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La classification des myopathies inflammatoires idiopathiques (MII) évolue, avec la prise en compte, en plus des critères clinico-pathologiques, de la présence d'auto-anticorps spécifiques des myosites (ASM) pour le diagnostic et l'identification de formes cliniques. Le diagnostic des dermatomyosites (DM) repose sur des critères cliniques, histologiques, d'imagerie et la mise en évidence d'ASM.Au sein de ces anticorps, les anti-TIF1γ ont une place à part en raison de leur forte prévalence dans la DM juvénile et dans les DM associées au cancer. Les anticorps anti-TIF1γ sont présents dans 7-31 % des DM de l'adulte. Les DM avec anti-TIF1γ sont associées à un cancer dans plus de la moitié des cas chez l'adulte (38-75 %), alors que ce n'est pas le cas dans les DM juvéniles. Le rôle de la protéine TIF1γ dans le processus tumoral et celui des auto-anticorps dans le déclenchement d'une DM font l'objet de nombreuses hypothèses, mais sans aucune conclusion définitive actuellement. Compte tenu de l'importance diagnostique et pronostique de la présence d'anticorps anti-TIF1γ, il est essentiel de disposer de tests fiables et adaptés à la pratique de routine pour les caractériser. La technique de référence, l'immunoprécipitation, n'est pas adaptée au laboratoire de routine. Les immunodots sont pratiques mais leurs performances ne sont pas optimales en termes de sensibilité et spécificité. L'Elisa et la technique automatisée type PMAT (technologie multi-analyte basée sur particule) semblent présenter des performances proches de l'immunoprécipitation mais avec une praticabilité adaptée à la routine. The classification of idiopathic inflammatory myopathies (IBD) is evolving, with the consideration, in addition to clinicopathological criteria, of the presence of autoantibodies specific to myositis for the diagnosis and identification of clinical forms. The diagnosis of dermatomyositis (DM) is based on clinical, histological, imaging criteria and the demonstration of myositis-specific autoantibodies (MSA). Within these antibodies, anti-TIF1γ have a special place due to their high prevalence in juvenile DM and in cancer-associated DM. Anti-TIF1γ antibodies are present in 7-31% of adult DMs. DMs with anti-TIF1γ are associated with cancer in more than half of cases in adults (38-75%), whereas this is not the case in juvenile DMs. The role of the TIF1γ protein in the tumor process and of autoantibodies in the triggering of DM are the subject of numerous hypotheses, but without any definitive conclusion currently. Given the diagnostic and prognostic importance of the presence of anti-TIF1γ antibodies, it is essential to have reliable tests suitable for routine practice to characterize them. The reference technique, immunoprecipitation, is not suitable for the routine laboratory. Immunodots are practical but their performance is not optimal in terms of sensitivity and specificity. Elisa and the particle-based multi-analyte technology (PMAT)-type automated technique seem to present performances close to immunoprecipitation but with a practicability adapted to routine use. [ABSTRACT FROM AUTHOR]

Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe, is becoming increasingly important due to its close proximity to humans, the increasing import of dogs from endemic regions and the impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA (GSD Frankfurt, Germany) was compared with three rKLi8.3-based diagnostic test systems, the rKLi8.3 ELISA (GSD Frankfurt, Germany), the INgezim® Leishma CROM (GSD Madrid, Spain) lateral flow test (LFT) and the VetBlot® Leishmania LineBlot (GSD Frankfurt, Germany). CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo- and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens; it showed very high specificity for healthy dogs from endemic regions in both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, serodiagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens, and the LFT has the advantage of providing a laboratory-independent, rapid and specific diagnosis of CanL. [ABSTRACT FROM AUTHOR]

The current Centers for Disease Control and Prevention (CDC) interpretive criteria for serodiagnosis of Lyme disease (LD) involve a two-tiered approach, consisting of a first-tier EIA, IFA, or chemiluminescent assay, followed by confirmation of positive or equivocal results by either immunoblot or a second-tier EIA. To increase overall sensitivity, single-tier alternative immunoblot assays have been proposed, often utilizing antigens from multiple Borrelia burgdorferi strains or genospecies in a single immunoblot; including OspA and OspB in their antigen panel; requiring fewer positive bands than permitted by current CDC criteria; and reporting equivocal results. Published reports concerning alternative immunoblot assays have used relatively small numbers of LD patients and controls to evaluate novel multi-antigen assays and interpretive criteria. We compared the two most commonly used alternative immunoblot interpretive criteria (labeled A and B) to CDC criteria using data from multiple FDA-cleared IgG and IgM immunoblot test kits. These single-tier alternative interpretive criteria, applied to both IgG and IgM immunoblots, demonstrated significantly more false-positive or equivocal results in healthy controls than two-tiered CDC criteria (12.4% and 35.0% for Criteria A and B, respectively, versus 1.0% for CDC criteria). Due to limited standardization and high false-positive rates, the presently evaluated single-tier alternative immunoblot interpretive criteria appear inferior to CDC two-tiered criteria. [ABSTRACT FROM AUTHOR]

Background: The usefulness of myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs) for the assessment of idiopathic inflammatory myopathies (IIMs) is acknowledged, but laboratory standardization remains a challenge. We detected MSAs/MAAs by multi-analytic line immunoassay (LIA) and particle-based multi-analyte technology (PMAT) in a multicenter cohort of patients with IIMs. Methods: We tested the sera from 411 patients affected with definite IIM, including 142 polymyositis (PM), 147 dermatomyositis (DM), 19 cancer-associated myositis, and 103 overlap myositis syndrome (OM), and from 269 controls. MSAs/MAAs were determined by 16Ags LIA in all sera, and anti-HMGCR by ELISA in 157/411 IIM sera and 91/269 control sera. The analytical specificity of LIA/HMGCR ELISA was compared with that of PMAT in 89 MSA+ IIM sera. Results: MSAs/MAAs were positive in 307/411 (75%) IIM patients and 65/269 (24%) controls by LIA (Odds Ratio 9.26, 95% CI 6.43–13.13, p < 0.0001). The sensitivity/specificity of individual MSAs/MAAs were: 20%/100% (Jo-1), 3%/99.3% (PL-7), 4%/98.8% (PL-12), 1%/100% (EJ), 0.7%/100% (OJ), 9%/98% (SRP), 5.6%/99.6% (TIF1γ), 4.6%/99.6% (MDA5), 8%/96% (Mi-2), 1.5%/98% (NXP2), 1.7%/100% (SAE1), 4%/92% (Ku), 8.5%/99% (PM/Scl-100), 8%/96% (PM/Scl-75), and 25.5%/79% (Ro52). Anti-HMGCR was found in 8/157 (5%) IIM patients and 0/176 (0%) controls by ELISA (p = 0.007). Concordance between LIA/HMGCR ELISA and PMAT was found in 78/89 (88%) samples. Individual MSAs detected by LIA were associated with IIM subsets: Jo-1 with PM and OM, PL-12 with OM, Mi-2, TIF1γ, and MDA5 with DM, SRP with PM, and PM/Scl-75/100 with OM (p < 0.001 for all). Conclusions: Since MSAs are mostly mutually exclusive, multi-specific antibody profiling seems effective for a targeted clinical-serologic approach to the diagnosis of IIMs. [ABSTRACT FROM AUTHOR]

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Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe, is becoming increasingly important due to its close proximity to humans, the increasing import of dogs from endemic regions and the impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA (GSD Frankfurt, Germany) was compared with three rKLi8.3-based diagnostic test systems, the rKLi8.3 ELISA (GSD Frankfurt, Germany), the INgezim® Leishma CROM (GSD Madrid, Spain) lateral flow test (LFT) and the VetBlot® Leishmania LineBlot (GSD Frankfurt, Germany). CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo- and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens; it showed very high specificity for healthy dogs from endemic regions in both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, serodiagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens, and the LFT has the advantage of providing a laboratory-independent, rapid and specific diagnosis of CanL.

The current Centers for Disease Control and Prevention (CDC) interpretive criteria for serodiagnosis of Lyme disease (LD) involve a two-tiered approach, consisting of a first-tier EIA, IFA, or chemiluminescent assay, followed by confirmation of positive or equivocal results by either immunoblot or a second-tier EIA. To increase overall sensitivity, single-tier alternative immunoblot assays have been proposed, often utilizing antigens from multiple Borrelia burgdorferi strains or genospecies in a single immunoblot; including OspA and OspB in their antigen panel; requiring fewer positive bands than permitted by current CDC criteria; and reporting equivocal results. Published reports concerning alternative immunoblot assays have used relatively small numbers of LD patients and controls to evaluate novel multi-antigen assays and interpretive criteria. We compared the two most commonly used alternative immunoblot interpretive criteria (labeled A and B) to CDC criteria using data from multiple FDA-cleared IgG and IgM immunoblot test kits. These single-tier alternative interpretive criteria, applied to both IgG and IgM immunoblots, demonstrated significantly more false-positive or equivocal results in healthy controls than two-tiered CDC criteria (12.4% and 35.0% for Criteria A and B, respectively, versus 1.0% for CDC criteria). Due to limited standardization and high false-positive rates, the presently evaluated single-tier alternative immunoblot interpretive criteria appear inferior to CDC two-tiered criteria.

Objectives Anti‐TIF1-gamma autoantibodies can be detected with immunoprecipitation (IP), line blot (LB) and ELISA. We compared assay performance in patients with DM and the potential of these assays to detect anti-TIF1-gamma positive cancer-associated DM (CADM). Methods We included sera from 131 patients with DM followed at Karolinska University Hospital, Stockholm, Sweden and 82 healthy controls. Serum samples taken at DM diagnosis were tested for anti-TIF1-gamma autoantibodies with IP, two ELISAs (in-house and commercial) and LB. Cancer diagnosis and dates were obtained from the Swedish national cancer register. CADM was defined as a malignancy that developed within 3 years of DM diagnosis. Results Anti-TIF1-gamma autoantibodies were detected in 19/101 (18.8%), 15/113 (13.2%), 34/131 (26%) and 45/131 (34.4%) of the patients with IP, LB, in-house and commercial ELISA, respectively. The anti-TIF1-gamma results from the in-house ELISA were confirmed with IP in 93 of 101 (92%) cases, κ = 0.76, with a commercial ELISA in 110 of 131 (84%) cases, κ = 0.63, and with LB in 101 of 113 (89.3%) cases, κ = 0.67. Anti-TIF1-gamma results with IP were confirmed with LB in 85 of 92 (92.4%) cases, κ = 0.73. For detecting CADM, the anti-TIF1-gamma in-house ELISA had a sensitivity of 58% and specificity of 86%, the commercial ELISA had a sensitivity of 63% and specificity of 82%, IP had a sensitivity of 52% and specificity of 92%, LB had a sensitivity of 40% and specificity of 96%. Conclusion The two anti-TIF1-gamma ELISA assays had advantages both for autoantibody detection and to identify anti-TIF1-gamma-positive CADM. [ABSTRACT FROM AUTHOR]

To confirm discrepant SARS-CoV-2-IgG results in four standard assays we applied for the first time a prototype of a coronavirus IgG-line-blot which employs antigens from seasonal coronaviruses, SARS-1 and SARS-CoV-2 combined with avidity testing as a confirmatory tool in a follow-up of five cases including pre-pandemic samples.

Background: The usefulness of myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs) for the assessment of idiopathic inflammatory myopathies (IIMs) is acknowledged, but laboratory standardization remains a challenge. We detected MSAs/MAAs by multi-analytic line immunoassay (LIA) and particle-based multi-analyte technology (PMAT) in a multicenter cohort of patients with IIMs. Methods: We tested the sera from 411 patients affected with definite IIM, including 142 polymyositis (PM), 147 dermatomyositis (DM), 19 cancer-associated myositis, and 103 overlap myositis syndrome (OM), and from 269 controls. MSAs/MAAs were determined by 16Ags LIA in all sera, and anti-HMGCR by ELISA in 157/411 IIM sera and 91/269 control sera. The analytical specificity of LIA/HMGCR ELISA was compared with that of PMAT in 89 MSA+ IIM sera. Results: MSAs/MAAs were positive in 307/411 (75%) IIM patients and 65/269 (24%) controls by LIA (Odds Ratio 9.26, 95% CI 6.43–13.13, p < 0.0001). The sensitivity/specificity of individual MSAs/MAAs were: 20%/100% (Jo-1), 3%/99.3% (PL-7), 4%/98.8% (PL-12), 1%/100% (EJ), 0.7%/100% (OJ), 9%/98% (SRP), 5.6%/99.6% (TIF1γ), 4.6%/99.6% (MDA5), 8%/96% (Mi-2), 1.5%/98% (NXP2), 1.7%/100% (SAE1), 4%/92% (Ku), 8.5%/99% (PM/Scl-100), 8%/96% (PM/Scl-75), and 25.5%/79% (Ro52). Anti-HMGCR was found in 8/157 (5%) IIM patients and 0/176 (0%) controls by ELISA (p = 0.007). Concordance between LIA/HMGCR ELISA and PMAT was found in 78/89 (88%) samples. Individual MSAs detected by LIA were associated with IIM subsets: Jo-1 with PM and OM, PL-12 with OM, Mi-2, TIF1γ, and MDA5 with DM, SRP with PM, and PM/Scl-75/100 with OM (p < 0.001 for all). Conclusions: Since MSAs are mostly mutually exclusive, multi-specific antibody profiling seems effective for a targeted clinical-serologic approach to the diagnosis of IIMs.

Detection of onconeural antibodies is important because establishes a definitive diagnosis of paraneoplastic neurological syndrome (PNS). The recommended method for diagnosis of onconeural antibodies is by immunohistochemistry on rodent brain sections and confirmation of results by immunoblot. However, in many diagnostic laboratories samples are only tested with commercial line blots. In this study we inquired whether this change in diagnostic methodology (line blot alone vs. combined immunohistochemistry and line blot) would affect the results. Among 439 samples examined by immunohistochemistry and a commercial line blot (Euroimmun, Lübeck, Germany) 96 (22%) were positive by line blot, and their clinical information was reviewed. Onconeural antibodies were detected by both assays in 46/96 (48%) patients (concordant group) whereas 50 (52%) were only positive by line blot (discordant group). In the concordant group 42/46 (91%) patients had a definite diagnosis of PNS whereas in the discordant group only 4/50 (8%) had PNS (p < 0.00001). None of the 14 patients with ZIC4 antibodies and 1/13 (8%) with Yo antibodies demonstrated only by line blot had PNS. These findings show a robust diagnostic value of combined diagnostic techniques, and both should be used to demonstrate onconeural antibodies, If antibody testing is performed only with line blot assay, positive bands should be confirmed by rodent brain immunohistochemistry. For ZIC4 or Yo antibody testing, line blot positivity with negative immunohistochemistry has no diagnostic significance, and for the rest of onconeural antibodies the predictive diagnostic value is low.

SOX1 autoantibodies are considered markers of small cell lung cancer (SCLC) and paraneoplastic neurological syndromes (PNS) and are usually determined by commercial line blot in many clinical services. Recent studies suggested that SOX1 autoantibodies also occur in patients with neuropathies unrelated to SCLC, questioning the value of SOX1 autoantibodies as paraneoplastic biomarkers. Here, we compared the specificity and sensitivity of a commercial line blot (Euroimmun, Lübeck, Germany) with those of an in house cell-based assay (CBA) with HEK293 cells transfected with SOX1. Overall, 210 patients were included in the study, 139 patients with polyneuropathies without SCLC, and 71 with disorders associated with SOX1 autoantibodies detected with the in-house CBA. Forty one of these 71 cases had been referred to our laboratory for onconeuronal antibody assessment and 30/71 were patients with known PNS and SCLC. None of the patients with polyneuropathies had SOX1 autoantibodies by either line blot or CBA (specificity of the immunoblot: 100%; 95%C.I.: 97.8–100). Among the 71 patients with CBA SOX1 autoantibodies, only 53 were positive by line blot (sensitivity: 74.6%; 95%C.I.: 62.9–84.2). Lung cancer was detected in 37/41 (90%; 34 with SCLC) patients referred for onconeuronal antibody assessment and 34 of them also had a PNS. Our study confirms the association of SOX1 autoantibodies with SCLC and PNS. The line blot test misses 25% of the cases; therefore, to minimize the frequency of false negative results we recommend the use of a confirmatory test, such as CBA, in patients suspected to have a SCLC-related PNS.

An adaptive immune response in less than 1% of people who develop cancer produces antibodies against neuronal proteins. These antibodies can be associated with paraneoplastic syndromes, and their accurate detection should instigate a search for a specific cancer. Over the years, multiple systems, from indirect immunofluorescence to live cell-based assays, have been developed to identify these antibodies. As the specific antigens were identified, high throughput, multi-antigen substrates such as line blots and ELISAs were developed for clinical laboratories. However, the evolution of assays required to identify antibodies to membrane targets has shone a light on the importance of antigen conformation for antibody detection. This chapter discusses the early antibody assays used to detect antibodies to nuclear and cytosolic targets and how new approaches are required to detect antibodies to membrane targets. The chapter presents recent data that support international recommendations against the sole use of line blots for antibody detection and highlights a new antigen-specific approach that appears promising for the detection of submembrane targets., (Copyright © 2024 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract Accelerated identification of autoantibodies associated with previously idiopathic neurological disease has provided insights into disease mechanisms, enhanced understanding of neurological function, and opportunities for improved therapeutic interventions. The role of the laboratory in the expanding field of neuroimmunology is critical as specific autoantibody identification provides guidance to clinicians in diagnosis, prognosis, tumor search strategies, and therapeutic interventions. The number of specific autoantibodies identified continues to increase and newer testing strategies increase efficiencies in the laboratory and availability to clinicians. The need for broadly targeted efficient testing is underscored by the variability in clinical presentation and tumor associations attributable to a specific autoantibody, and conversely the various autoantibody specificities that can be the cause of a given clinical presentation. While many of the antineural antibodies were first recognized in the setting of neoplastic disease, idiopathic autoimmune neurological disease in the absence of underlying tumor is increasingly recognized. Appropriation of therapeutic modalities used to treat autoimmune disease to treat these autoantibody mediated neurological diseases has improved patient outcomes. Interaction between clinicians and laboratorians is critical to our understanding of these diseases and optimization of the clinical benefits of our increasing knowledge in neuroimmunology. [ABSTRACT FROM AUTHOR]

Background Collection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. Methods We created serum pools from four different patient groups (systemic lupus erythematosus n = 85, rheumatoid arthritis n = 77, Sjögren's syndrome n = 91, systemic sclerosis n = 66) and one healthy control group (n = 67). Each serum pool was analyzed using three well-known immunoassays: enzyme-linked immunosorbent assay (ELISA), line blot, and immunofluorescence microscopy (anti-nuclear antibody (ANA) screening). The presence of Epstein-Barr virus (EBV) EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies was used to validate serum pools as an efficient tool for further investigations by comparison to previous findings in this area. Results The presence of EBV EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies in each pool was consistent within the obtained ELISA and line blot results, as increased titers of IgG against the four antigens were found in all patient serum pools and also in individual sera regarding gp350. These results correspond to previous findings on individual samples from patients with these diseases. The presence of ANAs was observed in all four patient serum pools and not in the HC pool by both line blots and immunofluorescence microscopy, which corresponds with the expectations and further corroborate the application of serum pools for screenings. Conclusion We developed and validated the use of serum pools that reliably and rapidly can confirm or deny hypotheses, which enables a more efficient research concentrating on the most evident factors. [ABSTRACT FROM AUTHOR]

Objective To analyze the performance of a line blot assay for the identification of autoantibodies in sera of patients affected by myositis, compared with immunoprecipitation (IP) as gold standard. Methods 66 sera of patients with myositis (23 polymyositis, 8 anti-synthetase syndromes, 29 dermatomyositis and 6 overlap syndromes) were tested by commercial LB (Euroimmun, Lubeck, Germany); 57 sera were analyzed also by IP of K562 cell extract radiolabeled with 35 S-methionine. Inter-rater agreement was calculated with Cohen's k coefficient. Results Myositis-specific antibodies (MSA) were detected in 36/57 sera (63%) by IP and in 39/66 sera (59%) by LB. The most frequent MSA found by LB were anti-Jo1 and anti-Mi2 found in 15% (10/66) of sera, followed by anti-NXP2 and anti-SRP detected in 106% (7/66) of sera. Anti-TIF1gamma and anti-MDA5 were found in 6 (9%) and 5 sera (7.6%), respectively. A good agreement between methods was found only for anti-TIF1γ, anti-MDA5 and anti-NXP-2 antibodies, while a moderate agreement was estimated for anti-Mi2 and anti-EJ. By contrast, a high discordance rate for the detection of anti-Jo1 antibodies was evident (k: 0.3). Multiple positivity for MSA were found in 11/66 (17%) by LB and 0/57 by IP (p: 0001). Comparing the clinical features of these 11 sera, we found total discrepancies between assays in 3 sera (27.3%), a relative discrepancy due to the occurrence of one discordant autoantibody (not confirmed by IP) in 5 cases (45.5%) and a total discrepancy between LB and IP results, but with a relative concordance with clinical features were found in other 3 sera (27.3%). The semiquantitative results do not support the interpretation of the data. Conclusions The use of LB assay allowed the detection of new MSA, such as anti-MDA5, anti-MJ and anti-TIF1gamma antibodies, previously not found with routine methods. However, the high prevalence of multiple positivities and the high discondant rate of anti-Jo1 antibodies could create some misinterpretation of the results from the clinical point of view. These data should be confirmed by enlarging the number of myositis cases. [ABSTRACT FROM AUTHOR]

Serum anti-Ro60 is one the most frequently encountered autoantibodies in the diagnostic immunopathology laboratory and in clinical practice. A large variety of assays are available to detect this including the popular multiplex line immunoblot (IB) assay. We evaluated the analytical performance of the IB for anti-Ro60 detection, using the counterimmunoelectrophoresis (CIEP) method as the 'gold standard'. We also undertook a survey of international laboratories, who use the IB, about their reporting practices for anti-Ro60. Using the manufacturer's reported cut-off of 15 units, the IB has an analytical sensitivity of 90.9% and specificity of 99.3% for anti-Ro60 detection. The optimal cut-off to balance sensitivity and specificity was determined to be 5 units with a sensitivity of 100% and specificity of 97.4%. Most laboratories use the manufacturer's specified cut-off (15 units) when determining a positive anti-Ro60 result. Whilst the commercial IB generally performs well, laboratorians need to be mindful of the limitations of IB in detecting antibodies that recognise conformational epitopes and what cut-offs they use. A vast majority of laboratories could potentially miss detection of this clinically important autoantibody., (Crown Copyright © 2022. Published by Elsevier B.V. All rights reserved.)

Onconeural antibodies are a group of autoantibodies present in patients with paraneoplastic syndromes (PNS), and are indicative for underlying malignancies. The use of indirect immunofluorescence assay (IFA) as the sole screening method for onconeural antibodies without line blot assay (LBA) could potentially miss a significant population of patients with PNS. However, testing each serum individually on LBA will pose significant economical and labour burdens to clinical laboratories. Based on the screening result from IFA, we developed a cost-effective pooling strategy for the detection of onconeural antibodies on LBA. Results of onconeural antibodies tested by IFA and LBA from 1887 serum samples received in the Central Sydney Immunology Laboratory were retrospectively analysed. Sera were pre-screened by IFA before proceeding to LBA. Sera with positive staining on IFA were tested individually on LBA while sera with negative IFA were examined by pooling. Agreements of antibody reactivity against onconeural antigens were evaluated for sera run by pooling and by individually. The estimate of the cost saving was also conducted for the pooling strategy. Antibody reactivity to each specific onconeural antigen from pooling run had over 95% qualitative result agreement with sera run individually on LBA. An excellent correlation (r = 0.88) of positive reactions quantitated by band signal intensity was also observed. Using our well-established sera pooling strategy for LBA, a cost saving of 50.1% was achieved for reagent alone. The sera pooling strategy for LBA is a reliable and cost-effective approach for testing low prevalence diseases such as PNS. [ABSTRACT FROM AUTHOR]

Objective. Serological testing for myositis-specific or associated autoantibodies [myositis-specific antibody (MSA) and myositis-associated antibody (MAA)] is useful for the diagnosis of idiopathic inflammatory myopathies (IIMs). However, available assays are neither standardized nor validated. The objective is to evaluate the accuracy of a commercial line blot assay for myositis diagnosis.Methods. IgG antibodies against Jo-1, PL-7, PL-12, PM/Scl, Ku, Mi-2 and Ro52 antigens were detected by a line blot and in-house RNA immunoprecipitation or immunoblot. We tested sera from 208 IIM patients, 50 healthy subjects and 180 control patients (11 non-autoimmune myopathy, 23 muscular dystrophy, 11 UCTD, 68 SLE, 36 SSc, 22 SS and 9 arthropathy).Results. MSAs or MAAs were detected in 98 (47%) out of the 208 IIM patients by line blot: anti-Jo-1 in 43 (21%), anti-PL-7 or anti-PL-12 in 8 (4%), anti-Mi-2 in 9 (4%), anti-PM/Scl in 9 (4%), anti-Ku in 10 (5%) and anti-Ro52 in 49 (24%). Overall specificity was: 100% for anti-Jo-1, anti-PL-7 or PL-12 and anti-PM/Scl; 96% for anti-Ku; 98% for anti-Mi-2; and 76% for anti-Ro52. In-house testing confirmed line blot results regarding anti-Jo-1, anti-PM/Scl and anti-Ku, while it was more accurate than line blot in detecting anti-Mi-2 (7 vs 4% sensitivity, 100 vs 98% specificity), and anti-aminoacyl-tRNA synthetase (anti-ARS) non-Jo-1 antibodies (11 vs 4% sensitivity, 97 vs 99% specificity).Conclusions. Line blot could be a suitable serological test in the diagnostic workup for myositis, and it represents a reliable alternative to more time-consuming procedures. Continuous effort is recommended in order to improve its accuracy. [ABSTRACT FROM PUBLISHER]

Autoantibody assays are reported in a variety of formats. Results only slightly above established cut-offs provide lower likelihood ratios; therefore, their clinical significance may be more uncertain, which is not readily communicated with dichotomous qualitative reporting. Line immunoassays (LIA) are a common method for detecting antibodies to extractable nuclear antigens (ENA) and myositis-associated antibodies. However, recommended positive cut-offs are contentious. We distributed a survey via e-mail to participants in the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) Immunology modules and to a dedicated immunology mailing list in Australasia. Questions explored general viewpoints surrounding autoantibody reporting, as well as current laboratory practices, with particular focus on interpretation and reporting of the most commonly used ENA LIA manufactured by Euroimmun. There were 31 responders, representative of at least 17 unique laboratories across Australia (8 public, 5 private) and New Zealand (4 laboratories). Responses suggest that autoantibody reporting is not standardised; there was variation in general viewpoints and reporting practices, particularly regarding the interpretation of and positive cut-offs used for the Euroimmun ENA LIA, which were contrary to the manufacturer's guidelines in a majority of the responses. Interpretative qualitative reporting based on results from other investigations and the clinical history was a common theme. There is large variation in the reporting of autoantibody assays within Australasia, especially by LIA. A majority of respondents report the most commonly used ENA LIA contrary to manufacturer's guidelines; alternative positive cut-offs are commonly utilised. LIA reports should indicate the level of positivity to enhance their relevance in the clinical decision-making process., (Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

To confirm discrepant SARS-CoV-2-IgG results in four standard assays we applied for the first time a prototype of a coronavirus IgG-line-blot which employs antigens from seasonal coronaviruses, SARS-1 and SARS-CoV-2 combined with avidity testing as a confirmatory tool in a follow-up of five cases including pre-pandemic samples., (Copyright © 2021. Published by Elsevier B.V.)

Detection of onconeural antibodies is important because establishes a definitive diagnosis of paraneoplastic neurological syndrome (PNS). The recommended method for diagnosis of onconeural antibodies is by immunohistochemistry on rodent brain sections and confirmation of results by immunoblot. However, in many diagnostic laboratories samples are only tested with commercial line blots. In this study we inquired whether this change in diagnostic methodology (line blot alone vs. combined immunohistochemistry and line blot) would affect the results. Among 439 samples examined by immunohistochemistry and a commercial line blot (Euroimmun, Lübeck, Germany) 96 (22%) were positive by line blot, and their clinical information was reviewed. Onconeural antibodies were detected by both assays in 46/96 (48%) patients (concordant group) whereas 50 (52%) were only positive by line blot (discordant group). In the concordant group 42/46 (91%) patients had a definite diagnosis of PNS whereas in the discordant group only 4/50 (8%) had PNS ( p < 0.00001). None of the 14 patients with ZIC4 antibodies and 1/13 (8%) with Yo antibodies demonstrated only by line blot had PNS. These findings show a robust diagnostic value of combined diagnostic techniques, and both should be used to demonstrate onconeural antibodies, If antibody testing is performed only with line blot assay, positive bands should be confirmed by rodent brain immunohistochemistry. For ZIC4 or Yo antibody testing, line blot positivity with negative immunohistochemistry has no diagnostic significance, and for the rest of onconeural antibodies the predictive diagnostic value is low., (Copyright © 2020 Ruiz-García, Martínez-Hernández, Saiz, Dalmau and Graus.)

SOX1 autoantibodies are considered markers of small cell lung cancer (SCLC) and paraneoplastic neurological syndromes (PNS) and are usually determined by commercial line blot in many clinical services. Recent studies suggested that SOX1 autoantibodies also occur in patients with neuropathies unrelated to SCLC, questioning the value of SOX1 autoantibodies as paraneoplastic biomarkers. Here, we compared the specificity and sensitivity of a commercial line blot (Euroimmun, Lübeck, Germany) with those of an in house cell-based assay (CBA) with HEK293 cells transfected with SOX1. Overall, 210 patients were included in the study, 139 patients with polyneuropathies without SCLC, and 71 with disorders associated with SOX1 autoantibodies detected with the in-house CBA. Forty one of these 71 cases had been referred to our laboratory for onconeuronal antibody assessment and 30/71 were patients with known PNS and SCLC. None of the patients with polyneuropathies had SOX1 autoantibodies by either line blot or CBA (specificity of the immunoblot: 100%; 95%C.I.: 97.8-100). Among the 71 patients with CBA SOX1 autoantibodies, only 53 were positive by line blot (sensitivity: 74.6%; 95%C.I.: 62.9-84.2). Lung cancer was detected in 37/41 (90%; 34 with SCLC) patients referred for onconeuronal antibody assessment and 34 of them also had a PNS. Our study confirms the association of SOX1 autoantibodies with SCLC and PNS. The line blot test misses 25% of the cases; therefore, to minimize the frequency of false negative results we recommend the use of a confirmatory test, such as CBA, in patients suspected to have a SCLC-related PNS.

The vast majority of the human adult population is infected with Epstein-Barr virus (EBV), and the majority of the EBV-infected individuals tolerates the infection well, without any further symptoms after primary infection. In cases of individuals which undergo primary infection in the form of an infectious mononucleosis, or which have undergone primary infection in their past, it is sometimes important to appraise symptomatic disease or differentiate infectious mononucleosis from other conditions. In these cases, serological methods, i.e., immunofluorescence, ELISA, or Western blot, are the methods of choice to come to an unequivocal diagnostic conclusion, while the detection and quantification of viral DNA through PCR plays a minor role.On the other hand, in a minority of the human population, EBV infection is associated or causally linked with autoimmune or malignant disease. Especially in the bone marrow or solid organ transplanted, or in otherwise severely immune-suppressed patients, prolonged EBV primary infection or EBV reactivation from latency may be a serious and life-threatening complication which needs to be diagnosed the faster the better, in order to take therapeutic steps in time. Determining the serostatus correctly is also important in these cases. However, the direct and quantitative detection of viral DNA are of importance for the diagnosis of serious EBV disease and its monitoring.In the following, we give an overview of diagnostic methods to accurately determine EBV serostatus and viral load. We evaluate the advantages and disadvantages of each method and report on the diagnostic significance of each and how to resolve diagnostic problems in case of uncertainties. For practical procedures, we refer to the detailed instruction manuals of the respective test kit manufacturers which have to be closely followed for reliable results.