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

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.

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 studies, for the following reasons:

• 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) that cover a variety of anatomical targets (Fig.2), 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 University of California, San Francisco (UCSF) (1).

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 400 patients or healthy subjects having been injected with HP ¹³C pyruvate as of January 2020 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 −20°C 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.

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.

Site #	Production Style	Facility	Release Procedure	pH	Pyruvate [mM]	Residual EPA [mM]	Polarization, Liquid state [%]	Temperature [° C]	Volume [mL]	Appearance test performed	Filter Integrity Test	Endotoxin Test [EU/mL]	Endotoxin Testing	Sterility Test	Sterility Testing
1	Sterile Preparation	Pharmacy	Other	4 – 9	200 – 300	<2.6	> 7.5	20 – 42	> 35	Yes	Yes	<3.7	Outside	No growth	Outside
2	Sterile Preparation	Radiopharmacy	Physician	5 – 9	200 – 280	<5.0	>5.0	25 – 37	> 40	Yes	No	<0.02	Outside	No growth	In House
3	Sterile Preparation	Pharmacy	Pharmacist	6.7– 8.3	N/A	<3.0	> 15	25 – 40	>38	Yes	No	<0.125	Outside	No growth	Outside
4	Terminal Sterilization	Hyperpolarized	Other	6.5– 8.5	150 – 280	<3.0	NA	25 – 40	6 ml + patient dose	NA	Yes	<5.0	Other	No growth	In House
5	Sterile Preparation	Pharmacy	Pharmacist	6.7– 8.2	220 – 280	<3.0	> 5	25 – 39	>40	Yes	No	<11	Outside	No growth	Outside
6	Sterile Preparation	Pharmacy	Pharmacist	6.5–8.2	210 – 280	<3.0	≥15	25 – 39	>40	Yes	Yes	<0.5	Outside	No growth	Outside
7	Terminal Sterilization	Pharmacy	Pharmacist	6.5–8.5	200 – 280	<3.0	> 15	25 – 40	> 38	Yes	Yes	<4.37	In House	No growth	In House
8	Sterile Preparation	Pharmacy	Physician	6.5–8.9	220 – 280	<3.0	> 15	25 – 37	> 40	Yes	No	<0.5	Outside	No growth	Outside
9	Terminal Sterilization	Hyperpolarized	Pharmacist	5 – 9	185 – 280	<5.0	NA	25 – 40	>38	Yes	Yes	<2.5	In House	No growth	In House
10	Sterile Preparation	Pharmacy	Physician	6.5–8.5	220 – 280	<3.0	≥10	25 – 37	>38	Yes	No	<0.1	Outside	No growth	Outside
11	Terminal Sterilization	Pharmacy	Physician	6.5–8.5	210 – 280	<3.0	> 15	25 – 37.4	>40	Yes	Yes	NA	Other	No growth	In House
12	Terminal Sterilization	Radiopharmacy	Pharmacist	6.7–8.6	220 – 280	<3.0	> 15	25 – 37	>40	Yes	Yes	<5.0	In House	No growth	In House
13	Sterile Preparation	Pharmacy/ Radiopharmacy	Pharmacist	5 – 9	220 – 280	<3.0	> 15	25 – 37	>38	NA	Yes	NA	In House	No growth	In House

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 ¹³C MRI human studies

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.

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.

Site	Primary Imaging System	13C Receive Channels	13C Peak Power [kW]	Phantom(s) - during study	Phantom(s) - before study	13C Frequency Calibration	Shimming	13C RF Transmit Power Calibration	13C RF Receive Gain Calibration
1	Philips 1.5T Achieva and 3T Ingenia	8	4	13C-urea, 13C-pyruvate, 13C-acetate, 13C-acetate, tubes	13C-urea, tube	Derived from 1H	Auto shimming	Power sweep on phantom before study	Not required
2	GE 3T Discovery MR750w	8	8	None	13C-urea doped with Gd, tube	Derived from 1H	Auto shimming	Bloch-Siegert	None
3	GE 3T Discovery MR750	8	8	13C-urea doped with Gd, sphere	13C-urea doped with Gd, sphere	Predetermined frequency	Auto shimming	Power sweep	Empirical
4	GE 3T Discovery MR750w	8	8	[1-13C]lactate doped with Gd None	13C-bicarbonate	Derived from 1H	PRESS box shim	Power sweep	Empirical
5	GE 3T Discovery MR750	32	8	13C-bicarbonate doped with Gd, sphere	ethylene glycol, head shape ethylene glycol, bottle dimethyl silicone, sphere & cuboid	Derived from 1H	Auto shimming High order shimming	Bloch-Siegert	Empirical
6	GE 3T Discovery MR750 and GE 3T Signa PET/MR	32	8	13C-bicarbonate doped with Gd, sphere 13C-urea doped with Gd, bottle or Eppendorf tube	dimethyl silicone, various shapes 13C-bicarbonate doped with Gd, sphere 13C-urea doped with Gd, bottle or Eppendorf tube	Derived from 1H	Auto shimming over PRESS box (breast, prostate) Auto shimming (abdomen) High order shimming (brain)	Bloch-Siegert	Empirical
7	GE 3T Discovery MR750w	8	8	13C-urea doped with Gd ethylene glycol	13C-urea doped with Gd, tube	Derived from 1H	Auto shimming followed by manual linear shimming	Power sweep	Empirical
8	GE 3T Signa Premier*	32	8	13C-urea doped with Gd, tube	ethylene glycol	Establishing protocol	Establishing protocol	Establishing protocol	Establishing Protocol
9	GE 3T Discovery MR750	32	11	13C-urea doped with Gd, tube	13C-urea doped with Gd, sphere ethylene glycol, head shape dimethyl silicone, sphere & cuboid	Derived from 1H	Auto shimming Specialized shimming	Power sweep	Empirical
10	Siemens 3T Biograph mMR	12		13C-urea doped with Gd, tube	13C-urea doped with Gd, tube ethylene glycol	Based on 13C phantom	Auto shimming	Power sweep	Automatic
11	GE 3T Signa Premier	32	8	13C-urea doped with Gd, tube	dimethyl silicone, cylinders	Based on 13C phantom	Auto shimming	RF pulse sweep	Maximum values
12	GE 3T Discovery MR750	8	8	13C-urea in D2O doped with Gd	dimethyl silicone, various shapes ethylene glycol, various shapes	Derived from 1H	Auto shimming	Power sweep Bloch-Siegert	Maximum values
13	GE 3T Discovery MR750w	8	8	13C-urea doped with Gd	ethylene glycol, sphere	Based on 13C phantom	Auto shimming	Bloch-Siegert, checked with power sweep	Maximum values

^*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.

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.

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.

Anatomical Target	13C TX-transmit coil type	13C RX-receive coil type	1H coil setup	Representative Reference
Abdomen/Pelvis	clamshell coil	16-channel bilateral array	Body coil	(4)
Abdomen/Pelvis	clamshell coil	2× 4-channel paddle arrays	Body coil or 32-channel abdomen array, repositioned	(37)
Abdomen/Pelvis	clamshell coil	8-channel array	32-channel abdomen array, repositioned	(46), Fig 4A, B
Abdomen/Pelvis	semi-flexible quadrature coil	2× 4-channel arrays	Body coil	(62)
Brain	clamshell coil	2× 4-channel paddle arrays	8-channel head array, repositioned	(5)
Brain	birdcage coil	32-channel array	Body coil	(14), Fig 4C
Brain	quadrature coil	8-channel array	quadrature TX/RX head coil	(50)
Brain	dual-tuned 1H/13C quadrature coil	dual-tuned 1H/13C quadrature coil	dual-tuned 1H/13C quadrature coil	(55)
Brain	clamshell coil	8-channel receive array	Body coil	(57)
Breast	2-channel TX/RX	2-channel TX/RX and 6-channel RX-only	Body coil or 8-channel breast array, repositioned	(43)
Heart	clamshell coil	2× 4-channel paddle arrays	not reported	(2)
Heart	Helmholtz 2-loop coil-pair	Helmholtz 2-loop coil-pair	Body coil	(36)
Heart	semi-flexible quadrature coil	2× 4-channel arrays	Body coil	(36)
Heart	clamshell coil	16-channel receive array	Body coil or 8-channel cardiac array, repositioned	(52)
Prostate	clamshell coil	dual 1H/13C endorectal coil	dual 1H/13C endorectal coil	(1)

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).

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.

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 (B₁⁺) 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.

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 (75). 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.

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.

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 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

The most common metrics used in HP [1–13c]pyruvate studies are illustrated in Fig. 7. These include metabolite images, metabolite ratios, metabolite timings, and pharmacokinetic 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) (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.

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.

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 multiparametric 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].

Figure 9:

Examples of different parameters, color schemes and anatomical reference used to visualize HP ¹³C 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 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 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).

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) have shown the most promise in this regard. HP ¹³C-pyruvate data has always been visualized with ¹H 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 ¹³C-pyruvate MRI, and we can probably learn from clinical PET practice where metabolism data is also visualized alongside anatomical reference images.

Discussion and Conclusions

Hyperpolarized [1-¹³C]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 highlights that the areas of common procedures include the use of the same clinical hyperpolarizer, ¹³C phantoms configurations for testing and calibration, time-resolved imaging with a trend towards 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 [1-¹³C]pyruvate than other sites, accounting for 25 out 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’s important to note that HP ¹³C-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.

Supplementary Material

Supinfo

Supporting Table S1: MRI system setup and calibration methods reported in HP ¹³C-pyruvate human study papers surveyed. The entries are blank when the methods were not reported in the manuscript or supplementary/supporting materials.

Supporting Table S2: Acquisition methods reported in HP ¹³C-pyruvate human study papers surveyed. Note that some papers reported multiple acquisition methods. The entries are blank when the methods were not reported in the manuscript or supplementary/supporting materials. If some values were not reported, they are denoted by a “?”. In some cases, the methods were ambiguous and have been inferred from the cited paper or its references, which are italicized and followed by “?”. “~” indicates a range of parameter values were reported. N/A = Not applicable. For the Start Time, some studies reported a single value, but it was not clear whether this was relative to the start of injection, end of injection, or other time, and these are shown as a single value and are italicized.

Supporting Table S3: Metrics reported in HP ¹³C-pyruvate human study papers surveyed.

HP 13C MRI Consensus Group

Aarhus University, Aarhus, Denmark: Esben Søvsø Szocska Hansen, Jack Miller, Lotte Bonde Bertelsen, Michael Vaeggemose Chang Gung Memorial Hospital, Taojuan City, Taiwan: Gigin Lin Technical University of Denmark, Copenhagen, Denmark: Andrea Capozzi ETH Zurich, Zurich, Switzerland: Max Fuetterer GE Healthcare, Waukesha, Wisconsin, USA: Albert Chen, Avi Leftin, Rolf Schulte Institute for Bioengineering of Catalonia, Barcelona, Spain: Irene Marco Rius Kings College London, London, UK: Thomas C Booth École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland: Mor Mishkovsky MD Anderson Cancer Center, Houston, Texas, USA: Christopher Michael Walker Memorial Sloan Kettering Cancer Center, New York, New York, USA: Kayvan Keshari MIAAI (Medical Image Analysis and AI), Danube Private University, Kerns-Stein an der Donau, Austria : Ramona Woitek National Institutes for Quantum Science and Technology, Chiba, Japan: Yuhei Takado National Institutes Health/National Cancer Institute, Bethesda, Maryland, USA: Crystal Vasquez NVision imaging technologies, Ulm, Germany: Christoph Müller Oxford Instruments, Oxford, UK: Joel Floyd Technical University of Munich, Munich, Germany: Martin Grashei, Geoff Topping, Frits van Heijster University of California - San Francisco, San Francisco, California, USA: Jenny Che, Yaewon Kim, John Kurhanewicz, Michael Ohliger, Renuka Sriram, Myriam Chaumeil University of Freiburg, Freiburg, Germany: Michael Bock, Andreas Schmidt University of Cambridge, Cambridge, UK: Ferdia Gallagher University of College London, London, UK: 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