Repository of intra- and inter-run variations of quantitative autoantibody assays: a European multicenter study.

OBJECTIVES: No reference data are available on repositories to measure precision of autoantibody assays. The scope of this study was to document inter- and intra-run variations of quantitative autoantibody assays based on a real-world large international data set. METHODS: Members of the European Autoimmunity Standardisation Initiative (EASI) group collected the data of intra- and inter-run variability obtained with assays quantifying 15 different autoantibodies in voluntary participating laboratories from their country. We analyzed the impact on the assay performances of the type of immunoassay, the number of measurements used to calculate the coefficient of variation (CVs), the nature and the autoantibody level of the internal quality control (IQC). RESULTS: Data were obtained from 64 laboratories from 15 European countries between February and October 2021. We analyzed 686 and 1,331 values of intra- and inter-run CVs, respectively. Both CVs were significantly dependent on: the method of immunoassay, the level of IQC with higher imprecision observed when the antibody levels were lower than 2-fold the threshold for positivity, and the nature of the IQC with commercial IQCs having lower CVs than patients-derived IQCs. Our analyses also show that the type of autoantibody has low impact on the assay' performances and that 15 measurements are sufficient to establish reliable intra- and inter-run variations. CONCLUSIONS: This study provides for the first time an international repository yielding values of intra- and inter-run variation for quantitative autoantibody assays. These data could be useful for ISO 15189 accreditation requirements and will allow clinical diagnostic laboratories to assure quality of patient results.

[Advice in autoimmunity biological diagnosis: guidelines for drafting comments on biological results]. / Prestation de conseil en auto-immunité : aide à la rédaction de commentaires pour les comptes rendus de résultats.

The ISO 15189 accreditation of biological analysis requires the presence of interpretation in the analysis report. The interpretation in the field of autoimmunity which includes many analyses and methods can be complex for biologists who may not have clinical data and for clinicians who may not be aware of technical difficulties. The French group of the european group EASI (European autoimmunity standardisation initiative) proposes a list of comments and advice in order to help biologists when interpreting auto-immune analyses results in several situations. These comments should be adapted to the clinical and biological situation (other biological results, clinical data…) and should alert the clinician. A dialogue between the biologist and the clinician is essential to adjust the interpretation on clinical data in order to provide a better health care for the patient.

Precision of autoantibody assays in clinical diagnostic laboratories: What is the reality?

BACKGROUND: ISO 15189 accreditation remains a challenge for specialized laboratories. In the field of autoimmunity, beside the crucial problem of absence of standardization, laboratories have to manage the analytical performances of the large panel of assays in terms of sensitivity and specificity, but also on their measurement precision for which no reference values are available on biorepositories. METHODS: As an initiative of the French EASI (European Autoimmunity Standardization Initiative) group, French clinical diagnostic laboratories were requested to participate in a survey aiming to analyze the coefficients of variation (CVs) of intra-run and inter-run variability obtained with assays quantifying 14 different autoantibodies. Two performance goals corresponding to the 90th percentile and the 50th percentile (lowest CV values reached by 90% and 50% of laboratories respectively) defined for three levels of concentration were calculated. The impact on the assay performances of the number of measurements, of the nature of the internal quality control (IQC) and the type of immunoassay, was also analyzed. RESULTS: 414 and 616 values of intra-run and inter-run CVs were collected, respectively. The 50th percentile performance goals were comprised between 1.0% and 8.9% for the intra-run CVs, and between 1.8% and 14.6% for the inter-run CVs. At 90th percentile, the performance goals were comprised between 3.2% and 13.5% for the intra-run CVs, and between 7.3% and 30.8% for the inter-run CVs. CVs calculated from 10 values were similar to those obtained from more values. Higher imprecision was observed when the antibody levels of the IQC was lower than 2 fold the positive threshold. Commercial IQCs gave lower CVs than IQCs derived from patient samples. CONCLUSION: Our results allow proposing some acceptability limits for the precision performances of the autoantibody assays, compatible with the reality of life in diagnostic laboratories and clinical care.

Interstitial Lung Disease-Complicated Anti-MDA5 Antibody in Clinically Amyopathic Dermatomyositis Patients: Report of Two Cases With Distinct Clinical Features.

Two patients presented simultaneously to our hospital with distinct clinical features associated with the presence of anti-MDA5 antibodies: the first one was admitted for a skin rash resembling to a toxic epidermal necrosis (Lyell syndrome) and the second one presented with pulmonary manifestations attributed to a diffuse fibrosing interstitial pneumonitis on chest CT-scan. In addition to the skin lesions involving 40% of the body surface area, the first patient developed a rapid diffuse interstitial pneumonitis with respiratory distress justifying the initiation of a systemic immunosuppressive treatment. However, she died 3 weeks after her admission from mesenteric thrombosis associated with septic shock. The second patient respiratory condition worsened despite an intensive immunosuppressive treatment with high doses of intravenous methylprednisolone and cyclophosphamide and plasmapheresis, and required lung transplantation. Anti-MDA5 antibody titer declined and disappeared on anti-rejection treatment. These two cases underline the diagnostic conundrum and the therapeutic difficulties in patients with anti-MDA5 antibodies and clinically amyopathic dermatomyositis (CADM) or interstitial lung disease (ILD), who may undergo rapidly-progressive and fatal outcome. Presence of anti-MDA5 antibodies should always be suspected when confronted to CADM patients with cutaneous ulcerations or ILD to allow a rapid and adapted treatment initiation.

Anti-factor H Autoantibodies Assay by ELISA.

The complement system is a part of the immune system implicated in host defense against pathogens and damaged cells and Factor H is the main regulatory protein of this powerful enzymatic cascade. Autoantibodies directed against Factor H (anti-FH antibodies) are implicated in different pathologies mainly atypical hemolytic and uremic syndrome and C3 glomerulopathies. The detection and quantification of these autoantibodies are crucial for the clinical management of the patients.Anti-Factor H antibodies are detected and quantified by an ELISA assay. The aim of this chapter is to describe the procedure to determine anti-FH autoantibodies and to provide information about their biological significance.

Analytical validation of an alternative method to quantify specific antibodies in 3 applications.

The detection and the quantification of specific antibodies represent essential tools for the diagnosis and for the biological monitoring of immune humoral response in many clinical situations in particular in autoimmune diseases or in the context of immunotherapy using monoclonal antibodies. This article focuses on the development of a specific antibody measuring method (Patent n°PCT/IB2014/064437). The principle of this method is based on the combined use of a monoclonal antibody as standard and the protein G as immunoglobulins detecting agent. We performed a complete analytical validation of this method for the quantification of antibodies in three different applications: autoantibodies, alloantibodies and therapeutic monoclonal antibody. The results showed good performances compatible with the use of these assays as diagnostic tools. This method allows avoiding the use of products from human origin as reagent that causes ethical and infectious concerns but also storage and long term stock management problems. Moreover, this approach is particularly useful when no commercial reagent is available, especially in the case of rare diseases.

Auto-immunité et gestion des toxicités des traitements par anti-check point inhibiteurs.

AUTOIMMUNITY AND MANAGEMENT OF THE IMMUNE-RELATED ADVERSE EFFECTS OF THE IMMUNE CHECKPOINT INHIBITORS: The immune checkpoint molecules such as CTLA-4 and PD-1 are involved in the tolerance mechanisms preventing the immune system to react against the self-antigens. When these receptors expressed on the lymphocyte membrane, bind to their ligands, they induce a negative signal to the cell which becomes unable to be completely activated in the presence of its antigen. In a context of tumor, the infiltrating T cells are frequently exhausted due to the expression of CTLA-4 and PD-1 ligands by the microenvironment impairing the antitumoral immunity. The use of antagonistic antibodies targeting these receptors or their ligands (called checkpoint inhibitors) aims to block their interaction unbalancing the negative regulation of the antitumoral lymphocytes. However, this effect affects all lymphocytes and may also disrupt the negative regulation of the peripheral autoreactive lymphocytes. Thus, a significant proportion of patients treated by these molecules develop immune-related symptoms affecting different tissues and organs due to lymphocyte activation. These symptoms are called immune-related adverse events (irAEs). This article aims to summarize the scientific data demonstrating the implication of these molecules in the tolerance mechanisms and in the autoimmune diseases. It also reports on the IrAEs observed in treated patients and gives an outline of guidelines to monitor and manage these patients.