Arduino Compass Navigation

Feel the way with a vibrotactile compass: Does a navigational aid aid navigation? Steven M. Weisberg, Daniel Badgio, & Anjan Chatterjee

Author Note

Work on this project was funded by a grant to the Spatial Intelligence and Learning Center from the National Science Foundation, SBE-1041707, and a grant from the National Institute of Health to S.M.W., F32-DC-015203.

Correspondence concerning this article should be addressed to Steven M. Weisberg, at .

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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 1, 2017.

Abstract

Knowing where north is provides a navigator with invaluable information for learning and recalling a space, particularly in places with limited navigational cues, like complex indoor environments. Although north is effectively used by orienteers, pilots, and military personnel, very little is known about whether non-expert populations can or will use north to create an accurate representation of an indoor space. In the current study, we taught people two non- overlapping routes through a complex indoor environment, with which they were not familiar – a vibrotactile compass on their arm, which vibrated continuously indicating the direction of north.

Along the other route, they were only told where north was at the start of the route. At the beginning, the end, and back at the beginning of each route, participants pointed to well-known landmarks in the surrounding city and campus (external landmarks), and newly-learned landmarks in the hospital (internal landmarks). We found improved performance with the compass only for external landmarks, driven by people’s use of the availability of north to orient these judgments. No such improved orientation occurred for the internal landmarks. These findings reveal the utility of vibrotactile compasses for learning new indoor spaces. We speculate that such cues help users map new spaces onto familiar spaces or to familiar reference frames.

Keywords: Spatial navigation; vibrotactile compass; reference frames; spatial memory; real world navigation. .

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Getting lost in complex indoor spaces, like hospitals, airports, or subways, can be dangerous, distressing, and costly. Unlike outdoor spaces, which typically offer long sightlines and distal landmarks, indoor spaces are often confined, undifferentiated, and labyrinthine.

Individual building layouts can be hard to learn if knowledge about direction with respect to the larger world is not easily attained. Confusing layouts, signs, and an inability to see outdoor landmarks compound these problems. Even in places with adequate signs or sight of outdoor landmarks, successful navigation by blind people and people with visual impairments is a substantial problem (Schinazi, 2008; Schinazi, Thrash, & Chebat, 2016). In the current study, we investigate whether providing non-visual directional information aids sighted people in learning an unfamiliar, complex indoor space. Specifically, we tested people using a commercially- available vibrotactile compass, which vibrates in the direction of north. Unlike visual compasses, people with visual impairments can use vibrotactile compasses. A non-visual aid might also limit the effects of divided attention, which complicates the use of visual aids (Gardony, Brunyé, & Taylor, 2015).

We tested two competing hypotheses in the current study. These hypotheses stem from various theoretical frameworks of navigational ability (Wolbers & Hegarty, 2010). 1. General: People would have access to more spatial information, and thus improve generally. In this case, providing a specific spatial cue decreases the error in the sensory signals, increasing the fidelity of the resulting spatial representations. We would expect improved performance across all aspects of navigation behavior.

2. Specific: People would have access to a specific spatial cue, and thus improve on tasks for which that cue is immediately helpful. In this case, providing a specific spatial cue .

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only improves access to specific information, but it does not create a more accurate general spatial representation. Addressing these questions offers insights into how spatial information is acquired in general, and provides insight into how vibrotactile compasses might aid blind or visually impaired navigators.

Why might people get lost in complex indoor spaces? One reason is that place-based strategies used by humans to navigate are thwarted by indoor spaces. Research on humans and rats reveals two navigational strategies (Hartley, Maguire, Spiers, & Burgess, 2003; Marchette, Bakker, & Shelton, 2011; McDonald & White, 1994; Morris, Garrud, Rawlins, & O’Keefe, 1982; Munn, 1950; Packard & McGaugh, 1996; Restle, 1957; Tolman, Ritchie, & Kalish, 1946).

First, a place-based strategy represents space as a cognitive map. A cognitive map is a flexible spatial representation, from which one can infer novel shortcuts. Second, a response strategy applies a stimulus-response approach to navigation. Choice points are identified and responses are recalled (e.g., turn left at the bank, then right at the big tree). The response strategy is relatively inflexible, and relies on associations between scenes and actions. In indoor environments, cues that support a place strategy, like distal landmarks and visual discriminability (Restle, 1957) are more likely to be absent. Instead, place strategies rely on path integration – tracking one's movement away from a starting location by attending to translations and rotations.

Unfortunately, unlike rodents, people are poor path integrators (Foo, Duchon, Warren, & Tarr, 2007; Loomis et al., 1993). Thus, in indoor spaces, if people adhere to response-based strategies, they are less likely to gain spatial knowledge about the overall environment.

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A second reason people easily get lost in complex indoor spaces could arise because of their use of preferred reference frames (Diwadkar & McNamara, 1997; McNamara, Rump, & Werner, 2003; Meilinger, Riecke, & Bülthoff, 2014; Meilinger, Frankenstein, Watanabe, Bülthoff, & Hölscher, 2015; Mou & McNamara, 2002; Mou, McNamara, & Zhang, 2013; Mou & Wang, 2015; Roskos-Ewoldsen, McNamara, Shelton, & Carr, 1998; Shelton & McNamara, 2004). A reference frame is a spatial representation in which objects or other representations of space are contained, or with respect to which they are ordered, oriented, located, or thought to move; a preferred reference frame refers to an individual's privileged reference frame, according to which a spatial layout is most easily recalled. In a global reference frame, properties do not change across different areas (e.g., global north). In familiar, large-scale (i.e., city-sized) spaces, reference frames can be aligned with north (Frankenstein, Mohler, Bülthoff, & Meilinger, 2011), or a salient organizing feature (like a street through a campus; Marchette, Yerramsetti, Burns, & Shelton, 2011; Yerramsetti, Marchette, & Shelton, 2013). Indoor environments, however, provide less access to global reference frames. In the absence of global cues (like distal landmarks), people typically organize space into several local reference frames, instead of one global reference frame (Meilinger et al., 2014). In the extreme, using local reference frames means each segment of a route is disconnected from the last, and results in a 1-dimensional representation of space (i.e., an ordered sequence of places, with no 2-dimensional spatial relations specified; Ishikawa & Montello, 2006). Being able to integrate across areas of a learned route to take novel shortcuts, for example, requires spatial knowledge about how to join local reference frames to each other to form a global reference frame – a difficult and demanding cognitive process (Weisberg & Newcombe, 2016; Weisberg, Schinazi, Newcombe, Shipley, & Epstein, 2014).

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If people directly sense a global spatial cue, like a cardinal direction, they might map an unfamiliar indoor space onto the larger external environment, and construct a more accurate spatial representation of this indoor space. In this way, place-based strategies and global reference frames become more useful in an unfamiliar indoor space. Vibrotactile compasses in particular can convey global north, with some success. König and colleagues (Kärcher, Fenzlaff, Hartmann, Nagel, & König, 2012; Kaspar, König, Schwandt, & König, 2014; König et al., 2016) developed a feelSpace belt, which provides tactile information about true north. Their work illustrates that, with extensive training (seven weeks), a vibrotactile compass improves basic homing tasks in sighted individuals (König et al., 2016). The focus of the current study is on how such information improves (or does not improve) specific and general aspects of navigation. That is, we do not know if such directional information is incorporated into different spatial representations, particularly in complex indoor spaces. Moreover, we wished to learn if this directional information could be used to learn a new environment with only rudimentary use of the device.

Participants

were highly familiar with the hospital (having spent over 30 hours in the environment within the last year). Of the remaining subjects, 18 had been in the hospital, but the maximum amount of time spent by any one subject was 4 hours, with an average of 1.97 hours. The resulting sample consisted of 48 participants (27 identifying as female). Sixteen participants self-reported as Asian, eight as African-American or Black, five as Hispanic or Latino, two as Other, and 17 as .

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Caucasian or White. Participants' average age was 21.73 years (SD = 3.43). This research was

Experimental Settings And Materials

Testing rooms. We used a small testing room to greet the participant, obtain informed consent, and have the participant fill out the initial questionnaires. The larger testing room was either the first or third author's office, and provided enough space for the participant to be blindfolded and disoriented without bumping into furniture or the walls.

hospital (Fig. 1 and Fig. 2A). In general, this part of the hospital has non-descript hallways and lacks windows or views of the outside. Occasional signs mention a street exit (e.g., to Spruce Street), but the majority of the signs consists of hospital building names and colors. The routes were on different floors so that they did not overlap. Each route took 3-4 minutes to walk, and contained three internal landmarks, or objects, about which the participant was instructed to learn certain details (see Measures and Procedure, below).

The starting and ending locations were in areas removed from heavy foot traffic (e.g., a small shipping alcove connected to a lesser-used section of hallway). These locations were also the points at which the participant would complete the pointing task (see Figure 1). The ground floor route consisted of ten turns (three choice points). The first floor route consisted of nine turns (four choice points).

Vibrotactile compass. We used a vibrotactile compass (Fig. 2B; NorthPaw 2.0; hereafter referred to as the compass), which was purchased pre-assembled from an online vendor (www.sensebridge.net). The compass consists of eight oscillating motors arranged approximately one inch apart around a cloth strap, and an Arduino circuit board and battery assembly. The cloth .

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strap was wrapped around the bicep of the participant’s non-dominant arm. The eight motors were programmed to vibrate based on whichever one was facing closest to true north, as measured by a compass sensor wired into the Arduino circuit board. Thus, as the participant moved and rotated around, the motor that was closest to north would vibrate. Because one motor is always the closest to north, exactly one motor vibrated the entire time the compass remained on. The experimenter explained this to the participant, and demonstrated the compass' functionality.

Pointing measurement compass. For the majority of participants1, to measure the participant's pointing judgments, we used a digital compass (purchased from Amazon.com https://www.amazon.com/Pyle-Sports-PSHTM24-Handheld-Chronograph/dp/B005G2SH8Y).

We measured pointing direction by placing a clipboard along the direction of the participant's arm as they pointed, then aligning the compass with the clipboard, and recording the direction to the nearest degree. This procedure was used so that the participant could not view the direction they were pointing.

Measures

Paper and pencil measures. Demographics. Demographics and self-report data were collected on age, ethnicity, sex, gender, education (in years), sexual orientation, English as a first language, and whether the subject was right-handed.

1 For two subjects, we used a custom built Arduino device which participants could hold in their hand, point in the direction, and press a button to indicate the direction. While this method had ergonomic benefits, and ensured that participants could not accidentally view the digital compass, calibration with the digital compass revealed that this method yielded less reliable measurements.

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Familiarity with hospital questionnaire. This questionnaire consisted of three questions: 1) Have you been inside the [hospital] before for any reason? 2) If yes, how long ago? 3) Approximately how many hours have you spent inside the hospital? Santa Barbara Sense of Direction Scale. (SBSOD; Hegarty, Richardson, Montello, Lovelace, & Subbiah, 2002)). This self-report measure of navigation ability consists of fifteen 7- point Likert-scale items such as "I am very good at giving directions," and "I very easily get lost in a new city." The average score for each participant has been shown to correlate highly with performance on behavioral navigation tasks in real and virtual environments (Hegarty et al., 2002; Weisberg et al., 2014), and with individual differences in neural structure and function (e.g., Epstein, Higgins, & Thompson-Schill, 2005; Schinazi, Nardi, Newcombe, Shipley, & Epstein, 2013).

Spatial Cognition, Navigation, and Experience Questionnaire. (SCNEQ). We developed this 7-question self-report survey to measure the extent to which each participant was accustomed to navigating similar urban environments. For example, two items were “I lived much of my life in a rural setting” and “I am used to the layout of a city whose roads are arranged in a grid.” See Supplementary Methods for the full versions of all questionnaires.

Navigation performance measures. Compass pre-test. The experimenter introduced the participant to the compass, explained and demonstrated how the mechanism worked, and described how it could be used to help maintain an orientation. The participant then completed a pre-test pointing task. The purpose of this pre-testing task was to familiarize them with the procedures required during the main experiment. Across participants, we counter-balanced the order of the pre-test. Half of the participants first completed the pointing without the compass, and the other half first completed .

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the pointing with the compass. The procedure was the same for both. The experimenter pointed in a pre-determined random direction (between 0-359, with 0 as true north), placed a blindfold on the participant, slowly spun them around clockwise and counterclockwise for 20-30 seconds, then asked the participant to point in what they thought was their original facing direction and recorded the judgment. This process was repeated twice, disorienting in the same way to record a second and third pointing judgment for the same direction. The participant remained blindfolded continuously throughout the duration of all three trials. The entire procedure was then executed with a new direction and with the compass turned on (or off, depending on what the participant had already completed).

Pointing judgments. Along each hospital route, participants completed a pointing task three times on each route: at the beginning before route learning, at the end of the route learning, and again at the beginning after route navigating. All three pointing sessions included the four cardinal directions (North, East, South, and West) and eight familiar external landmarks. Four of located in Philadelphia (External-City). The second and third pointing sessions included pointing judgments for three internal landmarks, which were located along that route. Participants pointed directly at each internal landmark (Internal-Location), and pointed in the direction they were facing when they viewed that landmark (Internal-Facing). The third pointing session of the participant’s second route also included judgments of the internal landmarks of the first route.

Route reversal. After learning the route in one direction, the participant navigated back to the beginning. If the participant made a navigational mistake (e.g., by making a wrong turn), the experimenter corrected it immediately. Along the way, they indicated and named the internal landmarks, and any apparent potential short cuts back to the start (e.g., doorway, hallway), .

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without actually taking them. Data were collected by the experimenter on numbers of navigational errors made, numbers of correctly indicated short cuts, and numbers of missed internal landmarks.

Sketch map. Following the third pointing task on each route, the participant sketched a map of the route they took. The maps were required to include the path of the route itself, the internal landmarks, and an arrow indicating north.

Post-route questionnaire. After the sketch map, participants answered four de-briefing questions about each route. 1) Explain how you tried to learn the route around the hospital and the locations of the objects. 2) Describe the route you took (in words). 3) What strategy did you use to perform the pointing task? 4) (Compass route only) How did the device help (or hurt) you

Procedure

Participants met the experimenter in a small testing room to provide informed consent and fill out four questionnaires (demographics, familiarity with the hospital, SBSOD, SCNEQ). The experimenter led the participant to a larger room for the compass familiarization and pre- test. The experimenter then took the participant from the meeting place to the hospital. The experimenter took the same path every time to enter the hospital.

At the hospital, the route order and compass-use order were counter-balanced between participants such that all four possibilities were equally represented in the sample. The experimenter led the participant to the beginning of her first route, and explained the general nature of the experiment (see supplemental methods for all experimenter scripts).

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Each route consisted of the same tasks in the same order. First, the experimenter pointed in the direction of north, and told the participant that she could use that information however she saw fit. The participant then pointed to the cardinal directions and all external landmarks.

The experimenter then outlined the participant's tasks along the route – to learn the order, locations, and facing orientations of three objects, and to learn the layout of the route with respect to the hospital and the broader external environment. For the Compass route, the experimenter strapped the compass on, and instructed the participant to use it as a helpful tool in maintaining her orientation throughout the environment. The experimenter then led the participant along the route, stopping at each object to name it. The experimenter also positioned the participant in front of each internal landmark in a consistent location, indicating the direction the participant was facing as the facing orientation to that internal landmark. At the end of the route, the participant performed the pointing task again, beginning with the cardinal directions and external landmarks, and including the internal landmark locations and facing orientations.

The participant also pointed back to the start of the route. The participant then led the way back from the end of the route to the beginning (the Route Reversal task), indicating each internal landmark along the way. Upon returning to the beginning, the participant performed all pointing judgments a third time, this time including pointing to the end of the route. If this was the second of the two routes, the participant then pointed to the internal landmark locations and facing orientations of the previous route. Finally, the participant drew a sketch map of the route, and answered the post-test questionnaire. If this was the first of the two routes, the experimenter would walk the participant over to the beginning of the second route and repeat the foregoing procedure.

Results

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Our primary interest was in differences when participants were wearing the compass and not. We also did not predict, and were not interested in interactions with route order or order of compass use. We thus collapsed data across subjects for route order and order of compass use.

To calculate the error for each pointing judgment we subtracted the angular distance between the correct answer and the participant's response, then corrected the difference to be less than 180°. Chance performance would average 90° error. For repeated-measures t-tests, we used the correction to Cohen's d suggested by Morris & DeShon (2002), which accounts for correlations between factors of the dependent variable. For repeated-measures effect sizes, we use generalized eta squared (Bakeman, 2005) provided by the ezAnova package in R, 3.0. For the sketch maps, we scanned and uploaded the hand-drawn maps, then calculated coordinates for the three landmarks, the starting point, and the finishing point, using the Gardony Map Drawing Analyzer (GMDA; Gardony, Taylor, & Brunyé, 2016).

Compass Pre-Test

Participants pointed to the indicated direction better than chance both with the compass, t(47) = 6.14, p < .0000001, d = 0.89, and without the compass, t(47) = 2.89, p < .006, d = 0.42. We compared performances using a within-subjects t-test on the pre-test and found that participants were significantly more accurate with the compass (M = 58.78, SD = 35.24) than without the compass (M = 76.74, SD = 31.81), t(47) = 2.81, p = .007, d = 0.41.

Route-Reversal

Overall, participants made fewer navigation errors when recreating the route with the compass (total errors = 27) than without the compass (total errors = 35). These did not differ significantly: t(47) = 1.31, p =.20, d = 0.19. Participants also did not make more mistakes naming the objects along the route, were not more likely to indicate the wrong direction for the .

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stairs, and did not indicate more shortcuts, all p's > .43. The additional input from the compass did not improve the participants' ability to effectively re-create the route, although we note that most participants were at a ceiling performance for this aspect of navigation.

Pointing Test Error

We assessed differences in pointing separately for each judgment type (cardinal directions, external landmarks, internal locations, and internal orientations) using a 2-factor (Compass x Route position) within-subjects ANOVA. Compass had two levels – with or without the compass. Route position also had two levels – End, and Beginning 2. We excluded judgments from the Beginning because a) participants were shown where north was during that pointing session, and b) no judgments were made to Internal Landmarks. Furthermore, no differences were observed between the two Beginning sessions, and the pattern of results is similar for the Cardinal Directions and External Landmarks when the two Beginning sessions are averaged together as when only Beginning 2 was used. For simplicity and brevity, we thus exclude Beginning judgments.

Cardinal Directions. We obtained a significant interaction between compass and route

Position, F(1,47) = 9.65, P = .003,

= 0.04. As predicted, follow-up contrasts revealed that pointing at the End was significantly better when participants wore the compass (M = 25.06, SD = 25.06) than when they did not (M = 52.33, SD = 54.22), t(47) = 3.46, p = .001, d = 0.55.

Participants performed equally well at Beginning 2 with the compass (M = 14.75, SD = 10.32) as without the compass (M = 16.75, SD = 8.56) t(47) = 1.58, p = .12, d = 0.23. The interaction resulted in a significant main effect of wearing the compass, F(1,47) = 13.95, p < .001,

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0.06, such that participants were more accurate when wearing the compass overall (M = 19.91, SD = 13.90) than without the compass (M = 34.54, SD = 27.50), and in a main effect of route .

Position, F(1,47) = 23.97, P < .001,

= 0.13, such that pointing to cardinal directions at Beginning 2 (M = 15.75, SD = 8.40) was significantly more accurate compared to the End (M = 38.70, SD = 32.21). Thus, participants became disoriented with respect to north after walking the route, and this disorientation was mitigated by use of the compass.

External Landmarks. We obtained a significant interaction between compass and route position for external landmark judgments, F(1,47) = 4.47, p = .04,

= 0.01. As Predicted,

follow-up contrasts revealed that pointing at the End was significantly better when participants wore the compass (M = 49.41, SD = 28.50) than when they did not (M = 61.58, SD = 39.69), t(47) = 2.34, p = .02, d = 0.35). There was no difference at Beginning 2 with the compass (M = 44.98, SD = 25.61) compared to without the compass (M = 44.89, SD = 27.87), t(47) = 0.04, p = .97, d = 0.005. This is not surprising because at the Beginning of both routes, participants were shown where north was, and could have used this information to reorient themselves. The interaction between compass and route position resulted in a significant main effect of wearing

The Compass, F(1,47) = 4.66, P = .036,

= 0.01, such that participants were more accurate with the compass (M = 47.19, SD = 24.34), than without the compass (M = 53.24, SD = 28.79). The interaction also resulted in a main effect of route position, F(1,47) = 9.31, p = .004,

= 0.03,

such that pointing to cardinal directions at the Beginning 2 (M = 44.94, SD = 25.51) was significantly more accurate compared to the End (M = 55.49, SD = 29.49). Thus, as with cardinal directions, participants lost their orientation with respect to the locations of external landmarks after walking the route, and the disorientation was mitigated by the use of the compass.

Internal Landmarks - Location. We did not obtain significant effects for the compass or route position, nor did we obtain an interaction. Participants were no better at pointing to the locations of the internal landmarks while wearing the compass at either the End (M = 49.59, SD .

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= 33.95) or at Beginning 2 (M = 58.64, SD = 38.78) compared to when they were not wearing the compass at the End (M = 51.35, SD = 35.99) or at Beginning 2 (M = 55.72, SD = 39.19), all p's > .12. Wearing the compass did not help pointing to the locations of internal landmarks.

Internal Landmarks - Orientation. We did not obtain significant effects for the compass or route position, nor did we obtain an interaction. Participants were no better at pointing to the orientations of the internal landmarks while wearing the Compass at either the End (M = 42.96, SD = 33.39) or at Beginning 2 (M = 47.92, SD = 33.95) compared to when they were not wearing the Compass at the End (M = 56.46, SD = 47.24) or at Beginning 2 (M = 46.94, SD = 44.28), all p's > .13. Again, the compass did not help pointing to the orientations of internal landmarks.

Pointing Reference Frames

By analyzing pointing errors, we found that participants were disoriented with respect to cardinal directions and external landmarks at the End of the route when they were not wearing the compass. From the pointing error analysis, we did not see improvement between compass and non-compass routes on the internal landmark orientation or location judgments. This result could be because participants were not able to use the compass to improve these judgments, despite attempting to align the representation of internal landmarks with north. Therefore, we also wanted to know whether the reference frame participants used for the internal landmarks mapped onto their reference frame for the external landmarks. If so, we would expect pointing judgments within a session to be internally consistent with each other. This would mean that each pointing judgment might have a large absolute error, but that overall, the pointing judgments might be internally consistent but with an orientation deviated from true north. We .

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tested this idea by finding the Best Fit Angle for each set of pointing judgments (see top panel of Figure 3). This approach allows us to infer the orientation of a set of pointing judgments. Note that we could use the direction that the participant pointed to when asked where north was. But this approach would not allow us to find an optimal solution for internal landmarks, which could be aligned with a local reference frame, or otherwise misaligned with where the participant thought north was, but still internally consistent.

The lower middle panel of Figure 3 illustrates an example wherein a participant believes that north is actually west, and was mapping other judgments according to that misaligned orientation. Each pointing judgment is thus off by approximately 90°. When the pointing judgments are rotated to minimize the error, true north lines up with Participant-north, suggesting that the participant's pointing judgments were consistent with where he thought north was. Applying the Best Fit Method allows us to see whether participants use self-consistent reference frames for external and internal landmarks.

To find the Best Fit Angle, we rotated each participant's pointing judgments one degree at a time and calculated the absolute error. For each set of pointing judgments, we recorded the angle at which the minimum absolute error was achieved (henceforth the Best Fit Angle). We did this separately for the external landmarks and internal landmarks to obtain separate Best Fit Angles for each set of judgments. Because participants were told where north was at the Beginning (and likely recalled this information at Beginning 2), we only used judgments from the End. All circular statistics were calculated using the CircStat package in Matlab (Berens, 2009).

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Error should be reduced overall for both routes, because finding the Best Fit Angle also minimizes error from noise. However, if participants pointed to landmarks based on a consistent frame, we would expect more error reduction for pointing without the compass compared to with the compass route. Consider the three examples in the bottom panel of Figure 3. In the example on the left, the participant knows where true north is, and has relatively accurate judgments for the landmarks. Rotating by the Best Fit Angle reduces the error by a very small amount. In the example in the middle, the participant does not know where true north is. He also does not know where the landmarks are with respect to north or with respect to each other. Thus, the Best Fit Angle does not line up with Participant-north. In this case, Best Fit Angle and Participant-north would be uncorrelated. Finally, in the example on the right, the participant thought north was actually west, and his landmark pointing judgments aligned with this "west-is-north" reference frame. In this case, the participant's Best Fit Angle is close to 90° (where 0° is north), which is also the angular difference between west and north. We expected this pattern to be common for the external landmarks when participants were not wearing the compass, because participants might become disoriented with respect to north but use their own sense of north to guide pointing to the external landmarks. We were most interested in the internal landmarks tasks. If participants used the compass to place and orient internal landmarks in terms of north, we expected the Best Fit Angle to substantially reduce the error more when participants did not wear the compass, and for the Best Fit Angle to correlate with Participant-north.

External Landmarks. For the external landmarks, one-sample t-tests revealed that rotating the pointing judgments, as expected, improved accuracy with the compass, t(47) = 4.24, p < .001, d = 1.24, and without the compass, t(47) = 4.95, p < .001, d = 1.44 (See Figure 4).

However, the improvement without the compass was greater (Mean Error Reduction = 26.08, SD .

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= 36.52) than with the compass (Mean Error Reduction = 13.88, SD = 22.68), t(47) = 2.36, p = .02, d = 0.36. The resulting Best Fit Error was the same with the compass, (M = 35.51, SD = 15.37) and without the compass (M = 35.51, SD = 17.73), t(47) = 0.001, p = .99, d < 0.01, confirming that despite being disoriented to true north participants remained consistent in their pointing based on an internal reference frame.

To determine whether the external landmark Best Fit Angle correlated with the direction which the participant judged as north, we obtained the circular correlation between the Best Fit Angle with Participant-north. The correlation was significant without the compass, r(48) = .61, p < .001, and marginally significant with the compass, r(48) = .33, p = .066. This suggests that the Best Fit Angle for the external landmarks brought participants' pointing judgments into alignment with where they thought north was.

Internal Landmarks. For the internal landmarks, we combined location and orientation judgments.2 One-sample t-tests revealed that rotating the pointing judgments resulted in improved accuracy with the compass, t(47) = 5.69, p < .001, d = 1.66, and without the compass, t(47) = 6.83, p < .001, d = 1.99 (See Figure 4). Unlike for the external landmarks, the non- Compass route (Mean Error Reduction = 16.50, SD = 25.22) was not significantly different from the Compass route (Mean Error Reduction = 12.50, SD = 15.22), t(47) = 0.35, p = .94, d = 0.14.

The resulting Best-fit error was the same for the Compass route, (M = 33.78, SD = 22.12) and the 2 Internal landmark judgments were combined so that the total number of pointing judgments used in the best fit analysis was more closely matched to the number of external landmarks (eight for external landmarks, six for internal landmarks). The start/end pointing judgments were excluded because these were not equated across time points. Simulations with random data reveal that fewer landmarks are easiest to fit with a best-fit analysis. With random data, the average error of three pointing judgments, after best-fit rotation, is approximately 45°; 6 pointing judgments have approximately 62°; 8 pointing judgments have approximately 66°. We determined that because error could be reduced more effectively with fewer judgments, that the number of judgments between internal and external landmarks should be as close as possible. Nevertheless we find the same results when location and orientation judgments are analyzed separately. This decision introduces the possibility that different best-fit solutions might exist for the two sets internal landmark judgments (orientations versus locations). However, in the current data, the best-fit rotations obtained for the locations and orientations separately were correlated highly (circular correlation r = .45, p = .006).

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non-Compass route (M = 37.42, SD = 20.67), t(47) = 1.06, p = .29, d = 0.15. This analysis confirms our basic finding that the compass did not help people in pointing accurately to the location of internal landmarks.

Again, we wondered whether Best-Fit Angle correlated with where the participant thought north was. Significant correlations would be evidence that participants are using participant north to guide their estimation of the internal landmark locations and orientations.

Using circular correlations, the Best Fit Angle did not correlate with Participant-north without the compass, r(48) = .04, p = .77, or with the compass, r(48) = -.12, p = .41.

Sketch Maps

We discarded the data of two participants who did not provide all the requested data for one of their maps, resulting in N=46. We wanted to know whether knowledge of the configuration of the internal landmarks in their sketches, including the starting point and ending point, improved when participants wore the compass. To measure configuration, we calculated a bidimensional regression for each map. Bidimensional regression (Tobler, 1994) corrects for deviations in scale, translation, and rotation, then provides a measure of similarity (here we use r2, which can be interpreted as the proportion of variance explained) between the actual configuration of points, and a reconstruction of those points. The configuration of internal landmarks was not significantly different with the compass (M = .73, SD = .25) than without the compass (M = .69, SD = .26), t(45) = 0.89, p = .38, d = 0.14. Data from the sketch maps corroborated the finding from the Internal Landmarks Location pointing judgments: the compass did not improve participants' ability to represent the locations of these landmarks.

Verbal Descriptions

.

20

In the verbal descriptions participants provided for each route, we coded for the amount of relative (right, left, ahead) and absolute (north, east, south, or west) spatial language used. The vibrotactile compass did not increase use of absolute spatial language in describing the routes.

Participants used a similar number of absolute spatial terms in the Compass (M = 1.75, SD = 3.31) and Non-Compass (M = 1.98, SD = 3.34) conditions, t(48) = 0.73, p = .47, d = 0.11. Participants also used a similar number of relative spatial terms in the Compass (M = 7.06, SD = 5.54) and Non-Compass (M = 6.27, SD = 4.91) conditions, t(48) = 1.66, p = .10, d = 0.25.

Individual Differences

The information provided by the compass may have been difficult to integrate and use as a navigational aid, except by navigators who were adept at using the information. We correlated SBSOD and SCNEQ scores with the difference in pointing error with the compass minus without the compass on each type of pointing judgment, for each session. We predicted that better navigators would have greater improvement between compass and non-compass pointing, resulting in a negative correlation. However, none of the correlations reached significance (all r's < |.25|). Improvement on the external landmark pointing judgments, specifically, appeared to be common, regardless of individual differences in navigation abilities.

As an exploratory analysis, we coded participants' responses to the post-test question about whether they found the compass useful and the reasons they found it useful. (This analysis was performed by authors: SW and DB). We used a consensus process whereby each coder separately rated the response as Useful, Not Useful, or Mixed. Mixed were a heterogeneous group, consisting of participants who responded that the compass A) neither helped nor hurt them, or B) helped them in some ways but hurt them in others. Disagreements (on 7 participants’ responses) were discussed and resolved. Each coder also rated what specific tasks the participant .

21

mentioned certain the compass being useful for, if any. We coded 6 participants' responses as being Not Useful, 16 as Mixed, and 26 as Useful. Of those who found the compass Mixed or Useful, 14 reported finding the compass useful specifically for Internal Landmarks. We did not observe any significant differences in performance as predicted by their self-assessment of the usefulness of the compass. Interestingly, we also did not observe any differences in self-reported sense of direction within each of these groups. Nor did we find that improvement between the non-compass and compass routes correlated with sense of direction or differed among these three groups.

Discussion

In this study, we assessed the use of a vibrotactile compass on real-world navigation. Our aim was to determine whether providing a global directional cue improved navigational performance generally across spatial tasks, or specifically on tasks for which the global direction was directly relevant. Our data support the specific hypothesis. We found selective improvement for pointing to external landmarks, which were situated in the larger urban area with which participants were already familiar. We did not find improvement on spatial knowledge of internal landmarks, either measured through pointing or with sketch maps. We also did not find better route learning or changes in use of spatial language as a function of wearing the compass.

Finally, we found that people used the compass more effectively to point to external landmarks because those landmarks were coded with respect to global north. These data suggest that vibrotactile compasses may help integrate location in a new space with a familiar, wider reference frame.

These data support the specific hypothesis – providing global directional cues resulted in selective improvement for navigational tasks that could be solved by knowing the location of .

Authors:

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

94143, Usa.

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

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

14Jlvmi Consulting Llc, Dousman, Wi, Usa

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

Abstract

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

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

Introduction

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

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

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

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

●

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

●

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

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

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

Hyperpolarized 13C-Pyruvate Preparation

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

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

General Considerations

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

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

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

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

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

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

Personnel

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

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

Equipment And Facility

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

Material Handling

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

Pharmacy Kit Filling And Assembling

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

Quality Control And Dose Release

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

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

The Final Dose Release And Injection

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

Some Key Challenges

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

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

Current Practices

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

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

In House

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

Summary

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

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

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

Mri System Setup And Calibrations

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

Imaging System

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

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

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

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

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

Rf Coils

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

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

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

Provide B1 Transmit Across The Fov (B1

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

B1

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

+ Profile But Has Been Used Because Of

relatively easy integration into the scanner bore. B1

+ Variation Results In Variations In The Flip

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

Homogeneous B1

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

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

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

(1)

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

Tx = Transmit

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

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

Phantoms

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

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

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

+) And Receive (B1

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

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

Prescan Calibration

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

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

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

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

+ Inhomogeneity As Well

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

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

Power [Kw]

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

8

13C-bicarbonate doped with dimethyl silicone, various

Maximum Values

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

Summary

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

+ Profiles. The

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

For Calibration Of B1

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

Acquisition And Reconstruction

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

+ Inhomogeneity,

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

Acquisition And Reconstruction Methods

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

Mrs/I Methods Specifically

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

Chemical Shift

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

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

Their Application To Different

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

The Majority Of

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

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

Prostate Studies

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

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

Heart Studies

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

Brain Studies

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

Abdomen And Breast Studies

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

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

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

1H Imaging

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

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

Reported Study Parameters

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

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

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

(B)

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

Summary

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

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

Data Analysis And Quantification

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

Metrics

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

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

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

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

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

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

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

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

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

Visualization

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

Metrics

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

Parameter Encoding

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

Anatomical Context

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

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