Anti-extractable Nuclear Antigen Research Articles

An Italian Multicenter Study for Application of a Diagnostic Algorithm in Autoantibody Testing

The presence in the serum of specific autoantibodies, such as antinuclear antibodies (ANA), anti-double-stranded DNA (anti-dsDNA), and antiextractable nuclear antigens (anti-ENA), is one of the diagnostic criteria for autoimmune rheumatic disease, and the requests for these tests in the last few years have grown remarkably. A guideline for reducing clinically inappropriate requests in autoantibody testing (ANA, anti-dsDNA, anti-ENA) has been applied in the Parma Hospital since 2007. The results for the period January-December 2007 were compared to those of the previous period January-December 2006, and a significant reduction in the number of anti-dsDNA (23.9%) and anti-ENA (20.7%) was found. The aim of this study was to assess the applicability of a similar guideline in a wide area (Parma, Modena, Piacenza, Reggio-Emilia) with reference to the diagnosis of autoimmune rheumatic disease. This project, supported by a regional grant for innovative research projects, was started in January 2008 and consists of three different steps: (1) a study group of clinicians and laboratory physicians to evaluate the diagnostic criteria, the analytical procedures, and the number of tests performed in different hospitals; (2) developing common guidelines for autoantibody testing that takes into account the different clinical needs with the aim of improving efficiency and clinical effectiveness of diagnosis and monitoring; and (3) assessing compliance with the guidelines in the different hospitals that are evaluating the second-level test (anti-dsDNA, anti-ENA) decrease. We think that the validation of guidelines for the laboratory diagnosis of autoimmune rheumatic disease can represent a tool for improving patients' outcomes and economic efficiency.

Concentration of antibodies to extractable nuclear antigens and disease activity in systemic lupus erythematosus

A hallmark of systemic lupus erythematosus (SLE) is the production of autoantibodies directed against intracellular antigens. Antibodies to double stranded DNA (dsDNA) are most closely associated with the clinical manifestations of the condition and appear to have a direct role in pathogenesis. On the contrary, the relationship between disease activity in SLE and anti-extractable nuclear antigen (ENA) antibodies has not been well demonstrated. Despite this, commercial assays for the quantification of anti-ENA antibodies are now widely available, although their usefulness in clinical practice is not known. The aim of this study was to investigate whether there is an association between disease activity in SLE and concentrations of individual anti-ENA antibodies. A prospective 2-year study of 45 patients with SLE, known to be positive for at least one anti-ENA antibody, was performed. Disease activity was assessed using the SLE Disease Activity Index (SLEDAI). Anti-ENA antibodies were quantified using a commercial antibody detection system. A total of 45 patients were studied over a 2-year period (median number of assessments 5, range 2-9). Of them 29 patients were positive for Ro, 8 for La, 9 for Sm and 27 for RNP antibodies. In the population as a whole, there was a weak relationship between peak SLEDAI score and anti-Sm concentration (r = 0.57, NS), but no relation with the other anti-ENA antibodies. In a small number of patients, there appeared to be either a positive (Ro, Sm) or negative (La, Sm, RNP) association between ENA antibody concentration and disease activity over time; however, this was not apparent for the majority of individuals. These results show that in SLE, clinically significant changes in disease activity do not correlate well with concentrations of anti-ENA antibodies, either within the population as a whole or on an individual basis. Repeated quantitative measurement of anti-ENA antibodies does not therefore appear to provide useful additional information in assessing disease activity in SLE. The widespread application of commercial quantitative assays to routine clinical practice is not recommended.

Letters to the Editor

Letters to the Editor

Clinicopathological evaluation ofin vivoepidermal nuclear fluorescence

Nuclear fluorescence in keratinocytes is an occasional phenomenon, often present in autoimmune diseases, especially in connective-tissue disease (CTD); however, its clinical significance remains unclear. To investigate the profile of patients with positive nuclear staining on direct immunofluorescence (DIF) of skin samples. A retrospective analysis of 28 patient records from our immunodermatology laboratory was performed between May 2003 and June 2006. Inclusion criteria were the presence of autoantibodies (IgG, IgA or IgM) or complement (C3) binding keratinocyte nuclei on DIF. The most prevalent diseases related to the nuclear keratinocyte DIF staining were systemic lupus erythematosus (n = 9), mixed CTD (n = 3), overlap syndrome (n = 3), Sjögren's syndrome (n = 1), and CREST (calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia) syndrome (n = 1). Serum antinuclear antibody (ANA) was positive in 20 of 28 patients, with titres varying from 1 : 160 to 1 : 1280. Of the 20 patients with positive anti-nuclear antibodies (ANA), 17 were positive for anti-extractable nuclear antigen antibodies, 12 had anti-SSA/Ro, 11 had anti-SSB/La and 8 had anti-ribonucleoprotein. Eight patients were negative for ANA. Positive predictive value of in vivo ANA for systemic CTDs was 75%. The present data suggest that in vivo ANA evaluation is an additional and feasible auxiliary tool for diagnosing CTDs.

Autoantibody Production during Chronic Graft-Versus-Host Disease Does Not Associate with Long-Term Persistence of Host B Cells in Humans

Background. Chronic graft-versus-host disease (cGvHD) is a common complication of allogeneic hemopoietic cell transplantation (allo-HCT). The pathogenesis of cGvHD is poorly understood. In cGvHD, the homeostasis of B lymphocytes is perturbed, as demonstrated by the production of autoantibodies. B-cell depletion with monoclonal antibodies (mAb) interferes with autoantibody production and ameliorates signs and symptoms of cGvHD. In mouse models, cGvHD and autoantibodies associate with the long-term persistence of host B cells after allo-HCT (Sylvain Perruche et al., Transplantation 2006). It has been postulated that host B cells may present alloantigens to donor T cells and, in turn, receive help for autoantibody production. This could be crucial to the pathogenesis of cGvHD. Aim. To investigate whether the long-term persistence of host B lymphocytes is associated with cGvHD and autoantibodies in humans.Patients and methods. We recruited 13 consecutive patients with active cGvHD (4 mild, 5 moderate, 4 severe according to NIH classification) with a median time of onset of 6 months (range 3–36) from HLA-identical sibling (9 patients) and HLA-matched unrelated (4) allo-HCT. As controls, we chose 10 patients that underwent HLAidentical sibling (2), HLA-matched unrelated (5) or haploidentical (3) allo-HCT and never experienced cGvHD. In the two groups, we studied:circulating autoantibodies, including anti-nuclear (ANA), anti-DNA, anti-extractable nuclear antigen, anti-beta2 glycoprotein, anti-neutrophil cytoplasm, anti-thyroid, anti-mytocondria antibodies, rheumatoid factor,absolute numbers of T (CD3+, CD4+, CD8+), conventional B (CD19+), B1 (CD5+/CD19+) and NK cells (CD16+/CD56+) in the graft and in the peripheral blood,microchimerism by short-tandem repeats (STR) on B, T and myeloid cells purified by immunomagnetic cell sorting (sensitivity 0,01%).Results. Patients with cGvHD had high-titer circulating ANA (>1:160) more frequently than controls (54% versus 10%, P<0,05). All other autoantibodies were negative. Peripheral T-cell counts were lower in patients with cGvHD than in controls (for CD8+ cells P<0,05). This was not due to a difference in the absolute numbers of T lymphocytes within the graft between the two groups. Peripheral counts of conventional B and B1 cells in patients with cGvHD were similar to controls. Autoantibodies and cGvHD were not associated with the persistence of host B lymphocytes, since the analysis of STR on purified B cells revealed that they were all of donor origin. T and myeloid cells were also of donor origin. Of interest, in univariate analysis, in vivo B-cell depletion with mAb for the prophylaxis against Epstein-Barr virus-related lymphoproliferative disease showed a trend towards a lower risk of cGvHD (P=0,06).Conclusions. This study indicates that autoantibody production during cGvHD does not associate with long-term persistence of host B cells in humans. Moreover, it suggests that the early depletion of donor B lymphocytes in vivo may be effective for GvHD prophylaxis

Drug-induced lupus erythematosus

Drug-induced lupus erythematosus (DILE) is defined as a lupus-like syndrome temporally related to continuous drug exposure which resolves after discontinuation of the offending drug. There are currently no standard diagnostic criteria for DILE and the pathomechanisms are still unclear. Similarly to idiopathic lupus, DILE can be diveded into systemic (SLE), subacute cutaneous (SCLE) and chronic cutaneous lupus (CCLE). Systemic DILE is characterized by typical lupus-like symptoms including skin signs, usually mild systemic involvement and a typical laboratory profile with positive antinuclear and anti-histone antibodies, while anti-double strand (ds) DNA and anti-extractable nuclear antigens antibodies are rare. High risk drugs include hydralazine, procainamide and isoniazid. Drug-induced SCLE is very similar to idiopathic SCLE in terms of clinical and serologic characteristic, and it is more common than the systemic form of DILE. Drugs associated with SCLE include calcium channel blockers, angiotensin-converting enzyme inhibitors, interferons, thiazide diuretics and terbinafine. Drug-induced CCLE is very rarely reported in the literature and usually refers to fluorouracile agents or non steroidal anti-inflammatory drugs. Recently, cases of DILE have been reported with anti-TNFalpha agents. These cases present with disparate clinical features including arthritis/arthralgia, skin rash, serositis, cytopenia and variable laboratory abnormalities. DILE to anti-TNFalpha agents differs in several ways to classic DILE. The incidence of rashes is higher compared to classical systemic DILE. In most cases of classic DILE visceral involvement is rare, whereas several cases of anti-TNFalpha DILE with evidence of renal disease have been reported. Low serum complement levels as well as anti-extractable nuclear antigen antibodies and anti-dsDNA antibodies are rarely present in classic DILE, whereas they are reported in half the cases of anti-TNFalpha DILE; in contrast, anti-histone antibodies are described in classic DILE more often than in anti-TNFalpha DILE. Recognition of DILE in patients receiving anti-TNFalpha therapy can be difficult due to the symptoms of their underlying disease. A temporal association (months to years) of the offending drug with characteristic or suggestive symptoms, and resolution of symptoms on drug withdrawal is the best evidence for this diagnosis of DILE.

Autoimmune response following influenza vaccination

The aim of this study was to assess autoimmune response following annual influenza vaccination in apparently healthy adults, staff at a children's hospital. 92 healthy adult subjects were tested for autoantibodies including antinuclear antibodies (ANA), anti-extractable nuclear antigen antibodies (anti-ENA), antiphospholipid antibodies (aPL), namely anticardiolipin antibodies (aCL), anti-beta2-glycoprotein I antibodies (aβ2-GPI) and lupus anticoagulant (LA). Blood samples were taken from each participant before annual influenza vaccination, one month and six months after vaccination. Before influenza vaccination 26% of participants were positive for ANA, 1% for anti-ENA, 16% for aCL, 7% for aβ2-GPI and 2% for LA. One month after influenza vaccination 76% of participants showed no change in autoantibodies titres. Six months after influenza vaccination 74% of participants showed no change in autoantibodies titres. Overall, there was no statistically significant difference in the percentage of positive ANA, aCL, aβ2-GPI and LA before and 6 months after the vaccination. Five participants developed autoantibodies 6 months after the vaccination and one who was initially low positive for ANA became highly positive (1:320). Eleven participants had only transiently increased autoantibodies. Persistently positive or progressively increased levels of autoantibodies during 6 months' follow up were observed in 6 persons (7%). Our study showed a high percentage of positive autoantibody testing among healthy adult staff at a children's hospital. There was no statistically significant difference in the percentage of positive autoantibodies before and after influenza vaccination. However, our study clearly demonstrated induction of autoantibodies production in selected subjects.

Prevalence and clinical associations of anti-Ku antibodies in patients with systemic sclerosis: a European EUSTAR-initiated multi-centre case–control study

Objectives:To determine the prevalence of anti-Ku antibodies in 625 patients with systemic sclerosis (SSc) from six European rheumatological centres and to evaluate their clinical and serological characteristics.Methods:Sera of 625 consecutive...

Carbimazole-induced juvenile dermatomyositis

E‐mail: stephflocohenbacrie@free.fr Conflicts of interest: none declared. Sir, Juvenile dermatomyositis (JD) is an inflammatory multisystem disease affecting skin and muscle, causing symmetric proximal weakness and characteristic skin rash. Drug‐related dermatomyositis has been reported rarely in adults but not in children.1, 2 This is the first report of JD induced by carbimazole. A 10‐year‐old girl was referred to the dermatology department at CHU Rangueil for an 11‐month history of skin lesions, asthenia and arthralgia. Past medical history included a diagnosis of Graves’ disease. She was treated with carbimazole and hyperthyroidism regressed after approximately 5 months. Three months after starting carbimazole, the patient developed arthralgia and asthenia. Three months later, skin lesions were noted. Five months later, she was referred to our department. Skin examination revealed symmetric erythematous and slightly scaly plaques on knees, elbows and medial malleolus. There were firm subcutaneous nodules on the extension aspect of both arms. She also had an eyelid erythema (Fig. 1a) and erythematous papules on the dorsal aspect of finger joints (Fig. 1b). She had symmetrical inflammatory pain of large and small joints, and weakness of the muscles without muscle pain. Rheumatological examination revealed stiffness of the knees and left elbow associated with flessum. Her fingers were swollen with a spindle aspect (Fig. 1b). Muscle strength testing showed a symmetric proximal weakness. She had high creatine kinase level of 230 U L−1 (0–140) and high aldolase level of 16 U L−1 (0–10). The antinuclear antibody titre was 1/320, without anti‐DNA antibody or antiextractable nuclear antigens. Complement, thyroid stimulating hormone and T4 were within normal range.

Prevalence of undiagnosed autoimmune rheumatic diseases in the first trimester of pregnancy. Results of a two‐steps strategy using a self‐administered questionnaire and autoantibody testing

To evaluate the prevalence of undiagnosed rheumatic diseases in the first trimester of pregnancy. We screened for rheumatic diseases in 1210 consecutive pregnant women during the first trimester of pregnancy using a 10-item questionnaire. A university hospital in northern Italy. One hundred and thirty-seven (11.3%) women who answered positively to at least one question constituted the cases and were compared with 107 negative controls. Cases and controls were tested for rheumatic autoantibodies (antinuclear antibody, anti-double-stranded DNA, anti-extractable nuclear antigen, anticardiolipin antibody, anti-beta2-glycoprotein I antibodies and lupus anticoagulant) and were evaluated by a rheumatologist for a definite diagnosis of rheumatic disease. Prevalence of undiagnosed rheumatic disease in the first trimester of pregnancy. The overall rate of positivity to the antibodies tested was 43.1% (59/137) among cases and 9.3% (10/107) in the controls (P < 0.001). A definitive diagnosis of rheumatic disease was made in 35 cases (25.5%) and in none of the controls (P <0.001). In stepwise logistic regression analysis, photosensitivity (adjusted OR 5.72; 95% CI 2.38-13.8), erythema or malar rash (adjusted OR 3.91; 95% CI 1.53-10) and history of two or more miscarriages (adjusted OR 5.6; 95% CI 1.55-20.6) were independent predictors of a definitive diagnosis of rheumatic disease (area under receiving operator curve = 0.814; 95% CI 0.76-0.86). Birthweight was lower (3180 g +/- 475 compared with 3340 g +/- 452, P= 0.008), and overall serious pregnancy complications (miscarriage, fetal growth restriction, delivery before 34 weeks of pregnancy and severe pre-eclampsia) were higher among cases (12/137) than controls (2/107) (adjusted OR 5.60; 95% CI 1.29-24.3; P= 0.021). A two-step screening process with a self-administered questionnaire proved to be a useful method to screen for undiagnosed rheumatic diseases during the first trimester of pregnancy.

Natural History and Clinical Impact of Cryoglobulins in Chronic Hepatitis C: 10-Year Prospective Study of 343 Patients

Serum cryoglobulins (CGs) are present in patients with chronic hepatitis C virus (HCV) infection, but their long-term clinical importance has not been established. We assessed the development rates, morbidity, and influence on the evolutionary course of hepatitis C of CG. A cohort of 343 HCV-RNA seropositive outpatients (173 men; age, 58 y; 82 with cirrhosis; 61 treated with interferon) with persistently increased aminotransferase levels and histologically defined liver disease was investigated. Patients initially were investigated for the presence, amount, and type of CG and prospectively followed up with clinical and laboratory examinations every 6 months. At enrollment, CGs were found in 163 (47%) patients at a mean level of 173 +/- 142 mg/L; 80% were type III, and associated to female sex (61% vs 40%, P = .0002) and cirrhosis (29% vs 19%, P = .04). Over the course of 17-130 months (median, 116 mo), de novo CG developed in 25 patients (2.3% per year), including 5 with cryoglobulinemic syndrome (.3% per year). The 10-year rates of progression to cirrhosis and of liver and extrahepatic complications were similar in CG (+) and CG (-) patients (32% vs 34%; 23% vs 16%; 5% vs 3%). The 10-year survival rates were lower for cirrhotic than for noncirrhotic patients (57% vs 91%, P < .00001), independently of CGs. CGs are common in patients with chronic HCV infection, mainly are type III, and do not influence the clinical course of hepatitis C during the first decades, except for the few rare cases of cryoglobulinemic syndrome.

Specific testing for “isolated” anti-52 kDa SSA/Ro antibodies during standard anti-extractable nuclear antigen testing is of limited clinical value

Aim: To ascertain whether specific testing for “isolated” anti-52 kDa SSA/Ro antibodies (a-SSA/Ro52) during standard anti-extractable nuclear antigen (ENA) testing is clinically useful. Methods: 1438 consecutive sera submitted for anti-ENA...

Outcome of infants from mothers with anti-SSA/Ro antibodies

To evaluate the incidence of electrocardiographic and laboratory abnormalities in neonates born from mothers with connective tissue disease and positive for anti-SSA/Ro antibodies. Electrocardiogram, blood cell counts, liver and renal function tests prospectively obtained from 51 infants born from anti-SSA/Ro-positive mothers with connective tissue disease were compared with those obtained from 50 control infants born from mothers with anti-extractable nuclear antigen (ENA)-negative connective tissue disease. One infant with congenital complete heart block was excluded from analysis. No infant showed sinus bradycardia. A first-degree atrioventricular block at birth was observed in five study group and no control group infants, P=0.023. Atrioventricular blocks spontaneously reverted or remained stable during the first year of life. Mean corrected QT value of infants born from anti-SSA/Ro-positive mothers was slightly prolonged as compared with the control group (0.404+/-0.03 s vs 0.395+/-0.02 s; P=0.060). Infants exposed to anti-SSA/Ro antibodies had a significantly higher prevalence of first-degree atrioventricular block. At variance with previous studies, we observed a low frequency of hematologic abnormalities and no cases of hepatobiliary disease.

Padrões de imunofluorescência do fator antinuclear (FAN) em células HEp-2 de soros reagentes para anti-SSA/Ro

OBJECTIVE: to evaluate the pattern at immunofluorescence of the antinuclear antibodies (ANA) detected by the indirect immunofluorescence (IIF) technique in positive samples for anti-SSA/Ro autoantibody and the clinical associations. METHODS: a retrospective transversal study was performed in a period of two years where the all the solicitations of testing for the presence of anti-extractable nuclear antigen (anti-ENA) antibodies delivered to the SPC/HCPA were analyzed. We selected the positive samples and identified which autoantibodies were involved (anti-SSA/RO, anti-SSB/La, anti-RNP, anti-Sm and anti-Scl70) as well as the immunofluorescence patterns by ANA testing and the clinical associations found in the patients presenting anti-SSA/Ro positive serum. IIF was used for ANA using HEp-2 cells and hemagglutination for anti-ENA antibodies detection. RESULTS: 90 out of the 392 solicitations analyzed were anti-ENA positive, with a predominance of women (86/91 - 94%) and the mean age was 42 years old. The most frequent autoantibody was anti-SSA/Ro (61/90 - 67.8%) and all samples that were anti-SSA/Ro positive were also ANA positive. Speckled nuclear immunofluorescence was the most frequent ANA pattern (42/61 - 68.9%) among the anti-SSA/Ro positive samples and systemic lupus erythematosus was the most common clinical diagnosis (31/61 - 50.8%). CONCLUSION: ANA testing by IIF using HEp-2 cells proved to be a good screening test for the detection of anti-SSA/Ro antibodies, that showed a strong positive association to the speckled nuclear IIF pattern. As opposed to what has been described in the literature, there was no ANA negative among the anti-SSA/Ro positive samples. At least in our experience, these data question the cost-effectiveness of performing routine screening for anti-SSA/Ro antibodies in ANA negative samples by IIF testing.

Serological and Clinical Comparison of Children and Adults with Anti-Endomysial Antibodies

We compared serological and clinical presentation of 38 adults (5 males, 33 females) and 37 children (15 boys, 22 girls) with anti-endomysial antibodies (AEA).AEA, antinuclear (ANA), anti-parietal (APA), anti-thyroid microsomal (ATMA), anti-thyreoglobulin (ATGA), anti-smooth muscle (SMA) and anti-mitochondrial (AMA) antibodies were detected by IIF. Anti-tissue transglutaminase (tTG), anti-extractable nuclear antigens (ENA) and anti-actin (AAA) antibodies were studied by ELISA. There were no differences in frequency of ANA, APA, ATGA, SMA, AMA and AAA in children and adults. ATMA (p < 0.001) and anti-ENA (p < 0.05) positivity were more frequently found in adults. Anti-Ro/SSA had 7/38 adults and 1/37 children (p < 0.05). Adults had more frequently silent form of celiac disease associated with autoimmune diseases (p < 0.001). We are the first to demonstrate that in spite of no difference in ANA positivity in adults and children, ANA in adults more frequently target ENA, especially Ro/SSA antigen. The reason for this ANA specificity could be the longer gluten exposure in adults.

Diagnostic accuracy of the FIDIS multiplex fluorescent microsphere immunodetection system for anti-extractable nuclear antigen (ENA) antibodies in connective tissue diseases

Anti-extractable nuclear antigen antibodies (ENA) are markers of connective tissue diseases (CTDs). We compared FIDIS reagents in the multiplex fluorescent microsphere immunodetection system to INNO-LIA and immunodiffusion for 174 antinuclear antibody-positive patients, 102 with well-defined CTDs and 72 disease controls. No significant differences were found in sensitivity or specificity between FIDIS and immunodiffusion, or between FIDIS and INNO-LIA for all anti-ENA in all CTD patients; nor were any differences found for individual anti-ENAs within distinct CTDs. The FIDIS sensitivity was 41% (anti-SSA) and 17% (anti-SSB) in lupus erythematosus (LE) or primary Sjögren's syndrome; 5% (anti-ribosome and anti-Sm) in LE; 17% (anti-RNP) in LE or mixed CTD; 21% (anti-Scl70) in systemic sclerosis; and 61% (anti-centromere) in limited systemic sclerosis. The specificity reached 88%-100%. Receiver operating characteristic curve areas did not differ between FIDIS and INNO-LIA. Agreement ranged from 91% (anti-SSB) to 99% (anti-Jo1) between FIDIS and INNO-LIA, and from 95% (anti-Scl70) to 100% (anti-Sm) between FIDIS and immunodiffusion. Samples scored positive with all techniques in 83% (anti-centromere), 70% (anti-RNP), 67% (anti-Jo1), 60% (anti-SSA), 40% (anti-SSB), 33% (anti-ribosome), 25% (anti-Sm) and 13% (anti-Scl70) of cases. The diagnostic performance of FIDIS anti-ENA reagents is comparable to immunodiffusion and INNO-LIA.

Application of a combined protocol for rational request and utilization of antibody assays improves clinical diagnostic efficacy in autoimmune rheumatic disease.

Abstract Context.—Because of a marked increase in the number of requests for antinuclear antibodies, anti–extractable nuclear antigen antibodies, and anti–double-stranded DNA antibodies for the diagnosis of autoimmune rheumatic disease, guidelines have been proposed for their appropriate use. Objective.—To evaluate in terms of clinical efficacy and cost-benefit ratio the outcome of applying a protocol for the diagnosis of autoimmune rheumatic disease. Design.—A diagnostic protocol for the rational utilization of second-level tests (anti–extractable nuclear antigen antibodies and anti–double-stranded DNA antibodies) was applied at Hospital Polyclinic beginning January 2004. The appropriateness of 685 consecutive requests received at the clinical pathology laboratory from January to June 2004 was assessed. Patients who underwent these laboratory tests were followed up for 12 months after blood sample drawing. Results.—Introduction of the protocol led to a significant reduction in the number of second-level ...

Application of a Combined Protocol for Rational Request and Utilization of Antibody Assays Improves Clinical Diagnostic Efficacy in Autoimmune Rheumatic Disease

Because of a marked increase in the number of requests for antinuclear antibodies, anti-extractable nuclear antigen antibodies, and anti-double-stranded DNA antibodies for the diagnosis of autoimmune rheumatic disease, guidelines have been proposed for their appropriate use. To evaluate in terms of clinical efficacy and cost-benefit ratio the outcome of applying a protocol for the diagnosis of autoimmune rheumatic disease. A diagnostic protocol for the rational utilization of second-level tests (anti-extractable nuclear antigen antibodies and anti-double-stranded DNA antibodies) was applied at Hospital Polyclinic beginning January 2004. The appropriateness of 685 consecutive requests received at the clinical pathology laboratory from January to June 2004 was assessed. Patients who underwent these laboratory tests were followed up for 12 months after blood sample drawing. Introduction of the protocol led to a significant reduction in the number of second-level tests prescribed (27.9% vs 49.5% for anti-extractable nuclear antigen antibodies; 27.5% vs 56.6% for anti-double-stranded DNA antibodies). After the period of observation, none of the 163 patients who had negative results on the first-level test and were asymptomatic, for whom second-level tests had not therefore been performed, were found to have autoimmune rheumatic disease. In 90.5% (77/85) of patients positive for the second-level tests, clinical confirmation of autoimmune rheumatic disease was obtained. Not only did application of the diagnostic protocol reduce the number of second-level tests performed but it also increased their specificity. Our data thus indicate that the use of shared guidelines by clinical and laboratory specialists yields satisfactory results.

B cell depletion therapy in systemic lupus erythematosus: Effect on autoantibody and antimicrobial antibody profiles

Autoantibody production in patients with systemic lupus erythematosus (SLE) is associated with abnormalities of B cell function and phenotype. Clinical responses to B cell depletion therapy (BCDT), based on rituximab, are encouraging. Therefore, we undertook this study to investigate the effect of BCDT on antibody profiles. Serial sera from 16 patients with active, refractory SLE were assayed for antinucleosome antibodies, anti-double-stranded DNA (anti-dsDNA), anti-extractable nuclear antigen, anti-tetanus toxoid, and antibodies to pneumococcal capsular polysaccharide for at least 1 year following BCDT. Anti-dsDNA antibodies derived from the V(H)4.34 immunoglobulin germ line gene (9G4+) were also measured. All patients achieved peripheral B cell depletion and improved clinically for at least 3 months. Antinucleosome and anti-dsDNA antibodies decreased to a mean +/- SD of 64 +/- 37% and 38 +/- 33% of baseline values, respectively, by 6-8 months post-BCDT. Levels of other autoantibodies and antimicrobial antibodies were generally unchanged. In the 9 of 16 patients who were still well at 1 year, anti-dsDNA antibodies fell to 42 +/- 36% of baseline values at 6-8 months and to 37 +/- 33% at 10-14 months. In patients who had disease flares within 1 year of BCDT, levels of these antibodies decreased to 60 +/- 40% and 83 +/- 93% of baseline values at 6-8 months and at 10-14 months, respectively. Circulating anti-dsDNA antibodies were positive for 9G4 expression in 4 of 6 patients tested, and flares in 2 of these patients were accompanied by rises in 9G4+ anti-dsDNA antibodies. These observations suggest that B cell clones committed to producing antinucleosome and anti-dsDNA antibodies, including the V(H)4.34 subpopulation of anti-dsDNA antibodies, have a relatively rapid turnover compared with B cell clones producing other antibodies. There was also a trend toward a greater and more sustained decrease in anti-dsDNA antibodies in patients with clinical benefit lasting >1 year.

Autoantibodies to Mitotic Apparatus: Association with Other Autoantibodies and Their Clinical Significance

The most important mitotic apparatus (MA) antigens are centrosome (CE), nuclear mitotic apparatus (NuMA-1, NuMA-2), midbody, and centromere F (CENP-F). We studied associations of anti-MA antibodies with other autoantibodies and their clinical significance. A total of 6270 patients were studied for the presence of anti-MA antibodies on HEp-2 cells. Sera positive for anti-MA were tested for anti-extractable nuclear antigens (ENA) antibodies. Anti-MA antibodies were detected in 56 (45 females and 11 males) of 6270 sera (0.9%). Of these 56, NuMA-1 was found in 23, NuMA-2 in 7, CE in 20, CENP-F in 5, and CENP-F/centrosome in 1 case. Anti-NuMA-1 were associated with anti-ENA antibodies (p < 0.001). Diagnoses were established in 43/56 patients: 22 connective tissue diseases, 7 infections, 6 autoimmune hepatitis, 3 vasculitis, 3 primary antiphospholipid syndrome, 1 malignancy, and 1 fever of unknown origin. The differential diagnosis of anti-NuMA-1-positive patients must include Sjögren's syndrome, while patients with anti-CE antibodies must be observed for HCV infection.