Splenic T1-mapping: a novel quantitative method for assessing adenosine stress adequacy for cardiovascular magnetic resonance

Background Perfusion cardiovascular magnetic resonance (CMR) performed with inadequate adenosine stress leads to false-negative results and suboptimal clinical management. The recently proposed marker of adequate stress, the “splenic switch-off” sign, detects splenic blood flow attenuation during stress perfusion (spleen appears dark), but can only be assessed after gadolinium first-pass, when it is too late to optimize the stress response. Reduction in splenic blood volume during adenosine stress is expected to shorten native splenic T1, which may predict splenic switch-off without the need for gadolinium. Methods Two-hundred and twelve subjects underwent adenosine stress CMR: 1.5 T (n = 104; 75 patients, 29 healthy controls); 3 T (n = 108; 86 patients, 22 healthy controls). Native T1spleen was assessed using heart-rate-independent ShMOLLI prototype sequence at rest and during adenosine stress (140 μg/kg/min, 4 min, IV) in 3 short-axis slices (basal, mid-ventricular, apical). This was compared with changes in peak splenic perfusion signal intensity (ΔSIspleen) and the “splenic switch-off” sign on conventional stress/rest gadolinium perfusion imaging. T1spleen values were obtained blinded to perfusion ΔSIspleen, both were derived using regions of interest carefully placed to avoid artefacts and partial-volume effects. Results Normal resting splenic T1 values were 1102 ± 66 ms (1.5 T) and 1352 ± 114 ms (3 T), slightly higher than in patients (1083 ± 59 ms, p = 0.04; 1295 ± 105 ms, p = 0.01, respectively). T1spleen decreased significantly during adenosine stress (mean ΔT1spleen ~ −40 ms), independent of field strength, age, gender, and cardiovascular diseases. While ΔT1spleen correlated strongly with ΔSIspleen (rho = 0.70, p < 0.0001); neither indices showed significant correlations with conventional hemodynamic markers (rate pressure product) during stress. By ROC analysis, a ΔT1spleen threshold of ≥ −30 ms during stress predicted the “splenic switch-off” sign (AUC 0.90, p < 0.0001) with sensitivity (90%), specificity (88%), accuracy (90%), PPV (98%), NPV (42%). Conclusions Adenosine stress and rest splenic T1-mapping is a novel method for assessing stress responses, independent of conventional hemodynamic parameters. It enables prediction of the visual “splenic switch-off” sign without the need for gadolinium, and correlates well to changes in splenic signal intensity during stress/rest perfusion imaging. ΔT1spleen holds promise to facilitate optimization of stress responses before gadolinium first-pass perfusion CMR. Electronic supplementary material The online version of this article (doi:10.1186/s12968-016-0318-2) contains supplementary material, which is available to authorized users.


Background
Adenosine stress perfusion cardiovascular magnetic resonance (CMR) accurately detects myocardial ischemia and guides clinical decision-making [1,2]. However, perfusion CMR has a reported false-negative rate of between 5 and 16% [2][3][4], which may lead to suboptimal management strategies. In the absence of poor image quality, inadequate adenosine stress response is the commonest cause of false-negative perfusion scans [4], because conventional hemodynamic markers of stress response, such as heart rate and systolic blood pressure, are unreliable predictors of myocardial vasodilatation and the achievement of maximal hyperemia [5].
Recently, the "splenic switch-off" sign was proposed as a CMR marker of adequate adenosine stress. It describes visually reduced splenic perfusion during stress imaging (spleen appears dark) compared to rest imaging (spleen appears bright) [6], and in retrospective analyses, failed splenic switch-off was more commonly observed in false-negative perfusion scans than true-negatives [6]. The physiological basis for this phenomenon is that splenic blood volume reduces significantly during exercise, due to splanchnic blood redistribution [7,8], and can manifest as splenic "disappearance" on nuclear imaging [9]. The degree of splenic blood volume reduction is proportional to exercise workload [7], independent of cardiac output [7], and is related to adenosine-mediated splenic vasoconstriction [10,11]. More recently, splenic switch-off has been shown to relate to higher myocardial T2 values during dipyridamole stress, further suggesting a connection between splenic and myocardial vascular biology [12].
A key limitation of splenic switch-off is that it can only be assessed after gadolinium first-pass perfusion imaging [6], at which point it is too late to optimize stress adequacy [13]. Repetition of inadequately stressed images would require a wait-period (10-15 min) for gadolinium "wash-out" from the LV cavity to optimize myocardialblood contrast during the subsequent (no longer firstpass) stress perfusion imaging, leading to longer scan durations, and exposes patients to additional adenosine and contrast agents [6]. Therefore, a method which can determine stress adequacy and offer opportunities for pre-emptive stress response optimization before gadolinium first-pass perfusion imaging is highly desirable.
Native T1-mapping enables quantitative characterization of tissue blood volumes without the need for gadoliniumbased contrast agents (GBCA) [14][15][16], and offers the potential to assess stress responses before GBCA first-pass perfusion. T1 (proton spin-lattice) relaxation time is a magnetic property of tissues measured in milliseconds [14], and each tissue type, including the spleen, has its own normal range of T1 values [14]. T1 is sensitive to changes in tissue water content or blood volume [15][16][17][18][19], and we recently showed that normal myocardial T1 increases by 6% during adenosine vasodilatory stress, due to expansions in myocardial blood volume [15,16]. Furthermore, stress-T1 appears sensitive to changes in normal, ischemic and infarcted myocardium, without the need for GBCA [15]. Contrary to its vasodilatory effects in the myocardium, adenosine causes splenic vasoconstriction, reducing the splenic blood volume, and thus expected to lower the splenic T1 (T1 spleen ). Conveniently, the spleen is typically visible on stress perfusion CMR and can be inspected without additional planning.
This study sought to evaluate stress and rest T1 spleen as a gadolinium-free CMR marker of adenosine stress responses by comparing with the existing "splenic switch-off" sign and hemodynamic markers. We hypothesized that: (i) T1 spleen will decrease significantly from resting values during adenosine stress, due to splenic blood volume reductions and; (ii) stress-related changes in T1 spleen (ΔT1 spleen ) correlate to changes in splenic perfusion on CMR (the "splenic switch-off" sign), but without the need for GBCA.

Methods
All study procedures received favourable opinions from local ethics committees, and all subjects gave written informed consent.

Study population
To establish the relationship between T1 spleen and splenic perfusion/switch-off, retrospective analysis was performed on CMR scans of 212 subjects; 104 subjects had CMR at 1.5 T (Magnetom Avanto, Siemens Healthcare, Erlangen, Germany) and 108 subjects had CMR at 3 T (Magnetom Trio a Tim system, Siemens Healthcare, Erlangen, Germany

CMR protocol
All subjects avoided adenosine antagonizers (e.g. caffeine) for ≥24 h before CMR. T1-mapping was performed using the Shortened Modified Look-Locker Inversion recovery (ShMOLLI) prototype sequence (WIP 561 and 448C) with inline map generation, which uses 9-heartbeats breathholds per T1-map acquisition and enables on-screen image reconstruction within 10 s [14].

T1-mapping analysis
Separate data files containing all T1-maps were created and anonymized before analysis by an observer (>3 years of T1-mapping analysis experience) blinded to perfusion images and clinical information. T1-maps were excluded from analysis if the spleen was not clearly visible (2%), had respiratory-motion artefacts on raw Inversion-Recovery-weighted images (3%) or had suboptimal goodness-of-fit R 2 -maps (2%) [17,20]. Overall, 738 T1-maps were included in final analysis, using dedicated in-house software MC-ROI (programmed by S.K.P. in IDL, version 6.1, Exelis Visual Information Solutions, Boulder, Colorado) [14][15][16][17][18]20]. To estimate mean native T1 spleen , regions of interest (ROIs) were manually placed on T1-maps to include as much splenic tissue as possible, avoiding partial volume effects from large splenic blood vessels and borders with neighbouring tissues (Fig. 1). ROIs were quality checked against corresponding Inversion-Recovery-weighted images and R 2 -maps. To derive thresholds suitable for direct application on the CMR console, splenic T1-reactivity to adenosine stress (ΔT1 spleen ) was expressed in absolute terms: ΔT1 spleen (ms) = StressT1 spleen -RestT1 spleen .

T1 spleen intra-scan variability assessments
Inter-slice variability in resting T1 spleen , stress T1 spleen and ΔT1 spleen were assessed in cases where matching stress and rest T1-maps were performed in ≥2 different short-axis slice positions. To assess for intra-slice T1 spleen variability, we re-analyzed healthy-volunteers data from the original ShMOLLI methods paper, where T1-maps were repeated >15 min apart in the same short-axis slice within the same scan [14]. Intra-scan variability was calculated as the standard deviation of differences-from-the-mean in each individual.

Splenic perfusion analysis
Splenic first-pass perfusion was analysed by an observer (>4 years of perfusion imaging analysis experience) blinded to T1-maps and clinical information, using CMR 42 software (Circle Cardiovascular Imaging Inc., Calgary, Canada). Splenic ROIs were placed on stress and rest perfusion images with frame-by-frame manual Fig. 1 Representative stress and rest splenic first-pass gadolinium perfusion and native T1-maps. Signal intensity (SI) curves represent splenic perfusion SI (y-axis, arbitrary units) over time (x-axis, 50-60 s). The maximum and minimum SI spleen are as indicated. Splenic regions of interests on perfusion images and T1-maps are outlined in red and black, respectively. Mean native T1 spleen and stress changes (ΔT1 spleen ) are as labelled. 3 T images were used for illustration (observed ΔT1 spleen and ΔSI spleen are field strength independent) correction for artefacts and respiratory motion, to generate curves showing mean splenic signal intensity (SI, arbitrary units) changes over time (50-60s). Peak splenic perfusion SI (SI spleen ) was estimated as the numerical difference between baseline-SI and maximal-SI during splenic first-pass perfusion as previously described [6]. Adenosine-induced changes in SI spleen compared to rest were expressed in percentages: ΔSI spleen (%) = (StressSI spleen -RestSI spleen ) ÷ RestSI spleen × 100%.
Splenic switch-off on perfusion imaging was visually assessed by 2 independent observers (>3 years clinical CMR perfusion experience). In the 5/212 cases where the 2 observers disagreed, adjudication was sought from a 3 rd independent observer (Fig. 1). Perfusion images were graded as previously described [6]: either displaying splenic switch-off (the spleen on rest imaging is clearly brighter than on stress imaging), or no switch-off (the spleen on rest imaging is of similar brightness compared to stress imaging).

Statistical analysis
Data are reported as mean ± SD, tests are 2-tailed and parametric, based on Kolmogorov-Smirnov normalitychecks. Differences in individual characteristics were tested using Student's t-tests, paired within individuals (e.g. stress vs rest T1 spleen ) and unpaired between groups (e.g. ΔT1 spleen in controls vs patients). Comparisons between ≥3 data groups were assessed using analysis of variance (ANOVA) with Bonferronicorrected post-hoc method. Linear correlations were assessed using Pearson's correlation coefficient (R) and non-linear correlations were assessed using Spearman's rank correlation coefficient (rho). Intrascan variability and inter-observer reproducibility of rest/stress T1 spleen and ΔT1 spleen were assessed using the Intra-class correlation coefficient (ICC), reporting 95% confidence intervals. The performance of ΔT1 spleen for replicating splenic switch-off was assessed using receiver-operating characteristics (ROC) curves [21], reporting area-under-the-curve (AUC ± SEM), and also sensitivity, specificity, diagnostic accuracy, positive predictive values (PPV) and negative predictive values (NPV), with 95% confidence intervals (CI). All analyses were performed on single measures per-subject, using MedCalc 12.7.8 (MedCalc Software, Ostend, Belgium). P < 0.05 denotes statistical significance.

Subject characteristics
Subject characteristics are summarised in Table 1. All subjects experienced at least one adenosine-related symptoms (e.g. chest-tightness, dyspnoea, flushing) [13], and >10 bpm increase in heart rate (HR) during adenosine stress, compared to rest. Significant blood pressure response (>10 mmHg SBP decrease during stress) was observed in 50% of subjects.
Mean stress HR was lower in 1.5 T patients compared to other subjects, despite similar resting HR, likely due to more frequent beta-blocker and non-dihydropyridine calcium channel antagonist administration in these patients (all AF/CAD, Table 1).
In pooled analysis, ΔT1 spleen did not appear to be significantly affected by field strength (1.
Visual splenic switch-off assessmentrelationships with perfusion, quantitative T1 spleen and hemodynamic parameters Subjects with visual splenic switch-off had greater stress ΔSI spleen and ΔT1 spleen values compared to those with no switch-off (Table 4 and Fig. 3). In contrast, there were no significant differences in stress-related  Values are n (%) or mean ± SD Abbreviations: RPP rate pressure product, TIA transient ischemic attack, ACEi angiotensin-converting enzyme inhibitors, ARB angiotensin receptor blockers, CCB calcium channel antagonist, DHP dihydropyridine *p < 0.05 compared to controls of corresponding field strength (1.5 T or 3 T). # p < 0.05 for comparisons between patient groups (1.

T vs 3 T)
haemodynamic changes (HR, SBP, RPP) between subjects with splenic switch-off and no switch-off (Table 4 and Fig. 3).

Discussion
This proof of principle study demonstrated that T1 spleen decreases significantly during adenosine stress compared to baseline. The magnitude of the stress-induced T1 spleen response (ΔT1 spleen ) is strongly correlated with splenic perfusion attenuation (ΔSI spleen ). From a clinical viewpoint, a native ΔT1 spleen threshold of ≥ −30 ms accurately replicated the "splenic switch-off" sign with a high positive predictive value of 98% and offers the potential to assess adenosine stress adequacy before GBCA firstpass perfusion imaging. From a practical viewpoint, assessment of T1 spleen takes~30 s (Fig. 5), which means it can be repeated as necessary "on-the-fly", to guide adenosine dosage up-titrations and optimize stress responses before injection of contrast agents (example protocol in Fig. 5). This pre-gadolinium approach may be advantageous over the retrospective and potentially gadolinium dose-sensitive splenic switch-off method for improving the quality of stress responses before firstpass perfusion imaging, which deserves further investigation in future studies to determine whether it decreases the number of false negative perfusion scans [6].

Stress/rest T1 spleen as a marker of adenosine stress response
Patients had lower resting native T1 spleen values compared to controls. This may be related to the presence of co-morbidities in patients, such as hypertension and peripheral vascular disease, which may induce peripheral vasoconstriction, with expected reductions in resting organ blood volumes and T1 spleen values. This observation deserves further investigation in larger future studies. Native T1-relaxation times of tissues are prolonged by increased blood volume (i.e. water content) [14,15,22]. Adenosine causes splenic artery vasoconstriction and  Fig. 2 Correlation between stress-induced reductions in peak splenic signal intensity (ΔSI spleen ) and splenic native T1 (ΔT1 spleen ). Pooled data of controls and patients at 1.5 T (blue) and 3 T (red), represented on per-subject basis (n = 212). Spearman's rank correlation coefficient (Rho) = 0.70, p < 0.0001 reduced blood volume [6][7][8][9][10][11], which shortens splenic T1-relaxation times. This is supported by our observation of significantly lower T1 spleen during adenosine stress compared to rest, in both controls and patients. The stress ΔT1 spleen was not significantly affected by different field strengths, age, gender and cardiovascular diseases, likely reflecting reproducible T1-estimations in this study [14,15,22]. The correlation between stress ΔT1 spleen and ΔT1 myocardium in normal controls suggests the vasoconstrictor effect of adenosine on the spleen is associated with vasodilatory effects in the myocardium. For the relationship between myocardial and splenic stress T1 in patients with cardiovascular disease, larger ongoing studies will offer reference ranges for ΔT1 in disease, and resolve the separate effects of regional myocardial differences and medication on stress T1 reactivity. The observed strong correlation between ΔT1 spleen and ΔSI spleen suggests that stress-induced changes in splenic blood volume are related to blood flow, which is regulated by alterations in the adenosine-mediated splenic arterial tone [10,11]. The lack of significant correlation between ΔSI spleen or ΔT1 spleen with rate pressure product is consistent with existing evidence showing dissociation between imaging and hemodynamic markers of stress response [5,6], and further suggests that stress responses during clinical CMR cannot be reliably assessed using hemodynamic observations alone [5]. This deserves further investigation.
A threshold of ≥30 ms decrease in T1 spleen replicated complete splenic switch-off with a high positive predictive value of 98%. The intra-scan variability in T1 spleen (inter-slice: ±10 ms; intra-slice: ±9 ms) was 3-times less than this proposed threshold ≥30 ms drop, with excellent T1-fit as evident on quality control R 2 -maps, despite the lack of dedicated image optimization (e.g. shimming) over the spleen. For stress T1 spleen responses <30 ms, further work is needed to determine whether adenosine dose-increments or waiting longer with the same infusion rate may improve the confidence of stress responses, and impact on diagnostic performance of stress CMR for the diagnosis of ischemia.

Limitations and future directions
This proof-of-concept study is based on ShMOLLI T1 spleen values derived from short-axis slices planned for myocardial perfusion CMR imaging; the spleen was not visible in a small proportion of T1-maps (~2%), and future applications of splenic T1-mapping may benefit  p<0.0001 p<0.0001 p=0.89 Fig. 3 Relations between different markers of stress adequacy. Subjects with the "splenic switch-off" sign had greater stress-induced reductions in a gadolinium-based splenic perfusion (ΔSI spleen , same technique) and b gadolinium-free splenic T1 (ΔT1 spleen , different technique) compared to subjects with no switch-off. There was no difference in stress-induced c hemodynamic changes in rate pressure product (RPP) between the splenic switch-off and the no switch-off subjects. Data are mean ± 1SD from a dedicated image planned through the spleen. Rapid on-scanner T1-map reconstructions, with the immediate availability of goodness-of-fit measures (such as R 2 -maps), are imperative to enable practical "on-the-fly" repetition of reliable T1 spleen estimations to guide stress response optimization (Fig. 5). Given the overall excellent R 2 -maps over the spleen and the narrow T1 spleen ranges obtained, data in this study suggest that stress/rest splenic T1-mapping can be feasibly included in CMR protocols without major technical adjustments. Practical in-vivo T1estimations are method-dependent, and demonstrate increasingly discrepant heart rate dependencies at longer T1-values [23]. Therefore, results achieved with ShMOLLI, in particular the splenic T1-thresholds replicating splenic switch-off, should be interpreted with care before directly translating to other T1-mapping techniques. Choosing methods that can withstand dynamic HRvariations and tachycardia without significant HRdependencies is therefore paramount when performing stress-T1 studies. The gadolinium-based splenic switchoff sign is only seen with non-selective adenosine receptor agonists (dipyridamole and adenosine), but was absent with cardio-selective vasodilators (e.g. regadenoson) or inotropic agents (e.g. dobutamine) [6]. Further work is needed to elucidate stress T1 spleen responses using pharmacological agents other than adenosine and during physical exercise. Patients in this study were unselected for diseases known to affect splenic blood volumes, e.g. venous portal hypertension, hematological malignancies and systemic inflammation; thus, further studies to characterize the effects of these diseases on T1 spleen will help to determine the general applicability of this technique. While we identified a cut-off of ≥30 ms drop in T1 spleen during stress for replicating complete splenic switch-off, the clinical utility of this threshold for detecting true stress adequacy needs to be validated against false-negative perfusion scans, determined by comparison to invasive coronary angiography and pressure-wire based assessments of functional ischemia, such as fractional flow reserve. This is topic of ongoing work.

Conclusions
Adenosine stress and rest splenic T1-mapping is a novel method for assessing stress responses, independent of conventional hemodynamic parameters. It Fig. 4 ROC curves of native ΔT1 spleen for replicating the gadolinium-based "splenic switch-off" sign. A ΔT1 spleen threshold of ≥ −30 ms replicated the "splenic switch-off" sign (AUC 0.90 ± 0.05, p < 0.0001), with high sensitivity 90%, specificity 88% and diagnostic accuracy 90% Fig. 5 Potential splenic ΔT1 spleen -guided protocol for real-time assessment and optimization of stress adequacy before gadolinium perfusion. Practical T1 spleen assessment using ShMOLLI typically takes around 30 s: breath-hold instructions (5 s), T1-map acquisition over 9-heart-beats (~10 s, shorter with higher stress heart rates), on-screen image reconstruction (5-10 s), splenic-ROI placement directly on CMR console screen by the operator (5 s) followed by immediate display of T1 spleen /SD estimations (as indicated). The ability of this protocol to improve the quality of stress responses deserves validation in future studies enables prediction of the visual "splenic switch-off" sign without the need for gadolinium, and correlates well to changes in splenic signal intensity during stress/rest perfusion imaging. ΔT1 spleen holds promise to facilitate optimization of stress responses before gadolinium first-pass perfusion CMR.

Additional files
Additional file 1: Figure S1. Description of data: Correlation between adenosine stress ΔT1 spleen and ΔT1 myocardium in 51 healthy controls. Data are presented per-subject. (DOCX 26 kb) Additional file 2: Table S1. Description of data: Effect of medication on ΔT1 spleen in patients with cardiovascular disease. ACE: angiotensin converting enzyme; ARB: angiotensin reception blocker; CCB: calcium channel blockers; DHP: dihydropyridine. (DOCX 13 kb) Additional file 3: Figure S2. Description of data: Bland Altman plot of ΔT1 spleen estimation by 2 independent blinded observers.