Search

The detection of cryoglobulins (CG) used to diagnose cryoglobulinemic vasculitis requires strict adherence to protocol, with emphasis on the preanalytical part. Our main objectives were to introduce a more sensitive and specific protocol for the detection of CG and to characterize CG in Slovenian patients diagnosed with cryoglobulinemic vasculitis, other vasculitides, connective tissue diseases or non-rheumatic diseases examined at the Department of Rheumatology (University Medical Centre Ljubljana). Samples were routinely analyzed for the presence of CG with the protocol using the Folin-Ciocalteu reagent. In the newly introduced protocol, the type of CG was determined by immunofixation on visually observed positive samples and the concentration of CG in the cryoprecipitate and rheumatoid factor (RF) activity were measured by nephelometry. RF, C3c and C4 were measured in patients` serum and a decision tree analysis was performed using all results. The agreement between negative and positive results between the two protocols was 86%. Of the 258 patient samples tested, we found 56 patients (21.7%) with positive CG (37.5% - type II, 62.5% - type III). The RF activity was observed in 21.4% of CG positive subjects. The median concentration of type II CG was significantly higher than that of type III CG (67.4 mg/L vs. 45.0 mg/L, p = 0.037). Patients with type II had lower C4 concentrations and higher RF compared to patients with type III CG. In the decision tree, C4 was the strongest predictor of cryoglobulinemia in patients. With the newly implemented protocol, we were able to improve the detection and quantification of CG in the samples of our rheumatology patients and report the results to adequately support clinicians., (© 2023. The Author(s).)

By monitoring serum concentrations of infliximab (IFX) and adalimumab (ADL) and levels of their antibodies in patients with inflammatory diseases, clinicians can adjust dose and increase safety and effectiveness of treatment. The aim was to develop and validate in-house enzyme-linked immunosorbent assays (ELISAs) for IFX and ADL, together with anti-IFX and anti-ADL ELISAs for routine detection and further analysis with acid dissociation of immune complexes. Furthermore, the objectives were to compare in-house assays with commercial ELISAs and reporter gene assays (RGAs) and to determine cross-reactivity between original Remicade®/Remsima™ and their antibodies. In-house ELISAs were validated (imprecision, accuracy among other criteria) and compared with commercial apDia ELISAs and RGAs. Correlation coefficients, intraclass correlation coefficients, agreement, and bias were calculated. All in-house ELISAs gave precise and repeatable results. The immune complexes between IFX and anti-IFX were found in 3% of samples, while complexes between ADL and anti-ADL were found in 14% of samples. Significant correlations were found between in-house and apDia ELISAs and RGA for IFX, ADL, anti-IFX, and anti-ADL results. Remicade®, Remsima™, and their antibodies could be accurately measured with either apDia or in-house IFX and anti-IFX ELISAs. Accurate and precise in-house ELISAs, highly comparable with commercial ELISAs and RGAs, were developed and validated for routine analysis of samples of patients treated with IFX (Remicade® or Remsima™) or ADL providing substantial cost benefit. Complex dissociation identified samples with anti-IFX-IFX (3%) and anti-ADL-ADL (14%) complexes indicating the benefit of adding acid dissociation to therapeutic drug monitoring of IFX and ADL.

About a third of patients with rheumatoid arthritis treated with adalimumab may develop anti-adalimumab antibodies. Anti-adalimumab antibodies are associated with reduced drug levels, loss of drug efficacy, clinical non-response and an increased risk of adverse effects. In case of suspected drug failure and in order to better define clinical efficacy, adalimumab as well as anti-adalimumab antibodies levels should be monitored. Sandwich or indirect enzyme-linked immunoassay is most commonly used for determining adalimumab, while bridging ELISA and antigen-binding test are most useful for determining anti-adalimumab antibodies. Most current assays cannot detect antibodies complexed with the adalimumab; however, methods for dissociation of the complexes using acid/temperature have been developed. The aim of this review is to report on the latest methodology for detecting adalimumab and anti-ADL antibodies, benefits of their detections in clinical practice, as well as expose problematic issues, such as different analytical sensitivity and specificity, standardization and validation. The main problem in measuring adalimumab or anti-ADL antibodies is high drug sensitivity, which can result in false-negative anti-ADL antibodies. Therefore, drug-tolerant assays have been developed. Cell-based assays, such as the reporter gene assay, are recommended for detection of functionally active adalimumab and their neutralizing anti-ADL antibodies.