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Background Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Patients with SLE exhibit multiple serum autoantibodies, including anti-neutrophil cytoplasmic antibodies (ANCAs). There are two main techniques to detect ANCAs: indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA). In this study, an attempt was made to determine the frequency and clinical associations of ANCAs in patients with SLE. Methods A cross-sectional study was conducted in a tertiary care hospital in Colombia that included 74 patients with SLE. The presence of ANCAs was assessed using IIF with ethanol-fixed slides, and ELISA was used to detect antibody specificities for myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA. Results Of the 74 patients with SLE evaluated, 60 (81.1%) of them were ANCA-positive by IIF. By contrast, only one patient showed specificity for PR3-ANCA by ELISA. The relevance of ANCA positivity by IIF and clinical and serological features was significant for renal involvement (p = .0174), and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (p = .0308). Conclusion ANCAs are common in the serum of patients with SLE, as detected by ethanol-fixed slides with IIF staining. However, detection of specificity to PR3 and/or MPO is rare, thus highlighting the importance of detecting these autoantibodies by different techniques.

Background Time and cost constraints lead to majority of clinical laboratories deviating away from an ideal practice of checking for antinuclear antibodies (ANAs) by indirect immunofluorescence (IIF) at multiple dilutions. Usage of screening dilution of 1:40 recommended by most manufacturers of commercial ANA kits results in numerous false positive-tests and misdiagnosis of connective tissue disorders (CTDs). We sought to study the ideal screening dilution for ANA by IIF for a diagnosis of ANA-related CTDs. Methods Serum samples of patients with ANA-related conditions (n = 233) and healthy controls (n = 154) were evaluated by IIF using Immuno Concepts Hep-2000 ® ANA kits at dilutions from 1:40 to 1:640. Accuracy for diagnosis of CTDs for each serum dilution was assessed by receiver operating curve (ROC) analysis. Results Antinuclear antibodies (ANA) positivity was observed in 19.5%, 10.4%, 4.55%, 0.65%, and 0% of healthy controls at dilutions of 1:40, 1:80, 1:160, 1:320, and 1:640, respectively. ANA positivity at 1:40 dilution was observed among 26.4% cases with mimics of CTDs. Prevalence of ANA positivity in ANA-related CTDs was 97.3%, 96.4%, 89.3%, 83.9%, and 71.4% at dilutions of 1:40, 1:80, 1:160, 1:320, and 1:640, respectively. ROC analysis revealed best test performance for distinction between healthy and ANA-related CTD populations at a serum dilution of 1 in 80. Conclusions Antinuclear antibodies (ANA) positivity at low titers (1:40) is highly prevalent in healthy population (19.5%) as well as amongst mimics of CTD (26.4%). Our study suggests a higher screening dilution of 1:80 for ANA by IIF for diagnosis of CTD maybe better. Combination of 1:80 and 1:160 dilutions provides optimum sensitivity and specificity for diagnosis of ANA-related disorders.

Introduction: IgA vasculitis is a systemic disease that results from the entrapment of circulating IgA-containing immune complexes in small-vessel walls in the skin, kidneys, and gastrointestinal tract. An excessive formation of neutrophil extracellular traps (NETs) is involved in the pathogenesis of vasculitis, especially in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. This study aimed to clarify whether NETs are implicated in IgA vasculitis. Methods: Twenty-two patients with IgA vasculitis and 4 healthy volunteers were enrolled in this study. Serum levels of myeloperoxidase (MPO)-DNA complex, a fragment derived from NETs, were determined by enzyme-linked immunosorbent assay (ELISA), and the association between MPO-DNA complex levels and clinical parameters was examined. The presence of the ANCA was also assessed by ELISA specific for MPO and proteinase 3 (PR3) and indirect immunofluorescence (IIF), followed by assessing the differences in clinical parameters with and without the ANCA. Results: Serum MPO-DNA complex levels were significantly higher in patients with IgA vasculitis than those in healthy controls. A significant positive correlation between the serum MPO-DNA complex and IgA levels was noted. Interestingly, 63.6% of IgA vasculitis patients were ANCA-positive in IIF with an atypical pattern, whereas neither MPO-ANCA nor PR3-ANCA was detected by ELISA. These findings indicated that some IgA vasculitis patients possessed the so called minor ANCA. Serum IgA and MPO-DNA complex levels and the frequency of hematuria in the minor ANCA-positive group were significantly higher than in the minor ANCA-negative group. Conclusion: The collective findings suggested that NETs are certainly involved in the pathogenesis of IgA vasculitis.

Aim To evaluate myositis line immunoassay (LIA) for diagnosis and sub-classification of suspected idiopathic inflammatory myopathy (IIM). To investigate if test performance is improved by increasing signal strength cut-off for myositis-specific antibody (MSA) or combining MSA with indirect immunofluorescence (IIF). Methods A retrospective, consecutive case series of patients investigated for MSAs from June 2013 to June 2020 for suspected IIM. Specificity, sensitivity, positive predictive value, and negative predictive value were calculated with 95% confidence intervals for diagnosis of IIM. Association of IIM diagnosis with increased signal strength and presence of an expected IIF pattern on Hep-2 cells was assessed by Fisher's exact test in MSA-positive patients. Results A total of 195 patients were evaluated. IIM was diagnosed in 32/195 (16.4%) patients. MSAs were detected in 41/195 (21%) patients, 18/41 (43.9%) patients with an MSA had a diagnosis of IIM. The probability of an IIM diagnosis was increased in MSA-positive patients with high compared with low signal strength (83.3% vs 43.5%; P = 0.01) and an expected compared with unexpected IIF pattern (61.5% vs 23.8%; P = 0.04). Specificity for IIM was not significantly improved by increasing signal strength cut-off (85.9% vs 93.8%). Positive predictive value of myositis LIA was only modest and not significantly improved by either increasing signal strength cut-off or requiring an expected IIF pattern for determination of MSA positivity (43.9% vs 60% vs 61.5%). Sub-classification of IIM correlated closely for respective MSAs (88.9%). Conclusion Increased MSA signal strength on myositis LIA and the presence of an expected IIF pattern were associated with IIM diagnosis. Test performance was non-significantly improved by these methods. Prevalence of IIM in this patient cohort was low; it is not excluded that LIA performance could be improved by these methods in a higher prevalence cohort.

Background Antineutrophil cytoplasmic antibodies (ANCA) are important biomarkers in the diagnosis of ANCA-associated vasculitis, and indirect immunofluorescence (IIF) had been the method of choice for its detection from the very beginning. However, international consensus on ANCA testing (2017) advocates the use of high-quality immunoassays as the primary screening method. The purpose of this study was to evaluate the diagnostic performance of enzyme-linked immunosorbent assay (ELISA) compared to IIF in detecting ANCA. Methods One-hundred eighty-nine serum samples of suspected or known cases of systemic vasculitis were screened for ANCA by IIF and proteinase-3- and myeloperoxidase-ELISA. In IIF, positive results were further divided into cytoplasmic pattern of ANCA and perinuclear pattern of ANCA, depending upon the pattern of fluorescence. McNemar’s chi-squared test was applied to check the equality of proportions of positive results, and Kappa statistics was used to measure the agreement between the two methods. Diagnostic performance of ELISA was evaluated taking IIF as reference. Results IIF detected ANCA in 17.5% cases and ELISA detected it in 11.6% cases. A good agreement between the overall performance of ELISA and IIF was observed (K-value: 6.8, p-value: < 001). However, a significant difference in the proportion of positive results by the two methods was observed in McNemar’s test (two-sided p-value: 0.007). Taking IIF as standard, ELISA showed 60.6% sensitivity, 98.7% specificity, and predictive value of positive and negative results of 90.9 and 92.2%, respectively. Conclusion The new generation antigen-specific ELISAs had high specificity but the chances of missing cases in primary screening due to the low sensitivity and high false negativity (39.4%) need to be dealt with.

Neospora spp. es un parásito protozoario causante de abortos y enfermedad del Sistema Nervioso Central (SNC) en diversas especies domésticas y silvestres. En equinos, se le ha involucrado como causa de aborto, mortalidad neonatal y enfermedades del SNC. La especie identificada en equinos es distinta a Neospora caninum y se denomina Neospora hughesi. El objetivo del presente, fue detectar la presencia de anticuerpos anti-Neospora spp., asociados a factores de riesgo en caballos de México. Se realizó una encuesta de cada cuadra y animales individuales de cuatro regiones del país (Centro, Norte, Occidente y Sur) para identificación de los factores de riesgo. Se obtuvo un total de 684 muestras de sueros de caballos de las diferentes regiones, 52.3 % (358) machos y 47.7 % (326) hembras. Los sueros fueron conservados a -20 °C. El diagnóstico se realizó mediante la técnica de inmunofluorescencia indirecta (IFI), los resultados de este fueron analizados para estimar la asociación entre la seropositividad y los factores de riesgo. La seroprevalencia a Neospora spp. fue de 2.34 % en la población estudiada; los casos positivos se encontraron principalmente en tres de las cuatro regiones incluidas, que presentaron una relación significativa a la presencia de anticuerpos anti-Neospora spp.; la convivencia de los caballos con otros animales obtuvo un valor de OR de 2.34 (IC 95%: 0.28 - 19.0; P

Pemphigoid gestationis (PG) is a rare autoimmune bullous skin disorder which usually presents with intense pruritus and urticarial lesions that may evolve into vesicles and tense blisters. In majority of patients, it starts in the second or third trimester of pregnancy and resolves spontaneously after delivery. Lesions appear in the periumbilical area in 90% of patients and rapidly spread centrifugally to other parts of the body. The diagnosis needs to be confirmed by direct immunofluorescence test (DIF) with indirect immunofluorescence test (IIF), ELISA and immunoblot techniques playing role in diagnosis and/or monitoring antibodies level. Mild symptoms of PG can be treated with topical therapy only, but in severe course of the disease the treatment may be escalated to oral corticosteroids. We present an unusual case of PG started 2 weeks after delivery with an updated overview on the epidemiology, pathology, clinical picture, treatment, and complications of the disease.

Bullous pemphigoid (BP) is an autoimmune blistering disease resulting from autoantibodies directed against BP180 and/or BP230 proteins. BP is traditionally diagnosed based on clinical features, histologic assessment of cutaneous biopsies, direct (DIF) and indirect immunofluorescence (IIF) studies, and/or enzyme-linked immunoassay (ELISA) analysis. We here report, 6 elderly patients who presented with multiple tense bullae, clinically mimicking BP in the absence of detectable autoantibodies by DIF, IIF, and ELISA for BP-180, BP-230, and type VII collagen autoantibodies. All patients in this case series had histological overlap between BP and eczema, potentially representing a unique form of bullous eczema. Patient demographics are listed in Table I along with clinical images (Fig 1). Patients were evaluated in the Autoimmune Blistering Disease Clinic at Emory University. All patients had at least 2 negative DIFs on 2 separate occasions. Only 1 patient was subject to systemic immune suppression at the time of biopsy, and no history of diabetes, peripheral edema, or arthropod assault was noted. Table I Clinical summary of patients 1 to 6

Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria (anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III). However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP) that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9) are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies. A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n = 32) and Italy (Careggi Hospital, Florence, n = 15) selected based on AC-9 HEp-2 IFA staining (> 1:640, clumpy nucleolar pattern) and 102 non-SSc controls (inflammatory bowel disease (IBD) n = 20, Sjögren’s syndrome (SjS) n = 20, infectious disease (ID) n = 7, systemic lupus erythematosus (SLE) n = 17, rheumatoid arthritis (RA) n = 17, and healthy individuals (HI) n = 21). All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples. The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0%, Italy) of samples preselected based on the AC-9 IIF pattern (difference p = 0.09). Collectively, the PMAT assay showed 91.5% (95% confidence interval (CI): 80.1–96.6%) sensitivity with 100.0% (95% CI: 96.4–100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman’s rho = 0.89, 95% CI: 0.77.9–0.95%, p

Objectives Currently available computer-aided diagnosis (CAD) systems for the detection of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) assay enable a standardized measurement of system-specific fluorescent intensity (FI) measures. We aimed to evaluate an internal quality control (iQC) program that controls the total ANA IIF process in routine practice. Methods In addition to the kit iQC materials, supplemental quality indicators were integrated in a total quality assurance (QA) program: patient-derived iQC’s samples (negative, 1/160 fine speckled and 1/160 homogeneous), median sample FI per run and percentage of ANA IIF positive samples per run. Analytical rejection criteria were based on the imprecision of the positivity index (PI) measure of the Zenit PRO system (Menarini). Clinical rejection criteria were based on changes in FI that correspond to a change in ANA IIF titer of ≥2. To evaluate the QA program, different artificial errors were introduced during the ANA IIF process. After every run, quality indicators were evaluated and compared to the pre-set target values. Results Rescanning the ANA IIF slides five times, using an old conjugate and a needle obstruction resulted in analytically and even clinically relevant errors in ANA IIF results. All errors were correctly detected by the different defined quality indicators. Traditional Westgard rules, including analytically (and clinically) defined rejection limits were useful in monitoring quality indicators. Conclusions The integration of a total process iQC program in CAD systems, based on the specific FI measurands and performance criteria of the system, adds value to QA.

Background: Anti-nuclear antibodies (ANA) are known to be associated with autoimmune diseases and most commonly with connective tissue diseases (CTD). Screening for ANA is the first investigation of choice in the diagnosis of autoimmune diseases and indirect immunofluorescence (IIF) test using Hep-2 cells has been considered the gold standard method. The prevalence of autoimmune diseases and hence the ANA positivity and ANA IIF patterns differ in various geographical regions. Aims and Objectives: This study was aimed to estimate the ANA positivity rates among patients with suspected or confirmed auto immune diseases and to determine the common ANA IIF patterns in the positive samples. Materials and Methods: This is a retrospective study including 235 patients with either clinical suspicion of, or suffering from autoimmune diseases attending a tertiary health care hospital in Puducherry, South India, between November 2018 and April 2020. ANA IIF test (Hep 20-10/ primate liver, Euroimmun, Germany) was performed with 1:100 dilutions of patient serum. Results: The ANA positivity rate was 14.5% (34 out of 235 samples were positive). ANA was positive in 10 (13.3%) males and 24 (15%) females. The mean age of ANA positive patients were 43.4 years.The most common pattern was nucleus fine granular (n=8) followed by cytoplasm fine granular (n=7) and centromere (n=4). Conclusion: A significant ANA positivity rate was found in our study and a detailed clinical study has to be conducted to draw further conclusions. Keywords: Antinuclear antibodies, Indirect immunofluorescence test, ANA patterns.

Antinuclear antibodies (ANAs) represent a spectrum of autoantibodies targeted for various nuclear and cytoplasmic components of the cells. Indirect immunofluorescence assay (IIF) is the main detection method for “antinuclear factor”. A positive ANA test is usually reported as a titer and a pattern of fluorescence. The ANA patterns refers to the distribution of staining produced by antibodies that react with antigens located in nucleus and cytoplasm of HEp-2 cells. To standardize nomenclature and descriptions of the various fluorescence patterns of antinuclear factor (ANF), the Initiative of the International Consensus on ANA Patterns (ICAP) group was developed in 2014. The aim of ICAP is to promote consensus regarding nomenclature of ANA patterns, a microphotograph database, as well as classification depending on the employee skills. Information on the main characteristics, as well as specific clinical associations of the patterns is available at www.ANApatterns.org. In ANA classification trees, the patterns are indicated by the #AC (anticell pattern) alphanumeric code, being divided into nuclear, cytoplasmic and mitotic groups. Depending on the clinical significance and/or ease of recognition, this nomenclature focuses on the differences between the patterns described by specialists at competent and expert levels. Of the nuclear types, the most significant are homogeneous, speckled, dense fine-speckled, centromere, nucleolar, nuclear dots. The cytoplasmic types may be discerned into fibrillar, speckled, mitochondrial, Golgi, rods and rings. On leaders, behalf of the ICAP translation team is headed by the Full Member of Russian Academy of Sciences, Professor A.A. Totolian, under the auspices of the Russian Research Society of Immunologists. In this article, we present the Russianlanguage adaptation of the ICAP nomenclature, in order to ensure unification and standardization of ANA detection results in the patients with autoimmune diseases.

Introduction: Autoimmune diseases (AID) are a manifestation of a self damaging immune response of the body to its own antigen. In any suspected AID, the first test to be done is ANA detection by indirect immunofluoresence using HeP 2 cells due to its sensitivity. A positive ANA test is further evaluated by performing immunoblot test to detect specific antigens causing autoimmunity. The present study was undertaken to study the prevalence of antinuclear antibodies in patient population of Central Madhya Pradesh as well as to study the prevalence of disease specific antigens. Materials and Methods: A total of 650 patients of all ages and both sexes coming to our diagnostic centre between January to June 2018 for ANA testing were included in the study. Serum ANA was determined by indirect immunofluorescence (IIFA) using Hep 2 cell lines (Euroimmun, Germany). A positive result by IIFA was further tested by immunoblot method (Euroimmun, Germany) when requested by the clinician. Result: Out of 650 patients ANA screen was positive in 280 (43.08%) patients and negative in 209(32.15%) patients. ANA blot was requested in 161 patients (57.5%) of the total 280 ANA screen positive patients. Out of the 161 patients, 69 had a positive Immunoblot test for one or more antigens. Total 53.69% patients were positive by both ANA screen and immunoblot test. The total coincidence rate was 44.5% between ANA screen and ANA blot test with a positive coincidence rate of 81.6% and negative coincidence rate of 79.1%. Conclusion: ANA IIF is considered to be the gold standard screening test for detecting autoantibodies and ANA blot is done as a confirmatory test to detect specific autoantibodies against antigens causing autoimmunity. The combination of ANA IIF and ANA blot is an effective method to diagnose autoimmune diseases. If used alone, a possibility of missing a diagnosis is high and so a combinbation of these two tests is of great benefit to both the clinicians as well as laboratorians.

Immediate post-thaw evaluation of membrane integrity has proven to yield overestimates of cell survival under conditions that preclude intracellular ice formation (IIF). However, prominent theories on the mechanisms of intracellular nucleation suggest a damaged membrane can reseal, prompting us to evaluate whether immediate post-thaw assessments of membrane integrity can in fact underestimate cell survival under conditions that promote IIF. HUVEC and HepG2 monolayers were treated with 1.4 M DMSO and frozen to −25 °C under conditions that formed either 0% or 100% IIF. Membrane integrity was evaluated both immediately and 24 h post-thaw, with metabolic activity assessments performed 24 h post-thaw as a secondary measure of survival. Treatment with 1.4 M DMSO and nucleation of 100% IIF resulted in a drastic increase in the relative percent of membrane intact cells following a 24 h culture period (HUVEC: 90.2% ± 0.7%; HepG2: 70.4% ± 4.0%), which correlated with 24 h post-thaw metabolic activity. These differences between the immediate and 24 h post-thaw membrane integrity assessments were significantly more than those seen in the absence of either IIF or DMSO treatment. Therefore, a high incidence of IIF in DMSO-treated monolayers may lead to erroneous underestimates of cell survival when conducting immediate post-thaw assessments of membrane integrity.

ANCA testing plays an established critical role in the diagnosis of ANCA Associated vasculitis (AAV). The spectrum of diseases associated with positive ANCA has recently broadened, thus calling into question the diagnostic implications of ANCA positivity in a hospital setting. We retrospectively studied all adult patients who had a positive ANCA test (by Indirect Immunofluorescence (IIF), ELISA or both) performed over the span of 19 years. Subjects were then divided into discordant (positive on one assay) and concordant ANCA (positive on both assays) groups based on their ANCA positivity status. The two groups were then compared with regards to their demographic, clinical and laboratory characteristics, the indication for ANCA testing in both groups and their final diagnoses. Of the 9189 ANCA tests ordered during the 19-year span of the study, 389 (4.2%) were positive. Two hundred and forty subjects met the exclusion criteria (patients aged less than 18 years or the lack of clinical and laboratory data in the medical file) thus resulting in a final cohort of 149 subjects. Of them, 122 subjects had discrepant ANCA results and 27 had matching ANCA results. Most cases in the discrepancy group were IIF positive and ELISA negative (86.8%). The diagnosis of AAV was highly unlikely in cases with discrepant IIF and ELISA serologies compared to cases with matching IIF and ELISA serologies (4.1% versus 44.4%, p value

Objective The objective was to assess the interobserver agreement rate, progression rates and malignancy rates in the assessment of complex renal cysts (≥ Bosniak IIF) using a population-based database. Methods A regional database identified 452 complex renal cysts in 415 patients between 2009 and 2019. Each patient was tracked and followed up using a unique identifier and deterministic linkage methodology. The interobserver agreement rate between radiologists was calculated using a weighted kappa statistic. Progression and malignancy rates of cysts (Bosniak ≥IIF) over the 11-year period were calculated. Results The linear-weighted kappa value was 0.69 for all complex cysts. The rate of progression and regression of Bosniak IIF cysts was 4.6% (7/151) and 3.3% (5/151), respectively. All malignant IIF cysts progressed within 16 months of diagnosis. The malignancy rate of surgically resected Bosniak III and IV cysts was 79.3% (23/29) and 84.5% (39/46), respectively. Of all malignant tumours, 73.8% and 93.7% were of low ISUP grade and low stage, respectively. Conclusions This study further confirms that there is a good degree of agreement between radiologists in classifying complex renal masses using the Bosniak classification. The progression rate of Bosniak IIF cysts is low, but the malignancy rates of surgically resected Bosniak IIF, III and IV cysts are high. Benign cysts are frequently resected, and a very high proportion of histopathologically confirmed cancers in complex renal cysts are of low grade and stage. Key Points •There is a good degree of agreement between radiologists in classifying complex renal masses using the Bosniak classification. •The rate of progression of Bosniak IIF cysts is low, and malignant cysts progress early during surveillance. Although the malignancy rates of resected Bosniak IIF, III and IV cysts are high, the rate of benign cyst resection is significant.

A line blot (LB) assay is a multi-analyte platform capable of simultaneously detecting multiple anti-nuclear antibody specificities. Here, we evaluated the performance of a commercial LB assay developed for the identification of myositis- or systemic sclerosis (SSc)-related autoantibodies (autoAbs).We screened 300 serum samples from patients with various connective tissue diseases using an LB assay and compared the results of myositis- or SSc-related autoAbs with those identified by RNA and protein immunoprecipitation (IP) assays or indirect immunofluorescence (IIF).The IP assays revealed anti-Jo-1 Abs in 14 patients, anti-EJ Abs in 12, anti-PL-7 Abs in 8, anti-PL-12 Abs in 4, anti-Mi-2 Abs in 6, anti-SRP Abs in 8, anti-topoisomerase I Abs in 54, anti-RNA polymerase III Abs in 24, anti-U3 RNP Abs in 9, anti-Th/To Abs in 9, anti-Ku Abs in 14 and anti-hUBF Abs in 4, whereas IIF identified anti-centromere in 35. Good agreement between the IP assays and the LB assay was found only for anti-Jo-1 and anti-centromere antibodies. When a cut-off was adjusted to reconcile with the results of IP assays, the detection performance of LB assay was improved for anti-EJ, anti-PL-7, anti-PL-12, anti-SRP, anti-topoisomerase I and anti-RNA polymerase III Abs. However, the results of anti-Mi-2, anti-U3 RNP, anti-Th/To, anti-hUBF and anti-Ku Abs remained discordant between the LB assay and IP assays at all cut-off levels.Detection of myositis- or SSc-related autoAbs using a commercial LB assay requires great caution since it can yield analytically false-positive or false-negative results. Key Points • A line blot (LB) assay is a multi-analyte platform capable of simultaneously detecting multiple antibodies with anti-nuclear specificities. • Detection of myositis- or systemic sclerosis-related autoantibodies using a commercial LB assay requires great caution since it can yield analytically false-positive or false-negative results.

Background Both enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence (IIF) are available for the diagnosis of autoimmune bullous diseases (AIBD). Many studies have reported on the performance of ELISAs and concluded that ELISAs could replace IIF. This study compares the diagnostic accuracy of ELISA and IIF for the detection of autoantibodies to desmoglein 1 (DSG1), desmoglein 3 (DSG3), bullous pemphigoid antigen 2 (BP180) and bullous pemphigoid antigen 1 (BP230) to support the diagnosis of pemphigus vulgaris (PV), pemphigus foliaceus (PF) and bullous pemphigoid (BP). Methods A literature search was performed in the PubMed database. The meta-analysis was performed using summary values and a bivariate random effect model. Results The five included studies on PV did not demonstrate significant differences between IIF and DSG3-ELISA (sensitivity 82.3% vs. 81.6%, p = 0.9284; specificity 95.6% vs. 93.9%, p = 0.5318; diagnostic odds ratio [DOR] 101.60 vs. 67.760, p = 0.6206). The three included studies on PF did not demonstrate significant differences between IIF and DSG1-ELISA (sensitivity 80.6% vs. 83.1%, p = 0.8501; specificity 97.5% vs. 93.9%, p = 0.3614; DOR 160.72 vs. 75.615, p = 0.5381). The eight included studies on BP showed that BP230-ELISA differed significantly from both IIF on monkey esophagus (MO) and BP180-ELISA with regard to DOR (11.384 vs. 68.349, p = 0.0008; 11.384 vs. 41.699, p = 0.0125, respectively) Conclusions Our meta-analysis shows that ELISA performs as well as IIF for diagnosing PV, PF and BP.

Background Anti-mitochondrial autoantibodies (AMA) detected by indirect immunofluorescence (IIF) on rodent tissues are the diagnostic marker of primary biliary cholangitis (PBC). However, up to 15% of patients with PBC are AMA-negative by IIF. In the effort to close the serological gap and improve the diagnostic sensitivity of PBC testing, recently, novel autoantibodies specific for PBC, such as kelch-like 12 (KLHL12, KLp epitope) and hexokinase 1 (HK1) have been described. In this study, we evaluated the autoantibody profile in a large cohort of PBC patients and in patients with other liver disease, including anti-HK1 and anti-KLp autoantibodies. Methods Sera of 194 PBC patients (126 AMA-IIF-positive and 68 AMA-IIF-negative) and 138 disease controls were tested for a panel of PBC-specific antibodies (MIT3, sp100, gp210, HK1, KLp) using a new automated particle-based multi-analyte technology (PMAT) assay on the Aptiva instrument (Inova). Results Selecting a cutoff yielding a specificity of >95% for all the markers, the sensitivity for anti-MIT3, anti-sp100, anti-gp210, anti-HK1 and anti-KLp in the PBC AMA-IIF-negative cohort was 20.6%, 16.2%, 23.5%, 22.0%, 17.6 and 13.2%, respectively. Six out of the 68 (8.8%) AMA-IIF negative sera were positive for anti-HK1 or anti-KLp alone. Using these new markers in addition to anti-MIT3, anti-sp100 and anti-gp210, the overall sensitivity in this cohort of AMA-IIF-negative patients increased from 53% to 61.8%, reducing the serological gap in AMA-negative PBC patients. Conclusions PBC antibody profiling, made possible by the new Aptiva-PMAT technology, allows recognition of a higher number of AMA-negative PBC patients than conventional immunoassays and may represent a useful tool to evaluate the prognostic significance of autoantibody association in PBC patients.

Systemic sclerosis is an autoimmune disease clinically characterized by vascular and immune dysfunction, leading to fibrosisthat can damage multiple organs. The presence of non-overlapping SSc-associated autoantibodies best presents the autoimmunenature of systemic sclerosis. The primary purpose of this study was to investigate the autoantibody profile in Iranian patientswith systemic sclerosis. Sera from 481 patients with systemic sclerosis were collected from 2013 to 2016. Levels of anti-nuclearantibodies (ANA) were quantitatively detected using the indirect immunofluorescence (IIF) method, and levels of specificautoantibodies including anti-topoisomerase I antibody (ATA), anti-centromere antibody (ACA) and anti RNA polymerase IIIantibody (anti-RNAP III) were determined qualitatively using the enzyme-linked immunosorbent assay (ELISA) technique.Among all patients evaluated, a predominance of females (86.7%) was found, and 434 (90.2%) patients showed positive ANAresults by IIF. ANA was detected in 87.3% and 92.0% of limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneoussystemic sclerosis (dcSSc) patients, respectively, which was not significantly different. The frequency of anti-RNAP III, ACA,and ATA was 5.19%, 6.09%, and 72.3%, respectively. Furthermore, anti-RNAP III, ATA, and ANA levels were correlated withdcSSc, whereas ACA levels were correlated with lcSSc. It was confirmed that ATA expression is significantly higher in dcSScpatients. This study had a lower frequency of ACA (6.09%) than most previous cohorts. The results demonstrated that theclinical subtype of systemic sclerosis may correlate positively with the presence of specific autoantibodies.

Cryoprotective effect of silver carp muscle hydrolysate (SCMH) on baker's yeast (Saccharomyces cerevisiae) was examined by analyzing the growth and survival of the yeast during freeze–thaw cycles, and the physicochemical properties [ultrastructure, intracellular proteins and fatty acids, external ice formation (EIF) and internal ice formation (IIF), freezable water content] of yeast cells with or without SCMH through transmission electron microscopy, SDS‐PAGE, GC‐MS, and differential scanning calorimetry. The 4% of SCMH treatment exhibited good yeast cryoprotective activity and increased the yeast survival rate from 0.71% to 90.95% after 1 freeze–thaw cycle as compared to the control. The results demonstrated that the addition of SCMH could attenuate the freeze damage of yeast cells, prevent the degradation or loss of soluble proteins, and increase the composition and absolute content of fatty acids. Besides, the addition of 4% SCMH caused a drop in the EIF peak temperature (from −17.95℃ to −25.14℃) and a decrease in the IIF and freezable water content of yeast cells.

Objective: The aim of this study was to test the efficacy of autoantibodies to desmoglein 1 and desmoglein 3 detected by ELISA and indirect immunofluorescence in the diagnosis of oral pemphigus and to correlate the antibody titres with the severity of the disease. Materials and Methods: We report a retrospective cohort study of 22 patients with oral pemphigus and 64 controls from a single tertiary centre. Data about histopathological examination, direct immunofluorescence, indirect immunofluorescence and ELISA were analysed. Global validation of ELISA and IIF both alone and combined was established by calculating sensitivity, specificity, accuracy and both positive predictive value and negative predictive value. The relationship between Oral Disease Severity Score values and ELISA titres was analysed using Pearson's coefficient. Results: The best diagnostic performance was observed for anti-desmoglein 3 ELISA. The sensitivity was 75% and specificity 100% and positive predictive value and negative predictive value were 92.5% and accuracy 93.9%. The level of agreement with histopathology+direct immunofluorescence was substantial (k=.758). Anti-desmoglein 3 titres showed a significant correlation with Oral Disease Severity Score (p&nbsp

Autoimmune diseases have been reported to develop months to years following drug rash with eosinophilia and systemic symptoms (DRESS) syndrome. However, the development of autoimmune bullous diseases in a patient with a history of DRESS syndrome is extremely rare. We here report the unusual case of mucous membrane epidermolysis bullosa acquisita (MM-EBA) presenting 2 years after lamotrigine-induced DRESS syndrome in a pediatric patient. The patient also developed nonpathogenic autoantibodies directed against BP 180 and BP 230.

In this study, we aimed to assess the prevalence of uncommon staining patterns found during testing for the presence of antinuclear antibodies (ANA) and to determine their association with certain antibodies and clinical diagnoses. Presence of ANA and the staining pattern was determined in 10955 samples using indirect immunofluorescence (IIF) on HEp-2 cells. ANA-positive samples were assessed for presence of 14 specific antibody types using a microbead based system. Demographic data (age, sex) and clinical diagnoses were collected from the referral documentation. Particular staining patterns were then compared with a representative comparison group comprised of samples with common staining patterns using these criteria. There were 22 patterns present in less than 3% of samples each and these were jointly present in 42.43% of ANA-positive samples. Specific antibodies were found in proportions similar to the comparison group (46.06%) and varied significantly between patterns. Likewise, there were significant differences in antibody distribution in particular patterns. Some patterns were associated with presence of rheumatic diseases or inflammatory arthropathies, while in others there was a concurrent diagnosis of liver disease, or a neoplastic process. Many of the uncommon IIF patterns have distinctive characteristics that warrant further investigation in order to determine their role in diagnosing various diseases, not limited only to the illnesses of the rheumatic spectrum. IIF on HEp-2 cells remains an irreplaceable method because of the diversity of ANA, only a number of which can be detected using other standardised methods.

Anti-neutrophil cytoplasmic antibodies (ANCAs) are autoantibodies that recognize neutrophil cytoplasmic antigens. The major ANCA antigens are myeloperoxidase and proteinase 3. Necrotizing small vessel vasculitis accompanied by ANCA production is called ANCA-associated vasculitis (AAV). In addition to AAV, ANCA is sometimes produced in patients with connective tissue diseases, such as systemic lupus erythematosus, and inflammatory bowel diseases. Indirect immunofluorescence (IIF) and enzyme immunoassay (EIA) have been used to detect ANCAs. Recently, the accuracy of EIA has improved and it has become the gold standard for ANCA detection. However, IIF does not lose its role in ANCA detection because EIA cannot detect ANCAs that recognize antigens other than those coated on the plate. For IIF, neutrophil substrates prepared with two different fixations, namely, ethanol fixation and formalin fixation, are used. There is a recommended protocol for ethanol fixation but not for formalin fixation. This study prepared neutrophil substrates according to the recommended protocol for ethanol fixation and protocols in the literature and original protocols for formalin fixation and then examined ANCA specificity and how storage period would influence the number of cells, antigen distribution, and antigenicity of the substrates. As a result, the number of cells and antigen distribution did not change after storage for up to 2 months regardless of fixation protocols, whereas a time-dependent decline in ANCA antigenicity and a fixation protocol-dependent difference in ANCA specificity were observed. How neutrophils are fixed on the glass slide needs to be checked upon evaluation of ANCAs by IIF.

Background The diagnosis of systemic autoimmune rheumatic diseases (SARD) is based on the detection of serum antinuclear antibodies (ANA) for which indirect immunofluorescence (IIF) is the golden standard. New solid‐phase immunoassays have been developed to be used alone or in combination with the detection of extractable antinuclear antibodies (ENA) to improve SARD diagnosis. The purpose of this study was to compare the clinical performances of different ANA screening methods alone or in combination with ENA screening methods for SARD diagnosis. Methods A total of 323 patients were screened for ANA by IIF, EliA™ CTD Screen, and ELISA methods. Agreements were calculated between the methods. Then, EliA™ CTD Screen positive samples were screened for ENA by line immunoassay (LIA) and fluorescence enzyme immunoassay (FEIA). Results The diagnostic accuracy of EliA™ CTD Screen (79% sensitivity and 91% specificity) was better than that of ELISA or IIF. The combination of EliA™ CTD plus IIF had the highest sensitivity (93%). ENA determination revealed that Ro52 and Ro60 were the most prevalent specificities. The use of IIF alone was not able of detecting up to 36% of samples positive for Ro52, and 41% for Ro60. Conclusions EliA™ CTD Screen has a better diagnostic performance when compared to IIF and ELISA. The combined use of EliA™ CTD Screen and IIF clearly improves the rate and accuracy of SARD diagnosis. The use of EliA™ CTD Screen as first‐line screening technique allows the detection of antibodies, which could not be detected by IIF alone., There is still room for improvement in ANA screening testing. Currently there are different methods available for systemic autoimmune rheumathic disease sift, but the optimization of screening protocols does not only rely on the selection of the best method–there is no perfect test‐ but in the detection of the optimal combination for the specific laboratory routine. In our study we compared the performance of two protocols in our routine samples: solid phase test+IIF and ELISA+IIF. IIF together with EliA CTD Screen (solid phase test) resulted in the highest sensitivity, while EliA CTD Screen showed the best sensitivity/specificity balance.

To investigate prevalence of anti-Pentraxin 3 (PTX3) antibodies in sera of anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients.Anti-PTX3 and PTX3 levels were analysed by enzyme-linked immunosorbent assays in sera from unselected patients with AAV and compared with patients with systemic lupus erythematosus (SLE, n = 130), other connective tissue diseases (CTDs, n = 97) and matched healthy controls (n = 97). Optical density (OD) cut-off for positive anti-PTX3 antibodies was determined by ROC curve analysis and set as 0.234. Indirect immunofluorescence (IIF) on fixed human granulocytes was used to analyze the fluorescence pattern of anti-PTX3 antibodies. Liquid-phase inhibition tests were conducted to assess potential interferences.We included 101 AAV patients (females 58%, median age 60[51-69] years) affected either with granulomatosis with polyangiitis (GPA, n = 51), microscopic polyangiitis (MPA, n = 12) or eosinophilic granulomatosis with polyangiitis (EGPA, n = 38). Anti-PTX3 antibodies were detected in 29.7% AAV patients, being significantly higher than in healthy controls (p 0.001) and CTDs (p = 0.030) but lower than in SLE (p = 0.004). Anti-PTX3 antibody prevalence was 44.7% in EGPA, 25% in MPA and 19% in GPA (p = 0.034). Among ANCA negative patients, 35.7% displayed positive anti-PTX3 antibodies. Anti-PTX3 were associated with a lower prevalence of systemic (p = 0.002), ear-nose-throat (p = 0.006) and renal manifestations (p = 0.016). Anti-PTX3 antibodies were characterized by a specific IIF pattern on fixed granulocytes. PTX3 serum levels resulted lower in AAV than healthy controls (p 0.001). PTX3 inhibited anti-PTX3 binding in a dose-dependent manner.Anti-PTX3 autoantibodies appear a promising novel biomarker of AAV, especially EGPA.

IMPORTANCE: An accurate diagnosis of mucous membrane pemphigoid (MMP) is essential to reduce diagnostic and therapeutic delay.OBJECTIVE: To assess the diagnostic accuracy of direct immunofluorescence microscopy on mucosal biopsy specimens and immunoserology in a large cohort of patients with suspected MMP.DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort study was carried out in a single tertiary care center for blistering diseases between January 2002 and March 2019. Eligible participants were patients with suspected MMP and paired data on at least a mucosal biopsy specimen for direct immunofluorescence microscopy (DIF) and indirect immunofluorescence microscopy (IIF) on a human salt-split skin substrate (SSS). In addition, an optional DIF test on a skin biopsy specimen and one or more performed routine immunoserologic tests were analyzed. Data analysis was conducted from April 2019, to June 2020.MAIN OUTCOMES AND MEASURES: Diagnostic accuracy of DIF, IIF SSS, and immunoblot for BP180 and BP230.RESULTS: Of the 787 participants, 121 (15.4%) received the diagnosis of MMP (50 men [41.3%], 71 women [58.7%]; mean [SD] age at diagnosis, 60.1 [17.7] years). Sixty-seven of the patients with MMP (55.4%) had monosite involvement, of which oral site was the most frequently affected (51 [42.1%]). No significant difference was found between the sensitivity of DIF on a perilesional buccal biopsy and a normal buccal biopsy (89.3% vs 76.7%). Three patients with solitary ocular involvement showed a positive DIF of only the oral mucosa. In 6 patients with a negative mucosal DIF, a skin biopsy confirmed diagnosis of MMP. Overall, IIF SSS was less sensitive (44.6%), but highly specific (98.9%). The sensitivity of immunoblot (66.1%) was higher compared to SSS, but with lower specificity (91.3%).CONCLUSIONS AND RELEVANCE: This comparative diagnostic accuracy study of a cohort of 787 patients found a high sensitivity of a mucosal DIF biopsy for diagnosis of MMP, and lower sensitivity of serologic analysis. A biopsy can be taken from either perilesional or normal buccal mucosa. An additional DIF biopsy of another mucosal site or of affected or unaffected skin may increase the diagnostic yield and is recommended in patients with negative DIF results and high clinical suspicion.

Feline morbillivirus (FeMV) was isolated for the first time in 2012 with an association with chronic kidney disease (CKD) suggested. This study aimed at investigating in cats from southern Italy FeMV prevalence and risk factors for exposure to FeMV, including the relationship with CKD, sequencing amplicons and analyzing phylogeny of PCR positive samples. Blood serum, K3EDTA blood and urine samples from 223 cats were investigated. Ten carcasses were also evaluated. FeMV RNA was detected in 2.4% (5/211) blood and 16.1% (36/223) urine samples. One carcass tested positive by qPCRFeMV from kidney, urinary bladder, and submandibular lymph nodes. Antibodies against FeMV were detected in 14.5% (28/193) cats. We followed up 27 cats (13 FeMV positive cats) and documented in some cases urine shedding after up to 360 days. Older and foundling cats and cats living in rescue catteries, were more frequently infected with FeMV. A significant correlation between FeMV and higher serum creatinine values or low urine specific gravity was found. FeMV positivity was significantly associated with retroviral infection, and the presence of some clinical signs apart from CKD clinicopathological markers. Our study highlights the possibility of a link between FeMV exposure and CKD and a general impairment of feline health.

Introduction Pemphigus vulgaris (PV) is an intraepidermal autoimmune bullous disease (AIBD) characterized by autoantibodies against desmosomal adhesion proteins, most commonly desmoglein (Dsg)3, leading to the suprabasal cleft formation and acantholysis. Areas covered Direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) studies display the intercellular deposition of IgG/C3 throughout the epidermis and presence of circulating autoantibodies respectively, as a net-like pattern. These methods help to differentiate intraepithelial from subepidermal blistering diseases. However, the target antigen remains unknown using immunofluorescence techniques. Thanks to the development of Dsg ELISA, using recombinant technology, circulating antibodies against Dsg1 and 3 could be detected sensitively. It is possible to differentiate PV from pemphigus foliaceus (PF) using this assay. BIOCHIP mosaic and multivariant ELISA are two novel serologic methods with the added value of the ability to screen several AIBDs simultaneously. Non-Dsg1/3 antigens are also involved in the pathogenesis of PV and investigated more deeply thanks to the protein microarrays technique. Additionally, patients with even high values of anti-Dsg1/3 may be lesion-free, suggesting the presence of non-pathogenic autoantibodies. Expert opinion Newer diagnostic methods to replace traditional techniques should possess high sensitivity and specificity and be widely available, noninvasive, and relatively cheap. The newly developed methods need to be further evaluated before they are recommended for routine use.

Canine visceral leishmaniasis (CVL) caused by Leishmania (Leishmania) infantum is transmitted by phlebotomine sandflies and a major zoonotic disease in Brazil. Due to the southward expansion of the disease within the country and the central role of dogs as urban reservoirs of the parasite, we have investigated the occurrence of CVL in two municipalities Erval Velho and Herval d’Oeste in the Midwest region of Santa Catarina state. Peripheral blood samples from 126 dogs were collected in both cities and tested for anti-L. infantum antibodies by indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence reaction (IIF) and for the presence of parasite DNA by polymerase chain reaction (PCR) in peripheral blood. From examined dogs, 35.71% (45/126) were positive for at least one of the three tests and two (1.6%) were positive in all performed tests. Twelve dogs (9.5%) were positive for both ELISA and IIF, while 21 dogs were exclusively positive for ELISA (16.7%), and 15 (11.9%) for IIF. L. infantum k-DNA was detected by PCR in 9 out of 126 dogs (7.1%) and clinical symptoms compatible with CVL were observed for 6 dogs. Taken together, these results indicate the transmission of CVL in this region, highlighting the needs for epidemiological surveillance and implementation of control measures for CVL transmission in this region. Resumo A Leishmaniose Visceral Canina (LVC) causada pela Leishmania (Leishmania) infantum e transmitida por flebotomíneos e é uma das principais zoonoses do Brasil que se encontra em expansão em estados da região sul do país, sendo os cães o principal reservatório urbano do parasito. O presente estudo investigou a ocorrência de LVC em dois municípios, Erval Velho e Herval d’Oeste localizados no meio-oeste de Santa Catarina. Para tanto, amostras de sangue periférico de 126 cães foram coletadas em ambas as cidades e submetidas à detecção de anticorpos anti-L. infantum por meio de testes de ELISA e imunofluorescência indireta (IFI), bem com a detecção de k-DNA pela reação em cadeia de polimerase (PCR). Além disso, também foram observados os sintomas clínicos e as condições ambientais associadas a esses animais. Dos cães examinados, 35,7% (45/126) foram positivos para pelo menos um dos três testes, dois cães (1,6%) foram positivos em todos os três testes, 12 cães (9,5%) foram positivos tanto no ELISA quanto na IFI, enquanto 21 cães (16,7%) foram positivos para ELISA e 15 (11,9%) para o IFI. A amplificação do k-DNA de L. infantum foi positiva em 9 dos 126 cães (7,1%). Entre os cães positivos seis apresentaram um ou mais sintomas clínicos correlacionados com a LVC. Esses resultados confirmaram a ocorrência de LVC na região e destacaram a importância do monitoramento e implementação de medidas de controle para a LVC nessa região

Gasdermin-D (GSDMD) is a member of the gasdermin (Gsdm) protein family, and its cleavage by inflammatory cysteine proteases (caspases, CASPs) is a critical event in cell pyroptosis. The role and functions of GSDMD on mice and humans are widely studied, but its expression, structure, and function in other species are less known. In the present work, rabbit anti-porcine GSDMD (pGSDMD) polyclonal antibody was prepared by immunizing New Zealand white rabbits with prokaryotic expressed recombinant pGSDMD (rpGSDMD). The prepared polyclonal antibody showed good specificity in Western blot and indirect immunofluorescence (IIF) assays. Western blot results showed that the polyclonal antibody could recognize overexpressed pGSDMD in human embryonic kidney cells (HEK293T) and endogenously expressed pGSDMD in cultured intestinal porcine enterocytes (IPEC-J2) and porcine kidney cells (PK-15). Western blot also revealed that pGSDMD was expressed in the heart, liver, lung, kidney, gallbladder, and jejunum of pigs. HEK293T cells overexpressing GSDMD showed green fluorescence in the IIF assay only after being treated with 0.3% Triton-X 100, which indicated that the full-length pGSDMD was located in the plasma but not on the cell membrane. This work provides a useful tool and basic information for further studies on pGSDMD.