Autoimmune Diagnostics Research Articles

#2646 Precision verification and method comparison of anti-MPO anti-hsPR3 immunoassay on Alegria2 instrument

Abstract Background and Aims Antibodies to myeloperoxidase (anti-MPO) and proteinase 3 (anti-PR3) are recognized as the only clinically relevant antineutrophil cytoplasmic antibodies (ANCA) specificities so far. ANCA testing can be used in different clinical settings, especially in situations that require rapid diagnosis like renal-pulmonary syndrome that require result within 24 hours. The aim of this study was to perform analytical verification of the new automated single test ELISA based method on Alegria2 instrument to test if the reagents and analyser were acceptable for the routine use. Method For precision assessment we tested two concentration levels of commercial control material in triplicate for five days in a row. We used EASI acceptable criteria for precision for anti-MPO as well as for anti-hsPR3. For method comparison we included 50 patients with history or suspicion of vasculitis. Anti-MPO and anti-PR3 were measured using 5 different reagents: Anti-Myeloperoxidase ELISA (IgG) and anti-PR3-hn-hr ELISA (IgG) on Euroimmun Analyzer I-2P (EUROIMMUN AG, Lubeck, Germany), MPO and PR3 II chemiluminiscent immunoassay (CLIA) on IDS iSYS (IDS ISYS, Pouilly en Auxois, France), QUANTA Flash® MPO and QUANTA Flash® PR3 CLIA on BIO-FLASH® (Inova Diagnostics Inc, San Diego, USA), Anti-MPO and anti-PR3 hs on Alegria2 ELISA based method (Orgentec, Mainz, Germany), and Anti-MPO and anti-PR3 ELISA (Orgentec, Mainz, Germany). Results were categorized as positive or negative and we calculated kappa statistics as well as intraclass correlation coefficient. Complete statistical analysis was done using MedCalc statistical software (MedCalc version 14.8.1, Ostend, Belgium). Results Within-laboratory precision (CV%) for anti-MPO for normal and pathological controls were 5.73% and 15.14% respectively. For anti-PR3 hs within-laboratory precision (CV%) for normal and pathological controls were 5.46% and 18.89% respectively. Intraclass correlation coefficient showed excellent degree of consistency in average measures (0.9778 (95% CI: 0.9665-0.9862)) for anti-MPO and good reliability on single ratings (0.8981 (95% CI: 0.8522-0.9344). For anti PR3 intraclass correlation coefficient showed excellent degree of consistency in average measures (0.9257 (95% CI: 0.8873-0.9539)) but only moderate consistency in single ratings (0.7136 (95% CI: 0.6115-0.8053)). Separate Cohen`s kappa testing revealed almost perfect agreement for most reagent combination for anti- MPO (kappa from 0.825 to 0.958). For anti-PR3 we found moderate agreement between QUANTA Flash® PR3 and PR3 II IDS iSYS (kappa 0.520) but mostly substantial agreement (kappa from 0.629 to 0.865). We also found almost perfect agreement between anti-PR3 hs on Alegria2 and QUANTA Flash® PR3 (kappa = 0.901). Conclusion Our results showed a rather small relative variability and met the precision criteria defined by EASI initiative. Satisfactory agreement between methods is very reassuring but due to the lack of standardisation in autoimmune diagnostics imposes that information about methodological differences must be clearly stated on laboratory report. According to our results the new method for anti-MPO and anti-PR3 proved to be suitable for the routine use.

Levels of Cell-Free DNA in Kidney Failure Patients before and after Renal Transplantation.

Circulating cell-free DNA (cfDNA) has diverse applications in oncological, prenatal, toxicological, cardiovascular, and autoimmune diseases, diagnostics, and organ transplantation. In particular, mitochondrial cfDNA (mt-cfDNA) is associated with inflammation and linked to early vascular ageing (EVA) in end-stage kidney failure (ESKF), which could be a noninvasive marker for graft rejection and organ damage. Plasma samples from 44 ESKF patients, of whom half (n = 22) underwent either conservative therapy (non-HD) or hemodialysis (HD) before kidney transplantation (KT). These samples were analyzed at baseline and two years after KT. cfDNA was extracted from plasma and quantified using the fluorometric method. qPCR was used to quantify and differentiate the fractions of mt-cfDNA and nuclear cfDNA (nc-cfDNA). mt-cfDNA levels in KT patients decreased significantly from baseline to two years post-KT (p < 0.0268), while levels of total cfDNA and nc-cfDNA did not differ. Depending on therapy modality (HD vs. non-HD) before KT, total cfDNA levels were higher in HD patients at both baseline (p = 0.0133) and two years post-KT (p = 0.0421), while nc-cfDNA levels were higher in HD only at baseline (p = 0.0079). Males showed a nonsignificant trend of higher cfDNA levels. Patients with assessed vascular fibrosis (p = 0.0068), either alone or in combination with calcification plus fibrosis, showed reduced mt-cfDNA post-KT (p = 0.0195). Changes in mt-cfDNA levels suggests the impact of KT on the inflammatory state of ESKF, as evidenced via its correlation with high sensitivity C-reactive protein after KT. Further studies are warranted to assess if cfDNA could serve as a noninvasive method for monitoring the response to organ transplantation and even for amelioration of EVA status per se.

Il metodo di immunofluorescenza per la ricerca di anticorpi ha i giorni contati?

The indirect immunofluorescence method (IIF) is the longest-lived in Laboratory Medicine. It has been used worldwide for more than 60 years for the search for autoantibodies in autoimmune diseases, both rheumatic and organ-specific. The huge technological evolution of the last decades is leading to reconsider its role in autoimmune diagnostics and accentuates its current limits towards faster, fully automated methods, with higher throughput, and often more accurate which may eventually replace IIF. In this article, I review the salient aspects, some of which still controversial, comparing the IIF method with the new technologies and with the new scenarios of autoimmune diagnostics.

Autoimmune diagnostics in nephrology and rheumatology

Autoimmune diagnostics plays a central role in the detection of various acute and/or chronic diseases in both nephrology and rheumatology, which are associated with high morbidity and mortality if left untreated or not detected in time. Patients are threatened with significant limitations in everyday skills and quality of life due to loss of kidney function and dialysis, immobilizing and destructive joint processes or also significant damage of organ systems. In all of these autoimmune diseases, early diagnosis and treatment is of central importance for the further course and prognosis of disease.Antibodies play an essential role in the pathogenesis of autoimmune diseases. Antibodies are either directed against organ or tissue-specific antigens, such as in primary membranous glomerulonephritis or Goodpasture's syndrome, or they lead to a systemic disease such as systemic lupus erythematosus (SLE) or rheumatoid arthritis.Knowledge of the sensitivity and specificity of antibodies is crucial for the interpretation of antibody diagnostics results. Antibody detection can precede the clinical onset of the disease, and antibody titers often reflect disease activity. However, there are also false positive results. Detection of antibodies in the absence of disease symptoms often leads to uncertainty and unnecessary further diagnostics. Therefore, an unfounded "antibody screening" is not recommended.A rational antibody diagnostics is an integral part of the diagnostics and during treatment of nephrological and rheumatological diseases like glomerulonephrititis, pulmorenal syndrome, SLE and other collagenosis, thrombotic microangiopathy (HUS/TTP) and rheumatoid arthritis.

An Italian nationwide survey on the evolution of autoantibody diagnostics in autoimmune rheumatic diseases.

The advent of new technologies and the discovery of new antigenic targets of autoantibodies has led to significant changes in autoimmune diagnostics worldwide. To address the extent to which autoimmunology laboratories adhere to such innovation in testing and reporting practices, the Italian Society of Clinical Pathology and Laboratory Medicine launched a national survey to assess the current status of autoimmune diagnostics and to provide direction for further harmonisation. A questionnaire covering topics related to the diagnosis of autoimmune systemic rheumatic diseases was distributed to 152 Italian autoimmunology laboratories. The 59 closed-answer questions were subdivided into four main sections: 1. the setting (university, hospital or private laboratory) and the number of tests carried out; 2. the technologies used and their level of automation; 3. the analytical phase (antibody tests and methods), including awareness of the International Consensus on ANA Patterns (ICAP) initiative; 4. reporting of results and clinician relations. A total of 121 laboratories (79.6%) responded to the survey (15% universities, 70 hospitals, and 15% private laboratories). Indirect immunofluorescence is used by 94.8% of respondents, chemiluminescence by 78.4%, fluoro-immuno-enzymatic assays by 67.5%, immunodot by 52.6%, line-immunoassay by 47.4%, addressable laser bead immunoassay by 10.3% and radioimmunological methods by 10.2%. The great majority of respondents implemented complete automation of the listed methodologies. 65% of participants state that they add an interpretative comment in the report. 45% of participants enjoy a collaborative relationship with clinicians; counselling activities are provided by almost half of participants. Survey results indicated that almost all respondent laboratories have broadened their antibody panel and that high-throughput technologies have been widely introduced. Gaps identified by the survey include a still incomplete compliance with guidelines in antibody profiles (e.g. in antiphospholipid syndrome and rheumatoid arthritis and reporting of test results. Awareness of these differences provides insights that may further contribute to achieving harmonisation in autoimmune diagnostics.

The Journal of Applied Laboratory Medicine Special Issue on Autoimmune Diagnostics.

The Journal of Applied Laboratory Medicine Special Issue on Autoimmune Diagnostics.

Проблемы диагностики аутоиммунной эпилепсии

Objective of the Review: To analyse and systematise knowledge in the problems with autoimmune epilepsy diagnostics. Main part. Clinical phenotypes of immune-mediated epilepsy depend on various types of antibodies. However, autoimmune epilepsy is diagnosed also in patients who are followed up for chronic refractory unexplained epilepsy, especially in initial epileptic status and late onset epilepsy without structural changes on brain imaging. Brain imaging changes may not be observed, especially in early disease. Very often a diagnostic challenge in autoimmune epilepsy is the difference between epileptic seizures and behavioural symptoms and the mental changes caused by involvement of limbic brain structures. An important role in detection of seizures and differential diagnosis is played by video-EEG-monitoring, which allows identifying the true number of seizures, epileptiform activity between seizures, behavioural changes not related to paroxysmal activity of cortical neurons. Any specific EEG signs for differentiation between various types of autoimmune epilepsy have not been found yet. Still, EEG can provide patterns that are unique for certain forms of autoimmune encephalitis. Conclusion. Video-EEG-monitoring significantly contributes to autoimmune epilepsy diagnostics, and some changes can be used as markers of disease severity. It is very important, especially in patients with impairment of consciousness, where identification of the clinical status and response to therapy is challenging. Keywords: autoimmune epilepsy, video-EEG-monitoring, epilepsy diagnostics

Prevalence of Autoimmune Diseases and Its Challenges in Diagnosis.

Autoimmune diseases (ADs) are the outcome of a malfunctioning immune system in which the immune system attacks self-antigens. These diseases are grouped into organ-specific and non-organ-specific types. Autoantibodies are important biomarkers used for confirming the diagnosis of ADs. Disease-specific autoantibodies are detected at a very early stage when typical clinical symptoms are not present in the patient, allowing prediction of the disease several years before the symptoms are visible. Diagnosis at an early stage is essential to decrease morbidity, disability, and mortality caused by ADs. Detection of autoantibodies, specific to particular phenotypes, helps to define these disorders as well as facilitate diagnosis, prognosis, and monitoring. In this review, we outline the present technologies used in autoimmune laboratories and the limitations of these methods along with future perspects of autoimmune diagnostics.

Rheumatoid factor isotype and Ro epitope distribution in primary Sjögren syndrome and rheumatoid arthritis with keratoconjunctivitis sicca.

Primary Sjögren syndrome (pS) is associated with autoantibodies such as rheumatoid factor (RF) and anti-nuclear antibodies such as anti-Ro (SS-A) and/or La (SS-B). Recent developments within autoimmune diagnostics allow quantitation of RF subclasses and anti-Ro epitopes. Will this refinement by autoimmune diagnostics help predicting development of extraglandular manifestations (EGM) in pS patients? A cohort of pS and rheumatoid arthritis (RA) patients with keratoconjunctivitis sicca (n = 35 and 16, resp) was included. Of the pS patients, 54% developed one or more EGM. Antibodies quantitated were IgM-RF, IgA-RF, IgG-RF, anti-Ro52, and anti-Ro60. Upon analysis of RF isotypes, pS patients generally displayed higher IgA-RF concentrations than RA patients (126 versus 49U/ml, p = 0.015), while the dominant RF isotype in RA patients was IgM-RF (82.5 versus 38U/ml, p = 0.012). No differences were observed regarding IgG-RF concentrations. In pS without/with EGM, the median RF IgM concentrations were similar, while RF IgA and IgG concentrations tended to be lower in pS patients with EGM > 1. Both Ro epitopes were strongly recognized by almost all pS patients, independent from EGM, while these antibodies were absent in RA patients. Primary Sjögren syndrome and RA patients have distinct serological profiles when analysing RF and Ro-specific antibodies. A longitudinal study of switched RF isotypes in pS patients is worthwhile from an immunological point of view, but its value is limited regarding identification of pS patients prone to developing EGM or RA patients prone to developing secondary sicca symptoms.

Development of an Ultra-Sensitive Multiplex (6-plex) Cytokine Digital Immunoassay with fg/ml Sensitivity

Abstract The future of immuno-oncology treatment and autoimmune diagnostics will involve the detection and quantification of inflammatory biomarkers. As immunotherapy becomes a more common approach for treating cancer and inflammatory disease, cytokines will not only play a role in determining a patient’s disease diagnosis, prognosis, and potential treatment options, but also may serve as a primary index of a therapy’s effectiveness. Incorporating these biomarkers into clinical practice, however, has been a challenge. Only a few cytokines have been studied in relation to inflammatory disease and immunotherapy, mainly because the biomarkers for these conditions are typically present in very small concentrations, and current research methods aren’t sensitive enough to detect them. It is critical that physicians have the ability to quantify these cytokines to track disease progression before and after treatment. We present development and characterization of a multiplex digital immunoassay to simultaneously detect and quantify six inflammatory biomarkers (IL-10, IL-12p70, IL-17A, IL-6, TNFa, IFNg) with fg/ml sensitivity. This advanced approach reduces assay volume requirements for multiple sample types and may be sensitive enough to detect circulating disease biomarkers before symptoms appear. As a result, it could potentially provide physicians with a new capability to track disease progression, and the effectiveness of treatments against diseases, which they previously could not monitor with such precision.

EASI - European Autoimmunity Standardisation Initiative: facing the challenges of diagnostics in autoimmunity.

Abstract The European Autoimmunity Standardisation Initiative (EASI) has been founded in order to improve autoimmune diagnostics by stimulating the interaction between the clinicians and laboratory specialists, by standardization of autoantibody tests, and by harmonization of testing algorithms. The ultimate goal of EASI is to utilize autoimmune diagnostics in the best way in order to optimize patient care. This mini-review gives an overview of the historical perspective of EASI and summarizes the major achievements.

Classification of indirect immunofluorescence images using thresholded local binary count features

Abstract Computer aided classification of HEp-2 cell based indirect immunofluorescence (IIF) images is a recommended procedure for standardising autoimmune disease diagnostics. In this work a novel feature, the thresholded local binary count (TLBC) has been proposed to classify IIF images into one among six classes. The TLBC is rotational invariant and is insensitive to pixel quantization noise. It characterizes the local binary gray scale pixel information in an image. The proposed feature along with global features such as area, entropy, illumination level and mean intensity, when classified using a support vector machine gave an accuracy of 86%. This feature could help in improving the diagnostics of autoimmune diseases which is highly clinically significant.

Challenges in the Standardization of Autoantibody Testing: a Comprehensive Review.

Standardization and harmonization are complementary tools to achieve higher testing quality in laboratory medicine. Both are of great relevance and are strongly needed in autoimmune diagnostics, due to the impressive advance in basic research and technological development observed in this diagnostic field in recent years that has led to the introduction of many new tests and new analytical methods. It is, therefore, essential that this strong innovative thrust is translated into clinical practice in a coordinated way to avoid confusion and the risk of potentially harmful errors for the patient. However, while standardization of antibody assays is a very complex task, harmonization of procedures and behaviors is a more feasible target and should necessarily include all the phases of the total testing process-in the pre-analytical phase, appropriateness of test requests, harmonization of autoantibody terminology, and adoption of uniform nomenclature for laboratory tests; in the analytical phase, harmonization of measurements, and sharing of test profiles and diagnostic algorithms; and in the post-analytical phase, harmonization of data reporting, and criteria for interpreting immunoserological results, especially harmonization of units, reference intervals, decision limits, and definition and notification of critical values. We here provide and discuss some examples of harmonization initiatives related to anti-nuclear antibodies, TSH receptor, and anti-thyroid peroxidase antibodies and to antibodies associated with autoimmune hepatitis and with celiac disease. These initiatives could be the starting steps to achieve a wider consensus and a closer interaction among stakeholders in the path of autoimmune diagnostics harmonization to enhance clinical effectiveness and provide greater patient safety.

The ANA-reflex test as a model for improving clinical appropriateness in autoimmune diagnostics.

Reflex tests are widely used in clinical laboratories, for example, to diagnose thyroid disorders or in the follow-up of prostate cancer. Reflex tests for antinuclear antibodies (ANA) have recently gained attention as a way to improve appropriateness in the immunological diagnosis of autoimmune rheumatic diseases and avoid waste of resources. However, the ANA-reflex test is not as simple as other consolidated reflex tests (the TSH-reflex tests or the PSA-reflex tests) because of the intrinsic complexity of the ANA test performed by the indirect immunofluorescence method on cellular substrates. The wide heterogeneity of the ANA patterns, which need correct interpretation, and the subsequent choice of the most appropriate confirmatory test (ANA subserology), which depend on the pattern feature and on clinical information, hinder any informatics automation, and require the pathologist’s intervention. In this review, the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine provides some indications on the configuration of the ANA-reflex test, using two different approaches depending on whether clinical information is available or not. We further give some suggestions on how to report results of the ANA-reflex test.

Development of a Certified Reference Material for myeloperoxidase-anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA)

A serum Certified Reference Material (CRM) for supporting reliable autoimmune diagnostics was recently released by the Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre of the European Commission. It was produced in collaboration with a Working Group on the Harmonisation of Autoimmune Tests of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC WG-HAT). This material is aimed at facilitating the standardisation of measurements of anti-myeloperoxidase immunoglobulin G antibodies. The CRM could be used as a common calibrant by clinicians and manufacturers thereby significantly improving the comparability of results from commercial immunoassays used for IgG anti-MPO measurements. This paper provides information on the new CRM and its intended use.

From autoantibody research to standardized diagnostic assays in the management of human diseases - report of the 12th Dresden Symposium on Autoantibodies.

Testing for autoantibodies (AABs) is becoming more and more relevant, not only for diagnosing autoimmune diseases (AIDs) but also for the differentiation of defined AID subtypes with different clinical manifestations, course and prognosis as well as the very early diagnosis for adequate management in the context of personalized medicine. A major challenge to improve diagnostic accuracy is to harmonize or even standardize AAB analyses. This review presents the results of the 12th Dresden Symposium on Autoantibodies that focused on several aspects of improving autoimmune diagnostics. Topics that are addressed include the International Consensus on ANA Patterns (ICAP) and the International Autoantibody Standardization (IAS) initiatives, the optimization of diagnostic algorithms, the description and evaluation of novel disease-specific AABs as well as the development and introduction of novel assays into routine diagnostics. This review also highlights important developments of recent years, most notably the improvement in diagnosing and predicting the course of rheumatoid arthritis, systemic sclerosis, idiopathic inflammatory myopathies, and of autoimmune neurological, gastrointestinal and liver diseases; the potential diagnostic role of anti-DFS70 antibodies and tumor-associated AABs. Furthermore, some hot topics in autoimmunity regarding disease pathogenesis and management are described.

Automation, consolidation, and integration in autoimmune diagnostics.

Over the past two decades, we have witnessed an extraordinary change in autoimmune diagnostics, characterized by the progressive evolution of analytical technologies, the availability of new tests, and the explosive growth of molecular biology and proteomics. Aside from these huge improvements, organizational changes have also occurred which brought about a more modern vision of the autoimmune laboratory. The introduction of automation (for harmonization of testing, reduction of human error, reduction of handling steps, increase of productivity, decrease of turnaround time, improvement of safety), consolidation (combining different analytical technologies or strategies on one instrument or on one group of connected instruments) and integration (linking analytical instruments or group of instruments with pre- and post-analytical devices) opened a new era in immunodiagnostics. In this article, we review the most important changes that have occurred in autoimmune diagnostics and present some models related to the introduction of automation in the autoimmunology laboratory, such as automated indirect immunofluorescence and changes in the two-step strategy for detection of autoantibodies; automated monoplex immunoassays and reduction of turnaround time; and automated multiplex immunoassays for autoantibody profiling.

EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics.

Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies.

Autoimmune diagnostics: the technology, the strategy and the clinical governance.

In recent years, there has been a profound change in autoimmune diagnostics. From long, tiring and inaccurate manual methods, the art of diagnostics has turned to modern, rapid and automated technology. New antibody tests have been developed, and almost all autoimmune diseases now have some specific diagnostic markers. The current need to make the most of available economic and human resources has led to the production of diagnostic algorithms and guidelines designated for optimal strategic use of the tests and to increase the diagnostic appropriateness. An important role in this scenario was assumed by the laboratory autoimmunologist, whose task is not only to govern the analytical phase, but also to help clinicians in correctly choosing the most suitable test for each clinical situation and provide consultancy support. In this review, we summarize recent advances in technology, describe the diagnostic strategies and highlight the current role of the laboratory autoimmunologist in the clinical governance of autoimmune diagnostics.

ANA HEp-2 cells image classification using number, size, shape and localization of targeted cell regions

The ANA HEp-2 medical test is a powerful tool in autoimmune disease diagnostics. The last step of this test, the interpretation of immunofluorescent images by trained experts, represents a potential source of errors and could theoretically be replaced by automated methods. Here we present a fully automatic method for recognition of types of immunofluorescent images produced by the ANA HEp-2 medical test. The proposed method makes use of the difference in number, size, shape and localization of cell regions that are targeted by the antinuclear antibodies – the humoral components of immune system that bind human antigens as a result of the immune system malfunction. The method extracts morphological properties of stained cell regions using a combination of thresholding-based and thresholding-less approaches and applies a conventional machine-learning algorithm for image classification.