Design of a non-interventional post-marketing study to assess the long-term safety and effectiveness of ocrelizumab in German real world multiple sclerosis cohorts - the CONFIDENCE study protocol.

BACKGROUND: Multiple sclerosis (MS) is a chronic disease that requires lifelong treatment. A highly effective drug not only for relapsing but also for progressive forms of MS with a favorable safety profile is needed to further improve overall patient outcomes. Ocrelizumab, a recombinant humanized monoclonal antibody that selectively targets CD20-expressing B-cells, is the first drug indicated for the treatment of adult patients with relapsing forms of MS (RMS) and primary progressive MS (PPMS). Its safety and effectiveness profile has yet to be studied in a large, real-world setting. CONFIDENCE aims to further characterize the safety profile of ocrelizumab in routine clinical practice. In addition, real-world effectiveness data will be collected to complement the efficacy data documented in the pivotal clinical trials. METHODS: CONFIDENCE is a non-interventional, prospective, multicenter, long-term study collecting primary data from 3000 RMS and PPMS patients newly treated with ocrelizumab and 1500 patients newly treated with other selected MS disease-modifying therapies (DMTs). Treatment must be in accordance with the local label and follow routine practice. Data will be collected at approximately 250 neurological centers and practices across Germany. The recruitment period of 30 months started in April 2018. The observation period per patient is planned 7.5 to 10 years, depending on the date of inclusion, regardless of whether patients discontinue treatment. Visits follow routine practice and will be documented approximately every 6 months. The primary endpoint is the incidence and type of uncommon adverse events and death. Statistical analyses will be mainly descriptive and exploratory. DISCUSSION: CONFIDENCE is a large, non-interventional, post-authorization safety study that assesses long-term safety and effectiveness of ocrelizumab and other DMTs in a real-world setting. Data collected in CONFIDENCE will also be integrated into studies that have been developed to fulfil international regulatory requirements.

Application of the SARS-CoV-2-S1 ACE-2 receptor interaction as the basis of the fully automated assay to detect neutralizing SARS-CoV-2-S1 antibodies in blood samples.

A quantitative, high throughput, fully automated diagnostic method for the detection of neutralizing anti-SARS-CoV-2 antibodies was developed on the Phadia system based on the interaction of SARS-CoV-2 S1 protein and the human ACE-2 receptor. This method was compared to the current state of the art plaque reduction neutralization test (PRNT) and a high correlation between the two methods was observed. Using a large cohort of blood samples from convalescent patients and controls the method displays very high sensitivity and specificity (99,8% and 99.99%, respectively). Neutralizing antibody titers of mRNA-1273 and BNT162b2-vaccinated persons can also be quantified with this method as well. This fully automated method provides the possibility to determine anti-SARS-CoV-2 neutralizing antibody concentrations in just 2  h.

Circulating calprotectin as biomarker in neutrophil-related inflammation: Pre-analytical recommendations and reference values according to sample type.

BACKGROUND: Calprotectin (CLP) is a promising biomarker for the evaluation of neutrophil-related inflammation. Our aim was to establish reference values for circulating CLP in different sample types and to study the effect of pre-analytical variables. METHODS: Reference values were determined in 100 healthy individuals. Pre-analytical variables were evaluated in 10 healthy controls and four rheumatoid arthritis patients with active disease and covered sample type (serum with/without gel separator, heparin, EDTA and citrate plasma), pre-centrifugation time (<2 h, 6 h, 24 h), storage condition (2-8 °C, 18-25 °C, 30 °C) and storage time (24 h, 72 h, 7 days). CLP measurements were performed with the EliA™Calprotectin 2 assay on Phadia™200 (Thermo Fisher Scientific). RESULTS: In healthy controls, baseline CLP concentrations in serum were more than double the concentration in EDTA and citrate plasma (0.909 µg/mL versus 0.259 µg/mL and 0.261 µg/mL respectively). Heparin, EDTA and citrate stabilized CLP concentrations for up to 6 h before centrifugation, whereas significant increases in CLP levels were observed when serum was left untreated during that time period. CONCLUSION: Clinical studies on circulating CLP need to apply sample type-specific reference values and decision limits. To obtain reproducible CLP results in serum, more stringent pre-analytical sample handling instructions are needed.

Recombinant protein to analyze autoantibodies to proteinase 3 in systemic vasculitis.

The presence of antineutrophil cytoplasmic autoantibodies with specificity for proteinase 3 (PR3-ANCA) usually is detected by enzyme-linked immunosorbent assay (ELISA) with purified PR3 as a substrate. We studied the technical performance of direct and capture ELISA using a recombinant proteolytically inactive form of PR3 produced in the baculovirus expression system for the detection of PR3-ANCA in 114 patients with systemic vasculitis at diagnosis. We found that ELISA using recombinant PR3 produced in insect cells is a promising alternative for ELISA with native PR3. We found a correlation between tests using recombinant or native PR3, as well as correlation of the ELISA results with ANCA titers measured by the indirect immunofluorescence technique. However, the specificity for ANCA-associated vasculitis of ELISA with recombinant PR3 was lower than ELISA using native PR3. Compared with the direct assay, capture ELISA is a more sensitive method for PR3-ANCA detection, with both native and recombinant PR3, and its results depend on the monoclonal antibody used to capture the antigen.