An Automated Assay for Growth DifferentiationFactor 15

Kai C. Wollert,1†* Tibor Kempf,1† Evangelos Giannitsis,2 Thomas Bertsch,3 Siegmund L. Braun,4

Harald Maier,5 Manfred Reim,6 and Robert H. Christenson7

Background: Growth differentiation factor 15 (GDF-15) can serve as a biomarker for cardiovascular disease burdenand risk. We evaluated a new, fully automated electrochemiluminescence immunoassay for measuring GDF-15.Methods: Six laboratories independently characterized the Elecsys® GDF-15 assay (Roche Diagnostics) under routineconditions. Within-run precision (repeatability), within-laboratory precision (intermediate precision), and between-laboratory precision (reproducibility) were assessed. Plasma-serumsample correlation, reagent lot-to-lot reproducibility,and instrument comparisons were performed. The Elecsys assay was compared to a research immunoradiometric assay(IRMA) and a commercially available ELISA. GDF-15 concentrations were measured with the Elecsys assay in 739 appar-ently healthy individuals.Results:CVs forwithin-run andwithin-laboratory precision ranged from0.7% to 7.7%and1.7% to 8.6%, respectively, forsamples containing 670–16039 ng/L. CVs for between-laboratory precision ranged from 7.1% to 8.9% (766–14289 ng/L).Recovery of GDF-15 was comparable for serum, Li-heparin plasma, K2- and K3-EDTA plasma, and citrated plasma,between 2 reagent lots, and on the cobas e 411 and cobas e 601 analyzers (Roche Diagnostics). GDF-15 concentrations inthe clinically relevant range (400–3000 ng/L) measured with the Elecsys assay showed a good correlation and agreementwith those measured by IRMA or ELISA. GDF-15 concentrations in apparently healthy individuals increased with age butdid not vary by sex.Conclusions: The Elecsys GDF-15 assay demonstrates a robust analytic performance under routine conditions andprovides an automated method for measuring GDF-15 concentrations in serum and plasma.

IMPACT STATEMENTGrowth differentiation factor 15 (GDF-15) is a biomarker of acute and chronic cellular stress that may inform

therapeutic decision-making in patients with cardiovascular disease. In the present study, 6 laboratories evaluated

a new, fully automated electrochemiluminescence immunoassay for GDF-15. The Elecsys®GDF-15 assay performed

with high within-run, within-laboratory, and between-laboratory precision. The recovery of GDF-15 was comparable

with different sample matrices, reagent lots, and laboratory analyzers. GDF-15 concentrations measured with the

Elecsys assay showed a good correlation and agreement with 2 previously described manual GDF-15 assays. This

study demonstrates a robust analytical performance of the first automated GDF-15 assay under routine conditions.

1Division of Molecular and Translational Cardiology, Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany;2Department of Internal Medicine III, Cardiology, University Hospital Heidelberg, Heidelberg, Germany; 3Institute of Clinical Chemistry, LaboratoryMedicine, and Transfusion Medicine, General Hospital Nuremberg, Paracelsus Medical University, Nuremberg, Germany; 4Institute of LaboratoryMedicine, German Heart Center Munich, Munich, Germany; 5Central Laboratory, District Hospital Altötting-Burghausen, Altötting, Germany;6Roche Diagnostics, Penzberg, Germany; 7Department of Pathology, University of Maryland School of Medicine, Baltimore, MD.

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Copyright 2017 by American Association for Clinical Chemistry.

Growth differentiation factor 15 (GDF-15)8 is astress-responsive member of the transforminggrowth factor-β cytokine superfamily (1). GDF-15 isexpressed and secreted in response to inflamma-tion, oxidative stress, hypoxia, telomere erosion,and oncogene activation. The circulating levels ofGDF-15 reflect these acute and chronic cellularstressors, which are associated with aging anddisease.Cardiovascular disease (CVD) is a major driver of

increased circulating levels of GDF-15 in community-dwelling individuals and patients. In community-dwelling individuals, higher concentrations ofGDF-15 are associated with increased prevalenceand incidence of CVD, chronic kidney disease, andcancer, independent of traditional risk factors, renalfunction, and other biomarkers (2–12). GDF-15 hasemerged as an independent marker of all-causemortality, cardiovascular, and noncardiovascularmortality, and nonfatal cardiovascular events in pa-tientswith coronary artery disease (13–20), heart fail-ure (21–24), and atrial fibrillation (25). GDF-15 isindependently associated withmajor bleeding in pa-tients receiving antithrombotic therapies (20,25) andhasbeen included inanewbleeding risk score,whichmay become useful for decision support (26).GDF-15 concentrations in human serum and

plasmahave beenmeasuredwith a research ELISA(27), a research immunoradiometric assay (IRMA)(28), an ELISA using antibodies from R&D Systems(16), and a Luminex sandwich assay developed byAlere (5). These assays are not approved for clinicaluse.More recently, an Elecsys® GDF-15 immuno-

assay, based on an electrochemiluminescencesandwich assay and biotin-streptavidin technol-

ogy, was released by Roche Diagnostics. Precom-mercial versions of the assay have been usedsuccessfully in a number of clinical studies (8–12,18–20, 23, 25, 26, 29).Here, we evaluated the analytic performance

of the Elecsys GDF-15 assay in multiple clinicallaboratories. We compared the assay with exist-ing methods for measuring GDF-15 and mea-sured GDF-15 in a cohort of apparently healthyindividuals.

MATERIALS AND METHODS

Study design

The study was conducted between April 2014and December 2015 at 5 sites in Germany (Han-nover Medical School, University Hospital Heidel-berg, General Hospital Nuremberg, German HeartCenter Munich, and District Hospital Altötting-Burghausen) and at 1 site in the US (University ofMaryland, Baltimore). At the beginning of thestudy, all participating laboratories performed a fa-miliarization experiment to confirm that the cobase analyzer, reagents, and controls worked prop-erly. The assay was calibrated at the start of allexperiments.Institutional review board/ethics committee ap-

proval was not required, since experiments wereperformed using either anonymized residual labo-ratory samples or banked serum samples from aprevious reference range study (Roche Diagnosticsstudy number 000788). The protocol for the earlierreference range study was previously approved bythe regional ethics committee, and all participantsprovided written informed consent.

*Address correspondence to this author at: Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.Fax +49-511-532-5307; e-mail wollert.kai@mh-hannover.de.†Kai C. Wollert and Tibor Kempf contributed equally to the work, and both should be considered as first authors.DOI: 10.1373/jalm.2016.022376© 2017 American Association for Clinical Chemistry8Nonstandard abbreviations:GDF-15, growth differentiation factor 15 ; IRMA, immunoradiometric assay; CVD, cardiovascular disease; LOB, limitof blank; LLOD, lower limit of detection; LLOQ, lower limit of quantification;MP,Master Pilot; PCC, PreciControl Cardiac reagent; HSP, human serumpool; MDP, medical decision point.

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Elecsys GDF-15 assay

The Elecsys GDF-15 assay is an electrochemi-luminescence immunoassay for use on the co-bas e series of chemistry analyzers. The assay isbased on the sandwich immunoassay principleand uses biotin-streptavidin technology. Sam-ples (35 μL) were incubated for 9 min with abiotinylated murine monoclonal anti–GDF-15antibody and a second murine monoclonal anti–GDF-15 antibody labeled with a ruthenium com-plex [Tris(2,2′-bipyridyl)ruthenium(II)], to form asandwich complex when GDF-15 is present.Streptavidin-coated microparticles were addedin a second 9-min incubation period. Unboundsubstances were subsequently removed using aProCell washing solution (Roche Diagnostics),and antigen–antibody complexes were detectedvia electrochemiluminescence using the cobas eanalyzers. Results were determined via an in-strument-specific calibration curve generated by2-point calibration and a master curve providedvia the reagent barcode.

Lower limits of measurement andlinearity

Linearity and lower limits of measurement wereassessed by Roche Diagnostics. The limit of blank(LOB), lower limit of detection (LLOD), and lowerlimit of quantification (LLOQ) were assessed ac-cording to the CLSI EP17-A2 guideline (30). TheLOB was determined from 60 measurements of azero calibrator over several independent series.Linearity was assessed according to the CLSIEP06-A guideline (31). For each experiment, 3 rep-licates of each dilution (n = 14–15; expectedGDF-15 concentration range 107–23333 ng/L)were analyzed on a single cobas e 411 and a singlecobas e 601 using the Master Pilot (MP) reagentlot. Experiments were repeated using 2 reagentlots (P02, P03), each with 1 serum and 1 plasmadilution set.

Precision

All precision assessments were performed on acobas e 411 and a cobas e 601 analyzers using 2assay controls containing recombinant GDF-15[PreciControl Cardiac reagents: PCC1 (1350 ng/L);PCC2 (7500 ng/L), Roche Diagnostics] and 5 hu-man serum pools (HSPs). Within-run precision andwithin-laboratory precision were evaluated ac-cording to the CLSI EP05-A3 guideline (32), using 4replicates of each sample pool (2 PCCs and 5HSPs). Samples were analyzed in random orderduring 2 runs per day (2 replicates of each sampleper run) for 21 days. The samples used for thedetermination of within-run and within-laboratoryprecision covered the following GDF-15 concen-tration ranges: HSP1, ≤1000 ng/L; HSP2, 1001–1800 ng/L; HSP3, 1801–3000 ng/L; HSP4, 3001–5000 ng/L; HSP5, >5000 ng/L. Between-laboratoryprecision was evaluated on cobas e 411 or cobas e601 analyzers using 5 replicates of each samplepool (2 PCCs and 5 HSPs). Samples were analyzedin 1 run per day for 5 days. The samples providedfor the between-laboratory precision had thefollowing predetermined (approximate) concen-trations: HSP1, approximately 729 ng/L; HSP2, ap-proximately 1511 ng/L; HSP3, approximately 2404ng/L; HSP4, approximately 4396 ng/L; HSP5, ap-proximately 13798 ng/L.

Sample matrix, instrument, and lot-to-lotcomparisons

Assay performance in different matrices wascompared using matched serum and plasma sam-ples containing lithium (Li)-heparin (n = 118), dipo-tassium (K2)-EDTA (n = 60), tripotassium (K3)-EDTA(n = 60), or citrate (n = 60) as anticoagulants.Matched serum and plasma pairs were analyzed inparallel, in a single run.An instrument comparison was performed on

cobas e 411 and cobas e 601 analyzers using se-rum or plasma samples with GDF-15 concentra-tions in the following ranges: <1000 ng/L (30%–40%

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of samples), 1000–5000ng/L (50%–60%), and>5000ng/L (5%–10%). Both measuring cells of the cobas e601 analyzer were used.Reagent lot-to-lot reproducibility was evaluated

using serum or plasma samples (GDF-15 concen-trations as per instrument comparison). Sampleswere tested using 2 different reagent lots (P02 andP03) using a single assay run per reagent lot.

Method comparison

SerumsampleswithGDF-15 concentrations cor-responding to the measuring range of the ElecsysGDF-15 assay (400–20000 ng/L; n = 95) were usedto compare the Elecsys assay with the previouslydescribed IRMA, and the Quantikine GDF-15 ELISAfrom R&D Systems. The IRMA was selected as theprimary reference method, since it has been vali-dated (28) and used in many biomarker investiga-tions (3, 13–15, 17, 21, 22). Interassay imprecisionof the IRMA ranges from 4.0% to 12.2% for sam-ples containing 232–39370 ng/L GDF-15 (28). Ac-cording to R&D Systems, the CVs for interassayprecision of the Quantikine assay are 6.0%, 4.7%,and 5.6% at mean GDF-15 concentrations of 225,442, and 900 ng/L, respectively.

Apparently healthy individuals

Serum samples were previously collected from739 apparently healthy individuals (see Table 1 inthe Data Supplement that accompanies the onlineversion of this article at http://www.jalm.org/content/vol1/issue5) who were recruited at a clini-cal research facility (Clinical Research Services, Kiel,Germany) in 2010. Eligible participants providedblood (after fasting for at least 2 h), and furtherlaboratory parameters were determined as part ofa health check analysis; samples were collectedand stored at −80 °C. Themedian agewas 49 years(25th–75th percentiles, 33–65 years; range, 20–79years). The cohort included 375 women (50.7%)and 364 men (49.3%); 495 individuals (67.0%) hadnever smoked, 180 (24.3%) were current smokers,

and 64 (8.7%) were past smokers. GDF-15 mea-surements were performed at 3 sites (approxi-mately 240 randomly chosen samples per site)using the cobas e 411 analyzer.

Data analysis and statistical methods

Measurements were analyzed using WinCAEvsoftware, and statistical analyses were performedusing SAS software (SAS Institute) and VCA soft-ware version 02.05.01 (Roche Diagnostics). TheLOB was calculated as the 95th percentile fromrepeated measurements of analyte-free samples(corresponding to the concentration below whichanalyte-free samples were foundwith a probabilityof 95%) (30). The LLOD was calculated based onthe LOB and the SD of low concentration samples,corresponding to the lowest analyte concentrationabove the LOB that could be detected with aprobability of 95%. The LLOQwas calculated as thelowest analyte concentration that could be repro-ducibly measured with a CV for intermediate pre-cision of ≤20%. For assay linearity evaluation,observed concentrations were plotted against ex-pected concentrations, and a regression modelwas selected according to CLSI EP06-A (31). Forprecision experiments, the CV was used as themeasure of assay imprecision. Serum vs plasma,lot-to-lot, instrument, and method comparisonswere assessed using Pearson correlation coeffi-cient (r), as determined by Passing–Bablok regres-sion analysis. Agreement between the methodswas assessedusing Bland–Altmanplots, which plotdifferences in GDF-15 concentration against meanGDF-15 concentration. Bias (deviation betweenthe comparator and reference results, as deter-mined by regression analyses) was reported at themedical decision points (MDPs) of 1200 ng/L and1800 ng/L, which have been used to identify pa-tients with coronary artery disease at low (<1200ng/L), intermediate (1200–1800 ng/L), and high(>1800 ng/L) risk of adverse outcomes (13–15, 18,19). Median, range, and the 10th, 25th, 75th, and90th percentiles were calculated for the GDF-15

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concentrations measured in apparently healthyindividuals, stratified by age, sex, and smoking sta-tus. The 95th percentiles with 95% CI were calcu-lated for different age-groups.

RESULTS

Lower limits of measurement and linearity

The LOB was 350 ng/L and the LLOD and LLOQwere both 400 ng/L.Using the serum dilution sets (MP reagent lot),

the Elecsys GDF-15 assay demonstrated linearityover the range 400–20000 ng/L (cobas e 411 ande 601 analyzers) (Fig. 1). Reagent lot (P02 vs P03)and samplematrix (serum vs plasma) did not influ-ence the linear range (data not shown).

Within-run and within-laboratory precision

Within-run and within-laboratory precision re-sults for mean GDF-15 concentrations rangingfrom 670 to 16039 ng/L (inclusive of the MDPs)are presented in Table 1. The CV for within-run

precision ranged from 0.8% to 1.5% on the co-bas e 601 analyzer and 0.7% to 7.7% on the co-bas e 411 analyzers. The CV for within-laboratoryprecision ranged from 2.7% to 3.4% on the co-bas e 601 analyzer and 1.7%–8.6% on the cobase 411 analyzers.

Between-laboratory precision

Between-laboratory precision results for meanGDF-15 concentrations ranging from 766 to 14289 ng/L are presented in Table 2. The CV for be-tween-laboratory precision ranged from 7.1% to8.9%.

Serum and plasma sample comparison

GDF-15 concentrations (ng/L) measured inmatched serum and plasma samples were closelycorrelated [serum vs K2-EDTA plasma, y = 0.939x +9, r = 0.998 (n = 1 site, 60 pairs); serum vs K3-EDTAplasma, y = 1.05x − 12, r = 0.994 (n = 1 site, 60pairs); serum vs citrate plasma, y = 0.936x − 16, r =0.993 (n = 1 site, 60 pairs); serum vs Li-heparin

Fig. 1. Elecsys GDF-15 assay linearity.(A), Assay linearity on the cobas e 411 analyzer. (B), Assay linearity on the cobas e 601 analyzer. Deviation limits (criteria forlinearity) were defined as ±160 ng/L (concentration range, LOD–800 ng/L) and ±20% (>800–20000 ng/L).

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plasma (n = 2 sites), y = 1.01x − 20, r = 0.970 (site 1,58 pairs) and y = 1.05x + 26, r = 0.997 (site 2, 60pairs)]. Bias ranged from 2.5% to 8.6% at the 1200ng/L MDP and from 2.5% to 7.6% at the 1800 ng/LMDP, for all comparisons.

Instrument comparison and reagentlot-to-lot reproducibility

GDF-15 concentrations in serum or plasmasamples measured with the cobas e 411 or cobas

e 601 analyzers were closely correlated (Passing–Bablok regression, y = 1.02x − 8; Pearson r = 0.998;n = 203). Bias at the lower and upper MDPs was3.0% and 3.4%, respectively.Serum or plasma samples were analyzed with

2 reagent lots (P02 and P03). A high level of con-sistency between lots was observed at all studysites. Bias at the lower and upper MDPs was≤4.5% at all sites, and results were consistentwhen reference and comparator lots wereswitched (see Table 2 in the online Data Supple-ment).

Comparison of methods for GDF-15quantification

There was a close correlation between GDF-15concentrations measured with the Elecsys assay(cobas e 411 analyzer) and the IRMA over theentire concentration range (400–20000 ng/L;Passing–Bablok regression, y = 1.31x − 442;Pearson r = 0.988; n = 95) (Fig. 2A) and withinthe clinically relevant range (400–3000 ng/L;Passing–Bablok regression, y = 1.11x − 215;

Table 1. Within-run and within-laboratory precision of the Elecsys GDF-15 assay.

Sample poolacobas eAnalyzer

Mean GDF-15, Within-run precisionb Within-laboratory precisionb

ng/L SD, ng/L CV, % SD, ng/L CV, %PCC1 411 1311 14–26 1.1–1.9 33–54 2.6–4.1

601 1294 10 0.8 36 2.8PCC2 411 7148 65–147 0.9–2.0 137–304 1.9–4.3

601 7390 70 0.9 199 2.7HSP1 411 670 9–55 1.3–7.7 18–61 2.8–8.6

601 679 10 1.5 23 3.4HSP2 411 1437 21–36 1.6–2.3 47–54 3.2–4.1

601 1234 16 1.3 38 3.0HSP3 411 2557 30–63 1.3–2.6 72–116 3.0–4.3

601 2192 24 1.1 65 3.0HSP4 411 4018 44–114 1.2–3.0 89–173 2.4–4.6

601 3808 57 1.5 120 3.1HSP5 411 10410 68–331 0.7–2.9 168–640 1.7–3.9

601 16039 216 1.4 487 3.0aMeasurements were performed on cobas e 411 (n = 4 sites) and cobas e 601 (n = 1 site) analyzers. HSPs were pooled by each site individually.b Data are presented as a range when experiments were performed at multiple sites.

Table 2. Between-laboratory precision of theElecsys GDF-15 assay.

Samplepoola

MeanGDF-15, ng/L SD, ng/L CV, %

PCC1 1345 106 7.9PCC2 7378 587 8.0HSP1 766 69 8.9HSP2 1582 115 7.3HSP3 2506 178 7.1HSP4 4539 333 7.3HSP5 14289 1047 7.3

aMeasurements were performed on the cobas e 411 analyzer (n = 5sites).

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Pearson r = 0.990; n = 61) (Fig. 2B). However, anincreasing deviation was observed at higherGDF-15 concentrations (Fig. 2A). Bland–Altmananalyses (Fig. 2, C and D) demonstrated that theElecsys assay tended to measure lower GDF-15values compared with the IRMA in the lower con-centration range (<1500 ng/L); the magnitude of

the absolute difference was generally <200 ng/L.For concentrations above 3000 ng/L, higher val-ues were measured with the Elecsys assay com-pared with the IRMA. The mean absolutedifference between both methods (Elecsys mi-nus IRMA) was 824 ng/L across the entire con-centration range (400–20000 ng/L; Fig. 2C) and

Fig. 2. Comparison of the Elecsys GDF-15 assay with the IRMA(A, B), Passing–Bablok regression analyses. (C, D), Bland–Altman plots; (A, C), GDF-15 concentration range 400–20000 ng/L.(B, D), GDF-15 concentration range 400–3000 ng/L.

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−71 ng/L over the clinically relevant range (400–3000 ng/L; Fig. 2D).There was a somewhat weaker correlation be-

tween the Elecsys assay (cobas e 601 analyzer) andthe Quantikine ELISA (GDF-15 concentrations, 400–6000ng/L; Passing–Bablok regression, y=1.09x−55;Pearson r = 0.852; n = 100) (Fig. 3A). Bland–Altmananalysis (Fig. 3B) showed that the direction of varia-tionbetweenbothmethodswasbalancedacross theexamined concentration range. Generally, the mag-nitude of negative absolute differences (i.e., lowerconcentrations measured with the Elecsys assay)was <200 ng/L, whereas the magnitude of positiveabsolute differences tended to be greater, exceed-ing 500 ng/L in some cases (Fig. 3B). Themean abso-lute difference between both methods (Elecsysminus ELISA) was 204 ng/L across the 400–6000ng/L concentration range.

GDF-15 concentrations in apparentlyhealthy individuals

Themedian GDF-15 concentration was 680 ng/L(25th–75th percentiles, 492–925 ng/L; range, 400–

3976 ng/L). GDF-15 concentration increased withage (Fig. 4). In individuals aged 20–29 and 30–39years, the median [95th percentile (95% CI)]GDF-15 concentrations were 429 ng/L [831 ng/L(687–1035 ng/L)] and 500 ng/L [852 ng/L (790–2337 ng/L)], respectively, compared with 1060ng/L [2199 ng/L (1920–3086 ng/L)] in individualsaged ≥70 years. Sex and smoking status didnot influence GDF-15 concentrations (data notshown). In the entire cohort, 87 out of 739 individ-uals (11.8%) had a GDF-15 concentration equal toor below the LLOQ of 400 ng/L; of these, 80 indi-viduals (92%)were younger than themedian age of49 years and 7 (8%) were ≥49 years old.

DISCUSSION

Evaluation in 6 clinical laboratories in Europeand the US demonstrated that the Elecsys GDF-15assay performs with a high level of within-run andwithin-laboratory precision (repeatability and in-termediate precision), with CVs in the range of0.7%–7.7% and 1.7%–8.6%, respectively. The

Fig. 3. Comparison of the Elecsys GDF-15 assay with the Quantikine ELISA.(A), Passing–Bablok regression analysis. (B), Bland–Altman plot.

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assay also demonstrated a high level of between-laboratory precision (reproducibility) with CVs inthe range of 7.1%–8.9%. Furthermore, the recov-ery of GDF-15 was comparable when using differ-ent sample matrices (serum, Li-heparin plasma,K2- and K3-EDTAplasma, citrated plasma), differentreagent lots, and different analyzers (cobas e 411,cobas e 601). The Elecsys GDF-15 assay thereforeprovides a technically robustmethodofmeasuringGDF-15 concentrations in different sample types.The Elecsys assay was compared with an IRMA

that had previously been used in many biomarkerinvestigations (3, 13–15, 17, 21, 22). GDF-15 con-centrations measured with both assays showed aclose correlation and good agreement in the clini-cally relevant range (400–3000 ng/L) although anincreasing deviationwas observed at concentrationsabove approximately 3000 ng/L. Comparison ofthe Elecsys assay with the Quantikine ELISA fromR&D Systems also yielded a good agreement, al-beit the correlation between GDF-15 concentra-

tions measured with the Elecsys assay and theQuantikine ELISA was somewhat weaker. Differ-ences between the assays may be because theElecsys assay is fully automated, whereas the IRMAand Quantikine assays require several manual pi-petting steps. Any variations in operator pipettingor washing techniques, incubation times or tem-perature, and other variables, such as differencesin antibody lots, recombinant GDF-15 standards,or assay diluents, may introduce noise. In addition,it is possible that antibody saturation in the IRMAand in the Quantikine ELISA contributed to the de-viations observed at higher concentrations ofGDF-15 as compared to the Elecsys assay. Of note,the IRMA and Quantikine ELISA are not approvedfor clinical use.Using the Elecsys assay to measure GDF-15 in

739 apparently healthy individuals with a medianage of 49 years (range, 20–79 years), we observedan age-dependent increase in circulating GDF-15concentrations. Sex and smoking status did nothave an impact on GDF-15. These findings are inagreement with previous studies using the IRMA(28) or a precommercial version of the Elecsys as-say (6) to measure GDF-15 in similar cohorts ofapparently healthy individuals. One of these earlierstudies suggested that apparently healthy individ-uals with GDF-15 levels at the upper end of thespectrummay have occult disease, as indicated bypositive correlations with C-reactive protein andcystatin C (28). Indeed, increased GDF-15 concen-trations in community-dwelling individuals are as-sociated with subclinical vascular and cardiacpathologies such as increased arterial stiffness,endothelial dysfunction, atherosclerotic plaqueburden, left ventricular hypertrophy, and left ven-tricular dysfunction (3, 7, 11, 12). Therefore, someindividuals included in our study, and in other ap-parently healthy cohorts (6, 28), probably hadsubclinical disease. The GDF-15 concentrationsmeasuredwith the Elecsys assay in our cohort pro-vide estimates of the GDF-15 concentrations thatmay be expected in similar cohorts. The definition

Fig. 4. Circulating GDF-15 concentrations in739 apparently healthy individuals.Boxes show median with interquartile range; whiskersare 10th and 90th percentiles; n = 119–127 individualsper age-group.

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of “normal” GDF-15 concentrations, which wouldbe associated with minimal risk of future diseasemanifestations (2, 4, 5, 7–10), will require largerreference cohorts and more vigorous screeningcriteria (e.g., based on imaging studies) toexclude individuals with subclinical disease(33).As shown here, the LLOQ of the Elecsys assay is

400 ng/L. In our cohort of apparently healthy indi-viduals with a median age of 49 years, 11.8% pre-sented with a GDF-15 concentration equal to orbelow that threshold. In a recent investigation thatused a precommercial version of the Elecsys assayto measure GDF-15 in 3428 community-dwellingindividuals from the Framingham Heart Study(mean age 59 years; 6% with a history of CVD), only2 individuals had a GDF-15 concentration below

400 ng/L (8). Likewise, GDF-15 concentrationsbelow 400 ng/L are rarely observed in patientswith acute coronary syndrome or heart failure(the principal intended uses of the assay) (13–18,20–24). The LLOQmay therefore represent a lim-itation of the assay when measuring GDF-15 inyoung and healthy individuals. However, theLLOQ is unlikely to be of clinical relevance for riskstratification and therapeutic decision-making inpatients with acute coronary syndrome or heartfailure.In conclusion, this multicenter evaluation dem-

onstrated a robust analytical performance of theautomated Elecsys GDF-15 assay under routineconditions. Further studies are warranted to ex-plore the usefulness of the assay in populationswith subclinical or manifest disease.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and havemet the following4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b)drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable forall aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriatelyinvestigated and resolved.

Authors’ Disclosures or Potential Conflicts of Interest:Uponmanuscript submission, all authors completed the author disclosureform. Employment or Leadership: M. Reim; Roche Diagnostics. Consultant or Advisory Role: R.H. Christenson, Roche Diag-nostics, Siemens Healthcare Diagnostics, and Abbott Diagnostics. Stock Ownership: None declared.Honoraria: R.H. Christen-son, Roche Diagnostics, Siemens Diagnostics, Philips Diagnostics, and Beckman Coulter. Research Funding: K.C. Wollert, T.Kempf, T. Bertsch, S.L. Braun, H.Maier, RocheDiagnostics; R.H. Christenson, RocheDiagnostics to institution. Expert Testimony:None declared. Patents: K.C. Wollert, T. Kempf, EP 2047275 B1 and US 8951742 B2.Other Remuneration: E. Giannitsis, RocheDiagnostics, Bayer Vital, and AstraZeneca.

Role of Sponsor: The funding organizations played a direct role in design and choice of enrolled patients.

Acknowledgments: The authors acknowledge David Evans (Gardiner-Caldwell Communications, an Ashfield Business, Maccles-field, UK) for medical writing assistance and thank Stefanie Gaupp (TRIGA-S, Habach, Germany) for contribution to, and criticalreview of, the manuscript.

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