Osteoporosis International

, Volume 28, Issue 9, pp 2541–2556 | Cite as

Use of CTX-I and PINP as bone turnover markers: National Bone Health Alliance recommendations to standardize sample handling and patient preparation to reduce pre-analytical variability

  • P. Szulc
  • K. Naylor
  • N. R. Hoyle
  • R. Eastell
  • E. T. Leary
  • for the National Bone Health Alliance Bone Turnover Marker Project
Position Paper

Abstract

Summary

The National Bone Health Alliance (NBHA) recommends standardized sample handling and patient preparation for C-terminal telopeptide of type I collagen (CTX-I) and N-terminal propeptide of type I procollagen (PINP) measurements to reduce pre-analytical variability. Controllable and uncontrollable patient-related factors are reviewed to facilitate interpretation and minimize pre-analytical variability.

Introduction

The IOF and the International Federation of Clinical Chemistry (IFCC) Bone Marker Standards Working Group have identified PINP and CTX-I in blood to be the reference markers of bone turnover for the fracture risk prediction and monitoring of osteoporosis treatment. Although used in clinical research for many years, bone turnover markers (BTM) have not been widely adopted in clinical practice primarily due to their poor within-subject and between-lab reproducibility. The NBHA Bone Turnover Marker Project team aim to reduce pre-analytical variability of CTX-I and PINP measurements through standardized sample handling and patient preparation.

Methods

Recommendations for sample handling and patient preparations were made based on review of available publications and pragmatic considerations to reduce pre-analytical variability. Controllable and un-controllable patient-related factors were reviewed to facilitate interpretation and sample collection.

Results

Samples for CTX-I must be collected consistently in the morning hours in the fasted state. EDTA plasma is preferred for CTX-I for its greater sample stability. Sample collection conditions for PINP are less critical as PINP has minimal circadian variability and is not affected by food intake. Sample stability limits should be observed. The uncontrollable aspects (age, sex, pregnancy, immobility, recent fracture, co-morbidities, anti-osteoporotic drugs, other medications) should be considered in BTM interpretation.

Conclusion

Adopting standardized sample handling and patient preparation procedures will significantly reduce controllable pre-analytical variability. The successful adoption of such recommendations necessitates the close collaboration of various stakeholders at the global stage, including the laboratories, the medical community, the reagent manufacturers and the regulatory agencies.

Keywords

Anti-osteoporotic treatment Bone turnover markers C-terminal telopeptide of type I collagen N-terminal propeptide of type I procollagen Osteoporosis 

Introduction

Bone turnover markers (BTMs) have been studied for over 30 years. They include two groups: markers of bone formation (chiefly N-terminal collagen type I extension propeptide (PINP), osteocalcin, bone alkaline phosphatase) and markers of bone resorption, mainly collagen I degradation products (e.g., C-terminal cross-linking telopeptide of type I collagen (CTX-I), N-terminal telopeptide of type I collagen, deoxypyridinoline, hydroxyproline). BTMs have been used in clinical research, but not widely adopted in clinical practice. The primary challenge to their adoption in routine practice has been the poor within-subject and between-lab reproducibility of many BTMs. Inadequately controlled pre-analytical variability and heterogeneity of the assays contribute to inconsistent BTM values. This renders interpretation of BTM results difficult [1, 2, 3].

The International Osteoporosis Foundation (IOF) and International Federation of Clinical Chemistry (IFCC) have recommended serum PINP and β-CTX-I as reference BTMs. The intent is to measure PINP and CTX by standardized immunoassays in observational and interventional studies in order to generate comparative data with alternative markers and to enlarge the global experience of BTM [1]. For BTMs to be useful in both clinical research and clinical practice, the pre-analytical sources of variability, along with the underlying disease process, must be identified, minimized, and controlled through carefully standardized patient preparation and sample handling procedures. An appreciation of the underlying disease processes may permit optimized sample collection and interpretation of the results.

The National Bone Health Alliance (NBHA) sets the goal for the writing group to review published information and provide concise recommendations for standardized sample handling, patient preparation, and reducing pre-analytical variability. The recommendations are directed at the laboratories and the manufacturers of reagents for the measurement of CTX-I and PINP, to clinicians, and to those who obtain and handle blood specimens.

The NBHA, IFCC, IOF, and others are also working toward additional interventions, which include the comparison of the assays and subsequent among-method standardization involving a harmonization process to achieve result agreement [1, 4].

N-terminal procollagen type I extension propeptide

The PINP is cleaved from type I pro-collagen during its extracellular processing when it is assembled into fibrils [5]. Serum PINP is generated during the synthesis of type I collagen, the most abundant bone protein (90% of matrix). Type I collagen may be synthesized in other connective tissues, particularly skin. Thus, since the metabolism of these tissues is slower than that of bone, the majority of serum PINP detected originates from bone. However, in rapid growth (childhood) and pathological fibrotic process, the contribution of PINP from non-bone tissues may be higher [6]. The quantities of PINP produced are equimolar to that of the resultant collagen incorporated into the bone matrix. Therefore, serum PINP levels are correlated significantly with histomorphometric measures of bone formation [7]. There are two forms of PINP in blood: in vivo cleaved trimeric peptide (“intact”) and low-molecular-weight dissociated peptides of α1 and α2 chains [8]. Circulating PINP is taken up by the scavenger receptor on endothelial cells of the liver, and then degraded [9].

C-terminal telopeptide of type I collagen

The CTX-I is a product of the breakdown of type I collagen containing pyridinium cross-links [5]. CTX-I is cleared by the kidney. Serum levels are correlated significantly with histomorphometric measures of bone resorption [7]. Immunoassays for CTX-I in blood use the antibody raised against an epitope on the α1 chain of type I collagen. Its amino acid sequence contains lysine and the aspartate-glycine sequence (DG). This sequence may undergo β-isomerization (generating the mature form, beta-CTX-I) and racemization. Available assays recognize the mature-bone epitope composed of two beta-CTX-I sequences on two chains connected by a cross-link [10, 11].

Pre-analytical variation

Current guidelines for patient preparation and sample handling for laboratory CTX-I and PINP testing lack consistency and clarity. Moreover, the interpretation of the BTM results should account for the clinical factors which cannot be controlled [12, 13].

Available commercial CTX-I assays include enzyme-linked immunosorbent assay (ELISA; Immunodiagnostic Systems; [IDS], Bolden, UK), automated electrochemiluminescence immunoassays (ECLIA; Roche Diagnostics, Mannheim, Germany), and automated chemiluminescence immunoassay (CLIA; IDS) [14, 15]. PINP measurements include intact PINP by radioimmunoassay (Orion Diagnostica, Espoo, Finland) which measures only the native trimeric peptide, total PINP with ECLIA by automated Roche Diagnostics platforms which measure both the trimeric PINP peptide and the low-molecular-weight peptides of α1 and α2 chains, and intact PINP with CLIA by automated IDS platform that measures native trimeric peptides only [8, 16, 17, 18].

It is not feasible in this review to assess the intrinsic behavior of each BTM independent of the testing method. Furthermore, differences in the design of each validation study such as number of subjects, demographics and disease states, time points studied, collection tube systems used, storage condition, freeze/thaw procedures, reagent formulation iterations, and criteria used to assess performance could contribute to apparent discrepancies in observed method performance. Since FDA clearance of diagnostic products requires the submission of stringently characterized data sets provided by the reagent/instrument manufacturer, the subsequent kit product inserts serve both as a useful and as an official sanctioned source of information. Thus, the summary and recommendations below are based on the authors’ extrapolation of available data including the product inserts where appropriate (Table 1).
Table 1

Recommendations for standardized sample handling and patient preparation for CTX-I and PINP measurements in blood

1. Collect blood sample after an overnight fast. Fasting is not necessary if only PINP is measured [19, 20, 21]. Avoid vigorous exercise on day prior to sampling [22].

2. Collect sample between 7:30 a.m. and 10 a.m. [21]. Random sample is acceptable for PINP [20].

3. Both serum and EDTA plasma are acceptable. Collect EDTA plasma for CTX-I if sample cannot be processed promptly [17, 23, 24, 25, 26].

4. Centrifuge and separate sample from red cells within 2 h of collection. Avoid hemolysis.

5. Store separated sample at≤−20 °C until analysis. For long-term storage (>3 months for CTX-I and >6 months for PINP), store at ≤−70 °C. Observe sample stability limits at RT and 4 °C [24, 25, 26]

6. Mix thawed sample thoroughly after frozen storage by inversion before analysis. Centrifuge sample to remove particulates if present.

7. Same sample type and handling conditions are necessary when monitoring a patient.

8. Freeze serial samples and analyze in the same batch if possible.

Sources of pre-analytical variability—sample handling

Sample type

Either serum or plasma samples may be used for CTX-I and PINP measurements [14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31]. EDTA plasma has the advantage of superior sample stability for CTX-I compared with serum. Tripotassium ethylenediaminetetraacetate (K3-EDTA), dipotassium EDTA (K2-EDTA), and sodium EDTA (Na-EDTA) plasma are acceptable [24, 28]. Lithium (Li) heparin plasma which has greater variability and decreased stability compared to serum and EDTA plasma may be used within its stability limits [23, 24, 27].

For PINP, serum and plasma perform equally well [17]. The current Orion UniQ PINP RIA package insert [32] specifies the use of serum only. However, previous insert (35554-DS4) and published literature have supported the use of serum and plasma in PINP measurements [17, 25, 29, 33]. It is the kit user’s responsibility to validate the acceptability of sample types or conditions which are not included in the package insert. It is obligatory that the same sample type be used consistently when monitoring a patient.

Sample collection and processing

CTX-I exhibits a circadian rhythm in blood which remains unchanged in various scenarios. CTX-I level peaks during the early morning hours (2–5 a.m.) and reaches a nadir between 11 a.m. and 2 p.m. The amplitude of the variation is 40 to 66% around the 24-h mean [19, 34]. The circadian pattern was consistent in young and older healthy male, in pre-menopausal and postmenopausal women with normal/low bone mass [19, 20, 34]. Although the serum CTX levels differ, the same circadian pattern is observed in different ethnic groups [20]. The pattern is not altered by 5 days of bed rest, absence of normal light cycle (blindness), absence of endogenous cortisol production, and during the anti-resorptive treatment [19].

The only known modulator having a major effect on the circadian rhythm of CTX-I is food intake [19, 20, 21, 34, 35] (Fig. 1). Overnight fasting markedly reduced the circadian variation of CTX-I from 36 to 8.7% in postmenopausal women and from 40 to 16% in pre-menopausal women [19, 35]. Therefore, blood samples for CTX-I measurement must be collected in a consistent fashion following an overnight fast during the morning between 7:30 a.m. and 10:00 a.m. A fasting morning collection is judged more manageable compared to early afternoon when less circadian variation is observed.
Fig. 1

Circadium rhythm in serum CTX-I in the fasting and non-fasting state in 15 postmenopausal women. In the non-fasting state, there is a circadian rhythm with the peak around 0500 and the nadir around 1200. This rhythm is significantly attenuated by fasting. Modified figure from Christgau S, Bitsch-Jensen O, Bjarnason NH, Henriksen EG, Qvist P, Alexandersen P, Henriksen DB. Serum Crosslaps for monitoring the response in individuals undergoing antiresorptive therapy. Bone 2000 26:505–11, reprinted with permission from Elsevier

Except for CTX-I, the clinical impact of feeding vs. fasting on BTM is small [21]. PINP is minimally affected by food intake and has minimal circadian rhythm [20, 21, 35]. The intra-individual variations of PINP in postmenopausal and pre-menopausal women are <10% [20, 36, 37]. To our knowledge, the effects of abnormal sleep patterns, stressed conditions, and shift work on the circadian rhythm of PINP and CTX-I have not been thoroughly studied.

Morning fasting sample should be used if both CTX-I and PINP are measured. If only PINP is measured, a random non-fasting sample is acceptable. PINP is often preferred in clinics where patients are seen throughout the day or in patients who may not be able to fast, such as diabetics. However, anti-resorptive drugs induce a more rapid and greater decrease in CTX-I than PINP. Thus, the risk of false negative findings could be higher if only PINP is used. Therefore, CTX-I should be measured, whenever possible.

Serial measurements of non-fasting CTX-I may be of some utility if all samples of a patient have been collected in the same conditions (time of the day, lapse of time after the meal, etc.). However, CTX-I values in non-fasting samples may not be compared with reference ranges or datasets which are determined in fasting early morning samples [19, 34, 38, 39, 40, 41].

Samples destined for CTX-I measurement should be frozen at ≤−20 °C preferably within 2 h of collection [23, 24, 38, 42, 43]. Due to the greater stability of PINP, immediate sample handling is not mandatory. It is critical to mix the thawed sample thoroughly to eliminate the density gradient that develops during the freezing. If measurement is to be made within 8 h of phlebotomy, the sample may be stored at 4 °C. Moderate hemolysis (>0.5 g/dL) should be avoided in CTX-I and PINP measurements, whereas icteria and lipemia are well tolerated in all methods [10, 17, 24, 25, 29, 30, 31, 32]. As each method (instrument and reagent combination) shows sensitivities to interference independent of the pure biochemical considerations, such as physical blocking of the sampling needle in hyperparaproteinemia, assay limitations in the kit package inserts must be observed.

Sample stability

CTX-I is more stable in EDTA plasma than in serum, regardless of measurement methods [23, 24, 26, 27, 28, 38]. The use of EDTA plasma for CTX-I is necessary when there is unavoidable delay in sample processing or unfavorable testing environment [20]. We recommend adopting the more conservative sample stability guidelines such as those stated in the Roche package inserts, i.e., CTX-I stability of 24 h at room temperature (RT, 20–25 °C), 8 days at 2–8 °C in EDTA plasma, and 6 h at RT and 8 h at 4 °C in serum [24]. Greater stability of CTX-I in both serum and plasma has been reported by others as has stability ≥4 h in non-centrifuged whole blood [10, 23, 38, 42].

PINP is more stable compared to CTX-I with stability of at least 24 h at RT and 5 days at 4 °C in both EDTA plasma and serum [25]. Longer sample stability up to 4–7 days at RT has been reported by others [8, 16, 18, 23, 40, 43]. Thermal degradation of the trimeric native structure can occur when samples are exposed to higher temperature such as during transport following collection. PINP has been reported to be unstable at 37 °C and above [16, 18, 44].

For long-term storage, stability of 3 months for CTX-I and 6 months for PINP at −20 °C is ensured for all methods [17, 24, 25, 26, 43]. Stability up to 3 years at −20 and −70 °C has been reported for CTX measured with the Roche method and the ELISA method [26]. At −20 °C, PINP was found to be stable for 24 months in the Roche method but increased by 41.3% after 2.5 years when measured with the IDS iSYS method [16, 17]. It is not clear if the discrepancy is due to method difference or differences in study design. For longer term research studies, the recommendation is to store aliquots of samples at −70 °C or below to allow analysis of all samples in a single batch at a later time.

Freeze/thaw cycle

Multiple freeze-thaw cycles are reported to be acceptable for CTX-I and PINP [10, 16, 17, 38]. Roche recommends “freeze only once” for CTX-I and “not more than 5 times for PINP.” However, IDS and Orion advice “avoid freezing” for their respective products. While repeated freezing and thawing of samples should in general be avoided, there is sufficient published data to support at least two freeze-thaw cycles for CTX and PINP. This information can be helpful especially in clinical research.

Table 1 summarizes recommendations for CTX-I and PINP sample handling which eliminate most of the controllable pre-analytical variability associated with their measurements. We recommend observing more conservative sample stability limits to allow a wider range of sample types and conditions. EDTA plasma is the preferred sample type for CTX-I measurement from the perspective of sample stability [23, 24, 27, 28]. Although plasma is more stable and offers the convenience of immediate sample processing, it is more susceptible to latent fibrin clot formation after frozen storage which may necessitate centrifugation prior to analysis. However, the decision whether to use plasma or serum depends on the specific scenario. If prompt sample processing and temperature-controlled sample transportation cannot be guaranteed, or if immediate access to refrigerated or frozen storage is an issue, the greater analyte stability offered by the use of EDTA plasma would outweigh the inconvenience of possible additional sample centrifugation. CTX-I and PINP are frequently measured together on automated platforms within one sample. Although PINP is a more robust analyte than CTX-I, if both are measured in the same sample, the conditions that ensure CTX-I integrity should be maintained.

Sources of pre-analytical variability—patient-related

Pre-analytical variability comprises controllable factors and uncontrollable factors (Table 2).
Table 2

Determinants of the pre-analytical variability of bone turnover—patient-related

Source of variability

Effect

Recommendation

Impact

Controllable determinants

 Circadian variation

Higher levels after midnight, lowest in early afternoon. CTX-I exhibits significant circadian rhythm, PINP small [19]

Collect samples from 7:30 a.m. to 10:00 a.m. consistently for CTX-I. Take longitudinal samples at same time of day

Very important

 Food intake

CTX-I significantly influenced by food intake (>20% decrease); PINP is minimally affected [19, 21]

For CTX-I, collect blood after an overnight fast before 10:30 a.m. Use fasting sample if both CTX-I and PINP are measured in same sample.

Very important

 Menstrual

PINP higher in luteal phase compared to follicular phase. CTX-I lower in luteal phase [45]

Collect sample during follicular phase in pre-menopausal women if feasible

Moderately important

 Seasonal

Mild effect for CTX-I [46, 47, 48]. Higher BTM in winter due to low 25OHD. More effect in older individuals

Consider vitamin D levels, particularly in longitudinal studies. Consider time of year for repeat collections

Moderately important

 Exercise

Intensive physical training produces moderate increase in CTX-I decrease in PINP [22]. Light exercise no effect [49].

Record details of exercise type and intensity (effect may be age dependent, chronic, and acute effects) [14]. Avoid vigorous exercise on day prior to sampling

Moderately important

 Lifestyle factors

Smoking, alcohol, diet

More data required

Moderately important

Uncontrollable determinants

 Age

High in infancy, decrease in childhood, and then increase during puberty. Lowest in fifth decade in men [50]; fourth in women, increase at menopause

Use appropriate reference range for age group. For children, use local established reference intervals or published data. Adolescent reference intervals should be based on pubertal status [13].

Very important

 Gender

Age-dependent differences

Separate reference ranges should be used for men and women.

Very important

 Menopausal status

BTM increase around the menopause [10]

Appropriate reference intervals for pre- and postmenopausal women should be used.

Very important

 Pregnancy and lactation

Elevated BTM; highest in third trimester, elevated during lactation [51, 52, 53, 54]

Results should be interpreted appropriately for stage of pregnancy or lactation.

Important

 Renal failure

High CTX-I in severe renal failure (GFR < 15 mL/min). Intact PINP normal; total PINP negatively correlated with GFR [55, 56, 57, 58, 59, 60].

Measure serum creatinine and assess GFR

Important

 Day to day variation

Short term and long term

Variability of analyte should be considered.

Important

 Geography and ethnicity

Differences exist for BMD and fracture incidence. Regional differences in BTM levels largely depend on lifestyle [61, 62]

For multicenter trials ethnicity and geographical location should be included in the data analysis.

Important

Conditions characterized by an acceleration of bone turnover

 Primary hyperparathyroidism, thyrotoxicosis, acromegaly, and hypogonadism

Increased BTM [1, 2, 3]

Record diagnosis in the medical record

Important

 Vitamin D deficiency

High BTM in vitamin D deplete individuals (<15 ng/mL) [63, 64, 65]. PINP and CTX-I data limited [20, 64, 66]

Measure 25OHD, consider seasonal variability

Important

 Immobility

Increase bone resorption [67]

Record recent bed rest or immobility and interpret results accordingly

Important

 Fracture

Increased BTM first 4 months, elevated for 12 months [68, 69, 70]

Details of fracture in the previous 12 months should be recorded.

Important

 Paget’s disease

High BTM levels including PINP and CTX-I [71, 72, 73]

Record the diagnosis and account for current activity of the disease

Important

Diseases characterized by a low bone turnover

 Hypothyroidism, hypoparathyroidism, hypopituitarism, growth hormone deficit

Low BTM levels (limited data on PINP/CTX-I) [74, 75, 76]

Record diagnosis in the medical record

Moderately important

Diseases characterized by a dissociation of bone turnover

 Rheumatoid arthritis (RA)

Elevated CTX-I, no increase in PINP in the acute phase [77, 78].

Record diagnosis in the medical record

Moderately important

 Multiple myeloma

Increased CTX-I, PINP—low normal, decreases with progressive disease [79, 80]

Record diagnosis in the medical record

Important

 Cushing’s disease

CTX-I slightly higher, PINP normal [81, 82]

Record diagnosis in the medical record

Moderately important

 Crohn’s disease

Higher CTX-I, PINP—inconsistent [83, 84, 85]

Record diagnosis in the medical record

Moderately important

 Liver disease

CTX-I higher, mainly in the acute phase, PINP—increases mainly in the late stages [6, 9, 86, 87]

Record diagnosis in the medical record

Moderately important

 HIV infection

Low bone formation, high bone resorption in the acute phase [88]

Record diagnosis, medical treatment, coexisting diseases, and adverse lifestyle in the medical record

Important

Patient-related controllable factors include circadian, menstrual and seasonal variabilities, exercise, and food intake. Controlling and standardizing food intake (fasting), time of sample collection, and sample handling procedure will reduce controllable pre-analytical variation, mainly for CTX-I. The impact of food and circadian rhythm on PINP is minor [20, 21].

Menstrual cycle

There is a small effect on BTMs during the menstrual cycle, mainly in the luteal phase [45]. The optimal time to collect samples in pre-menopausal women is the early-mid-follicular phase. Sex steroids are relatively low and BTM levels stable. Given the menstrual variability of bone turnover rate, blood collection in the early-mid-follicular phase is preferable in order to obtain comparable results.

Seasonal variation

There is a detectable, although minor, seasonal variation for CTX-I, mainly in older adults and those with severe vitamin D deficit [46, 47, 48, 89]. This is important for longitudinal research studies or repeat clinic visits. By contrast, a seasonal variation is not apparent for PINP.

Physical activities

Intensive physical training (e.g., elite soccer players) moderately increases serum CTX-I and slightly decreases PINP [22]. Light physical exercise has no significant effect on serum PINP or CTX-I levels [49]. Inconsistent data in the literature may be related to the age, type of physical exercise, and its chronic and acute effect [13]. Vigorous exercise should be avoided the day prior to sampling.

Age/sex

BTM levels are very high in newborns and infants, then decrease until puberty [90]. BTM levels are higher during early puberty (Tanner I to III), and then decrease earlier in girls than in boys [91, 92, 93]. In young adults, BTM levels are higher in men than women, then decrease (earlier in women) and achieve their lowest levels in the fourth decade in women and in the fifth decade in men [50]. Serum PINP and CTX-I increase at the menopause (greater change in CTX-I than in PINP) and then remain higher than before the menopause [2, 15]. By contrast, in older men, serum CTX-I and PINP levels remain stable or increase only slightly, generally after 70 years of age [94].

Pregnancy

During pregnancy, bone resorption markers increase progressively until parturition, whereas bone formation markers are low-normal during the first two trimeters and rapidly increase in the third trimester [51, 52, 53, 54]. After delivery, BTM levels decrease quickly but remain elevated during the postpartum period compared to non-pregnant age-matched women and are greater in lactating women vs. non-lactating women [66, 95].

Geography/locale

Geographic differences in BTM may be significant, although moderate, and largely depend on geographical latitude as well as ethnic and cultural differences in subject nutrition, lifestyle, and clothing [20, 61, 62]. Therefore, each region should establish its own reference interval.

Sources of variability, clinical implications of diseases, and medications

The above factors should be considered in the goal of attaining consistent, reproducible, and reliable BTM results. In addition, the disease and the medication administered may markedly influence the consistency of BTM levels detected (Table 2).

Accelerated bone turnover

BTMs are increased in metabolic bone diseases characterized by higher bone turnover such as primary hyperparathyroidism, acromegaly, and thyrotoxicosis [1, 2, 4].

Metastatic bone disease

Patients with bone metastases have high levels of all BTMs, including PINP and CTX-I [96, 97, 98]. High BTM levels are associated with high number of metastases, high risk of skeletal-related events (e.g., pathological fracture), and death. Bone metastases are characterized by rapid bone turnover in localized skeletal sites. However, serum β-CTX-I does not increase by as much as might be expected, probably because immature forms of CTX-I (i.e., alpha-CTX-I) are released and not detected by the β-specific immunoassays used [99].

Vitamin D deficiency

Vitamin D deficit is found in the elderly (due to lower metabolic capacity of the skin) and in case of low sunlight exposure (winter, home-bound patients, veil for religious reasons). Vitamin D depletion results in secondary hyperparathyoidism, mainly in people with low calcium intake. Elevated levels of parathyroid hormone (PTH) and BTMs are found mainly in the subjects with 25-hydroxycholecalciferol (25OHD) levels below 15 ng/mL [63, 64, 65, 100].

Seasonal variability of 25OHD levels (lowest values in winter) is mirrored by higher PTH and BTM levels in winter in older, but not younger, individuals (because older subjects have lower 25OHD levels) [48, 89, 101, 102, 103]. Very high PTH and BTM levels are found in the home-bound or institutionalized elderly [104]. However, this group has low physical activity and suffers from nutritional deficits [67, 105]. Thus, it is not possible to aportion the contribution of various components to the elevated BTM levels. These associations were found for various BTM including PINP and CTX-I; however, data are limited [48, 102, 106].

Bone fracture

Serum PINP and CTX-I levels increase steeply during the first weeks following bone fracture (up to 150%) [68, 69, 70]. CTX reaches a peak at 4 weeks after fracture and PINP a peak after 12 weeks; they then both decrease, but may remain elevated for more than 1 year postfracture. Fractures with larger surface of fracture and consolidation (e.g., pertrochanteric fracture) are associated with higher BTM levels.

Renal function

CTX-I and PINP monomers are partly degraded in the kidney or filtered in glomeruli. Therefore, severely impaired renal function is associated with higher CTX-I levels, due to their lower glomerular filtration and accumulation of their degradation products [55, 56]. Native trimeric intact PINP is not higher even in patients with severe renal failure (GFR < 15 mL/min) [56, 57, 58]. By contrast, “total” PINP (trimer and monomers) is negatively correlated with GFR and increased in patients with renal failure [57]. Serum CTX-I is markedly higher in patients with severe renal failure (GFR <15 mL/min) and in patients on hemodialysis [58, 59, 60]. In secondary hyperparathyroidism related to CKD, BTM levels are increased. Intact PINP and bone alkaline phosphatase are recommended for bone formation, whereas tartrate-resistant acid phosphatase 5b is recommended for bone resorption assessment [57]. Adynamic bone disease related to CKD is characterized by low BTM levels; however, specific data for PINP and CTX-I are missing. Other factors also affect BTM levels in patients with CKD, e.g., vitamin D status, state before or after dialysis, underlying or coexisting disease (diabetes mellitus, hypogonadism, myeloma), or treatment (mainly corticotherapy).

Paget’s disease

Active Paget’s disease is characterized by highly increased levels of bone resorption and bone formation markers, including PINP and CTX-I [71, 72, 73]. BTM levels are correlated with the metabolic activity of Pagets’ disease, e.g., BTMs are higher in the polyostotic vs. monoostotic disease. In addition, the release of immature α-CTX-I form and the α-CTX-I to β-CTX-I ratio are markedly increased in the active disease and decrease during remission [107].

Critically ill patients

Several studies show that critical illness requiring intensive care admission is associated with higher BTM levels reflecting accelerated bone turnover [108]. In particular, one study shows increased serum concentrations of PINP and CTX-I in surgical intensive care patients [109].

Low bone turnover

Bone formation and resorption are low normal or mildly decreased in diseases characterized by low bone turnover (hypoparathyroidism, hypothyroidism, hypopituitarism) [74, 75, 76]. However, data on PINP and serum CTX-I in these diseases are scant.

Dissociation of bone turnover

Diseases characterized by a dissociation of bone turnover include rheumatoid arthritis (RA), multiple myeloma and Cushing’s disease. The early acute phase of RA is characterized by elevated CTX-I, without parallel increase in bone formation [77, 78]. Greater clinical severity is associated with higher BTM levels, but in later stages of RA, BTM are close to normal reference values [100]. In multiple myeloma serums, CTX-I tends to increase, mainly in the progressive disease [79] while PINP is low-normal in the stable disease and decreases in the progressive disease [80]. In patients with Cushing’s disease, CTX-I is normal or increased, whereas PINP is normal [74, 76, 81, 82].

In patients with Crohn’s disease, bone resorption (including serum CTX-I) is increased [83, 84, 85]. Data on bone formation (including PINP) are inconsistent. Individual BTM levels depend on the disease activity and treatment.

Chronic liver diseases (e.g., alcoholic liver cirrhosis, primary biliary cirrhosis, cirrhosis related to viral hepatitis, hemochromatosis) may be associated with higher bone resorption (higher serum CTX-I) [86, 110, 111]. In later stages, impaired function of hepatocytes (which degrades PINP) and progressing liver fibrosis (characterized by type I collagen synthesis) result in higher PINP levels which are not related to abnormal bone turnover [6, 9, 86, 87]. Overall, CTX-I and PINP levels depend on the degree of the metabolic damage of the liver and on the underlying liver disease. Therefore, BTM levels in this group should be assessed cautiously.

HIV infection is associated with lower bone formation and increased bone resorption [88]. However, data are insufficient to provide useful guidelines as BTMs in this patient group are influenced by multiple factors such as treatment, coexisting diseases, and adverse lifestyle.

Effect of the anti-osteoporotic medications

Anti-resorptive drugs inhibit bone resorption (Table 3). The decrease in bone resorption markers is significant after 2–3 months during treatment with oral bisphosphonates, selective estrogen receptor modulators (SERMs), or 17β-estradiol, <1 week after administration of iv bisphosphonates (ibandronate, zoledronate), monthly ibandronate (150 mg po) or denosumab (sc), and <24 h after oral administration of cathepsin K inhibitors [116, 119, 124, 125, 163]. CTX-I may be used for monitoring the anti-resorptive therapy: after 3 months for oral bisphosphonates and after 1 month in case of iv bisphosphonates or denosumab. PINP may be used after 6 months in case of oral bisphosphonates, after 3 months in case of denosumab or iv bisphosphonates.
Table 3

Effect of medications on the serum levels of PINP and CTX-I

Medication

Effect

Anti-osteoporotic drugs

PINP

CTX-I

Inhibitors of bone resorption

Lower levels of bone resorption and formation markers

 17β-estradiol [112, 113]

−30%

−40 to −50%

 SERMs [114, 115]

−30 to −40%

−30 to −40%

 Alendronate [116, 117]

−50 to −60%

−70%

 Risedronate [118]

−50 to −60%

−40 to −50%

 Ibandronate [117, 119, 120, 121]

−50 to −60%

−50 to −70%

 Zoledronate [122, 123]

−50 to −60%

−75

 Denosumab [124]

−60 to −70%

−70 to −85%

 Cathepsin K inhibitors [125, 126]

−40

−60 to −70%

Stimulators of bone formation

Higher levels of bone resorption and formation markers

Teriparatide [127, 128]

+150%

+100%

Vitamin D and calcium [129, 130, 131, 132, 133]

Moderate, dose-dependent decrease in CTX-I and PINP. Effect varies with vitamin D status and nutritional calcium intake at baseline.

Other medications

 Corticosteroids (oral and parenteral) [134, 135, 136, 137]

Decrease PINP (2 days). CTX-I—data less consistent. Effect varies with dose, duration of treatment, and underlying disease.

 Inhaled corticosteroids [138]

No change in serum PINP and CTX-I levels (decreased serum osteocalcin concentration)

 Disease-modifying anti-rheumatic drugs (DMARDs) [139, 140, 141]

Decreases CTX-I (15–20%), no change in PINP

 Hormonal contraceptives [142, 143, 144]

Oral contraceptives—lower PINP and CTX-I (20–25%). Medroxyprogesterone acetate—higher PINP.

 Aromatase inhibitors [145, 146, 147]

Increase PINP and CTX-I 10–35%

 Anti-epileptic drugs (AED) [148, 149, 150, 151, 152]

CTX-I PINP slightly increased with some AED—discordant results obtained in small groups.

 Thiazolidinediones (TZDs) [153, 154, 155, 156, 157, 158]

Decrease PINP in some, but not all studies. Discordant results for CTX-I

 Thiazide diuretics [159, 160, 161]

Lower PINP and CTX-I (10–20%); the impact of the underlying disease to be taken into account

 Vitamin K antagonist [162]

No effect on PINP or CTX-I [116]

The decrease in bone resorption varies according to the drug and the dose. At the therapeutic dose, serum CTX-I decreases by 30–40% during the treatment with SERMs, 40–50% with risedronate or 17β-estradiol, 50–70% with oral and iv ibandronate, 60–70% with cathepsin K inhibitors, 70% with alendronate, 75% with zoledronate, and 70–85% with denosumab [112, 114, 116, 118, 120, 121, 122, 124, 126, 164]. At the therapeutic dose, serum PINP decreases by 30–40% during the treatment with SERMs, 30% with 17β-estradiol, 40% with cathepsin K inhibitors, 50–60% with bisphosphonates, and 60–70% with denosumab [113, 114, 115, 116, 117, 122, 123, 124, 126, 164].

Bone formation stimulating therapy (e.g., teriparatide) increases BTM levels. PINP levels increases during the first days of tratment and attains highest levels (∼150% above the initial level) after 1 to 3 months [127, 128]. Bone resorption markers (serum CTX-I) increase with a delay, more slowly and attain their highest levels (∼100% above the initial level) after 6 to 12 months. Afterward, PINP and CTX-I levels remain stable or slightly decrease, but remain elevated compared to the initial levels. PINP may be used for monitoring the anabolic therapy after 3 months.

Vitamin D and calcium supplementation decreased CTX-I and PINP levels moderately, dose-, and time-dependently [129, 130, 131, 132, 133]. The decrease was greater in women with vitamin D insufficiency, those with low nutritional calcium intake, and those living in the northern regions with low sunlight exposure.

Effect of other medications on BTM

Corticosteroids inhibit bone formation promptly and dose-dependently (Table 3). As they downregulate the osteocalcin expression, its level decreases rapidly [134]. In patients receiving oral or parenteral corticosteroids, the fall in osteocalcin is followed by a decrease in PINP [135, 136, 137]. Changes in bone resorption (also CTX-I) during long-term corticosteroid therapy are less consistent and depend on the schedule [135, 136, 137]. Inhaled corticosteroids do not influence the serum levels of PINP and CTX-I [138].

The effect of corticosteroids on BTMs depends on the dose, duration, and underlying disease. In the diseases without direct impact on bone (bronchial asthma, idiopathic thrombocytopenic purpura), changes in the BTMs reflect the effect of corticosteroids on bone. Conversely, inflammatory diseases (rheumatoid arthritis) affect bone themselves. In these patients, high bone resorption and suppressed bone formation at baseline and their treatment-induced normalization may confound the effect of corticosteroids on BTMs.

Rheumatoid arthritis (RA) is characterized by high levels of inflammatory cytokines, e.g., interleukin-1 (IL-1), IL-6, IL-17, or tumor necrosis factor alpha (TNFα) [165], which stimulate joint destruction and bone resorption. Thus, medications inhibiting inflammation in RA reduce bone resorption. Infliximab (anti-TNFa antibody) and tocilizumab (IL-6 receptor antagonist) decreased bone resorption (including CTX-I) by 15–20% [139, 140, 141]. By contrast, bone formation markers, including PINP, remained stable or presented mild fluctuations.

Women using oral contraceptives (containing estrogens) had 15–25% lower BTM levels (also PINP and CTX-I) vs. women who did not [142, 143]. By contrast, medroxyprogesterone acetate slightly stimulated bone turnover rate (e.g., increased PINP levels) [143, 144].

Aromatase inhibitors are used in the treatment of breast cancer. They reduce estrogen formation, further accelerating bone turnover (higher PINP and CTX-I levels) [145, 146, 147]. In treatment naive postmenopausal women with breast cancer, aromatase inhibitors increase serum PINP and CTX-I levels by 10–35% compared with baseline.

Anti-epileptic drugs (AED) modify bone turnover. Phenytoin, phenobarbital, carbamazepine, oxcarbazepine, or primidone stimulates the activity of hepatic cytochrome P450 hydroxylase increasing the synthesis of inactive 24-hydroxy-vitamin D metabolites [166]. AED may increase BTM levels [153, 154, 155]. However, data are limited. Serum CTX-I level increased in patients treated with topiramate or phenobarbital, but not in those treated with valproic acid, carbamazepine, or oxcarbazepine [152, 154, 155, 156]. PINP level was elevated in carbamazepine-treated patients [153]. However, these data are inconsistent; the study groups were small and composed of young patients. Treatment duration varied substantially.

Thiazolidinediones (TZDs) are agonists of the perioxisome proliferator-activated receptor (PPAR) [157]. TZD-induced activation of PPAR promotes differentiation of mesenchymal cells into adipocytes which decreases osteoblast number and activity. In most [158, 159, 160], not all [161, 162], studies, TZDs decreased PINP. Data on bone resorption are discordant. In patients receiving TZDs for >3 months, serum CTX-I increased [161, 162], decreased [163], or remained stable [158, 159, 160].

Thiazides transiently decrease BTM levels by 10–20% [164, 165]. In hypertensive women treated with thiazides, PINP and CTX-I levels were lower vs. normotensive controls and slightly, non-significantly, lower vs. hypertensive women treated with other drugs [166].

Vitamin K antagonists (oral anti-coagulants) inhibit γ-carboxylation of OC, but have no effect on PINP and CTX-I [167].

Discussion

In order to control and limit the impact of pre-analytical variability, several key aspects of patient and sample management must be considered and adhered to. The aspects considered “controllable” may be readily influenced by the clinician, phlebotomist and/or laboratorian.

Blood samples for CTX-I assay should be collected in a fasting state in the morning. Since serum PINP level does not display marked circadian variability nor is it affected by food, it is not critical for blood collection to take place in the fasting state nor in the morning.

The decision as to which biomarker should be measured—or possibly both—depends upon local practice and the clinical goal of BTM measurement in that patient. Patient medication status and whether other BTM-modifying processes are present in the patient should be considered. Certain diseases and therapies may have marked and specific effects upon serum BTM levels which may determine the choice of the marker. For instance, in patients with chronic liver disease with advanced fibrosis, CTX-I may be more informative than PINP, whereas in renal failure, intact but not total PINP is more useful than CTX-I.

Aside from sample collection time and fasting status, the correct processing of samples is necessary to ensure analyte integrity. EDTA plasma should be used for CTX-I measurements where adherence to sample handling conditions may be difficult e.g., when samples are sent to a remote laboratory. In the discussions of sample stability, the stability time intervals refer to the total time from blood collection to the time of measurement. This includes the combined resident time at RT, 4, −20, and −70 °C. Where CTX-I and PINP are measured within the same sample, the conditions that ensure the integrity of CTX-I, the more labile and variable analyte, should be adhered to. The availability of suitable sample affects the decision of which BTM to measure.

In routine practice, sample handling conditions provided by the reagent manufacturers should be followed. If a discrepancy exists between the recommendations provided here and those of the assay kit manufacturers, the user may follow the guidance that better support their specific testing environment supplemented by literature and data generated within the institution or country to satisfy local regulatory surveillance.

Aside from the diligent attention to limit controllable pre-analytical and analytical variability, uncontrollable factors including disease and treatments must be carefully considered in BTM interpretation. Some factors have a strong effect on BTM levels, and the information on these factors should be obtained in patients who have BTM measurements. They include recent fracture, chronic immobilization, renal failure, multiple myeloma, glucocorticoid therapy, and anti-osteoporotic treatment. Interpretation of BTM results should account for the medication, dose, duration, and adherence to treatment. Other factors have milder effect on BTM levels, including alcohol intake, smoking, liver diseases, AED, and TZDs. Their impact is weaker except for subjects with high intensity of a factor, e.g., in heavy smokers.

However, the successful adoption of standardized sample handling and patient preparation recommendations will depend on the close collaboration of various stakeholders at the global stage, including the reagent manufacturers, the laboratories, the medical community, and the regulatory agencies [168]. Reagent kit manufacturers and regulatory authorities could trigger a substantial improvement in comparability of results obtained from differing manufacturer kits, in conjunction with the development and use of international reference preparations for CTX-I and PINP to provide true assay equivalence.

Notes

Acknowledgements

The NBHA would like to acknowledge the efforts of the IFCC and IOF in BTM standardization. We fully support further joint initiatives toward achieving this common goal and believe that, ideally, the relevant commercial parties should be involved as well.

Compliance with ethical standards

R.E. received grants and consultancy payments from both Roche Diagnostics and Immunodiagnostic Systems. NRH was previously an employee of Roche and received routine remunerations.

Conflict of interest

None.

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Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2017

Authors and Affiliations

  1. 1.INSERM UMR 1033, Hôpital Edouard HerriotUniversity of LyonLyonFrance
  2. 2.Academic Unit of Bone Metabolism and Mellanby Centre for Bone ResearchUniversity of SheffieldSheffieldUK
  3. 3.MurnauGermany
  4. 4.ETL ConsultingSeattleUSA
  5. 5.Pacific BiomarkersSeattleUSA

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