Unveiling a new era with liquid chromatography coupled with mass spectrometry to enhance parathyroid hormone measurement in patients with chronic kidney disease

Abstract

Precise determination of circulating parathyroid hormone (PTH) concentration is crucial to diagnose and manage various disease conditions, including the chronic kidney disease–mineral and bone disorder. However, the lack of standardization in PTH assays is challenging for clinicians, potentially leading to medical errors because the different assays do not provide equivalent results and use different reference ranges. Here, we aimed to evaluate the impact of recalibrating PTH immunoassays by means of a recently developed LC-MS/MS method as the reference. Utilizing a large panel of pooled plasma samples with PTH concentrations determined by the LC-MS/MS method calibrated with the World Health Organization (WHO) 95/646 International Standard, five PTH immunoassays were recalibrated. The robustness of this standardization was evaluated over time using different sets of samples. The recalibration successfully reduced inter-assay variability with harmonization of PTH measurements across different assays. By recalibrating the assays based on the WHO 95/646 International Standard, we demonstrated the feasibility for standardizing PTH measurement results and adopting common reference ranges for PTH assays, facilitating a more consistent interpretation of PTH values. The recalibration process aligns PTH results obtained from various immunoassays with the LC-MS/MS method, providing more consistent and reliable measurements. Thus, establishing true standardization across all PTH assays is crucial to ensure consistent interpretation and clinical decision-making. Precise determination of circulating parathyroid hormone (PTH) concentration is crucial to diagnose and manage various disease conditions, including the chronic kidney disease–mineral and bone disorder. However, the lack of standardization in PTH assays is challenging for clinicians, potentially leading to medical errors because the different assays do not provide equivalent results and use different reference ranges. Here, we aimed to evaluate the impact of recalibrating PTH immunoassays by means of a recently developed LC-MS/MS method as the reference. Utilizing a large panel of pooled plasma samples with PTH concentrations determined by the LC-MS/MS method calibrated with the World Health Organization (WHO) 95/646 International Standard, five PTH immunoassays were recalibrated. The robustness of this standardization was evaluated over time using different sets of samples. The recalibration successfully reduced inter-assay variability with harmonization of PTH measurements across different assays. By recalibrating the assays based on the WHO 95/646 International Standard, we demonstrated the feasibility for standardizing PTH measurement results and adopting common reference ranges for PTH assays, facilitating a more consistent interpretation of PTH values. The recalibration process aligns PTH results obtained from various immunoassays with the LC-MS/MS method, providing more consistent and reliable measurements. Thus, establishing true standardization across all PTH assays is crucial to ensure consistent interpretation and clinical decision-making. Lay SummaryAccurate diagnosis and treatment of chronic kidney disease–related mineral and bone disorders (CKD-MBDs) hinge on measuring parathyroid hormone (PTH). Unfortunately, current PTH tests often yield inconsistent results, complicating clinical practice. To address this, standardizing PTH measurement methods is essential. Achieving this standardization requires advanced mass spectrometry techniques and understanding whether certain substances in the blood of chronic kidney disease and hemodialyzed patients affect PTH measurements. Recent advances in mass spectrometry revealed that a potentially problematic PTH fragment (7–84) was absent in patients’ blood, alleviating concerns. In addition, oxidized PTH was not detected and circulating fragments did not interfere PTH assays. As a result, we explored recalibrating 5 different PTH kits on to a precise liquid chromatography tandem mass spectrometry reference method. The outcomes were promising, aligning PTH results across various immunoassays with the reference method. This calibration process promises more reliable and consistent PTH measurements, ultimately enhancing patient care by reducing result variability. Accurate diagnosis and treatment of chronic kidney disease–related mineral and bone disorders (CKD-MBDs) hinge on measuring parathyroid hormone (PTH). Unfortunately, current PTH tests often yield inconsistent results, complicating clinical practice. To address this, standardizing PTH measurement methods is essential. Achieving this standardization requires advanced mass spectrometry techniques and understanding whether certain substances in the blood of chronic kidney disease and hemodialyzed patients affect PTH measurements. Recent advances in mass spectrometry revealed that a potentially problematic PTH fragment (7–84) was absent in patients’ blood, alleviating concerns. In addition, oxidized PTH was not detected and circulating fragments did not interfere PTH assays. As a result, we explored recalibrating 5 different PTH kits on to a precise liquid chromatography tandem mass spectrometry reference method. The outcomes were promising, aligning PTH results across various immunoassays with the reference method. This calibration process promises more reliable and consistent PTH measurements, ultimately enhancing patient care by reducing result variability. Beyond its paramount importance in diagnosing and managing various endocrine conditions such as primary and secondary hyperparathyroidism, hypoparathyroidism, and pseudohypoparathyroidism, the measurement of parathyroid hormone (PTH) is routinely conducted in patients with chronic kidney disease (CKD). The Kidney Disease: Improving Global Outcomes 2017 Clinical Practice Guideline Update for the Diagnosis, Evaluation, Prevention, and Treatment of Chronic Kidney Disease–Mineral and Bone Disorder recommends monitoring PTH levels in CKD patients beginning in CKD G3a and suggests maintaining PTH levels of hemodialyzed (HD) patients at ∼2 to 9 times the upper limit of the normal (ULN) values of the assay used.1Kidney Disease: Improving Global Outcomes (KDIGO) CKD-MBD Update Work GroupKDIGO 2017 Clinical Practice Guideline Update for the Diagnosis, Evaluation, Prevention, and Treatment of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD).Kidney Int Suppl (2011). 2017; 7: 1-59Abstract Full Text Full Text PDF PubMed Scopus (1166) Google Scholar Yet, a recommendation that treatments should be based on multiples of a ULN value is quite unique and is a circumvolution because of the lack of standardization of PTH assays.2Cavalier E. Vasikaran S. Bhattoa H.P. et al.The path to the standardization of PTH: is this a realistic possibility? A position paper of the IFCC C-BM.Clin Chim Acta. 2021; 515: 44-51Crossref PubMed Scopus (12) Google Scholar Such a lack of standardization is unfortunately a real source of confusion for clinicians in their daily practice, potentially leading to significant medical errors.3Sturgeon C.M. Sprague S. Almond A. et al.Perspective and priorities for improvement of parathyroid hormone (PTH) measurement—a view from the IFCC Working Group for PTH.Clin Chim Acta. 2017; 467: 42-47Crossref PubMed Scopus (46) Google Scholar,4Souberbielle J.-C. Boutten A. Carlier M.-C. et al.Inter-method variability in PTH measurement: implication for the care of CKD patients.Kidney Int. 2006; 70: 345-350Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Several reasons contributed so far to the current lack of standardization in PTH assays. The first one is the presence of circulating PTH fragments alongside the bioactive 1 to 84 PTH peptide.5Gardella T.J. Axelrod D. Rubin D. et al.Mutational analysis of the receptor-activating region of human parathyroid hormone.J Biol Chem. 1991; 266: 13141-13146Abstract Full Text PDF PubMed Google Scholar These fragments, which are N-terminal or C-terminal truncated forms of PTH, circulate in the blood owing to liver metabolism of the active peptide or direct secretion by the parathyroid glands.6Segre G.V. Perkins A.S. Witters L. Potts J.T. Metabolism of parathyroid hormone by isolated rat Kupffer cells and hepatocytes.J Clin Invest. 1981; 67: 449-457Crossref PubMed Scopus (75) Google Scholar, 7Hanley D.A. Ayer L.M. Calcium-dependent release of carboxyl-terminal fragments of parathyroid hormone by hyperplastic human parathyroid tissue in vitro.J Clin Endocrinol Metab. 1986; 63: 1075-1079Crossref PubMed Scopus (55) Google Scholar, 8Mayer G.P. Keaton J.A. Hurst J.G. Habener J.F. Effects of plasma calcium concentration on the relative proportion of hormone and carboxyl fragments in parathyroid venous blood.Endocrinology. 1979; 104: 1778-1784Crossref PubMed Scopus (177) Google Scholar These fragments, which are eliminated by the kidney, have a longer half-life than 1 to 84 PTH itself,9Yamashita H. Gao P. Cantor T. et al.Large carboxy-terminal parathyroid hormone (PTH) fragment with a relatively longer half-life than 1-84 PTH is secreted directly from the parathyroid gland in humans.Eur J Endocrinol. 2003; 149: 301-306Crossref PubMed Scopus (31) Google Scholar,10Yamashita H. Cantor T. Uchino S. et al.Sequential changes in plasma intact and whole parathyroid hormone levels during parathyroidectomy for secondary hyperparathyroidism.World J Surg. 2005; 29: 169-173Crossref PubMed Scopus (25) Google Scholar accumulate in the blood of CKD patients,11D’amour P. Lazure C. Labelle F. Metabolism of radioiodinated carboxy-terminal fragments of bovine parathyroid hormone in normal and anephric rats.Endocrinology. 1985; 117: 127-134Crossref PubMed Scopus (30) Google Scholar, 12D’Amour P. Circulating PTH molecular forms: what we know and what we don’t.Kidney Int. 2006; 70: S29-S33Abstract Full Text Full Text PDF Scopus (46) Google Scholar, 13Kritmetapak K. Losbanos L.A. Hines J.M. et al.Chemical characterization and quantification of circulating intact PTH and PTH fragments by high-resolution mass spectrometry in chronic renal failure.Clin Chem. 2021; 67: 843-853Crossref PubMed Scopus (16) Google Scholar and potentially interfere with second generation PTH assays (referred as “intact” PTH assay). Indeed, such assays are supposed to recognize, with various cross-reactivities, a family of large C-terminal fragments referred to as “non-(1 to 84)” PTH.14Lepage R. Roy L. Brossard J.H. et al.A non-(1-84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples.Clin Chem. 1998; 44: 805-809Crossref PubMed Scopus (335) Google Scholar This is not the case of third generation immunoassays (also referred as “whole” or “bioactive” PTH assays) because such assays incorporate an anti–N-terminal antibody directed toward the first 4 amino acids of the peptide, eliminating the issue of cross-reactivity with PTH fragments.15John M.R. Goodman W.G. Ping G. et al.A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure.J Clin Endocrinol Metab. 1999; 84: 4287-4290Crossref PubMed Google Scholar,16Gao P. Scheibel S. D’Amour P. et al.Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1-84: implications for improvement of accurate assessment of parathyroid function.J Bone Miner Res. 2001; 16: 605-614Crossref PubMed Scopus (344) Google Scholar The second reason for the lack of standardization in PTH assays is calibration. Indeed, despite the availability of the World Health Organization International Standard PTH 1-84, human, recombinant (NIBSC [National Institute for Biological Standards and Control] code: 95/646) (WHO 95/646 PTH IS), differences in calibration remain, which can be due to noncommutability of the WHO material and/or its incorrect use by assay the of the of the WHO 95/646 PTH can from to on the assay C.M. Sprague S. Almond A. et al.Perspective and priorities for improvement of parathyroid hormone (PTH) measurement—a view from the IFCC Working Group for PTH.Clin Chim Acta. 2017; 467: 42-47Crossref PubMed Scopus (46) Google Scholar the lack of a reference measurement a method providing true values which commercial assay be to the lack of standardization in PTH assays. In this we aimed at the impact of a recalibration of 5 PTH second and third PTH assays, on the liquid chromatography tandem mass spectrometry reference method we recently This method is calibrated the WHO 95/646 PTH and the to a reference measurement C. P. et of an LC-MS/MS method using for the quantification of 1-84 parathyroid toward a reference measurement Chem. Scopus Google Scholar the second and third generation PTH assays from on the the second and third generation assays from on the and the third generation PTH assay from on the The of methods are in 1 for reference. As we the LC-MS/MS method that we recently C. P. et of an LC-MS/MS method using for the quantification of 1-84 parathyroid toward a reference measurement Chem. Scopus Google Scholar this method by eliminating the use of during the from cross-reactivity concerns. is calibrated the WHO 95/646 PTH providing a reference LC-MS/MS method for 1 to 84 PTH from to and a measurement of of second and third generation PTH immunoassays as by the values antibody and antibody and a of of the WHO 95/646 PTH PTH 95/646 PTH PTH of human recombinant of human whole 95/646 PTH of International PTH, parathyroid World Health in a of International PTH, parathyroid World Health a calibration panel of of plasma samples by at different samples from daily to a The samples were on the of their value determined method third generation and were to the measuring panel we plasma from CKD patients from and from and and from G3a and from and from panel we plasma from CKD patients from from and from G3a from and from The plasma samples for panel were at for than a and not were at which the a WHO International Standard 1-84, human, recombinant (NIBSC code: 95/646) was to the calibration for the LC-MS/MS method. The measurements of the and were conducted at of a of the use of different for samples were in using the 5 immunoassays and the LC-MS/MS method. the of the results of the calibration we for of the immunoassays to the immunoassays on the LC-MS/MS method, and we the robustness of this calibration on the To evaluate the clinical impact of we the of and CKD patients using the LC-MS/MS method and the different immunoassays on the of a ULN we the of patients and recalibration by the Kidney Disease: Improving Global Outcomes Disease: Improving Global Outcomes (KDIGO) CKD-MBD Work GroupKDIGO clinical for the and treatment of chronic kidney bone (CKD-MBD).Kidney Int. 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