International Journal of Cardiology 181 (2015) 369–375

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A multimarker approach to diagnose and stratify heart failure Yunxia Wan a, Xi Xhang a,1, John J. Atherton b,c, Karam Kostner d, Goce Dimeski c,e, Chamindie Punyadeera a,⁎ a

The University of Queensland Diamantina Institute, The University of Queensland, The Translational Research Institute, Woolloongabba, Australia Department of Cardiology, Royal Brisbane and Women's Hospital, Australia c School of Medicine, The University of Queensland, Brisbane, Queensland, Australia d Department of Cardiology, Mater Adult Hospital, Brisbane, Queensland, Australia e Chemical Pathology Princess Alexandra Hospital, Brisbane, Queensland, Australia b

a r t i c l e

i n f o

Article history: Received 22 October 2014 Accepted 21 December 2014 Available online 23 December 2014 Keywords: NT-proBNP proBNP Classify heart failure

a b s t r a c t Background: We have previously demonstrated that circulating NT-proBNP is truncated at the N and C termini. Aims of this study are three-fold: firstly to determine whether the NT-proBNP levels correlate with NYHA functional classes when measuring with different antibody pairs; secondly to evaluate the diagnostic potential of ProBNP and; thirdly to investigate whether combining NT-proBNP assays with or without ProBNP would lead to better diagnostic accuracies. Methods: Plasma samples were collected from healthy controls (n = 52) and HF patients (n = 46). Customized AlphaLISA® immunoassays were developed and validated to measure the concentrations of proBNP and NTproBNP (with antibodies targeting 13–45, 13–76, 28–76). The diagnostic performance and predictive value of proBNP and NT-proBNP assays and their combinations were evaluated. Results: Plasma proBNP assay showed acceptable diagnostic performance. NT-proBNP13–76 assay is useful in diagnosing and stratifying HF patients. The diagnostic performance of NT-proBNP13–76 demonstrated improvement over commercial NT-proBNP tests. The combination of NT-proBNP13–76 with NT-proBNP28–76 assays gave the best diagnostic assay performance. Conclusion: Our results demonstrate that while there is major heterogeneity in circulating NT-proBNP, specific epitopes of the peptides are extraordinarily stable, providing ideal targets for clinically useful diagnostic assays. Future new clinical diagnostic clinical trials should include a multimarker approach rather than using a single marker to diagnose HF. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Heart failure (HF) is the leading cause of death worldwide, with 17.3 million deaths per year [1]. The incidence of HF is projected to increase by 2030 to 23.6 million due to an aging and a growing population [1]. Current medical therapy including angiotensin converting enzyme (ACE) inhibitors, beta blockers and mineralocorticoid receptor antagonists may slow the progression of disease and prolong survival [2], but are generally started late in the disease trajectory. Prevention of HF progression by early detection therefore becomes important in the reduction of HF disease burden. Echocardiography is the single most useful diagnostic test to evaluate a patient with suspected HF, including the evaluation of left ventricular (LV) systolic and diastolic dysfunction [2–4] to guide patient management. In addition, cardiac-specific biomarkers including plasma

⁎ Corresponding author at: Saliva Translational Research Group , Institute of Health and Biomedical Sciences, Queensland University of Technology, Room 603D, 60 Musk Avenue, Kelvin Grove, Queensland, 4059, Australia. E-mail address: [email protected] (C. Punyadeera). 1 Equal contributions.

http://dx.doi.org/10.1016/j.ijcard.2014.12.052 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

natriuretic peptides (N-terminal proBNP and BNP) are useful when the diagnosis is unclear and in circumstances where an echocardiogram cannot be performed in a timely fashion [5,6]. Ventricular wall stress and ischemia stimulate the cardiomyocytes to secrete proBNP [1–108 AA] [7], which is cleaved by furin (a ubiquitous serine protease), corin (cardiac serine protease) or other currently unknown proteases to BNP (77–108 AA) and NT-proBNP (1–76 AA) [8]. Plasma levels of BNP and NT-proBNP increase with disease severity and are associated with an adverse prognosis in patients with HF [5,6,9]. A recent review [10] summarized the diagnostic performance of the current Roche NTproBNP assay approved by the FDA (Roche Diagnostics, Basel, Switzerland) and it showed that the pooled sensitivity and specificity from 20 clinical studies were 80% and 61%, respectively, at the optimal cut points. BNP-32 and proBNP-108 (with and without glycosylation) have been detected in plasma from healthy controls and HF patients with atrial fibrillation [11]. We and others have identified various molecular forms of BNP and NT-proBNP in plasma including the presence and absence of glycoslation and truncated forms [12,13]. This molecular heterogeneity of NT-proBNP may be a confounding issue in measurements depending on the assay, and could affect diagnostic cut-offs, and reduce the ability to detect serial changes when using these peptide

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levels. Furthermore, recent studies have demonstrated that not only NTproBNP and BNP levels are increased in the plasma of patients with HF, but that the levels of the precursor molecule proBNP are elevated and correlate with NYHA functional class [11,14]. Indeed, the antibodies of the current BNP and to a lesser extent NT-proBNP immunoassays cross-react with proBNP [13,15]. A recent study has also confirmed previous findings by demonstrating that the antibody targeting BNP cross reacts with 50% of the total proBNP in plasma, but there was only 5% cross reactivity between the proBNP antibody and the NT-proBNP molecule [16]. Furthermore, when BNP and proBNP were measured in parallel, elevated levels of both of these peptides was a strong indicator for the development of HF [17]. Despite the benefits of measuring proBNP in circulation to diagnose HF, plasma proBNP is not used in clinical practice [18] and warrants further investigation. We have previously demonstrated that using antibody pairs targeting different parts of the full length NT-proBNP molecule (13–45 a.a.r, 13–76 a.a.r and 28–76 a.a.r) are present in plasma samples from HF patients facilitating HF diagnosis [12]. The aims of this study are three-fold: firstly to determine whether the plasma measurements of NT-proBNP levels using different antibodies pairs vary according to NYHA functional class (I–III), secondly to evaluate the diagnostic potential of plasma ProBNP and thirdly to determine whether by combining NT-proBNP levels or without ProBNP improves diagnostic accuracy compared to measuring full length NT-proBNP. 2. Materials and methods 2.1. Participants and sample collection The study complies with the 1975 Declaration of Helsinki. The project was approved by the University of Queensland Medical Ethical Institutional Board, Mater Medical Ethical Review Board, Royal Brisbane and Women's Hospital Research Governance. Two groups of healthy participants with no underlying cardiovascular diseases were recruited: young participants aged between 18 and 39 (years) and middle age group of participants aged N 40 years. Signed informed consents were obtained from all participants before sample collection. Cardiologists based on clinical symptoms, categorized HF patients according to their NYHA functional class (refer to Table 1). Volunteers were of European, Asian and Indian descent. Volunteers were asked to refrain from eating and drinking (except plain water) two hours prior to sample collection. Blood samples were collected in EDTA tubes (Greiner VACUETTE® # 454023, Greiner Bio-one, Graz, Austria) and centrifuged at 500 g at 24 °C for 15 min to separate plasma from blood cells. Each plasma aliquot was stored at −80 °C until analysis. 2.2. Biotinylation of proBNP and NT-proBNP monoclonal antibodies and coupling NT-proBNP monoclonal antibodies to acceptor beads for AlphaLISA® immunoassays A detailed description of the antibodies (manufacturer, immunogenicity, crossreactivity between natriuretic peptides) used for the AlphaLISA® immunoassays is listed in Supplementary Table 1. ProBNP monoclonal antibodies (mAbs) 50E1 and NT-proBNP mAbs 28F8 and 5B6 were biotinylated with N-hydroxysuccinimido-ChromaLink-biotin (2 mg/ml) (ProductNo: 9007-105K, Solulink, CA, USA) at molar ratio of 30:1 with an incubation time of 2 h. Unbound biotinylated proBNP and NT-proBNP monoclonal antibodies were removed using Zeba spin desalting columns (Product-No: 89882, Thermo Scientific Pierce, IL, USA) and biotinylated antibodies were stored at 4 °C. AlphaLISA® acceptor beads (Product-No: 6772003, Perkin Elmer®, Waltham, MA, USA) were mixed with 250 μL phosphate buffer solution and centrifuged at 16000 ×g for 15 min and the supernatant was discarded. For coupling of NT-proBNP monoclonal

antibodies 11D1 and 18H5 (0.1 mg, respectively) were added to acceptor beads, 1.25 μL of 10% Tween-20, 25 μg of NaBH3CN and PBS (0.13 M) and incubated for 24 h at 37 °C. After that, 10 μL of carboxy-methoxylamine (CMO) was added to the reaction and incubated for 1 h at 37 °C. Conjugated NT-proBNP monoclonal antibodies were collected by centrifugation at 16000 ×g for 15 min and the supernatants were discarded. The acceptor beads were washed twice in 1 mL of 0.1 M Tris–HCl (pH 8). Resuspension of conjugated NT-proBNP monoclonal antibodies to the acceptor beads in 1X PBS-0.05% Proclin 300 were performed before sonication. The purified NT-proBNP monoclonal antibodies conjugated to the acceptor beads were stored at 4 °C until analysis. 2.3. In-house development of AlphaLISA® immunoassays for the detection of proBNP, NT-proBNP13–76, NT-proBNP13–45, and NT-proBNP28–76 The AlphaLISA® assay technology (PerkinElmer®, USA) is a homogeneous, beadbased sandwiched immunoassay and offers many advantages over traditional EnzymeLinked Immunosorbent Assay (ELISA) including higher sensitivity (1 pg/mL), a requirement for only a low volume of samples, and enhanced reproducibility due to no wash steps [19]. In the presence of analytes (proBNP or NT-proBNP), the streptavidin coated donor beads and acceptor beads come into close proximity. Excitation of the donor beads will promote the release of oxygen singlet molecules thereby triggering a cascade of energy transfer to the acceptor beads, resulting in a sharp peak of light emission at 615 nm. NT-proBNP immunoreactivity were measured by using three NT-proBNP assays targeting different epitopes of the NT-proBNP molecule. The AlphaLISA® immunoassays for detecting proBNP and NT-proBNP in plasma were designed by targeting two different regions on proBNP and NT-proBNP molecules. The immunoassay nomenclature was based on the number of the first amino acid that the capture antibody binds to on the NT-proBNP molecule and the last amino acid that the detection antibody binds to on the NT-proBNP molecule of 1–76 amino acids: NT-proBNNP13–45 (18H513–20 and 11D128–45); NTproBNP28–76 (11D128–45 and 28F867–76); NT-proBNP13–76 (18H513–20 and 28F867–76). For the quantification of proBNP and NT-proBNP, a 12-point standard curve was generated using either the proBNP analyte (Product-No: NBC1-18491, Novus Biologicals®, UK) or NT-proBNP analyte (Product-No: 8NT2, Hytest Ltd, Finland) in pooled plasma from five healthy volunteers (to match the sample matrix) at a 1:1 ratio with HiBlock immunoassay buffer (PerkinElmer, Inc., USA). All the assays were performed in a 10 μL reaction volume (sample volume was 1 μL). For all immunoassays, the end concentration of acceptor beads and biotinylated antibodies were 10 μg/mL and 1 nM, respectively. Samples were loaded in triplicates in a 384 well ProxiPlates™ (Perkin Elmer®, Waltham, MA, USA). In brief the assay protocol for proBNP is as follows: firstly add acceptor beads to the sample and incubate for 30 min at room temperature (RT), followed by the addition of biotinylated proBNP and incubate for 1-hour. The final step is to add streptavidin donor beads, and incubate for 30 min at RT in the dark. Similarly, to determine the NT-proBNP concentrations in plasma, biotinylated antibody and acceptor beads were added to the samples and incubated for 1 h at RT followed by 30 minute incubation in the dark upon addition of streptavidin donor beads (final concentration 40 μg/mL). The only exception to the manufacturer protocol was to reduce total reaction volume from 50 μL to 10 μL. All experiments were performed by two trained individuals located in The University of Queensland Diamantina Institute. 2.4. Analytical performance of the in-house developed ProBNP and NT-proBNP AlphaLISA® immunoassays 2.4.1. Analyte recovery of proBNP, NT-proBNP13–76, NT-proBNP13−45 and NT-proBNP28–76 immunoassays To evaluate the suitability of AlphaLISA® immunoassays for measuring proBNP and NT-proBNP, two known concentrations of commercially available recombinant proBNP and NT-proBNP were spiked separately in pooled plasma collected from healthy controls (n = 5). Spiked and non-spiked pooled plasma were measured in the same AlphaLISA® immunoassay. The percentage recovery of the two spiked plasma samples was calculated with reference to corresponding un-spiked pooled plasma in a single AlphaLISA® immunoassay, using the following equation [20]

Percentage recovery ¼ Table 1 Clinical features of patients presenting with heart failure (HF). HF patients were classified according to the New York Heart Association functional classification. Two groups of healthy controls were selected to determine the influence of age on the circulating level of ProBNP and NT-proBNP fragment concentrations. Parameter

HF patients (n = 46)

Healthy controls (n = 52)

Age (years) N18 and b40 N40 Gender ratio (male: female) NYHA 1 NYHA 2 NYHA 3

70 (26–53) – 46 1.1 11 15 20

40 (21–49) 17 35 2.1 – – –

Calculated concentration of analyte in spiked samples − concentration of analyte in unspiked samples  100: Known concentration of spiked analyte

2.4.2. Intra-and inter-assay coefficient of variation of proBNP, NT-proBNP13–76, NT-proBNP13–45 and NT-proBNP28–76 immunoassays To determine intra- and inter-assay variations, triplicates of plasma samples from 46 healthy controls and 52 HF patients were analyzed in one AlphaLISA® proBNP and NTproBNP immunoassays and three independent AlphaLISA® proBNP and NT-proBNP immunoassays, respectively [21]. Intra- and inter-assay variations were expressed by intra- or inter-assay coefficient of variation (%CV). %CV was calculated using the following equation:

%CV ¼ ðMean of SDÞ  ðMeanÞ  100%:

Y. Wan et al. / International Journal of Cardiology 181 (2015) 369–375 2.4.3. Lower limit of detection for proBNP, NT-proBNP13–76, NT-proBNP13–45 and NT-proBNP28–76 immunoassays Limit of detection (LOD) for the proBNP and NT-proBNP immunoassays were calculated by taking the average count of blank samples made from pooled plasma of healthy controls plus 3 times the standard deviation of blank signal. The LOD for the plasma proBNP and NT-proBNP immunoassays were read from sigmoidal-dose response curves based on LOD signal counts derived from the equation [22]: LOD signal count ¼ ðaverage of blank signal countÞ þ 3 xðstandard deviation of blank signalÞ:

2.5. Stability of proBNP and NT-proBNP in plasma samples stored at room temperature and at 4 °C To determine the stability of proBNP and the NT-proBNP in the plasma, we spiked a known amount of recombinant proBNP and NT-proBNP in pooled plasma samples, respectively, and measured the levels of the two proteins in the samples at four different time points (0 h, 2 h, 8 h and 24 h) using our in house developed immunoassays. The first time point was chosen to parallel the assay incubation times. 2.6. Statistical analysis All statistical analyses were performed using GraphPad Prism 6 software version 6.01 (GraphPad Software Inc., La Jolla, CA, USA) and R (R Development Core Team. Vienna, Austria). All standard curves were generated by plotting the “raw” AlphaLISA® counts vs either the concentrations of proBNP or NT-proBNP standards using a 4-parameter logistic equation (sigmoidal dose–response curve with variable slope) and a 1/Y2 data weighting. For clinical characteristics (continuous variables) of the volunteers, Shapiro–Wilk normality test was performed in order to test for normal distribution before statistical analyses. Log transformation was performed to normalize the data. Ordinary one-way ANOVA and Holm–Sidak's multiple comparisons test were performed on unpaired and paired data with normal distribution to compare values between two groups. To investigate the relationship between proBNP and NT-proBNP levels, Pearson Correlation coefficients were calculated. The diagnostic accuracy of each AlphaLISA® immunoassays were described in terms of sensitivity, specificity, positive predictive value and negative predictive value obtained by generating ROC curves. Multivariate ROC curves [23] were generated to evaluate the diagnostic performance of the combination of two immunoassays.

3. Results 3.1. Participants A total of 52 healthy controls and 46 symptomatic HF patients were recruited from the University of Queensland, the Mater Adult Hospital or the Royal Brisbane and Women's Hospital in Brisbane, Australia from January 2012 to September 2013 (see Table 1). 3.2. proBNP and NT-proBNP stability in plasma To investigate the stability of NT-proBNP and proBNP in plasma, samples were spiked with NT-proBNP or proBNP and stored at RT and 4 °C. Aliquots of plasma samples were incubated at 0, 2, 8 and 24 h at both 4 °C and RT. Immunoreactive NT-proBNP was measured by assays targeting different epitopes of the peptide. Similarly, proBNP was also measured in the plasma. The stability of the analytes at each time point is presented as percentage recovery against its original concentration at 0 h (Supplementary Fig. 2). ProBNP and NT-proBNP analytes are stable for up to 8 h at 4 °C (less than 15% degradation). At RT these analytes are also stable for up to 2 h, which indicates that during the period of performing the assay, the analytes will not degrade and therefore have no influence on the overall assay performance. 3.3. Assay performance for proBNP, NT-proBNP13–76, NT-proBNP13–45 and NT-proBNP28–76 immunoassays The intra- and inter-assay coefficients of variations for the proBNP assay were below 10% and the LOD was below 1 pg/mL. For NTproBNP13–45, NT-proBNP28–76 and NT-proBNP13–76 immunoassays, the LOD were below 79 pg/mL, 27 pg/mL and 11 pg/mL respectively. The recoveries, inter- and intra-assay variations and LOD are summarized in Supplementary Table 2 for these immunoassays.

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3.4. The influence of age on the plasma concentrations of proBNP, NT-proBNP13–76, NT-proBNP13–45 and NT-proBNP28–76 It is well established that plasma NT-proBNP levels increase with age, especially in the elderly [24]. We therefore measured the proBNP and the NT-proBNP in the plasma collected from young and middle aged healthy controls. We found no significant difference in the plasma levels for the two proteins measured by proBNP and NT-proBNP13–45 immunoassays (p = 0.72 and p = 0.22 respectively) between young and middle aged people. However, significant differences were found in the plasma NT-proBNP concentrations using NT-proBNP28–76 and NT-proBNP13–76 immunoassays (p b 0.01 for these two assays) between young and middle aged individuals (see supplementary Fig. 3). 3.5. The concentrations of proBNP in plasma of healthy controls and HF patients ProBNP in healthy controls ranged from 1 pg/mL to 24 pg/mL with a median of 2 pg/mL (IQR, 1–7 pg/mL). ProBNP levels in HF patients ranged from 4–281 pg/mL (IQR 4–189 pg/mL; median = 5 pg/mL), 4–2273 pg/mL (IQR 4–120 pg/mL; median = 10 pg/mL) and 9–2068 pg/mL (IQR, 30–296 pg/mL; median = 81 pg/mL) for NYHA class 1, 2 and 3 respectively. There were significant differences in plasma ProBNP levels between the control and HF patients (Fig. 1). To evaluate the biological variation of the plasma proBNP in the participants, we calculated the %CV of plasma ProBNP level in each participant cohort [25]. The average plasma ProBNP %CV within the control group of 48 individuals was 119% and in 56 HF patients (all NYHA classes) was 54%. 3.6. The concentrations of NT-proBNP13–45, NT-proBNP28–76 and NT-proBNP13–76 in plasma from healthy controls and HF patients NT-proBNP 13–45 in healthy controls ranged from 5 pg/mL to 4322 pg/mL with a median of 134 pg/mL (IQR, 22–555 pg/mL). NT-proBNP13–45 levels in HF patients ranged from 18–3668 pg/mL (IQR 48–572 pg/mL; median = 107 pg/mL), 9–9770 pg/mL (IQR 66–1238 pg/mL; median = 294 pg/mL) and 165–14164 pg/mL (IQR, 841–3968 pg/mL; median = 2151 pg/mL) for NYHA class 1, 2 and 3 respectively. The significant difference can only be observed for healthy controls versus HF patients with NYHA class 3 (p b0.0001). (Fig. 2). The average plasma NT-proBNP13–45 %CV within the control group of 48 individuals was 59% and in 56 HF patients (all NYHA classes) was 28%. NT-proBNP 28–76 in healthy controls ranged from 9 pg/mL to 1957 pg/mL with a median of 67 pg/mL (IQR, 42–359 pg/mL). NTproBNP 28–76 levels in HF patients ranged from 22–1197 pg/mL (IQR 37–646 pg/mL; median = 156 pg/mL), 37–2869 pg/mL (IQR 257–820 pg/mL; median = 470 pg/mL) and 141–2995 pg/mL (IQR, 370–1339 pg/mL; median = 600 pg/mL) for NYHA class 1, 2 and 3 respectively. Significant differences for this assay were observed between healthy controls and HF patients with NYHA class 2 (p = 0.0181) and NYHA class 3 (p b 0.0001) symptoms (see Fig. 2). The average plasma NT-proBNP28–76 %CV within the control group of 48 individuals was 43% and in 56 HF patients (all NYHA classes) was 21%. NT-proBNP 13–76 in healthy controls ranged from 11 pg/mL to 3621 pg/mL with a median of 280 pg/mL (IQR, 27–267 pg/mL). NTproBNP13–76 levels in HF patients ranged from 55–26228 pg/mL (IQR 435–3127 pg/mL; median = 628 pg/mL), 165–71500 pg/mL (IQR 1404–6301 pg/mL; median = 2154 pg/mL) and 969–91458 pg/mL (IQR, 5045–28999 pg/mL; median = 14706 pg/mL) for NYHA class 1, 2 and 3 respectively. The concentrations of NT-proBNP13–76 in HF patients with NYHA class I (p b 0.0001), NYHA class II (p b 0.0001) and NYHA class III (p b 0.0001) were significantly higher than the levels in the controls. The average plasma NT-proBNP13–76 %CV within the control group

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Table 2 Diagnostic performance of proBNP, NT-proBNP13–45, NT-proBNP28–76, NT-proBNP13–76, and their combinations. The best combination of fragments (NT-proBNP13–76 and NTproBNP28–76) and their solo performances are highlighted in bold frame. Assay type

Sensitivity Specificity

Cutpoint AUC

Positive

Negative

predictive

predictive

(%)

(%)

proBNP

63.0

78.6

>0.547

0.86

72.1

70.8

NT-proBNP 13−45

47.8

85.7

>0.575

0.70

66.7

73.3

NT-proBNP 28−76

78.3

67.9

>0.388

0.74

79.2

66.7

NT-proBNP 13−76

89.1

89.3

>0.450

0.95

90.9

87.2

71.7

71.4

>0.447

0.85

75.5

67.4

60.7

>0.198

0.87

80.4

96.4

>0.579

0.95

87.1

95.0

NT-proBNP 13−76 + NT-proBNP 28−76

93.5

92.9

>0.390

0.97

94.5

91.5

NT-proBNP 13−45 + NT-proBNP 13−76

87.0

94.6

>0.619

0.96

89.8

93.0

NT-proBNP 13−45 + NT-proBNP 28−76

69.6

73.2

>0.442

0.74

74.5

68.1

proBNP + NTproBNP 13−76 + NTproBNP 28−76

91.3

94.6

>0.2247

0.96

93.0

92.7

proBNP + NTproBNP 13−45 + NTproBNP 13−76

84.8

92.9

>0.300

0.95

88.1

90.7

62.5

>0.247

0.86

value (%)

proBNP + proBNP 28−76

NT-

proBNP + proBNP 13−45

NT- 100

proBNP + proBNP 13−76

NT-

100

value (%)

67.6

and specificity for proBNP were 68% and 80% respectively; for NTproBNP13–76 it was 89% and 89% respectively (refer to Table 2). Using multivariate ROC model [23], we combined two NT-proBNP immunoassays and proBNP. We observed an improvement in the diagnostic performance (Fig. 3). The combination of NT-proBNP13–76 and, NTproBNP28–76 gave the best AUC value (0.97) compared with other combinations (refer to Table 2) (Fig. 3). 3.8. Correlations of proBNP with NT-proBNP The NT-proBNP level measured by our in-house immunoassays, NTproBNP13–45, NT-proBNP28–76 and NT-proBNP13–76, were all significantly correlated with proBNP levels (see Supplementary Fig. 4). The Pearson r values and P values for the correlation between the above mentioned NT-proBNP immunoassays were r = 0.41 and p b 0.0001, r = 0.72 and p b 0.0001, r = 0.45 and p b 0.0001, respectively. 4. Discussion

proBNP + NT- 100 proBNP 13−45 + NTproBNP 28−76

100

68.7

of 48 individuals was 35% and in 56 HF patients (all NYHA classes) was 18%. 3.7. The receiver operator characteristic curves for ProBNP and NT-proBNP immunoassays We compared proBNP and NT-proBNP immunoassays diagnostic performances by generating ROC curves (Figs. 1 and 2). NTproBNP13–76 gave the best diagnostic accuracy. The clinical sensitivity

Among CVDs, HF is projected to have the highest increase in population prevalence over the coming years and warrants early detection methods coupled to targeted therapy. The findings from this study, along with previously published work from our group [12] and others [26,27] have demonstrated that there is molecular heterogeneity of NT-proBNP in plasma samples from HF patients [13,28–30]. To our knowledge this is the first study to aim at detectin proBNP as well as NT-proBNP in circulation using immunoassays targeting different epitopes of the NT-proBNP molecule to diagnose and stratify HF patients. We have also demonstrated that the diagnostic performance of NTproBNP 13–76 immunoassay showed improvements over the commercial NT-proBNP1–76 assay based on previously published literature (pool sensitivity and specificity 80% and 61% respectively [12]). This is because we have chosen antibodies directed away from the N and C termini (truncated as we have previously published) as well as avoiding the glycosylated regions within the NT-ProBNP molecule [12,31]. When combining two of the NT-proBNP immunoassays (NT-proBNP 13–76 and NT-proBNP 28–76), we observed an improved diagnostic performance. In addition, plasma proBNP assay showed acceptable diagnostic performance. The widely accepted theory is that the “healthy” heart releases proteolytically processed BNP 1–32 and NT-proBNP, whereas the “diseased” heart secretes high amounts of unprocessed/glycosylated proBNP 1–108 or degraded BNPs which are devoid of biological activity [32]. In contrast, circulating proBNP1–108 has recently been identified in plasma collected from healthy individuals, indicating that the “healthy” heart also secretes unprocessed proBNP 1–108 [33]. However, a previous study [34] demonstrated that circulation BNP levels in HF patients measured by immunoassays does not correlate well with the full

Fig. 1. (A) plasma proBNP concentrations represented as Box and Whisker plots. Lower and upper ends of boxes represent the 25th percentile and the 75th percentile. Whiskers represent the 5th and 95th percentiles and filled circles are outliers. Significant differences in plasma proBNP concentrations between HF patients and healthy controls are indicated by *(p b0.05), **(p b 0.01), ***(p b 0.001) and ****(p b 0.0001). (B) Receiver operating characteristic curve for plasma proBNP concentrations.

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Fig. 2. Box and whisker plots for (A) NT-proBNP 13–45 (B) NT-proBNP 28–76 (C) NT-proBNP13–76. Lower and upper ends of boxes represent the 25th percentile and the 75th percentile. Whiskers represent the 5th and 95th percentiles and filled circles are outliers. Significant difference of NT-proBNP concentration against healthy controls are as indicated by *(p b0.05), **(p b 0.01), ***(p b 0.001) and ****(p b 0.0001); receiver operating characteristic curves for four NT-proBNP immunoassays (D) NT-proBNP 13–45 (E) NT-proBNP28–76 (F) NT-proBNP13–76 generated from AlphaLISA® immunoassays.

length BNP (1–32 a.a.r) measured by mass spectrometry, instead the immunoassay results correlated better with degraded BNP fragments (BNP3–32, BNP4–32 and BNP5–32). This indicated that in HF patients there may be limited presence of full length BNP with intact biological activity. We could perhaps speculate that the measurement of degraded

BNP fragments may provide better estimations of circulating BNP concentrations. Analytical problems have also been documented for circulating NT-proBNP, relating especially to assay specificity and analyte stability [26,27,35,36]. As an example, antisera are often produced raised against synthetic fragments of NT-proBNP without prior

Fig. 3. Receiver operating characteristic curves for combined NT-proBNP fragments and proBNP: (A) proBNP + NT-proBNP13–45 (B) proBNP + NT-proBNP28–76 (C) proBNP + NTproBNP13–76 (D) NT-proBNP13–76 + NT-proBNP28–76 (E) NT-proBNP13–45 + NT-proBNP13–76 (F) NT-proBNP13–45 + NT-proBNP28–76 (G) proBNP + NT-proBNP13–76 + NT-proBNP28–76 (H) proBNP + NT-proBNP13–45 + NT-proBNP13–76 (I) proBNP + NT-proBNP13–45 + NT-proBNP28–76.

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knowledge of their applicability in recognizing the full-length peptide. More importantly, the results generated using synthetic NT-proBNP peptides (unglycosylated forms) are not directly comparable with the endogenous NT-proBNP which is glycosylated. Furthermore, there is no consensus about the exact forms of circulating peptides derived from proBNP [37–39]. The stability of an analyte (NT-ProBNP or ProBNP) is also an important factor to ensure the reproducibility and reliability of a clinical test [40]. Our results demonstrated that the analytes (proBNP and NT-proBNP) were stable for the period of our in-house developed immunoassays. This indicated that our in-house developed assays have the realistic potential to be translated into clinical setting. Several recent studies have combined the measurements of BNP and proBNP levels with its derivatives [17,41,42] to address the above mentioned challenges. The rationale behind this approach is to observe significant changes in BNP/ProBNP levels and to determine whether the changes in one biomarker affect the concentration of other biomarkers in circulation. Dries et al. has incorporated BNP and proBNP to diagnose chronic systolic HF and has demonstrated that by combining proBNP and BNP tests provided additional information in determining risk of adverse clinical outcomes [17]. In addition, Nishikimi et al. compared proBNP to BNP ratio and found that BNP-32 and proBNP-108 was elevated in the plasma collected from HF patients and that the proBNP/ total BNP ratio increased during ventricular overload [11]. Similarly, in this study, we have also demonstrated relatively good assay performance when combing NT-proBNP immunoassays with ProBNP. Using multivariate ROC model, the combination of NT-proBNP13–76 and NT-proBNP28–76 assays gave an excellent diagnostic performance. This suggests that a multi-biomarker panel may be useful for the diagnosis of HF. In conclusion, our results demonstrated that while there is heterogeneity in circulating NT-proBNP, specific epitopes of the peptides are more stable, providing ideal targets for clinically useful diagnostic assays. Our NT-proBNP13–76 assay was able to stratify HF patients implicating that this specific area of NT-proBNP may be of importance in monitoring HF. In addition, the utility of using the precursor molecule, proBNP may provide another avenue for exploring the clinical utility of natriuretic peptides in clinical practice, and as demonstrated in this study the use of multimarkers panel needs to be explored further to better diagnose HF. Hence future new clinical diagnostic assays for HF should include multimarker approach rather than using a single marker to assess HF patients. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2014.12.052. Conflicts of interest None. Financial disclosure for all authors None. Acknowledgments The authors would like to acknowledge the financial support from the University of Queensland Foundation Research Excellence Scheme, Queensland Centre for Head and Neck Cancer, University of Queensland Diamantina Institute internal strategic funds and the Queensland Government Smart Futures Fellowship Program (QGSFF). In addition, authors wish to acknowledge the clinical support from the Mater Adult Hospital and Royal Brisbane Women Hospital. The authors would also like to acknowledge Mr. Muhammad Tarmizi Soleh for collecting some of the samples that were used in this study and Dr. Anne Bernard from QFAB Bioinformatics for providing guidance on statistical analysis.

References [1] A. Alwan, Global Status Report on Noncommunicable Diseases 2010, World Health Organization, 2011. [2] J.J. McMurray, S. Adamopoulos, S.D. Anker, A. Auricchio, M. Bohm, K. Dickstein, et al., ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC, Eur. Heart J. 33 (2012) 1787–1847. [3] M. Davies, F. Hobbs, R. Davis, J. Kenkre, A.K. Roalfe, R. Hare, et al., Prevalence of left-ventricular systolic dysfunction and heart failure in the Echocardiographic Heart of England Screening study: a population based study, Lancet 358 (2001) 439–444. [4] M.M. Redfield, S.J. Jacobsen, J.C. Burnett Jr., D.W. Mahoney, K.R. Bailey, R.J. Rodeheffer, Burden of systolic and diastolic ventricular dysfunction in the community: appreciating the scope of the heart failure epidemic, JAMA 289 (2003) 194–202. [5] K. Dickstein, A. Cohen-Solal, G. Filippatos, J.J. McMurray, P. Ponikowski, P.A. PooleWilson, et al., ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM), Eur. Heart J. 29 (2008) 2388–2442. [6] M. Jessup, W.T. Abraham, D.E. Casey, A.M. Feldman, G.S. Francis, T.G. Ganiats, et al., 2009 focused update: ACCF/AHA Guidelines for the Diagnosis and Management of Heart Failure in Adults: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: developed in collaboration with the International Society for Heart and Lung Transplantation, Circulation 119 (2009) 1977–2016. [7] M. Weber, C. Hamm, Role of B-type natriuretic peptide (BNP) and NT-proBNP in clinical routine, Heart 92 (2006) 843–849. [8] A.G. Semenov, N.N. Tamm, K.R. Seferian, A.B. Postnikov, N.S. Karpova, D.V. Serebryanaya, et al., Processing of pro-B-type natriuretic peptide: furin and corin as candidate convertases, Clin. Chem. 56 (2010) 1166–1176. [9] A.S. Maisel, K. Nakao, P. Ponikowski, W.F. Peacock, M. Yoshimura, T. Suzuki, et al., Japanese-Western consensus meeting on biomarkers, Int. Heart J. 52 (2011) 253–265. [10] R.A. Booth, S.A. Hill, A. Don-Wauchope, P.L. Santaguida, M. Oremus, R. McKelvie, et al., Performance of BNP and NT-proBNP for diagnosis of heart failure in primary care patients: a systematic review, Heart Fail. Rev. 19 (2014) 439–451. [11] T. Nishikimi, N. Minamino, M. Ikeda, Y. Takeda, K. Tadokoro, I. Shibasaki, et al., Diversity of molecular forms of plasma brain natriuretic peptide in heart failure–different proBNP-108 to BNP-32 ratios in atrial and ventricular overload, Heart 96 (2010) 432–439. [12] J.Y. Foo, Y. Wan, B.L. Schulz, K. Kostner, J. Atherton, J. Cooper-White, et al., Circulating fragments of N-terminal pro B-type natriuretic peptides in plasma of heart failure patients, Clin. Chem. 59 (2013) 1523–1531. [13] K.R. Seferian, N.N. Tamm, A.G. Semenov, A.A. Tolstaya, E.V. Koshkina, M.I. Krasnoselsky, et al., Immunodetection of glycosylated NT-proBNP circulating in human blood, Clin. Chem. 54 (2008) 866–873. [14] S.W. Waldo, J. Beede, S. Isakson, S. Villard-Saussine, J. Fareh, P. Clopton, et al., Pro-Btype natriuretic peptide levels in acute decompensated heart failure, J. Am. Coll. Cardiol. 51 (2008) 1874–1882. [15] H. Shimizu, K. Masuta, H. Asada, K. Sugita, T. Sairenji, Characterization of molecular forms of probrain natriuretic peptide in human plasma, Clin. Chim. Acta 334 (2003) 233–239. [16] F. Roubille, D. Delseny, J.-P. Cristol, D. Merle, N. Salvetat, C. Larue, et al., Depletion of proBNP1-108 in patients with heart failure prevents cross-reactivity with natriuretic peptides, PLoS One 8 (2013) e75174. [17] D.L. Dries, B. Ky, A.H. Wu, J.E. Rame, M.E. Putt, T.P. Cappola, Simultaneous assessment of unprocessed ProBNP1-108 in addition to processed BNP32 improves identification of high-risk ambulatory patients with heart failure, Circ. Heart Fail. 3 (2010) 220–227. [18] H.K. Gaggin, J.L. Januzzi Jr., Biomarkers and diagnostics in heart failure, Biochim. Biophys. Acta 2013 (1832) 2442–2450. [19] C. Punyadeera, G. Dimeski, K. Kostner, P. Beyerlein, J. Cooper-White, One-step homogeneous C-reactive protein assay for saliva, J. Immunol. Methods 373 (2011) 19–25. [20] K.M.T.J. Jaedicke, P.M. Preshaw, Validation and quality control of elisas for the use with human saliva samples, J. Immunol. Methods 377 (2012) 62–65. [21] B. Dieplinger, M Egger, C Gabriel, W Poelz, E Morandell, B Seeber, et al, Analytical characterization and clinical evaluation of an enzyme-linked immunosorbent assay for measurement of afamin in human plasma. Clin Chim Acta. 425 (2013) 236–241. [22] D.A.P.T. Armbruster, Limit of blank, limit of detection and limit of quantitation, Clin. Biochem. Rev. 29 (2008) S49–S52. [23] E.K. Shultz, Multivariate receiver-operating characteristic curve analysis: prostate cancer screening as an example, Clin. Chem. 41 (1995) 1248–1255. [24] P. Nadrowski, J. Chudek, T. Grodzicki, M. Mossakowska, M. Skrzypek, A. Wiecek, et al., Plasma level of N-terminal pro brain natriuretic peptide (NT-proBNP) in elderly population in Poland — the PolSenior study, Exp. Gerontol. 48 (2013) 852–857. [25] C.G. Fraser, E.K. Harris, Generation and application of data on biological variation in clinical chemistry, Crit. Rev. Clin. Lab. Sci. 27 (1989) 409–437.

Y. Wan et al. / International Journal of Cardiology 181 (2015) 369–375 [26] F.S. Apple, Standardization of cardiac markers, Scand. J. Clin. Lab. Invest. Suppl. 240 (2005) 107–111. [27] F.S. Apple, M. Panteghini, J. Ravkilde, J. Mair, A.H. Wu, J. Tate, et al., Quality specifications for B-type natriuretic peptide assays, Clin. Chem. 51 (2005) 486–493. [28] U. Schellenberger, J. O'Rear, A. Guzzetta, R.A. Jue, A.A. Protter, N.S. Pollitt, The precursor to B-type natriuretic peptide is an O-linked glycoprotein, Arch. Biochem. Biophys. 451 (2006) 160–166. [29] M. Ala-Kopsala, J. Magga, K. Peuhkurinen, J. Leipala, H. Ruskoaho, J. Leppaluoto, et al., Molecular heterogeneity has a major impact on the measurement of circulating Nterminal fragments of A- and B-type natriuretic peptides, Clin. Chem. 50 (2004) 1576–1588. [30] M. Ala-Kopsala, H. Ruskoaho, J. Leppaluoto, L. Seres, R. Skoumal, M. Toth, et al., Single assay for amino-terminal fragments of cardiac A- and B-type natriuretic peptides, Clin. Chem. 51 (2005) 708–718. [31] T. Nishikimi, M. Ikeda, Y. Takeda, T. Ishimitsu, I. Shibasaki, H. Fukuda, et al., The effect of glycosylation on plasma N-terminal proBNP-76 levels in patients with heart or renal failure, Heart 98 (2012) 152–161. [32] A. Clerico, S. Vittorini, C. Passino, Chapter 2 — circulating forms of the B-type natriuretic peptide prohormone: pathophysiologic and clinical considerations, in: S.M. Gregory (Ed.), Advances in Clinical Chemistry, Elsevier, 2012, pp. 31–44. [33] K.R. Seferian, N.N. Tamm, A.G. Semenov, K.S. Mukharyamova, A.A. Tolstaya, E.V. Koshkina, et al., The Brain Natriuretic Peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure, Clin. Chem. 53 (2007) 866–873. [34] W.L. Miller, M.A. Phelps, C.M. Wood, U. Schellenberger, A. Van Le, R. Perichon, et al., Comparison of mass spectrometry and clinical assay measurements of circulating

[35] [36] [37]

[38]

[39] [40] [41]

[42]

375

fragments of B-type natriuretic peptide in patients with chronic heart failure, Circ. Heart Fail. 4 (2011) 355–360. J. Ordonez-Llanos, P.O. Collinson, R.H. Christenson, Amino-terminal pro-B-type natriuretic peptide: analytic considerations, Am. J. Cardiol. 101 (2008) 9–15. A. Clerico, M. Emdin, Diagnostic accuracy and prognostic relevance of the measurement of cardiac natriuretic peptides: a review, Clin. Chem. 50 (2004) 33–50. P.J. Hunt, E.A. Espiner, M.G. Nicholls, A.M. Richards, T.G. Yandle, The role of the circulation in processing pro-brain natriuretic peptide (proBNP) to amino-terminal BNP and BNP-32, Peptides 18 (1997) 1475–1481. P.J. Hunt, T.G. Yandle, M.G. Nicholls, A.M. Richards, E.A. Espiner, The amino-terminal portion of pro-brain natriuretic peptide (Pro-BNP) circulates in human plasma, Biochem. Biophys. Res. Commun. 214 (1995) 1175–1183. J.P. Goetze, Biochemistry of pro-B-type natriuretic peptide-derived peptides: the endocrine heart revisited, Clin. Chem. 50 (2004) 1503–1510. F. Peters, Stability of analytes in biosamples — an important issue in clinical and forensic toxicology? Anal. Bioanal. Chem. 388 (2007) 1505–1519. F. Macheret, G. Boerrigter, P. McKie, L. Costello-Boerrigter, B. Lahr, D. Heublein, et al., Pro-B-type natriuretic peptide(1–108) circulates in the general community: plasma determinants and detection of left ventricular dysfunction, J. Am. Coll. Cardiol. 57 (2011) 1386–1395. T. Mueller, A. Gegenhuber, W. Poelz, M. Haltmayer, Head-to-head comparison of the diagnostic utility of BNP and NT-proBNP in symptomatic and asymptomatic structural heart disease, Clin. Chim. Acta 341 (2004) 41–48.