Investigation of the possible cause of over-estimation of human aldosterone in plasma, using a unique, non-synthetic human aldosterone-free matrix
-
Sheree Stoner
,
Peter E. Hickman
Abstract
Objectives
Aldosterone is over-estimated by direct immunoassay when compared to assay by LC-MS/MS. We have investigated the mechanism for this over-estimation, using dichloromethane (DCM) extraction and a unique matrix of non-synthetic aldosterone-free plasma (ALFP).
Methods
Samples used were from patients with normal estimated Glomerular Filtration Rate (eGFR). All samples were measured by direct DiaSorin immunoassay, followed by extraction using dichloromethane (DCM) and LC-MS/MS. Post-DCM samples were reconstituted in DiaSorin Liaison Endocrinology Diluent (DED), which is effectively a human serum albumin matrix, or in a unique matrix of non-synthetic human aldosterone-free plasma (ALFP). Patient plasma samples were selected to cover a wide range of aldosterone concentrations.
Results
Aldosterone direct immunoassay over-estimated aldosterone concentration in all samples, compared to LC-MS/MS. Post-DCM extraction and reconstitution with DED, aldosterone results matched well with LC-MS/MS results. Reconstitution with our unique ALFP plasma matrix rather than the DED, resulted in an over-estimation of aldosterone by approximately 50 pmol/L in all samples.
Conclusions
In this investigation, we have demonstrated that the most likely source of interference in the direct DiaSorin aldosterone immunoassay is an endogenous protein commonly found in patient samples. This protein probably interferes with the immunoassay by binding to the tracer.
-
Research ethics: The local Institutional Review Board deemed the study exempt from review.
-
Informed consent: Our institutional Ethics Committee approved all studies as a quality improvement exercise that did not require 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: None declared.
-
Conflict of interest: The authors state no conflict of interest.
-
Research funding: None declared.
-
Data availability: The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
1. Young, WFJ, Calhoun, DA, Lenders, JWM, Stowasser, M, Textor, SC. Screening for endocrine hypertension: an endocrine society scientific statement. Endocr Rev 2017;38:103–22. https://doi.org/10.1210/er.2017-00054.Search in Google Scholar
2. Mulatero, P, Monticone, S, Deinum, J, Amar, L, Prejbisz, A, Zennaro, MC, et al.. Genetics, prevalence, screening and confirmation of primary aldosteronism: a position statement and consensus of the Working Group on Endocrine Hypertension of the European Society of Hypertension. J Hypertens 2020;38:1919–28. https://doi.org/10.1097/hjh.0000000000002510.Search in Google Scholar PubMed
3. Cartledge, S, Lawson, N. Aldosterone and renin measurements. Ann Clin Biochem 2000;37:262–78. https://doi.org/10.1258/0004563001899401.Search in Google Scholar PubMed
4. Stowasser, M, Gordon, RD. Aldosterone assays: an urgent need for improvement. Clin Chem 2006;52:1640–2. https://doi.org/10.1373/clinchem.2006.073460.Search in Google Scholar PubMed
5. Schirpenbach, C, Seiler, L, Maser-Gluth, C, Beuschlein, F, Reincke, M, Bidlingmaier, M. Automated chemiluminescence-immunoassay for aldosterone during dynamic testing: comparison to radioimmunoassays with and without extraction steps. Clin Chem 2006;52:1749–55. https://doi.org/10.1373/clinchem.2006.068502.Search in Google Scholar PubMed
6. Rehan, M, Raizman, JE, Cavalier, E, Don-Wauchope, AC, Holmes, DT. Laboratory challenges in primary aldosteronism screening and diagnosis. Clin Biochem 2015;48:377–87. https://doi.org/10.1016/j.clinbiochem.2015.01.003.Search in Google Scholar PubMed
7. Lam, L, Chiu, WW, Davidson, JS. Overestimation of aldosterone by immunoassay in renal impairment. Clin Chem 2016;62:890–1. https://doi.org/10.1373/clinchem.2016.255737.Search in Google Scholar PubMed
8. Blocki, F, Zierold, C, Olson, G, Seeman, J, Cummings, S, Bonelli, F. In defense of aldosterone immunoassay: a broader perspective. Clin Chem Lab Med 2017;55:e87–9. https://doi.org/10.1515/cclm-2016-0707.Search in Google Scholar PubMed
9. Ray, JA, Kushnir, MM, Palmer, J, Sadjadi, S, Rockwood, AL, Meikle, AW. Enhancement of specificity of aldosterone measurement in human serum and plasma using 2D-LC–MS/MS and comparison with commercial immunoassays. J Chromatogr B 2014;970:102–7. https://doi.org/10.1016/j.jchromb.2014.08.042.Search in Google Scholar PubMed
10. Wiegand, M, Halsall, DJ, Cowan, SL, Taylor, K, Goudie, RJB, Preller, J, et al.. Unquantifiably low aldosterone concentrations are prevalent in hospitalised COVID-19 patients but May not be revealed by chemiluminescent immunoassay. Endocr Connect 2022;11:e220190. https://doi.org/10.1530/ec-22-0190.Search in Google Scholar PubMed PubMed Central
11. Eisenhofer, G, Kurlbaum, M, Peitzsch, M, Constantinescu, G, Remde, H, Schulze, M, et al.. The saline infusion test for primary aldosteronism: implications of immunoassay inaccuracy. J Clin Endocrinol Metab 2022;107:e2027–36. https://doi.org/10.1210/clinem/dgab924.Search in Google Scholar PubMed PubMed Central
12. SCIEX. Ultra-sensitive analysis of aldosterone in serum using the AB SCIEX triple quad™ 6500 LC/MS/MS system. Available from: https://sciex.com/content/dam/SCIEX/pdf/tech-notes/all/6500_Aldosterone.pdf [Accessed 9 May 2025].Search in Google Scholar
13. Badrick, T, Hickman, P, Ward, G. A graphical tool to investigate method validation. Clin Chem Lab Med 2023;61:e160–2. https://doi.org/10.1515/cclm-2023-0095.Search in Google Scholar PubMed
14. Sturgeon, CM, Viljoen, A. Analytical error and interference in immunoassay: minimizing risk. Ann Clin Biochem 2011;48:418–32. https://doi.org/10.1258/acb.2011.011073.Search in Google Scholar PubMed
15. Ward, G, Simpson, A, Boscato, L, Hickman, PE. The investigation of interferences in immunoassay. Clin Biochem 2017;50:1306–11. https://doi.org/10.1016/j.clinbiochem.2017.08.015.Search in Google Scholar PubMed
16. Ghazal, K, Brabant, S, Prie, D, Piketty, ML. Hormone immunoassay interference: a 2021 update. Ann Lab Med 2022;42:3–23. https://doi.org/10.3343/alm.2022.42.1.3.Search in Google Scholar PubMed PubMed Central
17. Wauthier, L, Plebani, M, Favresse, J. Interferences in immunoassays: review and practical algorithm. Clin Chem Lab Med 2022;60:808–20. https://doi.org/10.1515/cclm-2021-1288.Search in Google Scholar PubMed
18. Constantinescu, G, Bidlingmaier, M, Gruber, M, Peitzsch, M, Poitz, DM, van Herwaarden, AE, et al.. Mass spectrometry reveals misdiagnosis of primary aldosteronism with scheduling for adrenalectomy due to immunoassay interference. Clin Chim Acta 2020;507:98–103. https://doi.org/10.1016/j.cca.2020.04.019.Search in Google Scholar PubMed
19. Stoner, S, Browning, S, Young, K, Harrower, T, Bachmeier, C, etal, DK. Investigations of interference in aldosterone immunoassay in patient groups with abnormal levels of serum proteins. Clin Biochem Rev 2025;45:S1–135.Search in Google Scholar
20. Connolly, TM, Tibor, L, Gless, KH, Vecsei, P. Screening radioimmunoassay for aldosterone in preheated plasma without extraction and chromatography. Clin Chem 1980;26:41–5. https://doi.org/10.1093/clinchem/26.1.41.Search in Google Scholar
21. Brown, JM, Auchus, RJ, Honzel, B, Luther, JM, Yozamp, N, Vaidya, A. Recalibrating interpretations of aldosterone assays across the physiologic range: immunoassay and liquid chromatography-tandem mass spectrometry measurements under multiple controlled conditions. J Endocr Soc 2022;6:bvac049.https://doi.org/10.1210/jendso/bvac049.Search in Google Scholar PubMed PubMed Central
22. Micallef, JV, Hayes, MM, Latif, A, Ahsan, R, Sufi, SB. Serum binding of steroid tracers and its possible effects on direct steroid immunoassay. Ann Clin Biochem 1995;32:566–74. https://doi.org/10.1177/000456329503200609.Search in Google Scholar PubMed
23. Ozeki, Y, Tanimura, Y, Nagai, S, Nomura, T, Kinoshita, M, Shibuta, K, et al.. Development of a new chemiluminescent enzyme immunoassay using a two-step sandwich method for measuring aldosterone concentrations. Diagnostics. 2021;11:433.https://doi.org/10.3390/diagnostics11030433Search in Google Scholar PubMed PubMed Central
24. Fuld, S, Constantinescu, G, Pamporaki, C, Peitzsch, M, Schulze, M, Yang, J, et al.. Screening for primary aldosteronism by mass spectrometry versus immunoassay measurements of aldosterone: a prospective within-patient study. J Appl Lab Med 2024;9:752–66. https://doi.org/10.1093/jalm/jfae017.Search in Google Scholar PubMed
25. Ono, Y, Tezuka, Y, Omata, K, Morimoto, R, Yamazaki, Y, Oguro, S, et al.. Screening cutoff values for the detection of aldosterone-producing adenoma by LC-MS/MS and a novel noncompetitive CLEIA. J Endocr Soc 2024;8. Available from: https://doi.org/10.1210/jendso/bvae080.Search in Google Scholar PubMed PubMed Central
© 2025 Walter de Gruyter GmbH, Berlin/Boston
Articles in the same Issue
- Frontmatter
- Editorial
- Advancing diagnostic stewardship through claims-based utilization analysis: toward a system-wide vision of diagnostic excellence
- Review
- Biomarkers in body fluids and their detection techniques for human intestinal permeability assessment
- Mini Review
- Challenges of using natriuretic peptides to screen for the risk of developing heart failure in patients with diabetes: a report from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee on Clinical Applications of Cardiac Bio-Markers (C-CB)
- Opinion Papers
- Reference intervals in value-based laboratory medicine: a shift from single-point measurements to metabolic variation-based models
- Overview of laboratory diagnostics for immediate management of patients presenting to the emergency department with acute bleeding
- What Matters Most: an Age-Friendly approach to pathology and laboratory medicine
- No fault or negligence after an adverse analytical finding due to a contaminated supplement: mission impossible. Two examples involving trimetazidine
- General Clinical Chemistry and Laboratory Medicine
- Utilization analysis of laboratory tests using health insurance claims data: advancing nationwide diagnostic stewardship monitoring systems
- Evaluating large language models as clinical laboratory test recommenders in primary and emergency care: a crucial step in clinical decision making
- A novel corrective model based on red blood cells indices and haemolysis index enables accurate unhaemolysed potassium determination in haemolysed samples – Hemokalc project
- Validation of (self-collected) capillary blood using a topper collection system as alternative for venous sampling for 15 common clinical chemistry analytes
- Acoustophoresis-based blood sampling and plasma separation for potentially minimizing sampling-related blood loss
- Clinical validation of a liquid chromatography single quadrupole mass spectrometry (LC-MS) method using Waters Kairos™ Amino Acid Kit reagents
- Robustness of steroidomics-based machine learning for diagnosis of primary aldosteronism: a laboratory medicine perspective
- Investigation of the possible cause of over-estimation of human aldosterone in plasma, using a unique, non-synthetic human aldosterone-free matrix
- Performance of afternoon (16:00 h) serum cortisol for the diagnosis of Cushing’s syndrome
- MAGLUMI® Tacrolimus (CLIA) assay: analytical performances and comparison with LC-MS/MS and ARCHITECT Tacrolimus (CMIA) assay
- Assessment of 2023 ACR/EULAR antiphospholipid syndrome classification criteria in a Spanish cohort
- Comprehensive evaluation of antiphospholipid antibody testing methodologies in APS diagnosis: performance comparisons across assay systems and clinical subtypes
- Candidate Reference Measurement Procedures and Materials
- Exploring commutable materials for serum folate measurement: challenges in cross-method harmonization
- Reference Values and Biological Variations
- Reference ranges for ionized calcium in plasma in Danish children aged 0 days to 3 years using laboratory registry data
- A step forward in pediatric hemophagocytic lymphohistiocytosis and autoimmune disease: pediatric reference interval for serum soluble IL-2 receptor and soluble CD163
- Cancer Diagnostics
- Cellular expression of PD-1, PD-L1 and CTLA-4 in patients with JAK2V617F mutated myeloproliferative disorders
- Diabetes
- Serum N-glycans as independent predictors of the incidence of type 2 diabetes: a prospective investigation in the AEGIS cohort
- Infectious Diseases
- An assessment of molecular diagnosis of tuberculosis and multi-drug resistant tuberculosis testing and quality assessment: findings of an international survey
- Letters to the Editor
- Targeting low-value laboratory care
- Is time a significant factor in the release of potassium from lithium heparin plasma and serum?
- External quality assessment in resource-constrained laboratories: a survey of practices and perceptions in Nepal
- Is successfulness of platelet clump disaggregation by vortexing influenced by platelet measurement methods?
- Oligoclonal banding analysis: assessing plasma use and time interval requirements for paired CSF and blood
Articles in the same Issue
- Frontmatter
- Editorial
- Advancing diagnostic stewardship through claims-based utilization analysis: toward a system-wide vision of diagnostic excellence
- Review
- Biomarkers in body fluids and their detection techniques for human intestinal permeability assessment
- Mini Review
- Challenges of using natriuretic peptides to screen for the risk of developing heart failure in patients with diabetes: a report from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee on Clinical Applications of Cardiac Bio-Markers (C-CB)
- Opinion Papers
- Reference intervals in value-based laboratory medicine: a shift from single-point measurements to metabolic variation-based models
- Overview of laboratory diagnostics for immediate management of patients presenting to the emergency department with acute bleeding
- What Matters Most: an Age-Friendly approach to pathology and laboratory medicine
- No fault or negligence after an adverse analytical finding due to a contaminated supplement: mission impossible. Two examples involving trimetazidine
- General Clinical Chemistry and Laboratory Medicine
- Utilization analysis of laboratory tests using health insurance claims data: advancing nationwide diagnostic stewardship monitoring systems
- Evaluating large language models as clinical laboratory test recommenders in primary and emergency care: a crucial step in clinical decision making
- A novel corrective model based on red blood cells indices and haemolysis index enables accurate unhaemolysed potassium determination in haemolysed samples – Hemokalc project
- Validation of (self-collected) capillary blood using a topper collection system as alternative for venous sampling for 15 common clinical chemistry analytes
- Acoustophoresis-based blood sampling and plasma separation for potentially minimizing sampling-related blood loss
- Clinical validation of a liquid chromatography single quadrupole mass spectrometry (LC-MS) method using Waters Kairos™ Amino Acid Kit reagents
- Robustness of steroidomics-based machine learning for diagnosis of primary aldosteronism: a laboratory medicine perspective
- Investigation of the possible cause of over-estimation of human aldosterone in plasma, using a unique, non-synthetic human aldosterone-free matrix
- Performance of afternoon (16:00 h) serum cortisol for the diagnosis of Cushing’s syndrome
- MAGLUMI® Tacrolimus (CLIA) assay: analytical performances and comparison with LC-MS/MS and ARCHITECT Tacrolimus (CMIA) assay
- Assessment of 2023 ACR/EULAR antiphospholipid syndrome classification criteria in a Spanish cohort
- Comprehensive evaluation of antiphospholipid antibody testing methodologies in APS diagnosis: performance comparisons across assay systems and clinical subtypes
- Candidate Reference Measurement Procedures and Materials
- Exploring commutable materials for serum folate measurement: challenges in cross-method harmonization
- Reference Values and Biological Variations
- Reference ranges for ionized calcium in plasma in Danish children aged 0 days to 3 years using laboratory registry data
- A step forward in pediatric hemophagocytic lymphohistiocytosis and autoimmune disease: pediatric reference interval for serum soluble IL-2 receptor and soluble CD163
- Cancer Diagnostics
- Cellular expression of PD-1, PD-L1 and CTLA-4 in patients with JAK2V617F mutated myeloproliferative disorders
- Diabetes
- Serum N-glycans as independent predictors of the incidence of type 2 diabetes: a prospective investigation in the AEGIS cohort
- Infectious Diseases
- An assessment of molecular diagnosis of tuberculosis and multi-drug resistant tuberculosis testing and quality assessment: findings of an international survey
- Letters to the Editor
- Targeting low-value laboratory care
- Is time a significant factor in the release of potassium from lithium heparin plasma and serum?
- External quality assessment in resource-constrained laboratories: a survey of practices and perceptions in Nepal
- Is successfulness of platelet clump disaggregation by vortexing influenced by platelet measurement methods?
- Oligoclonal banding analysis: assessing plasma use and time interval requirements for paired CSF and blood
