Prevalence and Factors Associated with Positivity of Antinuclear Antibodies (ANA) Patterns, Native Anti-DNA and Extractable Nuclear Antigens (ENA) Antibodies: Experience from a Laboratory in Dakar

Background: Diagnosis of autoimmune diseases (AID) is challenging, due to overlapping features with other non-immune disorders. Anti-nuclear antibodies (ANA) are sensitive screening tests but anti-deoxyribonucleic acid-antibody (anti-DNA), and anti-extractable nuclear antigens (anti-ENA) are specific for AIDs. We aimed to look at ANA patterns in our patients and correlated them with anti-ENA for proper interpretation and better patient management cost-effectively. Methods: A retrospective study was conducted over 1 year from January to December 2022 who were tested for ANA at biology medical laboratory of Pasteur Institute of Dakar. Anti-ENA and anti-DNA results were also analyzed for ANA-positive patients. Statistical analysis was performed using STATA 14.0, p Results: 216 patients were analyzed. Women predominated at 79.2% and mean age was 48 years [CI 95%, 46 - 50], with extremes of 10 and 89. Most represented age group was [41 - 60] with 38%. ANA was positive in 27 (12.5%) of patients, 59.2% of whom were strongly positive (titer of 1/1000, 1/3200 or 1/6400). The most common pattern was nuclear speckled, which was found in 77.8% of samples. Anti-ENA and anti-DNA positivity in ANA-positive patients was found respectively in 63% (17/27) and 1.4% (3/27) of the samples analyzed. Most commonly identified anti-ENA was anti-Sm 29.6%, anti-SSA 29.6%, anti-Ro-52 25.9%, anti-RNP 18.5% and anti-SSB 14.8% which was associated with speckled pattern. Association results indicated a significant relationship between both tests and between ANA titer in the anti-ENA- and ANA-positive patients (p 0.001). Conclusions: ANA, Anti-ENA and anti-DNA antibodies are essential for AIDS diagnosis. However, the testing repertoire should follow an algorithm comprising of clinical features, followed by ANA results with nuclear, mitotic, and cytoplasmic patterns, anti-ENA, and anti-DNA for a more meaningful, and cost-effective diagnostic approach.

Detection of Antinuclear Antibodies in Canine Serum by Immunoperoxidase in MDCK and Indirect Immunofluorescence in HEp2 Cells

Antinuclear antibodies are found in animals suffering from Systemic Lupus Erythematosus (SLE) and some other diseases, their presence in the blood is determined by antinuclear antibody (ANA) test using indirect immunofluorescence (IF) with HEp2 cells as a substrate. In this work, an immunoperoxidase (IP) assay was developed to evaluate the ANAs in canine sera, using canine kidney cell lines (MDCK) and compared with a commercial immunofluorescence test on Hep2 cells for this system, a fluoresceinated anti-canine Ig antibody was standardized. The study was performed on 50 sera from dogs submitted to the laboratory with different clinical diagnoses of autoimmune-associated diseases. The procedures on both cells were unified to perform comparisons of the reactions, direct sera or at different dilutions were added to a monolayer of permeabilized MDCK cells, followed by a peroxidized anti-canine IgG conjugate, and a substrate for the IP reaction. The same sera were tested on the commercial IF assay on Hep2 cell system. In 22/50 cases, the presence of LE cells in peripheral blood was determined. A high correlation was found in the detection of antinuclear antibodies between both cell lines and techniques, however there were differences in the reaction patterns in the nucleus and cytoplasm between cell lines. The diffuse nuclear pattern observed in MDCK cells was more related to the presence of high percentages of LE cells in peripheral blood. The differences found in the results were possibly associated with the presence of homologous antigens between the MDCK cells and the dog. In addition, the methodology and standardization for the use and interpretation of a reference serum was developed to unify the interpretation criteria in the laboratory.

Role of autoantibodies in the clinical management of primary biliary cholangitis

Primary biliary cholangitis(PBC)is a chronic cholestatic liver disease characterized by immune-driven destruction of small intrahepatic bile ducts leading a proportion of patients to hepatic failure over the years.Diagnosis at early stages in concert with ursodeoxycholic acid treatment has been linked with prevention of disease progression in the majority of cases.Diagnosis of PBC in a patient with cholestasis relies on the detection of disease-specific autoantibodies,including anti-mitochondrial antibodies,and disease-specific anti-nuclear antibodies targeting sp100 and gp210.These autoantibodies assist the diagnosis of the disease,and are amongst few autoantibodies the presence of which is included in the diagnostic criteria of the disease.They have also become important tools evaluating disease prognosis.Herein,we summarize existing data on detection of PBC-related autoantibodies and their clinical significance.Moreover,we provide insight on novel autoantibodies and their possible prognostic role in PBC patients.

Clinical value of chemiluminescence method for detection of antinuclear antibody profiles

BACKGROUND Antinuclear antibodies(ANAs)are crucial in diagnosing autoimmune diseases,mainly systemic lupus erythematosus(SLE).This study aimed to compare the performance of chemiluminescence assay(CLIA)and line immunoassay(LIA)in detecting ANAs in patients with autoimmune diseases,evaluate their diagnostic accuracy for SLE,and develop a novel diagnostic model using CLIA-detected antibodies for SLE.Specimens from patients with autoimmune diseases and physical examination specimens were collected to parallel detect specific antibodies.Individual antibodies'diagnostic performance and a model combining multiple antibodies were assessed.The findings provide valuable insights into improving the diagnosis of SLE through innovative approaches.AIM To compare the performance of CLIA and LIA in detecting ANAs in patients with autoimmune diseases,assess their accuracy for SLE,and develop a novel diagnostic model using CLIA-detected antibodies for SLE.METHODS Specimens have been obtained from 270 patients with clinically diagnosed autoimmune disorders,as well as 130 physical examination specimens.After that,parallel detection of anti-double-stranded DNA(dsDNA)antibody,anti-histone(Histone)antibody,anti-nucleosome(Nuc)antibody,anti-Smith(Sm)antibody,anti-ribosomal P protein(Rib-P)antibody,anti-sicca syndrome A(Ro60)antibody,anti-sicca syndrome A(Ro52)antibody,anti-sicca syndrome(SSB)antibody,anticentromere protein B(Cenp-B)antibody,anti-DNA topoisomerase 1(Scl-70)antibody,anti-histidyl tRNA synthetase(Jo-1)antibody,and anti-mitochondrial M2(AMA-M2)antibody was performed using CLIA and LIA.The detection rates,compliance rates,and diagnostic performance for SLE were compared between the two methodologies,followed by developing a novel diagnostic model for SLE.RESULTS CLIA and LIA exhibited essentially comparable detection rates for anti-dsDNA antibody,anti-Histone antibody,anti-Nuc antibody,anti-Sm antibody,anti-Rib-P antibody,anti-Ro60 antibody,anti-Ro52 antibody,anti-SSB antibody,anti-Cenp-B antibody,anti-DNAScl-70 antibody,anti-Jo-1 antibody and anti-AMA-M2 antibody(P>0.05).The two methods displayed identical results for the detection of anti-dsDNA antibody,anti-Histone antibody,anti-Nuc antibody,anti-Sm antibody,anti-Ro60 antibody,anti-Ro52 antibody,anti-SSB antibody,anti-Cenp-B antibody,anti-Scl-70 antibody,and anti-AMA-M2 antibody(Kappa>0.7,P<0.05),but showed a moderate agreement for the detection of anti-Rib-P antibody and anti-Jo-1 antibody(Kappa=0.671 and 0.665;P<0.05).In addition,the diagnostic performance of these antibodies detected by both methods was similar for SLE.The diagnostic model's area under the curve values,sensitivity,and specificity,including an anti-dsDNA antibody and an anti-Ro60 antibody detected by CLIA,were 0.997,0.962,and 0.978,respectively.These values were higher than the diagnostic performance of individual antibodies.CONCLUSION CLIA and LIA demonstrated excellent overall consistency in detecting ANA profiles.A diagnostic model based on CLIA-detected antibodies can successfully contribute to developing a novel technique for detecting SLE.

Positive autoantibodies in living liver donors

BACKGROUND There is a nationwide shortage of organs available for liver transplantation.Living donors help meet this growing demand.Not uncommonly,donors will have positive autoantibodies.However,it is unclear whether donor positive autoantibodies are correlated with worse outcomes following living liver donor transplantations.AIM To analyze the significance of positive autoantibodies in donors on post-transplant outcomes in recipients.METHODS We performed a retrospective review of living liver donors who had undergone liver transplantation between January 1,2012 and August 31,2021.Demographic characteristics and pre-transplant data including antinuclear antibodies(ANA)and anti-smooth muscle antibody titers were collected in donors.Outcomes of interest were post-transplantation complications including mortality,biliary strictures,biliary leaks,infection,and rejection.Pediatric recipients and donors without measured pre-transplant autoantibody serologies were excluded from this study.RESULTS 172 living donor liver transplantations were performed during the study period,of which 115 patients met inclusion criteria.37(32%)living donors were autoantibody-positive with a median ANA titer of 1:160(range 1:80 to 1:1280)and median anti-SMA titer of 1:40(range 1:20 to 1:160).There were no significant differences in baseline demographics between the autoantibody positive and negative donors.Post-transplantation rates of death(P value=1),infections(P value=0.66),and overall rates of complications(P value=0.52)were similar between the autoantibody positive and negative groups.Higher incidences of anastomotic strictures and rejection were observed in the autoantibody positive group;however,these differences were not statistically significant(P value=0.07 and P value=0.30 respectively).CONCLUSION Isolated pre-transplant autoantibody positivity is not correlated to worse post-transplant outcomes in living liver donor transplants.

Wilson disease-the impact of hyperimmunity on disease activity:A case report

BACKGROUND In Wilson disease lack of biliary copper excretion causes hepatocellular injury by accumulation of free toxic copper.Its overspill to serum accounts for neuronal damage as second common manifestation.Therapy with copper chelators or zinc targets the removal of this free copper.However,in some patients liver disease persists for unknown reason despite normalized free copper.The discovery of a hyperimmunity as a contributing pathogenetic factor was discovered in this case report with implication also for other liver diseases.CASE SUMMARY A 9-year-old girl was diagnosed in August 2009 by family screening of having asymptomatic Wilson disease with elevated transaminases.Already at time of diagnosis antinuclear antibodies(ANA)were elevated without hyperimmunoglobulinemia(immunoglobulin G,IgG).After one year of therapy with Dpenicillamine transaminases normalized together with free serum copper.Under continuous therapy with copper chelators free copper remained normal until today,whereas transaminases raised to alanine aminotransferase values of 571 U/L in December 2019.For hyperimmunity a tentative steroid course on top of Dpenicillamine improved transaminases.Thus,hyperimmunity may have impact on liver inflammation after control of the metabolic disturbance.A retrospective cohort study confirmed the common association of elevated transaminases with ANA,but no IgG elevation.CONCLUSION This hyperimmune-triggered condition may represent a new entity which per se or on top of other liver diseases induces liver inflammation responsive to steroids.

Is autoimmune hepatitis a frequent finding among HCV patients with intense interface hepatitis?

AIM:To evaluate the overlap of autoimmune hepatitis in hepatitis C virus(HCV)-infected patients with intense interface hepatitis.METHODS:Among 1759 patients with hepatitis C submitted to liver biopsy,92(5.2%) presented intense interface hepatitis.These patients were evaluated regarding the presence of antinuclear antibody(ANA),anti-smooth muscle antibody(SMA) and anti-liver/kidney microsomal antibody(LKM-1),levels of γ-globulin and histological findings related to autoimmune hepatitis(plasma cell infiltrate and presence of rosettes).RESULTS:Among patients with hepatitis C and intense interface hepatitis there was a low prevalence of autoantibodies(ANA=12%,SMA=5%,LKM-1=0%) and the median γ-globulin level was within the normal range.Typical histological findings of autoimmune disease were observed in only two cases(2%).After applying the score for diagnosis of autoimmune hepatitis,only one patient was classified with a definitive diagnosis of autoimmune hepatitis.Since overlap with autoimmune hepatitis was not the explanation for the intense necroinflammatory activity in patients with chronic hepatitis C we sought to identify the variables associated with this finding.The presence of intense interface hepatitis was associated with more advanced age,both at the time of infection and at the time of the biopsy,and higher prevalence of blood transfusion and alcohol abuse.CONCLUSION:Although possible,overlap with autoimmune hepatitis is a very rare association in HCV-infected patients with intense interface hepatitis,an unusual presentation which seems to be related to other host variables.

Clinical evaluation of serum antimitochondrial antibody-negative primary biliary cirrhosis

BACKGROUND: Primary biliary cirrhosis (PBC) is cha- racterized by frequent presence of antimitochondrial anti- bodies (AMAs). The sensitivity and specificity of AMA for PBC are both greater than 90%-95%, so the presence of AMA in serum is the major hallmark in PBC. However, it has long been recognized that in 5%-10% of patients the clinical, biochemical and histological features are diagnos- tic for PBC, but their sera are consistently tested negative for AMA/AMA-M2. This study aimed to evaluate whether the presence of AMA alters the clinical, serological and his- tological features of the disease. METHODS: Clinical data of 70 patients clinically and/or histologically diagnosed with PBC were reviewed. AMA- negative and AMA-positive patients were compared in terms of clinical, biochemical, immunological and histo- logical features. RESULTS: At presentation, 11 patients were serum AMA/ AMA-M2 negative. At the initial visit, AMA-negative and AMA-positive patients were similar in terms of age, sex, clinical manifestations, liver biochemistries and histological findings. The mean level of serum immunoglobulin M (IgM) was significantly lower in AMA-negative PBC pa- tients than in AMA-positive PBC patients (2851±1418 mg/L vs 6361 ±4928 mg/L, P =0.033). Serum antinuclear anti- bodies ( ANA) and/or smooth muscle antibodies ( SMA) were more frequently positive in the AMA-negative PBC patients than in the AMA-positive patients (81.8% vs 40.7%, P =0.031). CONCLUSION: AMA-negative PBC patients are characte- rized by relatively lower levels of serum IgM and a higher prevalence of serum ANA/SMA and are not associated with substantial differences in the clinical, biochemical and histo- logical spectrum of the disease.

Recalcitrant paradoxical pustular psoriasis induced by infliximab:Two case reports

BACKGROUND Paradoxical psoriasis induced by tumor necrosis factor alpha antagonists is a rare side effect of those drugs and has similarities with and differences from classical psoriasis in clinical and pathological characteristics.Treating severe paradoxical psoriasis is challenging because the reported cases are rare,with treatment experience being only anecdotal.CASE SUMMARY We report 2 cases of paradoxical psoriasis caused by infliximab.Both cases manifested with a significant number of pustular lesions and had protracted and complicated clinical courses.In case 1,secukinumab alone could not control the eruptions,but colchicine supplementation markedly decreased disease activity.In case 2 miscellaneous medications were administered,including the systemic drug acitretin,the immunosuppressive drug cyclosporine,and the biologic agent ustekinumab.However,multiple applications of those medications failed to prevent new lesions from occurring.Both cases showed moderate-to-high antinuclear antibody titers.CONCLUSION Based on these cases,moderate-to-high anti-nuclear antibody titer seems to be a risk factor for paradoxical psoriasis.In addition,extensive pustular presentation may be a negative prognostic indicator and may portend a protracted clinical course refractory to therapy.

Transient positive antimitochondrial M2 in sera of patients with connective tissue diseases after intravenous immunoglobulin infusions

Background:Although antinuclear antibodies(ANAs),anti‐SSA and anti‐Ro52,are present in immunoglobulin preparations,it is unknown whether intravenous immunoglobulin(IVIG)therapy influences the testing of serum autoantibodies in patients with connective tissue diseases(CTDs).The present study aimed to investigate the dynamic change over time of serum ANA‐related autoantibodies in patients with CTDs receiving IVIG therapy.Methods:Serum ANA‐related autoantibodies were monitored in two patients with CTD before IVIG therapy and at different times after therapy.These autoantibodies were tested in different batches of immunoglobulin preparations from seven pharmaceutical companies.Results:One patient developed a new ANA pattern(cytoplasmic dense fine speckled pattern,AC‐19)just after IVIG therapy.Both patients developed de novo positivity for AMA‐M2 and anti‐SSA,but returned negative 1 month after IVIG therapy.The residual liquid in patients'immunoglobulin preparations showed positive ANAs with a high titer of AC‐19(1:640),a low titer of the nuclear fine speckled pattern(AC‐4,1:80),positive AMA‐M2,and positive anti‐SSA.ANA‐related autoantibodies were tested in 16 batches of immunoglobulin preparations and all had positive ANAs with two patterns:AC‐19(1:640 or 1:320)and AC‐4(1:80).AMA‐M2 and anti‐SSA were positive in 100%of the batches.Conclusion:Our study highlights high‐titer AMA‐M2 autoantibodies in immunoglobulin preparations and suggests their transient transfer into a patient's circulation via IVIG therapy.To avoid incorrect clinical decisions based on postinfusion antibody titers,our data recommend retesting 1–2 months after high‐dose IVIG immunomodulatory treatment.

Induction of lupus-like renal damages by double stranded DNA derived from Trypanosoma equiperdum

Lupus nephritis is the most common visceral complication in the patients with systemic lupus erythematosus （SLE）.It was evident that the anti-dsDNA antibodies were closely related to lupus nephritis, as seen in patients who had higher titers of serum anti-dsDNA antibodies had more severe renal lesions and even worse prognosis. So far it is still unknown how the dsDNA or anti-dsDNA antibody plays a role in the pathogenesis of lupus nephritis. The Trypanosoma equiperdum （TE） has uniformed dsDNA, no histone is found in both the cell nucleus and kinetonucleus. For this reason, TE became an optimal substrate used for detecting anti-dsDNA antibodies in SLE patients. It is proved that TE substrate is highly sensitive and specific. This reminds us to concern whether TE dsDNA share same SLE antigenic determinants with the pathogenic dsDNA in patients. Compared to the mammalian dsDNA, the kinetoplast DNA （kDNA） of TE has simpler molecular structure which makes it easier for purification. It offers us the possibility to establish lupus-like nephritis model by TE kDNA. We subcutaneously injected TE kDNA into normal mice. The result indicated lupus-like nephritis has been successfully induced by this simple and convenient protocol, which is useful to elucidate the particular role of dsDNA or anti-dsDNA antibody in lupus nephritis.