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Highlights • FDA-EUA approved anti-SARS-CoV-2 tests compare favorably with others. • longitudinal patterns of antibody development are subject to high individual variability. • comparing the assays of leading manufacturers, there are considerable differences in terms of achieved and stated sensitivity/specificity. • Immune response relies on disease severity, thus need to be considered for validation., Objective Numerous immunoassays for detecting antibodies directed against SARS-CoV-2 have rapidly been developed and released. Validations of these have been performed with a limited number of samples. The lack of standardization might lead to significantly different results. This study compares ten automated assays from six vendors in terms of sensitivity, specificity, and reproducibility. Methods This study compared ten fully automated IAs from the following vendors: Diasorin, Epitope Diagnostics, Euroimmun, Roche, YHLO, and Snibe. The retrospective part of the study included patients with a laboratory-confirmed COVID-19 infection and controls comprised patients with a suspected infection, in whom the disease was excluded. Furthermore, biobanked sera were taken as negative controls (n = 97). The retrospective part involved four classes: 1) laboratory-confirmed COVID-19 infection (n = 183); 1B) suspected COVID-19 infection (n = 167) without qRT-PCR result but positive serological results from at least two different assays, and suspected COVID-19 infection due to a positive serological result from the Roche assay (n = 295); 2) biobanked sera obtained from patients before the emergence of SARS-CoV-2 (n = 97) as negative controls. 2A) probably COVID-19-negative sera with negative serological results from at least two different assays (n = 152). Results Overall diagnostic sensitivities were: Euroimmun(IgA) 87%; Epitope Diagnostics(IgG) 83%; YHLO(IgG) 77%; Roche(IgM/IgG) 77%; Euroimmun(IgG) 75%; Diasorin(IgG) 53%; Epitope Diagnostics(IgM) 52%; Snibe(IgG) 47%; YHLO(IgM) 35%; Snibe(IgM) 26%. Diagnostic specificities were: YHLO(IgG) 100%; Roche, 100%; Snibe(IgM/IgG) 100%; Diasorin(IgG) 97%; Euroimmun(IgG), 94%; YHLO(IgM) 94%; Euroimmun(IgA) 83%. Conclusion Assays from different vendors vary substantially in terms of their performance. Our findings might facilitate the selection of appropriate serological assays.

Background/aim Exposure of the skin to sunshine is the major natural source of vitamin D. In order to imitate this natural production of vitamin D for patients with chronic kidney disease, hemodialysis patients were exposed three times a week to radiation of the front part of both legs to normalize the vitamin D status. Patients and methods Partial body UVB irradiation was performed during the routine dialysis session. Twenty-two patients took part, with a mean age of 61.7 (range=35-81) years. Results Serum levels of 25(OH)D3 and 1,25(OH)2D3 increased into the mid normal range. Intact parathyroid hormone decreased by 25% and osteocalcin by 45%. 24-Hour blood-pressure monitoring demonstrated decreases in systolic and diastolic blood pressure. Conclusion Partial body exposure to UVB radiation normalized not only the serum level of 25(OH)D3, but also that of 1,25(OH)2D3, which resulted in a significant decrease in parathyroid hormone, osteocalcin levels, and also in blood pressure. Sunshine imitating UVB exposure utilizes the capacity of the skin to convert extrarenally vitamin D3 to 25(OH)D3 and 1,25(OH)2D3.

This study compared features of the metabolic syndrome between healthy controls and depressed patients without activation of the hypothalamus-pituitary-adrenal (HPA) system. After exclusion of non-suppressors to 1mg dexamethasone, we included 20 depressed inpatients and 34 healthy controls in the analyses. We assessed HPA system activity (diurnal saliva cortisol profile, cortisol excretion), normetanephrine excretion as well as fasting glucose, lipid profile and blood pressure. With regard to body composition, we measured waist circumference as well as visceral fat and adrenal volume by magnetic resonance (MR) imaging. Five depressed patients (25%) and five healthy controls (15%) fulfilled the criteria of the metabolic syndrome according NCEP-ATP-III. Depression was significantly related with fasting glucose and negatively associated with mean blood pressure (BP) and, by trend, with low HDL-cholesterol. We conclude that depressed patients may have modest metabolic disturbances even in the complete absence of activation of stress-responsive systems. Hence some metabolic disturbances in depressed patients may not be explicable by HPA activation. Additional factors are required to mediate the link between affective and metabolic disorders.

In chronic kidney disease (CKD) a deficiency of 1,25-dihydroxyvitamin D is common. The aim of this review was to compare vitamin D status after oral supplementation of vitamin D3 to that of serial suberythemal irradiation in end-stage kidney disease (ESKD) patients.Ninety-five patients, with a mean age of 62 (range=35-82) years, were treated with a mean dose of 35,000 (20,000-60,000) IU vitamin D3 per week for a period of 18 months. Fourteen patients, with a mean age of 51 (range=41-57) years, were whole-body UVB irradiated for over 6 months. From 3 hemodialysis patients skin biopsies were performed.With oral supplementation 25(OH)D3 increased by 60%. With UV irradiation 25(OH)D3 increased by 400%. Gene expression analysis demonstrated an improvement in the vitamin D receptor (VDR) by 0.65 fold, in 1-alpha-hydroxylase (CYP27B1) by 1.0 fold, and in 25-hydroxylase (CYP2R) by 1.2 fold.Serial suberythemal UVB irradiation of patients with CKD on dialysis is capable to improve serum 25(OH)D3 and 1,25(OH)2D3 by enhancing the skin's ability to activate vitamin D.

Summary Background Most recently, a new rapid and fully automated electrochemiluminescence immunoassay for the determination of TSH receptor autoantibodies (TRAb) based on the ability of TRAb to inhibit the binding of a human thyroid-stimulating monoclonal antibody (M22) has been established. Objective To evaluate this assay system in clinical routine based on an international multicentre trial and to compare the results with other established TRAb assays. Patients and measurements Totally 508 Graves’ disease (GD), 142 autoimmune thyroiditis, 107 subacute thyroiditis, 109 nonautoimmune nodular goitre, 23 thyroid cancer patients and 446 normal controls were retrospectively evaluated. Results ROC plot analysis revealed an area under curve of 0·99 (95% CI: 0·99–1·0) indicating a high assay sensitivity and specificity. The highest sensitivity (99%) and specificity (99%) was seen at a cut-off level of 1·75 IU/l. Here, the calculated positive predictive value was 95%, whereas the negative predictive value was 100%. Applying the ROC plot-derived cut-off of 1·75 IU/l we found a sensitivity for TRAb positivity within the group of newly diagnosed GD patients of 97% which is in accordance to the sum of different nonautomated porcine TSH receptor-based assays with a sensitivity of 94% indicating an excellent analytical performance of the new assay format. Detailed comparison of the automated and the sum of manual assays revealed a near identical specificity. Conclusion Our results demonstrate that this new assay system has a high sensitivity for detecting GD and specificity for discriminating from other thyroid diseases. This assay may represent the future technology for rapid fully automated TRAb detection.

Background: Carbohydrate-deficient transferrin (CDT) is a promising biomarker of alcohol abuse. We describe the development and multicenter evaluation of N Latex CDT (Dade Behring), an automated, particle-enhanced, homogeneous immunonephelometric assay for directly determining CDT.Methods: N Latex CDT uses a monoclonal antibody that recognizes the structure of transferrin glycoforms lacking 1 or 2 complete N-glycans [i.e., disialo-, monosialo-, and asialotransferrins (CDT glycoforms)] in combination with a simultaneous assay for total transferrin. The Dade Behring BN II™ and BN ProSpec® systems automatically calculate the CDT value as a percentage of total transferrin (%CDT). No preanalytical sample treatment is used.Results: Total imprecision values for serum pools containing 1.8%–8.7% CDT were 3.4%–10.4% (mean, 6.8%). The mean (SD) %CDT for 561 serum samples from healthy control individuals was 1.76% (0.27%; range, 1.01%–2.85%). No marked sex or age differences were noted. The 97.5th percentile was at 2.35%. Transferrin genetic variants did not interfere with measurements. High transferrin concentrations did not falsely increase %CDT values, but increased %CDT values were noted for some samples with transferrin concentrations Conclusion: N Latex CDT is the first direct immunoassay for quantifying %CDT in serum. The specificity of N Latex CDT for identifying alcohol abuse may be higher than for immunoassays that use column separation, because transferrin genetic variants do not interfere with measurements.

Background Measuring plasma adrenocorticotropic hormone (ACTH) is a key step in the differential diagnosis of hypothalamic–pituitary–adrenal disorders. Methods The recently developed electrochemiluminescence Elecsys ACTH immunoassay (Roche Diagnostics, Mannheim, Germany) was evaluated at six clinical laboratories on the Modular E170 and/or the Elecsys 2010 (Roche Diagnostics) immunoanalysers. Results The within-run and between-run imprecision was ≤ 1.9% and ≤ 5%, respectively (concentration level 12–971 ng/L). Functional sensitivity was at least 1.8 ng/L. The Elecsys assay correlated well with two established ACTH immunoradiometric assays (Brahms ACTH RIA and Nichols ACTH IRMA) and with the Immulite ACTH assay. Reference values for the Elecsys ACTH assay, determined as the percentile 5 to 95 (2.5 to 97.5) interval in 354 adults without disorder with impact on the hypothalamic–pituitary–adrenal axis, were between 7.2 and 63 ng/L (5.6 and 76 ng/L). The clinical sensitivity of the Elecsys ACTH assay in 57 patients with confirmed disorders of the pituitary–adrenal axis was comparable to the established Brahms ACTH assay. Conclusion The non-isotopic Elecsys ACTH immunoassay provides precision and reliability in combination with reduced turnaround time.

Background To compare the effects of resistance training versus passive physical therapy on bone turnover markers (BTM) in the metastatic bone during radiation therapy (RT) in patients with spinal bone metastases. Secondly, to evaluate an association of BTM to local response, skeletal-related events (SRE), and number of metastases. Methods In this randomized trial, 60 patients were allocated from September 2011 to March 2013 into one of the two arms: resistance training (Arm A) or passive physical therapy (Arm B) with thirty patients in each arm during RT. Biochemical markers such as pyridinoline (PYD), desoxy-pyridinoline (DPD), bone alkaline phosphatase (BAP), total amino-terminal propeptide of type I collagen (PINP), beta-isomer of carboxy-terminal telopeptide of type I collagen (CTX-I), and cross-linked N-telopeptide of type I collagen (NTX) were analyzed at baseline, and three months after RT. Results Mean change values of PYD and CTX-I were significantly lower at 3 months after RT (p = 0.035 and p = 0.043) in Arm A. Importantly, all markers decreased in both arms, except of PYD and CTX-I in arm B, although significance was not reached for some biomarkers. In arm A, the local response was significantly higher (p = 0.003) and PINP could be identified as a predictor for survivors (OR 0.968, 95%CI 0.938–0.999, p = 0.043). BAP (OR 0.974, 95%CI 0.950–0.998, p = 0.034) and PINP (OR 1.025, 95%CI 1.001–1.049, p = 0.044) were related with an avoidance of SRE. Conclusions In this group of patients with spinal bone metastases, we were able to show that patients with guided resistance training of the paravertebral muscles can influence BTM. PYD and CTX-I decreased significantly in arm A. PINP can be considered as a complementary tool for prediction of local response, and PINP as well as BAP for avoidance of SRE. Trial registration Clinical trial identifier NCT 01409720. August 2, 2011.

Background: The carboxy-terminal cross-linking telopeptide of type I collagen (β-CrossLaps, β-CTX) is released into the circulation during degradation of type I collagen and serves as a marker of bone resorption. β-CTX is known to undergo a diurnal rhythm in normal individuals and to accumulate in chronic renal failure. β-CTX has a potential role in noninvasive diagnosis of renal bone disease. Methods: Serum β-CTX was compared to parathyroid hormone (PTH) and other biochemical bone markers in 90 unselected hemodialysis patients. Results: Mean β-CTX was elevated above the normal range (1.72 ± 0.93 µg/l); there were large individual variations. Serum β-CTX was significantly correlated with various PTH assays (r >0.56) and with tartrate-resistant acid phosphatase 5b (TRACP 5b, r = 0.629), bone-specific alkaline phosphatase (r = 0.404) and osteocalcin (r = 0.534, all correlations p < 0.001). The correlation between β-CTX and PTH was significantly higher than the correlation between TRACP 5b and PTH. Several factors which could confound interpretation of serum β-CTX were assessed in further studies: (i) There was no recognizable influence of the time of blood sampling (morning dialysis shift versus afternoon dialysis shift) on serum β-CTX. (ii) Serum β-CTX was not significantly related to residual diuresis of patients. Conclusions: We found a high association between β-CTX and other established markers of bone and calcium metabolism demonstrating the potential utility of β-CTX as marker of bone resorption in renal bone disease. However, further studies employing bone histology are still warranted to exactly define the influence of glomerular retention on serum β-CTX in end-stage renal disease.

Background The Roche Elecsys Vitamin D Total competitive protein-binding assay uses recombinant vitamin D binding protein for measuring 25-hydroxyvitamin D (25-OHD), which is different from commonly used antibody assays. Methods The assay, standardized against LC-MS/MS, was tested at four sites. Evaluation included precision; between-laboratory variability; functional sensitivity; correlation to LC-MS/MS, HPLC, and immunoassays; as well as robustness, traceability, and EQAS performance. Results Precision testing showed within-run coefficient of variations (CVs) of ≤7%, within-laboratory CVs of

CONTEXT: Relations between fibroblast growth factor-23 (FGF-23), soluble α-klotho (s-α-klotho), and kidney function in chronic kidney disease (CKD) are still unclear. Especially the role of s-α-klotho requires further study.OBJECTIVES: Our objectives were to analyze the relation of s-α-klotho to estimated glomerular filtration rate (eGFR), FGF-23, and other parameters of calcium-phosphate metabolism and to investigate the response of s-α-klotho to cholecalciferol.PATIENTS, DESIGN, AND SETTING: Twenty-four CKD (stage 1-5) patients participated in this 8-week randomized controlled trial (vitamin D and chronic renal insufficiency).INTERVENTIONS: Interventions included 40 000 IU cholecalciferol or placebo weekly.MAIN OUTCOME MEASURE: S-α-klotho was determined by ELISA with antihuman klotho antibodies 67G3 and 91F1.RESULTS: For all patients, s-α-klotho concentrations did not differ between CKD stages. When patients were subdivided based on FGF-23 concentrations, a positive association of s-α-klotho with eGFR became apparent in patients with lower than median FGF-23 concentrations but not in those above median value. Patients with s-α-klotho below 204 pg/mL showed higher age, lower phosphate clearance, and lower bone-specific alkaline phosphatase compared with patients with higher s-α-klotho. Treatment with cholecalciferol significantly increased 1,25-dihydroxyvitamin D. The increase of FGF-23 had only borderline significance. There was no significant effect of high-dose cholecalciferol administration for 8 weeks on plasma s-α-klotho.CONCLUSIONS: CKD patients with s-α-klotho below 204 pg/mL had higher age, lower phosphate clearance, and lower bone-specific alkaline phosphatase. An association of s-α-klotho with eGFR was observed only in the presence of close to normal, but not high, FGF-23 concentrations. Cholecalciferol treatment did not change s-α-klotho concentrations.

BACKGROUND: It was shown that nonoxidized PTH (n-oxPTH) is bioactive, whereas the oxidation of PTH results in a loss of biological activity.METHODS: In this study we analyzed the association of n-oxPTH on mortality in hemodialysis patients using a recently developed assay system.RESULTS: Hemodialysis patients (224 men, 116 women) had a median age of 66 years. One hundred seventy patients (50%) died during the follow-up period of 5 years. Median n-oxPTH levels were higher in survivors (7.2 ng/L) compared with deceased patients (5.0 ng/L; P = .002). Survival analysis showed an increased survival in the highest n-oxPTH tertile compared with the lowest n-oxPTH tertile (χ², 14.3; P = .0008). Median survival was 1702 days in the highest n-oxPTH tertile, whereas it was only 453 days in the lowest n-oxPTH tertile. Multivariable-adjusted Cox regression showed that higher age increased odds for death, whereas higher n-oxPTH reduced the odds for death. Another model analyzing a subgroup of patients with intact PTH (iPTH) concentrations at baseline above the upper normal range of the iPTH assay (70 ng/L) revealed that mortality in this subgroup was associated with oxidized PTH but not with n-oxPTH levels.CONCLUSIONS: The predictive power of n-oxPTH and iPTH on the mortality of hemodialysis patients differs substantially. Measurements of n-oxPTH may reflect the hormone status more precisely. The iPTH-associated mortality is most likely describing oxidative stress-related mortality.

Markers of bone formation determined in serum include alkaline phosphatase, bone-specific alkaline phosphatase, osteocalcin [bone gamma-carboxyglutamic acid peptide (BGP) and procollagen type I carboxyterminal propeptide. Recently, advances have been made in the immunoassay of bone-specific alkaline phosphatase. This is a marker for osteoblastic activity; it is very stable and is not primarily dependent on kidney function because it is degraded in the liver. BGP is not specific for bone formation because it increases in serum during bed rest (which involves increased bone resorption), and it is not stable. Furthermore, the elimination of BGP is dependent on glomerular filtration rate. Procollagen type I carboxyterminal propeptide is not as sensitive as bone-specific alkaline phosphatase because it increases less in women after the menopause. Urinary pyridinoline and deoxypyridinoline determined by high-performance liquid chromatography are regarded as the best methods for measuring bone resorption. These might be replaced by type I collagen crosslinked N-telopeptide or CrossLaps in the future in laboratories not equipped with a high-performance liquid chromatography system. Serum markers of bone resorption are currently under investigation. An immunoassay for the tartrate-resistant acid phosphatase in serum should be a very promising tool for the quantification of bone resorption.

The goal was to analyze the link between blood levels of calcidiol and all-cause, cardiac and infectious diseases, and mortality due to cancer in hemodialysis patients.This study retrospectively evaluated a representative sub-cohort (n=6,518) of German hemodialysis patients from the incidence cohorts 1997-2006.Most (58.8%) were found to be vitamin D deficient (25(OH)D20 ng/ml), with 41.2% being severely deficient (25(OH)D12.5 ng/ml). All-cause mortality risk more than doubled in patients with severe deficiency (adjusted odds ratio (aOR)=2.67; 95% confidence interval (CI)=2.30-3.10; p0.0001). Comparable data were obtained for mortality from cardiac disease (aOR=1.57; 95% CI=1.30-1.88; p0.0001), infectious disease (aOR=1.48; 95% CI=1.15-1.90; p=0.0026), and cancer (aOR=1.51; 95% CI=1.09-2.08; p=0.0121), respectively.These data highlight the need to ensure primarily adequate 25(OH)D levels in dialysis patients for an advantage of survival.

Oxidation of PTH at methionine residues results in loss of biological activity. PTH may be oxidized in patients with renal disease. The aim of this study was to develop an assay considering oxidation of PTH. Oxidized hPTH was analyzed by high resolution nano-liquid chromatography coupled to ESI-FTT tandem mass spectrometry (nanoLC-ESI-FT-MS/MS) directly and after proteolytic cleavage. The oxidized hPTH(1–84) sample shows TIC-peaks at 18–20 min and several mass peaks due to mass shifts caused by oxidations. No significant signal for oxidized hPTH(1–84) species after removal of oxidized PTH molecules by a specific column with monoclonal antibodies (MAB) raised against the oxidized hPTH was detectable. By using this column in samples from 18 patients on dialysis we could demonstrate that measured PTH concentrations were substantially lower when considering oxidized forms of PTH. The relationship between PTH concentrations determined directly and those concentrations measured after removal of the oxidized PTH forms varies substantially. In some patients only 7% of traditionally measured PTH was free of oxidation, whereas in other patients 34% of the traditionally measured PTH was real intact PTH. In conclusion, a huge but not constant proportion of PTH molecules are oxidized in patients requiring dialysis. Since oxidized PTH is biologically inactive, the currently used methods to detect PTH in daily clinical practice may not adequately reflect PTH-related bone and cardiovascular abnormalities in patients on dialysis.

Objectives A new electrochemiluminescence testosterone immunoassay (Elecsys® Testo II) was evaluated. Design and methods Within-run precision, total precision, and method comparisons to the former immunoassay and LC-MS/MS were investigated. Reference intervals were established for adults and children. Results Imprecision ranged from 0.9% to 8.5%. Method comparisons revealed slopes from 0.68 to 1.32 and coefficients of correlation from 0.893 to 0.991. Conclusions Improved agreement with LC-MS/MS was demonstrated, especially for female patients. The assay may be a suitable alternative for laboratories with no access to MS techniques.

Summary During and after prolonged bed rest, changes in bone metabolic markers occur within 3 days. Resistive vibration exercise during bed rest impedes bone loss and restricts increases in bone resorption markers whilst increasing bone formation. Introduction To investigate the effectiveness of a resistive vibration exercise (RVE) countermeasure during prolonged bed rest using serum markers of bone metabolism and whole-body dual X-ray absorptiometry (DXA) as endpoints. Methods Twenty healthy male subjects underwent 8 weeks of bed rest with 12 months follow-up. Ten subjects performed RVE. Blood drawings and DXA measures were conducted regularly during and after bed rest. Results Bone resorption increased in the CTRL group with a less severe increase in the RVE group (p = 0.0004). Bone formation markers increased in the RVE group but decreased marginally in the CTRL group (p

Extracorporeal shock wave therapy (ESWT) has recently been demonstrated to improve skin flap survival. In all these studies EWST was applied immediately after the surgical intervention. Thus, the purpose of this study was to determine the preoperative effect of ESWT as a noninvasive technique to precondition flap tissue in a rat epigastric skin flap model. EWST and control groups each contained 10 animals. ESWT was applied 7 days before the surgical intervention, whereas the control group received no treatment. Follow-up evaluation was performed on postoperative day 5. The mean area of flap necrosis, expressed as a percentage of the total flap area, was calculated. A significant reduction of the average flap necrosis area was observed in the ESWT group (27.2% +/- 9.6%) compared with the control group (46.1% +/- 7.9% (P < 0.05). In summary, this study indicates that preoperative ESWT may enhance skin flap survival in a rodent model.

Background: Hyperthyroidism in Graves' disease (GD) is often associated with the production of autoantibodies (TRAb) to the thyrotropin receptor (TSHR). Current manual second generation TRAb assays demonstrate high clinical sensitivity, but are labor-intensive and time consuming. Until recently, technical difficulties prevented the availability of an automatic TRAb assay. Methods: Development of a fast and fully automated TRAb assay on the Elecsys ® /cobas ® e electrochemiluminescence immunoassay platform. Results: A labeled thyroid-stimulating human monoclonal TSHR autoantibody (M22) was used in an automated M22-binding inhibition assay. TRAb are detected by their ability to competitively inhibit M22-binding to solubilized porcine TSHR (pTSHR). High clinical sensitivity could be maintained by assembling multiple TSHR binding sites within a soluble oligomeric immunocomplex for improved TRAb binding. Requirement of sufficient on board stability of the delicate TSHR structure in solution for several days was met by pre-complexation of the pTSHR with a capture antibody to its C-terminus in combination with the use of structure-stabilizing chemical chaperones. Total imprecision coefficient of variation (CV) at 1.71 (approximate cut-off) was found to be 11.4%. TRAb results were available within 30 min. Conclusions: Availability of a fast and automatic TRAb assay offers an attractive alternative to the manual TRAb assays for the differential diagnosis of hyperthyroidism.

Most recently, a new rapid and fully automated electrochemiluminescence immunoassay for the determination of TSH receptor autoantibodies (TRAb) based on the ability of TRAb to inhibit the binding of a human thyroid-stimulating monoclonal antibody (M22) has been established.To evaluate this assay system in clinical routine based on an international multicentre trial and to compare the results with other established TRAb assays.Totally 508 Graves' disease (GD), 142 autoimmune thyroiditis, 107 subacute thyroiditis, 109 nonautoimmune nodular goitre, 23 thyroid cancer patients and 446 normal controls were retrospectively evaluated.ROC plot analysis revealed an area under curve of 0.99 (95% CI: 0.99-1.0) indicating a high assay sensitivity and specificity. The highest sensitivity (99%) and specificity (99%) was seen at a cut-off level of 1.75 IU/l. Here, the calculated positive predictive value was 95%, whereas the negative predictive value was 100%. Applying the ROC plot-derived cut-off of 1.75 IU/l we found a sensitivity for TRAb positivity within the group of newly diagnosed GD patients of 97% which is in accordance to the sum of different nonautomated porcine TSH receptor-based assays with a sensitivity of 94% indicating an excellent analytical performance of the new assay format. Detailed comparison of the automated and the sum of manual assays revealed a near identical specificity.Our results demonstrate that this new assay system has a high sensitivity for detecting GD and specificity for discriminating from other thyroid diseases. This assay may represent the future technology for rapid fully automated TRAb detection.

Background Graves' disease (GD) is mediated by autoantibodies which bind to the TSH receptor (TRAb). The aim of the present study was to evaluate the technical performance of the first fully automated immunoassay for TRAb detection. Methods The Elecsys® Anti-TSHR immunoassay utilizes a porcine TSH receptor (TSHR) and the human thyroid stimulating monoclonal TSHR autoantibody M22. Results Intraassay and total imprecision CV were determined between 1.4%–14.9%, and 2.4%–28.8%, respectively. Using the 20% CV criteria the functional sensitivity was found at 0.73 IU/L. The median CV at the cut-off (1.75 IU/L) was found to be 11%. Comparison studies with five TRAb immunoassays yielded slopes and intercepts between 1.02–1.48, and − 0.74–0.56, respectively. Correlation coefficients were determined between 0.895 and 0.978. ROC plot analysis of patients with GD, patients with other thyroid disorders and healthy controls revealed an AUC of 0.99 resulting in a sensitivity of 97% and a specificity of 99% at a TRAb level of 1.75 IU/L. Conclusion The evaluation of the TRAb immunoassay generated homogeneous performance data and demonstrated a high degree of comparability to established TRAb assays. The automated TRAb assay represents a major improvement of thyroid testing in clinical practice.

Background The most reliable assessment of vitamin D status is measurement of plasma 25-hydroxyvitamin D (25[OH]D) concentration. High variability in 25(OH)D measurements due to utilized test and assay technologies and the lack of standardization against reference materials and reference method often confounds proper assessment of vitamin D status. Methods We evaluated the accuracy of six routinely available methodologies: high-performance liquid chromatography (HPLC), the IDS-radioimmunoassay (IDS-RIA) and enzyme immunoassay (IDS-EIA), the Nichols Advantage automated protein-binding assay (Advantage), two versions of the DiaSorin automated immunoassay (Liaison 1 and Liaison 2) – and one prototype automated immunoassay (Elecsys) for assessment of the 25(OH)D3 status in a cohort of 300 randomly selected patients' samples compared with the reference method liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results Passing-Bablok regression analysis demonstrated a slope for each method compared with LC-MS/MS that varied from 0.62 (IDS-EIA) to 1.0 (HPLC). The Advantage and the Liaison 1 showed significant deviation from linearity with highly variable individual results vs. the LC-MS/MS. Difference plots revealed a considerable persistent proportional bias for the IDS-RIA and IDS-EIA. All evaluated methods except HPLC demonstrated a more or less considerable deviation of individual 25(OH)D3 values compared with LC-MS/MS defined target concentrations. Conclusions Standardization of methods for the quantification of 25(OH)D on a human-based sample panel by means of LC-MS/MS would help to reduce the inter-method variability with respect to accuracy existing in 25(OH)D measurement considerably. However, there will still remain differences in the accuracy of methods utilizing sample purification before final quantification or immunological reaction when compared with those methods without separate sample purification.

Hyperprolactinaemia due to macroprolactin (MPRL) can lead to misdiagnosis and inappropriate treatment. We studied the new Roche Elecsys Prolactin assay (PRL II) which has been developed to reduce reactivity with MPRL. We investigated the performance of the PRL II assay at six laboratory sites to determine precision and establish reference intervals for total immunoreactive PRL and for monomeric PRL determined by precipitation with polyethylene glycol (PEG). We compared the reactivities with macroprolactin of the PRL II, the PRL I and seven other PRL assays. The PRL II assay reacted less strongly than the PRL I assay and similarly to the ADVIA Centaur assay with macroprolactin. PEG precipitation can be used with the PRL II assay to estimate the concentration of monomeric PRL.

Continuous monitoring of ciclosporin whole blood levels is essential to be aware of any change in drug pharmacokinetics due to interaction with additionally applied drugs, phytochemical drugs or nutritional and environmental factors. While monitoring ciclosporin levels in a 23-year old Caucasian woman diagnosed to have focal segmental glomerulosclerosis a 6-fold higher value than expected was measured. Because the kind and the dose of prescribed drugs were unchanged a drug to drug interaction could be ruled out. Additionally, phytochemical drugs, antibiotics, antimycotics or nutritional factors such as grapefruit juice as well as alcohol and tobacco were denied by the patient. She kept her protein and sodium poor diet free of citrus fruits, rare in meat and rich in vegetables unchanged before and during the course of the unexpected high ciclosporin levels. As the elevated ciclosporin levels were closely related to her cooking habits by means of a brand new steamer we advised her to refrain from preparing her vegetarian meals in this steamer. Since then ciclosporin levels were again in relation to the administered dosage without any episode of unexpected high ciclosporin values. Repeating the experiment some weeks later no episode of unexpected high ciclosporin values occurred. Unfortunately we could only identify the cause (steamer) but not the definite reason why. The report illustrates that the bioavailability of ciclosporin may not only be influenced by drug to drug interactions with prescribed or phytochemical drugs and herbal remedies but also by some yet unknown or unidentified factors or chemicals induced or altered by eating and cooking behavior or lifestyle in general. Therefore a careful assessment of the patient's anamnestic data and lifestyle combined with an unorthodox way of thinking as well as close-meshed drug monitoring is essential to obtain the desired pharmacologic effects and to avoid adverse toxic effects.

The E170 module was evaluated at 13 sites in an international multicentre study. The objective of the study was to assess the analytical performance of 49 analytes, and to collect feedback on the system's reliability and practicability. The typical, within-run coefficients of variation (CVs) for most of the quantitative assays ranged between 1 and 2% while a range of 2-4% was achieved with the infectious disease methods. Total precision CVs were found to be within the manufacturer's expected performance ranges, demonstrating good concordance of the system's measuring channels and a high reproducibility during the 2-4-week trial period. The functional sensitivity of 11 selected assays met the clinical requirements (e.g., thyreotroponin (TSH) 0.008 mU/l, troponin T 0.02 microg/l, total prostate-specific antigen (PSA) 0.03 microg/l). The E170 showed no drift during an 8-hour period and no relevant reagent carryover. Accuracy was confirmed by ring trial experiments and method comparisons vs. Elecsys 2010. The reliability and practicability of the system's hardware and software met with, or even exceeded, the evaluator's requirements. Workflow studies showed that E170 can cover the combined workload of various routine analysers in a variety of laboratory environment. Throughput and sample processing time requirements were achieved while personnel 'hands-on-time' could be reduced.

Introduction Biochemical markers of bone metabolism have been mainly determined manually until now and the precision and accuracy of these methods have not always been satisfactory. This has been shown in several external quality assessment schemes (EQAS). Objective and study design A study named BIOROSE was undertaken to evaluate new automated assays for serum markers of bone metabolism. The main focus was to evaluate the assay performance in a multicenter setting with 20 laboratories participating in Germany. The evaluation consists of a familiarization phase to determine precision and accuracy and an EQAS to evaluate the comparability between laboratories. Materials The parameters beta-CrossLaps (CTX), N-MID-Osteocalcin (OC) and intact parathyroid hormone (PTH) were measured with reagents including calibrators and control sera obtained from Roche Diagnostics, Mannheim, Germany, with electrochemiluminescence immunoassays (ECLIA) on the automated analyzer Elecsys 2010. Results We calculated for the control samples, PCB 1-3, the mean and median values from the measured values of all participating laboratories and used these as target values. From these target values, a recovery range for the participating laboratories was calculated for beta-CrossLaps, OC and intact PTH of better than 80-126% for PCB 2 and PCB 3, and for PCB 1 (low concentration range) for beta-CrossLaps 79-129%, OC 90-120% and intact PTH 78-126%. The between-day imprecision was 2.4-7.2% for beta-CrossLaps, 1.1-5.9% for OC and 1.7-5.5% for intact PTH in the elevated range (sample PCB 2). In the EQAS, the inter-laboratory imprecision for beta-CrossLaps in the sample with a value of 0.8 ng/ml (above the upper limit of normal, which is 0.6 ng/ml) was 9.8% on day 1 and 9.7% on day 2. Conclusion The performance evaluation of automated assays for beta-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone in the BIOROSE multicenter study showed that the participating laboratories had no problems in setting up these methods and they yielded results for precision and accuracy that are superior to results achieved in external quality assessment schemes for manually performed methods. In addition, at the clinically important decision level of the upper limit of the normal range, all three tested analytes gave precise results that improved medical decisions.

Determination of plasma parathyroid hormone (PTH) is routinely performed to diagnose and monitor renal bone disease. Recently, a new PTH assay ('whole PTH') using an antibody directed specifically against PTH(1-4) has been introduced. It was the aim of the current study to evaluate whole PTH and parameters derived from whole PTH in renal bone disease.The following measurements were carried out in blood samples from 141 unselected haemodialysis patients: three intact PTH assays (Nichols, Roche Elecsys), Scantibodies total); whole PTH (Scantibodies); bone-specific alkaline phosphatase (bAP); tartrate-resistant acid-phosphatase 5b (TRAP 5b); osteocalcin, 25-hydroxyvitamin D. Parameters derived from whole PTH were: (i) non-PTH(1-84), difference between intact PTH (Scantibodies assay) and whole PTH; (ii) whole PTH/non-PTH(1-84) ratio.The values generated by the intact PTH assays were comparable. The mean whole PTH concentration was lower than mean intact PTH concentrations (16.9+/-18.1 vs 26.4+/-30.5 pmol/l, Nichols, P0.05). The correlation coefficients between all four PTH assays were comparable and were very high (r0.96, ns). The rank order of values generated by the whole PTH assay was statistically not significantly different from the rank order generated by the Nichols intact PTH assay. The median non-PTH(1-84) concentration was 5.2 pmol/l (range 0-49.4). All PTH assays correlated highly significantly with non-PTH(1-84) (correlation coefficients 0.83-0.92). Corrected serum calcium was also associated with non-PTH(1-84) but the correlation was weaker (r=0.28). Regression analysis indicated that the non-PTH(1-84) concentration could be predicted by 76.6-84.6% by the prevailing intact PTH concentrations. Other parameters contributed only marginally to prediction of non-PTH(1-84). In the entire patient group, there was no statistically significant correlation between the whole PTH/non-PTH(1-84) ratio and any of the PTH assays or biochemical bone markers. Eight of 141 patients had a whole PTH/non-PTH(1-84) ratio1. TRAP 5b, bAP and osteocalcin had high correlations with intact PTH assays and the whole PTH assay (correlation coefficients 0.51-0.56, no significant difference). None of the PTH assays was superior to any other PTH assay in predicting serum concentrations of the bone markers. Therapy with active vitamin D metabolites (n=70) did not alter the results of our analyses.With respect to information about bone turnover we were not able to find differences between whole PTH and intact PTH assays. Our data also suggest that whole PTH and intact PTH assays give similar information. (i) The correlation between all PTH assays was very high. (ii) The rank order between whole PTH and Nichols intact PTH assays was comparable. (iii) The association between intact PTH assays and non-PTH(1-84) was very high. Albeit non-PTH(1-84) was mostly determined by the prevailing intact PTH concentration, diagnostic information on parathyroid activity provided by whole PTH or intact PTH, respectively, may differ in individual patients. How often this would happen cannot be answered with the currently available data. Unequivocal structural identification of the non-PTH(1-84) fraction would facilitate the answer to that question. The use of the whole PTH/non-PTH(1-84) ratio as a biochemical bone marker in renal bone disease requires further investigation.

The determination of parathyroid hormone (PTH) is of great clinical relevance in the assessment of calcium metabolic disorders. Although PTH was one of the first hormones measured by immunoassays, there are still many difficulties in its determination due to the low concentration of the hormone in blood and due to the heterogeneity of PTH resulting from different circulating hormone fragments. The aim of our multicenter-study was to evaluate the technical performance and the clinical validity of a new immunoassay for intact PTH measurement on the Elecsys Systems 2010 and 1010.The multicenter evaluation was performed in 11 clinical laboratories. The Elecsys PTH assay is a one step sandwich electrochemiluminescence immunoassay based upon the streptavidin-biotin technology. Two monoclonal antibodies are used in the assay providing detection of intact PTH. The imprecision study yielded within-run and between-days coefficients of variation of 3.1% - 6.6% and 3.4% - 15.6%, respectively using a three level control (PreciControl Bone, Roche Diagnostics) and human pool sera at two different concentrations (HS-low: 20 - 60 pg/ml, HS-high65 pg/ml). The analytical sensitivity calculated as the mean value plus 2 standard deviations of a within-run imprecision was below 2.70 pg/ml using zero calibrator matrix. Dilution linearity was observed up to 4890 pg/ml using zero calibrator matrix or human pool sera. Recoveries ranged between 85% - 115%. Serum, EDTA- and heparin plasma were evaluated for PTH measurement. Due to a better analyte stability (48h at 21 degrees C; 3d at 4 degrees C) EDTA plasma was recommended for PTH measurement. Results of the Elec sys PTH immunoassay correlated well (r = 0.926 - 0.994) with three different immunoradiometric assays (N-tact PTH SP, DiaSorin; Nichols Allegro Intact PTH, Nichols Institute Diagnostics; ELSA-PTH, CISBio International) and two different immunochemiluminometric assays (PTH-Intact-Immulite, DPC Biermann; Nichols Advantage Intact PTH, Nichols Institute Diagnostics) in technical and clinical method comparisons. The Passing/Bablok regression analysis yielded slopes of 0.692 - 1.729 and intercepts of -13.982 - +15.763 pg/ml. Deviations from slope 1.0 and intercept 0.0 were not unexpected due to differences in immunoassay standardization and probably due to the presence of different PTH fragments and a variable affinity of the used antibodies to these PTH fragments. Highly similar PTH concentration pattern of the Elecsys immunoassay and the Quick-Intraoperative Intact PTH immunoassay (Nichols Institute Diagnostics) obtained from specimens taken intraoperatively support the applicability of the Elecsys immunoassay to monitor the success of parathyroid resection. A reference range of 12.3 - 56.0 pg/ml calculated from PTH values of 43 apparently healthy individuals confirms reference limits published in the literature. The partition of collectives according to age showed, that individuals50 years have slightly higher PTH concentrations, independently of gender. This shift could be due to age itself or to an increased prevalence of individuals without obvious calcium metabolic disorders in this collective.The Elecsys PTH assay is a useful and reliable tool for determination of intact PTH. Our data support the intended use of the assay in clinical applications related to disorders of calcium metabolism.

Sir, Uraemic pruritus, now better known as chronic kidney disease-related pruritus (CKD-rP), is still a commonly experienced, tormenting and challenging symptom in patients with chronic kidney diseases [1]. There has been an ongoing discussion whether pruritus in chronic kidney disease is brought about by the common disturbance of calcium/phosphate homeostasis [2]. Recently it has been proposed that a vicious circle of metabolic derangements (malnourishment, inflammation, arteriosclerosis) may explain the exaggerated morbidity and mortality in a subset of haemodialysis patients [3]. Inflammation might be the most deleterious factor in this respect which, besides other factors, is related to the occurrence of CKD-rP and down-regulation of fetuin-A, an important calcium-binding circulating protein favouring tissue-calcifications in these patients [4]. Thus, we were interested in whether patients with CKD-rP display lower levels of serum fetuin-A. Ten patients in a hospital-based haemodialysis centre complaining about CKD-rP were compared to another 12 patients who did not report to have suffered from CKD-rP at least 6 months prior to the interview. Patients with CKD-rP were asked to score the intensity of current pruritus using a visual analogue scale (VAS) ranging from 0 to 10. In both groups, 10 ml of blood was taken immediately after puncture of the arterio-venous fistula, and fetuin-A, 25-hydroxyvitamin D3 (25[OH]D3), total protein, albumin, calcium (corrected for serum albumin), phosphate, and high-sensitivity CRP (hsCRP) were measured. Independent t-tests (continuous variables) and a χ2-test (gender) evaluated whether there were significant (P < 0.05) differences between patients with and without CKD-rP. The mean pruritus intensity of patients with CKD-rP was 5.9 ± 1.9. After identifying one univariate outlier (hsCKP = 35.6) in the group of patients without CKD-rP, CRP was significantly higher in patients with CKD-rP. We failed, however, to find significant differences in serum-calcium, phosphate, fetuin A and 25-OH-Vitamin D3 between the two groups (Table ​(Table1).1). Additionally, there was no relationship between the intensity of CKD-rP and the metabolic factors measured (including CRP) in patients suffering from pruritus. Table 1 Patient and clinical characteristics between the two groups. PTH (intact parathyreoid hormone), hsCRP (high-sensitivity C-reactive protein), 25(OH)D3 (25-hydroxyvitamin D3) Although our study approach has methodological limitations (small number of patients, no matched pairs) the results suggest that neither calcium-binding protein fetuin-A levels nor 25(OH)D3 values were noticeably different in patients with CKD-rP compared to patients without CKD-rP. On the other hand, the marker for inflammation, CRP, was found to be significantly higher in patients with CKD-rP as shown before [5]. We hence believe that other inflammation-driven processes need to be studied in the future in order to understand the pathomechanisms of CKD-rP. Conflict of interest statement. None declared.