Houshang A review 19F

transcript

MOLECULAR IMAGING

Cell tracking using 19F magnetic resonance imaging:Technical aspects and challenges towards clinical applications

Houshang Amiri & Mangala Srinivas & Andor Veltien &

Mark J. van Uden & I. Jolanda M. de Vries & Arend Heerschap

Received: 21 October 2013 /Revised: 3 October 2014 /Accepted: 16 October 2014# European Society of Radiology 2014

Abstract 19F MRI is emerging as a new imaging techniquefor cell tracking. It is particularly attractive because of itspotential for direct and precise cell quantification. The mostimportant challenge towards in vivo applications is the sensi-tivity of the technique, i.e. the detection limit in a reasonableimaging time. Optimal sensitivity can be achieved with ded-icated 19F compounds together with specifically adapted hard-ware and acquisition methods. In this paper we introduce the19F MRI technique focusing on these key sensitivity issuesand review the state-of-the-art of 19F MRI and developmentstowards its clinical use. We calculate 19F detection limitsreported in preclinical cell and clinical 19F drug studies interms of tissue concentration in a 1 cm3 voxel, as an alternateway to compare detection limits. We estimate that a tissueconcentration of a few millimoles per litre (mM) of 19F isrequired for a human study at a resolution of 1 cm3.Key Points•Direct and precise cell quantification can be done by 19FMRI.• 19F MRI sensitivity is the most important parameter towardsclinical application.

• A number of (technical) considerations can improve sensi-tivity significantly.

• A few millimoles per litre (mM) of 19F per voxel is requiredfor adequate detection.

Keywords 19Fmagnetic resonance imaging . Sensitivity .

Cell tracking . 19FMRI . Detection limit

AbbreviationsCA Contrast agent2D Two-dimensionalDy DysprosiumFDA Food and drug administrationFOV Field of viewF-uTSI Fluorine ultrafast turbo spectroscopic imagingGRE Gradient echoMRI Magnetic resonance imagingMRSI Magnetic resonance spectroscopic imagingPFC PerfluorocarbonPFOB Perfluorooctyl bromideRF RadiofrequencyROI Region of interestSAR Specific absorption rateSE Spin echoSNR/t Signal to noise ratio per unit scan timeSSFP Steady-state free precessionTE Echo timeTR Repetition timeUTE Ultrashort echo timeZTE Zero echo time

Introduction

Clinical imaging modalities such as magnetic resonance im-aging (MRI), positron emission tomography (PET) and singlephoton emission computed tomography (SPECT) allowin vivo monitoring of cells, e.g. in cellular therapy. Amongthose, MRI has been reported to have the highest resolutionand, in some cases, sensitivity for cell tracking [1]. Moreover,unlike PET and SPECT, MRI requires no radioactive materialand can acquire both anatomical and functional information.

H. Amiri (*) :A. Veltien :M. J. van Uden :A. HeerschapDepartment of Radiology, Radboud University Medical Center,Geert Grootplein 10, 6500 HB Nijmegen, The Netherlandse-mail: amiri.houshang@gmail.com

H. Amiri :M. Srinivas : I. J. M. de VriesDepartment of Tumor Immunology, Radboud Institute for MolecularLife Sciences, Radboud University Medical Center, Nijmegen, TheNetherlands

Eur RadiolDOI 10.1007/s00330-014-3474-5

MRI normally measures signals from protons (1H) in tissuewater. This provides high resolution images of soft tissueswith the contrast dependent on tissue-specific 1H spin relaxa-tion times and the variety of available imaging protocols.However, despite this high resolution and soft tissue contrast,it is generally not possible to distinguish individual cells. Thuscells of particular interest, such as those used in cellulartherapy, must be labelled to distinguish them from the back-ground. Labelling can be carried out in a number of ways suchas using conventional contrast agents (CAs) that alter 1Hrelaxation times, reporter genes that generate contrast bytrapping endogenous CAs, or by employing a nucleus otherthan 1H, e.g. fluorine-19 (19F).

CAs for MRI are typically either paramagnetic, usuallygadolinium-based [2, 3], or superparamagnetic [4, 5], usuallyiron oxide-based. They affect the 1H spin relaxation timeslocally and thus provide a regional change in contrast. Forcellular imaging, superparamagnetic CAs have been widelystudied and discussed [6, 7] with a variety of applications suchas lymph node imaging [8–10], cancer imaging [11–13] andcell tracking [14, 15]. The main advantage of thesuperparamagnetic CAs is their relatively high sensitivity;unfortunately, they do not possess high specificity. For in-stance, in particular disease models where disease progressionmay alter intrinsic contrast (e.g. by haemorrhage or vascularchanges), distinguishing between labelled cells and back-ground signal may become a major problem [16]. A solutionto this is to use nuclei other than hydrogen with minimalbackground in tissues. For this purpose, 19F has attractedmuch attention [17]. Another important issue is that clinicalsuperparamagnetic CAs for cell tracking have been taken offthe market [18] and this brings even more interest to 19F MRIas a high-potential technique.

The 19F nucleus has spin 1/2, 100 % natural abun-dance, sensitivity close to that of 1H, and owing to thelack of measurable endogenous fluorine, there is nobackground signal in tissue images. Moreover, contraryto the CAs in MRI that are measured indirectly via theirinteractions with water molecules, 19F is measured di-rectly and thus results in higher specificity. In order toperform 19F MRI experiments using a conventional MRIsystem, some modifications to both hardware and soft-ware are required. Once these modifications are accom-plished, in vivo experiments can be carried out, typically byacquiring an anatomical 1H MR image to be overlaid by a 19Fimage.

19F MRI is becoming of particular importance in the fieldof cellular imaging as the number of labelled cells in a regionof interest (ROI) is directly proportional to the 19F signalwhich enables cell quantification [19]. This quantitative tech-nique can be used to assess and optimize new approaches incellular therapy/diagnosis. However, typical fluorinated com-pounds, such as those used in MR spectroscopy [20], consist

of a relatively low number of fluorine atoms per molecule withlong 19F longitudinal relaxation times (T1). This may lead to arelatively low signal to noise ratio per unit scan time (SNR/t),giving rise to low sensitivity and long scan times, whichwould be a problem in the imaging of humans. Thus, high19F content, relatively short T1 and high magnetic fields arekey ingredients for successful in vivo 19F MRI. These require-ments lead to the use of perfluorocarbons (PFCs) for most celllabelling applications.

The overall objective of this work is to comprehensivelydiscuss the technical aspects of 19F MRI focusing on celltracking as a growing field with high potential for clinicalapplications. Towards this aim, we first introduce 19F MRIagents for cell labelling and then the 19F MRI technique ispresented, with particular attention to its sensitivity and otherchallenges in clinical translation.

19F agents for cell labelling

In cell tracking, typically cells are labelled either ex vivo,before being transferred into the subject, or in situ. The dif-ferent cell labels developed for 19F MRI have been reviewedelsewhere [1, 17, 19, 21]. In general, the 19F labels developedthus far consist of PFCs resulting in high 19F density permolecule. PFCs have been proposed as blood substitutes forseveral decades [22]; and new agents are still in clinical trials(e.g. Oxycyte by Oxygen Biotherapeutics Inc., MorrisvilleNC USA, is currently in phase II trials in several countries;http://clinicaltrial.gov/ct2/show/NCT00174980?term=perfluorocarbon&rank=5). Furthermore, some PFCs are alsoapproved for medical applications such as contrast-enhancedultrasound (e.g. SonoVue by Bracco Imaging SpA, Milan,Italy). Thus, overall PFCs are known to be well-toleratedin vivo. For MRI, PFCs with a single 19F resonance (or singledominant resonance) are preferable to avoid imaging artefacts.Preclinical and clinical research involving 19F MRI with PFCagents is ongoing.

Some considerations that arise with 19F cell labels are thestability of the agent, its localisation, sensitivity to oxygentension and number of resonance peaks within the receiverbandwidth. These factors are particularly important in quanti-tative imaging [19]. Specifically, it is assumed that all 19Fsignals measured are from the labelled cells (i.e. that no labelis lost to other cells or the extracellular space) and that the T1

of the compound is unchanged relative to the reference. Boththese assumptions may need to be validated experimentally.For instance, specific cellular localisation of the label is gen-erally validated using a secondary label, typically a fluores-cent dye [23], with ex vivo histology. This can also be used tostudy the fate of the label if the cells divide and the number ofcell divisions that may have occurred, when dividing cells areused. In practice, with cells such as dendritic cells and

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macrophages (the most commonly used cell types in celltracking studies), cell division is not an issue as these cellsdo not divide. Even in these cases, label can be lost to theextracellular space over time, especially with cell death. Thishas been shown to occur with fluorinated polylysine-labelledcells which lost nearly half the label within 7 days [24]. Thebest way to control for non-specific labelling is histologycoupled with a fluorescent dye.

Various types of cellular labels and cell labelling strategieshave been developed to optimize 19FMRI [21], althoughmostcell tracking work has focused on PFC-based emulsions [25].Commonly, these emulsions are added directly to the cellculture medium and taken up by the cells ex vivo. An alter-native approach is in vivo labelling of phagotypic cells, most-lymacrophages, through intravenous injection of the label(e.g. see [26–28]). In all cases, cell labelling is optimized tomaximize the 19F content per cell. The 19F content per cell andthe total number of cells transferred to the subject are often themain parameters that can be controlled in a particular in vivoexperimental model which directly affect the SNR/t.

19F MRI hardware

The hardware of a conventional MRI system requires somededicated parts for 19F imaging (see Fig. 1). Here we discussbriefly the key hardware components and their role in opti-mizing cell tracking effectively using 19F MRI.

Magnet

At higher magnetic fields, higher spatial and/or temporalresolutions are accessible because of the increase in SNR,i.e. 19F images with smaller numbers of 19F labelled cellscan be obtained in a shorter scan time. When using CAs,increasing the magnetic field may not always be beneficialas their efficiency is field-dependent. In contrast, 19F MRI, inwhich spin density is relatively low, always benefits fromhigher field strengths; however, some issues such as B1 andB0 homogeneity as well as prolonged relaxation times shouldbe considered.

A critical issue, particularly at higher fields, is the powerdeposition in the patient caused by radiofrequency (RF)pulses; this is proportional to B0

2 and defined by the specificabsorption rate (SAR). Power deposition in the tissue dependson a number of factors such as tissue characteristics, type ofsequence and its parameters. For instance, fast spin echo (SE)-based sequences usually are known as high SAR acquisitiontechniques since they include several refocusing (180°)pulses. More details on advantages and challenges of highmagnetic field, with a specific emphasis on safety issues, havebeen reviewed elsewhere [30].

Radiofrequency coils

For 19F measurements, a dedicated RF coil setup has to beinstalled, covering the relevant resonance frequency of 19F.The RF coils, to excite the spin systems, generate a B1 fieldthat ideally should be as homogeneous as possible for precisesignal quantification. In practice, this is not always the caseand corrections are needed [31–33].

In 19F MRI, low SNR is a serious concern as it may lead tolow sensitivity and long scan times which makes the transla-tion to the clinic challenging. Thus, selecting an appropriateRF coil is critical. For instance, surface coils are more efficientfor imaging ROIs close to the body surface, e.g. lymph nodes.The RF coils can be designed to operate as receive-only,transmit-only and transmit-receive (transceiver). In clinicalpractice, commonly, a volume coil (whole-body coil) is pres-ent in the magnet to provide the homogeneous transmit fieldand local coils are used as receive-only coils for high SNR,although this may not be the most appropriate for 19F imaging.

Fig. 1 a Schematic of a conventionalMR systemwith required hardwareto perform both 1H (black and white) and 19F (red) imaging. A 1H/19F coilis employed to record the response of the excited nuclear spins. Power RFamplifiers are needed to generate sufficient power for the excitationpulses at each nuclear frequency. The interface contains a 1H/19F filter,transmit-receive switches and a pre-amplifier. The final image is obtainedby overlaying 19Fmaps on the structural 1H image. bMR image ofmouselegs immediately after injection of perfluoro-[15]-crown-5 ether particlesin the footpad. The images shown are 2-mm coronal slices with 1H ingrayscale, 19F in false colour and an overlay (adapted with permissionfrom ref. [29])

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In cell tracking by 19F MRI, 19F and 1H anatomical imagesare acquired and overlaid to correlate the quantified cell num-bers with pathology of interest such as inflamed tissue. To dothis, selection of a tunable RF coil with a high bandwidth, toaccommodate both 1H and 19F resonance frequencies, couldbe an option but this may increase the receiver noise figure, aparameter which measures the reduction in SNR due to thelosses in the loop coil only. In addition, with tuning andmatching from 1H to 19F the sensitivity profiles of the RF coilat the two corresponding frequencies vary [34]. Image regis-tration also needs to be done accurately, especially if different1H and 19F coils are used.

Novel strategies of designing multinuclear coils for in vivoexperiments have been reported that allow changes in theresonance frequency using dedicated frequency switcheswhich potentially can be applied to 19F MRI as well [35].Another strategy is to use a double-tuned RF coil in whichboth 1H and 19F resonance frequencies are tuned and matchedonce in the beginning of the experiment [36]. In this way,registration of 1H and 19F images cannot change as a result ofthe coil and subject displacement; however, one may need toadjust the 90° excitation pulse for each nucleus channel sep-arately. Another reported approach is to use a dual frequency1H/19F coil able to collect both images simultaneously [34,37]. It is worth mentioning that parallel imaging using phasedarray coils [38] can also be beneficial in 19F imaging. Phasedarray coils increase the SNR and speed up the acquisitiontime. Moreover, since images of all elements are acquiredsimultaneously, larger regions of interest can be covered inthe same scan time.

MostMR systemmanufacturers provide spectrometers withmulti-nuclei options, including 19F; however, the standard coilpackage usually does not include 19F coils and these need to beacquired separately. Animal coils are mostly custom-built;although commercial ones are available upon request.

Gradient coils

Gradient coils are another important part of MRI hardware. Inquantitative cellular MRI, gradient instability may contributeto inaccurate quantification and experiment reproducibilityover time. A slice of interest is selected by employing sliceselective gradients. Selecting thicker slices improves the SNRin 19F imaging. Employing fast imaging sequences also im-proves the SNR/t significantly. In these cases, the slew rate,i.e. how quickly the gradient coils can be switched on or off, isa key parameter.

19F MRI acquisition methods

Spatial mapping of 19F spins has been done by magneticresonance spectroscopic imaging (MRSI), in which whole

19F spectra are acquired, and by MRI, in which the spectro-scopic dimension is discarded. In most cell tracking studies,this dimension is not relevant and thus MRI is more practical.

This section describes the influence of imaging parametersand sequences on the SNR in 19F MRI. In general, the SNRper voxel in two-dimensional (2D) is given by Eq. 1 [39]:

SNR ¼ CFOVð ÞxNx

⋅FOVð ÞyNy

⋅ Δz

� � ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiNx ⋅ NY ⋅ NA

rð1Þ

where (FOV)x and (FOV)y are the field of view in the x and ydirections. Nx and Ny are the number of frequency and phaseencoding steps, respectively. Δz is the slice thickness, NA isthe number of signal averages, and rBW is the readout(receiver) bandwidth. The constant C includes tissue intrinsicparameters (e.g. relaxation times), hardware characteristics(e.g. coil configuration), pulse sequences, imaging parametersetc.

Image acquisition parameters

According to Eq. 1, different imaging parameters such asrBW, FOV, matrix size, slice thickness, repetition time (TR)and echo time (TE) can affect SNR considerably. Some ofthese parameters are coupled and therefore it is always acompromise between high resolution, high SNR and shorterscan time. Here we will focus on the parameters that play amajor role in the optimization of SNR per unit time, as this isthe most challenging issue in 19F MRI for cell tracking(see Fig. 2).

For full recovery of magnetization between excitationpulses with maximum SNR, the pulse repetition TR shouldbe more than 5 times T1. However, for

19F compounds,specifically PFCs, usually the T1 is in the order of secondsand this makes the TR (and the scan time) long, leading to alow SNR/t. For instance, a T1 of 1,656±40 ms for biocom-patible perfluorooctyl bromide (PFOB), at 7T, has been re-ported [40]. Therefore, it is desirable to shorten T1, if possible.A reported strategy is to link a fluorinated compound to alanthanide (III) ion as a paramagnetic centre [41, 42]. Forexample, chemically linking fluorine to dysprosium (Dy)resulted in a T1 of about 7 ms at 7T [42].

In gradient echo (GRE) sequences, shorter TR andfaster imaging with optimum SNR/t is possible if theexcitation flip angle is set to the Ernst angle defined asθE=cos

−1[Exp(−TR/T1)]. If one employs a 19F compoundthat shortens T1 by a factor of 2 then, using the same exper-imental setup, TR can be decreased by the same factor leadingto a faster scan and therefore increase in the SNR/t.

The TE is the time between 90° excitation pulse and thesignal echo. As mentioned previously, a short T1 value for

19Fis desired but this is, usually, accompanied by shortening of

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the T2 relaxation time with signal loss. To compensate for this,TE is kept as short as possible in order to have a higher SNR.

The rBW is the frequency range in which the signal iscollected. By increasing the rBW in MR imaging, shorterTE is possible but it also decreases the SNR. Therefore, anincrease in rBW is not recommended unless a compound withmore than one resonance peak is employed in which caseincreasing the rBW will decrease the chemical shift artefact.

The FOV, voxel volume and matrix size are strongly inter-connected. Matrix size determines the pixel size in a givenFOV. The smaller the pixel size the higher the image resolu-tion. The slice thickness determines the third dimension of apixel to make a voxel. It is obvious that bigger voxels containmore 19F-labeled cells and thus provide more signal intensityand SNR, at the cost of lower resolution. In 19F MRI, highresolution images are not typically required; therefore, anincrease in voxel size by selecting a thick slice and/or largepixel size is often an easy way to improve the SNR. Forinstance, with dendritic cell tracking, keeping the slice thick-ness constant, reducing in-plane resolution by a factor of 4 andincreasing number of averages by a factor of 64, compared tothat of the 1H MRI acquisition, a reasonably good fluorineSNR in vivo has been achieved [43].

Imaging sequences

As with 1H MRI, GRE- or SE-based imaging techniques arefrequently used for cell tracking with 19F MRI. GRE

sequences result in an echo generation with a single excitationpulse and gradient reversal while in the SE imaging the 90°excitation pulse is followed by 180° refocusing pulses.Therefore a GRE sequence, with a low flip angle and conse-quently a shorter TR, provides faster imaging as well as lessRF power deposition. However, it is more sensitive to B0

heterogeneity and susceptibility effects with a faster T2* de-

cay. SE imaging, on the other hand, has a higher SNR (at thecost of longer scan time) but results in less susceptibilityartefacts. It is worth mentioning that relatively fast versionsof both GRE and SE sequences are widely available and usedin the clinical setting, known as FLASH (fast low-angle shot)and turbo SE, respectively.

The selection of an imaging sequence and its optimizationto get the highest SNR/t depend on the characteristics of the19F agent used. The highest SNR/t, for a given compound, isachieved when the T2

*/T1 ratio is close to 1. The generation ofcompounds with short relaxation times should go togetherwith imaging sequence optimization. For instance, by addingparamagnetic lanthanide tags to fluorine compounds relaxa-tion times in the range of 1–5 ms and a high T2

*/T1 ratio couldbe achieved [44]. However, to fully exploit this condition andrecover the full signal before it has decayed, the use of a shortTE is required. Using a zero echo time (ZTE) sequenceresulted in a 27-fold increase in the SNR, whereas this gainwas 11-fold when using a GRE sequence (see Fig. 3).

Some fluorinated compounds have multiple resonancepeaks and a broad range of frequencies, leading to chemical

Fig. 2 a Parameters with the most significant effect on the SNR (arrowsshow changes in a parameter which lead to an increase in the SNR;upward arrow increase, downward arrow decrease). b Coefficient factorapplied to the SNR by doubling some parameters assuming all experi-mental conditions unchanged. c Schematic of the effect of fluorineconcentration and slice thickness on detectability. If one selects a voxelas shown in dashed-dotted line, fluorine signal can be detected, whereas

an acquisition with a smaller voxel, e.g. dotted lines, may fail to detectany fluorine because of the lack of enough fluorine spins in that voxel. Itis noted that if fluorine atoms are only those existing in the dotted region,then none of the above voxel selections can detect a fluorine signal. As aresult of these considerations sensitivity measures are meaningful ifreported for a given voxel size. Therefore, the “bench mark” detectionlimit is proposed in Table 1

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shift artefacts which can make localisation and quantificationambiguous [45]. For these compounds different scenarios canbe applied. For instance, PFOB is such a compound that afluorine ultrafast turbo spectroscopic imaging (F-uTSI) tech-nique has been proposed to overcome its chemical shift arte-fact [45]. Using a short sampling time and taking advantage ofrelatively long T2 of PFOB (ca. 100 ms) allowed a highnumber of echoes per excitation to be acquired. In this way,SNR was increased up to 75 % with a 40 % shorter scan time,when using back projection [46]. In a 31P and 13C MRI study,images were obtained of multiple metabolites simulta-neously by selectively exciting different metabolite spins andexploiting their chemical shift dispersion [47]. By selectingproper bandwidth, imaging matrix size and FOV it was pos-sible to obtain different images associated with different res-onance peaks and even discard undesirable images corre-sponding to undesirable metabolites. This strategy would alsobe valuable in 19F cell tracking, for instance, to simultaneouslyimage different cell types labelled with different 19Fcompounds [48].

In addition to the imaging sequence, specific data acquisi-tion and reconstruction techniques can be used to increaseSNR/t or reduce the measurement time. 3D compressed sens-ing, for example, has been shown to decrease the acquisitiontime by a factor of 8 when imaging cells using 19F MRI [49].Another study has reported a two-fold increase in the detec-tion sensitivity while using a balanced steady-state free pre-cession (SSFP) sequence with an ultrashort echo time (UTE)and 3D radial readout [50].

Quantification and sensitivity in 19F MRI

In 19F MRI for cell tracking, cell quantification requires priorknowledge of the number of 19F atoms per cell. This can bedetermined by in vitro NMR experiments, on a known numberof labelled cells, before the cell implantation in vivo [51]. Thisis done by comparison of the peak integral of the 19F signalfrom the label with that from a reference with a known

concentration, within the same experiment. The same strategycan be applied for in vivo MRI experiments, i.e. a referencewith a known concentration of 19F is placed adjacent to thesubject to image both simultaneously. The number of 19Fatoms in the reference is proportional to its signal [52].Comparison of the reference signal and the signal obtainedfrom the subject allows calculation of the number of 19F atomsin the ROI. From this, the number of cells per voxel can thenbe calculated. It has to be taken into account that the actualvalue may be different owing to biological and/or physico-chemical phenomena such as cell division, cell death, loss oflabel, as well as undesirable changes in compound T1 and T2

after being administered into the tissue [19]. These issues canonly be addressed through additional ex vivo experiments,such as histology to study label localisation or flow cytometryto study cell division; nevertheless both techniques requireadditional agents such as a fluorescent dye to be included withthe 19F label. However, since most studies using 19F MRI forcell tracking thus far deal with non-dividing cells, label lossdue to cell division is not a major issue. However, nonspecificlabelling of resident macrophages can still influence the signaldetected.

Sensitivity in cell tracking is defined as the minimumnumber of detectable cells in a given scan time. As mostsensitivity studies with 19F-labelled cells to date have beenperformed in vitro or with animals, it is difficult to extrapolatethe sensitivity values of these studies to the conditions forhuman experiments. Moreover, it is also difficult to comparethe results of different preclinical studies as this value dependson a large number of parameters, in particular the number ofdetectable 19F atoms in the label used, cell loading, coilparameters (type and size), voxel size, field strength, acquisi-tion and reconstruction method, and scan time.

An alternate way to evaluate sensitivity and detection limitsis by comparing tissue concentrations of 19F atoms. In Table 1,values for the relevant parameters are listed for some in vitroand in vivo studies together with 19F tissue concentrations.Variations in the detection limit indeed reflect differences invalues for the above parameters, but also in the T2

*/T1 ratio of

Fig. 3 Diagram illustrating how T2*/T1 can be employed efficiently. Red

area is the acquisition window. a A fluorinated compound with longrelaxation times, acquired by a conventional sequence. Signal amplitudeafter excitation is low, as a result of the long T1. b A fluorinatedcompound with high T2

*/T1, acquired by a conventional sequence. Initial

signal is high, but it decays rapidly and is inefficiently sampled by aconventional sequence. c The same as b but acquired by a ZTE sequence.As TE is nearly zero, the initially high signal can be acquired. Reprinted,with permission, from ref. [44]

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the 19F spins. As voxel volume clearly is an important deter-minant of the detection limit, we also calculated and comparedthis limit as 19F tissue concentrations in a 1 cm3 voxel size.This bench mark resolution is selected as it represents a typicallower limit value in human 1H MRS at 3 T (1H has a similarintrinsic sensitivity as 19F), which can be measured in aclinically acceptable time and is estimated to be at sufficientresolution for the detection of 19F-labelled cells in humans.This resolution can be used to extrapolate values from pre-clinical studies to values required for human studies.

Previously, our group reported for an in vitro experiment adetection limit (at an SNR of 3) of 2,000 cells with about2×1013 19F atoms per cell at 7T [43]. This, at the resolutionof the experiment (2 mm3), translates to a tissue concentrationlimit of 33 mM corresponding to 0.066 mM in 1 cm3. If weextrapolate this to 3T, the detection limit would be 9,000 cells,which translates to a tissue concentration limit of about0.3 mM 19F at a spatial resolution of 1 cm3 (see Table 1). Sowe would roughly estimate that a few millimoles per litre 19Fwould be required for a successful human imaging study,dependant on the coil size and type. Interestingly, using ahead coil and a clinical 3T system, a detection limit of 4 mMhas been anticipated [57].

A realistic assessment of the feasibility of clinical applica-tions can be obtained from quantitative 19F magnetic reso-nance spectroscopy (MRS) studies in humans [58, 58], whichindeed have shown that a few millimoles per litre 19F isrequired for a human study at 3T, at a spatial resolution of1 cm3 (Table 1).

Challenges towards clinical applications

19FMRI has been widely used for cellular imaging in differentpathologies such as stem cell-based therapy [20, 23], immu-notherapeutic cell tracking [43, 51], cell migration in diabetesmodel [58], inflammation [26, 57, 58–62] as well as graftrejection assessment [25, 63]. Translation of preclinical celltracking procedures, using 19FMRI, into humans is possible ifsufficient sensitivity is achieved. However, most of the 19FMRI reports in the field of cellular imaging have been per-formed on animals, using custom-built RF coils, and mainly athigh and ultra-high magnetic fields. Indeed this improvessensitivity, but the SAR and acquisition times that are notlimitations for preclinical studies become important issues inhuman imaging. Therefore, careful consideration of systemhardware and imaging sequences and parameters is required.SAR limits should be carefully considered for the translationof fast imaging sequences and RF coils into clinics.

As discussed previously, the 19F agent used directly affectsimaging sensitivity. The generation of new agents certainlyalso requires the assessment of compound safety, toxicity andclearance before being applied to human imaging. In this

regard, PFCs have the advantage of prior clinical use,e.g. as oxygen carriers. A study in rats investigated thebiodistribution of a mixed fluorocarbon–hydrocarbon over24 h [64]. The authors found 70 % of the injected dose inthe liver, 17 % in the spleen, 4 % in the lungs, 2 % in thekidneys and 2 % in the blood, with no evidence of metabo-lism. The half-time retention of that particular compound(silicone-coated polychlorotrifluoroethylene capsules filledwith perfluoro-[65]-crown-5 ether) in the liver was estimatedto be 25±5 days. Recently, a superfluorinated molecular probehas been synthesized for cell tracking [66]. This probe pro-vides an intense single resonance peak owing to its 36 equiv-alent 19F atoms and possesses cellular compatibility.

Summary

19F MRI is emerging as a new imaging technique with partic-ular potential for cell tracking. Themost important issue of 19FMRI in this field is sensitivity, i.e. the detection limit in areasonable measurement time. Faster scans clearly lead to areduced cost but 19F MRI can also decrease the therapeuticcosts. For example, a major issue with the optimization ofcellular therapeutics is the dearth of knowledge about the fateof the cells in vivo [63]. For instance, dendritic cell therapy, asa novel cancer therapeutic approach, is very costly and cur-rently has a low success rate. This failure is partly due to themisinjection of dendritic cells, even when ultrasound-guided[65]. 19F MRI can potentially improve such a treatment bytracking the injected cells and therefore optimizing the injec-tion route. Importantly, subjects who do not show appropriatecell homing responses can be excluded from such studies atearly time points, thereby greatly reducing costs.

To optimize the sensitivity, the generation of dedicated 19Fcompounds together with improved hardware and acquisitionmethods is desirable. Compounds with many fluorine atoms, asingle resonance frequency and a T2

*/T1 close to 1 will beideal for a good SNR. Furthermore, 19F content per cell (cellloading) also directly affects detection sensitivity. Hardwareplays a key role in achieving higher sensitivity, in particularselecting an appropriate RF coil (right type and size) is veryimportant. An optimized imaging sequence is also essential.For example, using a ZTE sequence has been recommendedfor compounds with T2

*/T1 close to 1 [44], whereas a se-quence to selectively excite the peaks of interest has beenproposed for compounds with multi-resonance peaks [47].All optimizations must be accompanied by safety assessmentsbefore being applied in humans.

From clinical 19F drug MR reports it follows that a 19Ftissue concentration of a few millimoles per litre at a resolu-tion of about 1 cm3 is required for human 19F MRI, in areasonable measurement time. An extrapolation of the resultsof preclinical cell tracking studies and human examinations at

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3T show the great potential of 19F MRI for cell tracking inhumans. Indeed, a recent abstract reported the first successfuldendritic cell tracking in patients with colorectal cancer [67].To facilitate the use of published cell detection limits, as wellas having consistent parameters to assess clinical translation,we recommend reporting 19F tissue concentrations in celltracking studies.

Acknowledgements The scientific guarantor of this publication isProf. Arend Heerschap. The authors of this manuscript declare norelationships with any companies whose products or services may berelated to the subject matter of the article. This work was financiallysupported by the EuropeanUnion EU-FP7 ENCITE (HEALTH-F5-2008-201842) grant and Netherlands Institute for Regenerative Medicine(NIRM) FES0908. MS is supported by the Netherlands Organizationfor Scientific Research (NWO) VENI 700.10.409 and the EuropeanResearch Council (ERC) ERC-2014-StG-336454-CoNQUeST andJdV by NWO-VIDI 917.76.363. Methodology: performed at oneinstitution.

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Houshang A review 19F

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