Clinical Biochemistry 69 (2019) 36–44 Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem Analytical, biochemical and clearance considerations of soluble urokinase plasminogen activator receptor (suPAR) in healthy individuals T ⁎ Janice Chew-Harrisa, , Sarah Applebya, A. Mark Richardsa,b,c, Richard W. Troughtona,c, Christopher J. Pembertona a Christchurch Heart Institute, University of Otago, Christchurch, New Zealand b Cardiovascular Research Institute, National University of Singapore, Singapore c Department of Cardiology, Canterbury District Health Board, Christchurch, New Zealand ARTICLE INFO ABSTRACT Keywords: Background: Soluble urokinase plasminogen activator receptor (suPAR) is an emerging marker of cardiovascular suPAR disease burden. Appropriate assessment of assay performance and reference interval are required to enable Reference interval interpretation of results to facilitate its clinical application. Clearance Methods: suPAR was measured using the suPARnostic® ELISA in 155 healthy volunteers. Assay performance was Molecular weight assessed for anticoagulant effect, recovery, interference, linearity and cross-reactivity. The identity of im- munoreactive suPAR was confirmed by size-exclusion HPLC. To establish anatomical sites of release and uptake, we measured suPAR in regional samples from subjects undergoing cardiac catheterization. Results: The median concentration of suPAR was 2.1 ng/mL (IQR:1.7–2.3) in health. In comparison with EDTA, suPAR measurements were affected by lithium heparin (> 10% change) and increased with serum usage. suPAR reactivity also increased in the presence of haemolysis (10 g/L), but was suppressed with urokinase and lipids (4 g/L). In multiple regression analyses, suPAR associated independently with body weight, NT-proBNP and MR- proADM (P = .03) for healthy individuals. Regional plasma sampling showed lower suPAR concentrations in the coronary sinus and renal vein compared with concentrations in femoral arterial samples. Immunoreactive cir- culating suPAR species had Mr of 10–39 kDa. Conclusion: The suPARnostic® assay performs acceptably for a clinical assay but is limited in the presence of high levels of hemolysis, lipids and urokinase. We provide the first evidence for the heart and kidneys as organs of suPAR clearance in humans. Additional investigations are warranted to determine whether there is a need to compare the marker performance of differing circulating forms of suPAR. 1. Background plasmin through localization of urokinase, and as an orchestrator in dynamic substratum interactions such as cell adhesion, extracellular Recent evidence supports the role of soluble urokinase plasminogen matrix degradation and in cytoskeletal reorganization [10,11]. These activator receptor (suPAR) as an important marker of cardiovascular controlled actions are instrumental in promoting atherogenesis and in disease (CVD) burden. It has been known for some time that increased causing plaque vulnerability [12], and thus the measurement of suPAR concentrations of suPAR are predictive of outcomes in patients with is highly suggestive to correlate greater with CVD related burden than cancer [1,2], infectious disease [3,4] and in those at high risk of chronic other inflammatory markers which can be confounded by obesity [13]. kidney disease [5]. The extended discovery that suPAR also has ex- With growing interest in suPAR to potentially add to guiding per- cellent potential in prognosticating high mortality rates in CVD [6] and sonalized cardiovascular risk assessment and treatment, it is important in CVD worsening [7,8], is underpin by its ability in retaining similar to clarify analytical, biochemical, and performance characteristics of extracellular function to its cell bound pleiotropic receptor uPAR available assays, and to understand sources contributing to suPAR (NCBI:AAK31795) which is present in a variety of cell-types [9]. The bioavailability. Several methods of suPAR measurement are available actions of uPAR, highly expressed during immune system dysfunction, [14–16], however, one well used assay in clinical populations and the include; activating plasminogen conversion to the proteolytically active only CE-IVD certified commercially kit available is the suPARnostic® ⁎ Corresponding author at: Christchurch Heart Institute, Department of Medicine, University of Otago, 2 Riccarton Avenue, Christchurch 8011, New Zealand. E-mail address: [email protected] (J. Chew-Harris). https://doi.org/10.1016/j.clinbiochem.2019.05.010 Received 9 January 2019; Received in revised form 21 May 2019; Accepted 22 May 2019 Available online 23 May 2019 0009-9120/ © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. J. Chew-Harris, et al. Clinical Biochemistry 69 (2019) 36–44 two-site enzyme-linked immunosorbent assay (ELISA) from ViroGates, hour which was used for comparing fresh suPAR concentrations to that (Birkerød, Denmark) [17]. Apart from reported stability and the effect of samples that had undergone multiple freeze-thaw cycles. All samples of sample handling on suPAR measurements [14,18], there is limited for suPAR measurements were analysed in duplicate by one operator information on other key characteristics of this assay. Another im- (JCH) using the suPARnostic® ELISA kit, Lot #US1170 [17]. Any suPAR portant aspect of assay characteristics worth consideration too is in duplicate results which varied by > 10% were repeated. According to understanding the measuring capabilities of assay formats. In this the manufacturer, this assay has a coefficient of variation (CV) ≤ 6% at context, suPAR can circulate either as a full length protein (DI–DIII) or 2.3 ng/mL, detection limit of 0.1 ng/mL and an assay working range up in the truncated forms of DI and DII–DIII. Given that the suPARnostic® to 20.8 ng/mL. assay detects DIII of suPAR [14], implies that it should be capable of recognizing both the full length and DII–DIII but not in distinguishing 2.3. Assessment of assay performance, sample collection metrics and within- the amount of the different forms in circulation. individual suPAR variability Accordingly, we have used the suPARnostic® ELISA to (i) validate collection and analytical parameters relevant to suPAR measurement, We performed inter- and intra- batch CV analyses using the ii) document a suPAR reference interval using samples from 155 well- ViroGates curve control and a plasma pool in 11 assay runs over defined healthy subjects, (iii) characterize the molecular forms of im- 11 days. We estimated the limit of quantitation (LOQ) as ≥10% var- munoreactive suPAR in circulation as recognized by the assay and (iv) iation in repeated suPAR measurements in a set of samples with con- document trans-organ arterial and venous concentrations in catheter- centrations ranging from 0.1 ng/mL to 0.5 ng/mL. Linearity (sample ized patients to identify anatomical sites of production and clearance of parallelism) was determined by testing ViroGates standard A (20.8 ng/ suPAR. Our findings are reported here. mL), recombinant suPAR [Sino Biological (Life Technologies) Ref: 10925-H08H-50] (20.0 ng/mL) and sample from a HF patient with a 2. Materials and methods high suPAR concentration (19.0 ng/mL); each to the following dilu- tions; 1:2, 1:4, 1:8, 1:16 and 1:32 in assay buffer. The effect of collec- 2.1. Human plasma sample collection tion anticoagulants were assessed by comparing suPAR concentrations in fresh EDTA plasma (reference) with serum and lithium heparin Plasma samples were obtained from; (i) healthy volunteers aged 17 (n = 5) as comparators. Similarly, to assess the effect of freeze/thaw on to 70 years with no evidence of cardiovascular, endocrine or psychiatric suPAR concentrations, fresh EDTA plasma (n = 5) was compared with illness and taking no medications (n = 155), (ii) in 7 additional healthy plasma samples which had undergone multiple freeze/thaw cycles up to volunteers where within-individual suPAR variability were assessed, 5 cycles. For the assessment of daily suPAR variability and within-in- (iii) patients admitted to hospital with suspected acute heart failure dividual day variability, samples were assayed in the same batch. (HF) (n = 4), and (iv) patients undergoing clinically indicated cardiac To minimise matrix effects in the interference studies, the addition catheterization (n = 15). All patients had blood drawn into chilled of all exogenous materials/interferent was conducted in a ratio of vacutainer tubes without gel, centrifuged and stored at −80 °C (unless ≤1:10 dilution of plasma, followed by suPAR measurement. Sample specified) prior to analysis, apart from group (ii) in which suPAR recovery was assessed by spiking three patient's EDTA samples with measurements were also performed in a set of fresh plasma. For group standard A (ViroGates) and separately with recombinant suPAR. We (i), a brief physical examination including anthropometric measure- tested the effect of haemolysis (up to 10 g/L), lipaemia [Intralipid so- ments – weight, height, and blood pressure (BP) readings were under- lution Frisenius Kabi, Batch: 801G2069 (up to 6.0 g/L)], and icteria taken by trained staff. Samples were collected by standard vene- [(Bilirubin, Sigma Aldrich, Lot: 88C-0511) (up to 800 μmol/L)] on puncture between 7.30 and 10 am from fasting individuals. For group suPAR sample performance. To assess the effect of urokinase binding to (ii), blood was drawn daily at 9.30 am for 5 days in