The EUROFORS study is funded by Eli Lilly & Company, Europe. Drs Nickelsen, Marin, and Barker are full-time employees of Eli Lilly & Company. Dr Glüer is a consultant for and receives research funding from Eli Lilly & Company. All other authors state that they have no conflicts of interest.
Monitoring Teriparatide-Associated Changes in Vertebral Microstructure by High-Resolution CT In Vivo: Results From the EUROFORS Study†
Article first published online: 4 JUN 2007
Copyright © 2007 ASBMR
Journal of Bone and Mineral Research
Volume 22, Issue 9, pages 1426–1433, September 2007
How to Cite
Graeff, C., Timm, W., Nickelsen, T. N., Farrerons, J., Marín, F., Barker, C. and Glüer, C. C. (2007), Monitoring Teriparatide-Associated Changes in Vertebral Microstructure by High-Resolution CT In Vivo: Results From the EUROFORS Study. J Bone Miner Res, 22: 1426–1433. doi: 10.1359/jbmr.070603
- Issue published online: 4 DEC 2009
- Article first published online: 4 JUN 2007
- Manuscript Accepted: 30 MAY 2007
- Manuscript Revised: 22 MAY 2007
- Manuscript Received: 2 NOV 2006
- bone microstructure;
We introduce a method for microstructural analysis of vertebral trabecular bone in vivo based on HRCT. When applied to monitor teriparatide treatment, changes in structural variables exceeded and were partially independent of changes in volumetric BMD.
Introduction: Monitoring of osteoporosis therapy based solely on bone densitometry is insufficient to assess anti-fracture efficacy. Assessing bone microstructure in vivo is therefore of importance. We studied whether it is possible to monitor effects of teriparatide on vertebral trabecular microstructure independent of BMD by high-resolution CT (HRCT).
Materials and Methods: In a subset of 65 postmenopausal women with established osteoporosis who participated in the EUROFORS study, HRCT scans of T12, quantitative CT of L1–L3, and DXA of L1–L4 were performed after 0, 6, and 12 mo of teriparatide treatment (20 μg/d). We compared BMD and 3D microstructural variables in three groups of women, based on prior antiresorptive treatment: treatment-naïve; pretreated; and pretreated women showing inadequate response to treatment.
Results: We found statistically highly significant increases in most microstructural variables and BMD 6 mo after starting teriparatide. After 12 mo, apparent bone volume fraction (app. BV/TV) increased by 30.6 ± 4.4% (SE), and apparent trabecular number (app. Tb.N.) increased by 19.0 ± 3.2% compared with 6.4 ± 0.7% for areal and 19.3 ± 2.6% for volumetric BMD. The structural changes were partially independent of BMD as shown by a significantly larger standardized increase and a standardized long-term precision at least as good as DXA. Patients who had shown inadequate response to prior osteoporosis treatment did show improvements in BMD and structural measures comparable to treatment-naïve patients.
Conclusions: HRCT is a feasible method for longitudinal microstructural analysis of human vertebrae in vivo, offers information beyond BMD, and is sufficiently precise to show profound effects of teriparatide after 12 mo.
At present, the diagnostic assessment of bone status in clinical routine is limited to measurements of BMD, either with areal DXA or 3D quantitative CT (QCT), radiographic fracture assessment, some macroarchitectural bone geometry measures, and biochemical markers of bone turnover.
A majority of osteoporotic fractures occur in patients who are not osteoporotic according to the BMD criterion of the WHO.(1) Several studies have shown the shortcomings of BMD in monitoring the complex response of bone to drug treatment, especially when measured by DXA, because this method is not appropriate to fully explain the changes in material and structural properties of bone. Therefore, the treatment response of BMD does not adequately explain the observed fracture reduction, particularly when using antiresorptive agents.(2,3)
More complete information is expected by including aspects of bone quality, which comprises measures of bone architecture and bone material properties.(4) Biochemical bone markers,(5) whole bone geometry,(6,7) and the microstructure of trabecular bone(8) represent additional aspects of bone quality because they affect bone strength independent of BMD.
The assessment of the microstructure of the trabecular network in humans is currently limited to biopsies, the use of high-resolution MRI, or dedicated (pQCT) imaging systems for measurement in peripheral regions of the skeleton such as forearm or tibia. Bone biopsies can be analyzed at very high resolution but are invasive, cannot be repeated at the same location, represent a site not susceptible to fracture risk and suffer from limited sampling volumes. As can be expected because of the different loading conditions, structural measures from biopsies and peripheral sites show limited correlations with the clinically important fracture sites proximal femur and vertebrae.(9,10)
Recent advances in CT allow high-resolution imaging at central fracture sites such as the spine, with improved spatial resolution and faster scan acquisition because of multidetector technology.(11) Pilot studies have shown promising performance in discrimination of subjects with and without recent vertebral fractures.(12)
The aim of this work is to show (1) the feasibility of in vivo microstructural analysis of vertebral trabecular bone by HRCT; (2) the results of this analysis are partially independent of BMD; and (3) HRCT is suited to monitor structural and densitometric changes induced by teriparatide treatment over 1 yr in a multicenter study setting.
MATERIALS AND METHODS
Study design and participants
EUROFORS is a pan-European, multicenter, prospective, controlled, randomized, open-label clinical trial of 2-yr duration designed to compare three different sequential treatments involving teriparatide [rhPTH(1-34)]. In the first year of the study, all participants received subcutaneous injections of teriparatide (20 μg/d) in combination with supplements of calcium (500 mg/d) and vitamin D (400–800 IU/d).
Women ≥55 yr of age, with a T-score of ≤ −2.5 at the lumbar spine, total hip, or femoral neck, who had at least one known and documented preexisting fragility fracture in the last 3 yr were eligible for enrollment. Women with a known history of metabolic bone disease other than postmenopausal osteoporosis, elevated serum levels of calcium, alkaline phosphatase, or PTH, significantly impaired renal or hepatic function, or pretreatment with fluorides in the past 12 mo or with any other bone anabolic agent in the past 6 mo, were excluded. Prior treatment with antiresorptives (AR) had to be discontinued at baseline. Careful and detailed documentation of the prior AR therapy and the patient's clinical response to it was needed. Based on these data, patients were grouped into three subsets: (1) treatment-naïve patients without any previous anti-osteoporosis therapy; (2) AR-pretreated patients; (3) AR-pretreated patients with documented inadequate clinical outcome. Here, this was defined as any of the following: (1) sustaining at least one new clinical fragility fracture(s) despite AR therapy during at least the 12 mo before fracture; (2) a T-score ≤ −3.0, or (3) a BMD decrease of ≥3.5% at the lumbar spine, total hip, or femoral neck after 2 yr of AR therapy.
The EUROFORS Study was performed in 10 countries at 95 investigative centers and enrolled 865 evaluable subjects. Its primary endpoint is the change in lumbar BMD after 2 yr of treatment. Here we present results from the first year in the subset of 12 investigative centers from Germany and Spain participating in the HRCT study addendum.
Institutional Review Board approval was obtained from each of the clinical study sites, and written informed consents for the general study and the HRCT substudy were obtained from each participant.
Areal BMD was obtained from DXA scans of lumbar vertebrae 1–4 (aBMD L1–L4). Volumetric BMD of lumbar vertebrae 1–3 (vBMD L1–L3) was assessed by QCT (spiral CT, 3-mm slice thickness) using a solid calibration phantom and the software package QCT-Pro (Mindways, San Francisco, CA, USA). Elliptical volumes of interest (VOI) of up to 9 mm thickness—depending on the vertebral height—were placed manually. The LOCF method was applied to every vertebra individually to avoid discontinuities in the average calculation.
HRCT scan protocol
HRCT examinations at 120 kV and 360 mAs (estimated dose-equivalent 3 mSv) were obtained of the T12 vertebra or of L1, if T12 was fractured at baseline. CT scanner types included the Siemens Somatom 16 and Volume Zoom, the General Electric (GE) Lightspeed 16, and the Toshiba Asteion. Images were reconstructed with a voxel size in-plane of 187 (GE Lightspeed) or 156 μm (all other CT types). Slice thickness ranged from 300 to 500 μm (pitch 0.5). The different reconstruction kernels implemented on the CT scanner models caused additional variability.
HRCT image processing
All image processing was performed using the software “StructuralInsight” developed in-house. The elements of QCT phantom were extracted from a larger, low-resolution reconstruction of the HRCT scan with identical parameters except resolution (Fig. 1A). The high-resolution reconstruction (Fig. 1B) was calibrated to mineral scale. The complete trabecular bone volume was manually segmented excluding hyperdense degenerative calcifications and the thin cortical rim of the vertebral body. The segmentation was based on polygons manually defined by the user in multiple, nonadjacent slices (typically 4–8, time required: ∼5 min per case). These polygons were interpolated linearly to cover all reconstructed slices (typically 50–80). A set of subvolumes used for analysis of regional variability of the microstructural measures was calculated automatically consisting of the eight quadrants of the natural coordinate system of the segmented VOI.
Gray values were binarized to bone and marrow using a uniform threshold of 250 mg/ml for all patients. The threshold value was chosen according to three criteria evaluated in a subset of five patients for a range of thresholds: a continuous cortical shell, physiologically reasonable BV/TV values, and good separation of bone structure versus pseudostructure in soft tissue.
A 3D adaptation of the parallel plate model(13) and the mean intercept length method were used to calculate standard structural variables. A prefix “app.” for “apparent” was added to standard nomenclature of structural variables(14) to indicate the influence of limited resolution and signal-to-noise ratio. Variables studied included bone volume fraction (app. BV/TV), trabecular number (app. Tb.N), separation (app. Tb.Sp), thickness (app. Tb.Th), and degree of anisotropy (MIL DA). We validated the mathematical correctness of our implementation against a virtual phantom and tested the method ex vivo.(15)
Assessment of structural information
The long-term precision error in vivo was estimated from the SE of the estimate of linear regression over time, calculated for each patient and pooled as root mean square averages.(16) To adjust for differences in the variables' response rates, standardized precision errors were normalized to the ratio of mean absolute increases of each variable and DXA as the reference technique.(17) For this test, only patients with valid results in all three visits were included.
A multivariate regression model was calculated to study how much of the change in the microstructural variables could be explained by BMD. As a second test for independent information, we compared standardized changes of structural and densitometric variables scaled to SDs of baseline variability (commonly used percent changes were also calculated but these could be biased, e.g., for differently scaled variables).
Whether the independent information established by these two methods contained meaningful structural information was assessed by two additional tests. First, we studied “temporal consistency” of the structural information. For this, we studied whether each variable at baseline would be the best predictor of that same variable at a return visit, better than any other variable or BMD. For this purpose, partial correlations and multifactorial regression analysis of all directly measured structural variables (app. BV/TV, app. Tb.N, and app. MIL DA) and vBMDtot at baseline versus each of these variables at a follow-up visit were calculated. As a second test, we analyzed the “spatial consistency” of structural inhomogeneity across subject visits. For this test, each variable was computed in the eight quadrant subvolumes at each visit. All subvolumes at baseline were correlated to one of the subvolumes at a later visit to study if structural variables contain localized information preserved in the subsequent scans.
Only participants with a valid baseline and at least one valid follow-up HRCT measure after 6 or 12 mo were included in the analysis. If the 12-mo measure was missing the last observation was carried forward (LOCF).
All statistical analyses were performed with JMP 5.0.1 (SAS Institute, Cary, NC, USA). Significance of the treatment response was assessed by a two-sided t-test, increases in different variables by a paired t-test, and group differences by the Tukey-Kramer HSD test.
For the assessment of the response to treatment, outliers were excluded according to Grubbs' test(18) (i.e., an outlier deviates from the group mean by >3.2 SD; threshold for n = 60) of all cases, including the potential outliers.
A total of 119 participants were enrolled into the HRCT protocol. Sixty-seven patients fulfilled the above stated inclusion criteria. Reasons for exclusion were no follow-up visit (11 patients), scan protocol violations: inconsistent reconstruction kernel (31 patients), and fractures of the measured vertebra (10 patients). One of the fractures occurred between baseline and 6-mo follow-up, the others before study start.
For the assessment of the treatment effect, two more patients were excluded as statistical outliers fulfilling Grubb's criterion in their changes in structural variables. For a consistent comparison of BMD and structural data the two cases were also excluded from BMD analyses of treatment effects.
For the remaining 65 patients, clinical characteristics are shown in Table 1. After 6 mo, valid results were available for 64 participants (1 missing), and after 12 mo for 56 participants (9 missing but results carried forward from the 6-mo visit).
In single slices, trabecular elements were hard to distinguish from noise (Fig. 1B), particularly in severely osteoporotic subjects. Only larger trabeculae, especially those oriented perpendicular to the slices were visible without further image processing. Binarization with the selected threshold of 250 mg/ml eliminated most, but not all, noise structures outside the vertebral body. We assume a similar performance inside the vertebral body.
Figure 2 shows the image quality after calibration and segmentation in a time series of volume renderings for a single individual. For this, rectangular “virtual biopsies” were extracted from the volume at manually matched regions for each of the three visits. Two perpendicular views of each biopsy are displayed. The treatment effect was clearly visible. Certain structural elements as well as the general orientation and coarse distribution of the trabeculae could be consistently identified in each subsequent image. This shows the ability of HRCT to consistently depict larger trabecular structures over 1 yr.
Response to teriparatide treatment
Table 1 lists baseline values and changes after 6 and 12 mo of treatment. At baseline, no significant differences between pretreatment groups in age, BMD, and structural variables values were observed. The median of the lag time between AR treatment and onset of teriparatide differed between pretreated patients with inadequate response (1 mo) and pretreated patients (11 mo). All structural variables except for app. MIL DA showed a highly significant change from baseline in all patients (p < 0.001) after 6 and 12 mo.
Comparison of pretreatment groups: Increases from baseline for each pretreatment group were significant for all variables except for app. MIL DA (Table 1), but reached significance in the naïve group only after 12 mo of treatment in some variables (vBMD T12, app. Tb.N). DXA changes were larger (not significant) in the naïve group compared with the other groups. Volumetric BMD of L1–L3 and T12 showed similar results across all pretreatment groups. The structural variables showed larger (not significant) increases in the pretreated patients with an inadequate response.
Long-term precision: Long-term precision errors were 2.5% for aBMD L1–L4, 6.0% for vBMD L1–L3, 7.6% for vBMD T12, 9.5% for app. BV/TV, and 8.9% for app. Tb.N. Standardized to DXA response rates, precision errors were 2.5% for aBMD L1–L4, 2.9% for vBMD L1–L3, 2.6% for vBMD T12, 2.1% for app. BV/TV, and 3.1% for app. Tb.N. The monitoring time interval (MTI)(17) was 0.9 yr for app. BV/TV, 1.1 yr for aBMD L1–L4 and vBMD T12, 1.3 yr for vBMD L1–L3, and 1.4 yr for app. Tb.N.
Assessment of structural independence
Multivariate regression: The model used increases of vBMD T12 and aBMD L1–L4 to explain increases in either app. BV/TV or app. Tb.N. After 6 and 12 mo, volumetric BMD explained 60% and 68% of increases in app. BV/TV and 38% and 45% in app. Tb.N (all p < 0.0001); adding aBMD did not improve this model and, as a single determinant, aBMD explained only 5–20% of these structural variables.
Comparison of increases in standardized units: Standardized changes in structural variables were 0.39 ± 0.44 and 0.64 ± 0.68 (SD) in app. BV/TV and 0.30 ± 0.38 and 0.43 ± 0.53 in app. Tb.N after 6 and 12 mo, respectively. Densitometric measures increased by 0.24 ± 0.35 and 0.43 ± 0.47 in vBMD T12, 0.25 ± 0.39 and 0.40 ± 0.48 in vBMD L1–L3, and 0.28 ± 0.30 and 0.43 ± 0.37 in aBMD L1–L4. Standardized increases in app. BV/TV but not in app. Tb.N were significantly larger than increases in any of the BMD measures after 6 (p < 0.05) and 12 mo (p < 0.01; Fig. 3). Standardized increases in vBMD T12, vBMD L1–L3, and aBMD L1–L4 did not differ significantly.
Temporal consistency: Correlations of each variable at baseline with the same variable after 6 and 12 mo, and between months 6 and 12 showed a median of r2 = 0.86 (range: 0.83–0.86), r2 = 0.74 (range: 0.62–0.85), and r2 = 0.85 (range: 0.79–0.86), respectively. Partial correlation coefficients of a given variable at 6 or 12 mo with the same variable at baseline were always larger than with any other variable. Results are depicted in Fig. 4A. In multifactorial regression analyses, each structural variable after 6 and 12 mo of treatment was best predicted by its own baseline value at a significance level of p < 0.001. No other variable was a significant contributor except vBMD T12 for app. MIL DA (p < 0.05). Thus, structural variables contained information independent from each other and from BMD.
Spatial consistency: The average correlations between each of the independently measured variables in all eight subregions and every subvolume after 6 and 12 mo are shown in Fig. 4B. In 23 of 24 calculated correlations after 6 mo and in 17 of 24 cases after 12 mo, the subvolume was best predicted by its own baseline value. In 38 of the 48 multifactorial regression analyses that used all eight subvolumes at baseline to predict each subvolume after 6 and 12 mo, the identical subvolume was identified as the best contributor. The structural analysis therefore gave information specific to the subvolume within the vertebrae despite the treatment effect, limited image quality and manual subvolume matching.
To the best of our knowledge, this is the first publication showing the feasibility of HRCT-based structural analysis of the vertebral trabecular bone in vivo as a tool for monitoring osteoporosis treatment. Treatment with teriparatide was associated with significant improvements of structural variables and BMD. The increases observed in our study after 12 mo of treatment were in the order of 6% for aBMD, 20% for vBMD, and up to 30% for structural measures. The BMD increases were in agreement with earlier teriparatide studies that reported annual changes of 6–7% for aBMD(19,20) and ∼13% for vBMD.(20) The striking improvements of structural variables were consistent with the visual impression obtained from images of the vertebral microstructure in individual patients. Structural changes were partially independent of BMD.
Long-term precision expressed in a nonstandardized fashion could be misleading because higher response rates of the measures are not taken into account. Long-term precision errors as calculated here not only include technical imprecision but also true intraindividual nonlinear responses to treatment. As such they overestimate the true limits of reproducibility. Short-term precision could not be estimated because of radiation exposure constraints. Standardized precision showed comparable levels for structural and densitometric variables. The MTI was smaller than the recommended treatment duration of 18 mo in all variables, thus potentially allowing individual patient monitoring.
There are a few cross-sectional studies using clinical CT scanners to assess vertebral trabecular structure in vivo(21–23) aiming at fracture discrimination or diagnosis of osteoporosis. In comparison with our work, all of these studies used larger voxel sizes, in the order of 400 μm in plane, and a slice thickness of 1.0–1.5 mm. Structures were only analyzed in 2D.
Ito et al.(12) performed a cross-sectional study using HRCT with a resolution and dose comparable to our protocol. Volumetric microstructural analysis was used to separate women with a recent vertebral fragility fracture (n = 39) from a control group (n = 43). Structural variables performed significantly better than volumetric BMD.
When comparing the changes in structural and densitometric values, our study revealed pathophysiologically meaningful differences that were observed in vivo for the first time. The treatment response measured in app. BV/TV was significantly larger than BMD when expressing changes in percent and in standardized units. This was interpreted as the teriparatide-induced apposition of new, initially lower mineralized bone matrix. It contributes to BV/TV once its mineral concentration within a voxel exceeds the threshold. At a cut-off level of 250 mg/ml, which is far lower than the mineralization of mature bone, this happens when a substantial fraction of the voxel is filled by partially mineralized bone matrix. A human bone biopsy study using quantitative backscattered electron imaging showed that PTH therapy stimulates skeletal remodeling and thus increases the percentage of newly formed bone matrix of lower mineral density.(24) Interestingly, we found structural and density changes to be stronger in the periphery of the trabecular VOI than in the central ellipsoid traditionally used in QCT (results not shown).
Compared with the overall EUROFORS study,(25) we observed a similar pattern in the increases of lumbar aBMD across the pretreatment groups, despite the different lag times between the two pretreated groups not found in the overall study cohort. This pattern was slightly (not significant) different for HRCT variables but this may have been caused by the small sample size and cross-sectional bias. Patients who had inadequately responded to antiresorptive treatment before study start showed increases in vBMD and structural integrity that were of the same magnitude than those observed in treatment naïve patients. This indicates that teriparatide treatment is effective in this group of patients and suggests that initiating teriparatide treatment in subjects inadequately responding to antiresorptive treatment may be a valid option. Obviously, these findings are limited by the sample size and cross-sectional bias. The differences in the response of app. BV/TV across the three pretreatment groups with the smallest increase in pretreated women, and the largest in inadequate responders, lacks biological plausibility and is probably caused by the relatively small sample size of the antiresorptive pretreated subgroup.
This study has some limitations. The spatial resolution was characterized by voxel sizes larger than the typical diameter of individual trabeculae but lower than trabecular separation. One should consider that it was not the aim to measure individual trabeculae for which the sampling theorem limits resolution to twice the voxel size. However, for the averaged measures of the entire VOI that contain hundreds if not thousands of structures a substantially better resolution can be achieved, only limited by the level of noise present. In the peripheral skeleton microstructural assessments can be performed with better image resolution.(26) However, local bone structures differ because of distinct local loading,(10) and thus it is difficult to use peripheral measures to estimate structural changes in the spine. The various measurement techniques assessed different but adjacent vertebrae that will generally lower associations among techniques.(27) Our segmentation method with a fixed threshold is simple, but it was adequate to show a treatment effect in individual subjects. The number of different CT scanner types used resulted in variable image data quality. This may have affected cross-sectional analyses. However, each center contributed patients to almost all pretreatment groups and therefore the bias likely was limited. It is difficult to assess the accuracy of the results as gold standards are missing for measurements in vivo. However, all longitudinal analyses conducted were also calculated in SDs of baseline results. This is a conservative measure for an assessment of the responsiveness of structural variables because a larger noise contribution (which is expected when evaluating structural and densitometric variables) would enhance the standard deviation and thus lead to an underestimation of the standardized response rate of structural measures.
A further limitation was the lack of a control group not receiving teriparatide treatment, available only in the second year of the EUROFORS study. However, the densitometric increases measured in this 1-yr interim report were in good agreement with established conventional measures, such as aBMD measured by DXA and vBMD measured by QCT, from other clinical studies of teriparatide,(19,20) and all scanners were carefully calibrated over the entire study duration. Moreover, large changes were observed in a rather large study group for all variables except app. MIL DA and, therefore, it is unlikely that these are artifacts attributable to multiple statistical testing and chance.
In conclusion, this study showed that microstructural analysis of the human vertebra in vivo is feasible. When applied longitudinally in patients with severe osteoporosis who are being treated with teriparatide, the method offers information on treatment progress exceeding that provided by BMD alone. Further studies have to investigate whether HRCT also shows favorable performance for antiresorptive treatment. The new technology also provides insight into pathophysiological processes of bone turnover that hitherto were not measurable in vivo at central skeletal measurement sites like the spine.
Twelve months of treatment with teriparatide were associated with robust, statistically significant improvements in several measures of bone microarchitecture, including bone volume fraction, trabecular number, and trabecular separation.
Contributing Clinics and Physicians: Germany—Bruno Allolio, Eberhard Blind, Franz Jakob, Klinikum der Universität Würzburg; Klaus Badenhoop, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt; Reimar R. Fritzen, Med. Klinik für Endokrinologie des Universitätsklinikums Düsseldorf; Thorsten Hennings, Frankfurt; Christian Kasperk, Universitätsklinikum Heidelberg; Jörn Kekow, Fachkrankenhaus für Rheumatologie und Orthopädie, Vogelsang-Gommern; Hans-Peter Kruse, Universitäts-Krankenhaus Eppendorf, Hamburg; Heiner Moenig, Universitätsklinikum Schleswig-Holstein, Campus Kiel; Rüdiger Möricke, Magdeburg; Helmut Radspieler, München; Jutta Semler, Immanuel Krankenhaus Berlin-Wannsee; Wolfgang Spieler, Zerbst; Nikolaus Vollmann, München; Andreas Wagenitz, Berlin; Spain—César Díaz-López, Hospital Santa Creu i Sant Pau, Barcelona; Jordi Farrerons, Hospital Santa Creu i Sant Pau, Barcelona; José Andrés Roman Iborra, Hospital Universitario Dr. Pesset, Valencia; Javier del Pino, Hospital Clínico, Salamanca.
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- 252005 Response of BMD and back pain to 12 months of teriparatide (rhPTH1-34) in patients with and without prior antiresorptive treatment: One-year interim results from EUROFORS study. ASBMR 27th Annual Meeting, September 23–27, 2005, Nashville, TN, USA., , , , , , , ,