, and the Hyperpolarized MRI Technology Resource Center (NIH/NIBIB grant P41EB013598).

Abstract

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of hyperpolarized agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate - by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation, (2) MRI system setup and calibrations, (3) data acquisition and image reconstruction, and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the “HP 13C MRI Consensus Group” as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods & Equipment study groups. It further aims to provide a comprehensive reference for future consensus building as the field continues to advance human studies with this metabolic imaging modality.

Keywords: Hyperpolarized MRI, metabolic imaging, carbon-13, pyruvate, dissolution dynamic

Introduction

MRI with hyperpolarized 13C agents, also known as hyperpolarized (HP) 13C MRI, has shown great potential as a novel imaging modality, particularly for its ability to probe metabolic processes in real time. The first human studies with HP [1-13C]pyruvate were performed in 2011 in prostate cancer patients (1).

Since then, there have been over 60 papers published with imaging results of human subjects from 13 different sites, with applications including prostate cancer, brain tumors, breast cancer, kidney cancer, pancreatic cancer, metastatic disease, liver disease, ischemic heart disease, diabetes and cardiomyopathies. The vast majority of these studies used [1-13C]pyruvate (1–63), where [2-13C]pyruvate (64) and 13C-urea (56) have been demonstrated too.

As clinical HP 13C MRI advances, there is a growing need to build consensus for best practices, which are critical for comparing data across sites, performing multi-site trials,deploying methods to new sites, partnering with vendors, and potentially for obtaining broader regulatory approvals.

In March 2022, we initiated an effort to build consensus within the HP 13C MRI community with this opportunity in mind, and it was greeted with strong enthusiasm. The “HP 13C MRI Consensus Group”, containing over 55 members from 27 sites, identified the area of greatest need and opportunity for consensus building to be HP [1-13C]pyruvate human

●

Pyruvate is the most mature and widely used HP agent and has the most significant translational evidence emphasizing the potential clinical impact.

●

Clinical trials, particularly multi-site trials, have the strongest need for consensus methods to ensure that data can be combined across sites. This work is a Position Paper for which the goal is to describe current successful practices and study methods for HP [1-13C]pyruvate human studies along with justification to support those practices. This is divided into four major topic areas: (1) HP 13C-pyruvate preparation, (2) MRI system setup and calibrations, (3) data acquisition and image reconstruction, and (4) data analysis and quantification (Fig. 1). The current successful practices and study methods include a literature review of published peer-reviewed journal papers showing human HP [1-13C]pyruvate study data, up to September 2022 (1–63), as well as new unpublished information from surveys of HP 13C study sites. Based on this information, we also highlight the evidence gaps, strengths, and limitations of current practices which are summarized at the end of each section.

Figure 1: Illustration of the HP 13C MRI human study process, including the 4 major areas covered in this paper: Hyperpolarized 13C-pyruvate preparation, MRI system setup and calibration, Acquisition and Reconstruction, and Data Analysis and Quantification.

Figure 2: Anatomical targets of HP [1-13C]pyruvate MRI human studies published up to September 2022.

Hyperpolarized 13C-Pyruvate Preparation

This section covers the processes for creating the HP agent, 13C pyruvate, and will include many aspects and considerations that are needed to safely and effectively prepare doses for metabolic imaging studies in human subjects. These include material, personnel, equipment and facility, fluid path preparation, quality control, and release.

It is helpful to understand that the specifications of a dose of 13C pyruvate suitable for in vivo MR HP metabolic imaging were shaped in part by early preclinical studies performed by GE HealthCare summarized in Ref. (65). In short, the safety of the two novel drug components, 13C pyruvate and the electron paramagnetic agent (EPA) AH111501, were demonstrated in those studies. The more precise formulation of the dose suitable for human use was then determined from clinical studies (66) that included two Phase 1 clinical trials in young and elderly healthy volunteers without hyperpolarization of the 13C nuclei and another Phase 1/2a dose escalation and imaging feasibility study with HP 13C pyruvate in 31 prostate cancer patients at the With the exception of the first HP 13C imaging clinical trial, which utilized a prototype device in a cleanroom (1), all HP 13C studies performed in humans to date have utilized the SPINlab polarizer (manufactured by GE HealthCare). Consequently all doses of the HP 13C pyruvate delivered by SPINlab have been produced using the “SPINlab Pharmacy Kit” that serves as the container-closure system for the various drug components (13C pyruvic acid and EPA mixture, dissolution medium, and neutralization and dilution medium) during sample polarization, dissolution and quality control (QC) processes. Thus many aspects of the HP sample preparation considerations discussed below are related to the SPINlab instrument and the consumables designed to be used with it (67).

General Considerations

While more than 860 patients or healthy subjects having been injected with HP 13C pyruvate as of January 2022 without reports of any serious adverse events (68), HP 13C pyruvate injection remains an investigational MR contrast agent and can only be administered by those with Investigational New Drug (IND) exemption from the Food and Drug Administration (FDA) in the USA, a Clinical Trial Application (CTA) in Canada, approval from National Research Ethics Committee Services in the UK, or approval from the relevant local regulatory body. Thus, methods and processes involved to produce a dose should have patient safety as the first priority. Since utilizing dissolution dynamic nuclear polarization (dissolution-DNP) for human use is still a relatively new development, there are no existing published regulatory guidelines specifically for this method.

There are two major production styles that determine how various sites approach the agent preparation. In the US, the most common approach is to rely on a sterilizing filter (“Terminal Sterilization”) to ensure sterility of the final product, akin to PET tracer production, where a starting molecule with a radioisotope is processed using various other ingredients to make the final, desired and injectable contrast agent within a necessarily short amount of time (69). For these sites, sterilization of the components and accessories upstream of this filter are not required, although many of them were manufactured and tested following Good Manufacturing Practice (GMP) or Good Laboratory Practice (GLP) requirements. The filling process is usually performed under an ISO 5 laminar flow hood, but a clean room or an isolator is not required.

This approach is typically accompanied by testing the integrity of the sterilizing filter prior to release of the dose for injection. Typically, post release endotoxin and sterility tests are performed using an aliquot reserved from each released dose.

In the UK and EU, the most common approach is to more-closely follow sterile pharmaceutical compounding guidelines (70), where all components and ingredients are required to be sterile or manufactured under GMP guidelines and are assembled and filled within a clean room environment or an isolator system (“Sterile Preparation”). Typically a batch of Pharmacy Kits for HP 13C pyruvate injection are prepared together. The sterility of the final dose is also ensured by batch validation testing, in addition to the sterility of the ingredients and the sterile compounding process. The endotoxin and sterility testing are performed for the process validation but are not performed for each injected dose.

Some institutions fill and assemble the Pharmacy Kit required for a specific study on the same day or the day prior to polarization, dissolution, and patient administration, but others have also demonstrated the feasibility of preparing a batch of kits, keeping them in a -20ºC freezer and using them over a period of a few months.

Beyond the obvious requirements that the process and the facility has to ultimately produce a dose that is safe to inject into a human, regulatory authorities will also focus on the question “Are you in control of your processes?”. To be in control of your process requires an in-depth and broad understanding of all processes involved in pre, post, and during the production process.

Personnel

It is typical and may be required to have licensed personnel involved in the production process depending on local regulations.Typically a pharmacist, radiopharmacist or other similarly qualified person (QP), in charge of the facility where the Pharmacy Kit filling and preparation is taking place, is responsible for the overall process and the release of the injectable dose.

Qualified cleanroom technicians are often involved in the Pharmacy Kit filling under the supervision of the pharmacist or QP. As is required for pharmaceutical compounding or PET tracer production, training requirements and training records for all personnel need to be maintained and available for audit by the FDA or equivalent.

Equipment And Facility

The facility and all equipment need to have standard operating procedures (SOPs) that describe how equipment is used, maintained, and calibrated to comply with relevant legislation. Currently, almost all the filling of the Pharmacy Kit takes place within a compounding laminar flow hood or isolator (typically ISO 5). At some sites, the filling is conducted within a cleanroom, while at others, it is conducted in a dedicated non-cleanroom space, reflecting differences in cleanroom approach and specifications between regulators worldwide (71). Some equipment or facilities, such as the compounding hood or cleanroom, may require external certified laboratories for testing.

Material Handling

Material handling guidelines (69,70) require SOPs detailing a system to track all of the materials involved in the HP production process for a particular patient dose, similar to current good manufacturing practice (cGMP) requirements for material handling for drug compounding. This includes acceptance standards, storage conditions, amount used in the patient dose for each ingredient and materials used in the assembly of the fluid path and Pharmacy Kit. Currently some users choose to open and inspect and sometimes modify the Pharmacy Kits upon arrival, but some users keep them in the sealed packaging until they are required for dose preparation.

Pharmacy Kit Filling And Assembling

As required by an IND or its equivalent, the preparation of the doses of HP 13C agent are detailed in the Chemistry, Manufacturing, and Control (CMC) section of an applicable regulatory submission; an example of this has been made available (72). It describes the processes of filling the Pharmacy Kit with the different components that make up the final drug product, and of assembling the final kit for either storage or immediate use in the polarizer. Special attention should be given to the laser welding process in order to satisfy installation qualification (IQ) and operational qualification (OQ). Typically, the final developed process is validated by process qualification (PQ) runs, during which 3 or more Pharmacy Kits are filled and used and the final HP 13C products are tested for endotoxin and sterility and to confirm that they meet the dose specifications for injections (usually including pyruvate concentration, residual EPA concentration, pH, liquid state polarization level and dose temperature). The data from 3 consecutive PQ runs are submitted as part of the IND submission (or its equivalent), and are often also reviewed by the Institutional Review Board (IRB) where the studies are conducted.

Quality Control And Dose Release

The quality control (QC) and dose release can be separated into two aspects: one is the QC and release of the filled Pharmacy Kit, and second is the QC and release of the HP 13C agent for injection, after polarization and dissolution. For institutions filling a batch of kits and storing them to use over a period of time, typically the batch can be released based on initial validation, environmental monitoring data from the day of kit production, and if filters are used during preparation of any of the components, filter integrity testing. But in some cases one or more kits are used for validation before the batch of kits are released for future use. For institutions that fill only the kits required for specific studies shortly before the experiment, the filled kits often do not go through separate release tests before they are used.

The quality control of the HP 13C pyruvate solution post dissolution is primarily performed to ensure that the agent meets the dose specifications (Table 1) before it is administered to the subject. These specifications target both safety (pH, residual EPA, temperature) and efficacy (pyruvate concentration, polarization, volume). Typically, the pyruvate concentration, residual EPA concentration, pH, dose temperature, dose volume, and liquid state polarization are measured by the QC accessory associated with the SPINlab polarizer. Some users perform a secondary measurement for one of the parameters, such as pH, using a different instrument or pH paper. For sites that do not go through a separate release testing process for batch filled kits, the integrity of the sterilization assurance filter, a part of the Pharmacy Kit, is typically tested as a part of the dose release. It is also common for these users to preserve an aliquot of the final HP 13C pyruvate solution for post-release endotoxin and sterility testing. This testing cannot be completed fast enough to test an individual dose prior to injection, but this is why other processes such as PQ runs and validation testing are done to minimize the chance a subject could be injected with a contaminated dose.

The Final Dose Release And Injection

should be done under the supervision of a licensed professional, based on local regulations.

Some Key Challenges

Many of the challenges associated with HP 13C pyruvate preparation can be attributed to the conditions required for the dissolution-DNP method of high magnetic field (~3-7 T) and very low temperature (~1 K) during polarization, with pressurized and superheated water necessary for the rapid dissolution event. These extreme conditions are quite challenging for the design of the container-closure and fluid path system. In particular, the cryogenic temperature in the polarizer requires special attention to any moisture or ambient (moist) air introduced into that portion of the fluid path, which can form an ice block at ~1 K. This ice can lead to flow restriction during the dissolution event and reduce the strength of the laser welded bond between the cryovial and its cap. This can ultimately produce failures in the dissolution step, including variations in final pyruvate concentration and pH that may fail to meet QC release criteria as well as fluid path ruptures that provide no available dose and result in polarizer down-time.

The polarization of the HP 13C pyruvate sample decays quickly over the span of a few minutes after dissolution, and thus the process of dissolution, QC for release, and injection should be completed as fast as possible to preserve the high polarization level achieved. Any delays in the preparation process, such as transportation time or equipment malfunction, can significantly reduce the final polarization and result in lower quality imaging data.

Current Practices

A summary of data collected from all sites performing clinical trials with HP 13C-pyruvate is shown in Fig. 3 and Table 1, including the specification of the final dose and how the quality control and release of the final dose are performed. There is a split in the Production Style, described in the General Considerations section above, with 8/13 sites using Sterile Preparation versus 5/13 using Terminal Sterilization. While many of the dose specifications show notable differences in acceptable ranges, all of these variations listed in tables have been successfully and safely been used to perform HP 13C pyruvate studies in humans. Their differences depend on the institutions’ preferences, resources and their particular regulatory situation. There is high similarity in pyruvate ranges, temperature ranges, EPA limits, and volume limits. There is modest variability in pH ranges and large variability in the endotoxin test limit. There is a 3-fold difference in acceptable polarization levels, which are measured to ensure a futile dose is not injected since the polarization is directly proportional to SNR. This reflects the decision by several sites to believe that useful data can be still be obtained with suboptimal polarizations.

Figure 3: Hyperpolarized agent preparation methods reported by sites currently performing HP

In House

Table 1: HP 13C-pyruvate preparation parameters, methods, and dose specifications used for quality control testing and release as well as validation. These were obtained from a survey of all sites performing clinical trials with HP [1-13C]pyruvate. The parameters used for product release are noted in bold text, otherwise these parameters are measured for batch validation or other QC measurements. The endotoxin and sterility testing are performed during process validation of the batch and/or post-injection, and largely depends on the agent production approach.

Summary

The overall safety record of HP 13C-pyruvate has been very strong, and the SPINlab hyperpolarizer has proven to provide high polarizations at human sized doses while meeting numerous QC and release criteria. A weakness remains the failure modes of the SPINlab Phamacy Kits (e.g. ice blocks, path ruptures), which are placed under extreme requirements particularly during dissolution. The preparation process still requires a high degree of expertise.

Therefore, there is a significant need to improve the reliability, robustness, and ease of operation for generating HP 13C-pyruvate doses for human studies. Furthermore, there is a divide between manufacturing and sterile compounding style preparation as well as other site-specific practices, resulting in variations in SOPs and justification required to relevant regulatory bodies. There have also been no comparisons between these approaches. It is also unclear what release criteria and QC parameters are truly required to ensure patient safety.

However, all of the reported methods are acceptable and approved by the appropriate regulatory authorities, and have led to the rapid expansion of successful human studies in recent years.

Mri System Setup And Calibrations

This section covers the MRI system setup, including the imaging system, RF coils, phantoms, and prescan calibration methods.

Imaging System

The main prerequisite for a given MRI scanner to be capable of supporting studies with HP 13C is its “broadband” capability to transmit and receive radiofrequency (RF) signal at the frequency of 13C, which is around 4 times lower than 1H. This does not come as a default on clinical MR devices. The transmit power of the broadband amplifier should also be sufficient to support the intended flip angle and RF pulse shape with the employed transmission RF coil(s) for 13C. Most studies to date use relatively low flip angles (< 90 degrees) for HP 13C in order to preserve polarization for time-resolved imaging. The capability to receive 13C signal on multiple channels is also desirable to increase SNR, as discussed further in the “RF coils” section.

The choice of magnetic field strength is primarily dependent on the metabolites’ frequency separation due to chemical shift dispersion and 1H imaging. High field strengths do not enhance hyperpolarized 13C signal as they do for 1H because the signal strength in a HP experiment relies on manipulating the population of quantum energy states outside of the MRI scanner.

However, the injected HP 13C-pyruvate and its metabolic products have greater frequency separation at higher fields, and it may thus be easier to separate and quantify these resonances at higher fields. This comes at the cost of a reduction in the achievable T2* and often reduced T1. As the initial polarization is independent of the imaging field strength it has been proposed that the increased T2* at 1.5T can potentially be exploited to increase SNR by adapting the acquisition bandwidth or reduce off-resonance imaging effects in cases when the decay of the transverse magnetization is dominated by T2* (73). In practice, 3T has been used in all published human 13C-pyruvate studies surveyed (Supporting Table S1), and comprises the majority of scanners currently in use for human studies (Table 3). A field strength of 3T is well-suited for 1H MRI anatomical reference and correlative imaging.

Stronger and more rapidly slewing magnetic field gradients support more rapid spatial encoding, particularly for metabolite-specific single-shot imaging using echo-planar imaging (EPI) or spiral imaging (See “Acquisition and Reconstruction”). Although the spatial resolution acquired for HP 13C imaging is typically much coarser than for 1H MRI, the factor of ~4 in gyromagnetic ratio leads to the same reduction factor in performance of the gradient system, so 13C experiments are potentially more limited by gradient hardware performance. To date, all human studies have used the commercially-available integrated gradient systems provided in clinical MRI scanners.

Optimization of scanner design has understandably focused on minimization of artifacts in 1H MRI, where devices such as room lights, the gradient amplifiers, and the motors driving the patient bed are checked to ensure that they do not produce RF interference at the 1H frequency, but artifacts may arise at other frequencies. Eddy current compensation is also not always appropriately adjusted for nuclei at other frequencies (74). In order to optimize for 13C, many sites have performed checks on phantoms for RF interference, gradient artifacts, and eddy currents (74), including the use of post-hoc gradient impulse response function characterisation and correction, and some vendors have fixed these issues as well.

Rf Coils

For HP 13C imaging studies in humans, RF coils for both 1H and 13C nuclei are needed, with 1H MRI providing an anatomical reference for registration and optional additional multiparametric MRI readouts. At the Larmor frequency of 13C nuclei, the relative contributions from coil noise compared to sample noise increase compared to 1H (73,75), although sample noise still is likely the dominant contributor for human-sized coils at 32.1MHz - the resonance frequency of 13C nuclei at 3T.

The key requirement for human 13C-pyruvate RF coils are that the coil geometry and sensitive volume must cover the volume of interest in the subject. Table 2 and Figure 4 shows coil configurations that have been used and optimized for applications in different anatomic regions.

Volume resonators are most commonly used for transmit, as they surround the subject to

Provide B1 Transmit Across The Fov (B1

+). While 1H relies on a large birdcage (“body”) coil built into the scanner, 13C transmit coils must be placed inside the bore. This takes up valuable space within the magnet, and also has led to the use of designs with relatively inhomogeneous

B1

+. Many human studies have used Helmholz pair resonators for transmit, including the “clamshell coil”, which has a notably inhomogeneous B1

+ Profile But Has Been Used Because Of

relatively easy integration into the scanner bore. B1

+ Variation Results In Variations In The Flip

angles that control the use of the hyperpolarized magnetization and creates errors in common HP metrics (9,76). The exception are head coils, where birdcage designs with highly

Homogeneous B1

+ can be placed around the head while easily fitting inside the bore. As with 1H MRI, higher SNR can typically be achieved by smaller receive coil elements, such as surface coils or phased arrays, and the majority of 13C receive coils used have layouts similar to 1H phased arrays.

RF coil quality control is important to ensure proper functioning of the coils to provide consistent imaging quality, especially with limited natural abundance 13C signal in vivo. It typically involves 1) a physical integrity check of the coil cables and connectors and 2) phantom SNR tests to check the coil’s performance and to monitor it over time (see Phantoms below). An useful reference for RF coil quality control is outlined in the MRI accreditation program of the American College of Radiology (77) and can be adapted for 13C coils.

Notably, configurations for brain and prostate studies used dual-tuned 1H/13C coil designs, which greatly simplify workflow and registration of 1H and 13C images, as no switching of coils is needed.

(1)

Table 2: RF coil configurations reported for human HP [1-13C]pyruvate studies.

Tx = Transmit

coil, RX = receive coil. The commonly used “clamshell” TX coil is a Helmholz pair design. For 1H RF configurations, all used the Body coil for TX unless otherwise noted, and “repositioned” indicates the 13C coil was removed for 1H imaging. One representative reference is listed for each configuration. The RF coil configurations reported in the reviewed papers are shown in Supporting Table S1.

Figure 4: Examples of RF coil configurations used for human HP [1-13C]pyruvate brain studies. (A,B) 13C Clamshell TX (Helmholz pair) and 2× 4-channel paddle RX arrays. (C) 13C Birdcage volume TX and 32-channel RX array (RX array slides into TX coil). (D) 13C Birdcage volume TX and 24-channel RX array, combined with a 1H 8-channel RX array. Image reproduced with permission from Ref (16).

Phantoms

Since hyperpolarized magnetization is non-renewable, phantoms containing 13C nuclei are important to: 1) test the multi-nuclear capabilities of the imaging system, including all parts of the signal excitation and receive chain; 2) perform calibration measurements before a scan with hyperpolarized nuclei; and 3) perform necessary pre-scan adjustments (see “Prescan Calibration” section). The phantoms currently in use are listed in Table 3. Their composition must provide sufficient 13C signal, with additional considerations of conductivity, stability, chemical shift(s) present, potential for dynamic imaging, and cost. The phantom geometries are typically either compact, in order to be used alongside the subject during a HP scan, or large enough to mimic the inner volume of a RF coil for system testing.

One popular compact design contains enriched 13C-urea at high concentration, typically 8 M, which provides a single resonance, placed inside a small container ~1 mL. The most common recipe mixes 13C-urea in a 90% water/10% glycerol solution, with glycerol used to increase the urea solubility and doping with a Gd-based contrast agent to shorten T1 which increases the potential SNR per unit time. For example, when Dotarem is added at a 3:1000 volume ratio the 13C-urea T1 is around 500 ms and T2 is around 100 ms. However, when testing pulse sequences influenced by T1 and T2, doping should be used carefully. This phantom is suitable for frequency calibration, transmit gain calibration, sequence testing, and as a fiducial marker when placed next to a patient. However, enriched 13C-urea has a relatively high cost compared to natural abundance compounds.

For larger volumes (>100 ml), the phantoms most often used contain undiluted ethylene glycol, glycerol, or dimethyl silicone. These compounds have sufficiently high carbon concentrations to provide sufficient 13C signal even with the 1.1% natural abundance of 13C. These larger phantoms matching the inner volume of an RF coil are useful for coil testing, including transmit

+) And Receive (B1

-) coil profile mapping, as well as to mimic acquisitions using in vivo FOV requirements. In this case, size and conductivity should match the expected subject size in order to mimic coil loading and get a realistic estimation of B1+. Large-volume natural abundance urea phantoms have also been used by some sites, but suffer from higher conductivity compared to biological tissues. Typically, it is easier to increase the conductivity and hence coil loading of the non-conductive phantom by adding NaCl to match physiological loading (16,78).

Dynamic phantoms that aim to mimic metabolite kinetics have also been developed (79–81), and have the potential to more closely mimic the HP experiment, but so far these are not widely used.

Prescan Calibration

Prior to performing an MRI acquisition, the so-called prescan procedure is used to set the shim parameters to maximize B0 homogeneity over the field of view (FOV) or a specific region of interest (ROI), the scanner center frequency (CF), the RF transmit gain, and the receiver gain.

While this calibration procedure is usually automated for 1H, the lack of sufficient natural abundance 13C signal prevents use of automated methods. (Although natural abundance 13C lipid signal has been detected, there are so far no reports on using this signal for prescan.) Table 3 shows current practices across sites.

Maximizing B0 homogeneity is independent of the nucleus and is therefore performed prior to 13C imaging using the 1H water signal and existing shimming tools, such as by a standard automated process (“Auto Shimming”) or using high order shimming routines. Similarly, the 13C CF can be calculated from the 1H CF using a predetermined scaling factor that depends on the target chemical shift (82). Another common approach used is to have a small, high-concentration 13C phantom, e.g. 8M 13C-urea, integrated in the RF coil or placed next to the scan subject (1). The reference frequency can also be based on real-time measurements after the HP injection but prior to imaging (83). Both the CF and B0 shimming are critical when using spectrally-selective RF pulses, as inmetabolite-specific imaging methods, where the desired excitation bandwidths are typically very narrow and frequency offsets can lead to a failure mode that is only apparent after injection.

The calibration of the RF transmit power is typically performed on a small, high-concentration 13C phantom placed near the region of interest during the scan or on a large 13C phantom of similar size and coil loading as the subject, prior to the subject scan. Reference power is often done by sweeping the power in a pulse-acquire sequence (53,62), or the Bloch-Siegert method (52,84). When using a small phantom, the location of the phantom, B1

+ Inhomogeneity As Well

as any shielding effects, e.g., when the phantom is integrated into a coil (1), may degrade the accuracy. Other methods include real-time Bloch-Siegert method measurements after the HP injection (83), and using the stronger natural abundance 23Na signal that is close enough to the 13C resonance frequency to be detected by 13C coils (82).

The receiver gain is predetermined, either systematically based on independent phantom measurements and assuming the dose and polarization of the HP compound is known prior to injection, or based on past HP imaging studies.

Power [Kw]

Phantom(s) - during study Phantom(s) - before study 13C Frequency

8

13C-bicarbonate doped with dimethyl silicone, various

Power [Kw]

Phantom(s) - during study Phantom(s) - before study 13C Frequency

Maximum Values

Table 3: Summary of the imaging systems, phantoms, and prescan procedures used at sites currently performing HP 13C-pyruvate human studies. These were obtained from a survey of all sites performing clinical trials with HP [1-13C]pyruvate. *Previously performed studies with a Siemens 3T Tim Trio. The imaging systems, phantoms, and prescan procedures reported in the reviewed papers are shown in Supporting Table S1.

Summary

Commercially available 3T MRI systems are by far the most commonly used for human HP 13C-pyruvate studies, although a systematic investigation of the impact of B0 has only recently been investigated (73). The multi-nuclear RF transmit and receive chain has proven sufficient for current acquisition strategies, although many sites have observed artifacts due to RF interference, gradient interference, and residual eddy currents when operating at the 13C frequency. A variety of 13C RF coils, tailored for numerous anatomical targets, have been successfully demonstrated, with the main limitation that most transmit coils take up a lot of additional space inside the bore and provide relatively inhomogeneous B1

+ Profiles. The

phantoms used have converged into generally 2 categories - small phantoms containing 13C-enriched compounds that can be used during the study and human-sized phantoms containing compounds with high carbon concentrations but without 13C enrichment that are used to test and calibrate the coils. There are no standardized compositions or geometry, and dynamic phantoms that recapitulate in vivo kinetics would be desirable but are still an emerging area. Prescan calibration procedures were not well defined in most publications, so we surveyed individual sites to determine current practices. Calibration procedures for the B0 field (13C CF and shimming) for most sites take advantage of 1H signal and methods, while methods

For Calibration Of B1

+ is more variable across sites, likely a reflection of remaining challenges in how to perform this calibration. Standardization of both phantoms and calibration procedures would synergistically improve the robustness and reproducibility of HP 13C studies.

Acquisition And Reconstruction

Data acquisition strategies in human HP [1-13C]pyruvate MRI studies must account for multiple chemical shifts, efficiently utilize the non-renewable HP magnetization, and acquire data quickly relative to metabolism and relaxation decay processes. These studies require spectral encoding to separate metabolites, necessitating pulse sequences that efficiently encode up to 5D data (3 spatial + 1 spectral + 1 temporal dimension). RF pulses must efficiently sample without immediately saturating the non-renewable HP magnetization, and sequences must acquire data quickly and be robust to both experimental and physiologic variation (e.g. B1

+ Inhomogeneity,

variation in perfusion) to ensure reproducibility and minimize scan-to-scan variability. This section covers current successful practices for data acquisition in human [1-13C]pyruvate studies, and accompanying 1H imaging, from different anatomic regions, including scan parameters and image reconstruction.

Acquisition And Reconstruction Methods

The acquisition methods used in human [1-13C]pyruvate studies can be classified into 3 categories: 1) MR spectroscopy or MR spectroscopic imaging (“MRS/I”), 2) chemical shift encoding methods, and 3) metabolite-specific imaging (Fig. 5).

Mrs/I Methods Specifically

resolve a spectrum that can be analyzed to extract expected as well as unexpected resonances, making this approach very robust. It was used in many initial studies (1).

Chemical Shift

encoding methods, most commonly the Iterative Decomposition of water and fat with Echo Asymmetry and Least-squares estimation (IDEAL) method, use imaging sequences acquired with multiple TEs and rely on a model-based separation of expected chemical shifts (85).

Metabolite-specific imaging methods use specialized RF pulses that are spatially and spectrally selective to excite individual metabolites which are then typically imaged with fast k-space trajectories such as echo planar imaging (EPI) or spirals (86).

Their Application To Different

organ systems is described below. The image reconstruction methods used in human [1-13C]pyruvate studies have typically been conventional methods (e.g. FFT, non-uniform FFT, or equivalent). The incorporation of accelerated imaging and advanced reconstruction methods including parallel imaging (4,57,87) and compressed sensing (7) has also been applied in human studies for improved spatial resolution, temporal resolution and coverage, but have the potential for additional artifacts as well as SNR losses due to ill-conditioning of the reconstruction (e.g. g-factor).

The Majority Of

published studies do not use accelerated imaging indicating the resolution and coverage achievable without acceleration is currently adequate for successful data collection. Performing coil combination, even with fully sampled data has also been shown to have specific challenges for HP human images: using naive sum-of-squares methods suffer from high noise amplification in the relatively low SNR regime of HP [1-13C]pyruvate (compared to 1H), motivating several HP 13C-specific methods that include data-driven coil sensitivity estimation which have shown obvious improvements over sum-of-squares (11).

More recently denoising techniques have been applied as post-processing of human HP data(41,42,44). The techniques applied are based on spatial-temporal singular value decomposition for unsupervised estimation of signal and noise components. They have shown improvements in apparent SNR in the brain and liver, while care must be taken to choose parameters such as the rank threshold to avoid oversmoothing and overfitting to the estimated signal components.

Prostate Studies

Prostate cancer was the first human application of HP [1-13C]pyruvate (1), and data was acquired with MRS/I methods: 1D dynamic MRS, single-slice 2D dynamic echo-planar spectroscopic imaging (EPSI), and single time point 3D EPSI. Advances in imaging strategies led to the development and application of new acquisition schemes, including undersampled 3D EPSI with compressed-sensing (7), model-based chemical shift encoding methods that use a priori information (47,59), and metabolite-specific EPI (10), all of which can provide volumetric whole-organ coverage and dynamic acquisitions.

The pyruvate bolus arrival in the prostate can vary by ± 10 s between patients, necessitating dynamic imaging to reliably and consistently capture the pyruvate bolus (18). For this reason, all currently ongoing studies acquire dynamic data. While MRS/I, chemical shift encoding, and metabolite-specific imaging can all achieve dynamic imaging, chemical shift encoding and metabolite-specific imaging provide greater dynamic and volumetric coverage (85). For scan prescriptions, the FOV is designed to provide full prostate coverage and typically to match the orientation of the anatomic imaging used for registration. Flip angles used in current studies are constant through time, as quantification with a variable-through-time flip scheme is highly sensitive to bolus timing (8) and errors in the RF transmit (B1 +) field (76).

Heart Studies

Data acquisition methods for 13C imaging in the heart must be designed to meet the demands of significant cardiac motion and blood flow. To cope with the periodic cardiac motion, most human heart studies to date used gating to the diastolic window, the longest cardiac cycle interval, which has reduced motion (2,22,28,30,35,36,38,45,52). The duration of the diastolic window limits the available data sampling time, making cardiac acquisitions the most time-constrained of the HP 13C MRI applications. The most common acquisition approach is metabolite-specific imaging with spiral k-space trajectories (2). Their single-shot imaging capability makes these methods particularly robust to motion effects. Furthermore, spiral k-space trajectories provide rapid k-space coverage and relatively benign flow and motion artifacts. The majority of studies have used 2D multi-slice acquisitions, but 3D encoding has also been used successfully (35).

Brain Studies

For HP 13C MRI of the human brain, the majority of studies have also used 2D (slice selective) acquisitions (10–12,14,16,28,33,40,41,44,51,53,60), with a trend toward volumetric coverage using 2D multi-slice metabolite-specific imaging. 3D metabolite-specific imaging of the whole brain, with phase encoding of the slice direction (34,57), has been shown to provide similar SNR efficiency (88) compared with multislice imaging. A number of studies have employed MRS/I (5,6,29,31–33,50,55) resulting in a spectrum from each voxel, which has the advantage of not requiring a priori information about which peaks to encode. This was important in early brain studies when it was not known which peaks would be detectable. Chemical shift encoding, using a set of images with different echo times and an iterative reconstruction of the individual resonances (i.e. the IDEAL approach (85)), has also been used (12,49,54), with the drawback that coverage in the slice direction was limited due to the time required to acquire multiple echo time images.

Abdomen And Breast Studies

The fundamental approaches to data acquisition and reconstruction in the abdomen and breast are largely similar to the aforementioned applications, but demand attention to particular challenges associated with these anatomic regions, especially relating to respiratory motion.

Although it has been shown that a basic 2D MRSI approach based on phase encoding and FID readout can be successfully applied for HP 13C imaging in breast (15) and kidney (13), major advantages in terms of spatiotemporal resolution and coverage have been realized using tailored approaches based on metabolite-specific imaging (43,62) and chemical shift encoding (43), which have facilitated multi-slice or 3D dynamic acquisitions over large FOVs in the abdomen (4,37,46).

The significant respiratory motion encountered in these regions can directly blur 13C images, and has further favored these rapid acquisition strategies. Motion also degrades B0 homogeneity, which can shift frequency-selective excitation profiles and introduce artifacts into rapid imaging readouts. This makes accurate determination of the acquisition center frequency and shimming essential in these regions which often cover large FOVs. (See “Prescan Calibration” section for more information). In some studies, breath-holding was used to minimize motion effects and enforce frame-to-frame data consistency (42). A pragmatic and reasonably effective approach for dealing with respiratory motion during 13C data acquisition is an initial breath-hold (as long as can be tolerated), followed by free-breathing (46,62).

1H Imaging

Collection of 1H imaging data is essential both for prescribing the 13C acquisition and for interpretation of the resulting 13C data. Multi-planar 1H scouts are acquired prior to 13C acquisition to enable graphical prescription of the 13C imaging region. All human HP 13C-pyruvate imaging studies acquire conventional MRI scans (e.g. T1- and T2-weighted volumes) for anatomic reference, aiming to cover at least the full 13C FOV. Acquiring these anatomic scans as close as possible to the time of 13C imaging (immediately before or after) minimizes potential misregistration between the data sets. Depending on the application, other advanced 1H sequences are also acquired (e.g. diffusion-weighted imaging for cancer imaging).

When contrast-enhanced data is acquired, it is done after 13C imaging, as paramagnetic contrast agents will accelerate 13C relaxation.

Reported Study Parameters

Figures 5 and 6, and Supporting Table S2 shows the reported acquisition study parameters for human HP [1-13C]pyruvate studies published as of September 2022. Figure 5 shows a mixture of MRS/I, metabolite-specific imaging, and chemical shift encoding methods have been successfully used, where spectroscopy-based methods have become less prevalent in recent studies. Figure 6 shows the acquisition timing, including the important start time and interval/temporal resolution, is quite variable across studies.

Figure 5: Acquisition methods used in published HP [1-13C]pyruvate human studies published up to September 2022, classified into: MR spectroscopy and spectroscopy imaging (MRS/I); chemical shift encoding methods, such as IDEAL, that use multiple TEs and model-based reconstructions; and metabolite-specific imaging methods that use spectrally-selective excitation to image a single resonance at a time.

Figure 6: Temporal acquisition characteristics reported in HP [1-13C]pyruvate human studies published up to September 2022. (a) Reported referencing of acquisition start times.

(B)

Acquisition start times reported when using dynamic imaging and when timing was reported relative to the end of the injection. (c) Temporal resolutions. “Not Applicable” indicates dynamic imaging was not used.

Summary

Three general categories of acquisition strategies have been used successfully for human HP 13C-pyruvate studies: MRS/I, model-based chemical shift encoding (e.g. IDEAL) methods, and metabolite-specific imaging methods. These have enabled successful studies in the prostate, heart, brain, abdomen, and breast. Recent studies increasingly have used the imaging-based strategies of metabolite-specific imaging and chemical shift encoding which are the fastest methods, although a heads-to–head comparison between techniques has not been performed.

Metabolite-specific imaging is quite popular because of its speed and compatibility with single-shot imaging, but is sensitive to B0 field variations and thus requires careful calibrations. Nearly all studies surveyed acquired data dynamically, allowing measurement of the bolus and metabolite kinetics. The exact timings and associated flip angles vary quite widely across reported studies, with no consensus yet as to how to choose these parameters. Image reconstruction is typically done directly using Fourier Transform methods, and accelerated imaging strategies are uncommon.

Data Analysis And Quantification

This section covers the analysis of data from human HP [1-13C]pyruvate studies, including modeling and metrics, visualization, as well as considerations for how to store data and metadata. Depending on study design, the analysis may need to give quantitative or semi-quantitative output reflecting a biological process or may just reflect a contrast between different regions of interest for quantitative evaluation.

Metrics

Figure 7: HP [1-13C]pyruvate raw data (A) have typically been quantified using four categories of metrics depending on the acquisition. Data acquired as a single time point are often quantified using normalized metabolite images or metabolite ratios (B). Dynamic data can be quantified using normalized metabolite images or metabolite ratios (B), or with metabolite timings such as time-to-peak (TTP) or pharmacokinetic (PK) models (C). The latter two require the data to be time-resolved. [1-13C]alanine and 13C-bicarbonate are analyzed similarly to [1-13C]lactate but omitted here for display.

Metabolite images are commonly used as summary metrics for HP MRI data, often including some form of normalization as well as summed over time as an area under the time curve (AUC) (17). These are analogous to the visual evaluation that is most used for routine clinical work (89,90). In these metabolite images, we expect that the [1-13C]pyruvate AUC signal is predominantly weighted towards perfusion and uptake, while [1-13C]lactate, [1-13C]alanine and 13C-bicarbonate AUCs represent metabolic conversion. The strength of this approach lies in its simplicity and relatively few underlying assumptions. Limitations to the use of single-metabolite images or AUCs include sensitivity to inhomogeneous coil profiles (57,87,91), the acquisition strategy and acquisition parameters, pyruvate polarization and concentration level, and signal relaxation rates (92). Further, the reader must be careful to interpret all the images in conjunction to better understand the underlying biology; for example, increased [1-13C]lactate in the presence of decreased [1-13C]pyruvate delivery can have a very different meaning compared to increased [1-13C]lactate with increased [1-13C]pyruvate delivery.

In an attempt to address variations in coil sensitivity, polarization level, and pyruvate delivery, AUC images are often computed by normalizing to a specified parameter, such as the maximum pyruvate or average lactate signals, or presented as a ratio such as lactate/pyruvate or divided by “total Carbon” - the sum total of HP 13C signal observed across all metabolites. The AUC ratios between metabolites and pyruvate are proportional to the corresponding forward kinetic rates (81,93), but are not directly comparable to rate constants when magnetization loss rates (e.g. relaxation and losses due to signal excitation) differ between studies. Similarly, the ratios between the produced metabolites (e.g. bicarbonate/lactate) can reflect the balance between downstream metabolic pathways (12,55). Care must be taken to consider how AUC images are calculated and normalized before comparing values between studies.

To further quantify the interpretation, pharmacokinetic (PK) modeling approaches were developed to compute the apparent kinetics of pyruvate-to-metabolite exchange (92,94–99). These yield semi-quantitative to quantitative apparent rate constants, given in s-1. Some models require a vascular input function, while others avoid this requirement (95). PK models can explicitly account for acquisition-specific details such as excitation angle and repetition time, and thus may reduce the effects of these details on quantification. An input-less model, provided in the Hyperpolarized-MRI-Toolbox (https://github.com/LarsonLab/hyperpolarized-mri-toolbox) (100) and thus frequently employed for human data, has been shown to fit well and robustly to prostate and brain data (8,20). PK models are quantitative in nature, arguably provide more relevant biological information (8,20), and appear to be reproducible across sites (51). However, rate constants derived from PK models are still apparent rates, and likely do not reflect a single biological characteristic.

Some additional considerations include whether complex or magnitude data is used, as the noise behaviors will impact the analysis differently. Additionally, cut-off thresholds or other criteria may be used to identify and avoid voxels with insufficient SNR before analysis to improve robustness (20,41).

Regardless of the analysis approach, the underlying biology is not always clearly represented by the data; instead, the metrics may be influenced by perfusion, barrier permeability, intercellular shuttles, enzyme activities, co-substrate concentrations, or combinations thereof, depending on the organ and disease of interest (19,43,94,101–103). This may be addressed by incorporating complementary information. As an example, HP 13C pyruvate data is influenced by perfusion, and thus addition of perfusion MRI could be important for interpretation (98,104,105).

All the methods outlined above have been explored in clinical studies, described in Supporting Table 3 and summarized in Figure 8. As of September 2022, approximately 52% of studies involving human subjects report rate constants derived from a PK model with a few different models reported. A nearly equal fraction (51%) of the studies report AUC ratio values.

Approximately 66% of these studies report metabolite-specific images or AUC values. About 40% report SNR values; this metric is particularly frequent in manuscripts that describe technical developments for clinical HP MRI. Approximately 16% of these studies summarize model-free metrics, and 10% report measurements from a single timepoint. Most studies report a combination of quantities.

Figure 8: Reported metrics used for analysis in HP [1-13C]pyruvate human studies published up to September 2022.

Visualization

A wide variety of approaches have been used for visualizing data from human HP 13C-MRI studies. The challenges and practical considerations are: 1) choosing the appropriate metrics to display, 2) how to encode the parameters (e.g. the colormap), and 3) choosing how to provide anatomical context and other multi-parametric data. The choice of visualization also depends on the goal which could be for diagnostic interpretation, but also quality control, reproducibility among readers and publication.

Metrics

The choice of HP 13C metrics is described in detail above. At this stage in HP 13C development where there is no standardized metric, often a combination of metabolite images and ratios or PK model parameters are shown.

Parameter Encoding

The mapping function chosen should provide an adequate, often quantitative, impression of the parameter mapped. There is a consensus in the visualization field that perceptually uniform maps are best suited to visualize continuous parameters, like the greyscale typically used by radiologists as well as other monochrome (black to blue) and color ranges (fire-type, rainbow-type) (106,107). Multi-color heatmaps have been the most frequently employed method for HP 13C data, while greyscale has infrequently been used but it ensures there is no coloring-based bias as well as facilitating later reuse (Fig. 9a). Among the color schemes employed in the clinical HP 13C literature, fire-type scheme seems to be the most common [similar to “Plasma” or “Inferno” in matplotlib.org]. Next most commonly employed is the rainbow-type scheme [similar to “Rainbow” in matplotlib.org].

Anatomical Context

HP MRI faces the challenge that it does not necessarily depict the anatomical features, similar to PET, and thus requires an anatomical reference. Most often, a grayscale anatomical image is overlaid with a HP colormap (Fig. 9c,d). This approach is very intuitive, but can skew perception as the grey-scale anatomical reference may affect the brightness of the HP data (e.g. signal in the skull). This bias does not occur when showing adjacent maps (Fig. 9a, b). Here, anatomical outlines may help to provide reference (Fig. 9b).

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