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Reference intervals of circulating secretoneurin concentrations determined in a large cohort of community dwellers: the HUNT study

  • Kristin M. Aakre ORCID logo EMAIL logo , Magnus N. Lyngbakken , Arne L. Faaren , Helge Røsjø , Håvard Dalen and Torbjørn Omland
Published/Copyright: April 4, 2024
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 62 Issue 10

Abstract

Objectives

Secretoneurin (SN) is a novel cardiac biomarker that associates with the risk of mortality and dysfunctional cardiomyocyte Ca2+ handling in heart failure patients. Reference intervals for SN are unknown.

Methods

SN was measured with a CE-marked ELISA in healthy community dwellers from the fourth wave of the Trøndelag Health Study (HUNT4) conducted in 2017–2019. The common, sex and age specific 90th, 95th, 97.5th and 99th percentiles were calculated using the non-parametric method and outlier exclusion according to the Reed test. The applicability of sex and age specific reference intervals were investigated using Harris and Boyd test. We also estimated the percentiles in a subset with normal findings on echocardiographic screening.

Results

The total cohort included 887 persons (56.4 % women). After echocardiographic screening 122 persons were excluded, leaving a total of 765 persons (57.8 % women). The 97.5th percentile (95 % CI in brackets) of SN was 59.7 (57.5–62.1) pmol/L in the total population and 58.6 (57.1–62.1) pmol/L after echocardiography screening. In general, slightly higher percentiles were found in women and elderly participants, but less than 4 % in these subgroups had concentrations deviating from the common 97.5th percentile. Low BMI or eGFR was also associated with higher concentrations of SN.

Conclusions

Upper reference limits for SN were similar amongst healthy adult community dwellers regardless of prescreening including cardiac echocardiography or not. Women and elderly showed higher concentrations of SN, but the differences were not sufficiently large to justify age and sex stratified upper reference limits.

Keywords: secretoneurin; upper reference limit; reference interval; predictor

Introduction

Secretoneurin (SN) is a 33-amino acid peptide belonging to the granin protein family originating from neuroendocrine and myocardial tissue. SN is involved in different physiologic processes in the myocardium, including cardiomyocyte Ca2+ handling [1]. Recently, SN was proposed as a novel prognostic cardiac biomarker in patients with heart failure [2] and severe illness [3], as recent data suggest an association with mortality and ventricular arrhythmia in these patient groups [4]. SN also seems to have limited biological variability, which leads to high signal-to-noise ratio [5].

Correct interpretation and clinical use of novel biomarkers require certain basic information to be in place, and a crucial parameter necessary for clinical decision making is the reference interval. The reference interval of a biomarker is usually defined as the central 95 % distribution in a healthy cohort (≥2.5th to ≤97.5th percentile), although for one sided reference intervals (i.e., when only elevated or reduced concentrations are clinically important), the 95th percentile has sometimes been suggested. For prognostic markers there are examples that the 90th percentile has been suggested as upper cut-off for signaling increased risk [6].

Reference intervals describe the expected concentrations in healthy individuals [7], and a result within the reference interval can be interpreted as a normal physiological response and homeostasis. Healthiness is typically based on self-reporting (frequently questionnaires) in combination with measurement of relevant biomarkers [7], [8], [9], and more advanced investigations are rarely included for screening purposes. However, some studies on cardiac biomarkers have demonstrated that selection on normality based on advanced cardiac imaging like echocardiography influence the upper reference limits [10]. Other important metrics for interpretation in individual patients are related to systematic differences in concentrations based on physiological parameters like age, sex, and body mass index (BMI).

Accordingly, the primary aim of this study was to estimate upper reference limits (90th, 95th, 97.5th and 99th percentiles) for SN measured in a cohort of healthy adult community dwellers with and without prescreening using echocardiography. Secondly, we aimed to investigate if sex or age partitioned reference intervals were applicable for SN.

Materials and methods

Study population

The study sample encompasses a subset of participants from the fourth wave of the Trøndelag Health Study (HUNT4) who were investigated with echocardiography. HUNT is the largest Norwegian population study and has performed regular updates since 1984 with a total of 230,000 individuals included [11]. HUNT4 was conducted in 2017–2019 and included questionnaires, a basic clinical investigation collecting anthropometric measures and sampling of biological material including blood sampling from 56,044 participants [12]. The current study include data from a subset of 5,763 participants who were invited to the HUNT4Echo study, details have been described earlier [13], see Figure 1. Briefly, participants were invited if they had previously participated in the HUNT3 (2006–2008) Fitness or Echocardiography studies or if they had validated atrial fibrillation (AF) from HUNT3 or self-reported AF from HUNT4. In total, 3174 persons responded. SN measurements were available in 2,486 of responders and valid echocardiography data was available in 2,448 of these. We defined a healthy population subgroup according to self-reporting (questionnaire), clinical examination, and a biomarker screening (Supplemental Table 1). We also estimated percentiles in a subset of participants after excluding all participants who had echocardiographic findings indicating subclinical myocardial disease (left ventricular mass index>115 g/m2 for men and>95 g/m2 for women or left ventricular ejection fraction<55 %). A LVEF of 55 % was used as a cut-off to reduce the likelihood of including subjects with underlying heart failure or incipient heart failure in this subgroup.

Figure 1: Study overview. SN; secretoneurin.
Figure 1:

Study overview. SN; secretoneurin.

The study was approved by the Regional Committee for Medical and Health Research Ethics of Mid-Norway (REC ID 13083) and was conducted in compliance with the ethical principles of the Declaration of Helsinki. All participants provided informed written consent. Personal data security and data handling were approved by the institutional personal data protection officer at St Olav’s Hospital and Norwegian University of Science and Technology.

Laboratory analysis

Circulating SN was measured in serum samples using a CE-marked commercially available ELISA (CardiNor AS, Oslo, Norway). The average of duplicate measurements was calculated and reported as the final result. The within-plate analytical variation was 2.7 and 3.6 % at 32.6 and 71.5 pmol/L, respectively. The between-plate analytical variation was 4.8 and 5.0 % at 32.6 and 71.5 pmol/L, respectively. The within laboratory imprecision (run-to-run, day-to-day, and replicate-to-replicate variation) was 8.7 % at 32.6 pmol/L and 6.7 % at 71.5 pmol/L, respectively. The measuring range was 10–250 pmol/L [14].

C-reactive protein (CRP), total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, HbA1c and creatinine were measured using Architect ci8200 from Abbott Diagnostics. The creatinine assay was enzymatic and traceable to the IDMS reference method, and eGFR was estimated using the CKD-EPIcreat equation [15].

Echocardiography

Comprehensive details of the echocardiographic examinations have been published previously [13, 16, 17]. All echocardiograms were acquired using Vivid E95 scanners (GE Vingmed Ultrasound, Horten, Norway) with phased array transducers (M5S). All recordings and measurements were performed by experienced personnel according to the present recommendations [18]. Analyses and measurements were done using EchoPAC SWO (version 203; GE Ultrasound, Horten, Norway). Two-dimensional recordings included parasternal long- and short-axis views as well as standard apical views of the left ventricle, right ventricle and atria. All recordings were dedicated for the specific chamber and task. Shortly, left ventricular dimensions and wall thickness were measured at end-diastole and end-systole in parasternal long-axis view. LV mass was calculated from end-diastolic measurements using the Cube formula. Left ventricular volume was measured using the biplane Simpson’s (summation of discs) method from four- and two-chamber recordings with tracing of the endocardial borders at end-diastole and end-systole. Left ventricular ejection fraction was calculated as the difference between end-diastolic and end-systolic volumes divided by the end-diastolic volume.

Statistical analysis

Baseline characteristics of the study cohort are presented as absolute numbers with proportion or medians with interquartile range [IQR] unless otherwise stated. Between group comparisons for continuous variables were analyzed using the Mann–Whitney U test and categorical variables with the Fisher exact test. We assessed the crude associations between SN concentration and age, eGFR, and BMI by restricted cubic splines with knots placed at the 5th, 27.5th, 50th, 72.5th, and 95th sample percentiles. The 90th, 95th, 97.5th and 99th percentiles for SN concentrations were calculated for men and women separately and combined, and according to age (subgroups stratified according to the cohort median age or arbitrary chosen age groups (<40 years, 40–59 years and ≥60 years)), using the non-parametric method and outlier exclusion according to the Reed test, as described by the Clinical & Laboratory Standards Institute (CLSI) [9]. The applicability of sex and age specific reference intervals were investigated as suggested by Harris and Boyd (i.e. if >4 % of a specific population subgroup exceed the common 97.5th percentile, stratified reference intervals could be recommended) [9].

Results

A total of 887 persons (56.4 % women) from the general population were defined as a healthy population subgroup and included in the study. Baseline characteristics according to quartiles of SN are provided in Table 1. Persons in the upper quartile were more frequently older females with lower eGFR and BMI and showed higher HDL cholesterol. Baseline characteristics for the healthy population subgroup and after echocardiography screening (n=765) are shown in Supplemental Table 2. BMI and eGFR were linearly associated with SN concentrations (Figure 2).

Table 1:

Baseline characteristics of the healthy population subgroup according to quartiles of secretoneurinconcentrations.

Quartile 1 Quartile 2 Quartile 3 Quartile 4
n=222 n=222 n=222 n=221 p-Value
Secretonaurin, pmol/L 30.8 (28.3–32.5) 36.7 (35.5–37.9) 42.1 (40.6–43.8) 50.0 (47.4–53.9) <0.001
Male sex, n (%) 132 (59.5 %) 105 (47.3 %) 88 (39.6 %) 62 (28.1 %) <0.001
Age, years 54.1 (45.8–63.6) 54.0 (47.1–63.3) 54.6 (46.9–63.2) 58.1 (50.5–65.0) 0.024
Higher education, n (%) 106 (47.7 %) 135 (60.8 %) 150 (67.6 %) 122 (55.5 %) <0.001
Body mass index, kg/m2 25.9 (24.5–27.9) 24.8 (23.0–27.4) 24.7 (22.7–26.5) 24.3 (21.9–26.6) <0.001
Waist-to-hip ratio 0.92 (0.88–0.97) 0.91 (0.87–0.96) 0.90 (0.86–0.96) 0.89 (0.86–0.95) <0.001
Heart rate, bpm 66 (60–74) 67 (60–74) 66 (61–73) 68 (60–75) 0.82
Systolic blood pressure, mmHg 121 (114–128) 121 (113–128) 119 (111–127) 119 (112–127) 0.14
Diastolic blood pressure, mmHg 72 (67–77) 73 (66–79) 72 (66–77) 71 (66–76) 0.19
Total cholesterol, mmol/L 5.6 (4.7–6.2) 5.5 (4.8–6.2) 5.6 (5.0–6.3) 5.6 (5.0–6.4) 0.25
HDL cholesterol, mmol/L 1.4 (1.2–1.6) 1.5 (1.2–1.8) 1.5 (1.2–1.7) 1.6 (1.3–1.9) <0.001
HbA1c, % 5.1 (5.0–5.3) 5.2 (5.0–5.3) 5.2 (5.0–5.4) 5.2 (5.0–5.3) 0.46
eGFR, mL/min/1.73m2 94.0 (87.0–102.0) 93.0 (85.0–101.0) 91.0 (81.0–100.0) 87.0 (78.0–96.0) <0.001

  1. HDL, high-density lipoprotein; HbA1c, hemoglobin A1c; eGFR, estimated glomerular filtration rate.

Figure 2: Associations between body mass index, estimated glomerular filtration rate (eGFR), and secretoneurin concentrations, adjusted for age and sex.
Figure 2:

Associations between body mass index, estimated glomerular filtration rate (eGFR), and secretoneurin concentrations, adjusted for age and sex.

The distributions of SN concentrations are shown in Supplemental Figures 1 and 2 and the upper reference limit percentiles for SN in the healthy population subgroup are shown in Table 2. The number of outliers excluded based on the Reed test is outlined in Supplemental Table 3.

Table 2:

Common upper reference limit percentiles for secretoneurin (with Reed outlier detection).

Secretoneurin, pmol/L (95 % CI)
Ordinary screening Ordinary screening + echocardiography screening
Total (n=886) Total (n=764)
99th percentile 65.9 (61.1–85.3) 66.1 (60.6–85.3)
97.5th percentile 59.7 (57.5–62.1) 58.6 (57.1–62.1)
95th percentile 54.8 (53.5–57.7) 54.4 (53.3–57.5)
90th percentile 51.2 (50.3–52.7) 51.1 (50.1–52.7)

  1. Ordinary screening, excluding subjects with established cardiovascular disease (angina pectoris, myocardial infarction, heart failure, atrial fibrillation, stroke), BMI <18 kg/m2 or >35 kg/m2, on lipid lowering therapy, on antihypertensive therapy or with baseline systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, with established diabetes mellitus or baseline HbA1c ≥48 mmol/mol, with established renal failure or baseline estimated glomerular filtration rate <60 mL/min/1.73m2, with obstructive lung disease, with history of malignancy, or current smoker. Echocardiography screening, excluding subjects with left ventricular mass index >115 g/m2 for men and >95 g/m2 for women or left ventricular ejection fraction <55%.

The sex-neutral percentiles were similar after ordinary and echocardiographic screening. There was a trend towards lower percentiles in men, except for the 99th percentile when estimated in the ordinary screening group (Table 3). Similar trends were observed for age, with generally higher percentiles of SN in study participants with age above the cohort median (Table 4), and lower reference intervals in the age group <40 years (Supplemental Table 4). Adjusting for age, mean concentrations of SN were 41.1 (95 % CI 40.3 to 41.9) pmol/L in women and 37.3 (95 % CI 36.4 to 38.1) pmol/L in men (p for comparison<0.001). SN values were above the common 97.5th percentile in 3.0 % of women and 1.8 % of men (p<0.001). Similarly, 1.4 % of study participants with age below the median and 3.6 % of study participants with age above the median had SN values above the common 97.5th percentile (p=0.003). For the arbitrary chosen age groups, 1.1 % (<40 years), 2.4 % (40–59 years) and 3.0 % (≥60 years) was above the common 97.5 percentile, respectively.

Table 3:

Upper reference limit percentiles for secretoneurin, stratified in men and women (with Reed outlier detection).

Secretoneurin, pmol/L (95 % CI)
Ordinary screening Ordinary screening + echocardiography screening
Female (n=499) Male (n=386) Female (n=441) Male (n=322)
99th percentile 65.7 (60.6–75.3) 68.4 (58.1–101.3) 65.4 (60.6–75.3) 64.7 (55.4–101.3)
97.5th percentile 60.2 (57.8–63.8) 57.4 (51.8–65.9) 59.8 (57.7–63.8) 55.3 (51.2–65.9)
95th percentile 56.4 (54.3–59.3) 51.4 (49.5–55.4) 56.4 (54.3–58.6) 51.1 (48.1–54.0)
90th percentile 52.8 (51.3–54.3) 47.6 (46.5–49.9) 52.9 (51.3–54.3) 47.0 (45.6–49.2)

  1. Ordinary screening, excluding subjects with established cardiovascular disease (angina pectoris, myocardial infarction, heart failure, atrial fibrillation, stroke), BMI<18 kg/m2 or >35 kg/m2, on lipid lowering therapy, on antihypertensive therapy or with baseline systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, with established diabetes mellitus or baseline HbA1c ≥48 mmol/mol, with established renal failure or baseline estimated glomerular filtration rate <60 mL/min/1.73m2, with obstructive lung disease, with history of malignancy, or current smoker. Echocardiography screening, excluding subjects with left ventricular mass index >115 g/m2 for men and >95 g/m2 for women or left ventricular ejection fraction <55%.

Table 4:

Upper reference limit percentiles for secretoneurin, stratified according to median age (with Reed outlier detection).

Secretoneurin, pmol/L (95 % CI)
Ordinary screening Ordinary screening + echocardiography screening
Below median (n=444) Above median (n=442) Below median (n=388) Above median (n=376)
99th percentile 62.3 (57.1–156.8) 69.2 (62.1–101.3) 64.0 (56.4–156.8) 68.3 (60.9–101.3)
97.5th percentile 56.3 (53.2–60.5) 61.4 (58.5–67.7) 55.3 (53.0–63.8) 60.8 (58.1–67.7)
95th percentile 52.7 (51.0–54.6) 58.1 (54.5–60.6) 52.7 (51.1–54.6) 58.0 (54.3–60.6)
90th percentile 49.4 (47.6–51.1) 53.0 (51.1–54.5) 49.6 (47.6–51.1) 53.0 (51.0–54.3)

  1. Median age 55.4 years. Ordinary screening, excluding subjects with established cardiovascular disease (angina pectoris, myocardial infarction, heart failure, atrial fibrillation, stroke), BMI <18 kg/m2 or >35 kg/m2, on lipid lowering therapy, on antihypertensive therapy or with baseline systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, with established diabetes mellitus or baseline HbA1c ≥48 mmol/mol, with established renal failure or baseline estimated glomerular filtration rate <60 mL/min/1.73m2, with obstructive lung disease, with history of malignancy, or current smoker. Echocardiography screening, excluding subjects with left ventricular mass index >115 g/m2 for men and >95 g/m2 for women or left ventricular ejection fraction <55%.

Discussion

The current study reports the upper reference limits of SN in presumably healthy subjects recruited from the general population. We report several findings important for future clinical use and interpretation of SN concentrations.

First, we report the reference intervals for SN as determined in a healthy population. Our data clearly demonstrate that a clinical screening using questionnaires and common biomarkers is sufficient to provide robust estimates of the upper reference limit, this includes a sex-neutral cut-off and all percentiles from 90th through the 99th percentile. The upper reference limits for SN were similar amongst healthy adult community dwellers regardless of prescreening including cardiac echocardiography or not. Compared to cardiac troponins, SN appears to be less influenced by structural myocardial disease as identified by echocardiography [10, 19]. This observation suggests that in a healthy population neuroendocrine activity may contribute quantitatively more to circulating SN than cardiac production [20], [21], [22].

Secondly, SN concentrations are influenced by physiological factors, but the effect is moderate [23]. It is known that natriuretic peptides are higher in women, increase with age and that lower concentrations are associated with higher BMI [24, 25]. SN showed a similar trend, however findings were less pronounced and are unlikely to have a major effect on clinical interpretation of test results as differences were not large enough to justify sex or age stratified reference intervals [9]. Higher SN concentrations were associated with lower eGFR, indicating a renal dependent clearance.

Clinical implications

Reference intervals are used to characterize a physiological condition. Measuring a concentration outside the reference interval may be interpretated as a pathological condition or disease, but this will depend on applicable diagnostic definitions and medical consensus regarding interpretation of abnormal values. Current recommendations for cardiac markers show some diversity and both upper reference limits derived from a general healthy population and clinical cut-offs derived from large clinical trials are used for signaling disease or need of intervention. For cardiac troponin, which is integrated as a main criterion in the Universal Definition of Myocardial Infarction [26], the 99th percentile is used as upper reference limit. This cut-off was chosen to increase the specificity of the diagnosis [8]. Lately, the use of 99th percentiles has been questioned, in particular when cardiac troponins are used to investigate other conditions than myocardial infarction. Moreover, in children the robustness of the clinical screening procedure and statistical calculations are substantially lower for estimating the 99th percentile compared to the 97.5th percentile [27]. In the emergency room, clinical cut-offs derived from observational and randomized clinical trials are used for early prediction of low (rule-out) and high (rule-in) risk of myocardial infarction, and thereby improved logistics and patient flow [28], [29], [30]. For other well-known cardiac biomarkers like natriuretic peptides, the 97.5th percentile is used as upper reference limit although typically aligned with relevant clinical cut-offs being derived from large clinical studies [31], [32], [33]. These clinical cut-offs are used to predict high and low risk of heart failure, and thereby decide subsequent clinical investigations, both in the chronic and acute setting [33]. For another prognostic biomarker, growth differential factor 15, the 90th percentile is commonly used as cut-off for signaling increased long-term risk of cardiovascular events or death [6]. Based on current laboratory practice and expectation of potential future clinical utility of SN as reflecting risk, possibly via link to cardiac arrythmia, we suggest that the sex-neutral 97.5 percentile is the most applicable upper reference limit for SN. Also, even though we in this study report the concentrations down to one decimal place (for academic purposes), laboratories my find it feasible to report the concentrations as whole numbers if SN implemented into routine practice. If this is the most applicable clinical cut-off for signaling disease or future risk of disease should be determined in future clinical studies. Of note, we recently reported SN concentrations in patients with chronic heart failure and the patients with concentrations above 48.7 pmol/L (approximately corresponding to the 90th percentile reported here) had a poor prognosis [2]. That long-term cardiovascular prognosis is predicted by concentrations below the upper reference limit is in line with the prognostic cut-offs that have been reported for cardiac markers, like troponins and natriuretic peptides [21, 34].

Strength and limitations

The major strength in this study is the inclusion of a large cohort allowing for a thorough evaluation of physiological and clinical variables that potentially could influence SN concentrations. The high number of included subjects increases the accuracy of the reported percentiles. All participants were investigated with echocardiography allowing for exclusion of subclinical structural myocardial disease, hereby providing robust estimates for the upper percentiles reported.

The main limitation is the use of an ELISA that might be too labor intensive for later implementation in routine laboratory practice. If SN measurements are implemented in clinical practice in the future, it is likely that automated applications of the assay become available. The current reference intervals should not be directly transferred to another assay unless good agreement with the current assay has been sufficiently documented. Finally, it should be noted that the subgroup <40 years were smaller than what is recommended when 99th and 97.5 percentiles are calculated, and accordingly the estimates should be seen as suggestive.

Conclusions

Upper reference limits for SN were similar amongst healthy adult community dwellers regardless of pre-screening including cardiac echocardiography or not. Women, persons with the lowest BMI and eGFR and elderly showed higher percentiles of SN. Even though sex and age influence SN concentrations, the differences were not sufficiently large to justify age and sex stratified upper reference limits.

Corresponding author: Kristin M. Aakre, MD, PhD, Department of Medical Biochemistry and Pharmacology, Haukeland University Hospital, Jonas Lies vei 65, Bergen, Norway; Department of Heart Disease, Haukeland University Hospital, Bergen, Norway; and Department of Clinical Science, University of Bergen, Bergen, Norway, E-mail:

Funding source: K.G. Jebsen Center for Cardiac Biomarkers

Award Identifier / Grant number: SKGJ-MED-024

Funding source: CardiNor AS

Acknowledgments

The Trøndelag Health Study (HUNT) is a collaboration between HUNT Research Centre (Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology NTNU), Trøndelag County Council, Central Norway Regional Health Authority, and the Norwegian Institute of Public Health. We also acknowledge the generous support by Stiftelsen Kristian Gerhard Jebsen (K.G. Jebsen Center for Cardiac Biomarkers, grant number SKGJ-MED-024).

  1. Research ethics: The study was approved by the Regional Committee for Medical and Health Research Ethics of Mid-Norway (REC ID 13083) and was conducted in compliance with the ethical principles of the Declaration of Helsinki.

  2. Informed consent: Informed consent was obtained from all individuals included in this study, or their legal guardians or wards.

  3. Author contributions: The authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Competing interests: HR and TO have intellectual property rights for the use of SN as a biomarker in CVD, HR and TO have stocks in CardiNor AS, which holds the patent for SN as a CV biomarker, and HR and TO have received consultant fees from CardiNor AS. TO has received speaker and/or consultancy honoraria from Abbott Diagnostics, Bayer, CardiNor, Roche Diagnostics and Siemens Healthineers, and has received research support from Abbott Diagnostics, Novartis, Roche Diagnostics, via Akershus University Hospital. KMA has served on advisory board for Roche Diagnostics and SpinChip, consultant honoraria form CardiNor, lecturing honorarium from Siemens Healthineers and Snibe Diagnostics and have received research grants from Siemens Healthineers and Roche Diagnostics, she is Associate Editor of Clinical Biochemistry and Chair of the IFCC Committee of Clinical Application of Cardiac Bio-markers. ALF is an employee and own stocks in CardiNor AS.

  5. Research funding: K.G. Jebsen Center for Cardiac Biomarkers, Grant number SKGJ-MED-024, CardiNor AS.

  6. Data availability: Aggregated data can be obtained on request from the corresponding author, raw data may not be made available according to Norwegian legislation.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2024-0154).

Received: 2024-01-31
Accepted: 2024-03-07
Published Online: 2024-04-04
Published in Print: 2024-09-25

© 2024 the author(s), published by De Gruyter, Berlin/Boston

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

Abstract

Objectives

Secretoneurin (SN) is a novel cardiac biomarker that associates with the risk of mortality and dysfunctional cardiomyocyte Ca2+ handling in heart failure patients. Reference intervals for SN are unknown.

Methods

SN was measured with a CE-marked ELISA in healthy community dwellers from the fourth wave of the Trøndelag Health Study (HUNT4) conducted in 2017–2019. The common, sex and age specific 90th, 95th, 97.5th and 99th percentiles were calculated using the non-parametric method and outlier exclusion according to the Reed test. The applicability of sex and age specific reference intervals were investigated using Harris and Boyd test. We also estimated the percentiles in a subset with normal findings on echocardiographic screening.

Results

The total cohort included 887 persons (56.4 % women). After echocardiographic screening 122 persons were excluded, leaving a total of 765 persons (57.8 % women). The 97.5th percentile (95 % CI in brackets) of SN was 59.7 (57.5–62.1) pmol/L in the total population and 58.6 (57.1–62.1) pmol/L after echocardiography screening. In general, slightly higher percentiles were found in women and elderly participants, but less than 4 % in these subgroups had concentrations deviating from the common 97.5th percentile. Low BMI or eGFR was also associated with higher concentrations of SN.

Conclusions

Upper reference limits for SN were similar amongst healthy adult community dwellers regardless of prescreening including cardiac echocardiography or not. Women and elderly showed higher concentrations of SN, but the differences were not sufficiently large to justify age and sex stratified upper reference limits.

Keywords: secretoneurin; upper reference limit; reference interval; predictor

Introduction

Secretoneurin (SN) is a 33-amino acid peptide belonging to the granin protein family originating from neuroendocrine and myocardial tissue. SN is involved in different physiologic processes in the myocardium, including cardiomyocyte Ca2+ handling [1]. Recently, SN was proposed as a novel prognostic cardiac biomarker in patients with heart failure [2] and severe illness [3], as recent data suggest an association with mortality and ventricular arrhythmia in these patient groups [4]. SN also seems to have limited biological variability, which leads to high signal-to-noise ratio [5].

Correct interpretation and clinical use of novel biomarkers require certain basic information to be in place, and a crucial parameter necessary for clinical decision making is the reference interval. The reference interval of a biomarker is usually defined as the central 95 % distribution in a healthy cohort (≥2.5th to ≤97.5th percentile), although for one sided reference intervals (i.e., when only elevated or reduced concentrations are clinically important), the 95th percentile has sometimes been suggested. For prognostic markers there are examples that the 90th percentile has been suggested as upper cut-off for signaling increased risk [6].

Reference intervals describe the expected concentrations in healthy individuals [7], and a result within the reference interval can be interpreted as a normal physiological response and homeostasis. Healthiness is typically based on self-reporting (frequently questionnaires) in combination with measurement of relevant biomarkers [7], [8], [9], and more advanced investigations are rarely included for screening purposes. However, some studies on cardiac biomarkers have demonstrated that selection on normality based on advanced cardiac imaging like echocardiography influence the upper reference limits [10]. Other important metrics for interpretation in individual patients are related to systematic differences in concentrations based on physiological parameters like age, sex, and body mass index (BMI).

Accordingly, the primary aim of this study was to estimate upper reference limits (90th, 95th, 97.5th and 99th percentiles) for SN measured in a cohort of healthy adult community dwellers with and without prescreening using echocardiography. Secondly, we aimed to investigate if sex or age partitioned reference intervals were applicable for SN.

Materials and methods

Study population

The study sample encompasses a subset of participants from the fourth wave of the Trøndelag Health Study (HUNT4) who were investigated with echocardiography. HUNT is the largest Norwegian population study and has performed regular updates since 1984 with a total of 230,000 individuals included [11]. HUNT4 was conducted in 2017–2019 and included questionnaires, a basic clinical investigation collecting anthropometric measures and sampling of biological material including blood sampling from 56,044 participants [12]. The current study include data from a subset of 5,763 participants who were invited to the HUNT4Echo study, details have been described earlier [13], see Figure 1. Briefly, participants were invited if they had previously participated in the HUNT3 (2006–2008) Fitness or Echocardiography studies or if they had validated atrial fibrillation (AF) from HUNT3 or self-reported AF from HUNT4. In total, 3174 persons responded. SN measurements were available in 2,486 of responders and valid echocardiography data was available in 2,448 of these. We defined a healthy population subgroup according to self-reporting (questionnaire), clinical examination, and a biomarker screening (Supplemental Table 1). We also estimated percentiles in a subset of participants after excluding all participants who had echocardiographic findings indicating subclinical myocardial disease (left ventricular mass index>115 g/m2 for men and>95 g/m2 for women or left ventricular ejection fraction<55 %). A LVEF of 55 % was used as a cut-off to reduce the likelihood of including subjects with underlying heart failure or incipient heart failure in this subgroup.

Figure 1: Study overview. SN; secretoneurin.
Figure 1:

Study overview. SN; secretoneurin.

The study was approved by the Regional Committee for Medical and Health Research Ethics of Mid-Norway (REC ID 13083) and was conducted in compliance with the ethical principles of the Declaration of Helsinki. All participants provided informed written consent. Personal data security and data handling were approved by the institutional personal data protection officer at St Olav’s Hospital and Norwegian University of Science and Technology.

Laboratory analysis

Circulating SN was measured in serum samples using a CE-marked commercially available ELISA (CardiNor AS, Oslo, Norway). The average of duplicate measurements was calculated and reported as the final result. The within-plate analytical variation was 2.7 and 3.6 % at 32.6 and 71.5 pmol/L, respectively. The between-plate analytical variation was 4.8 and 5.0 % at 32.6 and 71.5 pmol/L, respectively. The within laboratory imprecision (run-to-run, day-to-day, and replicate-to-replicate variation) was 8.7 % at 32.6 pmol/L and 6.7 % at 71.5 pmol/L, respectively. The measuring range was 10–250 pmol/L [14].

C-reactive protein (CRP), total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, HbA1c and creatinine were measured using Architect ci8200 from Abbott Diagnostics. The creatinine assay was enzymatic and traceable to the IDMS reference method, and eGFR was estimated using the CKD-EPIcreat equation [15].

Echocardiography

Comprehensive details of the echocardiographic examinations have been published previously [13, 16, 17]. All echocardiograms were acquired using Vivid E95 scanners (GE Vingmed Ultrasound, Horten, Norway) with phased array transducers (M5S). All recordings and measurements were performed by experienced personnel according to the present recommendations [18]. Analyses and measurements were done using EchoPAC SWO (version 203; GE Ultrasound, Horten, Norway). Two-dimensional recordings included parasternal long- and short-axis views as well as standard apical views of the left ventricle, right ventricle and atria. All recordings were dedicated for the specific chamber and task. Shortly, left ventricular dimensions and wall thickness were measured at end-diastole and end-systole in parasternal long-axis view. LV mass was calculated from end-diastolic measurements using the Cube formula. Left ventricular volume was measured using the biplane Simpson’s (summation of discs) method from four- and two-chamber recordings with tracing of the endocardial borders at end-diastole and end-systole. Left ventricular ejection fraction was calculated as the difference between end-diastolic and end-systolic volumes divided by the end-diastolic volume.

Statistical analysis

Baseline characteristics of the study cohort are presented as absolute numbers with proportion or medians with interquartile range [IQR] unless otherwise stated. Between group comparisons for continuous variables were analyzed using the Mann–Whitney U test and categorical variables with the Fisher exact test. We assessed the crude associations between SN concentration and age, eGFR, and BMI by restricted cubic splines with knots placed at the 5th, 27.5th, 50th, 72.5th, and 95th sample percentiles. The 90th, 95th, 97.5th and 99th percentiles for SN concentrations were calculated for men and women separately and combined, and according to age (subgroups stratified according to the cohort median age or arbitrary chosen age groups (<40 years, 40–59 years and ≥60 years)), using the non-parametric method and outlier exclusion according to the Reed test, as described by the Clinical & Laboratory Standards Institute (CLSI) [9]. The applicability of sex and age specific reference intervals were investigated as suggested by Harris and Boyd (i.e. if >4 % of a specific population subgroup exceed the common 97.5th percentile, stratified reference intervals could be recommended) [9].

Results

A total of 887 persons (56.4 % women) from the general population were defined as a healthy population subgroup and included in the study. Baseline characteristics according to quartiles of SN are provided in Table 1. Persons in the upper quartile were more frequently older females with lower eGFR and BMI and showed higher HDL cholesterol. Baseline characteristics for the healthy population subgroup and after echocardiography screening (n=765) are shown in Supplemental Table 2. BMI and eGFR were linearly associated with SN concentrations (Figure 2).

Table 1:

Baseline characteristics of the healthy population subgroup according to quartiles of secretoneurinconcentrations.

Quartile 1 Quartile 2 Quartile 3 Quartile 4
n=222 n=222 n=222 n=221 p-Value
Secretonaurin, pmol/L 30.8 (28.3–32.5) 36.7 (35.5–37.9) 42.1 (40.6–43.8) 50.0 (47.4–53.9) <0.001
Male sex, n (%) 132 (59.5 %) 105 (47.3 %) 88 (39.6 %) 62 (28.1 %) <0.001
Age, years 54.1 (45.8–63.6) 54.0 (47.1–63.3) 54.6 (46.9–63.2) 58.1 (50.5–65.0) 0.024
Higher education, n (%) 106 (47.7 %) 135 (60.8 %) 150 (67.6 %) 122 (55.5 %) <0.001
Body mass index, kg/m2 25.9 (24.5–27.9) 24.8 (23.0–27.4) 24.7 (22.7–26.5) 24.3 (21.9–26.6) <0.001
Waist-to-hip ratio 0.92 (0.88–0.97) 0.91 (0.87–0.96) 0.90 (0.86–0.96) 0.89 (0.86–0.95) <0.001
Heart rate, bpm 66 (60–74) 67 (60–74) 66 (61–73) 68 (60–75) 0.82
Systolic blood pressure, mmHg 121 (114–128) 121 (113–128) 119 (111–127) 119 (112–127) 0.14
Diastolic blood pressure, mmHg 72 (67–77) 73 (66–79) 72 (66–77) 71 (66–76) 0.19
Total cholesterol, mmol/L 5.6 (4.7–6.2) 5.5 (4.8–6.2) 5.6 (5.0–6.3) 5.6 (5.0–6.4) 0.25
HDL cholesterol, mmol/L 1.4 (1.2–1.6) 1.5 (1.2–1.8) 1.5 (1.2–1.7) 1.6 (1.3–1.9) <0.001
HbA1c, % 5.1 (5.0–5.3) 5.2 (5.0–5.3) 5.2 (5.0–5.4) 5.2 (5.0–5.3) 0.46
eGFR, mL/min/1.73m2 94.0 (87.0–102.0) 93.0 (85.0–101.0) 91.0 (81.0–100.0) 87.0 (78.0–96.0) <0.001

  1. HDL, high-density lipoprotein; HbA1c, hemoglobin A1c; eGFR, estimated glomerular filtration rate.

Figure 2: Associations between body mass index, estimated glomerular filtration rate (eGFR), and secretoneurin concentrations, adjusted for age and sex.
Figure 2:

Associations between body mass index, estimated glomerular filtration rate (eGFR), and secretoneurin concentrations, adjusted for age and sex.

The distributions of SN concentrations are shown in Supplemental Figures 1 and 2 and the upper reference limit percentiles for SN in the healthy population subgroup are shown in Table 2. The number of outliers excluded based on the Reed test is outlined in Supplemental Table 3.

Table 2:

Common upper reference limit percentiles for secretoneurin (with Reed outlier detection).

Secretoneurin, pmol/L (95 % CI)
Ordinary screening Ordinary screening + echocardiography screening
Total (n=886) Total (n=764)
99th percentile 65.9 (61.1–85.3) 66.1 (60.6–85.3)
97.5th percentile 59.7 (57.5–62.1) 58.6 (57.1–62.1)
95th percentile 54.8 (53.5–57.7) 54.4 (53.3–57.5)
90th percentile 51.2 (50.3–52.7) 51.1 (50.1–52.7)

  1. Ordinary screening, excluding subjects with established cardiovascular disease (angina pectoris, myocardial infarction, heart failure, atrial fibrillation, stroke), BMI <18 kg/m2 or >35 kg/m2, on lipid lowering therapy, on antihypertensive therapy or with baseline systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, with established diabetes mellitus or baseline HbA1c ≥48 mmol/mol, with established renal failure or baseline estimated glomerular filtration rate <60 mL/min/1.73m2, with obstructive lung disease, with history of malignancy, or current smoker. Echocardiography screening, excluding subjects with left ventricular mass index >115 g/m2 for men and >95 g/m2 for women or left ventricular ejection fraction <55%.

The sex-neutral percentiles were similar after ordinary and echocardiographic screening. There was a trend towards lower percentiles in men, except for the 99th percentile when estimated in the ordinary screening group (Table 3). Similar trends were observed for age, with generally higher percentiles of SN in study participants with age above the cohort median (Table 4), and lower reference intervals in the age group <40 years (Supplemental Table 4). Adjusting for age, mean concentrations of SN were 41.1 (95 % CI 40.3 to 41.9) pmol/L in women and 37.3 (95 % CI 36.4 to 38.1) pmol/L in men (p for comparison<0.001). SN values were above the common 97.5th percentile in 3.0 % of women and 1.8 % of men (p<0.001). Similarly, 1.4 % of study participants with age below the median and 3.6 % of study participants with age above the median had SN values above the common 97.5th percentile (p=0.003). For the arbitrary chosen age groups, 1.1 % (<40 years), 2.4 % (40–59 years) and 3.0 % (≥60 years) was above the common 97.5 percentile, respectively.

Table 3:

Upper reference limit percentiles for secretoneurin, stratified in men and women (with Reed outlier detection).

Secretoneurin, pmol/L (95 % CI)
Ordinary screening Ordinary screening + echocardiography screening
Female (n=499) Male (n=386) Female (n=441) Male (n=322)
99th percentile 65.7 (60.6–75.3) 68.4 (58.1–101.3) 65.4 (60.6–75.3) 64.7 (55.4–101.3)
97.5th percentile 60.2 (57.8–63.8) 57.4 (51.8–65.9) 59.8 (57.7–63.8) 55.3 (51.2–65.9)
95th percentile 56.4 (54.3–59.3) 51.4 (49.5–55.4) 56.4 (54.3–58.6) 51.1 (48.1–54.0)
90th percentile 52.8 (51.3–54.3) 47.6 (46.5–49.9) 52.9 (51.3–54.3) 47.0 (45.6–49.2)

  1. Ordinary screening, excluding subjects with established cardiovascular disease (angina pectoris, myocardial infarction, heart failure, atrial fibrillation, stroke), BMI<18 kg/m2 or >35 kg/m2, on lipid lowering therapy, on antihypertensive therapy or with baseline systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, with established diabetes mellitus or baseline HbA1c ≥48 mmol/mol, with established renal failure or baseline estimated glomerular filtration rate <60 mL/min/1.73m2, with obstructive lung disease, with history of malignancy, or current smoker. Echocardiography screening, excluding subjects with left ventricular mass index >115 g/m2 for men and >95 g/m2 for women or left ventricular ejection fraction <55%.

Table 4:

Upper reference limit percentiles for secretoneurin, stratified according to median age (with Reed outlier detection).

Secretoneurin, pmol/L (95 % CI)
Ordinary screening Ordinary screening + echocardiography screening
Below median (n=444) Above median (n=442) Below median (n=388) Above median (n=376)
99th percentile 62.3 (57.1–156.8) 69.2 (62.1–101.3) 64.0 (56.4–156.8) 68.3 (60.9–101.3)
97.5th percentile 56.3 (53.2–60.5) 61.4 (58.5–67.7) 55.3 (53.0–63.8) 60.8 (58.1–67.7)
95th percentile 52.7 (51.0–54.6) 58.1 (54.5–60.6) 52.7 (51.1–54.6) 58.0 (54.3–60.6)
90th percentile 49.4 (47.6–51.1) 53.0 (51.1–54.5) 49.6 (47.6–51.1) 53.0 (51.0–54.3)

  1. Median age 55.4 years. Ordinary screening, excluding subjects with established cardiovascular disease (angina pectoris, myocardial infarction, heart failure, atrial fibrillation, stroke), BMI <18 kg/m2 or >35 kg/m2, on lipid lowering therapy, on antihypertensive therapy or with baseline systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, with established diabetes mellitus or baseline HbA1c ≥48 mmol/mol, with established renal failure or baseline estimated glomerular filtration rate <60 mL/min/1.73m2, with obstructive lung disease, with history of malignancy, or current smoker. Echocardiography screening, excluding subjects with left ventricular mass index >115 g/m2 for men and >95 g/m2 for women or left ventricular ejection fraction <55%.

Discussion

The current study reports the upper reference limits of SN in presumably healthy subjects recruited from the general population. We report several findings important for future clinical use and interpretation of SN concentrations.

First, we report the reference intervals for SN as determined in a healthy population. Our data clearly demonstrate that a clinical screening using questionnaires and common biomarkers is sufficient to provide robust estimates of the upper reference limit, this includes a sex-neutral cut-off and all percentiles from 90th through the 99th percentile. The upper reference limits for SN were similar amongst healthy adult community dwellers regardless of prescreening including cardiac echocardiography or not. Compared to cardiac troponins, SN appears to be less influenced by structural myocardial disease as identified by echocardiography [10, 19]. This observation suggests that in a healthy population neuroendocrine activity may contribute quantitatively more to circulating SN than cardiac production [20], [21], [22].

Secondly, SN concentrations are influenced by physiological factors, but the effect is moderate [23]. It is known that natriuretic peptides are higher in women, increase with age and that lower concentrations are associated with higher BMI [24, 25]. SN showed a similar trend, however findings were less pronounced and are unlikely to have a major effect on clinical interpretation of test results as differences were not large enough to justify sex or age stratified reference intervals [9]. Higher SN concentrations were associated with lower eGFR, indicating a renal dependent clearance.

Clinical implications

Reference intervals are used to characterize a physiological condition. Measuring a concentration outside the reference interval may be interpretated as a pathological condition or disease, but this will depend on applicable diagnostic definitions and medical consensus regarding interpretation of abnormal values. Current recommendations for cardiac markers show some diversity and both upper reference limits derived from a general healthy population and clinical cut-offs derived from large clinical trials are used for signaling disease or need of intervention. For cardiac troponin, which is integrated as a main criterion in the Universal Definition of Myocardial Infarction [26], the 99th percentile is used as upper reference limit. This cut-off was chosen to increase the specificity of the diagnosis [8]. Lately, the use of 99th percentiles has been questioned, in particular when cardiac troponins are used to investigate other conditions than myocardial infarction. Moreover, in children the robustness of the clinical screening procedure and statistical calculations are substantially lower for estimating the 99th percentile compared to the 97.5th percentile [27]. In the emergency room, clinical cut-offs derived from observational and randomized clinical trials are used for early prediction of low (rule-out) and high (rule-in) risk of myocardial infarction, and thereby improved logistics and patient flow [28], [29], [30]. For other well-known cardiac biomarkers like natriuretic peptides, the 97.5th percentile is used as upper reference limit although typically aligned with relevant clinical cut-offs being derived from large clinical studies [31], [32], [33]. These clinical cut-offs are used to predict high and low risk of heart failure, and thereby decide subsequent clinical investigations, both in the chronic and acute setting [33]. For another prognostic biomarker, growth differential factor 15, the 90th percentile is commonly used as cut-off for signaling increased long-term risk of cardiovascular events or death [6]. Based on current laboratory practice and expectation of potential future clinical utility of SN as reflecting risk, possibly via link to cardiac arrythmia, we suggest that the sex-neutral 97.5 percentile is the most applicable upper reference limit for SN. Also, even though we in this study report the concentrations down to one decimal place (for academic purposes), laboratories my find it feasible to report the concentrations as whole numbers if SN implemented into routine practice. If this is the most applicable clinical cut-off for signaling disease or future risk of disease should be determined in future clinical studies. Of note, we recently reported SN concentrations in patients with chronic heart failure and the patients with concentrations above 48.7 pmol/L (approximately corresponding to the 90th percentile reported here) had a poor prognosis [2]. That long-term cardiovascular prognosis is predicted by concentrations below the upper reference limit is in line with the prognostic cut-offs that have been reported for cardiac markers, like troponins and natriuretic peptides [21, 34].

Strength and limitations

The major strength in this study is the inclusion of a large cohort allowing for a thorough evaluation of physiological and clinical variables that potentially could influence SN concentrations. The high number of included subjects increases the accuracy of the reported percentiles. All participants were investigated with echocardiography allowing for exclusion of subclinical structural myocardial disease, hereby providing robust estimates for the upper percentiles reported.

The main limitation is the use of an ELISA that might be too labor intensive for later implementation in routine laboratory practice. If SN measurements are implemented in clinical practice in the future, it is likely that automated applications of the assay become available. The current reference intervals should not be directly transferred to another assay unless good agreement with the current assay has been sufficiently documented. Finally, it should be noted that the subgroup <40 years were smaller than what is recommended when 99th and 97.5 percentiles are calculated, and accordingly the estimates should be seen as suggestive.

Conclusions

Upper reference limits for SN were similar amongst healthy adult community dwellers regardless of pre-screening including cardiac echocardiography or not. Women, persons with the lowest BMI and eGFR and elderly showed higher percentiles of SN. Even though sex and age influence SN concentrations, the differences were not sufficiently large to justify age and sex stratified upper reference limits.

Corresponding author: Kristin M. Aakre, MD, PhD, Department of Medical Biochemistry and Pharmacology, Haukeland University Hospital, Jonas Lies vei 65, Bergen, Norway; Department of Heart Disease, Haukeland University Hospital, Bergen, Norway; and Department of Clinical Science, University of Bergen, Bergen, Norway, E-mail:

Funding source: K.G. Jebsen Center for Cardiac Biomarkers

Award Identifier / Grant number: SKGJ-MED-024

Funding source: CardiNor AS

Acknowledgments

The Trøndelag Health Study (HUNT) is a collaboration between HUNT Research Centre (Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology NTNU), Trøndelag County Council, Central Norway Regional Health Authority, and the Norwegian Institute of Public Health. We also acknowledge the generous support by Stiftelsen Kristian Gerhard Jebsen (K.G. Jebsen Center for Cardiac Biomarkers, grant number SKGJ-MED-024).

  1. Research ethics: The study was approved by the Regional Committee for Medical and Health Research Ethics of Mid-Norway (REC ID 13083) and was conducted in compliance with the ethical principles of the Declaration of Helsinki.

  2. Informed consent: Informed consent was obtained from all individuals included in this study, or their legal guardians or wards.

  3. Author contributions: The authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Competing interests: HR and TO have intellectual property rights for the use of SN as a biomarker in CVD, HR and TO have stocks in CardiNor AS, which holds the patent for SN as a CV biomarker, and HR and TO have received consultant fees from CardiNor AS. TO has received speaker and/or consultancy honoraria from Abbott Diagnostics, Bayer, CardiNor, Roche Diagnostics and Siemens Healthineers, and has received research support from Abbott Diagnostics, Novartis, Roche Diagnostics, via Akershus University Hospital. KMA has served on advisory board for Roche Diagnostics and SpinChip, consultant honoraria form CardiNor, lecturing honorarium from Siemens Healthineers and Snibe Diagnostics and have received research grants from Siemens Healthineers and Roche Diagnostics, she is Associate Editor of Clinical Biochemistry and Chair of the IFCC Committee of Clinical Application of Cardiac Bio-markers. ALF is an employee and own stocks in CardiNor AS.

  5. Research funding: K.G. Jebsen Center for Cardiac Biomarkers, Grant number SKGJ-MED-024, CardiNor AS.

  6. Data availability: Aggregated data can be obtained on request from the corresponding author, raw data may not be made available according to Norwegian legislation.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2024-0154).

Received: 2024-01-31
Accepted: 2024-03-07
Published Online: 2024-04-04
Published in Print: 2024-09-25

© 2024 the author(s), published by De Gruyter, Berlin/Boston

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

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  1. Frontmatter
  2. Editorial
  3. Six years of progress – highlights from the IFCC Emerging Technologies Division
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  5. Skin in the game: a review of single-cell and spatial transcriptomics in dermatological research
  6. Bilirubin measurements in neonates: uniform neonatal treatment can only be achieved by improved standardization
  7. Validation and verification framework and data integration of biosensors and in vitro diagnostic devices: a position statement of the IFCC Committee on Mobile Health and Bioengineering in Laboratory Medicine (C-MBHLM) and the IFCC Scientific Division
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  17. Targeted ultra performance liquid chromatography tandem mass spectrometry procedures for the diagnosis of inborn errors of metabolism: validation through ERNDIM external quality assessment schemes
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  24. Verification of bile acid determination method and establishing reference intervals for biochemical and haematological parameters in third-trimester pregnant women
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  27. Exploring evolutionary trajectories in ovarian cancer patients by longitudinal analysis of ctDNA
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  29. Evaluation of effects from hemoglobin variants on HbA1c measurements by different methods
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  33. The routine coagulation assays plasma stability – in the wake of the new European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) biological variability database
  34. Improving HCV diagnosis following a false-negative anti-HCV result
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