| 1. |
- Andrade, Luis E. C., et al.
(författare)
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International consensus on antinuclear antibody patterns : definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I
- 2018
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Ingår i: Clinical Chemistry and Laboratory Medicine. - : WALTER DE GRUYTER GMBH. - 1434-6621 .- 1437-4331. ; 56:10, s. 1783-1788
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Tidskriftsartikel (refereegranskat)abstract
- The indirect immunofluorescence assay (IFA) on HEp-2 cells is the reference method for autoantibody screening. The HEp-2 IFA pattern provides useful information on the possible autoantibodies in the sample. The International Consensus on Antinuclear Antibody Patterns (ICAP) initiative seeks to define and harmonize the nomenclature of HEp-2 IFA patterns. The most relevant and usual patterns have been assigned an alphanumeric code from anti-cell (AC)-1 to AC-28 and were organized into a classification algorithm (www.ANApatterns.org). The systemic sclerosis-associated autoantibodies to DNA topoisomerase I (Topo I) produce a peculiar composite 5-element HEp-2 IFA pattern (Topo I-like pattern) comprising the staining of the nucleus, metaphase chromatin plate, nucleolar organizing region, cytoplasm and nucleolus. In a recent assessment of the European Consensus Finding Study Group on autoantibodies, a well-defined anti-Topo I sample was blindly analyzed and classified according to ICAP AC patterns by 43 participant laboratories across Europe. There were wide variations among these laboratories in reporting nuclear, nucleolar and cytoplasmic patterns, indicating the inadequacy of the existing AC patterns to report the Topo I-like pattern. Several ICAP member laboratories independently demonstrated the overall consistency of the HEp-2 IFA Topo I-like pattern using HEp-2 slides from different manufacturers. The ICAP committee reviewed 24 candidate images and selected the four most representative images to be available on the ICAP website. The proper recognition of the AC-29 pattern should trigger suspicion of the presence of anti-Topo I antibodies, which may engender appropriate analyte-specific reflex tests to confirm the autoantibody specificity.
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| 2. |
- Bonroy, Carolien, et al.
(författare)
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Detection of antinuclear antibodies : recommendations from EFLM, EASI and ICAP
- 2023
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Ingår i: Clinical Chemistry and Laboratory Medicine. - : Walter de Gruyter. - 1434-6621 .- 1437-4331. ; 61:7, s. 1167-1198
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA).Methods: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group “Autoimmunity Testing”; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP).Results: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations.Conclusions: These recommendations are an important step to achieve high quality ANA testing.
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| 3. |
- Dellavance, Alessandra, et al.
(författare)
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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
- 2019
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Ingår i: Clinical Chemistry and Laboratory Medicine. - : WALTER DE GRUYTER GMBH. - 1434-6621 .- 1437-4331. ; 57:11, s. 1754-1763
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Tidskriftsartikel (refereegranskat)abstract
- 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 mu L-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 antiDFS70 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.
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