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.

Cyclosporine induced biochemical remission in childhood autoimmune hepatitis.

The conventional treatment of autoimmune hepatitis (AIH) in children, which includes prednisone alone or in combination with azathioprine, induces remission in most cases but is often associated with poorly tolerated side effects. To avoid the adverse effects, Alvarez et al. introduced an alternative treatment regimen, using cyclosporine A (CyA) as primary immunosuppression. We carried out a retrospective study to evaluate the efficacy and tolerance of CyA treatment in children and adolescents with AIH treated in our center. During 2000-2010 period, nine children (6 female) aged 5-17.5 years, were diagnosed with AIH according to established international criteria. Following the suggested protocol, CyA was administered orally and when the transaminases tended to normalise, dose was adjusted to lover serum levels. Conversion to low dose of prednisone and azathioprine was started after 6 months, with gradual tapering and discontinuation of CyA. All nine patient had elevated transaminases and gammaglobulin levels, with proven histological changes typical for AIH in 8 patients that underwent liver biopsy (in one patient biopsy was contraindicated due to the prolonged prothrombin time). Serum ANA/SMA autoantibodies were positive in all but one patient, who had positive anti-LKM1. Complete or near complete and persistent normalisation of transaminase activity was observed in 8/9 patients within first 6 to 12 months. In one patient with partial response, an overlap syndrome was established. After ursodeoxycholic acid was added complete remission was observed. All patients had excellent clinical course and histological improvement. During the long-term follow-up (1.5-9 yrs; median 4.5 yrs), biochemical relapse occured in one patient after discontinuation of maintenance corticosteroid dose. Despite registered improvement, none of the patients fulfilled the criteria for therapy discontinuation, so all of them are still receiving maintenance doses of prednisone or azathioprine. The applied protocol allowed for the control of the liver inflammatory disease in all of our patients and protected them from the side effects related to steroid treatment. Side effects of CyA were minimal and were well tolerated.

Antibodies to mutated citrullinated vimentin and antibodies to cyclic citrullinated peptides in juvenile idiopathic arthritis.

BACKGROUND: The goal of the study was to assess the presence of antibodies to mutated citrullinated vimentin (anti-MCV) and cyclic citrullinated peptides (anti-CCP) in patients with juvenile idiopathic arthritis (JIA) compared with patients with other juvenile onset rheumatic diseases. METHODS: The study included 56 patients who fulfilled the International League of Associations for Rheumatology (ILAR) classification criteria for JIA, and 17 control patients with other juvenile onset rheumatic diseases. Data on six core outcome variables and the Sharp score were collected for patients with JIA. Sera and synovial fluid, if available, were tested for anti-CCP and anti-MCV antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). RESULTS: Anti-MCV antibodies were positive in 3/56 (5.4%) and anti-CCP in 1/56 (1.8%) of patients with JIA. Two out of three anti-MCV positive patients (one of them also anti-CCP positive) were found to be rheumatoid factor (RF)-positive with polyarticular disease. Within the control group, anti-MCV was positive in 4/17 (23.5%) patients, while anti-CCP positivity was not observed. No correlation between anti-MCV with anti-CCP antibody levels was found for any of the six core outcome variables or for the adapted Sharp score. CONCLUSIONS: Our results show that antibodies targeting citrullinated proteins are not a useful diagnostic marker for JIA, but can indicate severe patterns of disease in JIA.

Henoch-Schönlein purpura in the third trimester of pregnancy.

Henoch-Schönlein purpura (HSP) is an IgA-mediated small vessels' vasculitis that affects the skin, intestines and kidneys. Pregnancy has been reported as an exacerbating factor. We present the case of a 24-year-old primigravida with HSP that occurred in the third trimester and lasted up to the end of the successful delivery. She had pruritic maculopapular exanthema on her legs. Biopsy of a cutaneous lesion was performed for histopathologic features and direct immunofluorescence (DIF) for the presence of perivascular IgA deposition. Histopathology of the cutaneous lesion confirmed leukocytoclastic vasculitis. A DIF examination of the skin lesion confirmed deposits of fibrinogen in the small blood vessel walls. Six weeks following delivery, the skin lesions almost completely disappeared. Control laboratory findings were normal. This case of HSP might have been primarily associated with a preceding respiratory infection but this should first be carefully investigated due to a possible severe immunological disease in the patient's background requiring special attention since nephrotic symptoms may occur.

Current practice in laboratory diagnostics of autoimmune diseases in Croatia. Survey of the Working group for laboratory diagnostics of autoimmune diseases of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

INTRODUCTION: With the trend of increasing incidence of autoimmune diseases, laboratories are faced with exponential growth of the requests for tests relating the diagnosis of these diseases. Unfortunately, the lack of laboratory personnel experienced in this specific discipline of laboratory diagnostic, as well as an unawareness of a method limitation often results in confusion for clinicians. The aim was to gain insight into number and type of Croatian laboratories that perform humoral diagnostics with the final goal to improve and harmonize laboratory diagnostics of autoimmune diseases in Croatia. MATERIALS AND METHODS: In order to get insight into current laboratory practice two questionnaires, consisting of 42 questions in total, were created. Surveys were conducted using SurveyMonkey application and were sent to 88 medical biochemistry laboratories in Croatia for the first survey. Out of 33 laboratories that declared to perform diagnostic from the scope, 19 were selected for the second survey based on the tests they pleaded to perform. The survey comprised questions regarding autoantibody hallmarks of systemic autoimmune diseases while regarding organ-specific autoimmune diseases was limited to diseases of liver, gastrointestinal and nervous system. RESULTS: Response rate was high with 80 / 88 (91%) laboratories which answered the first questionnaire, and 19 / 19 (1.0) for the second questionnaire. Obtained results of surveys indicate high heterogeneity in the performance of autoantibody testing among laboratories in Croatia. CONCLUSIONS: Results indicate the need of creating recommendations and algorithms in order to harmonize the approach to laboratory diagnostics of autoimmune diseases in Croatia.

Unusually difficult clinical presentation of an infant suffering from congenital Cytomegalovirus (CMV) infection combined with alpha 1-antitrypsin (A1AT) deficiency.

Congenital Cytomegalovirus (CMV) infection and alpha 1-antitrypsin (A1AT) deficiency are separately well described entities, but their simultaneous occurrence can pose a special challenge to a clinician, especially dealing with optimal diagnostic as well as therapeutic approach. Congenital CMV infection is the most common vertically transmitted infection in developed countries. In 85-95% of newborns it runs asymptomatic, while in others it is presented with jaundice, petechias, hepatosplenomegaly and central nervous system damage. A1AT deficiency is on the other hand, the most common genetic liver disease in children, and the clinical spectrum varies from the accidentally detected increased levels of transaminases through to the severe infant cholestasis that can progress to cirrhosis. The following case report describes a two-month old male with severe clinical presentation of congenital CMV infection probably exacerbated due to A1AT deficiency comorbidity. The clinical manifestations and unusually difficult clinical signs this infant presented lead to assumption that the additional liver damage exists. Extensive laboratory analyses were performed, including PCR for CMV DNA, A1AT serum concentration, A1AT genotyping, followed and confirmed with phenotyping. Patient was treated parenteral with ganciclovir, what continued with oral valganciclovir and supportive therapy. Intensive and thorough supportive treatment of the infant resulted in satisfactory progress and excellent outcome. Patient was followed-up till the age of 18 months. The presented case provides excellent example about successful overcoming obstacles in differential diagnosis of A1AT in neonates and infants. Medical charts analysis was the methodology used in making this report.

Serological markers of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a heterogeneous group of chronic inflammatory disorders of the gastrointestinal tract with two main distinguishable entities, Crohn's disease (CD) and ulcerative colitis (UC). IBD-unclassified (IBD-U) is a diagnosis that covers the "grey" zone of diagnostic uncertainty between UC and CD. Current diagnosis of IBD relies on the clinical, endoscopic, radiological, histological and biochemical features, but this approach has shortcomings especially in cases of overlapping symptoms of CD and UC. The need for a diagnostic tool that would improve the conventional methods in IBD diagnosis directed the search towards potential immunological markers, since an aberrant immune response against microbial or endogenous antigens in a genetically susceptible host seems to be implicated in IBD pathogenesis. The spectrum of antibodies to different microbial antigens and autoantibodies associated with IBD is rapidly expanding. Most of these antibodies are associated with CD like anti-glycan antibodies: anti-Saccharomices cerevisiae (ASCA) and the recently described anti-laminaribioside (ALCA), anti-chitobioside (ACCA), anti-mannobioside (AMCA), anti-laminarin (anti-L) and anti-chitin (anti-C) antibodies; in addition to other antibodies that target microbial antigens: anti-outer membrane porin C (anti-OmpC), anti-Cbir1 flagellin and anti-12 antibody. Also, autoantibodies targeting the exocrine pancreas (PAB) were shown to be highly specific for CD. In contrast, UC has been associated with anti-neutrophil cytoplasmic autoantibodies (pANCA) and antibodies against goblet cells (GAB). Current evidence suggests that serologic panels of multiple antibodies are useful in differential diagnosis of CD versus UC and can be a valuable aid in stratifying patients according to disease phenotype and risk of complications.

Mutated citrullinated vimentin antibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most common inflammatory systemic autoimmune disease, primarily affecting the peripheral joints. The past decade has been marked with revolutionary changes both in the therapeutic and diagnostic perspectives of RA. The discovery of an RA-specific citrullination-driven immune reaction gave a substantial contribution in the diagnostic approach to RA. Efforts directed towards the identification of the antigenic target specifically recognized by these autoantibodies resulted in the identification of vimentin in citrullinated form as the potential native antigen, among other proteins. Furthermore, it was found that the mutation of vimentin represents an independent trigger of antigenic properties, in addition to citrullination. As a result of this discovery, a commercial ELISA using mutated citrullinated vimentin (MCV) was developed. Increasingly, data now support the use of anti-MCV in RA diagnosis and prognosis for errosion. This review summarizes the research to date on the use of anti-MCV in RA diagnosis and prognosis and its potential use as a therapeutic marker. The pathologic role of these antibodies in RA disease is also discussed.