82Rb and [15O]H2O myocardial perfusion PET imaging: a prospective head to head comparison

Background 82Rb PET and [15O]H2O PET are both validated tracers for myocardical perfusion imaging but have not previously been compared clinically. During our site’s transition from 82Rb to [15O]H2O PET, we performed a head-to-head comparison in a mixed population with suspected ischemic heart disease. Methods A total of 37 patients referred for perfusion imaging due to suspicion of coronary stenosis were examined with both 82Rb and [15O]H2O PET on the same day in rest and during adenosine-induced stress. The exams were rated by two blinded readers as normal, regional ischemia, globally reduced myocardial perfusion, or myocardial scarring. For [15O]H2O PET, regional ischemia was defined as two neighboring segments with average stress perfusion ≤ 2.3 mL/(min·g). Further, we evaluated a total perfusion deficit (TPD) of ≥ 10% as a more conservative marker of ischemia. Results [15O]H2O PET identified more patients with regional ischemia: 17(46%) vs 9(24%), agreement: 59% corresponding to a Cohen’s kappa of .31 [95%CI .08-.53], (P < .001). Using the more conservative TPD ≥ 10%, the agreement increased to 86% corresponding to a kappa of .62 [95%CI .33-.92], (P = .001). For the subgroup of patients with no known heart disease (n = 18), the agreement was 94%. Interrater agreement was 95% corresponding to a kappa of .89 [95%CI .74-1.00] (P < .001). Conclusions In clinical transition from 82Rb to [15O]H2O PET, it is important to take into account the higher frequency of patients with regional ischemia detected by [15O]H2O PET. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s12350-023-03372-7.


INTRODUCTION
Identification of patients with coronary artery disease (CAD) that will most likely benefit from revascularization is guided by non-invasive work-up e.g.myocardial perfusion imaging. 1 For decades, myocardial perfusion imaging was used with single photon emission computed tomography (SPECT) with e.g.[ 99m Tc]Sestamibi or other irreversible tracers which are extracted into the myocardium dependent on the level of perfusion and not significantly redistributed back into the vascular system.For these irreversible tracers, regionally reduced perfusion during stress but not during rest is referred to as stress-induced ischemia.Reduced perfusion during both stress and rest, matched defects, are considered to be scarring.
A number of sites have switched to PET using 82 Rb eluted from an 82 Sr generator.The PET technology results in high-resolution images, and due to the short half-life of 82 Rb (76 s), rest and stress tests can be performed shortly after each other reducing the entire examination from a two-day protocol to no more than 30 minutes. 82Rb is actively transported into the myocytes through the Na ?/K ?pump where the tracer is trapped allowing for static imaging.Hence, the resulting perfusion images are comparable to SPECT images but with better resolution.Further, the software packages for 82 Rb PET support the quantification of myocardial blood perfusion (MBF).The increase in MBF referred to as the coronary flow reserve (CFR) allows detection of globally reduced perfusion e.g. in triple-vessel disease (balanced ischemia) or microvascular disease. 2 The quantification method differs between software packages, 3 but are generally based on a one-tissue compartment model: C T is the myocardium time-activity-curve and C A is the arterial input function both obtained from the dynamic PET series and denotes the convolution operation.K 1 and k 2 are the influx and efflux rate constants.For the irreversible 82 Rb, k 2 is close to zero.The measured influx K 1 represents MBF and is subsequently corrected for partial volume, motion and arterial blood volume by software package specific corrections. 3nother PET perfusion tracer, [ 15 O]H 2 O is a cyclotron product with a short physical half-life (122 s) requiring an on-site cyclotron.With the rapidly developing cyclotron technology, our site has established a mini-cyclotron GENtrace (GE healthcare, Uppsala, Sweden) dedicated for production of 15 O and subsequent synthesis to [ 15 O]H 2 O.In contrast to the irreversible PET tracer 82 Rb, [ 15 O]H 2 O is a freely diffusible tracer.The software package calculation for [ 15 O]H 2 O is also based on the one-tissue compartment model (Eq. 1) but contrary to 82 Rb, the efflux rate constant k 2 is used to achieve a more robust estimation for MBF. 4,5k 2 represents the clearance of tracer from the tissue and is-at least in theory-more independent of partial volume, motion, 4,6 and even attenuation correction 5 which affect only the influx as k 2 is measured relative to the influx.Thus, [ 15 O]H 2 O PET allows for parametric images with robust MBF estimates at the expense of static robust high-resolution images obtained using 82 Rb PET.
While water is freely diffusible with an extraction fraction of 1 in myocardial tissue allowing for direct measurements of perfusion, 82 Rb has a limited extraction which is reduced at higher perfusion rates and correction for the lower extraction is necessary.The extraction corrections are based on the Renkin-Crone model 7,8 : 2][13] In fact, early studies on FFR used [ 15 O]H 2 O PET as the reference standard. 14or [ 15 O]H 2 O PET, the most accurate metric for predicting an FFR-positive coronary stenosis in patients without previous cardiac disease is MBF during stress (MBF stress ) in two neighboring segments B 2.3 mL/ (minÁg) in a 17 segment model. 15he clinical transition from [ 99m Tc]Sestamibi SPECT to 82 Rb PET is straightforward with better image quality and patient experience, while the clinical transition to [ 15 O]H 2 O PET may be more complicated due to the aforementioned differences in uptake mechanisms. 82Rb PET and [ 15 O]H 2 O PET are both validated for heart perfusion but have not previously been compared for clinical performance in a prospective cohort of patients suspected for myocardial ischemia.During our clinical transition from 82 Rb PET to [ 15 O]H 2 O PET, we performed a head-to-head comparison in a mixed population with suspected myocardial ischemia and a high fraction of patients with previous heart disease.We hypothesized that there is no significant difference in the overall occurrence of detected clinically relevant myocardial ischemia as determined with 82 Rb PET and [ 15 O]H 2 O PET and that in the vast majority of cases, the patients would be assessed similarly.

Study population
We prospectively and consecutively included patients referred from the Department of Cardiology, Copenhagen University Hospital Bispebjerg from January to April 2022.The study was initiated when [ 15 O]H 2 O production was approved at our site and during a four month overlap with continued clinical use of 82 Rb.Thus all [ 15 O]H 2 O PET scans were performed during the startup period.The center has a highthroughput with around 1,500 heart PET examinations per year 16 and during the startup period, a number of exams were co-reviewed by experts from Turku PET Centre to ensure quality.To increase the likelihood of ischemia in the cohort, we included patients with typical anginal chest pain and with at least one of the following risk factors: a family history of cardiac disease (\ 55 years for male and \ 65 years for female family members), smoking, diabetes, hypertension, BMI [ 30 or hyperlipidemia.Exclusion criteria were unstable angina, significant chronic obstructive lung disease or asthma, claustrophobia, acute severe illness or a significant language barrier.The study was approved by the Research Ethics Committee of the Capital Region of Denmark (ID: H-21016899) and written consent to participate were obtained from all individuals after receiving oral and written information according to the Helsinki declaration.All data were handled according to regulations by The Danish Data Protection Agency.
was produced in two steps.First, a target gas mixture (97.5% 15 N 2 , 2.5% O 2 ) was continuously bombarded for a few minutes with a 7.8 MeV proton beam in a GenTrace cyclotron (GE, Uppsala, Sweden) dedicated to the production of 15 O. Second, for administration of [ 15 O]H 2 O, the target was mixed with the transport gas (N 2 with 4% H 2 ) and pushed into an oven with a 400 °C hot palladium wire.The resulting radioactive water vapor was fed into a bedside automated production system (Hidex RWG, Hidex Oy, Turku, Finland) consisting of a dual membrane system to mix physiological saline with [ 15 O]H 2 O.The resulting radioactive saline solution was injected into the patient without further user interaction.

PET scans
All subjects refrained from using caffeine-containing beverages and food or theophylline-containing medication for 24 hours before examination.Furthermore, phosphodiesterase type 5 inhibitors were withheld five days before examination, antithrombotic medicine containing Dipyridamole or Nicorandil two days before, extended-release nitrates 12 hours before and shortacting nitroglycerin two hours before examination.All patients were scanned using a Discovery 710 PET/CT scanner (GE Healthcare, Milwaukee, WI, USA).After a CT scan for attenuation correction and for anatomical localization, a 5 minutes dynamic emission scan in list mode was performed during resting condition after intravenous injection of 1100 MBq of 82 Rb eluted from an 82 Sr/ 82 Rb generator (CardioGen-82; Bracco, Princeton, NJ).[ 15 O]H 2 O PET in resting condition was mean performed earliest 10 minutes after 82 Rb PET, and a dose of 394 MBq (range: 345-563 MBq) [ 15 O]H 2 O was injected intravenously using a synthesis and injection system, Hidex RadioWaterGenerator (Hidex, Turku, Finland) and a 5-minutes scan was initiated simultaneously with the bolus arrival.After a 10 minutes interval to allow for decay of radioactivity, an identical PET sequence was performed during stress conditions induced by intravenous adenosine infusion (140 lg/kg/ min) for 6 minutes.Adenosine was started 2 minutes prior to the stress PET scans to achieve maximum hyperaemia.We did not randomize the order of 82 Rb and [ 15 O]H 2 O PET as several patients could not cooperate to two adenosine infusions and we aimed to ensure a clinically useful 82 Rb PET examination.Reconstruction of dynamic PET images was performed using ordered subset expectation maximization (OSEM) with Time of Flight (ToF) (2 iterations, 24 subsets and 6.4 mm in-plane filtering).For 82 Rb a static reconstruction for the last 150 s was used.The Corridor 4DM software version 2018 (Invia Medical Imaging Solutions, Ann Arbor, MI, USA) was used for the analysis of 82 Rb PET data, while CarimasCE software version 1.3.1.(Turku, Finland) was used for [ 15 O]H 2 O PET data.Corridor 4DM estimates quantitative MBF from 82 Rb K 1 measurements using a modified version of Equation 2according to Lortie 17 : PET interpretation 82 Rb PET 82 Rb PET was evaluated as part of daily clinical routine by a nuclear medicine specialist with [ 10 years of experience in myocardial imaging.Previous studies have shown good inter-observer agreement. 18,19 82Rb PET were assessed visually using the 'splash' images using polar plots with relative differences compared to a normal database.The degree of relative defects within the myocardium was rated using 17 segments and 4 degrees of reduction, i.e. maximum total score 68.A stress defect score of 7 or more (i.e.&10%) of the myocardial wall was considered significant.Based on the clinical readings, the 82 Rb PET was classified into four groups (normal, regional ischemia, globally reduced myocardial perfusion and myocardial scarring, see Table 1 for definitions).To further simplify data, the classifications were reduced to two groups of normal and regional ischemia, the latter included global reduction with suspicion of triple-vessel disease.The vascular territory involved was noted as left anterior descending artery (LAD), left circumflex artery (LCX) or right coronary artery (RCA).
[ 15  period as we quickly realized that the threshold of two segments B 2.3 mL/(minÁg) identified too many patients with ischemia in the current patient population, which was also demonstrated in a recent study in patients with previous myocardial infarction or PCI. 20 1 for definitions).The involved vascular territories (LAD, LCX and/or RCA) were noted.In case of discrepancy between the readers, consensus reading was performed.If a scan was classified with both a regional ischemia and global perfusion reduction or scarring, the regional defect overruled the other findings.To assess how previous heart disease affected the agreement, we performed a subgroup analysis of patients without known heart disease.

Statistics
Data are reported with mean and standard deviation or median and interquartile range.Differences in heart rate response during adenosine infusion were compared using a paired Student's t-test.Using SPSS (IBM SPSS Statistic version 25).Agreement was defined as the number of identical classification divided by the total number of patients.Cohen's kappa was used to compare agreement between methods and between readers for the two and four group comparisons when sample sizes were sufficient.Kappa-values were evaluated according to Altman: j B .2: poor, .2\ j \ .4:fair, .4\ j \ .6:moderate, .6 \ j \ .8:good, j [ .8:very good. 21cNemar's test were used to test for agreement between tracers with the 2 9 2 contingency tables.Pearson's correlation coefficient was used to compare the measured perfusion with the two methods.The K 1 values were calculated from 82 Rb MBF estimates using Equation 3 to compare the apparent 82 Rb uptake in static images to the parametric [ 15 O]H 2 O PET MBF images.

RESULTS
A total of 57 patients were included.A number of subjects were subsequently excluded due to cancellation (n = 1), [ 15 O]H 2 O production failure (n = 3), intravenous access failure (n = 1), not able to receive adenosine twice (n = 4), or technical failure (n = 5) leaving 43 patients for the analysis.During reading, 6 additional examinations were excluded due to excessive movement artefacts (n = 4) or insufficient vasodilatation (n = 2) leaving 37 patients with diagnostic scans with both tracers (Suppl.Figure 1).3A), and 72% for patients with no previous heart disease (Table 4A).
Rating into two groups with and without a TPD [ 10%, the number of positive findings was reduced from 17 to 10 for [ 15 O]H 2 O PET and from 9 to 8 for 82 Rb PET, indicating the importance of selecting a proper cut-off.The agreement increased to 86% for the entire population corresponding to a good kappa of .62 [95%CI .33-.92], (P = .001)(Table 3B) and 94% for the patients with no previous heart disease (Table 4B).No significant differences between tracers was found for the entire population or the patients with no previous heart disease (McNemar, P = .16and P = .50,respectively).A total of six patients had a TPD[ 10% for both tracers and five of these had identical scoring of vascular territory.
Table 5 shows the cases with discrepancies, and Figure 1 shows examples of agreement and disagreement.The inter-reader agreement of [ 15 O]H 2 O PET was 84% for four group classification corresponding to a good kappa of .73[95%CI .54-.91] (P \ .001),and for classification in two groups, the agreement was 95% corresponding to a very good kappa of .89[95%CI .74-1.00] (P \ .001).
MBF obtained with the two methods correlated significantly but lower values were measured at high perfusion for 82 Rb compared to [ 15  The CFR obtained with the two tracers also correlated significantly (Figure 3) (r = .66[95%CI .42-.81], P \ .0001)and also with systematically lower values for 82 Rb compared to [ 15 O]H 2 O.Note that the cut-off from the literature as depicted by the solid lines is also lower for 82 Rb.A total of 6 patients (marked red) would be classified differently in terms of CFR by the two tracers and one of these had different final ratings.with 82 Rb but still identified three more subjects with ischemia.

DISCUSSION
Part of the disagreement between 82 Rb and [ 15 O]H 2 O can be explained by the application of the cut-off of 2.3 mL/(minÁg) which was determined in patients without prior CAD 15 that were slightly younger (61 vs 66 years), weighed less (80 vs 86kg) and included more women (42% vs 20%).Thus, the cut-off is likely to identify too many patients with ischemia in a mixed population.This finding is in accordance with the PACIFIC 2 study, 20 showing reduced specificity of [ 15 O]H 2 O PET in patients with previous ischemic heart disease.However, in the subpopulation of the patients with no previous heart disease, [ 15 O]H 2 O PET also seem to find regional ischemia more frequently than 82 Rb PET (Table 4A).The solely use of relative perfusion defects for 82 Rb may be less sensitive.We do not know to what degree [ 15 O]H 2 O PET identifies patients with signs of hemodynamically significant coronary artery stenosis as determined by invasive coronary angiography (ICA) since only patients with stress-induced defects on 82 Rb PET were considered for ICA.None of the patients identified with perfusion defects by [ 15 O]H 2 O PET who had a normal 82 Rb PET scan had subsequent angiographic imaging or were subsequently hospitalized suspected for myocardial infarction but the limited number of patients and short time of follow-up of 12 months does not rule out that the patients did indeed have significant coronary artery stenosis (see Table 5 for details of the patients with discrepancy).We anticipate that a less dichotomous interpretation of [ 15 O]H 2 O PET than simply applying an MBF cut-off value of 2.3 mL/ (minÁg) will be needed in a mixed population, e.g.taking into account other factors potentially contributing to globally decreased stress myocardial perfusion such as previous revascularization, atrial fibrillation, decreased left ventricular ejection fraction, diabetes, and other conditions involving microvascular dysfunction.Segmental relative CFR calculation could be a possible tool to evaluate perfusion in these patients. 22 82 b has a flow-dependent lower extraction of tracer compared to the freely diffusible [ 15 O]H 2 O.This is partly corrected in the quantitative measures, achieving a high correlation between MBF and CFR measured with the two methods (Figures 2, 3), although a flowdependent bias is still clearly visible in Figure 2a.The bias in our study is more pronounced than previous studies have reported 23 and must be kept in mind when comparing MBF measured with the two methods.But more importantly, the 82 Rb images which are visually assessed using 'splash' images or polar plots are indeed images of tracer uptake, i.e. uncorrected K 1 values which show a much more pronounced bias.Figure 2b shows that differences in 82 Rb K 1 correspond to much larger differences in the k 2 -derived MBF using [ 15 O]H 2 O, especially for stress images, which have a slope of only .10,i.e. visual differences are about 10 times greater for [ 15 O]H 2 O compared to 82 Rb.This is not only true between subjects as in Figure 2b but also within subjects.Thus, the higher extraction likely explains the higher frequency of patients with regional ischemia detected by [ 15  patients with no prior heart disease.Indeed, in some sites only stress images are obtained resulting in a highly efficient workflow.At our own institution, half of our patients had previous heart disease ( O]H 2 O-even after correction for the lower extraction of 82 Rb (see Figure2): Figure 2a: Rest: r = .75[95%CI .56-.86], P \ .000001;stress: r = .62[95%CI .37-.79], P \ .0001;and Figure 2b for uncorrected 82 Rb K 1 and [ 15 O]H 2 O MBF values: Rest: r = .77[95%CI .59-.87], slope = .33,P \ .0000001and stress: r = .62[95%CI .38-.79], slope = .10,P \ .0001.

Table 1 .
O]H 2 O PET [ 15 O]H 2 O PET was assessed according to Danad 15 with a cut-off of two neighboring segments with MBF stress B 2.3 mL/(minÁg)., and very severely reduced (MBF stress B 1.4; score 4).TPD C 10% was tentatively considered significant for ischemia.TPD is not a validated measure in [ 15 O]H 2 O PET but was introduced in our clinic during the training Definitions of classifications All [ 15 O]H 2 O PET images were evaluated after the startup period had ended and [ 15 O]H 2 O PET had been clinical routine for 2 months.Readers had [ 10 years of experience in nuclear cardiology (UT and MK).[ 15 O]H 2 O PET images were randomized and readers were blinded to the result of the 82 Rb PET but with full access to all clinical data to simulate daily clinical routine.As for 82 Rb, each [ 15 O]H 2 O PET examination was classified into four groups based on the two segment threshold with MBF stress B 2.3 mL/(minÁg) and two groups based on the aforementioned TPD of 10% (see Table

Table 2 .
Table 2 lists demographic and clinical data of the included patients.Please note the high number of patients with significant previous heart disease.Infusion of adenosine elicited a heart rate response from on average 67.0 (± SD 9.1) to 89.6 (± 12.1) during 82 Rb scan and a slightly diminished (p \ .005)response from 69.6 (± 11.7) to 87.8 (± 13.1) during the [ 15 O]H 2 O scan, which was probably due to habituation.Please refer to Table 3 for comparison of the 82 Rb PET and consensus [ 15 O]H 2 O PET ratings for the entire Demographic and clinical data of the 37 included patients IQR, interquartile range; BMI, body mass index; CAD, cardiovascular disease; PCI, percutaneous coronary intervention; CABG, coronary artery bypass graft surgery; EDV, end-diastolic volume normalized to body surface; LVEF, left ventricle ejection fraction Previous heart disease includes: heart failure (LVEF\50%), atrial fibrillation, second or third degree atrioventricular block, (acute) myocardial infarction, PCI or CABG group and Table 4 for patients without previous heart disease.We found more positive findings with [ 15 O]H

Table 3 .
15reement between ratings based on [15O]H 2 O and 82 Rb for the entire population Bold represents identical ratings.A: Agreement between the tracers when rating into four groups.Note that three patients were classified with scarring using 82 Rb PET and none using [15O]H 2 O PET. B: Rating into two groups with and without a TPD[ 10%.For discrepancies, see Table 5 TPD, total perfusion defect (see methods for definition).

Table 4 .
O]H 2 O.It was shown by Danad et al 15 that rest images of [ 15 O]H 2 O PET were not prognostic in a setting of Agreement between ratings based on [ 15 O]H 2 O and 82 Rb for patients with no prior heart disease Bold represents identical ratings.A: Agreement between the tracers when rating into four groups.B: Rating into two groups with and without a TPD [ 10%.TPD, total perfusion defect (see methods for definition)

Table 2 )
15d traditionally, rest82Rb images were used to differentiate ischemia from previous infarcts/fibrosis/scarring.Two factors reduce the clinical usefulness of rest [15O]H 2 O images for differentiating between scarring and ischemia.Firstly, as explained above, [15O]H 2 O PET shows higher variation in MBF and differentiating between true infarcts and minor relative changes may be difficult.Secondly, and more importantly, the efflux constant k 2 is used instead of the influx constant K 1 to assess MBF (see Equation1).While the influx K 1 is highly sensitive not only to movement and attenuation artefacts but also to tissue defects and old infarcts, k 2 is a PaƟent A.82Rb PET, stress [ 15 O]H 2 O PET, stress PaƟent B. 82 Rb PET, stress [ 15 O]H 2 O PET, stress Figure 1.Patient A: Example of agreement. 61year old man with angina but without prior known CAD. Boh tracers showed large perfusion defects ( 82 Rb: 25% and [ 15 O]H 2 O: 54%) during stress in LAD and RCA.Patient B. Example of disagreement.74 year old man with angina, a number of risk factors for CAD and dilated left atrium.82 Rb showed homogenous tracer uptake while [ 15 O]H 2 O showed a defect of 15% in LCX and/or RCA.CFR compared for the two tracers (r = .66[95%CI .42-.81], P \ .0001).Note that the cut-offs for reduced CFR are different 15 .It is noteworthy that four subjects (marked red) were regarded normal according to the 82 Rb scan and had too low CFR according to the [ 15 O]H 2 O scan, and additional two subjects (also marked red) were opposite below normal for

Table 5 .
Discrepancies from Table 3 , invasive coronary angiography; TPD, total perfusion deficit; CAD, coronary artery disease; LVEF, left ventricle ejection fraction; EDV, end diastolic volume; PCI, percutaneous coronary intervention; LCX, left circumflex artery; RCA, right coronary artery; AMI, acute myocardial infarction NEW KNOWLEDGE GAINED In a clinical setting, we found only moderate agreement between [ 15 O]H 2 O and 82 Rb perfusion PET as [ 15 O]H 2 O PET identifies a higher frequency of patients with regional ischemia.CONCLUSION [ 15 O]H 2 O perfusion PET is a sensitive imaging modality for myocardial ischemia with a high interrater agreement.However, a number of differences exist between 82 Rb and [ 15 O]H 2 O perfusion PET.Primarily, we found only a moderate agreement as [ 15 O]H 2 O PET identifies a higher frequency of patients with regional ischemia especially if using the literature cut-off of 2.3 mL/(minÁg) determined in individuals without prior CAD.An improved agreement was found using the more conservative TPD of C 10% but future studies are warranted to establish [ 15 O]H 2 O PET interpretation criteria in a mixed population.Secondly, matched rest and stress perfusion defects using 82 Rb interpreted as scarring are not always detected by [ 15 O]H 2 O PET which likely detects only transmural scars. ICA