Effect of antinuclear antibody positivity on antineutrophil cytoplasmic antibody results by indirect immunofluorescence assay.

BACKGROUND: The indirect immunofluorescence assay (IIFA) utilizing antineutrophil cytoplasmic antibodies (ANCA) is widely used as a diagnostic test for autoimmune vasculitis. The presence of antinuclear antibodies (ANA) might lead to a misleading interpretation of ANCA. This study aims to explore the impact of the presence of ANA on the interpretation of ANCA. METHODS: This retrospective research examined samples negative for antiMPO and antiPR3 ANCA by IIFA and explored correlations between the ANA-IIFA results and the ANCA interpretation frequencies. Our analysis involved the use of suitable statistical methods, including Chi-square and kappa statistics. RESULTS: Up to 75.2% of the ANCA-IIFA-positive samples exhibited a positive p-ANCA pattern when using the ethanol-fixed substrate, with c-ANCA positivity at 24.8%. In the ANA-IIFA-positive samples, ~77.3% displayed p-ANCA patterns on ethanol-fixed substrates. A comparison between the ANA-IIFA titers and the p-ANCA results revealed that p-ANCA positivity was notably more common in samples with higher titers, and this correlation was found to be statistically significant. CONCLUSION: Positive ANA results by IIFA tests are linked to a higher incidence of p-ANCA interpretation, particularly in cases with higher titer patterns. This insight aids laboratories in establishing effective workflows to investigate potential p-ANCA interference.

Retrospective analysis of the clinical significance of Ro52/TRIM21 antibody and specific antinuclear antibody patterns by indirect immunofluorescence.

Objectives: To determine the clinical significance of Ro52 protein/tripartite motif-containing 21 antibody and specific antinuclear antibody patterns using indirect immunofluorescence technique. METHODS: The retrospective study was conducted at the clinical laboratory of the First Affiliated Hospital of Chongqing Medical University, China, and comprised data from January 2017 to December 2021 of patients who underwent antinuclear antibody and anti-extractable nuclear antigen antibody detection. Inpatients with Ro52 antibody-positive status were taken as the cases, while anti-Ro52 negative patients with clear clinical diagnosis were taken as the controls. Data was analysed using SPSS 19. RESULTS: There were 1802 cases and 1211 controls. Positive Ro52 showed significantly greater frequency in patients with primary Sjogren's syndrome, systemic lupus erythematosus, inflammatory myositis, dry eyes and interstitial lung disease (p<0.05). Ro52 antibody showed high positive predictive value for primary Sjogren's syndrome 25(96.15%), systemic lupus erythematosus 259(91.20%), connective tissue disease-associated interstitial lung disease 45(86.67%) and inflammatory myositis 60(86.67%). Antinuclear antibody indirect immunofluorescence patterns most frequently detected were nuclear speckled 128(40.89%) and cytoplasmic speckled 126(40.26%) (p<0.05). Interstitial lung disease was associated with the presence of cytoplasmic speckled antinuclear antibody indirect immunofluorescence pattern 24(19.2%), while tumours 47(36.5%) and hepatitis B 26(20.3%) seemed to be more frequent with nuclear speckled pattern (p<0.05). The simultaneous reactivity extractable nuclear antigen antibodies most frequently detected were antinuclear antibody+Ro52+anti-Sjogren's syndrome A+ 558(33.96%). CONCLUSIONS: Ro52 antibody positivity was found to be associated with Sjogren's syndrome, systemic lupus erythematosus, inflammatory myositis, dry eye and interstitial lung disease. The antinuclear antibody immunofluorescence pattern of Ro52 positive was single and primarily granular cytoplasm type. Antinuclear antibody negative and Ro52 positive in the serum of patients also had certain significance in auxiliary disease diagnosis.

Recognition of rare antinuclear antibody patterns based on a novel attention-based enhancement framework.

Rare antinuclear antibody (ANA) pattern recognition has been a widely applied technology for routine ANA screening in clinical laboratories. In recent years, the application of deep learning methods in recognizing ANA patterns has witnessed remarkable advancements. However, the majority of studies in this field have primarily focused on the classification of the most common ANA patterns, while another subset has concentrated on the detection of mitotic metaphase cells. To date, no prior research has been specifically dedicated to the identification of rare ANA patterns. In the present paper, we introduce a novel attention-based enhancement framework, which was designed for the recognition of rare ANA patterns in ANA-indirect immunofluorescence images. More specifically, we selected the algorithm with the best performance as our target detection network by conducting comparative experiments. We then further developed and enhanced the chosen algorithm through a series of optimizations. Then, attention mechanism was introduced to facilitate neural networks in expediting the learning process, extracting more essential and distinctive features for the target features that belong to the specific patterns. The proposed approach has helped to obtained high precision rate of 86.40%, 82.75% recall, 84.24% F1 score and 84.64% mean average precision for a 9-category rare ANA pattern detection task on our dataset. Finally, we evaluated the potential of the model as medical technologist assistant and observed that the technologist's performance improved after referring to the results of the model prediction. These promising results highlighted its potential as an efficient and reliable tool to assist medical technologists in their clinical practice.

Harmonization of ANA testing challenge: quantification strategy to accurately predict end-point titers avoiding serial dilution.

Despite the advantages of automated systems for antinuclear antibody (ANA) analysis, the prediction of end-point titers avoiding serial dilutions is still in progress. The aims of this study were to set a conversion table providing discriminant ranges of fluorescence signal intensity values (FI) corresponding to the end-point titers and validate this tool in a real-life laboratory setting. Eight hundred ninety-four serum samples were analyzed for ANA using Image Navigator System. In order to classify FI into non-overlapping groups corresponding to conventional end-point titers, statistical discriminant analysis was used. Validation study was performed calculating agreement and error rates between visual readings and conversion table of 1119 routine ANA positive samples. Setting of FI ranges corresponding to the end-point titers for different staining patterns was computed. For samples showing single pattern, the overall agreement between visual readings and conversion table was 98.4% for all titers ranging from 1:160 to 1:2560, of which 68.0% had the same titer and 30.4% were within ± one titer difference. Concordance rates according to ANA patterns were as follows: (1) nuclear 98.4%, of which 67.0% had the same titer and 31.4% ± one titer; (2) cytoplasmic 100%, of which 72.7% had the same titer and 27.3% than ± one titer; (3) mitotic 66.6%, of which 33.3% had more ± one titer. Our study developed a quantification method for autoantibodies titers assessment based on just one single sample dilution instead of traditional serial dilution approach, providing significant advantages in routine laboratory in terms of reduction in hand-on time and harmonization of results.

A novel way to evaluate autoantibody interference in samples with mixed antinuclear antibody patterns in the HEp-2 cell based indirect immunofluorescence assay and comparison of conventional microscopic and computer-aided pattern recognition.

BACKGROUND: A major challenge of the HEp-2 cell-based indirect immunofluorescence (IIF) assays is the correct identification of the individual anti-nuclear antibodies (ANAs) if more than one is present in a sample. We created artificial mixes by pooling two different samples with a single autoantibody in different titers. Comparison of the expected and observed patterns and titers clarifies the interference between the two tested ANAs. METHODS: Serum samples with a single homogeneous or speckled ANA pattern were serially diluted and mixed in 16 combinations, providing end-point titers of 1:5,120 to 1:80 for both patterns. These mixes were tested by a HEp-2 IIF assay and were evaluated by conventional evaluation, the EUROPattern (EPa) system and on-screen analysis. RESULTS: Homogeneous pattern can alter the identification of the speckled pattern much more than vice versa, but both has an interfering effect on the other. The effect of the interfering on the tested pattern was higher if the titer of the former one was higher. The pattern recognition efficacy of conventional and the on-screen evaluation was similar and superior compared to the EPa analysis. CONCLUSIONS: The application of artificial mixed samples can help the evaluation of the efficacy of manual and computer-aided ANA HEp-2 pattern recognition.

Longitudinal study of patients with discrepant results in CLIFT and a solid-phase dsDNA antibody assay: does a gold standard dsDNA assay exist?

OBJECTIVE: Antidouble-stranded DNA (dsDNA) antibodies are essential for diagnosis and follow-up of systemic lupus erythematous (SLE). To ensure the best diagnostic approach, most healthcare laboratories opt for a combination of highly sensitive methods, such as solid-phase immunoassays, and highly specific methods, such as the Crithidia luciliae indirect immunofluorescence test (CLIFT). Even so, discordant results are common, thus hindering the diagnostic process. Therefore, this study aimed to characterise a cohort of patients with discrepant results for a dsDNA fluorescence enzyme immunoassay (FEIA) and CLIFT during 2016-2018 and to follow patients up until December 2021. METHODS: We performed an observational, longitudinal and retrospective study on 417 samples from 257 patients who had been referred for suspected connective tissue diseases or followed up after diagnosis. All of them were positive for antinuclear antibodies (ANAs) using an indirect immunofluorescence assay (IFA) on Hep-2 cells, the entry criterion in our laboratory, and positive for FEIA dsDNA. Samples were then tested with CLIFT according to our routine protocol, which includes CLIFT testing after FEIA dsDNA results ≥10 UI/ml. After the assessment of data quality, the final analysis was based on 222 patients. RESULTS: Eighty-three patients (37.4%) had positive results in both tests and met the diagnostic criteria for SLE. However, 139 patients (62.6%) had discrepant results (FEIA+, CLIFT-). Of these, 58 patients (41.7%) had a diagnosis of SLE, with 47 (33.8%) having been previously diagnosed and under treatment. The remaining 11 patients (7.9%) had a new diagnosis of SLE, which was made up within 4 years of the initial screening. A total of 81 of the 139 patients (57.5%) with discrepant results did not meet lupus criteria during the follow-up period. CONCLUSIONS: The study showed that CLIFT could be negative in both treated and newly diagnosed SLE, thus underlining the importance of follow-up of dsDNA-positive results using solid-phase tests. Therefore, quantitative tests such as FEIA could add value to the diagnosis and management of patients with suspected SLE.

Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate.

The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.

A Novel Application of Zinc Oxide Nanoparticles for Inhibition of Molluscum contagiosum Virus Infection.

Molluscum contagiosum virus (MCV) is an infection caused by the molluscum contagiosum virus. Antiviral medications used to treat MCV infections have several problems, including drug-resistant and toxicity. As a result, improving safe, innovative, and effective antiviral drugs is critical. Therefore the current study aimed to investigate ZnO-NPs effects on M. contagisum infection and molluscum contagiosum virus replication, among the main exciting viruses that menace human health. The antiviral activity of zinc oxide nanoparticles (ZnO-NPs) against MCV infection was investigated in this work. FESEM and TEM electron microscopy were used to examine the nanoparticles. The cytotoxicity of the nanoparticles was assessed using the MTT assay, and anti-influenza effects were detected using RT-PCR and TCID50. An indirect immunofluorescence experiment was used to investigate the inhibitory effect of nanoparticles on viral antigen expression. In all tests, acyclovir was employed as a control. Compared to virus control, post-exposure of MCV with ZnO nanoparticles at the highest dose but is not toxic (100 g/mL) resulted in 0.2, 0.9, 1.9, and 2.8 log10 TCID50 reductions in infectious diseases virus titer (P=0.0001). This ZnO-nanoparticles level was accompanied by an inhibition percentage (17.8%, 27.3%, 53.3%, 62.5 %, and 75.9%), respectively, measured based on viral load compared with the virus control. Compared to the positive control, fluorescence emission intensity in virally infected cells that administrated ZnO nanoparticles was statically decreased. Our findings demonstrated that ZnO-NPs have antiviral effects against the MCV. This property indicates that ZnO-NP has a high potential for usage in topical formulations to treat facial and labial lesions.

Investigation of the Inhibition Activity of Aluminium Oxide Nanoparticles for Herpes Simplex Type 1.

Several studies have shown that Herpes simplex type 1 )HSV-1 (is one of the viruses resistant to medications, so potential antiherpetic agents need to be evaluated. This study aimed to evaluate the impact of Aluminum Oxide Nanoparticles (Al2O3-NPs) on HSV-1 infection. Characterization of Al2O3-NPs was performed using field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and high-resolution transmission electron microscopy (HRTEM). The MTT test was used to investigate the toxicity action of Al2O3-NPs on viable cells. Quantitative Real-Time PCR (qRT-PCR)and TCID50 assays were used to achieve the antiherpetic performance Al2O3-NPs.Indirect immunofluorescence assay (IFA) was performed to determine the inhibitory impact of Al2O3-NPs on viral antigen expression, and acyclovir was utilized as a standard agent in all tests. HSV-1 subjected to Al2O3-NPs at the maximum non-toxic concentration (100 µg / mL) leads to a decrease of 0.1, 0.7, 1.8, and 2.5 log10 TCID50 in the infectious titer relative to virus control (P<0.0001). This concentration of Al2O3-NPs was correlated with 16.9%, 47.1 %, 61.2 %, 72.5 % and 74.6 % inhibition rates, calculated based on HSV-1 viral load compared to virus control. Our results have shown that Al2O3-NPs have a robust antiviral activity against HSV-1. This function demonstrates excellent potential for using Al2O3-NP in topical formulations for treating orolabial or genital herpetic lesions.

A novel magnetic bead-based immunoprecipitation method for anti-dense fine speckled 70 antibodies.

The indirect immunofluorescence assay (IIFA) on HEp-2 cells has been widely used for screening anti-nuclear antibodies (ANA) that are associated with systemic autoimmune rheumatic diseases (SARD). Sera containing ANA display multiple distinct fluorescence patterns on HEp-2 cells. Among them, a dense fine speckled (DFS) pattern caused by anti-DFS70 antibodies has been reported to have higher prevalence in healthy individuals than in patients with SARD. This DFS pattern is often difficult to distinguish amongst other SARD-associated ANA patterns, in particular a mixed homogeneous and speckled pattern. Furthermore, a strong DFS pattern can mask other SARD-associated patterns. Hence, we developed a novel immunoprecipitation method using magnetic beads to remove anti-DFS70 antibodies in serum prior to running IIFA. We also aimed to confirm the presence of anti-DFS70 and to uncover any SARD-associated ANA patterns masked by a strong DFS pattern. The sera used in our study were from 70 individuals who had routine ANA screen, of which 35 sera had an isolated DFS pattern with monospecific anti-DFS70 antibodies confirmed by a complementary assay, and 35 were control sera without a DFS pattern. An immunoprecipitation method using magnetic beads coated with recombinant human full length DFS70 protein was developed. The performance of this new method was evaluated in comparison to an immunoadsorption method using the same DFS70 protein. Our newly developed immunoprecipitation method demonstrated excellent sensitivity (91.4%) and specificity (100%) in confirming the DFS pattern associated with anti-DFS70 in sera. Additionally, our method was able to remove anti-DFS70 and uncover SARD-associated ANA patterns masked by a strong DFS pattern. It also showed a clearer background on IIFA than that of the immunoadsorption method. The novel magnetic bead-based immunoprecipitation method demonstrated satisfactory performance in removing anti-DFS70 without interfering with the detection of other antibodies. It can be easily integrated with IIFA to confirm anti-DFS70 associated DFS pattern. Additionally, it can simultaneously unmask other ANA patterns, which cannot be achieved by a conventional protocol that requires a complementary anti-DFS70 specific assay to be performed. Therefore, the novel method provides a more effective and accurate solution for ANA screening.

The significance of dense fine speckled pattern in antinuclear antibody-associated rheumatic disease and coexisting autoantibodies: A propensity score-matched cohort study.

AIM: To investigate the relationship between the prevalence of antinuclear antibody (ANA) -associated rheumatic diseases (AARD) and the presence of dense fine speckled (DFS) and homogeneous patterns in ANA tests. METHODS: This retrospective study enrolled adult patients with either a DFS or homogeneous pattern in their ANA test. A mixed pattern was defined as the presence of more than one pattern reported in the test. The presence of anti-DFS70 antibodies and other common autoantibodies were detected using EUROLINE ANA Profile 23. A 1:2 propensity score matching was applied to control for demographic and other interfering factors. RESULTS: A total of 59 patients with a DFS pattern were enrolled and compared with a matched homogeneous group. The DFS group had a significantly lower prevalence of AARD (3.4% vs. 16.9%, p = .008) and the subgroup with anti-DFS70 antibodies showed an even lower prevalence (2% vs. 20%, p = .002). Among the 33 patients with monospecific anti-DFS70 antibodies, five had a mixed pattern, and all patients with common autoantibodies had an isolated DFS pattern. CONCLUSIONS: The findings of this study suggest that patients with a DFS pattern in their ANA test may have a lower prevalence of AARD compared with those with a homogeneous pattern. However, an isolated DFS pattern in ANA testing does not necessarily indicate the presence of monospecific anti-DFS70 antibodies or AARD. Confirmatory testing for the monospecific anti-DFS70 antibody is mandatory to exclude AARD.

Antieosinophil Antibodies Alone or in Combination with Antineutrophil Cytoplasmic Antibodies (ANCA) Detected in Different Autoimmune Conditions.

Circulating antieosinophil antibodies (AEOSA) have been associated with various autoimmune conditions affecting the liver, kidneys, lungs, and joints but are not part of routine clinical diagnostics. While analyzing human sera for antineutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence (IIF) on granulocytes, 0.8% of analyzed samples were found to be reactive with eosinophils. Our aim was to determine the diagnostic relevance and antigenic specificity of AEOSA. AEOSA were seen either in combination with an myeloperoxidase (MPO)-positive p-ANCA (44%; AEOSA+/ANCA+) or on their own (56%; AEOSA+/ANCA-). AEOSA/ANCA positivity was seen in patients with thyroid disease (44%) or vasculitis (31%), while AEOSA+/ANCA- pattern was more common in patients with autoimmune disorders of the gastrointestinal tract and/or liver. Eosinophil peroxidase (EPX) was the main target recognized in 66% of the AEOSA+ sera by enzyme-linked immunosorbent assay (ELISA). Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) were also identified as target antigens but less frequently and only in combination with EPX. In conclusion, we confirmed that EPX is a major target of AEOSA, illustrating the high antigenic potential of EPX. Our results also demonstrate the presence of concomitant AEOSA/ANCA positivity in a defined patient group. Further research should aim to elucidate the association of AEOSA with autoimmunity.

Laboratory and clinical practices in antinuclear antibody detection and related antigens: recommendations from a Spanish multicentre survey.

Antinuclear antibodies (ANA) are the most widely used immunological test for the diagnosis of autoimmune diseases. Despite the recommendations of experts, there is some variability in performing and interpreting this test in routine practice. In this context, the Spanish Group on Autoimmune Diseases (GEAI) of the Spanish Society of Immunology (SEI) conducted a national survey of 50 autoimmunity laboratories. Here we report the survey results on ANA testing, detection of related antigens, and our recommendations. The survey showed that most of the participating laboratories use a similar approach for most key practices: 84% perform ANA by indirect immunofluorescence (IIF) on HEp-2 cells as the screening methodology while the other laboratories use IIF to confirm positive screens; 90% report ANA test results as either negative or positive with titer and pattern; 86% indicated that the ANA pattern conditioned follow-up testing for specific antigen-related antibodies; and 70% confirm positive anti-dsDNA. However, testing practices were highly heterogeneous for certain items, such as sera dilutions and the minimum time period for repeating ANA and related antigen determinations. Overall, this survey shows that most autoimmune laboratories in Spain use a similar approach but that further standardization of testing and reporting protocols is needed.

A population-based characterization study of anti-mitochondrial M2 antibodies and its consistency with anti-mitochondrial antibodies.

OBJECTIVE: This study aims to estimate the prevalence of anti-mitochondrial antibody subtype M2 (AMA-M2) and assess its consistency with AMA in a general population. METHODS: A total of 8954 volunteers were included to screen AMA-M2 using enzyme-linked immunosorbent assay. Sera with AMA-M2 >50 RU/mL were further tested for AMA using an indirect immunofluorescence assay. RESULTS: The population frequency of AMA-M2 positivity was 9.67%, of which 48.04% were males and 51.96% were females. The AMA-M2 positivity in males had a peak and valley value of 7.81% and 16.88% in those aged 40 to 49 and ≥70 years, respectively, whereas it showed a balanced age distribution in females. Transferrin and immunoglobulin M were the risk factors for AMA-M2 positivity and exercise was the only protective factor. Of 155 cases with AMA-M2 >50 RU/mL, 25 cases were AMA-positive, with a female-to-male ratio of 5.25:1. Only 2 people, with very high AMA-M2 of 760 and >800 RU/mL, met the diagnostic criteria of primary biliary cholangitis (PBC), making the prevalence of PBC 223.36 per million in southern China. CONCLUSION: We found that AMA-M2 has a low coincidence rate with AMA in the general population. A new decision-making point for AMA-M2 is needed to improve consistency with AMA and diagnostic accuracy.

Negative ANA-IIF in SLE patients: what is beyond?

The antinuclear antibody (ANA) test has high sensitivity in diagnosing and classifying systemic lupus erythematosus (SLE). OBJECTIVES: To describe the immunological pattern of SLE patients through investigating specific antinuclear autoantibodies by enzyme dot immunoassay and studying their frequency in both positive and negative ANA indirect immunofluorescence assay (IIF) cases. METHODS: In a cross-sectional study, blood samples from 393 newly diagnosed SLE patients were analyzed using (IIF) on HEp-2 cells and ANA dot immunoassay by automated enzyme immunoassay (EIA) to detect 19 antibodies. RESULTS: Ninety-one percent of the patients are females; their mean age was 37 ± 12.28. Antinuclear antibody (ANA) was detected by IIF in 82.4% of cases, with 181 (46.1%) speckled and 167 (42.4%) homogeneous ANA patterns. The majority of patients (96%) demonstrated autoantibodies via EIA. Among the ANA-IIF-negative patients, 97.2% demonstrated autoantibodies. There was a significant difference in the frequency of certain autoantibodies between SLE patients with negative and positive ANA-IIF (1.44 0.73, 3.12 2.09, p = 0.00) respectively. CONCLUSION: The results of analyzing 19 autoantibodies with the ANA staining pattern increased the significance of analyzing the immune profile even if IIF is negative when clinical symptoms strongly suggest SLE diagnosis. Certain autoantibodies may evade staining by the IFA approach while they are present in the patient's serum, and they may not be detected by the ANA EIA profile if it does not contain that antigenic substrate. Key Points • Indirect immunofluorescence on Hep-2 is the conventional method for ANA detection and is regarded as the "gold standard" for testing in clinical practice for SLE. • In our study, ANA profile dot enzyme immunoassay (EIA)-based test was performed to evaluate 19 autoantibodies in SLE patients either positive or negative for ANA-IIF. • The presence of anti-dsDNA with ANA-IIF-negative serum in 32.4% of SLE patients provides evidence that not all anti-dsDNA antibodies are identified on standard HEp-2 substrates. • certain autoantibodies can evade staining by the ANA-IIF method despite being present in the SLE patient's blood; this supports the ANA profile enzyme dot immunoassay as a more sensitive test.

Diagnostic Performance of Anti-dsDNA Tests by Indirect Immunofluorescence and Enzyme-linked Immunosorbent Assay in Patients with Systemic Lupus Erythematosus.

BACKGROUND: Several laboratory techniques for anti double-stranded (ds) DNA detection in systemic lupus erythematosus (SLE) are available, with variable diagnostic performance. We aimed to evaluate anti-dsDNA's diagnostic performance by indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (EIA). METHODS: We conducted a single-center retrospective (2015 to 2020) study. Patients with anti-dsDNA tests by IIF and EIA were included. We evaluated the indications, applications, concordance, positive predictive value (PPV) of anti-dsDNA to confirm SLE diagnosis or flares, and associations of disease manifestations with positivity with each technique. RESULTS: A total of 1368 reports of anti-dsDNA tests by IIF and EIA and the corresponding medical records of the patients were analyzed. The main indication for anti-dsDNA testing was to help in the diagnosis of SLE in 890 (65%) of the samples, and the main application after obtaining the results was SLE exclusion in 782 (57.2%) cases. The combination with the highest frequency was the negativity result by both techniques in 801 (58.5%) cases (Cohen kappa 0.57). Both methods were positive in 300 patients with SLE (Cohen kappa 0.42). The PPVs of anti-dsDNA tests to confirm diagnosis/flare was 79.64% (95% CI, 75.35-83.35) by EIA, 78.75% (95% CI, 74.27-82.62) by IIF, and 82% (95% CI, 77.26-85.93) when both were positive. CONCLUSIONS: Anti-dsDNA detection by IIF and EIA are complementary and may indicate different clinical patterns in patients with SLE. The detection of anti-dsDNA antibodies by both techniques has a higher PPV than either separately for confirming SLE diagnosis or flares. These results highlight the need for evaluating both methods in clinical practice.

Usefulness of immunofluorescence overlay antigen mapping in the identification of autoantigen in anti-p200 pemphigoid.

Anti-p200 pemphigoid is a rare subepidermal blistering disease showing immunoglobulin G (IgG) autoantibodies reactive with a 200-kDa protein. In most patients, serum IgG antibodies react with laminin γ1. The diagnosis of anti-p200 pemphigoid is occasionally difficult, mainly due to the lack of standardized tests. We performed fluorescence overlay antigen mapping by laser scanning confocal microscopy (FOAM-LSCM) to identify autoantigens in an anti-p200 pemphigoid patient and assessed its usefulness for the diagnosis. A 71-year-old man presented with blisters and erosions on the bilateral forearms. No mucosal lesions were observed. Laboratory examinations revealed mild leukocytosis and antinuclear antibody negativity. A histopathological examination showed subepidermal blisters with neutrophil infiltration. Direct immunofluorescence showed linear IgG staining along the basement membrane zone. Indirect immunofluorescence using 1 M NaCl-split skin sections revealed IgG reactivity on the dermal side. Immunoblotting detected circulating IgG autoantibodies that reacted with a 200-kDa protein. Accordingly, anti-p200 pemphigoid was diagnosed. FOAM-LSCM revealed that the patient's IgG signals were co-localized with laminin γ1 but were observed above type VII collagens. A direct immunofluorescent analysis for IgG deposition patterns showed an n-serrated pattern. Thus, FOAM-LSCM may be useful for diagnosing anti-p200 pemphigoid.

Computer-aided classification of indirect immunofluorescence patterns on esophagus and split skin for the detection of autoimmune dermatoses.

Autoimmune bullous dermatoses (AIBD) are rare diseases that affect human skin and mucous membranes. Clinically, they are characterized by blister formation and/or erosions. Depending on the structures involved and the depth of blister formation, they are grouped into pemphigus diseases, pemphigoid diseases, and dermatitis herpetiformis. Classification of AIBD into their sub-entities is crucial to guide treatment decisions. One of the most sensitive screening methods for initial differentiation of AIBD is the indirect immunofluorescence (IIF) microscopy on tissue sections of monkey esophagus and primate salt-split skin, which are used to detect disease-specific autoantibodies. Interpretation of IIF patterns requires a detailed examination of the image by trained professionals automating this process is a challenging task with these highly complex tissue substrates, but offers the great advantage of an objective result. Here, we present computer-aided classification of esophagus and salt-split skin IIF images. We show how deep networks can be adapted to the specifics and challenges of IIF image analysis by incorporating segmentation of relevant regions into the prediction process, and demonstrate their high accuracy. Using this semi-automatic extension can reduce the workload of professionals when reading tissue sections in IIF testing. Furthermore, these results on highly complex tissue sections show that further integration of semi-automated workflows into the daily workflow of diagnostic laboratories is promising.