Surface plasmon resonance assays for the therapeutic drug monitoring of infliximab indicate clinical relevance of anti-infliximab antibody binding properties.

OBJECTIVES: The therapeutic antibody infliximab (IFX) has improved the life quality of numerous autoinflammatory disease patients. However, IFX can trigger the generation of anti-drug antibodies (ADA), whose optimal evaluation and management are currently subject of controversial discussions. We present two novel surface plasmon resonance (SPR) biosensor assays for therapeutic drug monitoring of IFX and characterization of ADA and investigated the diagnostic value of ADA binding properties. METHODS: IFX and ADA were quantified via developed SPR biosensor assays (IFXmon and ADAmon, respectively) and diagnostics-approved ELISA in sera from inflammatory bowel disease patients. Pre-analytic ADA enrichment with magnetic beads enabled analytical drug tolerance of the ADAmon assay. The dissociation ratio (DissR) as an index for ADA:IFX binding stability was calculated from the SPR sensorgrams of ADA quantification runs. RESULTS: IFX levels determined by IFXmon assay and ELISA showed high agreement, whereas ADA quantification concordance between ADAmon assay and ELISA was poor. In patients, DissR was predominantly constant over time and differed significantly between therapy outcomes. A DissR cut-off of 1.524 indicated undetectable IFX levels with 71.4% sensitivity and 88.9% specificity. Additionally, the SPR reference surface was exploited as serum-individual negative control to check result plausibility within multi-sample run sequences. CONCLUSIONS: Overall, both SPR biosensor assays exhibited reliable quantitative performance with accuracies superior to their ELISA counterparts and precision inferior to ELISA only for ADAmon. DissR presented itself as promising ADA binding parameter and could contribute to both earlier and more tailored therapeutic decisions.

Dynamics of serum concentrations of antibodies to infliximab: a new approach for predicting secondary loss of response in inflammatory bowel diseases.

BACKGROUND: Antibodies to infliximab (ATI) in serum are associated with secondary loss of response (LOR) to infliximab (IFX) therapy in patients with inflammatory bowel disease (IBD). However, feasible ATI-related predictors of therapy success are lacking and knowledge about individual ATI dynamics is limited. Therefore, this study analyzed whether ATI dynamics are able to predict LOR to IFX therapy and compared their predictive power with known predictors of LOR to IFX. METHODS: This was a retrospective study of patients with Crohn's disease (CD) or ulcerative colitis (UC) on IFX maintenance therapy and proactive IFX and immunogenicity monitoring in an outpatient clinic in Germany. Slopes of ATI (S ATI) and IFX levels (dynamic parameters) and medians of ATI, IFX, C-reactive protein, and fecal calprotectin (static parameters) were calculated over a defined period of time after ATI emergence. Dynamic and static parameters were analyzed for associations with end points infliximab discontinuation due to secondary LOR and total IFX discontinuation. RESULTS: In all, 500 visits from 38 IBD patients (28 CD, 10 UC) with a median IFX maintenance duration of 68.2 weeks were evaluated. Grouping by S ATI (ATI-N = ATI nondetectable, ATI- ↓ = negative S ATI, ATI- ↑ = positive S ATI) yielded significant differences for outcomes LOR (p = 0.004) and total IFX discontinuation (p = 0.01). Patients in the ATI-↓ group survived significantly longer LOR-free compared with the ATI-↑ group (p = 0.02). Cox regression confirmed S ATI to be a significant risk factor for LOR (p = 0.002). An S ATI cut-off of approximately 2.0 AU mL-1 week-1 was determined to predict LOR with 83.3% sensitivity and 93.8% specificity. CONCLUSION: The ATI slope-based index S ATI is a new feasible diagnostic predictor of LOR in IBD patients. S ATI may facilitate quick therapeutic decisions after ATI emerge.

Evaluation of the sensitivity and specificity of a novel line immunoassay for the detection of criteria and non-criteria antiphospholipid antibodies in comparison to established ELISAs.

BACKGROUND: Persistent antiphospholipid antibodies (aPL) constitute the serological hallmark of the antiphospholipid syndrome (APS). Recently, various new assay technologies for the detection of aPL better suited to multiplex reaction environments than ELISAs emerged. We evaluated the diagnostic performance of such a novel line immunoassay (LIA) for the simultaneous detection of 10 different aPL. METHODS: Fifty-three APS patients and 34 healthy controls were investigated for criteria (antibodies against cardiolipin [aCL], ß2-glycoprotein I [aß2-GPI]) and non-criteria aPL (antibodies against phosphatidic acid [aPA], phosphatidyl-choline [aPC], -ethanolamine [aPE], -glycerol [aPG], -inositol [aPI], -serine [aPS], annexin V [aAnnV], prothrombin [aPT]) IgG and IgM by LIA. Criteria aPL were additionally determined with the established Alegria (ALE), AcuStar (ACU), UniCap (UNI), and AESKULISA (AES) systems and non-criteria aPL with the AES system. Diagnostic performance was evaluated with a gold standard for criteria aPL derived from the results of the four established assays via latent class analysis and with the clinical diagnosis as gold standard for non-criteria aPL. RESULTS: Assay performance of the LIA for criteria aPL was comparable to that of ALE, ACU, UNI, and AES. For non-criteria aPL, sensitivities of the LIA for aPA-, aPI-, aPS-IgG and aPA-IgM were significantly higher and for aPC-, aPE-, aAnnV-IgG and aPC- and aPE-IgM significantly lower than AES. Specificities did not differ significantly. CONCLUSIONS: The LIA constitutes a valuable diagnostic tool for aPL profiling. It offers increased sensitivity for the detection of aPL against anionic phospholipids. In contrast, ELISAs exhibit strengths for the sensitive detection of aPL against neutral phospholipids.

External quality assessment schemes for glucose measurements in Germany: factors for successful participation, analytical performance and medical impact.

BACKGROUND: Determination of blood glucose concentration is one of the most important measurements in clinical chemistry worldwide. Analyzers in central laboratories (CL) and point-of-care tests (POCT) are both frequently used. In Germany, regular participation in external quality assessment (EQA) schemes is mandatory for laboratories performing glucose testing. METHODS: Glucose testing data from the two German EQAs "Reference Institute for Bioanalytics" (RfB) and "INSTAND - Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien" (Instand) were analyzed from 2012 to 2016. Multivariable odds ratios (OR) for the probability to reach a "good" result were calculated. Imprecision and bias were determined and clinical risk of measurement errors estimated. RESULTS: The device employed was the most important variable required for a "good" performance in all EQAs. Additional participation in an EQA for CL automated analyzers improved performance in POCT EQAs. The reciprocal effect was less pronounced. New participants performed worse than experienced participants especially in CL EQAs. Imprecision was generally smaller for CL, but some POCT devices reached a comparable performance. Large lot-to-lot differences occurred in over 10% of analyzed cases. We propose the "bias budget" as a new metric to express the maximum allowable bias that still carries acceptable medical risk. Bias budgets were smallest and clinical risks of errors greatest in the low range of measurement 60-115 mg/dL (3.3-6.4 mmol/L) for most devices. CONCLUSIONS: EQAs help to maintain high analytical performances. They generate important data that serve as the foundation for learning and improvement in the laboratory healthcare system.

Stronger Together: Aggregated Z-values of Traditional Quality Control Measurements and Patient Medians Improve Detection of Biases.

BACKGROUND: In clinical chemistry, quality control (QC) often relies on measurements of control samples, but limitations, such as a lack of commutability, compromise the ability of such measurements to detect out-of-control situations. Medians of patient results have also been used for QC purposes, but it may be difficult to distinguish changes observed in the patient population from analytical errors. This study aims to combine traditional control measurements and patient medians for facilitating detection of biases. METHODS: The software package "rSimLab" was developed to simulate measurements of 5 analytes. Internal QC measurements and patient medians were assessed for detecting impermissible biases. Various control rules combined these parameters. A Westgard-like algorithm was evaluated and new rules that aggregate Z-values of QC parameters were proposed. RESULTS: Mathematical approximations estimated the required sample size for calculating meaningful patient medians. The appropriate number was highly dependent on the ratio of the spread of sample values to their center. Instead of applying a threshold to each QC parameter separately like the Westgard algorithm, the proposed aggregation of Z-values averaged these parameters. This behavior was found beneficial, as a bias could affect QC parameters unequally, resulting in differences between their Z-transformed values. In our simulations, control rules tended to outperform the simple QC parameters they combined. The inclusion of patient medians substantially improved bias detection for some analytes. CONCLUSIONS: Patient result medians can supplement traditional QC, and aggregations of Z-values are novel and beneficial tools for QC strategies to detect biases.

Chromogranin A and neurone-specific enolase serum levels as predictors of treatment outcome in patients with metastatic castration-resistant prostate cancer undergoing abiraterone therapy.

OBJECTIVE: To determine the impact of elevated neuroendocrine serum markers on treatment outcome in patients with metastatic castration-resistant prostate cancer (mCRPC) undergoing treatment with abiraterone in a post-chemotherapy setting. PATIENTS AND METHOD: Chromogranin A (CGa) and neurone-specific enolase (NSE) were determined in serum drawn before treatment with abiraterone from 45 patients with mCRPC. Outcome measures were overall survival (OS), prostate-specific antigen (PSA) response defined by a PSA level decline of ≥50%, PSA progression-free survival (PSA-PFS), and clinical or radiographic PFS. RESULTS: The CGa and NSE serum levels did not correlate (P = 0.6). Patients were stratified in to low- (nine patients), intermediate- (18) or high-risk (18) groups according to elevation of none, one, or both neuroendocrine markers, respectively. The risk groups correlated with decreasing median OS (median OS not reached vs 15.3 vs 6.6 months; P < 0.001), decreasing median clinical or radiographic PFS (8.3 vs 4.4 vs 2.7 months; P = 0.001) and decreasing median PSA-PFS (12.0 vs 3.2 vs 2.7 months; P = 0.012). In multivariate Cox regression analysis the combination of CGa and NSE (≥1 marker positive vs both markers negative) remained significant predictors of OS, clinical or radiographic PFS, and PSA-PFS. We did not observe a correlation with PSA response (63% vs 35% vs 31%; P = 0.2). CONCLUSION: Chromogranin A and NSE did not predict PSA response in patients with mCRPC treated with abiraterone. However, we observed a correlation with shorter PSA-PFS, clinical or radiographic PFS, and OS. This might be due to an elevated risk of developing resistance under abiraterone treatment related to neuroendocrine differentiation.

Establishment of triglyceride cut-off values to detect chylous ascites and pleural effusions.

OBJECTIVES: Lipoprotein electrophoresis is the gold standard for the detection of chylous ascites and pleural effusions. It is, however, not suitable as a front-line test and not widely available. Most clinicians must rely solely on the quantitative determination of lipids. The aim of this work was to establish lipid cut-off values for the presence of chylomicrons in pleural and peritoneal fluid. DESIGN AND METHODS: Triglyceride and cholesterol levels from 113 peritoneal and 154 pleural fluid samples investigated for chylomicrons via lipoprotein electrophoresis were considered. Receiver operating characteristic analyses were performed and cut-off levels determined. RESULTS: 54 peritoneal and 59 pleural fluid samples were positive for chylomicrons. In peritoneal fluid, triglycerides and triglycerides/cholesterol ratio exhibited areas under the curve (AUC) not significantly different from each other, but significantly larger than cholesterol alone. The AUC for triglycerides in pleural fluid was significantly larger than the AUCs for cholesterol and the triglycerides/cholesterol ratio. Triglyceride cut-offs with maximum Youden-Index, sensitivity >95%, and specificity >95% were calculated to be 187, 148, and 246mg/dl (2.13, 1.69, and 2.80mmol/l) for peritoneal fluid, and 240, 94, and 240mg/dl (2.74, 1.07, and 2.74mmol/l) for pleural fluid. CONCLUSIONS: Triglyceride levels are the best parameter to detect chylous body fluids when lipoprotein electrophoresis is not available. Single-point triglyceride cut-offs of 187 and 240mg/dl (2.13 and 2.74mmol/l) or alternatively equivocal ranges of 148-246 and 94-240mg/dl (1.69-2.80 and 1.07-2.74mmol/l) were established for peritoneal and pleural fluid, respectively.

Evaluation of antiphospholipid antibody assays using latent class analysis to address the lack of a reference standard.

BACKGROUND: Method evaluation of new assays for the detection of antiphospholipid antibodies (aPL) such as anti-cardiolipin (aCL) or anti-ß2-glycoprotein I (aß2-GPI) is challenging, as no internationally accepted reference material is available yet. Besides a lack of standardization, unacceptable inter-laboratory comparability of established tests is regularly observed. Owing to the absence of a commonly accepted reference standard, the evaluation of two research surface plasmon resonance (SPR) biosensor assays was performed using statistical methods from latent class analysis (LCA). METHODS: aCL and aß2-GPI IgG and IgM were measured in sera from 63 antiphospholipid syndrome patients, fulfilling the Sydney criteria, and in 34 healthy controls with four commercial assays. LCA was performed on the results and sera were assigned to the antibody-positive or antibody-negative group. Sera were subsequently evaluated in the SPR assays for aCL and aß2-GPI. Optimal cutoffs and diagnostic performances of the research systems were established employing the LCA-derived gold standard. RESULTS: With area under the curve results of 0.96 and 0.89 for the detection of aCL and aß2-GPI, the research SPR assays discriminated well between antibody-positive and antibody-negative sera. Their sensitivities and specificities were comparable to the investigated commercial immunoassays. CONCLUSIONS: SPR assays are a suitable tool for the detection of aCL and aß2-GPI with diagnostic performances not different from currently available commercial tests. LCA enabled the calculation of sensitivities and specificities for aPL assays in absence of a reference standard.

The biomarker sex hormone-binding globulin - from established applications to emerging trends in clinical medicine.

Sex hormone-binding globulin (SHBG) is a serum glycoprotein exhibiting the unique feature of binding sex steroids with high affinity and specificity. Its serum levels are regulated not only by androgens and estrogens but also by thyroid hormones and other metabolic factors. Several disease conditions are accompanied by altered SHBG levels such as hyper- and hypoandrogenism, thyroid disorders, pituitary diseases, liver disorders, and breast as well as prostate cancer. Additionally, several drugs and alcohol consumption influence serum concentrations of SHBG. In some cases, altered SHBG levels are a specific result of the underlying pathology. In others, they merely constitute an epiphenomenon, which still might offer the possibility of using serum measurements of SHBG as surrogate marker. This review article portrays the different disorders associated with altered SHBG levels and discusses the usefulness of SHBG as disease biomarker from a clinicians as well as from an endocrinological researchers point of view.

Clinically relevant lot-to-lot reagent difference in a commercial immunoturbidimetric assay for glycated hemoglobin A1c.

OBJECTIVES: Hemoglobin A1c (HbA1c) is employed for diagnosis and therapy monitoring of diabetes mellitus. The effect of a change of reagent lot on the measured values of a commercial immunoturbidimetric HbA1c assay (A1C3) was investigated. DESIGN AND METHODS: Comparison measurements of A1C3 and an automated affinity chromatography method (VIIT) were performed in 15 samples for the initial and in 20 samples for the subsequent A1C3 lot. The results of 27 and 19 measurements of a normal and of 28 and 20 of a pathological control (before and after the switch of the A1C3 reagent lot, respectively) were evaluated. Finally, the results of 6463 patient samples that had been measured with the initial and 434 that had been measured with the subsequent A1C3 lot were investigated. RESULTS: VIIT yielded significantly higher results than the initial A1C3 lot (bias: 0.41% HbA1c, 4.5mmol/mol) but agreed well with the subsequent lot (bias: -0.01% HbA1c, -0.1mmol/mol). Changing to the subsequent reagent lot resulted in significant increases of the mean of the normal control of 0.316% HbA1c (3.5mmol/mol) and of the pathological control of 0.749% HbA1c (8.2mmol/mol). The median of patient samples measured with the subsequent lot was significantly higher by 0.40% HbA1c (4.4mmol/mol). CONCLUSIONS: The subsequent A1C3 reagent lot yields significantly higher measurement results than the initial by approximately 0.5% HbA1c (5.5mmol/mol). This difference is considered as clinically relevant. A combined effort of manufacturers and notified bodies is necessary to minimize lot-to-lot variation.

Diagnostic performance study of an antigen microarray for the detection of antiphospholipid antibodies in human serum.

BACKGROUND: The parallelization of clinically relevant antigens in a microarray format is of growing importance due to the ability to measure multiple antigen-antibody interactions. With the development of a microarray for the detection of antiphospholipid antibodies we focussed on one important autoimmune disease that is still diagnostically challenging. Reasons are the heterogeneity of the autoantibodies and the unspecific clinical symptoms. METHODS: For the covalent immobilization of antigenic structures, glass transducers were coated with 11-aminoundecyltrimethoxysilane (11-AUTMS). In total 35 antiphospholipid syndrome (APS) patients, six patients with lupus erythematosus and 24 healthy controls were investigated on a microarray format using polarized imaging reflectometric interference spectroscopy. RESULTS: The novel surface modification based on the short derivative 11-AUTMS resulted in a selective biosensor allowing a clear differentiation of patient and control samples. It combined proteinogenic as well as phospholipid-derived antigens, namely ß2-glycoprotein I (ß2-GPI), prothrombin, cardiolipin (CL) and a ß2-GPI/CL complex. With optimized regeneration conditions, up to 20 consecutive measurements could be performed on one chip. Sensitivity was determined to be 0.800-0.929, specificity was between 0.733 and 0.969, depending on the respective antigen. CONCLUSIONS: Multiplexed determination of serological parameters has a great potential. We have shown that our biosensor is capable of detecting four different APS relevant antibodies in parallel exhibiting a sensitivity and specificity comparable to existing ELISA methods.