Towards optimising the provision of laboratory services for bone turnover markers
Vasikaran, Samuel D.1,2; Chubb, S. A. Paul1,2,3; Schneider, Hans-Gerhard4,5
1Department of Clinical Biochemistry, PathWest Laboratory Medicine, Royal Perth and Fremantle Hospitals, Perth
2School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands
3School of Medicine and Pharmacology, University of Western Australia, Nedlands, WA
4Clinical Biochemistry Unit, Alfred Pathology Service, Melbourne
5Monash University, Melbourne, Vic, Australia
Address for correspondence: Professor S. D. Vasikaran, Royal Perth Hospital, Department of Clinical Biochemistry, GPO Box X2213, Perth, WA 6847, Australia. E-mail: Samuel.firstname.lastname@example.org
Received 5 December, 2013
Revised 30 January, 2014
Accepted 30 January, 2014
Bone turnover markers (BTMs) are either secreted by osteoblasts during bone formation or released by degradation of the collagen matrix of bone during bone resorption, and may be measured in blood or urine to provide an estimate of the rate of bone remodelling. Increased bone remodelling rate is often associated with bone loss which can result in osteoporosis; however, lack of data preclude the inclusion of BTMs in fracture risk algorithms. The changes in BTMs following therapy for osteoporosis may be useful for monitoring. Serum procollagen type I amino-terminal propeptide (s-PINP) and serum carboxy-terminal cross-linking telopeptide of type I collagen (s-βCTX) have been designated as reference standard markers of bone formation and resorption respectively in osteoporosis; further research is needed for their routine use in osteoporosis. BTMs are useful in diagnosing and monitoring Paget's disease of bone and other bone diseases associated with abnormal bone formation and/or resorption. Standardised patient preparation is required to mitigate the effect of biological variation, and appropriate sample handling and storage are important to minimise sample degradation. Significant inter-method differences exist for BTMs, and harmonisation of methods for the reference BTMs is being pursued. This will help develop universally accepted decision limits and treatment goals. Australian consensus reference intervals have been developed for some methods for s-PINP and s-βCTX.
NORMAL BONE PHYSIOLOGY AND BONE REMODELLING
Once growth is complete, the adult bone may stop growing, but remains a dynamic organ throughout life, continually renewed by the process of bone remodelling which occurs in discrete areas on bone surface. The functional anatomy of this process has been defined as the bone remodelling unit (BRU). BRUs provide a mechanism for repair of micro-damage and for adaptation to stresses, as well as for the maintenance of calcium homeostasis.1 Cortical or compact bone makes up approximately 80% of the skeleton, and is found especially in the shafts of long bones. Trabecular or cancellous bone is more porous and makes up most of the vertebral bodies. Due to its relatively large surface area, trabecular bone accounts for the majority of bone remodelling activity. The bone remodelling cycle starts with attraction of osteoclast precursors to a particular bone surface area to be remodelled, where they fuse to form multinucleated osteoclasts, which resorb a pocket of bone. This is followed by the reversal phase where pre-osteoblasts migrate to the newly resorbed site and differentiate into osteoblasts and form new bone (osteoid, the amount of new bone being equal to that resorbed). Following mineralisation with calcium, phosphate and carbonate, arranged predominantly as hydroxyapatite crystals, the bone surface returns to its quiescent state. In the process, osteoblasts undergo transformation into osteocytes and become embedded in the osteoid or die by apoptosis.
Bone remodelling is influenced by mechanical strain, local and systemic hormones, growth factors and cytokines. The key molecules secreted by osteoblasts and stromal cells that regulate osteoclasts are macrophage colony stimulating factor (M-CSF), receptor activator of nuclear factor κB ligand [RANKL, tumour necrosis factor (TNF) ligand superfamily member 11], and its decoy receptor osteoprotegerin (OPG, TNF receptor superfamily member 11B).2,3 RANKL interacts directly with its receptor RANK on the surface of osteoclasts and their precursors to activate them.2,3 Similarly, osteoblast differentiation and activation is promoted by ligands of the Wnt signalling pathway, and inhibited by Wnt pathway inhibitors such as sclerostin.4
COMMON SUBTYPES OF BONE DISEASE
Bone formation and resorption are ‘coupled’ and, in health, the net cumulative effect of all the bone remodelling cycles in the adult skeleton is the maintenance of bone volume. Disturbances of coupling where formation does not equal resorption can lead to net bone loss, and over time, osteoporosis. Conversely, excessive bone formation can lead to osteosclerosis, such as is seen in Paget's disease of bone,5 or in relation to osteoblastic bony metastases from prostate or breast cancer. The majority of cancer-related bone diseases are osteolytic in nature and result in net loss of bone, leading to osteoporosis when this is generalised, or lytic lesions when the lesions are discrete.6 Multiple myeloma is an example of malignant bone disease leading to both lytic lesions as well as generalised bone loss.7 The generalised increase in bone resorption in multiple myeloma is not associated with an increase in bone formation; i.e., there is uncoupling of resorption and formation in this condition.
Osteoporosis is defined as a disease characterised by low bone mass and micro-architectural deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk.8 The World Health Organization (WHO) diagnostic criterion for osteoporosis is a bone mineral density (BMD) measurement equal to or more than 2.5 standard deviations (SD) below the young reference mean (T-score ≤−2.5 SD).9 The majority of cases are found among post-menopausal women where oestrogen deficiency is implicated. Bone loss is also related to ageing and the prevalence increases with age in both women and men. In addition, osteoporosis may be associated with medications such as high dose glucocorticoids, and several diseases, such as rheumatoid arthritis, hypogonadism (in males), hyperparathyroidism and hyperthyroidism, conditions associated with malabsorption of calcium and/or vitamin D and malignant bone disease such as multiple myeloma. Paget's disease is a focal disorder of increased local bone remodelling which results in expansion and structural weakness of bone leading to pain, deformity and increased fracture risk, and is more common in older people.5 The osteogenesis imperfectas are a group of inherited diseases characterised by defective connective tissue and decreased bone formation due to a deficiency of type-I collagen caused by mutations in the COL1A1 and COL1A2 genes and resulting in growth abnormalities and/or brittle bones.10
Severe vitamin D deficiency leads to lack of mineralisation of bone and in children causes rickets, characterised by soft bones and skeletal deformities.11 The adult form of the disease is osteomalacia and is generally associated with serum 25-hydroxyvitamin D <12.5 nmol/L.12 Osteoid calcification is impaired and both bone resorption and formation are increased. Mild to moderate forms of vitamin D deficiency (serum 25-hydroxyvitamin D levels of 25–50 nmol/L and 12.5–25 nmol/L respectively), which are much more common in developed countries, are associated with secondary hyperparathyroidism and increased bone loss, which can result in osteoporosis.12 Primary hyperparathyroidism is also associated with osteoporosis due to increased bone resorption and consequent bone loss.13
Renal osteodystrophy, which is associated with chronic kidney disease, may be characterised by one or more of the following:14
- Osteitis fibrosa cystica, in which bone turnover is increased due to secondary hyperparathyroidism.
- Adynamic bone disease, in which bone turnover is low, mostly due to excessive suppression of parathyroid hormone secretion, although aluminium toxicity was implicated in the past.
- Mixed uraemic osteodystrophy, a mixture of both high and low bone turnover together with marrow fibrosis and increased unmineralised osteoid.
Further discussion of renal osteodystrophy is beyond the scope of this review.
OVERVIEW OF BTMS AND THE THEORY AND EVIDENCE FOR THEIR CLINICAL UTILITY
BTMs are classified as bone formation markers or bone resorption markers. Their concentration in blood and in urine may reflect bone remodelling rate, although the accuracy with which they reflect bone turnover may be influenced by their tissue specificity for bone and a number of physiological and pathological factors, some of which may be disease-specific. All BTMs to some extent reflect both bone formation and resorption since bone formation and resorption are ‘coupled’ in most cases.
Bone formation markers are peptides or enzymes produced by osteoblasts during the different phases of bone formation. Osteoblasts synthesise type 1 procollagen molecules during the early stages of formation of bone matrix. The amino- and carboxy-terminal propeptides (PINP and PICP) of the procollagen molecules are cleaved off when collagen is laid down, and released into blood where their concentrations reflect bone formation rate.15–18 Bone-specific alkaline phosphatase (BALP) is an enzyme produced by osteoblasts and its concentration in blood reflects osteoblast activity.19,20 Osteocalcin is a non-collagenous bone matrix protein, the concentration of which in blood also reflects osteoblast activity.21
Bone resorption markers are degradation products of bone collagen such as the amino-terminal and carboxy-terminal cross-linking telopeptides of type I collagen (NTX and CTX) and deoxypyridinoline (DPD), or enzymes secreted by osteoclasts such as tartrate-resistant acid phosphatase (TRACP5b).22–28 Whilst the former reflect type 1 collagen breakdown, mainly bone resorption rate, TRACP5b reflects osteoclast numbers.
Other molecules such as the inflammation marker highly sensitive C-reactive protein (hs-CRP) might be useful to predict risk of future fracture,29 but are not considered markers of bone turnover, nor are they currently utilised in clinical practice, and will not be further discussed here.
BTMS IN CURRENT USE
Although several BTMs have been studied for their utility in the investigation and management of bone diseases, the formation markers that are used in practice are s-PINP, s-BALP and osteocalcin. The resorption markers that are in current use are s-βCTX, urine NTX (u-NTX) and u-DPD. Currently available commercial immunoassays for s-BALP, s-PINP and osteocalcin as well as for s-βCTX, u-NTX and u-DPD are adequately bone-specific; however, their clinical utility in terms of diagnostic efficiency and utility for monitoring disease progress and response to treatment may vary between different bone diseases.
DISEASE-SPECIFIC ISSUES FOR BTMS
BTMs have been studied in metabolic and metastatic bone diseases and their ability to reflect bone turnover or activity in different disease states has been empirically determined. Recommendations for their clinical use for diagnosis, prognostication, monitoring disease progression and response to treatment have been based on the findings of these studies.30
Osteoporosis is a silent disease, with fracture being the clinically important outcome. Whilst the diagnosis of osteoporosis is based on BMD measurement, the latter is just one of many risk factors for fracture; other important independent risk factors for fracture include age, sex, a history of prior fractures, family history of fracture, body mass index, height, ethnicity, smoking, alcohol use, glucocorticoid use, rheumatoid arthritis and other diseases which lead to bone loss as well as a propensity to fall. The contributions of these risk factors to fracture risk have been quantified and the absolute fracture risk for a patient can be estimated with fracture risk calculators [e.g., FRAX ( http://www.shef.ac.uk/FRAX), or the Garvan risk calculator ( http://www.garvan.org.au/bone-fracture-risk/)], in order to select patients who need anti-osteoporosis treatment. There are inadequate data from prospective studies on any single BTM for their inclusion in fracture risk algorithms; therefore, the use of BTMs in fracture risk assessment is currently not recommended by most osteoporosis guidelines. Whilst there is more general acceptance of the use of BTMs for monitoring therapy in osteoporosis, their utility in improving outcomes need confirmation.
The International Osteoporosis Foundation (IOF) and International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) joint Working Group on Bone Marker Standards has recommended that s-PINP and s-βCTX be designated as reference standard markers of bone formation and resorption respectively in osteoporosis.31 They recommended that these two markers be included in all clinical trials and in observational studies in order that adequate data can be accumulated for their application for osteoporosis in clinical practice. In the US, the National Bone Health Alliance (NBHA) has endorsed the choice of s-βCTX and s-PINP as the reference standards for bone resorption and formation respectively in osteoporosis, and has embarked on an effort to harmonise commercial assays for these two markers in the US and to determine reference intervals for clinical use.32
The utility of bone markers for the diagnosis and monitoring of Paget's disease is well established.5 Total alkaline phosphatase is adequate for this purpose in most cases of Paget's disease due to the major increases seen in this condition. Rarely, in localised (mono-ostotic) disease, total alkaline phosphatase may not be sensitive enough to detect an increase in disease activity, and therefore BALP or PINP may be used as well as u-NTX as a marker of bone resorption. s-βCTX (commercial assays detect the beta isomerised form of CTX) is a less sensitive marker of Paget's disease activity; osteocalcin is also not recommended.
The large intra-individual variations seen in some BTMs due to diurnal variation and the effect of food are well recognised, and need to be taken into consideration in the selection of the marker for clinical use and in the sample collection procedure; the time of collection and fasting status may need to be controlled to minimise their effects.33,34
s-βCTX has a large circadian variation of between ±30% and ±35%, with the peak βCTX in the early hours of the morning and nadir concentrations in the middle of the day.35 This is in part mediated by food intake which decreases s-βCTX.31 Fasting reduces the diurnal variation and also the between-day within-individual biological variability. Recreational exercise is unlikely to be a significant confounding factor in most clinical situations in which s-βCTX will be measured.35 Therefore, collection of sample for s-βCTX is recommended to be in the fasting state in the morning at a well-defined time to minimise the within-individual variation.35 The preferred sample is EDTA plasma (stable unseparated for at least 24 h at room temperature) with rapid centrifugation and either immediate analysis, or freezing of the plasma (once separated stable for 48 h at room temperature and for 7 days at 4°C). βCTX is less, but still sufficiently, stable in serum (stability at room temperature 4 h if unseparated and 8 h if separated, stability at 4°C, 24 h if unseparated and 48 h if separated). If a clotted sample is collected it should be centrifuged immediately and analysed or frozen within 4 h. S-βCTX is stable after multiple freeze-thaw cycles, an important observation for retrospective analyses of clinical trials.35
s-PINP shows little diurnal or seasonal variation within the individual.36 s-PINP is not significantly influenced by food intake and, therefore, patients do not need to fast for PINP measurement.34 Both serum and heparin plasma are acceptable for PINP measurement. PINP is stable in serum for at least 5 days at room temperature and for at least 4 weeks at 4°C.37 In frozen serum, PINP is stable at −18°C for 24 months and at −70°C for 19 months, and is also stable after four freeze-thaw cycles.38
u-NTX and DPD also show significant diurnal variation and some effect of feeding.34 Sampling is best standardised by collecting without preservatives the second or third void (spot) urine in the morning after an overnight fast (a drink of water is allowed to facilitate urine flow). The concentration of urine BTM is expressed as a ratio to creatinine in order to correct for the effect of dilution. u-NTX is stable for at least 48 h at 23–25°C, 7 days at 2–8°C, 3 months when frozen at −20°C, and for at least 1 year at −80°C.39 Urine DPD is not stable at room temperature, but is stable for at least 7 days at 2–8°C, for at least 3 months at −20°C, and for at least 1 year at −80°C.39
Within- and between-individual variation data for BTMs are summarised in Table 1, along with desirable performance specifications and reference change values.
The effect of renal failure on the accumulation of some BTMs in blood significantly impacts on their use in patients with this condition.40 Among formation markers, serum intact but not total PINP appears to be least affected by renal failure, whilst s-BALP may also be acceptable.41 Osteocalcin, on the other hand, accumulates in serum in renal failure rendering it less useful in such patients.40 Concentrations of urine markers are almost always affected by the effect of renal failure and are therefore difficult to interpret in such cases. The resorption marker that is least affected by renal failure and therefore of most use in examining bone resorption rate in patients with significant renal impairment is TRACP5b.40
ASSAYS AVAILABLE CURRENTLY FOR THE RECOMMENDED BTMS, AND THEIR CHARACTERISTICS
Significant inter-method differences exist for both s-PINP and for s-βCTX, making it difficult to use different laboratories for following a patient or for performing multicentre clinical trials and impossible to recommend universally applicable cut-points and decision limits in clinical guidelines (Box 1).31,32
Although all commercial assays for s-βCTX (CrossLaps; Roche, Switzerland) measure the 8-amino acid peptide (EKAHD-βGGR) of cross-linked CTX dimers, there are differences in results produced by different assays, which have been demonstrated in the results of external quality assurance (QA) programs (College of American Pathologists and UK National External Quality Assurance Programmes). There is currently no QA program for BTMs available in Australia, and a number of laboratories use sample exchange. The inter-method biases between s-βCTX assays may be explained by calibration differences. However, until harmonisation of methods is achieved, results from different methods cannot be used interchangeably for clinical care or in research studies, and patients should be monitored by the use of the same method over time.
REFERENCE INTERVALS AND TREATMENT TARGETS
Consensus reference intervals for s-PINP and s-βCTX were developed by a group of Clinical Biochemists and Endocrinologists with expertise in bone turnover markers from around Australia under the umbrella of the Australasian Association for Clinical Biochemistry (AACB) Reference Intervals Harmonisation Project (Tables 2–7).
Australian consensus reference intervals for s-PINP
There was felt to be adequate data in the published literature for the Roche (total) s-PINP assay for the age groups listed in Table 2.42–50 Provisional reference intervals are provided for females >70 years and both sexes <25 years age in Tables 3 and 4.
Data are awaited for intact PINP assays, although there seems to be reasonable agreement for PINP values reported by intact and total PINP assays in subjects with normal renal function and without metastatic bone disease.41,52,53
Australian consensus reference intervals for s-βCTX
There was felt to be adequate data in the published literature for the Roche s-βCTX assay based on a fasting morning sample for the age groups listed in Table 5.42,43,45–47,50,54 Provisional reference intervals are provided for females >70 years and <20 years and males <25 years age in Tables 6 and 7.
Data are awaited for s-βCTX values by IDS iSYS.52
In addition to reference intervals, knowledge of expected changes in BTMs with treatment may be useful in monitoring. In Paget's disease of bone where BTMs are increased above the upper reference limit in active disease, normalisation of BTMs well into the reference interval is expected following successful treatment.55 Recurrence of disease activity is heralded by a significant increase of the BTMs from the nadir.55
Although the reference intervals reflect the 95% confidence limits for a healthy population, in osteoporosis BTMs may not always be raised above the upper reference limit.31 Conversely, the menopausal reference interval for BTMs may encompass values from women with high rates of bone loss. Decision limits and target intervals for BTMs based on fracture rates are currently lacking, and more data are needed. Incorporation of BTMs in fracture risk calculations require correlation of BTM levels as a gradient of fracture risk (i.e., increase in fracture risk per SD increase in BTM).31
Knowledge of the least significant change (reference change value) for a BTM is useful in confirming response to treatment, and this is suggested to be around 30% for serum markers and close to 50% for urine markers (Table 1).56 Following anti-resorptive treatment, a decrease of BTM into the lower half of the premenopausal interval 1–3 months after intravenous treatment or 3–6 months after oral therapy has been suggested as a goal of treatment.56 This is achieved in most cases where patients adhere to therapy; for example, s-βCTX decreases to <250 ng/L in these patients.57 Conversely, following treatment of osteoporosis with an anabolic agent such as teriparatide, an increase in PINP by >10 μg/L from baseline within 1–3 months has been suggested as an indication of response to treatment.58,59 Lack of response in BTMs following therapy may be due to non-compliance with treatment or the presence of a secondary cause for continuing bone loss.60 BTMs are not useful for monitoring strontium ranelate treatment.
Although the use of s-βCTX has been suggested to be useful in predicting the occurrence of osteonecrosis of the jaw following treatment with bisphosphonates, the studies on which this suggestion was based were not properly designed and the use of s-βCTX for this purpose is not recommended.61–63
BTMs are widely used in bone research including therapeutic trials of new medications for osteoporosis and other bone disease. Whilst further data are awaited to confirm their utility in the clinical management of osteoporosis, they are currently used in specialist clinical practices, especially in monitoring treatment. Laboratories need to be able to provide a service to such practices. The use of BTMs is well established for other bone diseases such as Paget's disease and shows promise for malignant bone disease. The proposed harmonisation of commercial assays and further data on reference intervals where hiatuses exist will help improve the current status of laboratory services for BTMs.
Consensus reference intervals for s-PINP and s-βCTX were reached by a group of Clinical Biochemists and Endocrinologists with expertise in BTMs from around Australia under the umbrella of the AACB reference intervals harmonisation project: Sam Vasikaran (Chair), Paul Chubb, Peter Ebeling, Nicole Jenkins, Graham Jones, Mark Kotowicz, Howard Morris, Hans Schneider, Markus Seibel (s-PINP only), Greg Ward.
Conflicts of interest and sources of funding: PC has received research support from Roche Diagnostics by way of discounted reagent kits for research studies.
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Amino-terminal cross-linking telopeptide of type I collagen; bone formation; bone remodelling; bone resorption; carboxy-terminal cross-linking telopeptide of type I collagen; deoxypyridinoline; osteocalcin; osteoporosis; Paget's disease of bone; procollagen type I amino-terminal propeptide; tartrate-resistant acid phosphatase
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