Current Methods for Hyperpolarized [1-13C]pyruvate MRI Human Studies

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.


INTRODUCTION
MRI with hyperpolarized 13 C agents, also known as hyperpolarized (HP) 13 C 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- 13 C]pyruvate were performed in 2011 in prostate cancer patients. 1Since 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- 13 C]pyruvate,  where [2- 13 C]pyruvate 64 and 13 C-urea 56 have been demonstrated too.
As clinical HP 13 C 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 13 C MRI community with this opportunity in mind, and it was greeted with strong enthusiasm.
The "HP 13 C 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- 13 C]pyruvate human studies, for the following reasons: 1. Pyruvate is the most mature and widely used HP agent and has the most significant translational evidence emphasizing the potential clinical impact.2. 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- 13 C]pyruvate human studies along with justification to support those practices.This is divided into four major topic areas: (1) HP 13 C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification (Figure 1).The current successful practices and study methods include a literature review of published peer-reviewed journal papers showing human HP [1- 13 C]pyruvate study data, up to September 2022,  that cover a variety of anatomical targets (Figure 2), as well as new unpublished information from surveys of HP 13 C study sites. Bad on this information, we also highlight the evidence gaps, strengths, and limitations of current practices, which are summarized at the end of each section.

HP 13 C-PYRUVATE PREPARATION
This section covers the processes for creating the HP agent, 13 C 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 (QC), and release.
It is helpful to understand that the specifications of a dose of 13 C pyruvate suitable for in vivo MR HP metabolic imaging were shaped in part by early preclinical studies performed by GE HealthCare summarized in Vanderheyden. 65In short, the safety of the two novel drug components, 13 C 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 13 C nuclei and another Phase 1/2a dose escalation and imaging feasibility study with HP 13 C pyruvate in 31 prostate cancer patients at the University of California, San Francisco (UCSF). 1ith the exception of the first HP 13 C imaging clinical trial, which used a prototype device in a cleanroom, 1 all HP 13 C studies performed in humans to date have used the SPINlab polarizer (manufactured by GE HealthCare).Consequently, all doses of the HP 13 C pyruvate delivered by SPINlab have been produced using the "SPINlab Pharmacy Kit" that serves as the container-closure system for the various drug components ( 13 C pyruvic acid and EPA mixture, dissolution medium, and neutralization and dilution medium) during sample polarization, dissolution, and QC processes.Therefore, 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
Although more than 860 patients or healthy subjects having been injected with HP 13 C pyruvate as of January 2022 without reports of any serious adverse events, 68 HP 13 C pyruvate injection remains an investigational MR contrast agent and can only be administered by those with Investigational New Drug (IND) exemption from the Illustration of the hyperpolarized (HP) 13 C MRI human study process, including the 4 major areas covered in this paper: HP 13 C-pyruvate preparation, MRI system setup and calibration, acquisition and reconstruction, and data analysis and quantification.

F I G U R E 2
Anatomical targets of hyperpolarized [1- 13 C]pyruvate MRI human studies published up to September 2022.
United States (US) Food and Drug Administration (FDA) in the United States, a Clinical Trial Application (CTA) in Canada, approval from National Research Ethics Committee Services in the United Kingdom (UK), or approval from the relevant local regulatory body.Therefore, methods and processes involved to produce a dose should have patient safety as the first priority.Because using 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 United States, 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. 69For 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 before 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 United Kingdom and the European Union (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 13 C pyruvate injection is 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 before polarization, dissolution, and patient administration, but others have also demonstrated the feasibility of preparing a batch of kits, keeping them in a −20 • C freezer and using them over a period of a few months.
Beyond the obvious requirements that the process and the facility have 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, whereas at others, it is conducted in a dedicated non-cleanroom space, reflecting differences in cleanroom approach and specifications between regulators worldwide. 71Some 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 on 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 13 C agent are detailed in the Chemistry, Manufacturing, and Control (CMC) section of an applicable regulatory submission; an example of this has been made available. 72It 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 to satisfy installation qualification (IQ) and operational qualification (OQ).Typically, the final developed process is validated by process qualification (PQ) runs, during which three or more Pharmacy Kits are filled and used and the final HP 13 C 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 three consecutive PQ runs are submitted as part of the IND submission (or its equivalent), and are often also reviewed by the Institutional Review Board where the studies are conducted.

QC and Dose Release
The 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 13 C 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.However, 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 QC of the HP 13 C 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, and temperature) and efficacy (pyruvate concentration, polarization, and 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 13 C pyruvate solution for post-release endotoxin and sterility testing.This testing cannot be completed fast enough to test an individual dose before 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 13 C 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 13 C pyruvate sample decays quickly over the span of a few minutes after dissolution, and therefore, 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 13 C-pyruvate is shown in Figure 3 and Table 1, including the specification of the final dose and how the QC 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 of 13 sites using Sterile Preparation versus 5 of 13 using Terminal Sterilization.Although 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 13 C 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 threefold difference in acceptable polarization levels, which are measured to ensure a futile dose is not injected because the polarization is directly proportional to SNR.This reflects the decision by several sites to believe that useful data can still be obtained with suboptimal polarizations.

Summary
The overall safety record of HP 13 C-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 Pharmacy 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 13 C-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 HP 13 C-pyruvate preparation parameters, methods, and dose specifications used for quality control testing and release as well as validation.

F I G U R E 3
Hyperpolarized (HP) agent preparation methods reported by sites currently performing HP 13 C MRI human studies.
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 13 C is its "broadband" capability to transmit and receive RF signal at the frequency of 13 C, which is approximately four times lower than 1 H.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 used transmission RF coil(s) for 13 C.Most studies to date use relatively low flip angles (<90 • ) for HP 13 C to preserve polarization for time-resolved imaging.The capability to receive 13 C 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 because of chemical shift dispersion and 1 H imaging. High field strengths do not enhance HP 13 C signal as they do for 1 H 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 13 C-pyruvate and its metabolic products have greater frequency separation at higher fields, and it may, therefore, be easier to separate and quantify these resonances at higher fields.This comes at the cost of a reduction in the achievable T 2 * and often reduced T 1 .As the initial polarization is independent of the imaging field strength it has been proposed that the increased T 2 * at 1.5 T 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 T 2. 73 In practice, 3 T has been used in all published human 13 C-pyruvate studies surveyed (Table S1), and comprises the majority of scanners currently in use for human studies (Table 2).A field strength of 3 T is well-suited for 1 H 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 EPI or spiral imaging (see Acquisition and Reconstruction).
Although the spatial resolution acquired for HP 13 C imaging is typically much coarser than for 1 H MRI, the factor of ∼4 in gyromagnetic ratio leads to the same reduction factor in performance of the gradient system, therefore, 13 C 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 1 H 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 1 H frequency, but artifacts may arise at other frequencies.Eddy current compensation is also not always appropriately adjusted for nuclei at other frequencies. 74To optimize for 13 C, 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 characterization and correction, and some vendors have fixed these issues as well.

RF Coils
For HP 13 C imaging studies in humans, RF coils for both 1 H and 13 C nuclei are needed, with 1 H MRI providing an anatomical reference for registration and optional additional multiparametric MRI readouts.At the Larmor frequency of 13 C nuclei, the relative contributions from coil noise compared to sample noise increase compared to 1 H, 75,76 although sample noise still is likely the dominant contributor for human-sized coils at 32.1 MHz-the resonance frequency of 13 C nuclei at 3 T.
The key requirement for human 13 C-pyruvate RF coils is that the coil geometry and sensitive volume must cover the volume of interest in the subject.Table 3 and Figure 4 show 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 B 1 transmit across the FOV (B 1 + ).Although 1 H relies on a large birdcage ("body") coil built into the scanner, 13 C 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 B 1 + .Many human studies have used Helmholtz pair resonators for transmit, including the "clamshell coil," which has a notably inhomogeneous B 1 + profile, but has been used because of relatively easy integration into the scanner bore.B 1 + variation results in variations in the flip angles that control the use of the HP magnetization and creates errors in common HP metrics. 9,76The exception are head coils, where birdcage designs with highly homogeneous B 1 + can be placed around the head while easily fitting inside the bore.
As with 1 H MRI, higher SNR can typically be achieved by smaller receive coil elements, such as surface coils or phased arrays, and the majority of 13 C receive coils used have layouts similar to 1 H phased arrays.
RF coil QC is important to ensure proper functioning of the coils to provide consistent imaging quality, especially with limited natural abundance 13 C 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).A useful reference for RF coil QC is outlined in the MRI accreditation program of the American College of Radiology 77 and can be adapted for 13 C coils.
Notably, configurations for brain and prostate studies used dual-tuned 1 H/ 13 C coil designs, which greatly simplify workflow and registration of 1 H and 13 C images, as no switching of coils is needed.

Phantoms
Because HP magnetization is non-renewable, phantoms containing 13 C 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 HP nuclei; and (3) perform necessary pre-scan adjustments (see Prescan Calibration).The phantoms currently in use are listed in Table 2. Their composition must provide sufficient 13 C signal, with additional considerations of conductivity, stability, chemical shift(s) present, potential for dynamic imaging, and cost.The phantom geometries are typically either compact, 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 13 C-urea at high concentration, typically 8 M, which provides a single resonance placed inside a small container ∼1 mL.The most common recipe mixes 13 C-urea in a 90% water/10% glycerol solution, with glycerol used to increase the urea solubility and doping with a gadolinium-based contrast agent to shorten T 1 , which increases the potential SNR per unit time.For example, when Dotarem is added at a 3:1000 volume ratio the 13 C-urea T 1 is ∼500 ms and T2 is ∼100 ms.However, when testing pulse sequences The commonly used "clamshell" TX coil is a Helmholtz pair design.For 1 H RF configurations, all used the body coil for TX unless otherwise noted, and "repositioned" indicates the 13 C coil was removed for 1 H imaging.One representative reference is listed for each configuration.The RF coil configurations reported in the reviewed papers are shown in Table S1.Abbreviations: HP, hyperpolarized; RX, receive coil; TX, transmit coil.

F I G U R E 4
Examples of RF coil configurations used for human hyperpolarized [1-13 C]pyruvate brain studies.(A,B) 13 C Clamshell TX (Helmholtz pair) and 2× 4-channel paddle RX arrays.(C) 13 C Birdcage volume TX and 32-channel RX array (RX array slides into TX coil).(D) 13 C Birdcage volume TX and 24-channel RX array, combined with a 1 H 8-channel RX array.Image reproduced with permission from Autry et al. 16 RX, receive coil; TX, transmit coil.influenced by T 1 and T 2 , 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 13 C-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 13 C signal even with the 1.1% natural abundance of 13 C.These larger phantoms matching the inner volume of an RF coil are useful for coil testing, including transmit (B 1 + ) and receive (B 1 − ) 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 to mimic coil loading and get a realistic estimation of B 1 +.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. 16,78ynamic phantoms that aim to mimic metabolite kinetics have also been developed, [79][80][81] and have the potential to more closely mimic the HP experiment, but so far these are not widely used.

Prescan Calibration
Before performing an MRI acquisition, the so-called prescan procedure is used to set the shim parameters to maximize B 0 homogeneity over the FOV or a specific region of interest (ROI), the scanner center frequency (CF), the RF transmit gain, and the receiver gain.Although this calibration procedure is usually automated for 1 H, the lack of sufficient natural abundance 13 C signal prevents use of automated methods.(Although natural abundance 13 C lipid signal has been detected, there are so far no reports on using this signal for prescan.)Table 2 shows current practices across sites.
Maximizing B 0 homogeneity is independent of the nucleus and is, therefore, performed before 13 C imaging using the 1 H water signal and existing shimming tools, such as by a standard automated process ("Auto Shimming") or using high order shimming routines.Similarly, the 13 C CF can be calculated from the 1 H CF using a predetermined scaling factor that depends on the target chemical shift. 82Another common approach used is to have a small, high-concentration 13 C phantom (e.g., 8 M 13 C-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 before imaging. 37Both the CF and B 0 shimming are critical when using spectrally selective RF pulses, as in metabolite-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 13 C phantom placed near the ROI during the scan or on a large 13 C phantom of similar size and coil loading as the subject before the subject scan.Reference power is often done by sweeping the power in a pulse-acquire sequence, 53,62 or the Bloch-Siegert method. 52,83When using a small phantom, the location of the phantom, B 1 + 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, 84 and using the stronger natural abundance 23 Na signal that is close enough to the 13 C resonance frequency to be detected by 13 C coils. 82he receiver gain is predetermined, either systematically based on independent phantom measurements and assuming the dose and polarization of the HP compound is known before injection, or based on past HP imaging studies.

Summary
Commercially available 3 T MRI systems are by far the most commonly used for human HP 13 C-pyruvate studies, although a systematic investigation of the impact of B 0 has only recently been investigated. 77The multi-nuclear RF transmit and receive chain has proven sufficient for current acquisition strategies, although many sites have observed artifacts because of RF interference, gradient interference, and residual eddy currents when operating at the 13 C frequency.A variety of 13 C 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 B 1 + profiles.The phantoms used have converged into generally two categories-small phantoms containing 13 C-enriched compounds that can be used during the study and human-sized phantoms containing compounds with high carbon concentrations, but without 13 C 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; therefore, we surveyed individual sites to determine current practices.Calibration procedures for the B 0 field ( 13 C CF and shimming) for most sites take advantage of 1 H signal and methods, whereas methods for calibration of B 1 + 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 13 C studies.

ACQUISITION AND RECONSTRUCTION
Data acquisition strategies in human HP [1- 13 C]pyruvate MRI studies must account for multiple chemical shifts, efficiently use 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., B 1 + 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- 13 C]pyruvate studies and accompanying 1 H imaging, from different anatomic regions, including scan parameters and image reconstruction.

Acquisition and Reconstruction Methods
The acquisition methods used in human [1- 13 C]pyruvate studies can be classified into three categories (1) MR spectroscopy or MRSI; (2) chemical shift encoding methods; and (3) metabolite-specific imaging (Figure 5).MRSI 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. 85Metabolite-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 EPI or spirals. 86Their application to different organ systems is described below.
The image reconstruction methods used in human [1- 13 C]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 10,57,87 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 because of 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- 13 C]pyruvate (compared to 1 H), motivating several HP 13 C-specific methods that include data-driven coil sensitivity estimation, which have shown obvious improvements over sum-of-squares. 11ore recently denoising techniques have been applied as postprocessing of human HP data. 41,42,44The 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, whereas 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 [1-13 C]pyruvate, 1 and data was acquired with MRSI 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. 18or this reason, all currently ongoing studies acquire dynamic data.Although MRSI, 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. 85For 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 (B 1 + ) field. 76

Heart Studies
Data acquisition methods for 13 C 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. 2,22,28,30,35,36,38,45,52The duration of the diastolic window limits the available data sampling time, making cardiac acquisitions the most time-constrained of the HP 13 C MRI applications.The most common acquisition approach is metabolite-specific imaging with spiral k-space trajectories. 2Their 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 multislice acquisitions, but 3D encoding has also been used successfully. 35

Brain Studies
For HP 13 C MRI of the human brain, the majority of studies have also used 2D (slice selective) acquisitions, [10][11][12]14,16,28,33,[40][41]44,51,53,60 with a trend toward volumetric coverage using 2D multislice metabolite-specific imaging. Three dimensional metabolite-pecific 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 used MRSI 5,6,29,[31][32][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 TEs 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 because of the time required to acquire multiple TE 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 13 C 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 multislice or 3D dynamic acquisitions over large FOVs in the abdomen. 4,37,46he significant respiratory motion encountered in these regions can directly blur 13 C images and has further favored these rapid acquisition strategies.Motion also degrades B 0 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 and Calibration for more information).In some studies, breath-holding was used to minimize motion effects and enforce frame-to-frame data consistency. 42A pragmatic and reasonably effective approach for dealing with respiratory motion during 13 C data acquisition is an initial breath-hold (as long as can be tolerated), followed by free-breathing. 46,62

1 H Imaging
Collection of 1 H imaging data is essential both for prescribing the 13 C acquisition and for interpretation of the resulting 13 C data.Multi-planar 1 H scouts are acquired before 13 C acquisition to enable graphical prescription of the 13 C imaging region.All human HP 13 C-pyruvate imaging studies acquire conventional MRI scans (e.g., T 1 -and T 2 -weighted volumes) for anatomic reference, aiming to cover at least the full 13 C FOV. Acquiring these anatomic scans as close as possible to the time of 13 C imaging (immediately before or after) minimizes potential misregistration between the data sets.Depending on the application, other advanced 1 H sequences are also acquired (e.g., diffusion-weighted imaging for cancer imaging).When contrast-enhanced data is acquired, it is done after 13 C imaging, as paramagnetic contrast agents will accelerate 13 C relaxation.

Reported Study Parameters
Figures 5 and 6, and Table S2 show the reported acquisition study parameters for human HP [1- 13 C]pyruvate studies published as of September 2022.Figure 5 shows a mixture of MRSI, 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.

Summary
Three general categories of acquisition strategies have been used successfully for human HP 13 C-pyruvate studies, MRSI, 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 head-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 B 0 field variations and therefore, 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- 13 C]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 ROIs for quantitative evaluation.

Metrics
The most common metrics used in HP [1- 13 C]pyruvate studies are illustrated in Figure 7.These include metabolite images, metabolite ratios, metabolite timings, and pharmacokinetic (PK) modeling.
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). 17These are analogous to the visual evaluation that is most used for routine clinical work. 89,90In these metabolite images, we expect that the [1-13 C]pyruvate AUC signal is predominantly weighted toward perfusion and uptake, whereas [1-13 C]lactate, [1- 13 C]alanine and 13 C-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. 92Further, the reader must be careful to interpret all the images in conjunction to better understand the underlying biology; for example, increased [1- 13 C]lactate in the presence of decreased [1-13 C]pyruvate delivery can have a very different meaning compared to increased [1- 13 C]lactate with increased [1- 13 C]pyruvate delivery (Figure 7).
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 13 C 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 because of signal excitation) differ between studies.Similarly, the ratios between the produced metabolites (e.g., bicarbonate/lactate) can reflect the balance between downstream metabolic pathways. 12,55are must be taken to consider how AUC images are calculated and normalized before comparing values between studies.
5][96][97][98][99] These yield semi-quantitative to quantitative apparent rate constants, given in s −1 .Some models require a vascular input function, whereas others avoid this requirement. 95PK models can explicitly account for acquisition-specific details such as excitation angle and repetition time, and therefore, 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) 99 and therefore, frequently used for human data, has been shown to fit well and robustly to prostate and brain data. 8,20PK models are quantitative in nature, arguably provide more relevant biological information, 8,20 and appear to be reproducible across sites. 51However, 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, cutoff thresholds or other criteria may be used to identify and avoid Hyperpolarized [1- 13 C]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- 13 C]alanine and 13 C-bicarbonate are analyzed similarly to [1-13 C]lactate, but omitted here for display.voxels with insufficient SNR before analysis to improve robustness. 20,411][102] This may be addressed by incorporating complementary information.][105] All the methods outlined above have been explored in clinical studies, described in Table S3 and summarized in Figure 8.As of September 2022, ∼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.Approximately 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.

Visualization
A wide variety of approaches have been used for visualizing data from human HP 13 C-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 QC reproducibility among readers and publication.

Metrics
The choice of HP 13 C metrics is described in detail above.At this stage in HP 13 C 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). 106,107ulti-color heatmaps have been the most frequently used method for HP 13 C data, whereas greyscale has infrequently been used, but it ensures there is no coloring-based bias as well as facilitating later reuse (Figure 9A).Among the color schemes used in the clinical HP 13 C literature, fire-type scheme seems to be the most common (similar to "Plasma" or "Inferno" in matplotlib .org).Next most commonly used is the rainbow-type scheme (similar to "Rainbow" in matplotlib.org).

F I G U R E 8
Reported metrics used for analysis in hyperpolarized [1- 13 C]pyruvate human studies published up to September 2022.

F I G U R E 9
Examples of different parameters, color schemes, and anatomical reference used to visualize hyperpolarized 13 C MRI. (A) Cardiac study showing each metabolite (SNR) on one monochromatic grayscale image and separate anatomic reference, no overlays. 2

(B)
Preclinical cardiac study showing one image per metabolite in monochrome (red/blue) on outlines of anatomy, and additional annotated anatomical image in separate panel. 108(C) Brain study where each metabolite area under the time curve (AUC) (multicolor) was fused with an anatomic image, plus one anatomical image. 28(D) Prostate study showing k PL as multicolor overlay on anatomic image. 26

Anatomical context
HP MRI faces the challenge that it does not necessarily depict the anatomical features, similar to PET, and therefore, requires an anatomical reference.Most often, a grayscale anatomical image is overlaid with a HP colormap (Figure 9C,D).This approach is very intuitive, but can skew perception as the gray-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 (Figure 9A,B).Here, anatomical outlines may help to provide reference (Figure 9B).

Summary
Three general categories of analysis metrics have been reported: metabolite images (often normalized), metabolite ratios, and PK model parameters.There is no apparent convergence to one of the categories, and many studies report multiple metrics in their analysis.A standard metric that is robust to experimental variations (known or unknown) would be highly desirable to compare results across studies.PK modeling of conversion rates (k PL , k PB ) has shown the most promise in this regard.HP 13 C-pyruvate data has always been visualized with 1 H anatomical reference images, allowing for localization of metabolism information.Most papers show HP data as color overlays, but using a variety of colormaps.Using a consistent, perceptually uniform colormap would facilitate better translation of HP 13 C-pyruvate MRI, and we can probably learn from clinical PET practice where metabolism data is also visualized alongside anatomical reference images.

DISCUSSION AND CONCLUSIONS
HP [1-13 C]pyruvate MRI has made great strides in recent years, with an explosion of human research studies covering a broader range of applications and performed by more sites worldwide.We have systematically surveyed the methods for HP agent preparation, system setup and calibration, acquisition and reconstruction, and data analysis used in previous and current studies.The results of this in this paper highlight that the areas of common procedures include the use of the same clinical hyperpolarizer, 13 C phantoms configurations for testing and calibration, time-resolved imaging with a trend toward imaging-based acquisitions, and the use of area-under-curve and PK model based metrics.It also highlights that the areas of variations include the fluid path preparation approach and agent release criteria, system and experiment calibration procedures, choice of imaging-based acquisition and acquisition timing, and numerous metrics applied for analysis.See the summary for each section above for additional details.
In preparing this paper, we also observed notable omissions in reporting of study methods that we believe are relevant to the study results.It was often difficult to determine the calibration procedures used.HP pyruvate preparation processes were often minimally described or left out entirely.In some studies, the key parameter of acquisition timings was not reported.Based on these findings, we believe it would be valuable to define guidelines for reporting of studies to ensure a minimum set of methods is described.
There is some potential bias in our findings and reported methods because one site (UCSF) has published more human HP [1- 13 C]pyruvate than other sites, accounting for 25 of 63 of the papers reviewed.To mitigate this bias, we show the results of current practices submitted from different sites and have included named authors representing a variety of sites as well.
The next steps we propose are formal consensus building, in which a voting process will be used in the hopes of creating a concrete set of recommendations and best practices, as well as clearly identify where there is a lack of consensus and need for further development.It is important to note that HP 13 C-pyruvate MRI is still a research study, and therefore, the methods described and recommended will be treated as research from a regulatory point of view.With the development of guidelines and best practices, we hope to improve the reliability and reproducibility of results in future studies and facilitate the translation of this technology to clinical applications.
Rafat Chowdhury, Yangcan Gong, Shonit Punwani University of Florida, Gainesville, Florida, USA: Matthew Merritt University of Oxford, Oxford, UK: James Grist, Damian Tyler University of Texas-Southwestern, Dallas, Texas, USA: Fatemeh Khashami, Craig Malloy, Jae Mo Park, Vlad Zaha This work was also supported by the ISMRM Hyperpolarized Media MR Study Group and the ISMRM Hyperpolarization Methods & Equipment Study Group.
These were obtained from a survey of all sites performing clinical trials with HP [1-13 C]pyruvate.The parameters used for product release are noted in bold text, otherwise these parameters are measured for batch validation or other quality control (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.Abbreviations: HP, hyperpolarized; NA, not available.

F I G U R E 5
Acquisition methods used in published hyperpolarized [1-13 C]pyruvate human studies published up to September 2022, classified into: MR spectroscopy and spectroscopy imaging (MRSI); chemical shift encoding methods, such as iterative decomposition of water and fat with echo asymmetry and least-squares estimation (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.

F I G U R E 6
Temporal acquisition characteristics reported in hyperpolarized [1-13 C]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.
13mmary of the imaging systems, phantoms, and prescan procedures used at sites currently performing HP 13 C-pyruvate human studies.These were obtained from a survey of all sites performing clinical trials with HP [1-13C]pyruvate.The imaging systems, phantoms, and prescan procedures reported in the reviewed papers are shown in TableS1.
T A B L E 2Abbreviations: Gd, gadolinium; HP, hyperpolarized.a Previously performed studies with a Siemens 3 T Tim Trio.