How to report the antinuclear antibodies (anti-cell antibodies) test on HEp-2 cells: guidelines from the ICAP initiative.

Results of the anti-nuclear antibodies-indirect immunofluorescence assay (anti-cell antibodies test) on HEp-2 cell substrates should be communicated to clinicians in a standardized way, adding value to laboratory findings and helping with critical clinical decisions. This paper proposes a test report based on the practices informed by 118 laboratories in 68 countries, with recommendations from the International Consensus on ANA Patterns (ICAP) group. Major focus is placed on the report format containing endpoint titers, immunofluorescence patterns together with anti-cell (AC) nomenclature, remarks on follow-up or reflex testing, and possible other autoantibody associations. ISO 15,189 directives were integrated into the test report. Special situations addressed include serum screening dilutions and endpoint titers, relevance of immunofluorescence patterns with special attention to cytoplasmic patterns, mixed and compound patterns, and how to report different titers corresponding to multiple patterns or autoantibodies in the same sample. This paper suggests a subtitle for the HEp-2-IIFA, namely anti-cell antibodies test, which could gradually substitute the original outdated ANA nomenclature. This ICAP pro forma report represents a further step in harmonizing the way relevant clinical information could be provided by laboratories.

Strong Association of the Myriad Discrete Speckled Nuclear Pattern With Anti-SS-A/Ro60 Antibodies: Consensus Experience of Four International Expert Centers.

Introduction: The morphological patterns in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) reflect the autoantibodies in the sample. The International Consensus on ANA Patterns (ICAP) classifies 30 relevant patterns (AC-0 to AC-29). AC-4 (fine speckled nuclear pattern) is associated to anti-SS-A/Ro, anti-SS-B/La, and several autoantibodies. Anti-SS-A/Ro samples may contain antibodies to Ro60 and Ro52. A variation of AC-4 (herein designated AC-4a), characterized by myriad discrete nuclear speckles, was reported to be associated with anti-SS-A/Ro. The plain fine speckled pattern (herein designated AC-4b) seldom was associated with anti-SS-A/Ro. This study reports the experience of four expert laboratories on AC-4a and AC-4b. Methods: Anti-Ro60 monoclonal antibody A7 was used to investigate the HEp-2 IFA pattern. Records containing concomitant HEp-2 IFA and SS-A/Ro tests from Durand Laboratory, Argentina (n = 383) and Fleury Laboratory, Brazil (n = 144,471) were analyzed for associations between HEp-2 IFA patterns and disease-associated autoantibodies (DAA): double-stranded DNA, Scl-70, nucleosome, SS-B/La, Sm, and U1-RNP. A total of 381 samples from Dresden Technical University (TU-Dresden), Germany, were assayed for HEp-2 IFA and DAA. Results: Monoclonal A7 recognized Ro60 in Western blot and immunoprecipitation, and yielded the AC-4a pattern on HEp-2 IFA. Analyses from Durand Laboratory and Fleury Laboratory yielded compatible results: AC-4a was less frequent (8.9% and 2.7%, respectively) than AC-4b (26.1% and 24.2%) in HEp-2 IFA-positive samples. Reactivity to SS-A/Ro occurred in 67.6% and 96.3% of AC-4a-pattern samples against 23% and 6.8% of AC-4b pattern samples. Reciprocally, AC-4a occurred in 24% and 47.1% of anti-SS-A/Ro-positive samples, and in 3.8% and 0.1% of anti-SS-A/Ro-negative samples. Data from TU-Dresden show that the AC-4a pattern occurred in 69% of 169 anti-SS-A/Ro-monospecific samples (62% of all anti-SS-A/Ro-positive samples) and in 4% of anti-SS-A/Ro-negative samples, whereas anti-SS-A/Ro occurred in 98.3% of AC-4a samples and in 47.9% of AC-4b samples. In all laboratories, coexistence of anti-SS-B/La, but not other DAA, in anti-SS-A/Ro-positive samples did not disturb the AC-4a pattern. AC-4a was predominantly associated with anti-Ro60 antibodies. Conclusions: This study confirms the association of AC-4a pattern and anti-SS-A/Ro in opposition to the AC-4b pattern. The results of four international expert laboratories support the worldwide applicability of these AC-4 pattern variants and their incorporation into ICAP classification under codes AC-4a and AC-4b, respectively. The AC-4 pattern should be maintained as an umbrella pattern for cases in which one cannot discriminate AC-4a and AC-4b patterns. The acknowledgment of the AC-4a pattern should add value to HEp-2 IFA interpretation.

Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera.

Background International autoantibody standards, traditionally based on material obtained from plasmapheresis of single subjects, represent individual immune response and may not comprehend the heterogeneity of the general population. The anti-DFS70 autoantibody yields a characteristic dense fine speckled (DFS) nuclear pattern on indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and speaks against autoimmunity. We propose a novel strategy for developing autoantibody reference standards, based on stepwise pooling of serum samples from hundreds of individuals with anti-DFS70 antibodies. Methods Within a 2-year period, serum samples were selected from routine HEp-2 IFA according to the following criteria: DFS HEp-2 IFA pattern at titer ≥1:640; anti-DFS70 reactivity in three analyte-specific tests (Western blot [WB], enzyme-linked immunosorbent assay [ELISA] and chemiluminescent immunoassay [CLIA]). Aliquots of individual samples were combined into progressively larger pools with stepwise validation of intermediary pools as for individual samples. Validated intermediary pools were merged into a final pool for lyophilization. Results A total of 741 validated samples yielded a 750 mL final pool that was lyophilized into thousands of 200 µL-aliquots. Reconstituted aliquots yielded the expected anti-DFS70 reactivity in ELISA, CLIA and WB, as well as high-titer DFS HEp-2 IFA pattern. The appropriate anti-DFS70 reactivity of the lyophilized pool was confirmed by seven international expert centers, using HEp-2 IFA, ELISA, WB and immunoprecipitation. Conclusions This proof-of-concept study provides an innovative and efficient strategy to build serum reference standards for autoantibody testing. The anti-DFS70 standard will integrate the panel of standards of Autoantibody Standardization Committee (ASC, www.autoab.org), contributing to education for proper assay validation and interpretation of the DFS pattern and other HEp-2 IFA patterns.

Anti-rods/rings autoantibody generation in hepatitis C patients during interferon-α/ribavirin therapy.

Chronic inflammation associated with hepatitis C virus (HCV) infection can lead to disabling liver diseases with progression to liver cirrhosis and hepatocellular carcinoma. Despite the recent availability of more effective and less toxic therapeutic options, in most parts of the world the standard treatment consists of a weekly injection of pegylated interferon α (IFN-α) together with a daily dose of ribavirin. HCV patients frequently present circulating non-organ-specific autoantibodies demonstrating a variety of staining patterns in the indirect immunofluorescence assay for antinuclear antibodies (ANA). Between 20% to 40% of HCV patients treated with IFN-α and ribavirin develop autoantibodies showing a peculiar ANA pattern characterized as rods and rings (RR) structures. The aim of this article is to review the recent reports regarding RR structures and anti-rods/rings (anti-RR) autoantibody production by HCV patients after IFN-α/ribavirin treatment. Anti-RR autoantibodies first appear around the sixth month of treatment and reach a plateau around the twelfth month. After treatment completion, anti-RR titers decrease/disappear in half the patients and remain steady in the other half. Some studies have observed a higher frequency of anti-RR antibodies in relapsers, i.e., patients in which circulating virus reappears after initially successful therapy. The main target of anti-RR autoantibodies in HCV patients is inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme involved in the guanosine triphosphate biosynthesis pathway. Ribavirin is a direct IMPDH2 inhibitor and is able to induce the formation of RR structures in vitro and in vivo. In conclusion, these observations led to the hypothesis that anti-RR autoantibody production is a human model of immunologic tolerance breakdown that allows us to explore the humoral autoimmune response from the beginning of the putative triggering event: exposure to ribavirin and interferon.

Implications in the difference of anti-Mi-2 and -p155/140 autoantibody prevalence in two dermatomyositis cohorts from Mexico City and Guadalajara.

INTRODUCTION: Autoantibodies and clinical manifestations in polymyositis/dermatomyositis (PM/DM) are affected by both genetic and environmental factors. The high prevalence of DM and anti-Mi-2 in Central America is thought to be associated with the high UV index of the area. The prevalences of autoantibodies and the clinical manifestations of PM/DM were evaluated comparing two cohorts in Mexico. METHODS: Ninety-five Mexican patients with PM/DM (66 DM, 29 PM; 67 Mexico City, 28 Guadalajara) were studied. Autoantibodies were characterized by immunoprecipitation using 35S-methionine labeled K562 cell extract. Clinical information was obtained from medical records. RESULTS: DM represented 69% of PM/DM and anti-Mi-2 was the most common autoantibody (35%), followed by anti-p155/140 (11%); however, anti-Jo-1 was only 4%. The autoantibody profile in adult-onset DM in Mexico City versus Guadalajara showed striking differences: anti-Mi-2 was 59% versus 12% (P = 0.0012) whereas anti-p155/140 was 9% versus 35% (P = 0.02), respectively. A strong association of anti-Mi-2 with DM was confirmed and when clinical features of anti-Mi-2 (+) DM (n = 30) versus anti-Mi-2 (-) DM (n = 36) were compared, the shawl sign (86% versus 64%, P < 0.05) was more common in the anti-Mi-2 (+) group (P = 0.0001). Levels of creatine phosphokinase (CPK) were higher in those who were anti-Mi-2 (+) but they responded well to therapy. CONCLUSIONS: Anti-Mi-2 has a high prevalence in Mexican DM and is associated with the shawl sign and high CPK. The prevalence of anti-Mi-2 and anti-p155/140 was significantly different in Mexico City versus Guadalajara, which have a similar UV index. This suggests roles of factors other than UV in anti-Mi-2 antibody production.

Longitudinal study of a human drug-induced model of autoantibody to cytoplasmic rods/rings following HCV therapy with ribavirin and interferon-α.

BACKGROUND: A novel pattern in the indirect immunofluorescence antinuclear antibody assay on HEp-2 cells (IIF-HEp-2) characterized by cytoplasmic rods and rings (RR) was reported in HCV patients, but stringent disease specificity studies and longitudinal analysis are lacking. We investigated the clinical significance of anti-RR in an HCV cohort with up to a 12-month treatment follow up. METHODOLOGY/RESULTS: 597 patients (342 HCV, 55 HCV/HIV, 200 non-HCV) were screened and titered for anti-RR. Serial samples were available from 78 of 176 treated and 27 of 166 untreated patients. Anti-RR was detected in 14.1% of 342 HCV patients, 9.1% of 55 HCV/HIV, 3.4% of 29 Hepatitis B, and none of 171 non-HCV (p<0.0001; HCV versus non-HCV). Anti-RR was present in 38% of 108 patients receiving interferon-α/ribavirin, but none in 26 receiving either interferon-α or ribavirin, or 166 untreated patients (p<0.0001). Other IIF-HEp-2 patterns were more frequently associated with interferon-α treatment alone (52.2%) as compared to interferon-α/ribavirin (25%), ribavirin alone (33.3%), and no therapy (26.5%). Anti-RR frequency was not associated with sex, age, ethnicity, HCV genotype or viral load. Anti-RR occurred only after initiation of treatment, beginning as early as 1 month (6%), but by the sixth month >47% tested positive for anti-RR. The anti-RR titer generally increased with sustained treatment and remained high in 53% of patients. After treatment, anti-RR titer was negative in 41%. Non-responders to HCV therapy were 77% in anti-RR-positive versus 64% in anti-RR-negative patients. Response to treatment was not associated with anti-RR titer or the dynamics of anti-RR reactivity during and after treatment. CONCLUSIONS: The exquisite association of anti-RR reactivity with combined interferon-α/ribavirin therapy in HCV patients represents a unique model for drug-induced autoantibody generation in humans as demonstrated by the fact that a significant fraction of patients who have anti-RR during therapy becomes anti-RR-negative after completion of therapy.