Objectives The prognostic significance of an afterload-integrated diastolic index, the ratio of diastolic elastance (Ed) to arterial elastance (Ea) (Ed/Ea=[E/e′]/[0.9×systolic blood pressure]), is valid for 1 year after discharge in older patients with heart failure with preserved ejection fraction (HFpEF). We aimed to clarify the association with changes in Ed/Ea from enrolment to 1 year and prognosis thereafter in patients with HFpEF.
Setting A prospective, multicentre observational registry of collaborating hospitals in Osaka, Japan.
Participants We enrolled 659 patients with HFpEF hospitalised for acute decompensated heart failure (men/women: 296/363). Blood tests and transthoracic echocardiography were performed before discharge and at 1 year after.
Primary outcome measures All-cause mortality and/or re-admission for heart failure were evaluated after discharge.
Results High Ed/Ea assessed before discharge was a significant prognostic factor during the first, but not the second, year after discharge in all-cause mortality or all-cause mortality and/or re-admission for heart failure. When re-analysis was performed using the value of Ed/Ea at 1 year after discharge, high Ed/Ea was significant for the prognosis during the second year for both end points (p=0.012 and p=0.033, respectively). The poorest mortality during 1‒2 years after enrolment was observed in those who showed a worsening Ed/Ea during the first year associated with larger left ventricular mass index and reduced left ventricular ejection fraction. In all-cause mortality and/or re-admission for heart failure, the event rate during 1‒2 years was highest in those with persistently high Ed/Ea even after 1 year.
Conclusions Time-sensitive prognostic performance of Ed/Ea, an afterload-integrated diastolic index, was observed in older patients with HFpEF.
Trial registration number UMIN000021831.
- heart failure
- cardiac epidemiology
Data availability statement
Data are available on reasonable request. Yes.
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Strengths and limitations of this study
The ratio of diastolic elastance (Ed) to arterial elastance (Ea), Ed/Ea, is a novel index of an afterload-integrated diastolic function and left atrial (LA) pressure overload.
The clinical significance of prognostic factors related to haemodynamics in patients with heart failure and preserved ejection fraction (HFpEF) may differ according to the follow-up period.
The prognostic significance of Ed/Ea for all-cause mortality is only valid for 1 year after discharge in HFpEF.
The limitations of our study are that all-cause mortality rather than cardiac death was examined and the sample size was small.
Evaluation of severity of diastolic dysfunction is useful for assessing the prognosis of patients with heart failure (HF) with preserved ejection fraction (HFpEF).1 However, there is important crosstalk between afterload and diastolic function.2 Blood pressure shows a circadian pattern that is mainly affected by autonomic nerve activity. Various changes occur throughout the day and affect the cardiovascular system, leading to various alterations in cardiac function according to time and circumstances. Diastolic relaxation is reduced with acute increases in afterload.3–5 Unfortunately, none of non-invasive diastolic indices consider the afterload.
Early studies suggested that E/e′ could be used to reliably estimate left ventricular (LV) filling pressure in the clinical setting of diastolic HF.6 7 The correlation between E/e′ and direct left atrial (LA) pressure or pulmonary capillary wedge pressure is significant in a stable state.8 9 Among several indices evaluated using Doppler echocardiography, E/e′-related indices such as E/e′ itself and (E/e′)/stroke volume (SV), that is, operant diastolic elastance (Ed), reportedly reflect LV diastolic function.10 11 The effective arterial elastance (Ea) was calculated as (0.9×systolic blood pressure)/SV.10 We previously reported age-related and sex-related differences in LV diastolic function relative to arterial elasticity among hypertensive patients with preserved LV ejection fraction (LVEF) and no history of HF.12 13 We found that the afterload-integrated diastolic index, Ed/Ea=(E/e′)/(0.9×systolic blood pressure), was significantly increased in older (aged ≥75 years) hypertensive women and was coincident with cardiac structural alterations. Recently, we reported that Ed/Ea is highly sustained during admission in patients with HFpEF,14 and the prognostic significance of Ed/Ea for all-cause mortality is valid for 1 year after discharge.15 During the follow-up period, the value of prognostic factors may change, especially in older patients, and the altered extent may affect prognosis. Therefore, we aimed to clarify the association with changes in Ed/Ea and prognosis in patients with HFpEF. The survival analysis was performed for 2 years by a landmark analysis.
Of the 771 patients with prognostic data recruited (2016.6–2019.4) from the Prospective Multicentre Observational Study of Patients with Heart Failure with Preserved Ejection Fraction (PURSUIT HFpEF) registry,16 we excluded 112 patients with poor or missing echocardiographic data, or with no measurement of systolic blood pressure around the examination of echocardiography. Therefore, we enrolled 659 patients (men/women, 296/363; mean age, 81 years) at discharge during the index hospitalisation with acute decompensated heart failure (ADHF); patients were enrolled based on the Framingham criteria, and if they met the criteria of LVEF ≥50% on transthoracic echocardiography (TTE) and N-terminal pro-brain natriuretic peptide (NT-proBNP) ≥400 pg/mL on admission. The PURSUIT HFpEF registry is being conducted with a prospective multicentre observational design, in which collaborating hospitals including one university hospital in the Osaka region of Japan collect demographic, clinical and outcome data from patients hospitalised due to congestive HFpEF (UMIN ID: UMIN000021831).16 We excluded patients with severe aortic stenosis, aortic regurgitation, mitral stenosis or mitral regurgitation due to structural changes in valves detected by TTE on admission from the first.
Data collection and follow-up/clinical outcome
We collected data on age, sex, height, weight, body mass index; data on comorbidities, including atrial fibrillation, hypertension, diabetes mellitus, dyslipidaemia and history of coronary artery disease were also collected. Oral medications were evaluated before discharge and 1 year after discharge.
Research cardiologists and specialised research nurses recorded patient data during hospital stays, and designated visits after discharge. After discharge, all patients were followed up at each hospital. Survival data were obtained by dedicated coordinators and investigators through direct contact with patients, their physicians at the hospital or in an outpatient setting, or via a telephone interview with their families or by mail. Data collection was performed using an electronic data capture system integrated into electronic medical records developed at the Osaka University.17 In-hospital data were entered into the system and were transferred to the data collection centre via a secure internet connection for processing and analysis. The primary end point of this study was all-cause mortality, or all-cause mortality and/or re-admission for HF. Collaborating hospitals were encouraged to enrol consecutive patients with HFpEF irrespective of treatment.
Patient laboratory data and echocardiography examination
Serum NT-proBNP and albumin levels, haemoglobin concentration and the estimated glomerular filtration rate were examined when patients were stable before discharge and at 1 year after discharge. TTE parameters were also obtained immediately before discharge (n=659) and at 1 year after discharge in some patients (n=344). The measurement of blood pressure (systolic and diastolic) and heart rate were performed around the examination of echocardiography, which were obtained according to the American Society of Echocardiography or European Society of Echocardiography guidelines.18 19 Volumetry was standardised using the modified Simpson’s rule. As a relative marker of LA pressure overload for estimating LV diastolic function, we examined an afterload-integrated Ed/Ea ([E/e′]/[0.9×systolic blood pressure]).12 As the relative markers of LAV overload, we evaluated LAV index (LAVI) and the ratio of SV to LAV.20
Patient and public involvement
Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Continuous variables are expressed as mean±SD, whereas categorical variables are presented as frequencies and percentages. Differences in categorical variables between the groups were assessed using the χ2 test or Fisher’s exact test, while those in continuous variables were assessed using the Student’s or Welch’s t-tests, as appropriate. Cut-off points of the prognostic factors for all-cause mortality and/or re-admission for HF were evaluated using a receiver operating characteristic (ROC) curve analysis. Survival curves were estimated using a Kaplan-Meier survival analysis, and the groups were compared using a log-rank test. A landmark analysis was performed for 2 years per year after discharge. A Cox proportional hazards regression analysis was evaluated by adjusting for age, sex, LAVI and left ventricular mass index (LVMI). The significance of Ed/Ea at 1 year after enrolment on prognosis was re-evaluated during the second year after discharge in Kaplan-Meier and Cox regression analyses as a categorical variable. P values <0.05 were considered statistically significant. All statistical analyses were performed using the EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).
Clinical and laboratory characteristics of patients with low and high Ed/Ea before discharge and 1 year after discharge
The cut-off point of Ed/Ea was evaluated in the ROC curve analysis before discharge for the prediction of all-cause mortality and/or re-admission for HF. Online supplemental table 1 shows the comparison of clinical and laboratory characteristics between patients with low and high Ed/Ea using the cut-off point for all-cause mortality before discharge and at 1 year after discharge. Before discharge, the differences in patient characteristics were nearly similar to the results shown previously.15 In terms of echocardiographic parameters, the LAVI, LVMI and E wave were significantly larger, and the ratio of SV to LAV and mean e′ were significantly smaller in patients with high Ed/Ea than in those with low Ed/Ea. LVEF did not differ significantly between patients with low and high Ed/Ea. We observed no significant differences in medications in each phase between the two groups (online supplemental table 1). When patients were re-divided into two groups by the value of Ed/Ea at 1 year after discharge, differences similar to those by the value of Ed/Ea before discharge were observed during the second year, although the number of patients examined and the extent of the differences observed were reduced. When the same examinations were performed with emphasis on low and high Ed/Ea using the cut-off point for all-cause mortality and/or re-admission for HF, nearly the same tendency of differences was observed before discharge and at 1 year after (online supplemental table 2). Online supplemental table 3 shows the differences in patient characteristics between those initially recruited at the time of their hospital admission (n=659) and those reviewed at 1 year after in outpatient department for all-cause mortality (n=344); the latter showed significantly higher blood pressure and LVEF, but lower Ed/Ea ratio. There were no differences in age (80±9 vs 81±10 years, p=0.356), male sex (46% vs 44%, p=0.809) and systolic blood pressure (129±19 vs 127±20 mm Hg, p=0.609) between patients with and without the data of Ed/Ea at 1 year after discharge.
Prognostic analysis using the value before discharge
A median follow-up time was 558 days. During the first year after enrolment, 71 patients (men/women: 28/43) had all-cause mortality, and 182 patients (men/women: 73/109) had all-cause mortality and/or re-admission for HF (online supplemental tables 1, 2). There were no between-sex differences in the incidence of all-cause mortality and all-cause mortality and/or re-admission for HF.
The Kaplan-Meier survival curve analysis during the first year (figure 1A) revealed that Ed/Ea was a significant prognostic factor for all-cause mortality (log-rank test, p<0.001). In a univariable Cox hazard analysis, Ed/Ea was also significant (table 1, p<0.0001). In the components of Ed/Ea, E (HR 2.346, 95% CI 1.286 to 4.281, p=0.005) and mean e′ (HR 0.552, 95% CI 0.339 to 0.898, p=0.016) levels were also significant prognostic factors for all-cause mortality in a univariable Cox hazard analysis. When a multivariable Cox hazard analysis was performed with adjustments for age, sex, LAVI and LVMI, the significance of Ed/Ea as a prognostic index was also observed (HR 2.409, 95% CI 1.414 to 4.104, p=0.001). The LAVI (log-rank test, p=0.104) and LVMI (log-rank test, p=0.186) were not significant for prognosis in the Kaplan-Meier analysis.
The results of the prognostic analysis for all-cause mortality and/or re-admission for HF were nearly the same as those for all-cause mortality (table 1): high Ed/Ea was a significant prognostic factor in a Kaplan-Meier survival analysis (figure 2A) and a Cox hazard analysis with adjustments for age, sex, LAVI and LVMI (HR 1.759, 95% CI 1.195 to 2.589, p=0.004). The mortality rate was significantly higher in patients with high Ed/Ea than in those with low Ed/Ea (online supplemental table 1). In patients with high Ed/Ea before discharge, no significant differences were observed in LVMI, and LVEF between those with and without all-cause mortality (table 2), or all-cause mortality and/or re-admission for HF (table 3).
In contrast, during 1‒2 years after discharge, high Ed/Ea before discharge was no longer a significant prognostic factor for all-cause mortality (Kaplan-Meier analysis, p=0.553, figure 1B; a univariable Cox hazard analysis, p=0.554, table 1) or all-cause mortality and/or re-admission for HF (Kaplan-Meier analysis, p=0.521, figure 2B; a univariable Cox hazard analysis, p=0.521, table 1).
Prognostic analysis using the Ed/Ea value at 1 year after discharge
During the second year after enrolment, 24 patients (men/women: 14/10) had all-cause mortality, and 43 patients (men/women: 19/24) had all-cause mortality and/or re-admission for HF among those who underwent echocardiographic examination at 1 year after discharge (online supplemental tables 1, 2).
When a landmark analysis was performed using the Ed/Ea value at 1 year after discharge, high Ed/Ea was still a significant prognostic factor during the second year in a Kaplan-Meier analysis for both all-cause mortality (p=0.009, figure 1C) and all-cause mortality and/or re-admission for HF (p=0.029, figure 2C). A Cox hazard analysis also revealed the prognostic significance of high Ed/Ea for all-cause mortality (p=0.012, table 1) and all-cause mortality and/or re-admission for HF (p=0.033, table 1). In patients with high Ed/Ea at 1 year after discharge, there were differences in LVMI and LVEF, but not LV volume, between those with and without all-cause mortality (table 2), or with and without all-cause mortality and/or re-admission for HF (table 3), although the incidence of hypertension was significantly lower in event-positive patients with high Ed/Ea. No differences were observed in LVMI (p=0.079) and LVEF (p=0.975), and the incidence of hypertension (p=0.855) between those with (n=10) and without (n=219) all-cause mortality in patients with low Ed/Ea at 1 year after discharge.
To assess changes in Ed/Ea related to prognosis, we divided patients into four groups according to changes in Ed/Ea from the value before discharge to that at 1 year after. The poorest group for all-cause mortality during the second year was that with low Ed/Ea before discharge and high Ed/Ea at 1 year after (group 2, figure 1D), and the best prognosis group during the second year was that with low Ed/Ea both before discharge and at 1 year after (group 0, figure 1D). Although no significant differences were observed in age, systolic blood pressure, the incidence of male sex and comorbidities, LVEF and LAVI between patients with low (group 0, figure 1D) and high (group 2, figure 1D) Ed/Ea assessed at 1 year after discharge among those with low Ed/Ea before discharge, LVMI was significantly higher in patients with high Ed/Ea than in those with low Ed/Ea (p=0.002) (table 4). There were no significant differences in Ed/Ea and LVMI at 1 year between group 2 patients with and without all-cause mortality. However, LVEF was significantly lower in group 2 patients with all-cause mortality than in those without all-cause mortality (46%±14% vs 61%±8%, p=0.007).
In the case of all-cause mortality and/or re-admission for HF, the prognosis of the divided groups was significantly different in the Kaplan-Meier analysis (p=0.036, figure 2D) and a univariable Cox hazard analysis (HR 1.312, 95% CI 1.015 to 1.697, p=0.038). The poorest group had high Ed/Ea both before discharge and at 1 year after, and the event rate in these patients was significantly higher than those with high Ed/Ea only before discharge (group 1 vs group 3, p=0.047, figure 2D).
The prognostic significance of Ed/Ea was valid only for 1 year in older patients with HFpEF in all-cause mortality and/or re-admission for HF. When re-analysis was performed using the value of Ed/Ea at 1 year, Ed/Ea was still a significant prognostic factor during the next 1 year.
Validity of an afterload-integrated diastolic index
Advanced age and female sex are associated with increases in arterial and ventricular stiffness even in the absence of cardiovascular disease.10 Increases in LV filling pressures owing to exercise correlate with changes in diastolic relaxation rates and arterial afterload.21 The linear slope of the single-beat diastolic pressure-volume relationship is defined as Ed.22 Exercise induces an increase in Ed evaluated invasively21 and non-invasively ([E/e′]/SV).23 However, in individual subjects, the non-invasive index E/e′ does not reliably track changes in left-side filling pressures induced by volume change24 or exercise,9 although these results were not evaluated according to changes in afterload. Arterial afterload could be assessed using effective arterial elastance (Ea=end-systolic pressure/SV).10 25 Exercise also increases Ea, but Ed/Ea does not seem to change significantly after stress according to the results of Borlaug et al.21 Changes in Ea in addition to those in diastolic elastance are compromised in HFpEF,26 and these changes are beyond the changes associated with ageing or hypertension.27 We recently reported that the LAVI and Ed/Ea are high in patients with HFpEF.14 Ed/Ea reflects the LA pressure relative to the systemic pressure,20 which can change minimally all day long under various circumstances with preserved LVEF. Although blood pressure can be significantly influenced by antihypertensive treatment or circadian rhythm, the E/e′ ratio would change accordingly, resulting in a subtle change in Ed/Ea. Thus, the Ed/Ea ratio may reflect global left-side heart function, including the atrioventricular-arterial interaction, under preserved LVEF conditions.
Difference in prognosis in relation to the follow-up duration in HFpEF
The pathology of HFpEF is complex and includes alterations in cardiac structure and function, systemic and pulmonary vascular abnormalities and comorbidities.28 The prevalence of and hospitalisation related to HFpEF are increasing and the growing older population causes further worsening of this trend. To determine the difference in prognosis in relation to the follow-up duration, we performed survival analysis using two different time points; during the first year after enrolment and 1‒2 years after enrolment. High Ed/Ea before discharge was a significant prognostic factor during the first year after discharge, but not during the 1‒2 years after discharge. However, using the Ed/Ea value at 1 year after discharge, high Ed/Ea was still significant for prognosis during the next year. The poorest prognosis for all-cause mortality during 1‒2 years after enrolment was observed in patients with low Ed/Ea before discharge and high Ed/Ea at 1 year after. These patients with high Ed/Ea values first observed after 1 year showed larger LVMI than those with low Ed/Ea even after 1 year. Furthermore, systolic function was reduced in patients with all-cause mortality. HFpEF generally does not transition to other conditions, such as HF with reduced LVEF or with mid-range LVEF, especially within 1 year in patients with relatively younger age (mean, 72 years).29 However, in some older patients with HFpEF, LVEF may be progressively reduced and poor prognosis may occur. The cause of death may differ between those in the first and second years. In the case of all-cause mortality and/or re-admission for HF, patients with persistent high Ed/Ea for 1 year after discharge showed the poorest prognosis during the second year.
The heterogeneity of the cardiac structure in patients with HFpEF is well known. Which type of clinical features is a candidate for pharmacological intervention to improve the prognosis of HFpEF remains undefined. The clinical significance of prognostic factors related to haemodynamics in patients with HFpEF may differ according to the follow-up period. In this sense, the role of NT-proBNP30 31 and LVEF29 32 in prognosis may be the same as that of Ed/Ea. In older patients, pathophysiological haemodynamic changes may markedly occur during 1 year after discharge, possibly leading to different haemodynamic conditions and prognosis that could not be estimated during enrolment. These issues are in accordance with the report that the most recent Kansas City Cardiomyopathy Questionnaire score is most strongly associated with subsequent death and cardiovascular hospitalisation in serial health status evaluations of patients with HFpEF.33
All-cause mortality rather than cardiac death was examined because the precise determination of cardiac death is challenging in older patients. The number of patients with obvious cardiac death was 31 out of 71 (44%) during the first year. In patients with HFpEF, the cause of death in >20% of the European Society of Cardiology Heart Failure Long-Term registry was unknown for 1 year,34 although the mortality rate was nearly the same as that in our results. However, our mortality rate was lower than that reported in other studies of patients with ADHF in Japan.35 This relatively low mortality rate may have affected our results regarding the prognostic significance of Ed/Ea.
We need to pay attention to precisely measure E/e′. The R-R interval is irregular in atrial fibrillation, and we measured the mean value of E/e′ among several beats in patients with atrial fibrillation in association with blood pressure that is not fixed in its value. However, E/e′ could change similar to blood pressure, and a large difference in the ratio of E/e′ to blood pressure does not occur under stable conditions. E/e′ exhibits a relative and not an absolute value of LA filling pressure, and Ed/Ea could show the performance of left-sided heart under preserved LVEF. The cut-off point of Ed/Ea (0.132) observed in the ROC curve analysis for all-cause mortality in patients with HFpEF was higher than that in patients with preserved LVEF without HF (mean±SD value of Ed/Ea, 0.100±0.030, mean age 80 years),12 indicating the accuracy of the cut-off point. Large-scale prospective studies are required to investigate the differences in the clinical significance of Ed/Ea for prognosis between younger patients with HFpEF and real-world older patients.
Time-sensitive prognostic performance of Ed/Ea, an afterload-integrated diastolic index, was observed in older patients with HFpEF. Measurement of serial non-invasive index such as Ed/Ea in clinical care can provide an updated assessment of prognosis.
Data availability statement
Data are available on reasonable request. Yes.
Patient consent for publication
The PURSUIT HFpEF registry was managed in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the ethics committee of each participating hospital. The protocol (Osaka University Clinical Research Review Committee, R000024414) was approved by the ethics committee of Yao Municipal Hospital (2016-No.0006). All participants provided written informed consent regarding the design and conduct of the study during the indexed hospitalisation. We performed only essential examinations in routine clinical practice.
The authors thank Nagisa Yoshioka, Kyoko Tatsumi, Satomi Kishimoto, Noriko Murakami and Sugako Mitsuoka for their excellent assistance with data collection.
Collaborators The OCVC-Heart Failure Investigators Shunsuke Tamaki, Tetsuya Watanabe and Takahisa Yamada, Osaka General Medical Center, Osaka, Japan; Takaharu Hayashi and Yoshiharu Higuchi, Osaka Police Hospital, Osaka, Japan; Masaharu Masuda, Mitsutoshi Asai and Toshiaki Mano, Kansai Rosai Hospital, Amagasaki, Japan; Hisakazu Fuji, Kobe Ekisaikai Hospital, Kobe, Japan; Daisaku Masuda, Yoshihiro Takeda, Yoshiyuki Nagai and Shizuya Yamashita, Rinku General Medical Center, Izumisano, Japan; Masami Sairyo, Yusuke Nakagawa and Shuichi Nozaki, Kawanishi City Hospital, Kawanishi, Japan; Haruhiko Abe, Yasunori Ueda, Masaaki Uematsu and Yukihiro Koretsune, National Hospital Organization Osaka National Hospital, Osaka, Japan; Kunihiko Nagai, Ikeda Municipal Hospital, Ikeda, Japan; Masamichi Yano, Masami Nishino and Jun Tanouchi, Osaka Rosai Hospital, Sakai, Japan; Yoh Arita and Shinji Hasegawa, Japan Community Health Care Organization Osaka Hospital, Osaka, Japan; Takamaru Ishizu, Minoru Ichikawa and Yuzuru Takano, Higashiosaka City Medical Center, Higashiosaka, Japan; Eisai Rin, Kawachi General Hospital, Higashiosaka, Japan; Yukinori Shinoda and Shiro Hoshida, Yao Municipal Hospital, Yao, Japan; Masahiro Izumi, Kinki Central Hospital, Itami, Japan; Hiroyoshi Yamamoto and Hiroyasu Kato, Japan Community Health Care Organization, Osaka Minato Central Hospital, Osaka, Japan; Kazuhiro Nakatani and Yuji Yasuga, Sumitomo Hospital, Osaka, Japan; Mayu Nishio and Keiji Hirooka, Saiseikai Senri Hospital, Suita, Japan; Takahiro Yoshimura and Yoshinori Yasuoka, National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Japan; Akihiro Tani, Kano General Hospital, Osaka, Japan; Yasushi Okumoto and Hideharu Akagi, Kinan Hospital, Tanabe, Japan; Yasunaka Makino, Hyogo Prefectural Nishinomiya Hospital, Nishinomiya, Japan; Toshinari Onishi and Katsuomi Iwakura, Sakurabashi Watanabe Hospital, Osaka, Japan; Nagahiro Nishikawa and Yoshiyuki Kijima, Japan Community Health Care Organization, Hoshigaoka Medical Center, Hirakata, Japan; Takashi Kitao and Hideyuki Kanai, Minoh City Hospital, Minoh, Japan; Wataru Shioyama and Masashi Fujita, Osaka International Cancer Institute, Osaka, Japan; Koichiro Harada, Suita Municipal Hospital, Suita, Japan; Masahiro Kumada and Osamu Nakagawa, Toyonaka Municipal Hospital, Toyonaka, Japan; Ryo Araki and Takayuki Yamada, Otemae Hospital, Osaka, Japan; Akito Nakagawa and Yoshio Yasumura, Amagasaki Chuo Hospital, Amagasaki, Japan; and Taiki Sato, Akihiro Sunaga, Bolrathanak Oeun, Hirota Kida, Takayuki Kojima, Yohei Sotomi, Tomoharu Dohi, Kei Nakamoto, Katsuki Okada, Fusako Sera, Shinichiro Suna, Hidetaka Kioka, Tomohito Ohtani, Toshihiro Takeda, Daisaku Nakatani, Hiroya Mizuno, Shungo Hikoso, Yasushi Matsumura and Yasushi Sakata, Osaka University Graduate School of Medicine, Suita, Japan.
Contributors SHo: conceptualisation, investigation, validation, writing—reviewing and editing; SHi: project administration, resources, software; YS: formal analysis, writing—original draft preparation; KT: data curation, visualisation; TM: visualisation, investigation; TS: validation, visualisation; MY: data curation, methodology; TH: data curation, resources; AN: writing—reviewing and editing; YN: data curation, methodology; TY: methodology, validation; YY: methodology, data curation; DN: resources, software; YS: project administration, supervision. Final approval of the version to be submitted: all authors.
Funding This work was funded by Roche Diagnostics and Fuji Film Toyama Chemical.
Competing interests None declared.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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