There is a need for cardiac biomarkers for the diagnosis of heart failure and to assist risk stratification and monitoring of therapy. Natriuretic peptides are currently widely used to assist diagnosis. The new marker sST2 has potential to provide prognostic information and to monitor therapy.

by Dr Stuart J. Bennett and Dr Ruth M. Ayling

Introduction
Heart failure (HF) is a complex clinical syndrome of symptoms and signs that suggest impairment of the heart supporting physiological circulation and affects approximately 900 000 people in the UK [1]. The incidence and prevalence increase with age and the prevalence is expected to rise in future as a result of the ageing population and improved survival of people with ischaemic heart disease. The symptoms (e.g. dyspnoea, fatigue and ankle swelling) and signs (e.g. pulmonary crackles) are not sensitive or specific for HF so diagnosis remains challenging. Investigations such as chest X-ray and echocardiogram are used for this purpose but attention is increasingly being focused on cardiac biomarkers as a tool to assist diagnosis and management.

An ideal biomarker would enable underlying, and hence potentially reversible, causes of HF to be identified and would differentiate between the presence and absence of HF. It would also enable an estimation of severity and disease prognosis and could be used for monitoring of treatment. In addition, an ideal marker should be widely available at reasonable cost and short notice and the assay should be appropriately robust. Various cardiac biomarkers have been proposed and represent a wide range of pathophysiological mechanisms of cardiovascular disease, for example markers of myocardial stress (natriuretic peptides), myocyte injury (troponin), inflammation (C-reactive protein) and myocyte remodelling (galectin-3, sST2). Of these, only the natriuretic peptides are in routine clinical use as markers of HF and this review will describe their use in more detail, together with that of the new biomarker, sST2.

The natriuretic peptides
The natriuretic peptides B-type natriuretic peptide (BNP) and N-terminal (NT)-proBNP are currently the most commonly used markers of HF. BNP is derived from a 134 amino acid precursor, preproBNP, which is synthesized in cardiac myocytes in response to ventricular stretch and stress. On release, a 26 amino acid signal peptide is cleaved from the N-terminus to produce proBNP, which is then further cleaved by a membrane-bound protease into a 76 amino acid N-terminal-proBNP (NT-proBNP) and the active C-terminal 32 amino acid hormone (BNP). The most common use of natriuretic peptides is in the diagnosis of HF. In the ‘breathing not properly’ trial, BNP was found to have a sensitivity of 90% and a specificity of 76%, using a cut-off of 100 ng/L, for diagnosing HF in patients presenting to the emergency department with breathlessness [2]. The National Institute for Health Care Excellence (NICE) suggests that in new suspected acute HF, a BNP concentration <100 ng/L or NT-proBNP concentration <300 ng/L are appropriate thresholds to rule out the diagnosis [3]. In chronic HF, a BNP concentration <100 ng/L or NT-proBNP of <400 ng/L makes HF unlikely [4]. Defining rule-in cut-offs for HF is more complicated; a cut-off of >400 ng/L has been proposed for BNP and age-related cut-offs for NT-proBNP of >450 ng/L for <50 years, >900 ng/L for 50–75 years and >1800 ng/L for >75 years [5]. As natriuretic peptide concentrations can provide prognostic information, NICE advise referral for further investigation within 6 weeks if BNP is 100–400 ng/L or NT-proBNP is 400–2000 ng/L or within 2 weeks if BNP is >400 ng/L or NT-proBNP is >2000 ng/L. There is some evidence to suggest that measurement of natriuretic peptides may be of use in monitoring therapy. A meta-analysis of six randomized controlled trials found a reduction in all-cause mortality with natriuretic-peptide-guided therapy [6]. However, optimal monitoring schedules and targets are not yet established.

NT-proBNP may have certain practical advantages as, unlike BNP, it can be measured in serum as well as plasma and has superior stability. However, BNP and NT-proBNP concentrations should always be interpreted with due regard to the clinical setting. In addition to age and female sex, factors other than HF that may increase baseline concentrations include myocardial ischemia, left ventricular hypertrophy, pulmonary embolism, liver failure, sepsis and renal failure. Conversely, BNP and NT-proBNP concentrations may be lowered in the presence of obesity (BMI >35kg/m2) and some medications (e.g. angiotensin converting enzyme inhibitors, β-blockers, angiotensin receptor blockers, aldosterone antagonists).

Soluble ST2
ST2 (growth stimulating expressed gene 2) is a member of the interleukin (IL)-1 receptor family and has both membrane-bound (ST2L) and soluble (sST2) forms, both forms can bind IL-33, which is released in response to stretch. The source of circulating sST2 was presumed to be the myocardium but it may be that in cardiac disease the major source is vascular endothelium. When circulating sST2 is low, its ligand, IL-33, binds to ST2L which has a protective effect. When sST2 concentrations are raised there is competitive binding to IL-33, with less binding to ST2L reducing the amount available for cardioprotection. This leads to fibrosis and hypertrophy with reduced cardiac function. Binding to ST2L promotes signalling that protects against fibrosis and hypertrophy, whereas binding to sST2 acts as a decoy receptor, tending to promote fibrosis and hypertrophy. The potential clinical use of sST2 was first highlighted in animal studies by the findings of induced sST2 mRNA in cultured heart muscle after mechanical strain and raised circulating concentrations after myocardial infarction [7]. In human subjects, raised sST2 was associated with poor outcome after myocardial infarction but was not of value for diagnosis of the condition [8], leading to a focus on its use as a biomarker for HF.

Various methods of measurement have been described for sST2. The Presage® assay (Critical Diagnostics, CA, USA) has been extensively evaluated [9] and has received FDA and CE approval. However, neither this nor other commercially available assays are rigorously standardized. The Presage® assay is a quantitative sandwich ELISA using two monoclonal antibodies to mouse ST2. Either serum or plasma is suitable for analysis and samples remain suitable for analysis if stored for up to 48 hours at 20°C. A suggested cut-off for sST2 in chronic HF is 35 ng/mL but more recently sex-related differences, higher in males, have been reported [10]. A recent development is the availability of a point-of-care test, allowing rapid sST2 testing.

In the PRIDE study, sST2 was not found to be a useful tool for the diagnosis of HF but does have potential for risk stratification in undiagnosed dyspnoea [11]. A number of studies have examined the use of sST2 in acute HF and found it to be associated with the severity of HF and with poor outcome and to provide independent and additive prognostic information in addition to other markers, e.g. natriuretic peptides and troponins [12]. In chronic HF, elevated sST2 concentrations are strongly associated with HF severity and with increased risk of cardiac death, cardiovascular events and hospitalization [13]. sST2 has been shown to be equivalent to natriuretic peptides in classifying risk in chronic HF and if used in addition improves risk stratification [14].
The usefulness of serial measurements of sST2 has been examined for monitoring HF [15], suggesting it correlates with clinical course and has potential as a marker for monitoring response to therapy. sST2 appears not to have particular advantages in the diagnosis of HF but can add value in identifying patients at high risk and in whom advanced disease management may be advantageous.

Conclusion
The importance of biochemical tests in contributing to HF diagnosis is evidenced by the incorporation of BNP and NT-proBNP into current NICE guidelines. There is a desire to find suitable markers for use in prognosis and monitoring enabling intensified management of high risk patients and tailoring of treatment regimens. sST2 is a marker of myocardial fibrosis and cardiac stretch and data exist to demonstrate its prognostic value, alone or in combination with natriuretic peptides. The existence of an ELISA method that can be used on a standalone analyser in the setting of a clinical laboratory means its routine use in HF management is now a distinct possibility and the advent of a point-of-care assay is likely to lead to further clinical opportunities for its use.

References
1. Cleland J, Dargie H, Hardman S, McDonagh T, Mitchell P. National Heart Failure Audit. British Society for Heart Failure. 2012; https://www.ucl.ac.uk/nicor/audits/heartfailure/documents/annualreports/hfannual11-12.pdf.
2. Maisel AS, Krishnaswamy, Nowak RM, McCord J, Hollander JE, Duc P, Omland T, Storrow AB, Abraham WT, et al. Rapid measurement of B-type natriuretic peptide in emergency diagnosis of heart failure. N Eng J Med. 2002; 347: 161–167.
3. NICE. Acute Heart Failure: diagnosis and management. 2014; https://www.nice.org.uk/guidance/cg187.
4. NICE. Chronic heart failure in adults: management. 2010; https://www.nice.org.uk/Guidance/CG108.
5. Maisel A, Mueller C, Adams K Jr, Anker SD, Aspromonde N, Cleland JG, Cohen-Solal A, Dahlstrom U, DeMaria A, et al. State of the art: using natriuretic peptide levels in clinical practice. Eur J Heart Fail. 2008; 10: 824–839.
6. Felker GM, Hasseblad V, Hernandez A, O’Connor CM. Biomarker-guided therapy in chronic heart failure: a meta-analysis of randomized controlled trials. Am Heart J. 2009; 158: 422–430.
7. Weinberg EO, Shimpo M, DeKeulenaer GW, MacGillivray C, Shin-Ichi T, Solomon SD, Rouleau JL, Lee RT. Expression and regulation of ST2, an interleukin-1 receptor family member, in cardiomyocytes and myocardial infarction. Circulation 2002; 106: 2961–2966.
8. Brown Am, Wu AH, Clopton P, Robey JL, Hollander JE. ST2 in emergency department chest pain patients with potential acute coronary syndromes. Ann Emerg Med. 2007; 50: 153–158.
9. Dieplinger B, Januzzi JL Jr, Steinmair M, Gabriel C, Poelz W, Haltmeyer M, Mueller T. Analytical and clinical evaluation of a novel high-sensitivity assay for measurement of soluble ST2 in human plasma–the Presage ST2 assay. Clin Chim Acta 2009; 409: 33–40.
10. Coglianese EE, Larson MG, Vasan RS, Ho JE, Ghorbani A, McCabe EL. Cheng S, Fradley MG, Kretschman D. et al. Distribution and clinical correlates of the interleukin receptor family member soluble ST2 in the Framingham Heart Study. Clin Chem. 2012; 58: 1673–1681.
11. Januzzi JL Jr, Peacock WF, Maisel AS, Chae CU, Jesse RL, Baggish AL. Measurement of the interleukin family member ST2 in patients with acute dyspnea: results from the PRIDE (Pro-Brain Natriuretic Peptide Investigation of Dyspnea in the Emergency Department) study. Am Coll Cardiol. 2007; 50: 607–613.
12. Pascual-Figal DA, Manzano-Fernandez S, Boronat M, Casa T, Garrido IP, Bonaque JC. Soluble ST2, high-sensitivity troponin T- and N-terminal pro-B-type natriuretic peptide: complementary role for risk stratification in acutely decompensated heart failure. Eur J Heart Fail. 2011; 13: 718–25.
13. Dieplinger B, Mueller T. Soluble ST2 in heart failure. Clin Chim Acta 2015; 443: 57–70.
14. Ky B, French B, McCloskey K, Rame JE, McIntosh E, Shahi P, et al. High-sensitivity ST2 for prediction of adverse outcomes in chronic heart failure. Circ Heart Fail .2011; 4: 180–187.
15. Bayes-Genis A, Pascual-Figal D, Januzzi JL, Maisel A, Casas T, Valdas Chavarri M. Soluble ST2 monitoring provides additional risk stratification for outpatients with decompensated heart failure. Rev Esp Cardiol. 2010; 63: 1171–1178.

The authors
Stuart J. Bennett PhD, Ruth M. Ayling* PhD, FRCP, FRCPath
Department of Clinical Biochemistry, Pathology and Pharmacy Building, Royal London Hospital, Bart’s Health NHS Trust, London, UK

*Corresponding author
E-mail: Ruth.Ayling@bartshealth.nhs.uk

Lp(a) is a complex macromolecule synthesized in the liver, it presents similarities with Low Density Lipoprotein (LDL) as it possesses a cholesterol-phospholipid core and a closely associated protein, apoB100. Lp(a) differs however from LDL in that each lipoprotein particle contains one copy of the glycoprotein apolipoprotein (a) [apo (a)] covalently bound to apoB100 by a single disufide bond [1]. Studies indicate the association of elevated levels of plasma Lp(a) and risk of coronary heart disease [2,3].  A study reported that elevated plasma Lp(a) and small apolipoprotein (a) increased the risk of recurrent arterial ischemic stroke in children [4]. Moreover, the structural similarities between apo(a) and plasminogen highlighted the importance of Lp(a) in both atherosclerosis and thrombogenesis [5-7]. The intra and inter-individual size heterogeneity of apo(a) is genetically determined, this size variation constitutes a challenge for the immunochemical measurement of Lp(a) in plasma. The use of a 5-point calibrator which take into account the heterogeneity of Lp(a) for each of the levels would reduce result discrepancies. This study reports the performance characteristics of an immunoturbidimetric assay for the determination of Lp(a) in serum or plasma with minimum apo(a) size related bias.

Methodology
In this immunoturbidimetric assay agglutination occurs due to an antigen-antibody reaction between Lp(a) in a sample and anti-Lp(a) antibody adsorbed to latex particles. The agglutination is detected as an absorbance change at 700 nm proportional to the concentration of Lp(a) in the sample. The reagents are stable and ready to use. Lp(a) Calibrator Series, Lp(a) Control and Lipid Controls were used (Randox Laboratories Limited, Crumlin, UK). The assay is applicable to a variety of clinical chemistry analysers, for the results of this study the RX Daytona Plus analyser was used (Randox Laboratories Limited, Crumlin, UK).

Results
Assay range
This immunoturbidimetric assay presented a reportable range of 3 to 106 mg/dL.

Sensitivity
The Limit of Quantitation (LOQ), the Limit of Detection (LOD) and the Limit of Blank were determined consistent with CLSI guidelines EP17-A (table 1).

Prozone
Antigen excess effects were not noted until Lp(a) levels approached 493 mg/dL.

Within run and total precision
Within run precision and total precision, expressed as CV(%), were <4.00 and <4.5 respectively (Table 2). Serum/plasma comparison
The assay was used to compare serum to plasma samples (n=56) collected into tubes containing Li heparin, Na heparin, Na EDTA, K EDTA or citrate. The data was subjected to linear regression analysis and in all cases the correlation coefficient (r) was > 0.996.

Conclusion   
The immunoturbidimetric assay reported here for the determination of Lp(a) in serum/plasma with minimum apo (a) size related bias showed optimal analytical performance. The assay utilises ready- to –use stable reagents which facilitates the application in test settings by simplifying the experimental procedure and reducing handling errors. Its applicability to different automated analysers ensures the reliability, the accuracy of the measurements and facilitates the testing procedure. This represents an excellent analytical tool to facilitate clinical investigation.

References
1. Utermann G, Weber W. Protein composition of Lp(a) lipoprotein from human plasma. FEBS Lett. 1983; 154: 357-361.
2. Bennet A, Di Angelantonio E, Erqou S, Eirikdottir G, Sigurdsson G, Woodward M, Rumley A, Lowe G.D, Danesh J, Gudnason V. Lipoprotein (a) levels and risk of future coronary heart disease: large-scale prospective data. Arch. Intern. Med. 2008; 168: 598-608.
3. Emerging Risk Factors Collaboration, Erqou S, Kaptoge S, Perry P.L, Di Angelantonio E, Thompson A, White I.R, Marcovina S.M, Collins R, Thompson S.G, Danesh J. Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality. JAMA. 2009; 302: 412-423.
4. Goldenberg N.A, Bernard T.J, Hillhouse J, Armstrong-Wells J, Galinkin J, Knapp-Clevenger R, Jacobson, L, Marcovina S.M, Manco-Johnson M.J. Elevated lipoprotein(a), small apolipoprotein (a), and the risk of arterial ischemic stroke in North American children. Haematol. 2013; 98: 802-807.
5. McLean J.W, Tomlinson J.E, Kuang W.J, Eaton D.L, Chen E.Y, Fless G.M, Scanu A.M, Lawn R.M. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature. 1987; 300: 132-137.
6. Tate J.R, Rifai N, Berg K, Couderc R, Dati F, Kostner G.M, Sakurabayashi I, Steinmetz A. International Federation of Clinical Chemistry standardization project for the measurement of lipoprotein(a). Phase I. Evaluation of the analytical performance of lipoprotein(a) assay systems and commercial calibrators. Clin Chem. 1998; 44: 1629-1640.
7. Hajjar K.A, Nachman R.L. The role of lipoprotein(a) in atherogenesis and thrombosis. Annu Rev Med. 1996; 47: 423-442.

Randox Laboratories Limited, Diamond Road, Crumlin, County Antrim, N. Ireland, BT29 4QY, UK
www.randox.com

Acute chest pain remains the most common reason for emergency hospital admissions in the West, accounting for around 10% of visits. The majority of these patients do not have a life-threatening condition, but around 17% will be diagnosed with acute myocardial infarction (AMI). A physical examination and an ECG or serial ECGs remain essential. Diagnosis is straightforward in patients with typical cardiac symptoms and notable ST-segment deviation, but biomarker testing is necessary in patients with atypical symptoms and non-diagnostic ECGs. Despite huge and sustained efforts by the scientific community during the last six decades, a perfect cardiac biomarker to detect which of these patients have AMI has not yet been found. The cardiac troponin immunoassay, first developed in 1989, has now given rise to a fifth generation hs-cTn immunoassay that is currently used to facilitate the triage of chest pain patients, but is there any scope for improvement in either cardiac biomarker tests or their role in patient management?
The perfect cardiac biomarker would be present in significant concentration in the myocardium but not in any other tissues, and be released rapidly into the blood when MI occurs. It would persist for sufficient duration to allow diagnosis via a rapid and relatively inexpensive assay. Current hs-cTn assays can detect cardiac troponin release within 3 hours and MI can be ruled out in the approximately 60% of chest pain patients who have undetectable levels, or levels below the 99th percentile upper reference limit of a healthy population; the negative predictive value is nearly 100%. Tests are cost-effective and fairly rapid: central labs are able to provide results within an hour, and POC test results can be available within 10 to 20 minutes. However, predictably, the increased sensitivity of hs-cTn assays lowers specificity, resulting in values above normal in patients with conditions other than MI, including atrial fibrillation, hypertension, hepatic and renal disorders, acute and chronic pulmonary disease and even some allergic reactions. Using the currently set diagnostic cut-off for MI, the low positive predictive value results in approximately 22% of chest pain patients without MI remaining in hospital under observation.
Is cTn the best cardiac biomarker that will ever be available, or is it possible that an ever-increasing knowledge of the pathophysiology of acute cardiac disease together with current technological advances may eventually discover the perfect biomarker? Until this happens hs-cTn assays, with probable refinements in their use, will remain an integral part of suspected MI patient management.