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Bone-turnover marker (BTM) measurements in the blood or urine reflect the bone-remodeling rate and may be useful for studying and clinically managing metabolic bone diseases. Substantial evidence supporting the diagnostic use of BTMs has accumulated in recent years, together with the publication of several guidelines. Most clinical trials and observational and reference-interval studies have been performed in the Northern Hemisphere and have mainly involved Caucasian populations. This review focuses on the available data for populations from the Asia-Pacific region and offers guidance for using BTMs as diagnostic biomarkers in these populations. The procollagen I N-terminal propeptide and β-isomerized C-terminal telopeptide of type-I collagen (measured in plasma) are reference BTMs used for investigating osteoporosis in clinical settings. Premenopausal reference intervals (established for use with Asia-Pacific populations) and reference change values and treatment targets (used to monitor osteoporosis treatment) help guide the management of osteoporosis. Measuring BTMs that are not affected by renal failure, such as the bone-specific isoenzyme alkaline phosphatase and tartrate-resistant acid phosphatase 5b, may be advantageous for patients with advanced chronic kidney disease. Further studies of the use of BTMs in individuals with metabolic bone disease, coupled with the harmonization of commercial assays to provide equivalent results, will further enhance their clinical applications.

Background: Well defined reference intervals are central to the utility of serum C-terminal telopeptide of type 1 collagen (CTX) and N-terminal propeptide of type I procollagen (P1NP), designated as reference markers in osteoporosis, and useful for monitoring therapeutic response in that condition. This study reports the reference intervals for plasma CTX and serum P1NP in a multi-ethnic Malaysian population., Methods: Ethnic Malay, Chinese or Indian subjects aged 45-90 years old were recruited from Selangor, Malaysia from June 2016 to August 2018. Subjects with known medical conditions (e.g., bone disorders, malnutrition, immobilisation, renal impairment, hormonal disorders) and medications (including regular calcium or vitamin D supplements) that may affect CTX and P1NP were excluded. Additionally, subjects with osteoporosis or fracture on imaging studies were excluded. The blood samples were collected between 8 a.m. and 9 a.m. in fasting state. The CTX and P1NP were measured on Roche e411 platform in batches., Results: The 2.5th-97.5th percentiles reference intervals (and bootstrapped 90%CI) for plasma CTX in men (n = 91) were 132 (94-175) - 775 (667-990) ng/L; in post-menopausal women (n = 132) 152 (134-177) - 1025 (834-1293) ng/L. The serum P1NP reference intervals in men were 23.7 (19.1-26.4) - 83.9 (74.0-105.0) µg/L, and in post-menopausal women, 25.9 (19.5-29.3) - 142.1 (104.7-229.7) µg/L., Conclusion: The reference intervals for plasma CTX and serum PINP for older Malaysian men and post-menopausal women are somewhat different to other published studies from the region, emphasising the importance of establishing specific reference intervals for each population.

Functional reference limits describe key changes in the physiological relationship between a pair of physiologically related components. Statistically, this can be represented by a significant change in the curvature of a mathematical function or curve (e.g., an observed plateau). The point at which the statistical relationship changes significantly is the point of curvature inflection and can be mathematically modeled from the relationship between the interrelated biomarkers. Conceptually, they reside between reference intervals, which describe the statistical boundaries of a single biomarker within the reference population, and clinical decision limits that are often linked to the risk of morbidity or mortality and set as thresholds. Functional reference limits provide important physiological and pathophysiological insights that can aid laboratory result interpretation. Laboratory professionals are in a unique position to harness data from laboratory information systems to derive clinically relevant values. Increasing research on and reporting of functional reference limits in the literature will enhance their contribution to laboratory medicine and widen the evidence base used in clinical decision limits, which are currently almost exclusively contributed to by clinical trials. Their inclusion in laboratory reports will enhance the intellectual value of laboratory professionals in clinical care beyond the statistical boundaries of a healthy reference population and pave the way to them being considered in shaping clinical decision limits. This review provides an overview of the concepts related to functional reference limits, clinical examples of their use, and the impetus to include them in laboratory reports.

Objectives: The exploration of the metabolites in the degradation pathways of vitamin D (VTD) has gained importance in recent years and simultaneous quantitation of twenty-five-hydroxy vitamin D (25(OH)D) mass concentration together with 24,25-dihydroxyvitamin D (24,25(OH)2D) has been proposed as a newer approach to define VTD deficiency. Yet, no data are available on 24,25(OH)2D biological variation (BV). In this study, we evaluated 24,25(OH)2D's BV on the European Biological Variation Study (EuBIVAS) cohort samples to determine if analytical performance specifications (APS) for 24,25(OH)2D could be generated., Methods: Six European laboratories recruited 91 healthy participants. 25(OH)D and 24,25(OH)2D concentrations in K 3 -EDTA plasma were examined weekly for up to 10 weeks in duplicate with a validated LC-MS/MS method. The Vitamin D Metabolite Ratio (24,25(OH)2D divided by 25(OH)D × 100) was also calculated at each time point., Results: Linear regression of the mean 24,25(OH)2D concentrations at each blood collection showed participants were not in steady state. Variations of 24,25(OH)2D over time were significantly positively associated with the slopes of 25(OH)D concentrations over time and the concentration of 25(OH)D of the participant at inclusion, and negatively associated with body mass index (BMI), but not with age, gender, or location of the participant. The variation of the 24,25(OH)2D concentration in participants over a 10 weeks period was 34.6%. Methods that would detect a significant change linked to the natural production of 24,25(OH)2D over this period at p<0.05 would need a relative measurement uncertainty ( u %)<14.9% while at p<0.01, relative measurement uncertainty should be <10.5%., Conclusions: We have defined for the first time APS for 24,25(OH)2D examinations. According to the growing interest in this metabolite, several laboratories and manufacturers might aim to develop specific methods for its determination. The results presented in this paper are thus necessary prerequisites for the validation of such methods., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Background: Between-subject biological variation (CV g ) is an important parameter in several aspects of laboratory practice, including setting of analytical performance specification, delta checks and calculation of index of individuality. Using simulations, we compare the performance of two indirect (data mining) approaches for deriving CV g ., Methods: The expected mean squares (EMS) method was compared against that proposed by Harris and Fraser. Using numerical simulations, d the percentage difference in the mean between the non-pathological and pathological populations, CV i the within-subject coefficient of variation of the non-pathological distribution, f the fraction of pathological values, and e the relative increase in CV i of the pathological distribution were varied for a total of 320 conditions to examine the impact on the relative fractional of error of the recovered CV g compared to the true value., Results: Comparing the two methods, the EMS and Harris and Fraser's approaches yielded similar performance of 158 conditions and 157 conditions within ± 0.20 fractional error of the true underlying CV g , for the normal and lognormal distributions, respectively. It is observed that both EMS and Harris and Fraser's method performed better using the calculated CV i rather than the actual ('presumptive') CV i . The number of conditions within 0.20 fractional error of the true underlying CV g did not differ significantly between the normal and lognormal distributions. The estimation of CV g improved with decreasing values of f, d and CV i CV g ., Discussions: The two statistical approaches included in this study showed reliable performance under the simulation conditions examined., (Copyright © 2022 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Indirect reference intervals and biological variation studies heavily rely on statistical methods to separate pathological and non-pathological subpopulations within the same dataset. In recognition of this, we compare the performance of eight univariate statistical methods for identification and exclusion of values originating from pathological subpopulations., Methods: The eight approaches examined were: Tukey's rule with and without Box-Cox transformation; median absolute deviation; double median absolute deviation; Gaussian mixture models; van der Loo (Vdl) methods 1 and 2; and the Kosmic approach. Using four scenarios including lognormal distributions and varying the conditions through the number of pathological populations, central location, spread and proportion for a total of 256 simulated mixed populations. A performance criterion of ± 0.05 fractional error from the true underlying lower and upper reference interval was chosen., Results: Overall, the Kosmic method was a standout with the highest number of scenarios lying within the acceptable error, followed by Vdl method 1 and Tukey's rule. Kosmic and Vdl method 1 appears to discriminate better the non-pathological reference population in the case of log-normal distributed data. When the proportion and spread of pathological subpopulations is high, the performance of statistical exclusion deteriorated considerably., Discussions: It is important that laboratories use a priori defined clinical criteria to minimise the proportion of pathological subpopulation in a dataset prior to analysis. The curated dataset should then be carefully examined so that the appropriate statistical method can be applied., (Copyright © 2022 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Objectives: Within-subject biological variation ( CV i ) is a fundamental aspect of laboratory medicine, from interpretation of serial results, partitioning of reference intervals and setting analytical performance specifications. Four indirect (data mining) approaches in determination of CV i were directly compared., Methods: Paired serial laboratory results for 5,000 patients was simulated using four parameters, d the percentage difference in the means between the pathological and non-pathological populations, CV i the within-subject coefficient of variation for non-pathological values, f the fraction of pathological values, and e of the pathological distribution. These parameters resulted in a total of 128 permutations. Performance of the Expected Mean Squares method (EMS), the median method, a result ratio method with Tukey's outlier exclusion method and a modified result ratio method with Tukey's outlier exclusion were compared.CV i of the pathological distribution. These parameters resulted in a total of 128 permutations. Performance of the Expected Mean Squares method (EMS), the median method, a result ratio method with Tukey's outlier exclusion method and a modified result ratio method with Tukey's outlier exclusion were compared., Results: Within the 128 permutations examined in this study, the EMS method performed the best with 101/128 permutations falling within ±0.20 fractional error of the 'true' simulated CV , followed by the result ratio method with Tukey's exclusion method for 78/128 permutations. The median method grossly under-estimated the i . The modified result ratio with Tukey's rule performed best overall with 114/128 permutations within allowable error.CV i . The modified result ratio with Tukey's rule performed best overall with 114/128 permutations within allowable error., Conclusions: This simulation study demonstrates that with careful selection of the statistical approach the influence of outliers from pathological populations can be minimised, and it is possible to recover CV i values close to the 'true' underlying non-pathological population. This finding provides further evidence for use of routine laboratory databases in derivation of biological variation components., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Correct interpretation of pathology results is a requirement for accurate diagnosis and appropriate patient management. Clinical Pathologists and Scientists are increasingly focusing on providing quality interpretative comments on their reports and these comments are appreciated by clinicians who receive them. Interpretative comments may improve patient outcomes by helping reduce errors in application of the results in patient management. Thyroid function test (TFT) results are one of the areas in clinical chemistry where interpretative commenting is practised by clinical laboratories. We have provided a series of TFT reports together with possible interpretative comments and a brief explanation of the comments. It is felt that this would be of help in setting up an interpretative service for TFTs and also assist in training and continuing education in their provision., Competing Interests: We have no conflict of interest to declare., (© 2021 Published by Elsevier B.V.)

Background: Biochemical bone turnover markers are useful tools to assess bone remodeling. C-terminal telopeptide of type I collagen (ß-CTX) has been recommended as a reference marker for bone resorption in research studies., Methods: We describe the results of a multicenter study for routine clinical laboratory assays for ß-CTX in serum and plasma. Four centers (Athens GR, Copenhagen DK, Liege BE and Sheffield UK) collected serum and plasma (EDTA) samples from 796 patients presenting to osteoporosis clinics. Specimens were analyzed in duplicate with each of the available routine clinical laboratory methods according to the manufacturers' instructions. Passing-Bablok regressions, Bland-Altman plots, V-shape evaluation method, and Concordance correlation coefficient for ß-CTX values between serum and plasma specimens and between methods were used to determine the agreement between results. A generalized linear model was employed to identify possible variables that affected the relationship between the methods. Two pools of serum were finally prepared and sent to the four centers to be measured in 5-plicates on 5 consecutive days with the different methods., Results: We identified significant variations between methods and between centers although comparison results were generally more consistent in plasma compared to serum. We developed univariate linear regression equations to predict Roche Elecsys®, IDS-iSYS, or IDS ELISA ß-CTX results from any other assay and a multivariable model including the site of analysis, the age, and weight of the patient. The coefficients of determination (R 2 ) increased from approximately 0.80 in the univariate model to approximately 0.90 in the multivariable one, with the site of analysis being the major contributing factor. Results observed on the pools also suggest that long-term storage could explain the difference observed with the different methods on serum., Conclusion: Our results show large within- and between-assay variation for ß-CTX measurement, particularly in serum. Stability of the analyte could be one of the explanations. More studies should be undertaken to overcome this problem. Until harmonization is achieved, we recommend measuring ß-CTX by the same assay on EDTA plasma, especially for research purposes in large pharmacological trials where samples can be stored for long periods before they are assayed.

Parathyroid hormone (PTH) determination is of greatest importance for patients suffering from parathyroid gland disorders and for the follow-up of bone turnover in patients suffering from chronic kidney disease (CKD). Two generations of PTH assays are simultaneously present on the market for PTH quantification. As these assays are not yet standardized, this results in a significant level of confusion in the care of CKD patients. One key objective of the IFCC Committee for Bone Metabolism is to improve this situation. In this position paper, we will highlight the current state of PTH testing and propose a pathway to ultimately overcome issues resulting from PTH assay variability., (Copyright © 2020. Published by Elsevier B.V.)

Currently the 25-hydroxy vitamin D (25(OH)D) concentration is thought to be the best estimate of the vitamin D status of an individual. Unfortunately, its measurement remains complex, despite recent technological advances. We evaluated the biological variation (BV) of 25(OH)D in order to set analytical performance specifications (APS) for measurement uncertainty (MU). Six European laboratories recruited 91 healthy participants. The 25(OH)D concentrations in K 3 -EDTA plasma were examined weekly for up to 10 weeks in duplicate on a Lumipulse G1200 (Fujirebio, Tokyo, Japan). The linear regression of the mean 25(OH)D concentrations at each blood collection showed that participants were not in a steady state. The dissection of the 10-sample collection into two subsets, namely collections 1-5 and 6-10, did not allow for correction of the lack of homogeneity: estimates of the within-subject BV ranged from 5.8% to 7.1% and the between-subject BV ranged from 25.0% to 39.2%. Methods that would differentiate a difference induced by 25(OH)D supplementation at p < 0.05 should have MU < 13.6%, while at p < 0.01, the MU should be <9.6%. The development of APS using BV assumes a steady state of patients. The findings in this study suggest that patients are not in steady state. Therefore, APS that are based on MU appear to be more appropriate.

Whatman No. 1 chromatography paper is widely used as a substrate for cellulose-based immunoassays. The immobilized proteins are used to capture target biomarkers for detection. However, alternative paper substrates may facilitate mass production of immunoassays as diagnostic tests. Here, we assessed the physical characteristics and protein immobilization capabilities of different commercial papers. Some substrates fulfilled our design criteria, including adequate flow rate and sufficient protein immobilization for efficient target capture. This study demonstrates that a variety of paper substrates can be bioactivated and used to capture target biomarkers, enabling development of affordable diagnostic tests from a range of starting materials.

Background: India, with diabetes mellitus (DM) prevalence of nearly 7%, contributes 20% of the DM population in the world. The diagnosis and management of DM is largely dependant on laboratory parameters. We aimed to survey the laboratory testing practices for DM in this country., Methods: A survey of 890 practising Laboratorians in India was conducted through Survey Monkey., Results: A total of 310 (35%) complete responses were received. The majority of respondents worked in academic institutions, public hospital laboratories and private hospital laboratories. HbA1c was approved for diagnosis in 75% of laboratories. The HbA1c method was NGSP (National Glycohaemoglobin Standardisation Programme) certified in 70% of laboratories only. Oral glucose tolerance testing (OGTT) was recommended for diagnosis of gestational diabetes (GDM) in 56% of respondents. Fifty-nine percent respondents recommended an early morning urine sample for microalbuminuria testing whilst 39% and 2% opted for 24 hour urine and timed overnight sample respectively. Sixty-six percent participated in proficiency testing (PT) for both glucose and HbA1c. Twelve percent and 4% respondents respectively participated in PT for glucose only and HbA1c only, and 9% participated in PT for neither., Conclusions: Based on the above survey we recommend that Scientific bodies and Professional Associations in India should educate Laboratorians to adopt NGSP certified methods for HbA1c testing and morning spot sample for microalbuminuria testing. DIPSI (Diabetes in Pregnancy Study Group in India) guidelines for diagnosis of Gestational diabetes since it is a simple, single step procedure, non-fasting, cost effective, feasible method should be implemented., (Copyright © 2020 International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). All rights reserved.)

Background Biochemical bone turnover markers (BTM) are useful tools to assess bone remodeling at the cellular level. N-terminal propeptide of type I procollagen (PINP) has been recommended as a reference marker for bone formation in research studies. Methods We describe the results of a multicenter study for routine clinical laboratory assays for PINP in serum and plasma. Four centers (Athens, Greece [GR], Copenhagen, Denmark [DK], Liege, Belgium [BE] and Sheffield, United Kingdom [UK]) collected serum and plasma (EDTA) samples from 796 patients presenting to osteoporosis clinics. Specimens were analyzed in duplicate with each of the available routine clinical laboratory methods according to the manufacturers' instructions. Passing-Bablok regressions, Bland-Altman plots, V-shape evaluation method and the concordance correlation coefficient for PINP values between serum and plasma specimens and between methods were used to determine the agreement between results. A generalized linear model was employed to identify possible variables that affected the relationship between the methods. Results We showed that both EDTA plasma and serum were suitable for PINP determination. We observed a significant proportional bias between Orion radioimmunoassay and the automated methods for PINP (Roche Cobas and IDS iSYS), which both gave very similar results. The multivariate model did not improve the excellent correlation that was observed between the methods. Conclusions Harmonization of PINP assays is possible by applying a correction factor or correctly assigning the values of the calibrators. This work will benefit from further collaboration between assays manufacturers and clinical laboratory professionals.

Background Plasma-free metanephrines (PFM) or urinary fractionated metanephrines (UFM) are the preferred biochemical tests for the diagnosis of phaeochromocytoma and paraganglioma (PPGL). Borderline increased results should be followed up to either exclude or confirm diagnosis. Methods We extracted all PFM and UFM results reported by our laboratory over a six-month period from the laboratory information system. We categorized patients with borderline increased results according to whether follow-up testing had been performed as suggested in the initial laboratory report. Questionnaires were then sent to all requesting doctors and medical notes reviewed where available. Results Two hundred and four patients with borderline increased PFM or UFM were identified. Sixty-five (38.5%) of 169 patients with borderline increased PFM had a repeat test out of which 36 were normal and 29 did not normalize. Of 35 patients with borderline increased UFM, 17 (48.6%) had subsequent PFM measurement, out of which 15 were normal. Questionnaires were returned to 106 (52%) patients. Of these, the most frequent indication for testing was hypertension ( n = 50); 15 patients had an incidental adrenal mass and two of these patients were diagnosed with a phaeochromocytoma. Conclusion Only 38% of patients with borderline increased PFM had a repeat PFM measurement. This was not significantly higher when compared with the 28% in a previous audit that we reported in 2010 ( P = 0.10). Forty-nine per cent of patients with a borderline increased UFM had a repeat UFM or PFM measurement. There remains a substantial possibility of missed detection of PPGL.

Bone turnover marker (BTMs) concentrations in blood and urine reflect bone-remodelling activity, and may be useful adjuncts in the diagnosis and management of metabolic bone diseases. Newer biomarkers, mainly bone regulatory proteins, are currently being investigated to elucidate their role in bone metabolism and disease and may in future be useful in clinical diagnosis and management of metabolic bone disease. BTM concentrations increase around menopause in women, and at a population level the degree of increase in BTMs reflect bone loss. However, lack of adequate data precludes their use in individual patients for fracture risk assessment in clinical practice. The rapid and large changes in BTMs following anti-resorptive and anabolic therapies for osteoporosis treatment indicate they may be useful for monitoring therapy in clinical practice. The offset of drug effect on BTMs could be helpful for adjudicating the duration of bisphosphonate drug holidays. BTMs may offer useful additional data in skeletal diseases that are typically characterised by increased bone remodelling: chronic kidney disease (CKD), primary hyperparathyroidism (PHPT) and Paget's disease. In CKD, bone specific alkaline phosphatase (bAP) is currently endorsed for use for the assessment of mineral bone disease. The role of BTMsin predicting the bone mineral density response to successful parathyroidectomy in PHPT shows some utility but the data are not consistent and studies are limited in size and/or duration. In Paget's disease of bone, BTMs are used to confirm diagnosis, evaluate extent of disease or degree of activity and for monitoring the response to bisphosphonate treatment. Whilst BTMs are currently used in specific clinical practice instances when investigating or managing metabolic bone disease, further data are needed to consolidate their clinical use where evidence of utility is limited., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aim of this study was to ascertain the impact of gender specific hs-TnI thresholds in a clinical setting and determine the clinical characteristics and discharge diagnosis for individuals presenting to the Emergency Department (ED) with elevated troponin I with the Abbott high-sensitivity troponin I (hs-TnI) assay, but non-elevated troponin I on the previous generation assay (STAT TnI-II). Medical records of individuals presenting to the Royal Perth Hospital ED with elevated hs-TnI between 12 November 2013 and 24 December 2013 were retrospectively reviewed. The 99th percentile hs-TnI thresholds were ≥26 ng/L for males and ≥16 ng/L for females. TnI-II assays were performed concomitantly. In total, 1449 individuals [855 (59%) males] had 3580 troponin measurements. hs-TnI was elevated in 1569 (43.8%) measurements. Elevated hs-TnI with normal TnI-II was found in 120 (8.3%) individuals: 77 (64%) females and 43 (36%) males. Eight (6.7%) individuals were diagnosed with acute coronary syndrome (ACS): four (9.3%) males and four (5.2%) females. Other cardiac aetiologies were found in 33 (42%) females and 17 (40%) males. Individuals with elevated hs-TnI had high rates of hypertension (80%), diabetes mellitus (33%), cardiac failure (23%), aspirin use (53%) and lipid lowering therapy (52%). Significantly fewer females than males with discrepant troponin I results had previous ischaemic heart disease. The hsTnI assay identifies 8% more individuals with elevated troponin in an acute setting, with a female predominance (64%). However, only 6.7% of these individuals with multiple cardiovascular risk factors were diagnosed with ACS, a ∼0.5% increase overall. Outcome studies are required to determine if the Australian hs-TnI thresholds are clinically appropriate., (Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Current evidence continues to support the potential for bone turnover markers (BTM) to provide clinically useful information particularly for monitoring the efficacy of osteoporosis treatment. Many of the limitations identified earlier remain, principally in regard to the relationship between BTM and incident fractures. Important data are now available on reference interval values for CTX and PINP across a range of geographic regions and for individual clinical assays. An apparent lack of comparability between current clinical assays for CTX has become evident indicating the possible limitations of combining such data for meta-analyses. Harmonization of units for reporting serum/plasma CTX (ng/L) and PINP (μg/L) is recommended. The development of international collaborations continues with an important initiative to combine BTM results from clinical trials in osteoporosis in a meta-analysis and an assay harmonization program are likely to be beneficial. It is possible that knowledge derived from clinical studies can further enhance fracture risk estimation tools with inclusion of BTM together with other independent risk factors. Further data of the relationships between the clinical assays for CTX and PINP as well as physiological and pre-analytical factors contributing to variability in BTM concentrations are required., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The provision of interpretative advice on laboratory results is a post-analytic activity and an integral part of clinical laboratory services. It is valued by healthcare workers and has the potential to prevent or reduce errors and improve patient outcomes. It is important to ensure that interpretative comments provided by laboratory personnel are of high quality: comments should be patient-focused and answer the implicit or explicit question raised by the requesting clinician. Comment providers need to be adequately trained and qualified and be able to demonstrate their proficiency to provide advice on laboratory reports. External quality assessment (EQA) schemes can play a part in assessing and demonstrating the competence of such laboratory staff and have an important role in their education and continuing professional development. A standard structure is proposed for EQA schemes for interpretative comments in clinical chemistry, which addresses the scope and method of assessment including nomenclature and marking scales. There is a need for evidence that participation in an EQA program for interpretative commenting facilitates improved quality of comments. It is proposed that standardizing goals and methods of assessment as well as nomenclature and marking scales may help accumulate evidence to demonstrate the impact of participation in EQA for interpretative commenting on patient outcome.

Objective: We undertook to identify levels for plasma β isomerised carboxy-terminal telopeptides of type I collagen (p-βCTX-I) that are comparable to currently used urine amino-terminal telopeptides of type I collagen (u-NTX) cut-points and treatment targets in osteoporosis., Design and Methods: Fasting morning samples were collected from patients attending tertiary hospitals and clinics for investigation of metabolic bone disease. Patients with Paget's disease or <20years of age were excluded. Second void spot urine for NTX and plasma (EDTA) samples were utilised. Urine was analysed routinely and plasma stored at -20C until analysis by enzyme-linked immunosorbent assay (ELISA) (Immunodiagnostic Systems plc), E170 (Roche Diagnostics) and IDS-iSYS (Immunodiagnostic Systems plc) methods. The relationship of u-NTX with each p-βCTX-I method's results was assessed by Passing and Bablok regression, and p-βCTX-I levels equivalent to u-NTX cut-points and targets were interpolated., Results: One hundred and forty six patients were included. Spearman correlation coefficients ranged from 0.71 to 0.75 for the three βCTX-I assays. The equivalent βCTX-I concentrations for NTX/Cr values of 21 (fracture risk reduction target following risedronate therapy), 27 (healthy pre-menopausal women's mean value), and 38 (threshold for reduction of BMD on calcium alone) nmol BCE/mmol were 230, 312 and 462ng/L for the automated Roche assay and 271, 395 and 624ng/L for the automated IDS i-SYS assay respectively., Conclusions: The p-βCTX-I equivalent to the only available fracture outcome based absolute treatment threshold of 21nmol BCE/mmol established for u-NTX, is close to 250ng/L but will vary between p-βCTX-I assays., (Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The aim of this report was to summarize the clinical performance of two reference bone turnover markers (BTMs) in the prediction of fracture risk. We used an updated systematic review to examine the performance characteristics of serum procollagen type I N propeptide (s-PINP) and serum C-terminal cross-linking telopeptide of type I collagen (s-CTX) in fracture risk prediction in untreated individuals in prospective cohort studies. We excluded cross-sectional studies. Ten potentially eligible publications were identified and six included in the meta-analysis. There was a significant association between s-PINP and the risk of fracture. The hazard ratio per SD increase in s-PINP (gradient of risk [GR]) was 1.23 (95 % CI 1.09-1.39) for men and women combined unadjusted for bone mineral density. There was also a significant association between s-CTX and risk of fracture, GR = 1.18 (95 % CI 1.05-1.34) unadjusted for bone mineral density. For the outcome of hip fracture, the association between s-CTX and risk of fracture was slightly higher, 1.23 (95 % CI 1.04-1.47). Thus, there is a modest but significant association between BTMs and risk of future fractures.

Introduction: A standard short Synacthen test (SST) is the conventional diagnostic test for primary hypoadrenalism. Measuring simultaneous plasma cortisol and adrenocorticotrophin hormone (ACTH) and using the cortisol: ACTH ratio as a first-line test may be safer and more convenient than performing a SST., Methods: A retrospective study of 349 patients who had a SST with simultaneous baseline plasma cortisol and ACTH performed between 2005 and 2010 in two separate Australian health centres. The plasma cortisol: ACTH ratio was calculated for each patient and their final diagnosis was determined based on their SST result and a review of their clinical notes., Results: Eighteen patients had primary hypoadrenalism, 46 patients had secondary hypoadrenalism and 285 patients had normal adrenal function. All the patients with primary hypoadrenalism had a plasma cortisol: ACTH ratio <3, while none of the patients with normal adrenal function or secondary hypoadrenalism had a cortisol: ACTH ratio <3. Therefore, a cortisol: ACTH ratio <3 had a 100% sensitivity and specificity for the diagnosis of primary hypoadrenalism. Patients with secondary hypoadrenalism had a cortisol: ACTH ratio >3, while subjects with normal adrenal function had a cortisol: ACTH ratio >15. There was overlap in cortisol: ACTH ratios of patients with secondary hypoadrenalism and normal adrenal function., Conclusions: Although the cortisol: ACTH ratio predicts primary hypoadrenalism, its value is limited to diagnosing primary hypoadrenalism as it does not distinguish secondary hypoadrenalism from normal adrenal function. Larger prospective studies that include patients with early primary hypoadrenalism are needed to confirm the reliability of plasma cortisol: ACTH ratio as a diagnostic test for primary hypoadrenalism.

Context: Heterophilic antibodies are a well-described interferent but poorly appreciated and are often not a recognized problem affecting most immunoassays. We describe for the first time heterophilic antibodies interference affecting an adrenocorticotropic hormone (ACTH) assay in a patient with Cushing's syndrome due to bilateral nodular adrenal hyperplasia., Case: A 60-year-old retired female nurse underwent extensive invasive investigations, which were ultimately unnecessary, as a result of initial analytical interference in the ACTH assay, which could not be resolved using a proprietary heterophilic binding reagent., Results: This case highlights the inherent difficulty of diagnosing Cushing's syndrome and the large emphasis placed on laboratory tests. The consequence of not initially identifying interference in this patient's laboratory test results led to unnecessary and costly investigations with potentially adverse outcomes., Conclusions: Clinicians and the laboratory community need to be continuously vigilant and view laboratory results with caution when they are inconsistent with the clinical picture. This approach is paramount, especially at a time of increasing automation and ever-diminishing scientist involvement in sample processing.

What Is Already Known About This Subject: • Paracetamol is commonly used in deliberate self poisoning (DSP) and this requires blood sampling to refine risk assessment. If saliva concentrations agreed with plasma concentrations, then this could support the development of non-invasive testing. Our pilot work supports this hypothesis, but was largely confined to nontoxic concentrations., What This Study Adds: • We found agreement between the indications for treatment of paracetamol DSP based on plasma and saliva paracetamol concentrations. Saliva may hold promise as a non-invasive method to risk stratify paracetamol poisoning., Aims: Paracetamol is commonly used in deliberate self poisoning (DSP) and requires blood sampling to refine risk assessment. We aimed to test the agreement between plasma and saliva paracetamol concentrations in the toxic range in DSP., Methods: Contemporaneous paired plasma and saliva paracetamol concentrations were measured. Saliva was collected using a Sarstedt Salivette® device and the concentration was measured using a colorimetric method., Results: Fifty-six patients (44, 78% female) median age 26 years (IQR 20-41) were enrolled. The median reported paracetamol ingestion was 10 g (IQR 6-14). Specimens were collected at a median of 4 h (IQR 4-5.3) post ingestion. The median plasma and saliva paracetamol concentrations were 29 mg l(-1) (IQR 8-110) and 38 mg l(-1) (IQR 10-105) respectively [mean difference 8 mg l(-1) , 95% confidence interval (CI) 2, 14]. Lin's concordance correlation was 0.97 (95% CI 0.96, 0.98). There were 15 patients who were treated with N-acetylcysteine. Their median reported paracetamol ingestion was 14 g (IQR 10-23) and samples were collected at a median of 4 h post ingestion. The median plasma and saliva paracetamol concentrations were 167 mg l(-1) (IQR 110-200) and 170 mg l(-1) (IQR 103-210) respectively (mean difference 15 mg l(-1) , 95% CI -4, 35). Lin's concordance correlation was 0.94 (95% CI 0.88, 0.99). No patient needing treatment would have been missed using saliva concentrations only., Conclusions: The agreement between the indications for treatment of paracetamol DSP based on plasma and saliva paracetamol concentrations extends into the toxic range, but with slightly lower agreement. Saliva may hold promise as a non-invasive method to risk stratify paracetamol poisoning., (© 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.)

Osteoporosis is a major health problem worldwide, and is projected to increase exponentially due to the aging of the population. The absolute fracture risk in individual subjects is calculated by the use of algorithms which include bone mineral density (BMD), age, gender, history of prior fracture and other risk factors. This review describes the laboratory investigations into osteoporosis which include serum calcium, phosphate, creatinine, alkaline phosphatase and 25-hydroxyvitamin D and, additionally in men, testosterone. Parathyroid hormone (PTH) is measured in patients with abnormal serum calcium to determine its cause. Other laboratory investigations such as thyroid function testing, screening for multiple myeloma, and screening for Cushing's syndrome, are performed if indicated. Measurement of bone turnover markers (BTMs) is currently not included in algorithms for fracture risk calculations due to the lack of data. However, BTMs may be useful for monitoring osteoporosis treatment. Further studies of the reference BTMs serum carboxy terminal telopeptide of collagen type I (s-CTX) and serum procollagen type I N-terminal propeptide (s-PINP) in fracture risk prediction and in monitoring various treatments for osteoporosis may help expedite their inclusion in routine clinical practice.

The International Osteoporosis Foundation (IOF) and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Working Group on Bone Marker Standards (WG-BMS) has evaluated the clinical potential of bone turnover markers (BTMs) in the prediction of fracture risk and for monitoring treatment. Research evidence suggests that BTMs may provide information on fracture risk independently from BMD, so that fracture risk prediction might be enhanced by their inclusion in assessment algorithms. The potential use of BTMs to predict the response to treatments for osteoporosis in the individual patient is also of great interest. Treatment-induced changes in specific markers account for a substantial proportion of fracture risk reduction. However, there is still a need for stronger evidence on which to base practice in both situations. IOF/IFCC recommends one bone formation marker (serum procollagen type I N propeptide, s-PINP) and one bone resorption marker (serum C-terminal cross-linking telopeptide of type I collagen, s-CTX) to be used as reference markers and measured by standardised assays in observational and intervention studies in order to enlarge the international experience of the application of markers to clinical medicine and to help resolve uncertainties over their clinical use.

The best indirect evidence that increased bone turnover contributes to fracture risk is the fact that most of the proven therapies for osteoporosis are inhibitors of bone turnover. The evidence base that we can use biochemical markers of bone turnover in the assessment of fracture risk is somewhat less convincing. This relates to natural variability in the markers, problems with the assays, disparity in the statistical analyses of relevant studies and the independence of their contribution to fracture risk. More research is clearly required to address these deficiencies before biochemical markers might contribute a useful independent risk factor for inclusion in FRAX(®)., (Copyright © 2011. Published by Elsevier Inc.)

Unlabelled: The International Osteoporosis Foundation (IOF) and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommend that a marker of bone formation (serum procollagen type I N propeptide, s-PINP) and a marker of bone resorption (serum C-terminal telopeptide of type I collagen, s-CTX) are used as reference analytes for bone turnover markers in clinical studies., Introduction: Bone turnover markers (BTM) predict fracture risk, and treatment-induced changes in specific markers account for a substantial proportion of fracture risk reduction. The aims of this report were to determine their clinical potential in the prediction of fracture risk and for monitoring the treatment of osteoporosis and to set an appropriate research agenda., Methods: Evidence from prospective studies was gathered through literature review of the PUBMED database between the years 2000 and 2010 and the systematic review of the Agency for Healthcare Research and Quality up to 2001., Results: High levels of BTMs may predict fracture risk independently from bone mineral density in postmenopausal women. They have been used for this purpose in clinical practice for many years, but there is still a need for stronger evidence on which to base practice. BTMs provide pharmacodynamic information on the response to osteoporosis treatment, and as a result, they are widely used for monitoring treatment in the individual. However, their clinical value for monitoring is limited by inadequate appreciation of the sources of variability, by limited data for comparison of treatments using the same BTM and by inadequate quality control. IOF/IFCC recommend one bone formation marker (s-PINP) and one bone resorption marker (s-CTX) to be used as reference markers and measured by standardised assays in observational and intervention studies in order to compare the performance of alternatives and to enlarge the international experience of the application of markers to clinical medicine., Conclusion: BTM hold promise in fracture risk prediction and for monitoring treatment. Uncertainties over their clinical use can be in part resolved by adopting international reference standards.

Background: The effects of vitamin D2 and D3 supplementation on circulating concentrations of 25(OH)D3 require reliable analytical tools for specific determination of 25(OH)D3 and 25(OH)D2. We have developed a highly specific 25-OH Vitamin D3 ELISA with negligible cross-reactivity towards 25(OH)D2., Methods: 25(OH)D3 concentrations were measured in several study participants; 1) 641 healthy men and women; 2) 39 postmenopausal women receiving 400-800 IU vitamin D3 daily for 4 months; 3) 45 men and women with hip fracture receiving 1000 IU vitamin D2 daily for 3 months., Results: This 25-OH Vitamin D3 ELISA had minimal cross-reactivity to 25(OH)D2, (0.7%), and demonstrated a high correlation (r2 = 0.93) with 25(OH)D3 determined by HPLC. 25(OH)D3 increased by 14% in subjects receiving vitamin D3 for 4 months (p < 0.01), whereas there was no significant change in 25(OH)D3 levels in those receiving vitamin D2., Conclusions: We report that 25(OH)D3 ELISA was used for evaluation of 25(OH)D3 concentrations in subjects receiving vitamin D2 and D3 supplementation. The increase of 25(OH)D3 in circulation with vitamin D3 supplementation and lack of increase with vitamin D2 supplementation suggest that this assay has sufficient sensitivity and specificity to be used as a reliable measurement of nutritional vitamin D3 status in humans.

* Clinical laboratories should be able to offer interpretation of the results they produce. * At a minimum, contact details for interpretative advice should be available on laboratory reports.Interpretative comments may be verbal or written and printed. * Printed comments on reports should be offered judiciously, only where they would add value; no comment preferred to inappropriate or dangerous comment. * Interpretation should be based on locally agreed or nationally recognised clinical guidelines where available. * Standard tied comments ("canned" comments) can have some limited use.Individualised narrative comments may be particularly useful in the case of tests that are new, complex or unfamiliar to the requesting clinicians and where clinical details are available. * Interpretative commenting should only be provided by appropriately trained and credentialed personnel. * Audit of comments and continued professional development of personnel providing them are important for quality assurance.

Aim: To determine the correlation between plasma and saliva paracetamol levels following paracetamol deliberate self-poisoning., Methods: Paired plasma and saliva paracetamol levels were measured. Saliva analysis was performed contemporaneously using a colorimetric method., Results: 21 patients (76% female) mean age 28.3 +/- 12.9 years (range 15-55) were enrolled. Mean reported paracetamol ingestion was 10.3 g (range 2-20 g). Specimens were collected at a mean of 6.2 +/- 3.1 hours post-ingestion (range 4-13 hours) and mean plasma and saliva paracetamol levels were 48 mg/L and 62 mg/L respectively (mean difference 14; 95% CI 5-22; p < 0.004); Pearson's correlation r = 0.95 (p < 0.0001). No patient needing treatment would have been missed using saliva levels only., Conclusion: There is concordance between the indications for treatment of paracetamol deliberate self-poisoning based on plasma and saliva paracetamol levels. Saliva paracetamol levels are typically higher than plasma levels. Further studies involving larger numbers of patients, comparing plasma and saliva paracetamol levels in patients with potentially toxic plasma paracetamol concentrations, would be useful in determining the potential clinical value of this method.

Objective: The objective of this study is to determine the association between established cardiovascular risk factors and depression among older men., Methods: The authors conducted a cross-sectional study of a community-representative sample of 5,439 men aged 71-89 years. Cardiovascular disease and risk factors assessed included history of diabetes, hypertension, angina, myocardial infarction, and stroke; current smoking; total cholesterol and fractions; triglycerides; total plasma homocysteine; and MTHFR677 genotype. Depression was defined by a Geriatric Depression Scale 15 items score of 7 or greater., Results: A complete data set was available for 4,204 men, of whom 212 were depressed (5%). Men who were depressed reported higher frequency of diabetes (23.1% versus 13.2%), angina (30.2% versus 20.4%), myocardial infarction (26.2% versus 16.0%), and stroke (23.6% versus 9.1%) than nondepressed men. Participants with depression were also more likely to have plasma homocysteine above 15 mumol/L (39.1% versus 25.5%) and high triglycerides (32.1% versus 20.9%) than nondepressed subjects. Depressed older men were also more likely to be active smokers (9.9% versus 4.8%). The other factors measured in the study were not significantly associated with depression. Estimation of the population-attributable fraction (PAF) after logistic regression showed that high plasma homocysteine had the highest PAF for depression (PAF:15%, 95% confidence interval [95% CI]: 5%-23%) followed by high triglycerides (PAF: 11%, 95% CI: 2%-18%), angina (PAF: 9%, 95% CI: 2%-15%), stroke (PAF: 8%, 95% CI: 3%-13%), diabetes (PAF: 7%, 95% CI: 1%-13%), myocardial infarction (PAF: 5%, 95% CI: 0%-11%), and smoking (PAF: 5%, 95% CI: 1%-9%)., Conclusions: High plasma homocysteine and triglycerides appear to account for a considerable proportion of cases of depression in older men. The successful management of these risk factors may contribute to decrease the prevalence of depression in later life.

The results of several observational studies suggest that the use of estrogen replacement is associated with better mood, cognitive function and quality of life. Such findings are consistent with those of laboratory-based research showing that estrogen promotes neuronal sprouting, enhances cholinergic activity in the brain, decreases brain and plasma levels of beta-amyloid, increases serotonin postsynaptic responsivity and the turnover of noradrenaline, and inhibits monoamine oxidase activity. However, the findings from the Women's Health Initiative controlled trial showed that hormone replacement (estrogen plus progestin) not only failed to improve mood, cognition and quality of life but also increased the risk of dementia. At present, there is limited information about the effect of unopposed estradiol replacement therapy (ERT) on the mental health outcomes of women at increased risk of cognitive decline (aged 70 years and over). We designed the present randomized, double-blind, placebo-controlled trial to clarify this issue. One hundred and fifteen women were randomized to treatment with estradiol (n=58; 2mg per day) or placebo for a total period of 20 weeks. The outcomes of interest in this study included changes in the Beck Depression Inventory (BDI) scores between baseline and week 20, as well as changes in quality of life scores (as measured by the SF-36) and cognitive function (CAMCOG, Block Design, Memory for Faces, California Verbal Learning Test (CVLT) and verbal fluency (VF)). Nineteen women treated with estradiol and 10 of those treated with placebo discontinued the use of the medication during trial, most frequently due to adverse reactions (OR=4.11, 95% CI=1.29-15.37). Intention-to-treat analysis showed that the active and placebo groups did not differ in their response to treatment in any of the outcome measures (p>0.05). A separate analysis restricted to women who completed the 20-week-trial produced similar negative results. The results of this trial indicate that the use of a relatively high dosage of unopposed estrogen replacement for 20 weeks is not associated with significant changes in cognitive function, mood and quality of life. Other more efficacious and safer interventions need to be devised with the aim of improving the mental state and quality of life of older women.

Objective: The authors investigated the frequency distribution of well-established risk factors for dementia--high plasma homocysteine and the apolipoprotein E epsilon4 allele (APOE epsilon4)--among older women with subjective memory complaints (SMC) but no cognitive impairment, and with mild cognitive impairment (MCI)., Methods: This was a cross-sectional, community-based study., Results: Women with MCI had higher total plasma homocysteine than healthy-comparison subjects. There was also a nonsignificant excess of APOE epsilon4 carriers in the MCI than in the healthy group. Participants with SMC had higher depression and anxiety scores than healthy-comparison subjects, but did not differ from subjects in the healthy-comparison group in relation to their total plasma homocysteine and APOE epsilon4 distribution., Conclusions: MCI seems to be more closely related to well-established risk factors for dementia than is SMC.

Homocysteine (Hcy) is harmful to neurons and blood vessels, including the cerebral microvasculature. It is possible that such effects contribute to the cascade of events that leads to cognitive decline, dementia, and depression in later life. Hcy is produced during the metabolism of the essential amino-acid methionine, which also involves a methyl group transfer derived from folate and choline metabolism. Its plasma level can be influenced by factors such as age, vitamin deficiency, renal function, and a common mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, where cytosine is replaced by thymidine (C-->T) at nucleotide position 677. Subjects with the TT genotype have higher homocysteine levels and may be particularly prone to experiencing depression as a result of high plasma Hcy and dysfunction of methylation metabolic pathways critical to the synthesis of noradrenaline and serotonin. We designed the present study to investigate whether older women with the TT genotype would have higher depression and lower cognitive scores than women with CT and CC genotypes. A total of 240 community-dwelling women aged 70 years or over volunteered to take part in the study - 29 carried the TT genotype, 113 the CT and 98 the CC genotype. The Beck Depression Inventory (BDI) score for subjects with the TT genotype was statistically similar to the other groups (P = 0.609). Plasma Hcy showed a modest and significant correlation with BDI scores (r = 0.21) that was independent from age, B12 and folate levels. There was no association between beck anxiety inventory (BAI) scores and MTHFR genotype or homocysteine levels. The cognitive assessment of participants included measures of verbal memory, memory for faces, verbal fluency, visuo-spatial abilities and the cognitive section of the Cambridge Examination For Mental Disorders Of The Elderly (CAMCOG)-MTHFR genotype had no clear association with cognitive scores. These results indicate that, in isolation, the MTHFR C677T gene variation does not play an important role in the modulation of mood and cognitive performance in later life.

Second and third generation bisphosphonates are the treatment of choice for Paget's disease of bone. These drugs are more effective than calcitonin and etidronate, but there have been no head to head, randomized controlled trials comparing potent bisphosphonates. We conducted a 2-year, randomized, open-label trial comparing oral alendronate and intravenous pamidronate in 72 subjects with Paget's disease. Randomization was stratified according to baseline plasma total alkaline phosphatase (ALP) and previous bisphosphonate treatment (yes or no). All previously treated patients had received pamidronate but not alendronate. Assigned treatments were pamidronate (60 mg) every 3 months as a single infusion or alendronate (40 mg) daily in 3-month blocks, continued until biochemical remission (defined as both ALP and urine deoxypyridinoline (DPD)/creatinine ratio in the reference range) or a clear plateau effect was observed. At 1 year, nonresponders to pamidronate were crossed over to alendronate treatment. At 1 year, 31/36 (86%) subjects randomized to alendronate achieved biochemical remission compared with 21/36 (56%) for pamidronate (P = 0.017). There was a significantly greater reduction in ALP (P < 0.001) and DPD/creatinine ratio (P < 0.001) for alendronate compared with pamidronate treatment. In previously untreated patients, alendronate resulted in remission in 20/22 (91%) subjects compared with 19/22 (86%) of pamidronate-treated subjects, which was not significantly different; however, alendronate resulted in a significantly greater reduction in ALP (P = 0.014) and DPD/creatinine ratio (P < 0.001). In previously treated patients, alendronate resulted in remission in 11/14 (79%) subjects compared with 2/14 (14%) for pamidronate (P < 0.001), with a significantly (P < 0.001) greater reduction in both ALP and DPD/creatinine ratio. Of subjects crossed over from pamidronate to alendronate, 10/14 (71%) achieved remission, including 9/11 (82%) previously treated patients. We conclude that, in patients with previously untreated Paget's disease of bone, alendronate and pamidronate have similar efficacy in achieving biochemical remission. In patients previously treated with pamidronate, alendronate is more effective.

Background and Objectives: To determine the factors contributing to changes in bone mineral density (BMD) over time in HIV-infected patients receiving highly active antiretroviral therapy (HAART)., Methods: Analyses of lumbar spine BMD in 183 male Caucasian participants in the Western Australian HIV Cohort study, comprising a longitudinal analysis of data from 54 patients on stable HAART regimens, and a cross-sectional analysis comparing data from 131 protease inhibitor (PI)-treated patients and 52 PI-naive (including 28 antiretroviral treatment-naive) patients., Results: Average lumbar spine BMD remained stable or increased over the time frame considered. Although there was no evidence of a change of average BMD over time in patients receiving nelfinavir (P = 0.92), there was evidence of increasing bone density in the indinavir group (average increase, 0.31 z-score per year; P < 0.001). Lower initial z-scores in the longitudinal analysis were significantly associated with lower pre-HAART BMI (P = 0.003), consistent with results of the cross-sectional analysis in which lowest BMI prior to initial dual X-ray absorptiometry scan was associated with decreased BMD (P = 0.02, overall group). Although PI therapy was also associated with decreased BMD in a univariate analysis of the cross-sectional data (P = 0.04), this effect was abrogated in a multiple linear regression analysis (P = 0.11) with lowest BMI remaining significant (P = 0.04)., Conclusions: We found no evidence, overall, of accelerated bone loss in patients treated with nelfinavir- or indinavir-containing HAART regimens, and propose that indinavir therapy may be associated with an increase in bone mineral density over time. Pre-HAART BMI was an independent and powerful determinant of an individual's initial z-score in the longitudinal analysis, and adjustment for this effect in a cross-sectional analysis abrogated the association between PI therapy and decreased lumbar spine z-score.

Twenty-five years after the first paper on etidronate in Paget's disease, there are few published papers that address bisphosphonate resistance as a specific clinical phenomenon. We report our data from two studies. Study 1 is a retrospective study of 20 patients with moderate to severe disease who were treated with intravenous (iv) pamidronate (221 +/- 18 mg [SEM]; range 60-360 mg), and after biochemical remission and relapse were retreated with generally larger iv dosage (293 +/- 28 mg; range 180-600 mg). The nadir bone turnover values were similar: plasma alkaline phosphatase (pAP) in 20 patients was 243 +/- 40 IU/l (mean +/- SEM) after the first course, and 267 +/- 44 IU/l after the second (reference range [RR] 35-135 IU/l). Likewise, fasting urinary hydroxyproline excretion (HypE) in 14 of the 20 patients was 4.5 +/- 1.1 micromol/LGF and 4.1 +/- 0.9 micromol/LGF, respectively (RR 0.40-1.92 micromol/LGF). However the minimum duration of biochemical remission was significantly shorter after the second course-10.9 +/- 1.7 months (first) and 5.6 +/- 0.9 months (second) (p < 0.03; Friedman's ANOVA n = 17). A subgroup of 10 patients who were followed for three courses showed a significantly higher pAP nadir in the third course. Study 2 is a prospective study of 40 patients, 23 previously untreated (NILPREV) and 17 previously treated with iv pamidronate (PAMPREV) and in biochemical relapse, who were randomly allocated to either oral alendronate 40 mg daily in 3 month units, or iv pamidronate 60 mg every 3 months. Treatment was continued until pAP and fasting urinary deoxypyridinoline/creatinine (Dpy/Cr) ratios (RR 5-27 micromol/mol) were both in the reference range, or a clear plateau in each marker developed. At baseline, there were no significant differences in either marker between the two NILPREV groups and between the two PAMPREV groups. Using log-transformed data, in NILPREV the pAP reductions were significant and similar over the first 6 months. However, although each Dpy/Cr reduction was also significant, the difference in responses favored alendronate (p < 0.015). In PAMPREV both markers showed no significant response to pamidronate; comparison showed a significantly greater response to alendronate (pAP p < 0.02; Dpy/Cr p < 0.002). Using two-way ANOVA, the pAP responses to alendronate in NILPREV and PAMPREV were similar and those to pamidronate were different (p = 0.034). The percentage of patients with both markers in the RR at 6 months or earlier were identical in NILPREV patients: alendronate 87% and pamidronate 87%. However in PAMPREV they were different: alendronate 83% and pamidronate 0% (p = 0.003). These data indicate: 1) patients treated with the same aminobisphosphonates for two courses show similar nadir values of bone turnover markers but a shorter remission time after the second course. In a third course the nadirs are significantly higher; and 2) in the alendronate/pamidronate comparison, NILPREV and PAMPREV patients showed similar pAP responses to alendronate, but significantly different responses to pamidronate. Thus, patients showing acquired partial resistance to one aminobisphosphonate (usually after two or more previous courses) are still capable of remission after exposure to another compound of the same class.