Why are bone turnover markers still not widely used?

A: The plasma bone turnover markers N-terminal propeptide of procollagen type I (PINP) and C-terminal telopeptide of collagen type I (CTX-I) can help clinicians manage treatment for patients with osteoporosis and other bone turnover-related diseases. The International Osteoporosis Foundation and the International Federation of Clinical Chemistry and Laboratory Medicine have also identified PINP and CTX-I as the reference markers for studies of bone turnover.

These markers have not been widely adopted, however, due to within-subject variability and lack of reproducibility between labs. To address this issue, the National Bone Health Alliance (NBHA) recently released recommendations for reducing preanalytical variability of PINP and CTX-I via standardized sample handling and patient preparation.

What patient variables impact bone turnover marker levels?

The effect of circadian rhythm is significant for CTX-I, with this marker’s peak concentrations occurring between 2-5 a.m. and nadir occurring at midday. Food intake also has a major effect on CTX-I levels, to the point that overnight fasting actually reduces circadian variation. For PINP, on the other hand, the effects of food and circadian rhythm are minimal.

Renal function influences clearance rates of CTX-I, so this analyte should be used with caution when a patient’s estimated glomerular filtration rate is <30 mL/min/1.73 m2. For PINP, the effect of renal function is more complicated. Both monomeric and trimeric forms of PINP exist in serum, and while the monomeric form is cleared by the kidneys, the trimeric form, referred to as intact PINP, is metabolized by the liver. Therefore, intact PINP is a better measurement for patients with kidney disease.

Other conditions or diseases can significantly influence bone turnover rate. These include intensive physical training, immobilization, bone fracture or bone disease, vitamin D deficiency, thyrotoxicosis, and primary hyperparathyroidism.

Certain drugs also have significant effects on PINP and CTX-I. For example, selective estrogen receptor modulators, 17 β-estradiol, bisphosphonates, denosumab, and cathepsin K inhibitors all decrease levels of bone turnover markers, while teriparatide increases levels. Drugs with a more moderate effect on bone turnover include hormonal contraceptives, corticosteroids, thiazide diuretics, and aromatase inhibitors. Disease-modifying antirheumatic drugs have been shown to decrease CTX-I, but not PINP.

What are the NBHA recommendations for patient preparation and acceptable sample types?

Due to the effects of food and circadian rhythm, NBHA recommends sample collection between 7:30-10 a.m. after an overnight fast. However, random sampling is acceptable if only measuring PINP. Providers should also instruct patients to avoid vigorous exercise on the day prior to the draw.

Labs may use either serum or plasma samples for PINP and CTX-I. However, CTX-I is more stable in EDTA plasma than in Li heparin plasma or serum and is preferred if a lab will not be able to process a sample immediately.

What are the NBHA recommendations for storing and handling of samples?

Labs should centrifuge samples and separate serum or plasma from red blood cells within 2 hours of collection, avoiding hemolysis. CTX-I has limited stability at room temperature and when refrigerated at 4°C, so if labs can’t process samples immediately, NBHA recommends freezing at -20°C. PINP, on the other hand, is more stable than CTX-I and is useful for at least 24 hours at room temperature and 5 days at 4°C, with -20°C recommended for short-term storage and -70°C recommended for long-term storage. Labs should mix any frozen samples thoroughly after thawing them and before analyzing them.

For improved within-subject results, NBHA recommends using the same sample type and handling conditions. Batch analysis is also preferred for serial samples from the same patient.

Overall, NBHA hopes that better knowledge of and adherence to these preanalytical recommendations will lead to more reproducible results and meaningful result interpretation for PINP and CTX-I, as well as establishment of appropriate thresholds for patient management.

Jennifer L. Powers, PhD, is director of the Core Laboratory for Clinical Studies and assistant professor in the Division of Endocrinology, Metabolism and Lipid Studies at Washington University in Saint Louis. +Email: powers.jennifer.l@wustl.edu