Introduction: Bone turnover markers (BTMs) have been recognized as useful tools in clinical practice, but have some short‐ and long‐term fluctuations related to biology and technical variability that have limited their clinical use to date. Among the contributors to the variation in BTM results include analytical methods (e.g., calibration), sample handling (e.g., sample collection) and laboratory performance (e.g., sample analysis). To address these issues, among the NBHA’s major efforts is the implementation of a number of complementary activities around the harmonization and standardization of the use of these markers that includes the participation of leading academic experts, the diagnostic and pharmaceutical industries, federal government representatives (from the Centers for Disease Control and Prevention, National Institute for Standards and Technology and U.S. Food and Drug Administration) and a number of both commercial and academic laboratories.
These efforts build upon the recommendations of the International Osteoporosis Foundation (IOF)/ International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Bone Marker Standards Working Group position paper published in late 2011 in Clinical Chemistry Laboratory Medicine, which identified one bone resorption marker, serum C-terminal telopeptide (s‐CTX) and one bone formation marker, type I procollagen N-terminal (s‐PINP) most applicable as reference markers to compare the performance of alternative markers and enlarge the international experience of the application of markers to clinical medicine. In July 2012, a selection of experts from the NBHA Bone Turnover Marker Standardization Project Team published the position paper “The National Bone Health Alliance Bone Turnover Marker Project: current practices and the need for US harmonization, standardization, and common reference ranges” in the journal Osteoporosis International.
This manuscript elucidates the needs in this area to be executed by the NBHA, which include the following investigator-initiated studies:
The Inter-Laboratory Comparison Study seeks to determine the laboratory reproducibility of contemporary bone turnover markers (BTM). To accomplish this identical aliquots of frozen serum from 3 pools (containing BTM levels typically observed in patients with low, medium, and high bone turnover) are being sent to 4 U.S. commercial laboratories, 5 times over 6 months (to assess longitudinal reproducibility). On the date of final shipment, identical specimens from each pool will be sent to each laboratory (to assess within-run reproducibility). The within and between laboratory reproducibility of serum CTX will be assessed by calculating the coefficient of variation (CV) using all determinations. Furthermore, specimens will be collected and archived to repeat this study at a future date when automated serum P1NP becomes available. This information is needed to effectively improve the accuracy and reliability of these BTM tests and ultimately helps to standardize the universal use of these markers as clinical measures of bone formation and bone resorption.
Status: The University of California San Francisco has initiated the study. Douglas Bauer and Hubert Vesper serve as co-PIs of the study. The first three sets of specimens have been sent to the four clinical labs for CTX analysis, and results from the first and second set of specimens have been received. Two additional sets of specimens will be sent to the clinical labs within the next few months. Initial results indicate there is a difference in CTX levels between post- and pre-menopausal females and that samples have quite a bit of variability but are all within the normal range. The project is ahead of schedule and the investigators hope to submit a late-breaking abstract of the results for presentation during the 2016 ASBMR Annual Meeting (September 16-19, Atlanta, GA).
The Drug Holiday Study seeks to determine the utility of using biochemical markers to establish the effect a drug holiday has on bone turnover for patients that have been on continuous bisphosphonate therapy for 5 years or more. Bisphosphonates are widely used in the prevention and treatment of osteoporosis in both postmenopausal women and older men. Recently, there has been concern about the risk of adverse events after several years of using these agents. This has resulted in a publication from the Food and Drug Administration that suggested that, for many individuals, a holiday from bisphosphonates might be considered after 4-5 years of continuous use. In that publication there was little if any guidance on how clinicians should proceed.
Bisphosphonates likely work by first binding to the hydroxyapatite crystal, and when the crystal is dissolved in the acid medium created by osteoclasts in the process of bone resorption, the bisphosphonate is released, and is incorporated into the osteoclast where it inhibits farnesyl pyrophosphate synthase and interferes with intracellular protein trafficking. The consequence is loss of osteoclast resorptive activity and in some cases osteoclast apoptosis. Thus the potency of any particular bisphosphonate is dependent on independent processes, such as the binding affinity to hydroxyapatite and the potency of enzyme inhibition. As patients remain on bisphosphonates more and more of the drug becomes incorporated into the skeleton. When the drugs are discontinued, they gradually leach from the skeleton. The off rate should be dependent on the binding affinity, and continued pharmacologic effect will also be dependent on potency of enzyme inhibition. Thus, each bisphosphonate will likely have a unique duration of the off effect. Data from some of the clinical trials support that concept, but there are no head to head data, and the clinical trial data are limited in their clinical utility.
The overall aim of this study is to determine the utility of biochemical markers in establishing the off rate for bisphosphonates after 5 years or more of continuous therapy. To accomplish that we have 7 specific aims:
Status: Drs. Stuart Silverman and Paul Miller are serving as co-PI’s of the study. Funding has been secured for this project. The study has been granted IRB approval and recruitment began in 2015. Currently, approximately 30 patients are active in the study; some of these patients have reached past the 6 month point and no fractures have occurred.
The Reference Population Database study seeks to develop a common-consistent young and older normal reference population database between Roche Diagnostics and Immunodiagnostic immunoassay devices. Both bone turnover markers (BTM) can be accurately measured on either the Roche Diagnostic or Immunodiagnostic immunoassay machines. The commercial laboratory companies throughout the United States use either machines, which have their separate and independent normal reference ranges for both CTX and PINP. To complicate matters even more, some commercial laboratories even using the same manufacturer assay machine yet have their own independent normal reference ranges, which may differ depending on the commercial laboratory. For example, “normal” reference ranges may differ between Lab Corp or Quest devices for CTX or PINP even using the same (e.g. Roche Diagnostic assay machine).
The most robust normal reference range established to date for (Glover S et al JBMR 2009 124(3): 389-397) utilized only the Roche Diagnostic radioimmunoassay device but even the Glover database is not the database utilized in the commercial application of the immunoassay machines in The United States.
One of the results of this lack of a consistent database for normal values is the creation of confusion and distrust of the results by clinicians attempting to manage patients with osteoporosis since they receive different values for CTX/PINP from commercial laboratories; and, in addition, difficulties in obtaining reimbursement for BTM by commercial insurance due, in part, to their distrust in inconsistent results.
Hence, the aim of the NBHA common reference population database project is to develop a common normal reference population database for both CTX and PINP done simultaneously on the same young normal and older normal women and men on both the Roche Diagnostic and the Immunodiagnostic automated immunoassay machines. This database will be owned by the NBHA who will then oversee the future research proposals for scientific investigation on additional data that may be derived from the database; and, by working through their (NBHA) consortium of scientific, professional, governmental, and industry consortium encourage this common and consistent reference population database inclusion into all commercial immunoassay devices providing services for measurements of bone turnover markers.
Status: Dr. Paul Miller is the PI of the study. The majority of funding (~65%) has been secured for this project. 250 patient visits have occurred; the screen fail rate is running slightly higher in the 60-75 year old group mostly due to undiagnosed osteoporosis and a few with hyperparathyroidism. It is anticipated as we see more of the younger age group this will go down. Samples have been run multiple times on both machines and results are consistent with what is expected for each age range both of the machines are running well with no complications.
The Patient Sample Collection Procedure Standardization Project seeks to derive a standardized sample handling procedure from careful examination of existing data as the foundation for all BTM measurements.
BTM levels may vary significantly as a result of diurnal variation and meal status. Furthermore, BTM sample stability may vary between sample types, sample storage conditions and measurement methods. Thus, pre-analytical sources of variation must be carefully standardized to allow the comparison of BTM results obtained at different patient visits, from different laboratories and to permit the use of universal reference ranges. A standardized sample handling procedure recommendation will be derived from careful examination of existing data as the foundation for all BTM measurements.
A working group consisting of the following members has submitted a draft of the manuscript to NBHA. The group has a goal to submit the manuscript by mid-2016.
Note: NBHA supports the efforts of the IOF/IFCC working group in regards to the inter-method comparison of CTX and PINP.
 Vasikaran S, et al. Clin Chem Lab Med. 2011;49(8):1271-4.
 Bauer D, et al. Osteoporos Int. 2012;23(10):2425-33.
Materials and Publications: