Scholarly article on topic 'Reference values of galectin-3 and cardiac troponins derived from a single cohort of healthy blood donors'

Reference values of galectin-3 and cardiac troponins derived from a single cohort of healthy blood donors Academic research paper on "Chemical sciences"

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{"Cardiac troponin" / "Evaluation study" / Galectins / "Natriuretic peptides" / "Reference values" / "Upper reference limits"}

Abstract of research paper on Chemical sciences, author of scientific article — Thomas Mueller, Margot Egger, Isabella Leitner, Christian Gabriel, Meinhard Haltmayer, et al.

Abstract Background Here we describe the determination of upper reference limits (URL) for galectin-3, high-sensitivity cardiac troponin I (hs-cTnI) and high-sensitivity cardiac troponin T (hs-cTnT) in a single cohort of healthy blood donors using routine assays. Methods For this reference value study, we used a cohort of 402 consecutive blood donors (64% were male and 36% were female). The median individuals' age was 35.0years (range, 18.0–64.4). Individuals of this reference population were free of cardiovascular disease, diabetes mellitus, renal disease, cancer, current infection and chronic inflammatory disease. Plasma concentrations of galectin-3 were measured with the “routine Galectin-3” assay (Abbott Diagnostics), of hs-cTnI with the “STAT High Sensitive Troponin-I” assay (Abbott Diagnostics), and of hs-cTnT with the “Troponin T hs” assay (Roche Diagnostics). URLs were calculated by using a non-parametric percentile method. Results The 97.5th percentile URL for galectin-3 was 16ng/mL in males and 17ng/mL in females; the 99th percentile URL for hs-cTnI was 39ng/L in males and 24ng/L in females; and the 99th percentile URL for hs-cTnT was 14ng/L in males and 11ng/L in females. Those individuals with hs-cTnI values ≥15ng/L (n=8) were different from those individuals with hs-cTnT values ≥10ng/L (n=7). Of the 402 individuals, none had galectin-3 values below the limit of detection (LOD, <1.0ng/mL), 290 (72%) had hs-cTnI values below the LOD (i.e., 1.9ng/L), and 359 (89%) had hs-cTnT values below the LOD (i.e., 5.0ng/L). Conclusion Plasma concentrations of galectin-3, hs-cTnI and hs-cTnT and corresponding 99th percentile URLs were rather low in our cohort of healthy blood donors compared with previously published data. In our reference population, analyte plasma concentrations above the LOD were detectable in 100% of the individuals with the Abbott galectin-3 assay, but only in less than 50% for both the Abbott hs-cTnI assay and the Roche hs-cTnT assay.

Academic research paper on topic "Reference values of galectin-3 and cardiac troponins derived from a single cohort of healthy blood donors"

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Clinica Chimica Acta

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CLINICA CHIMICA

Reference values of galectin-3 and cardiac troponins derived from a single cohort of healthy blood donors

Thomas Mueller a,*< Margot Egger a, Isabella Leitner a, Christian Gabrielb, Meinhard Haltmayer a, Benjamin Dieplinger a

a Department of Laboratory Medicine, Konventhospital Barmherzige Brueder Linz, Linz, Austria b Red Cross Transfusion Service of Upper Austria, Linz, Austria

ARTICLE INFO

ABSTRACT

Article history:

Received 11 January 2016

Received in revised form 21 February 2016

Accepted 21 February 2016

Available online 24 February 2016

Keywords: Cardiac troponin Evaluation study Galectins

Natriuretic peptides Reference values Upper reference limits

Background: Here we describe the determination of upper reference limits (URL) for galectin-3, high-sensitivity cardiac troponin I (hs-cTnI) and high-sensitivity cardiac troponin T (hs-cTnT) in a single cohort of healthy blood donors using routine assays.

Methods: For this reference value study, we used a cohort of 402 consecutive blood donors (64% were male and 36% were female). The median individuals' age was 35.0 years (range, 18.0-64.4). Individuals of this reference population were free of cardiovascular disease, diabetes mellitus, renal disease, cancer, current infection and chronic inflammatory disease. Plasma concentrations of galectin-3 were measured with the "routine Galectin-3" assay (Abbott Diagnostics), of hs-cTnI with the "STAT High Sensitive Troponin-I" assay (Abbott Diagnostics), and of hs-cTnT with the "Troponin T hs" assay (Roche Diagnostics). URLs were calculated by using a non-parametric percentile method.

Results: The 97.5th percentile URL for galectin-3 was 16ng/mLinmalesand 17 ng/mL in females; the 99thpercen-tile URL for hs-cTnI was 39 ng/L in males and 24 ng/L in females; and the 99th percentile URL for hs-cTnT was 14 ng/L in males and 11 ng/L in females. Those individuals with hs-cTnI values > 15 ng/L (n = 8) were different from those individuals with hs-cTnT values >10 ng/L (n = 7). Of the 402 individuals, none had galectin-3 values below the limit of detection (LOD, < 1.0 ng/mL), 290 (72%) had hs-cTnI values below the LOD (i.e., 1.9 ng/L), and 359 (89%) had hs-cTnT values below the LOD (i.e., 5.0 ng/L).

Conclusion: Plasma concentrations of galectin-3, hs-cTnI and hs-cTnT and corresponding 99th percentile URLs were rather low in our cohort of healthy blood donors compared with previously published data. In our reference population, analyte plasma concentrations above the LOD were detectable in 100% of the individuals with the Abbott galectin-3 assay, but only in less than 50% for both the Abbott hs-cTnI assay and the Roche hs-cTnT assay. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Galectin-3 is a unique member of chimera type galectins and is involved in a large number of disease processes [1]. Galectin-3 participates in cell adhesion, activation, proliferation, apoptosis as well as cell migration [1,2]. It plays an important role in cancer [2] and inflammation [1,3]. Even in the pathophysiology of cardiac disease, galectin-3 is thought to play a biological role through inflammation and fibrosis [4, 5]. As a consequence, the biomarker galectin-3 has been included in the 2013 ACCF/AHA guideline for additive risk stratification of patients with acute and chronic heart failure [6].

To date, only few and conflicting data have been published on upper reference limits (URL) of circulating galectin-3. Thus, the aim of this

* Corresponding author at: Department of Laboratory Medicine, Konventhospital Barmherzige Brueder, Seilerstaette 2-4, A-4020 Linz, Austria. E-mail address: thomas.mueller@bs-lab.at (T. Mueller).

study was to establish reference values for galectin-3. As reference values for cardiac troponins are still under debate [7-14], we additionally aimed at evaluating URLs of high-sensitivity cardiac troponin I (hs-cTnI) and high-sensitivity cardiac troponin T (hs-cTnT) in a single cohort of healthy blood donors.

2. Materials and methods

2.1. Study protocol

For the present reference value study, we used a cohort of 559 consecutive Caucasian blood donors as described previously [15]. This cohort was recruited at the Red Cross organization in Linz, Austria, in 2008. All study participants completed questionnaires detailing their medical histories and medications in order to confirm their good health before blood collection according to Austrian laws. This healthy reference population was, thus, defined as individuals who were free of

http: //dx.doi.org/10.1016/j.cca.2016.02.014

0009-8981/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

cardiovascular disease (no arterial hypertension, no history of any cardiac disease, cardiac treatment or cardiac intervention, no history of stroke or transient ischemic attack, no history of peripheral artery disease, no history venous thromboembolism). Additional exclusion criteria included, e.g., pregnancy, age > 65.0 years, history of diabetes mellitus, renal disease and cancer, or current infection and any chronic inflammatory disease. The study protocol was approved by the local ethics committee in accordance with the Declaration of Helsinki, and all study participants gave informed consent.

Blood was obtained by conventional venipuncture avoiding venous stasis. Using VACUETTE® polyethylene terephthalate glycol (PET) blood collection tubes (Greiner Bio-One, Kremsmuenster, Austria), EDTA anticoagulated blood and serum were collected. C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), creatinine, and B-type natriuretic peptide (BNP) were quantified within 4 h of blood collection in all study participants from fresh samples. In addition, serum and plasma samples were frozen in several aliquots at — 80 °C within 4 h. One of these frozen aliquots from each study participant was thawed to determine hs-cTnT, another aliquot to determine hs-cTnI, and a third aliquot to determine galectin-3. Of note, each of the aliquots was subjected to one freeze-thaw cycle only.

Criteria for the 559 blood donors to be included into the present reference value study were CRP serum concentrations < 1.0 mg/dL, PCT serum concentrations < 0.5 ng/mL, IL-6 serum concentrations < 15.0 pg/mL, and BNP plasma concentrations < 100 pg/mL (which are reference values used in our laboratory and are also generally accepted URLs for those same analytes as described in the literature).

Data were statistically analyzed with the SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) and the MedCalc 13.0.0.0 package (MedCalc Software, Mariakerke, Belgium). Comparisons of continuous variables (i.e., galectin-3, hs-cTnI and hs-cTnT) between different reference value groups were performed with the non-parametric Mann-Whitney U test or the non-parametric Kruskal-Wallis test, as appropriate. Obtained p values were not adjusted for multiple comparisons and are therefore descriptive only. URLs were calculated by using a right-sided non-parametric percentile method according to the Clinical and Laboratory Standards Institute (CLSI) guideline EP28-A3c [16]. Ninety percent confidence intervals (CI) were provided for the URLs, if possible.

22. Galectin-3, hs-cTnI and hs-cTnT measurements

In December 2009, we measured hs-cTnT plasma concentrations of the blood donors in one batch with the "Troponin T hs" assay (Roche Diagnostics, Mannheim, Germany, reagent lot number 156 593-01) [17,18] on a Modular platform because we started to use this assay at this time in clinical routine in our hospital. As stated in the package insert, this assay has a measurement range of3.0-10,000 ng/L (defined as limit of blank to the maximum value of the master curve) and a limit of detection of 5.0 ng/L. Accordingly, hs-cTnT concentrations < 5.0 ng/L were reported as 5.0 ng/L in the present work. The manufacturer reports a 10% coefficient of variation at a hs-cTnT concentration of 13.0 ng/L (i.e., limit of quantification). We confirmed all results with plasma concentrations > 15 ng/L by a second measurement; no testing for heterophilic antibodies was performed in these samples.

In February 2013, hs-cTnI plasma concentrations of the blood donors were measured in one batch with the "STAT High Sensitive Troponin-I" assay (Abbott Diagnostics, Vienna, Austria, reagent lot number 23911JN00) [18,19] on an ARCHITECT I2000SR analyzer because in 2013 we switched to this assay for clinical routine use in our hospital. As described in the package insert, this assay has a limit of blank of 0.7-1.3 ng/L, a limit of detection of 1.1 -1.9 ng/L, and a measurement range up to 50,000 ng/L. Thus, we decided to report observed hs-cTnI concentrations < 1.9 ng/L as 1.9 ng/L in this work. The manufacturer claims a 10% coefficient of variation at an hs-cTnI concentration of 4.0-10.0 ng/L (i.e., limit of quantification). We confirmed all results with plasma concentrations >25 ng/L by a second measurement; no testing for heterophilic antibodies was performed in these samples.

In November 2015, we measured galectin-3 plasma concentrations of the blood donors in one batch with the "routine Galectin-3" assay (Abbott Diagnostics, Vienna, Austria, reagent lot number 48441M500) [20,21] on an ARCHITECT I2000SR analyzer. According to the package insert this assay has a measurement range of 4-114 ng/mL, and the limit of detection is claimed to be < 1.0 ng/mL by the manufacturer. As previously reported [22], the Abbott galectin-3 assay has a total coefficient of variation of < 5.0% for analyte concentrations > 16 ng/mL, and the manufacturer describes a 10% coefficient of variation at galectin-3 concentrations of <4.0 ng/mL (i.e., limit of quantification). We confirmed all results with plasma concentrations > 20 ng/mL by a second measurement.

2.3. Further biochemical analyses

Serum concentrations of CRP were measured with the standard laboratory method Tina-quant CRP (Latex) (Roche Diagnostics, Mannheim, Germany), a particle-enhanced immunoturbidimetric assay, processed on a Modular platform. PCT serum concentrations were determined with an immunofluorescent assay by using the B.R.A.H.M.S. PCT assay (B.R.A.H.M.S. AG, Hennigsdorf, Germany) on the KRYPTOR analyzer. Serum concentrations of IL-6 were measured with a solid-phase, enzyme-labeled, chemiluminescent sequential immunometric assay on an IMMULITE 2000 instrument (Siemens Medical Solutions Diagnostics, Vienna, Austria). Creatinine serum concentrations were measured by a kinetic colorimetric assay according to the Jaffe method (calibrators IDMS traceable; Roche Diagnostics, Mannheim, Germany) on a Modular platform. Estimated glomerular filtration rate (eGFR) was calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation [23]. Plasma BNP concentrations were measured by using the ARCHITECT BNP assay (Abbott Diagnostics, Vienna, Austria) which is a chemilumines-cent microparticle immunoassay on an ARCHITECT I2000SR analyzer.

3. Results

Of the 559 blood donors recruited for establishing reference values, 31 did not fulfill the inclusion criteria and, thus, 528 were finally enrolled into this reference value study for determining upper reference limits of galectin-3, hs-cTnI and hs-cTnT. Of the 528 individuals, 338 (64%) were male and 190 (36%) were female. Biomarker concentrations of all three analytes differed significantly between male and female individuals (Mann-Whitney-U tests for each analyte, p < 0.001). In

Analyte concentrations of the entire reference value cohort (n = 528) according to renal function.

Analyte Individuals with eGFR >90 mL/min/1.73 m2 Individuals with eGFR <90 mL/min/1.73 m2 p value (n = 402) (n = 126)

Galectin-3 [ng/mL] 10.7 (range, 5.0-21.9; 25th-75th percentiles, 9.3-12.4) 12.1 (range, 6.0-24.4; 25th-75th percentiles, 10.2-14.1) <0.001

hs-cTnI [ng/L] 1.9 (range, 1.9-101.1; 25th-75th percentiles, 1.9-2.0) 1.9 (range, 1.9-48.7; 25th-75th percentiles, 1.9-2.5) 0.005

hs-cTnT [ng/L] 5.0 (range, 5.0-20.1; 25th-75th percentiles, 5.0-5.0) 5.0 (range, 5.0-25.3; 25th-75th percentiles, 5.0-5.2) <0.001

Abbreviations: hs-cTnI, high-sensitivity cardiac troponin I (Abbott); hs-cTnT, high-sensitivity cardiac troponin T (Roche).

Data are presented as median (range; 25th-75th percentiles). Difference of the analyte concentrations of both groups was calculated with Mann-Whitney-U tests (two-sided p values were not corrected for multiple comparisons).

addition, when stratifying the 528 individuals into two groups by renal function, those with an eGFR > 90 mL/min/1.73 m2 did have significantly different galectin-3, hs-Tnl and hs-TnT plasma concentrations compared to those with an eGFR <90 mL/min/1.73 m2 (Mann-Whitney-U tests for each analyte, p < 0.005). Analyte concentrations of the entire reference value cohort (n = 528) according to renal function are shown in Table 1.

Thus, we decided to calculate sex-specific upper reference limits by excluding those individuals with an eGFR < 90 mL/min/1.73 m2. As a consequence, we had 402 individuals with an eGFR >90 mL/min/ 1.73 m2 for our present evaluation; 259 were male (64%) and 143 female (36%). The median individuals' age was 35.0 years (range, 18.064.4, 25th-75th percentiles, 25.9-45.1), and the median individuals' body mass index was 24.5 kg/m2 (range, 17.3-38.4,25th-75th percentiles, 22.3-27.2). The distributions of galectin-3, hs-cTnI and hs-cTnT plasma concentrations across different age groups are depicted in Fig. 1 for male and female individuals separately. The results of our reference value calculations are detailed in Table 2. Because it has been posed that the future metric for cardiac troponins may shift from the 99th percentile to the more conventional 97.5th percentile as used in laboratory medicine for most analytes [10], we decided to report several measures of distribution for galectin-3, hs-cTnI and hs-cTnT.

The 97.5th percentile URL for galectin-3 was 16 ng/mL in male and

17 ng/mL in female individuals; the 99th percentile URL for cTnI was 39 ng/L in male and 24 ng/L in female individuals; and the 99th percen-tile URL for cTnT was 14 ng/L in male and 11 ng/L in female individuals. For our reference value analyses, we did not exclude any potential outliers because those individuals with hs-cTnI plasma concentrations

> 15 ng/L (n = 8) were different from those individuals with hs-cTnT plasma concentrations > 10 ng/L (n = 7); in addition there was no overlap of those individuals with galectin-3 plasma concentrations

> 16 ng/mL (n = 12) with the individuals of the former two groups.

Of the 402 individuals of the present evaluation, none had galectin-3 plasma concentrations below the limit of detection of this assay (limit of detection of < 1.0 ng/mL for this assay, lowest value of 5.0 ng/mL in this reference value cohort). In contrast, 290 (72%) had hs-cTnI plasma concentrations < 1.9 ng/L (i.e., limit of detection of the Abbott assay), and 359 (89%) had hs-cTnT plasma concentrations < 5.0 ng/L (i.e., limit of detection of the Roche assay).

The distribution of analyte concentrations across different age groups indicated a rather weak but statistically significant association of galectin-3 plasma concentrations with age groups (Kruskal-Wallis tests, p = 0.008 for male and p = 0.006 for female individuals) but a much smaller phenomenon for hs-cTnI plasma concentrations (Kruskal-Wallis tests, p = 0.009 for male and p = 0.186 for female individuals) and for hs-cTnT plasma concentrations (Kruskal-Wallis tests, p = 0.106 for male and p = 0.388 for female individuals).

4. Discussion

To date, two studies evaluating reference values for galectin-3 in adults using the Abbott assay have been published, however, with conflicting results [20,21]. La'ulu et al. reported 97.5th percentile URLs of

18 ng/mL for males and 20 ng/mL for females in a cohort of 242 self-reported cardiac healthy individuals [20]. In contrast, Gaze et al. found higher values in male individuals compared with female individuals and reported 97.5th percentile URLs of 33 ng/mL for males and 27 ng/mL for females in a cohort of 627 apparently healthy individuals [21]. Our present reference value study on 402 healthy blood donors revealed higher values in females compared with males rather supporting the results of La'ulu et al. [20]. Even the manufacturer describes higher median galectin-3 concentrations and higher URLs for females compared with males in the package insert of the galectin-3 assay. Our results of higher galectin-3 concentrations in female individuals are further supported by previously published data from community based studies using another galectin-3 assay [24,25]. In our present

Fig. 1. Distribution of (A) galectin-3; (B) hs-cTnI; and (C) hs-cTnT plasma concentrations in the reference value cohort according to age groups. In each age group all data were plotted as open squares (male individuals) or as open circles (female individuals), and the horizontal lines represent the median analyte concentrations for males and females of each age group. Abbreviations: hs-cTnI, high-sensitivity cardiac troponin I (Abbott); hs-cTnT, high-sensitivity cardiac troponin T (Roche).

evaluation, we found 97.5th percentile URLs of 16 ng/mL for males and 17 ng/mL for females, which are lower than in both previously published reference value studies on the Abbott galectin-3 assay [20,21 ] and also lower than the values reported in the package insert. We would like

Table 2

Reference values for the cohort of individuals with an eGFR > 90 mL/min/1.73 m2 (n = 402).

Analyte Gender Median 25th-75th percentiles 95th percentile URLa 97.5th percentile URLa 99th percentile URLa

Galectin-3 [ng/mL] Male (n = 259) 10.4 9.1-12.1 14.7(90% CI, 13.9-15.7) 15.8 (90% CI, 14.7-16.1) 16.2

Female (n = 143) 11.6 9.5-13.1 16.2(90% CI, 15.2-16.9) 16.9 (90% CI, 16.0-21.9) 20.0

hs-cTnI [ng/L] Male (n = 259) 1.9 1.9-2.2 7.1 (90% CI, 4.9-14.8) 16.4(90% CI, 7.5-36.0) 39.0

Female (n = 143) 1.9 1.9-1.9 4.1 (90% CI, 2.7-14.0) 10.1 (90% CI, 3.9-29.7) 23.5

hs-cTnT [ng/L] Male (n = 259) 5.0 5.0-5.0 7.3 (90% CI, 6.5-9.6) 9.9(90% CI, 7.3-11.7) 13.9

Female (n = 143) 5.0 5.0-5.0 5.0 (90% CI, 5.0-5.0) 5.0 (90% CI, 5.0-16.1) 11.3

Abbreviations: CI, confidence interval; hs-cTnI, high-sensitivity cardiac troponin I (Abbott); hs-cTnT, high-sensitivity cardiac troponin T (Roche); URL, upper reference limit.

a We followed the Clinical and Laboratory Standards Institute (CLSI) guideline EP28-A3c for estimating right-sided percentiles and their 90% CI. In these guidelines, percentiles are calculated as the observations corresponding to rank r = p * (n + 1). Also for the 90% CI of the reference limits, the CLSI guidelines were followed and conservative CI was calculated using integer ranks (and therefore the CI is at least 90% wide). No potential outliers were excluded for galectin-3, hs-cTnI or hs-cTnT.

to suggest that our reference value cohort was more "healthy" due to our stringent exclusion criteria (regarding not only cardiovascular disease, but also renal disease, diabetes mellitus, cancer, current infection and chronic inflammatory disease; diseases known to be associated with circulating galectin-3 concentrations). Probably, the situation for galectin-3 is the same as with, e.g., cardiac troponins, where the reference population selection strategy (coning) significantly influences the reference values for troponin assays [8,9,26].

In the past, numerous studies have been published on reference values for hs-cTnI determined with the "STAT High Sensitive Troponin-I" assay from Abbott Diagnostics [9,17,18] and for hs-cTnT determined with the "Troponin T hs" assay from Roche Diagnostics [8,9,18, 19,26]. In principle, our work is confirming the results of previously published reference value studies on the same assays with respect to the magnitude of the URLs obtained. Considering that our URLs were derived without any "outlier" exclusion procedure, we are able to argue that we had very stringent selection criteria and, thus, rather low reference values for hs-cTnI and hs-cTnT. If we had, for example, excluded one "outlier" in our male cohort and one "outlier" in the female cohort, the 99th percentile URLs would have dropped significantly for hs-cTnI (male, from 39 ng/L to 29 ng/L; female from 24 ng/L to 15 ng/L) and also for hs-cTnT (male, from 14 ng/L to 11 ng/L; female from 11 ng/L to 5 ng/L). However, as stated in the Results section, those individuals with hs-cTnI values > 15 ng/L (n = 8) were different from those individuals with hs-cTnT values > 10 ng/L(n = 7), indicating that the substantial increased values produced by both assays in different individuals were not real in nature (in the sense of myocardial injury) but represent non-specific rises of hs-cTnI or hs-cTnT. This observation was the reason why we decided not to exclude potential "outliers" from our reference value calculation for both analytes. Our advantage was that we had measured hs-cTnI or hs-cTnT (and also galectin-3) in a single cohort of healthy individuals. Thus, we complied with an important requirement recently claimed by several experts in the field [11,12,18,27].

Most importantly, with respect to the "STAT High Sensitive Troponin-I" assay from Abbott Diagnostics and the "Troponin T hs" assay from Roche Diagnostics, we found that of the 402 healthy blood donors, 290 (72%) had hs-cTnI plasma concentrations < 1.9 ng/L (i.e., limit of detection of the Abbott assay), and 359 (89%) had hs-cTnT plasma concentrations <5.0 ng/L (i.e., limit of detection of the Roche assay). Recently, it has been postulated that high-sensitivity assays should measure cardiac troponins above the limit of detection in > 50% of healthy individuals [13,27]. Thus, at least in our reference population both the Abbott hs-cTnI assay and the Roche hs-cTnT assay failed to comply with this demand.

In conclusion, plasma concentrations of galectin-3, hs-cTnI and hs-cTnT and corresponding 99th percentile URLs were rather low in our cohort of healthy blood donors compared with previously published data. In our reference population, analyte plasma concentrations above the limit of detection were detectable in 100% of the individuals with the Abbott galectin-3 assay, but only in less than 50% for both the Abbott hs-cTnI assay and the Roche hs-cTnT assay.

We acknowledge that hs-cTnT, hs-cTnI and galectin-3 have been measured in the same cohort but after approximately one, five and seven years from sample collection, respectively. At present, there are no published studies reporting on the long-term in-vitro stability of galectin-3 and hs-cTnI as measured with the Abbott assays and of hs-cTnT as measured with the Roche assay at — 80 °C. According to the package inserts and to otherwise published work [21,28-30], however, in-vitro stabilities of all three analytes stored at <—10 °C are evident for up to several months. Because we have subjected frozen aliquots to one freeze-thaw cycle only as outlined in the Materials and methods section and, thus, met the accepted pre-analytical requirements for frozen samples, we assume analyte stabilities at — 80 °C even for several years.

It might be considered another limitation or our work that our reference value population was mainly composed by young people with a median individuals' age of 35.0 years (range, 18.0-64.4; 25th-75th percentiles, 25.9-45.1). As a consequence, we cannot entirely exclude the possibility that this might have contributed to the observation that the tests for hs-cTnI and hs-cTnT did not match the definition for "high sensitive". Nevertheless, we saw remarkably increased troponin concentrations predominantly in the younger individuals as shown in Fig. 1, and because these values did significantly contribute to the 99th percentile URLs of hs-cTnI and hs-cTnT in our work, evaluating rather elderly individuals might have contributed to lower 99th percentile URLs of both analytes. Notwithstanding the above, we think that in the case of establishing reference values for cardiac troponins and other cardiac bio-markers rigor in subject selection is mandatory. We would like to suggest that it would be prudent to use highly selected population groups when performing reference interval studies. Selection criteria should include a detailed questionnaire and several biomarker measurements (maybe also electrocardiography and echocardiography) to reliably exclude individuals with any cardiovascular disease, renal disease and inflammatory/infectious disease. With such rigid criteria one would probably be forced to rely on rather young individuals for a reference value study.

A last possible limitation deserves also clarification: According to the Materials and methods section, the three analytes of interest have been determined in EDTA plasma samples; according to the package inserts of Abbott and Roche, galectin-3 hs-cTnI and hs-cTnT concentrations can be evaluated in both EDTA plasma and serum. However, according to a recent publication [19], slight but significantly different values may be obtained measuring hs-cTnI in heparin plasma vs. EDTA plasma or serum. In this publication, the slopes and intercepts obtained for serum vs. lithium heparin and for EDTA vs. lithium heparin were approximately 1.15 and <0.5 ng/L, respectively, by Passing Bablok but not significantly different by Bland-Altman and, thus, can be considered negligible for clinical practice.

Conflict of interest statement

Author's conflict of interest disclosure: Abbott Diagnostics and Roche Diagnostics provided reagents for measurement of hs-cTnI and hs-cTnT plasma concentrations, respectively, free of charge. The companies

(1) did not play a role in the study design; (2) in the collection, analysis, and interpretation of data; (3) in writing of the report; (4) or in the decision to submit the report for publication. Research funding: None declared. Employment or leadership: None declared.

Honoraria: Thomas Mueller has received speaking fees from Abbott Diagnostics, B.R.A.H.M.S. AG, and Roche Diagnostics. Benjamin Dieplinger has received speaking fees from Abbott Diagnostics and Roche Diagnostics.

Nonstandard abbreviations

BNP B-type natriuretic peptide

CI confidence interval

CRP C-reactive protein

eGFR estimated glomerular filtration rate

hs-cTnI high-sensitivity cardiac troponin I

hs-cTnT high-sensitivity cardiac troponin T

IL-6 interleukin-6

PCT procalcitonin

URL upper reference limit

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