How accurate is your sclerostin measurement?:Comparison between three commercially available sclerostin ELISA kits

Sclerostin, bone formation antagonist is in the spotlight as a potential biomarker for diseases presenting with associated bone disorders such as chronic kidney disease (CDK-MBD). Accurate measurement of sclerostin is therefore important. Several immunoassays are available to measure sclerostin in serum and plasma. We compared the performance of three commercial ELISA kits. We measured sclerostin concentrations in serum and EDTA plasma obtained from healthy young (18-26 years) human subjects using kits from Biomedica, TECOmedical and from R&D Systems. The circulating sclerostin concentrations were systematically higher when measured with the Biomedica assay (serum: 35.5 ± 1.1 pmol/L; EDTA: 39.4 ± 2.0 pmol/L; mean ± SD) as compared with TECOmedical (serum: 21.8 ± 0.7 pmol/L; EDTA: 27.2 ± 1.3 pmol/L) and R&D Systems (serum: 7.6 ± 0.3 pmol/L; EDTA: 30.9 ± 1.5 pmol/L). We found a good correlation between the assay for EDTA plasma (r > 0.6; p < 0.001) while in serum, only measurements obtained using TECOmedical and R&D Systems assays correlated significantly (r = 0.78; p < 0.001). There was no correlation between matrices results when using the Biomedica kit (r = 0.20). The variability in values generated from Biomedica, R&D Systems and TECOmedical assays raises questions regarding the accuracy and specificity of the assays. Direct comparison of studies using different kits is not possible and great care should be given to measurement of sclerostin, with traceability of reagents. Standardization with appropriate material is required before different sclerostin assays can be introduced in clinical practice

Assessment of C3-Epi-25-Hydroxyvitamin D concentration in adult serum: LC-MS/MS determination using [2H3] 3-epi-25OHD3 internal standard and NIST traceable commercial 3-epi-25OHD calibrators.

Background: The C-3 Epimer of 25 Hydroxyvitamin D3 (3-Epi-25OHD3) is produced in the liver by the epimerisation pathway of 25-hydroxy vitamin D3. It differs from 25OHD3 in configuration of the hydroxyl group at the third carbon (C-3) position. Despite the fact that little is known regarding its clinical significance, concerns have been raised that isobaric interference may result in over-estimation of total 25OHD when measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Objective: The aim of the study was to assess the occurrence of 3-Epi-25OHD3 in adult serum samples. A LC-MS/MS technique was developed to resolve and quantify 3-Epi-25OHD3 from 25OHD3. The newly available NIST (SRM972a) traceable 3-Epi-25OHD commercial standards were used to ensure assay accuracy. Method: Serum was precipitated with zinc sulphate and acetonitrile containing [2H3]-3-epi-25OHD3 as internal standard. The extract was chromatographed using a 2.6µm 100 x 2.1mm I.D. solid core particle column. Mass detection and quantification were performed by positive electrospray ionization with MS/MS in multiple reaction monitoring mode. Results: The method was able to fully resolved 3-Epi-25OHD3 from 25OHD3. The intraassay CVs for the epimer were 6.3% and 4.1% at 25.4 and 62.1 nmol/L respectively; and interassay CVs were 8.3% and 6.5% at 27.6 and 63.2 nmol/L, respectively. In our sample cohort with 25OHD3 ranged between 3.4 – 165 nmol/L, 3-Epi-25OHD3 was detected in 91.9% of samples (mean = 3.8 nmol/L). No detectable 3-Epi-25OHD2 was found in our sample study. One patient sample had total 25OHD3 of 187 nmol/L that was shown to contain 141 nmol/L of 25OHD3 and 44 nmol/L of 3-Epi-25OHD3. This patient was receiving a high dose of vitamin D supplementation. Conclusion: Using [2H3]-3-epi-25OHD3 as internal standard and NIST aligned calibrators enabled us to obtain an accurate assessment of 3-epi-25OHD concentration in adult serum. Although the concentration of serum 3-epi-25OHD3 was found to be low the presence was observed in the majority of our samples. The findings in this study showed that 3-epi-25OHD3 contributed to the overestimation of 25OHD3 that could potentially resulted in misinterpretation of total vitamin D status

Interference of Asfotase alfa in immunoassays employing alkaline phosphatase technology

BACKGROUND: Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.) is the only approved treatment for patients with pediatric-onset hypophosphatasia, a disease caused by a mutation in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. ALP is often used as signaling system in routine immunoassays. Because asfotase alfa contains the active site of the full ALP enzyme, it can catalyze the substrate as the antibody-conjugated ALP would within an assay. Therefore, its presence in a treated patient's sample may generate false positive or false negative results. We investigated whether the presence of asfotase alfa within a sample induced interference in immunoassays that utilize ALP or alternative detection systems. METHODS: Asfotase alfa was added to samples at concentrations from 0.08-5 µg/mL and analysed on various immunoassays following manufacturer's instructions. RESULTS: Asfotase alfa was detected in all ALP assays but ALKP1 (RayBiotech). We observed no changes in normetanephrine and noradrenaline (IBL) at any asfotase alfa concentration. However, asfotase alfa notably interfered in an oxytocin (ENZO) assay in nonextracted samples. Extraction using a C18 column eliminated the interference. No interference was observed on automated analyzers using alternative detection system (COBAS fT4 and TSH; Advia Centaur FSH, fT4; Architect LH; FSH). Immulite 2000 fT4, TSH, testosterone and hCG (ALP-based) showed no interference. However, the presence of asfotase alfa resulted in a dose-dependent increase of Troponin I signal. CONCLUSION: The presence of asfotase alfa must be taken into consideration when analyzing blood samples in treated patients to avoid any risk of misinterpretation of false positive/negative results. It is essential that assays be tested for this possible interference

Measurement of autoantibodies against osteoprotegerin in adult human serum: development of a novel ELISA assay

Introduction: In 2009, neutralizing autoantibodies against OPG (α-OPGAb) blocking the inhibitory effect of OPG on RANK signaling pathway were identified in a man with celiac disease associated with severe osteoporosis. Although this finding was not reproduced in thirty patients presenting coeliac disease and low bone mineral density, Hauser et al (2013) recently detected the presence of α-OPGAb in patients presenting Rheumatoid Arthritis, Systemic Lupus Erythematosus, Spondyloarthritis and Osteoporosis. There is a growing focus on OPG autoantibodies as primary cause of high bone turnover in disorders with unknown etiology. Objective: To develop an enzyme linked immunosorbent assay (ELISA) for detection and quantification of α-OPGAb in patient serum samples. Method: A full-length human recombinant OPG is immobilized on a plate to allow capture of the antibodies from the sera. In a two-step reaction, the αOPGAb is detected using a biotinylated antibody and a horseradish peroxidase-labelled streptavidin. Substrate is incubated in a timed reaction and color development measured in a spectrophotometric microtiter plate reader. The concentration of human α-OPGAb in the samples is determined directly from a 4PL-fit standard curve. Results: Intra-assay imprecision was <5% at 274.4 ± 18.8 and 98.5 ± 2.9 ng/mL. Inter-assay imprecision was <20% at 324.2 ± 53.3 and 166.8 ± 30.6 ng/mL. Linear range was 0-500ng/mL. Lower and upper limit of quantification were 3.9 and 500 ng/mL. Cross reactivity was assessed against human sera containing raised thyroid antibody and RANKL to ensure assay specificity. Using the method presented, we established that the adult population would be considered positive with a titer above the cut-off limit (95%) of 68ng/mL. Our preliminary data suggested that 14% of our sample population (n=136) presented elevated α-OPGAb. Conclusion: We presented a novel ELISA assay for the detection and measurement of anti-OPG autoantibodies in human serum. The validated method showed excellent assay characteristics and is suitable for use in research and clinical hospital laboratories. In patients with severe form of osteoporosis, measurement of OPG autoantibodies could help clinicians identify appropriate treatment options for this particular subgroup of patients

Assessment of vitamin D status using MitraTM volumetric absorptive microsampling (VAMS) device

Introduction: The use of dried blood spot (DBS) sampling for general wellness assessment and in clinical diagnostics has gained popularity as a convenient and less invasive alternative to venous sampling. Collection of blood samples from a finger/heel prick using conventional filter paper suffers from variability in sample volume and spot sizes which undermine the quality of results. We describe the use of a volumetric absorptive microsampler (VAMS), called MitraTM (Torrance, CA, USA) for measurement of 25OHD3 and interpretation of vitamin D status according to current international guidelines.  Method: A liquid-chromatography mass spectrometry (LC-MS/MS) method was used for measurement of 25OHD3 (Tang et al. ASBMR 2015, LB-MO0026). We compared results from patient samples (n=97) collected by VAMS and Whatman® 903 cards extracted as whole spot (wDBS) and sub-punches (spDBS) against plasma 25OHD3 concentration. We investigated the volume displacement effects of haematocrit (Hct) on DBS 25OHD3 measurements and described the use of DBS-to-plasma equivalence value (PEV) to allow accurate interpretation of vitamin D status.  Results: VAMS showed the best assay precision CV (<8.2%) compared to wDBS (<16.6%) and spDBS (<15.1%) across the assay range of 0.1-125 nmol/L, the least variability in recovery and lowest LLoQ (Figure 1). We observed a decrease in DBS 25OHD3 concentration in proportion to the reduction in plasma volume and increase in packed cell volume. The displacement effect of Hct resulted in a strong but negatively biased correlation (r2=0.893, -39.3%) between raw DBS values and plasma concentrations, that was dependent upon the level of Hct present in sample. We demonstrated the use of simple linear regression model to transform raw DBS values into PEVs. In a subsequent cohort of patient samples (n=70), PEVVAMS produced the most accurate interpretation of vitamin D status compared to PEVwDBS and PEVspDBS.  Discussion: We present data supporting the use of VAMS for measurement of 25(OH)D3, particularly in circumstances where venesection may be impossible or difficult and where sample volume may be limited. Although the recovery of analyte remains Hct-dependent, the use of DBS-to-plasma equivalence values improves the clinical applicability and broadens the utility of DBS as a sampling technique

Effectiveness of parathyroid hormone (PTH) analogues on fracture healing: a meta-analysis

Purpose: This meta-analysis evaluated the evidence of Parathyroid Hormone (PTH) analogues in fracture healing. The use of PTH analogues to prevent osteoporotic fractures is well investigated and studies are emerging on extended indications. One such indication receiving increasing attention is the effect of PTH in fracture healing; however, the overall degree of efficacy remains inconclusive. Methods: A systematic electronic database search of MEDLINE, EMBASE and the Cochrane Library was conducted for relevant articles in August 2019 with no date restrictions. Randomised controlled trials of adults with acute fractures treated with a PTH analogue were included. PTH was compared with a comparator intervention, placebo, or no treatment. Results: PTH analogue treatment improved functional outcomes in a range of fracture types but did not affect the fracture healing rate or reduce pain. Most trials included in this review were in elderly patients with osteoporosis. There was no evidence that PTH treatment caused harm or impeded fracture healing. Conclusions: Meta-analysis of published data supports the use of PTH analogues to improve functional outcomes but not fracture healing rate or pain for different fracture types. The evidence for PTH analogue use in fracture healing is less clear in younger, nonosteoporotic patient populations. Trial design was heterogeneous and of limited quality justifying further original trials

Reference intervals for serum 24,25-Dihydroxyvitamin D and the ratio with 25-Hydroxyvitamin established using a newly developed LC-MS/MS method

24,25(OH)2D is the product of 25(OH)D catabolism by CYP24A1.The measurement of serum 24,25(OH)2D concentration may serve as an indicator of vitamin D catabolic status and the relative ratio with 25(OH)D can be used to identify patients with inactivating mutations in CYP24A1. We describe a LC-MS/MS method to determine: 1) the relationships between serum 24,25(OH)2D and 25(OH)D; 2) serum reference intervals in healthy individuals; 3) the diagnostic accuracy of 24,25(OH)2D measurement as an indicator for vitamin D status; 4) 24,25(OH)2D cut-off value for clinically significant change between inadequate and sufficient 25(OH)D status. Serum samples of healthy participants (n=1996) from Army recruits and patients (n=294) were analysed. The LC-MS/MS assay satisfied industry standards for method validation. We found a positive, concentration-dependent relationship between serum 24,25(OH)2D and 25(OH)2D concentrations. The 25(OH)D:24,25(OH)2D ratio was significantly higher (p4.2 nmol/L was identified as a diagnostic cut-off for 25(OH)D replete status. One patient sample with an elevated 25(OH)D:24,25(OH)2D ratio of 32 and hypercalcaemia who on genetic testing confirmed to have a biallelic mutation of CYP24A1. Our study demonstrated the feasibility of a combined 24,25(OH)2D and 25(OH)D assessment profile. Our established cut-off value for 24,25(OH)2D and ratio reference ranges can be useful to clinicians in the investigation of patients with an impaired calcium/phosphate metabolism and may point towards the existence of CYP24A1 gene abnormalities