Concentrations and agreement over 10 years with different assay versions and analyzers for troponin T and N-terminal pro-B-type natriuretic peptide

Peter A. Kavsak; Lorna Clark; Andrew Worster; Sukhbinder Dhesy-Thind

doi:10.1515/cclm-2024-1382

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Concentrations and agreement over 10 years with different assay versions and analyzers for troponin T and N-terminal pro-B-type natriuretic peptide

Peter A. Kavsak , Lorna Clark , Andrew Worster and Sukhbinder Dhesy-Thind

Published/Copyright: December 10, 2024

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 63 Issue 5

Keywords: high-sensitivity cardiac troponin; N-terminal pro-B-type natriuretic peptide; stability; assay versions; different analyzers

To the Editor,

Retrospective analyses on samples stored frozen have contributed largely to the knowledge base of cardiac troponin (cTn) and the B-type natriuretic peptides (NPs) [1], 2]. The impact of sample type, storage conditions and degradation has been well documented for the NPs (i.e., with BNP being more susceptible to degradation as compared to N-terminal pro-BNP; NTproBNP), with less information on these parameters being known for the various cTn assays [2], 3]. The recent publication by Henricks and colleagues further details the impact of matrix and stability for cTnT over six years, with serum as opposed to lithium heparin yielding fewer discordant results on repeat measurements over this period [4]. We sought to further assess agreement of cTnT (as measured with a high-sensitivity assay) and NTproBNP over different assay versions and analyzers on samples stored frozen for 11 years.

Briefly, EDTA plasma was stored (−80 °C) from patients who consented to provide plasma for cardiac biomarker measurements (research ethics approved) with cTnT (Elecsys Troponin T hs) and NTproBNP (Elecsys proBNP II) measurements first performed in 2012 on the Roche Modular E170 analyzer [5]. In 2012, the cTnT concentrations were obtained from a reagent lot that recovered lower values (approximately 5–7 ng/L lower), with Roche indicating that future lots will recover these biases [6], 7]. An additional aliquot was measured in 2018 for NT-proBNP and cTnT in 2020 on the Roche e602 analyzer, with a second thaw performed on these aliquots in 2023 with measurements performed on the Roche e411 for these analytes. Of note, in 2021 the cTnT assay underwent a revision to further mitigate the effect of biotin on measurements [8]. Correlation, Passing-Bablok regression analyses, and median differences (95 % confidence intervals; CI)) between the analyzers were determined with values <3 ng/L (i.e., below the limit of blank) for cTnT recorded as 2 ng/L for this analysis (Analyse-it statistical software). Agreement between results was considered acceptable if differences were within 30 % for NTproBNP [2] and ±3 ng/L <10 ng/L or ±30 % ≥10 ng/L for cTnT [9], 10]; with the 30 % criterion also being proposed for hs-cTn assays by the Study Group on Cardiac Biomarkers from the Italian Society of Biochemical Chemistry (SIBioC) and European Ligand Assay Society (ELAS) [11].

There were 102 plasma aliquots from 11 patients that were measured in 2012, 2018, 2020 and 2023. For the NTproBNP assay, the range of concentrations was 15–1,019 ng/L in 2012 and 16–1,137 ng/L in 2023. Regression analyses yielded the following equation: NTproBNP 2023 (e411)=1.09 (95 % CI: 1.07–1.11) × NTproBNP 2012 (E170) − 3.6 ng/L (95 % CI: −4.5 to −2.2), r=0.999 with the median difference being 4.9 % (95 % CI: 3.2–7.0). The median differences for NTproBNP ranged from 0.3 % (e411 in 2023 vs. e602 in 2018) to 6.0 % (e602 in 2018 vs. E170 in 2012), with the range of discordant results being 0–5.8 % (Figure 1A–C).

Figure 1:

Testing 102 EDTA plasma samples over 11 years for NTproBNP and cTnT. For NTpro BNP. (A) Difference plot between 2018 e602 analyzer vs. 2012 E170 analyzer; (B) difference plot between 2023 e411 analyzer vs. 2018 e602 analyzer; (C) difference plot between 2023 e411analyzer vs. 2012 E170 analyzer and cTnT with a high-sensitivity assay: (D) difference plot between 2018 e602 analyzer vs. 2012 E170 analyzer; (E) difference plot between 2023 e411 analyzer vs. 2018 e602 analyzer; (F) difference plot between 2023 e411 analyzer vs. 2012 E170 analyzer.

For cTnT, the range of concentrations were 2.0–111 ng/L in 2012 and 3.7–97.6 ng/L in 2023. Regression analyses yielded the following equation: cTnT 2023 (e411)=0.90 (95%CI: 0.85 to 0.96)) × cTnT 2012 (E170) + 4.3 ng/L (95 % CI: 3.8–4.9), r=0.996 with the median difference being 3.4 ng/L (95 % CI: 3.2 to 3.8) (Figure 1F). In 2012, 40 % of the cTnT results were <3 ng/L with all results being ≥3 ng/L in 2020 and 2023. Between 2023 (e411) and 2020 (e602) the median difference was −1.4 ng/L (95 %CI: −1.9 to −0.9) with no discordant results (Figure 1E). However, assessing differences (cTnT in 2023 – cTnT in 2020) around the manufacturer’s listed 99th percentile concentrations (i.e., 9 ng/L which is the female 99th percentile outside the United States to 22 ng/L which is the male 99th percentile in the United States), the median difference was −1.7 ng/L (95 % CI: −2.2 to −1.4) (n=50, range in 2020 for cTnT being 9.0–22.4 ng/L with corresponding range being 7.7–20.4 ng/L in 2023). Comparing 2020 (e602) vs. 2012 (e411) the results were the most divergent for cTnT with the median difference being 5.1 ng/L (95 % CI: 4.6–5.4) with 77 % of the result being discordant (Figure 1D).

With studies like this, there are always two potential causes of deviations: i) change in the true concentration of the analyte and ii) change in the assays (i.e., calibration, instrumentation, etc.), with multiple freeze/thaws having minimal effect on stability of these analytes [12], 13]. The findings of our study are noteworthy insofar that NTproBNP measured on three different analyzers and lots over a span of 11 years yielded very close agreement in concentrations. However, differences existed between cTnT as measured on different analyzers, lots, and assay versions over this same period. Importantly, these data confirm Roche’s initial technical bulletin that post 2012, cTnT levels will be higher at the low end by approximately 5 ng/L, and importantly further revisions to the assay to mitigate the impact of biotin have not changed the analytical agreement, if using the ±3 ng/L or ±30 % criteria. However, minor absolute differences near the 99th percentile concentrations exist between 2023 and 2020, with additional studies needed to identify if these differences can be corrected (i.e., with different calibrations etc.). Nevertheless, these data extend previous stability work in this area [4] and support that results obtained in EDTA plasma after 2012 for both cTnT and NTproBNP are in close agreement when measured on different Roche analyzers and after 10 or more years of storage below −80 °C.

Corresponding author: Dr. Peter A. Kavsak, Juravinski Hospital and Cancer Centre, 711 Concession Street Hamilton, ON, L8V 1C3, Canada, E-mail: kavsakp@mcmaster.ca

Acknowledgments

Roche Diagnostics for providing the reagents for testing.

Research ethics: Research ethics approval was obtained.
Informed consent: Patients provided informed consent.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Use of Large Language Models, AI and Machine Learning Tools: No LLM, AI or ML tools were used.
Conflict of interest: Dr. Kavsak has received grants/reagents/consultant/advisor/honoraria from Abbott Laboratories, Abbott Point of Care, Beckman Coulter, Ortho Clinical Diagnostics, Randox Laboratories, Roche Diagnostics, Quidel, Siemens Healthcare Diagnostics and Thermo Fisher Scientific. McMaster University has filed patents with Dr. Kavsak listed as an inventor in the acute cardiovascular biomarker field.
Research funding: Roche Diagnostics for providing the reagents for testing.
Data availability: Contact the corresponding author.

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Received: 2024-11-25

Accepted: 2024-12-02

Published Online: 2024-12-10

Published in Print: 2025-04-28

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/cclm-2024-1382

Keywords for this article

high-sensitivity cardiac troponin; N-terminal pro-B-type natriuretic peptide; stability; assay versions; different analyzers

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