Osteoporosis International

, Volume 28, Issue 7, pp 2103–2113 | Cite as

Comparison of two automated assays of BTM (CTX and P1NP) and reference intervals in a Danish population

  • N. R. JørgensenEmail author
  • L. T. Møllehave
  • Y. B. L. Hansen
  • N. Quardon
  • L. Lylloff
  • A. Linneberg
Original Article



Bone turnover markers are used for monitoring osteoporosis treatment. Therefore, we evaluated the agreement between different assays for CTX and PINP and established reference intervals in a cohort of 2300 individuals. We found poor agreement between assays and different reference intervals. This highlights the importance of harmonization of the assays.


Two reference markers for bone turnover have been proposed: CTX bone resorption and P1NP for bone formation. The purpose of the current study was to establish reference intervals for the two markers in a Danish cohort and to determine the agreement on the two platforms.


Fasting sera from 2308 individuals (1250 males and 1058 females, age range 24–76 years) participating in the Health2006 study were analyzed for CTX and P1NP using the automated IDS-iSYS analyzer and the automated Cobas e411 analyzer. Participants in anti-osteoporotic treatment were excluded, while subjects on hormonal contraceptives were included.


There was significant disagreement between both the two P1NP assays with a mean difference of −3 μg/L (LoA −19 to 14) (p < 0.001) and the two CTX assays with a mean difference of 13 ng/L (LoA−187 to 214) (p < 0.001). For CTX, there was a systematic bias: at low values, Cobas measured a higher value than iSYS and at higher concentrations, iSYS measured increasingly higher values than Cobas. Based on the results, we propose three reference intervals for each sex: 25–29, 30–39, and 40–80 years for men, and 25–29, >30 (pre-menopausal), and >30 years (post-menopausal) for women.


There is significant disagreement between the IDS-iSYS and Roche Cobas assays for both reference markers. Consequently, the reference intervals for an adult, healthy population are different depending on the analysis method used. Therefore, repeated measurements of patient samples used for monitoring of treatment should be done on the same assay. Moreover, assay-specific reference intervals should be used. Harmonization of assays for BTM is highly warranted.


BTM CTX P1NP Reference intervals 



This work was supported by Immunodiagnostic Systems, plc, Tyne and Wear, UK, and Roche Diagnostics GmbH, Mannheim, Germany, who donated reagents for the measurements of BTM.

Author contributions

Concept and design of the study: AL, NRJ. Conducting experiments: NQ. Set up and validation of analyses: ALL, NQ, NRJ, YBLH. Analyzing data: AL, LTM, NRJ. Writing, critically reviewing, and approving the manuscript: AL, ALL, LTM, NQ, NRJ, YBLH.

Compliance with ethical standards

Ethics statement

The study was approved by the Ethical Committee of The Capital Region (approval “H-2-2013-080”) and performed in accordance with the Helsinki Declaration as revised in 1983.

Conflicts of interest

Line Tang Møllehave, Young Bae Lee Hansen, and Nadia Quardon and Louise Lylloff declare that they have no conflict of interest. Niklas Rye Jørgensen and Allan Linneberg declare that they have received the assays for the study as a donation from IDS Plc and Roche but have no further conflict of interest.

Supplementary material

198_2017_4026_MOESM1_ESM.docx (16 kb)
Supplemental Table 1 (DOCX 15 kb)
198_2017_4026_MOESM2_ESM.docx (16 kb)
Supplemental Table 2 (DOCX 15 kb)


  1. 1.
    Naylor K, Eastell R (2012) Bone turnover markers: use in osteoporosis. Nat Rev Rheumatol 8:379–389CrossRefPubMedGoogle Scholar
  2. 2.
    Vasikaran S, Eastell R, Bruyere O, Foldes AJ, Garnero P, Griesmacher A, McClung M, Morris HA, Silverman S, Trenti T, Wahl DA, Cooper C, Kanis JA (2011) Markers of bone turnover for the prediction of fracture risk and monitoring of osteoporosis treatment: a need for international reference standards. Osteoporos Int 22:391–420CrossRefPubMedGoogle Scholar
  3. 3.
    Johansson H, Oden A, Kanis JA, McCloskey EV, Morris HA, Cooper C, Vasikaran S (2014) A meta-analysis of reference markers of bone turnover for prediction of fracture. Calcif Tissue Int 94:560–567CrossRefPubMedGoogle Scholar
  4. 4.
    Morris HA, Eastell R, Jorgensen NR, Cavalier E, Vasikaran S, Chubb SA, Kanis JA, Cooper C, Makris K (2016) Clinical usefulness of bone turnover marker concentrations in osteoporosis. Clin Chim Acta. doi: 10.1016/j.cca.2016.06.036 CrossRefPubMedGoogle Scholar
  5. 5.
    Bauer DC, Black DM, Garnero P, Hochberg M, Ott S, Orloff J, Thompson DE, Ewing SK, Delmas PD (2004) Change in bone turnover and hip, non-spine, and vertebral fracture in alendronate-treated women: the fracture intervention trial. J Bone Miner Res 19:1250–1258CrossRefPubMedGoogle Scholar
  6. 6.
    Vasikaran S, Cooper C, Eastell R, Griesmacher A, Morris HA, Trenti T, Kanis JA (2011) International Osteoporosis Foundation and International Federation of Clinical Chemistry and Laboratory Medicine position on bone marker standards in osteoporosis. Clin Chem Lab Med 49:1271–1274CrossRefPubMedGoogle Scholar
  7. 7.
    Bauer D, Krege J, Lane N, Leary E, Libanati C, Miller P, Myers G, Silverman S, Vesper HW, Lee D, Payette M, Randall S (2012) National Bone Health Alliance Bone Turnover Marker Project: current practices and the need for US harmonization, standardization, and common reference ranges. Osteoporos Int 23:2425–2433CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Horowitz GL (2010) Estimating reference intervals. Am J Clin Pathol 133:175–177CrossRefPubMedGoogle Scholar
  9. 9.
    Guañabens N, Filella X, Monegal A, Gómez-Vaquero C, Bonet M, Buquet D, Casado E, Cerdá D, Erra A, Martinez S, Montalá N, Pitarch C, Kanterewicz E, Sala M, Surís X, Torres F, LabOscat Study Group (2016) Reference intervals for bone turnover markers in Spanish premenopausal women. Clin Chem Lab Med 54:293–303CrossRefPubMedGoogle Scholar
  10. 10.
    Thuesen BH, Cerqueira C, Aadahl M, Ebstrup JF, Toft U, Thyssen JP, Fenger RV, Hersoug LG, Elberling J, Pedersen O, Hansen T, Johansen JD, Jorgensen T, Linneberg A (2014) Cohort profile: the Health2006 cohort, research centre for prevention and health. Int J Epidemiol 43:568–575CrossRefPubMedGoogle Scholar
  11. 11.
    Fenger RV, Vidal C, Gonzalez-Quintela A, Husemoen LL, Skaaby T, Aadahl M, Linneberg A (2014) The association of the ‘additional height index’ with atopic diseases, non-atopic asthma, ischaemic heart disease and mortality: a population-based study. BMJ Open 4(2):e003933. doi: 10.1136/bmjopen-2013-003933 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Horwitz A, Skaaby T, Karhus LL, Schwarz P, Jorgensen T, Rumessen JJ, Linneberg A (2015) Screening for celiac disease in Danish adults. Scand J Gastroenterol 50:824–831CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Rosengren A, Wilhelmsen L (1997) Physical activity protects against coronary death and deaths from all causes in middle-aged men. Evidence from a 20-year follow-up of the primary prevention study in Goteborg. Ann Epidemiol 7:69–75CrossRefPubMedGoogle Scholar
  14. 14.
    Saltin B, Grimby G (1968) Physiological analysis of middle-aged and old former athletes. Comparison with still active athletes of the same ages. Circulation 38:1104–1115CrossRefPubMedGoogle Scholar
  15. 15.
    Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1:307–310CrossRefPubMedGoogle Scholar
  16. 16.
    Solberg HE (1987) International Federation of Clinical Chemistry. Scientific committee, clinical section. Expert Panel on Theory of Reference Values and International Committee for Standardization in Haematology Standing Committee on Reference Values. Approved recommendation (1986) on the theory of reference values. Part 1. The concept of reference values. Clin Chim Acta 165:111–118CrossRefPubMedGoogle Scholar
  17. 17.
    Solberg HE (1987) International Federation of Clinical Chemistry (IFCC), scientific committee, clinical section, Expert Panel on Theory of Reference Values, and International Committee for Standardization in Haematology (ICSH), Standing Committee on Reference Values. Approved recommendation (1986) on the theory of reference values. Part 1. The concept of reference values. J Clin Chem Clin Biochem 25:337–342PubMedGoogle Scholar
  18. 18.
    Chubb SA, Mandelt CD, Vasikaran SD (2015) Comparison of results from commercial assays for plasma CTX: the need for harmonization. Clin Biochem 48:519–524CrossRefPubMedGoogle Scholar
  19. 19.
    Wheater G, Goodrum C, Tuck SP, Datta HK, van Laar JM (2014) Method-specific differences in beta-isomerised carboxy-terminal cross-linking telopeptide of type I collagen and procollagen type I amino-terminal propeptide using two fully automated immunoassays. Clin Chem Lab Med 52:e135–e138CrossRefPubMedGoogle Scholar
  20. 20.
    Schafer AL, Vittinghoff E, Ramachandran R, Mahmoudi N, Bauer DC (2010) Laboratory reproducibility of biochemical markers of bone turnover in clinical practice. Osteoporos Int 21:439–445CrossRefPubMedGoogle Scholar
  21. 21.
    Michelsen J, Wallaschofski H, Friedrich N, Spielhagen C, Rettig R, Ittermann T, Nauck M, Hannemann A (2013) Reference intervals for serum concentrations of three bone turnover markers for men and women. Bone 57:399–404CrossRefPubMedGoogle Scholar
  22. 22.
    Morovat A, Catchpole A, Meurisse A, Carlisi A, Bekaert AC, Rousselle O, Paddon M, James T, Cavalier E (2013) IDS iSYS automated intact procollagen-1-N-terminus pro-peptide assay: method evaluation and reference intervals in adults and children. Clin Chem Lab Med 51:2009–2018CrossRefPubMedGoogle Scholar
  23. 23.
    Li M, Li Y, Deng W, Zhang Z, Deng Z, Hu Y, Xia W, Xu L (2014) Chinese bone turnover marker study: reference ranges for C-terminal telopeptide of type I collagen and procollagen I N-terminal peptide by age and gender. PLoS One 9:e103841. doi: 10.1371/journal.pone.0103841 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Jenkins N, Black M, Paul E, Pasco JA, Kotowicz MA, Schneider HG (2013) Age-related reference intervals for bone turnover markers from an Australian reference population. Bone 55:271–276CrossRefPubMedGoogle Scholar
  25. 25.
    Olmos JM, Hernandez JL, Martinez J, Pariente E, Llorca J, Gonzalez-Macias J (2010) Bone turnover markers in Spanish adult men the Camargo cohort study. Clin Chim Acta 411:1511–1515CrossRefPubMedGoogle Scholar
  26. 26.
    Chubb SA, Byrnes E, Manning L, Golledge J, Ebeling PR, Flicker L, Yeap BB, Vasikaran SD (2016) Bone turnover markers: defining a therapeutic target. Clin Biochem Sep. doi: 10.1016/j.clinbiochem.2016.09.010 CrossRefGoogle Scholar
  27. 27.
    Ardawi MS, Maimani AA, Bahksh TA, Rouzi AA, Qari MH, Raddadi RM (2010) Reference intervals of biochemical bone turnover markers for Saudi Arabian women: a cross-sectional study. Bone 47:804–814CrossRefPubMedGoogle Scholar
  28. 28.
    Gossiel F, Finigan J, Jacques R, Reid D, Felsenberg D, Roux C, Glueer C, Eastell R (2014) Establishing reference intervals for bone turnover markers in healthy postmenopausal women in a nonfasting state. Bonekey Rep. doi: 10.1038/bonekey.2014.68 PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Howe TE, Shea B, Dawson LJ, Downie F, Murray A, Ross C, Harbour RT, Caldwell LM, Creed G. (2011) Exercise for preventing and treating osteoporosis in postmenopausal women. Cochrane Database Syst Rev CD000333: doi:  10.1002/14651858.CD000333.pub2
  30. 30.
    Gomez-Cabello A, Ara I, Gonzalez-Aguero A, Casajus JA, Vicente-Rodriguez G (2012) Effects of training on bone mass in older adults: a systematic review. Sports Med 42:301–325CrossRefPubMedGoogle Scholar

Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2017

Authors and Affiliations

  • N. R. Jørgensen
    • 1
    • 2
    Email author
  • L. T. Møllehave
    • 3
  • Y. B. L. Hansen
    • 1
  • N. Quardon
    • 1
  • L. Lylloff
    • 4
  • A. Linneberg
    • 3
    • 5
    • 6
  1. 1.Research Center for Ageing and Osteoporosis, Department of Clinical BiochemistryRigshospitaletCopenhagenDenmark
  2. 2.OPEN, Odense Patient data Explorative Network, Odense University Hospital/Institute of Clinical ResearchUniversity of Southern DenmarkOdenseDenmark
  3. 3.Research Centre for Prevention and Health, Centre for Health, Capital Region of DenmarkCopenhagenDenmark
  4. 4.Department of Clinical BiochemistryCopenhagen University Hospital HvidovreHvidovreDenmark
  5. 5.Department of Clinical Experimental ResearchRigshospitaletCopenhagenDenmark
  6. 6.Department of Clinical Medicine, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations