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.

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

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

Comparison of current methods for anti-dsDNA antibody detection and reshaping diagnostic strategies.

Anti-double-stranded DNA antibodies (anti-dsDNA) are considered a specific marker for systemic lupus erythematosus (SLE). Though the Farr technique was once the reference method for their detection, it has been almost entirely replaced by more recently developed assays. However, there is still no solid evidence of the commutability of these methods in terms of diagnostic accuracy and their correlation with the Crithidia luciliae immunofluorescence test (CLIFT). Anti-dsDNA antibody levels were measured in 80 subjects: 24 patients with SLE, 36 disease controls drawn from different autoimmune rheumatic diseases (14 systemic sclerosis, 10 Sjögren's syndrome, nine autoimmune myositis, three mixed connective tissue disease), 10 inflammatory arthritis and 10 apparently healthy blood donors by eight different methods: fluorescence enzyme immunoassay, microdot array, chemiluminescent immunoassay (two assays), multiplex flow immunoassay, particle multi-analyte technology immunoassay and two CLIFT. At the recommended manufacturer cut-off, the sensitivity varied from 67% to 92%, while the specificity ranged from 84% to 98%. Positive agreement among CLIFT and the other assays was higher than negative agreement. Mean agreement among methods assessed by the Cohen's kappa was 0.715, ranging from moderate (0.588) to almost perfect (0.888). Evaluation of the concordance among quantitative values by regression analysis showed a poor correlation index (mean r2, 0.66). The present study shows that current technologies for anti-dsDNA antibody detection are not fully comparable. In particular, their different correlation with CLIFT influences their positioning in the diagnostic algorithm for SLE (either in association or sequentially). Considering the high intermethod variability, harmonization and commutability of anti-dsDNA antibody testing remains an unachieved goal.

Current technologies for anti-ENA antibody detection: State-of-the-art of diagnostic immunoassays.

BACKGROUND: Autoantibodies against extractable nuclear antigens (ENA) play a pivotal role in the diagnosis and classification of systemic autoimmune rheumatic diseases (SARD). In recent years, newly developed methods have enabled the simultaneous and quantitative detection of multiple anti-ENA reactivities. However, data regarding the comparability of results obtained using different technologies across different platforms are scarce. In this study we compared eight different immunoassays, commonly used in current laboratory practice for detection of anti-ENA antibodies. METHODS: Sixty patients suffering from different SARD, 10 inflammatory arthritis patients (disease controls) and 10 healthy blood donors were included in this comparative study. Sera were collected in 15 centers belonging to the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine. We evaluated the analytical sensitivity, specificity and diagnostic accuracy of each method for antibodies to Sm, RNP, Ro60, Ro52, Scl70, CENP-B and Jo1. Cohen's kappa was used to analyze the agreement among methods. RESULTS: Average agreement among methods was 0.82, ranging from substantial (k = 0.72) to almost perfect (k = 0.92). However, while the specificity was very good for all methods, some differences emerged regarding the analytical sensitivity. CONCLUSIONS: Diagnostic performance of current technologies for anti-ENA antibody detection showed good comparability. However, as some differences exist among methods, laboratory scientists and clinicians must be aware of the diagnostic accuracy of the testing method in use.

Impact of the COVID-19 Pandemic on the Appropriateness of Diagnostic Pathways of Autoimmune Rheumatic Diseases.

OBJECTIVE: Early diagnosis of autoimmune rheumatic diseases (ARDs) is key to achieving effective treatment and improving prognosis. The coronavirus disease 2019 (COVID-19) pandemic has led to major changes in clinical practice on a global scale. We aimed to evaluate the impact of the COVID-19 pandemic on rheumatological clinical practice and autoimmunity testing demands. METHODS: Data regarding the first rheumatological visits and new diagnoses, together with the autoimmunity laboratory testing volumes related to the COVID-19 pandemic phase (January-December 2020), were collected from medical records and the laboratory information system of a regional reference hospital (Basilicata, Italy) and compared with those obtained during the corresponding period in 2019. RESULTS: A significant decrease in the 2020 autoimmunity laboratory test volume was found when compared with the same period in 2019 (9912 vs 14,100; P < 0.05). A significant decrease in first rheumatological visits and diagnosis (1272 vs 2336; P < 0.05) was also observed. However, an equivalent or higher percentage of positive autoimmunity results from outpatient services was recorded during 2020 when compared to the prepandemic state. Of note, COVID-19-associated decline in new diagnoses affected mainly less severe diseases. In contrast, ARDs with systemic involvement were diagnosed at the same levels as in the prepandemic period. CONCLUSION: The COVID-19 pandemic has affected access to health services. However, our study highlighted that during the outbreak, greater appropriateness of the requests for laboratory tests and visits emerged, as shown by a greater percentage of positive test results and new diagnoses of more severe ARDs compared to the prepandemic period.

A First Step for the Molecular Characterization of Neurological Involvement of Behçet Syndrome: an Italian Pivotal Study.

Behçet syndrome (BS) is a vasculitis characterized by several clinical manifestations including the rare neurological involvement (neuro-BS, NBS). The aim of our pivotal study was to investigate the mutational status of several inflammation-related genes in a cohort of Italian patients with and without the neurological involvement (20 NBS vs 40 no-NBS patients). The preliminary in silico single nucleotide polymorphism (SNP) selection and primer design were performed by NCBI Primer-Blast tool. Genomic DNA was isolated and amplified using PCR. PCR amplicons were sequenced and bioinformatically analysed. Twelve tagSNPs were selected and genotyped: ERAP1 rs30187, rs17482078, and rs27044; IL10 rs1800872 and rs1518111, IL12A rs17810546, IL23R rs17375018, IL23R-IL12RB2 rs924080, STAT4 rs7572482, CCR1 rs7616215, KLRC4 rs2617170, and UBAC2 rs3825427. ERAP1 and IL23R SNPs showed statistically significant higher frequencies in NBS group than no-NBS. ERAP1 rs30187 AA was more common in no-NBS patients (20.0% NBS vs 47.5% no-NBS; p < 0.05), while rs17482078 GA frequency was higher in NBS patients (55.0% NBS vs 22.5% no-NBS; p < 0.05, OR: 4.21). IL23R rs17375018 GG was more frequent in NBS group (65.0% NBS vs 40.0% no-NBS; p < 0.05), according to a previous finding. No other statistically significant differences were found. In conclusion, ERAP1 and IL23R SNPs were found associated with neurological involvement of BS. Additional and larger analyses were required to verify our preliminary findings.

Serum amyloid A in healthy subjects: assessment of reference value using ELISA method.

Serum amyloid A (SAA) is a family of acute-phase reactants. The rise of SAA concentration in blood circulation during the acute-phase response is a clinical marker of active inflammation. Despite its practical and analytical advantages, SAA measurement by enzyme-linked immunosorbent assay (ELISA) has been used mainly as a research tool rather than for the routine laboratory testing. This may be partly explained by the lack of robust reference data in the literature for the different commercially available immunoassays. Using the recommended procedures for the production of reference intervals published by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), we developed the SAA reference interval for a well-defined Italian healthy population and investigated the correlation among SAA and C-reactive protein (CRP), the commonly used acute-phase marker. After data normalization, the reference cutoff was calculated as 225 ng/ml. A good correlation between SAA and CRP was found (P < .05). No statistically significant differences was found between males and females when the means of SAA values were compared, suggesting that not gender-partitioned reference range is recommended for this analyte. This study allowed to define a widely accepted reference cutoff for the SAA detected by ELISA, responding to an unmet need of laboratory medicine.

A new diagnostic algorithm for pattern-oriented autoantibody testing according to the ICAP nomenclature: A pilot study.

The commercial tests currently available as second-level tests to detect ANA sub-specificities are generally used independently from the ANA immunofluorescence (IIF) pattern. The aim of this study was to evaluate the efficacy of the use of a customizable pattern-oriented antigenic panel by immunoblot (IB) using the International Consensus on ANA Patterns (ICAP) classification scheme, in order to introduce a novel and updated autoimmune diagnostic flowchart. 710 sera referred for routine ANA testing were selected on the basis of the ANA pattern according to the ICAP nomenclature (nuclear speckled AC-2,4,5; nucleolar AC-8,9,10,29; cytoplasmic speckled AC-18,19,20) and on an IIF titer ≥1:320. They were then assayed by three experimental IB assays using a panel of selected antigens. ICAP-oriented IB detected 515 antibody reactivities vs. 457 of traditional anti-ENA in the nuclear speckled pattern group, 108 vs. 28 in the nucleolar pattern group, and 43 vs. 34 in the cytoplasmic speckled pattern. This pilot study may lead the way for a new approach introducing an ICAP pattern-oriented follow up testing as a valid alternative to the existing standard panels, thus enabling more patients with autoimmune rheumatic disease to be accurately diagnosed.

Correlation of Tumor Necrosis Factor-α -308G>A Polymorphism with Susceptibility, Clinical Manifestations, and Severity in Behçet Syndrome: Evidences from an Italian Genetic Case-Control Study.

To investigate the association between a functional drug-response tumor necrosis factor (TNF)α gene polymorphism (at the positions of -308; rs1800629; NG_007462.1:g.4682G>A) and both disease susceptibility and clinical manifestations in a cohort of 130 Italian patients with Behçet syndrome (BS). A group of 100 ethnically, age, and gender matched healthy controls (HC) was also recruited. Genotyping was performed using molecular (amplification and direct sequencing) and in silico methods. The genotype distribution of BS patients and HC underlined a lower percentage of wild-type GG genotype in BS patients versus HC (106/130 patients, 81.5% vs. 91/100 HC, 91%; p < 0.05), while the heterozygous genotype (GA) was identified in 24/130 patients (18.5%) versus 9/100 HC (9%) (p < 0.05). GA genotype was significantly associated with the disease (odds ratio = 2.29, 95% confidence interval 1.01-5.18). No significant association was recognized between the single nucleotide polymorphism (SNP) and the BS clinical manifestations, as well as with disease severity (Krause's index). We found statistically significant higher frequency of TNFα rs1800629 GA genotype in patients than in controls. No significant association was recognized between the polymorphism and the clinical parameters, as well as between the SNP and the disease severity. Our data need to be confirmed in larger cohort of patients and matched controls.

Anti-DFS70 antibodies detected by specific methods in patients with thrombosis or recurrent pregnancy loss: no evidence of an association.

A dense fine speckled pattern (DFS) caused by antibodies to the DFS70 kDa nuclear protein is a relatively common finding while testing for anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cells. However, despite many efforts and numerous studies, the clinical significance of anti-DFS70 antibodies is still unknown as they can be found in patients with various disorders and even in healthy subjects. In this study we aimed at verifying whether these antibodies are associated with thrombotic events or with unexplained recurrent pregnancy loss (RPL). We studied 443 patients with venous or arterial thrombosis or RPL and 244 controls by IIF on HEp-2 cells and by a DFS70-specific chemiluminescent immunoassay (CIA). The DFS pattern was observed in IIF in 31/443 (7.0%) patients and in 6/244 (2.5%) controls (p = 0.01) while anti-DFS70 specific antibodies were detected by CIA in 11 (2.5%) patients and in one (0.4%) control (p = 0.06). Positive samples, either by IIF or by CIA, were then assayed by a second DFS70-specific line-immunoassay (LIA) method: 83.3% of the CIA positive samples were confirmed DFS70 positive versus only 29.7% of the IIF positive samples. These findings show that IIF overestimates anti-DFS70 antibody frequency and that results obtained by specific CIA and LIA assays do not indicate that venous or arterial thrombosis or RPL are linked to a higher prevalence of anti-DFS70 antibodies.

Assessing vitamin D levels in an anti-DFS70 positive population: New insights emerging.

Background: Anti-dense fine speckled 70 (DFS70) autoantibodies have more often been described in apparently healthy individuals than in patients with systemic autoimmune rheumatic diseases (SARD). The aim of this study was to explore the link between anti-DFS70 autoantibodies and vitamin D (25(OH)D) levels in an Italian adult cohort.Methods: Serum samples from 34 (five males and 29 females) anti-DFS70 positive patients (index cases), 34 ANA-negative healthy controls, 34 ANA-positive anti-DFS70 negative SLE patients, both groups age- and gender-matched with the index cases, 23 ANA-positive anti-DFS70 negative healthy blood donors and six female SARD patients showing mixed DFS positive pattern were collected and tested for 25(OH)D levels. Relevant demographics and lifestyle practices, body mass index (BMI), comorbidities, and use of medication were recorded for patients and healthy controls.Results: Mean serum levels of 25(OH)D were significantly higher in anti-DFS70 positive subjects (mean ± SD: 22.1 ± 9.8 ng/ml) than in ANA-negative healthy controls (mean ± SD: 17.3 ± 6.7 ng/ml; p = .03), ANA-positive healthy controls (mean ± SD: 15.2 ± 6.8 ng/ml; p = .01), SLE patients (16.6 ± 11.0 ng/ml; p = .01) and in patients with SARD (15.0 ± 5.6 ng/ml; p = .01). No statistically relevant differences in BMI, clinical, or demographic parameters were found.Conclusions: Our findings showed higher levels of vitamin D in anti-DFS70 positive subjects than in the controls, which is compatible with the hypothesis of the "benign" nature of anti-DFS70 antibodies.

From structure to function for the characterization of ERAP1 active site in Behçet syndrome. A novel polymorphism associated with known gene variations.

INTRODUCTION: ERAP1 has been recently proposed as risk marker of Behçet syndrome (BS). Gene single nucleotide polymorphisms (SNPs) could affect the enzymatic activity and the conserved active site is pivotal for the aminopeptidase function. This study aims to characterize the ERAP1 active site in a cohort of BS patients vs healthy controls (HC) integrating genomics, transcriptomics and bioinformatics approach. MATERIALS AND METHODS: We recruited 109 consecutive Italian BS patients (63M:46 F; mean age: 45.07 ± 12.28 years) and 106 matched HC (55M:51 F; mean age: 42.57 ± 12.29 years). DNA was isolated and amplified using PCR with home made-primer pairs. PCR products were directly sequenced and computational analyses were performed to search active site SNPs (NCBI-BlastN tool), to predict SNPs functional effect (PolyPhen-2 software) and to obtain protein 3D modelling (Protean3D software). In a second phase of analysis, RNA was extracted and reverse transcribed. Quantitative Real-Time PCR (qPCR) was performed to assess ERAP1 mRNA level in presence (target) and in absence (control) of gene polymorphisms. The Fold change was calculated for the relative quantification of gene expression. RESULTS: A novel coding variation (NG_027839.1:g.25637 T > G; NP_057526.3:p.Phe360Cys, HGSV nomenclature) was found in heterozygosity state in 5/109 BS patients (4.59 % of cases) and none of HC. It was recognized in association with rs2287987, rs30187, rs17482078, and rs27044 BS-related polymorphisms for 4 out of 5 patients. All patients carrying the novel SNP were HLA-B*51-positive. The novel SNP was released in GenBank database with MK140632.1 ID. The SNP was predicted to be damaging and resides within the Zn-binding HEXXH(X)18E region of the active site, changing the structurally conserved region for the amminopeptidase function. In fact, the change in energy (ΔE) score between wild-type and SNP-containing protein showed a less stable protein in presence of p.Cys360 (ΔE:3.584) (Protean3D prediction). Preliminary qPCR results underlined a significant difference in fold change value when target and control values were compared (p < 0.05), suggesting a reduced expression of ERAP1 mRNA in presence of the novel SNP. CONCLUSIONS: Our study strengthens the association between ERAP1 and BS. The most significant point was the localization of the novel p.Phe360Cys SNP within the Zn-binding region of protein active site that was predicted to affect its function, causing protein destabilization. Our findings need to be tested in larger genetic studies.

Identification of a de novo NLRP3 gene variation in an Italian Behçet syndrome patient.

A novel nonsynonymous variation of NLRP3 was identified in an Italian patient with Behçet syndrome using both bioinformatics and molecular methods. This variation was a thymine to guanine polymorphism responsible for the isoleucine to serine amino acid change at position 348. The novel variation was predicted to be a pathogenic allele.

Distribution of rs17482078 and rs27044 ERAP1 polymorphisms in a group of Italian Behçet's syndrome patients: a preliminary case-control study.

The Endoplasmic reticulum aminopeptidase protein 1 (ERAP1) trims N-terminal amino acids from epitope precursors for Major Histocompatibility Complex class I presentation. Genome-wide association studies demonstrated that ERAP1 gene single nucleotide polymorphisms (SNPs) are associated with Behçet's syndrome (BS). This study was conducted on the two most consistently BS-associated ERAP1 polymorphisms, rs17482078 (NG_027839.1:g.35983G>A) and rs27044 (NG_027839.1:g.35997C>G) to analyse their distribution in 55 Italian BS patients and 65 ethnically matched controls (healthy controls, HC) and to test their association with BS risk. SNPs were detected by isolation, amplification of genomic DNA and direct sequencing. SNPs functional effects were predicted by bioinformatics software. The odds ratio (OR) with 95% confidence intervals was calculated to assess the strength of BS association for genotypes and alleles, also validated by logistic regression (LR). LR was used to test the association between both SNPs and patients HLA genetic data. Bonferroni correction was also applied. Comparing patients and controls, we found a significant higher frequency of rs17482078 A allele (32.73% BS vs 17.69% HC, p = 0.007) and AA genotype (18.18% BS vs 0% HC; p = 0.0003) and rs27044 G allele (63.64% BS vs 46.92% HC; p = 0.0096) in BS group after Bonferroni correction. No association was found between HLA-B*51 and both ERAP1 SNPs. Although preliminary, our data show a stronger association of rs17482078 with BS compared to rs27044 by means of case-control genetic analysis and bioinformatics prediction of protein structure change. A larger series of patients and controls is required to confirm our preliminary findings.

Prevalence and serological profile of anti-DFS70 positive subjects from a routine ANA cohort.

Anti-Dense Fine Speckled 70 (DFS70) antibodies are a common finding in clinical laboratory referrals. High prevalence of DFS70 autoantibodies in healthy population and usual negative association with Antinuclear Antibody (ANA)-associated autoimmune rheumatic diseases (AARD) were reported. The aim of this study was to evaluate the prevalence of DFS70 autoantibodies and their association with other autoantibodies in the context of a routine ANA referral cohort. Consecutive sera submitted for ANA screening were analyzed for anti-DFS70 antibodies by indirect immunofluorescence (IIF) (n = 3175, 1030 men and 2145 women) then confirmed by immunoblotting. Anti-DFS70 positive samples were also assayed for a large spectrum of other circulating autoantibodies. The prevalence of anti-DFS70 antibodies was 1.7% in the whole population and 4.6% in the ANA-positive samples. Comparison between DFS70 IIF and immunoblotting showed an excellent correlation between the two methods. The prevalence of anti-DFS70 positive was significantly higher in females (2.1%, 45/2145) than in males (1.0%, 10/1030). Of note, no concomitant autoantibodies were found in the DFS70-positive male group compared with DFS70-positive females group that showed other serum autoantibodies in the 51% of cases. Anti-DFS70 reactivity in male population may represent an useful biomarker predicting the absence of other autoantibodies. On the contrary, the serological profile of DFS70-positive females required further investigations in order to define the presence of concomitant disease-marker autoantibodies.