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

IntroductionThe 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.MethodsAnti-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.ResultsMonoclonal 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.ConclusionsThis 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.

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The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory performance, pattern recognition and interpretation

Autoimmunity Highlights ◽

10.1186/s13317-020-00146-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anne E. Tebo ◽

Robert L. Schmidt ◽

Kamran Kadkhoda ◽

Lisa K. Peterson ◽

Edward K. L. Chan ◽

...

Keyword(s):

Indirect Immunofluorescence ◽

Clinical Laboratory ◽

Immunofluorescence Assay ◽

Indirect Immunofluorescence Assay ◽

Medical Laboratory ◽

Nuclear Staining ◽

International Consensus ◽

In Vitro Diagnostic ◽

Traditional Pattern

Abstract Background To evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IFA) using HEp-2 substrates based on common practice and guidance by the International Consensus on ANA patterns (ICAP). Method Participants included two groups [16 clinical laboratories (CL) and 8 in vitro diagnostic manufacturers (IVD)] recruited via an email sent to the Association of Medical Laboratory Immunologists (AMLI) membership. Twelve (n = 12) pre-qualified specimens were distributed to participants for testing, interpretation and reporting HEp-2 IFA. Results obtained were analyzed for accuracy with the intended and consensus response for three main categorical patterns (nuclear, cytoplasmic and mitotic), common patterns and ICAP report nomenclatures. The distributions of antibody titers of specimens were also compared. Results Laboratories differed in the categorical patterns reported; 8 reporting all patterns, 3 reporting only nuclear patterns and 5 reporting nuclear patterns with various combinations of other patterns. For all participants, accuracy with the intended response for the categorical nuclear pattern was excellent at 99% [95% confidence interval (CI): 97–100%] compared to 78% [95% CI 67–88%] for the cytoplasmic, and 93% [95% CI 86%–100%] for mitotic patterns. The accuracy was 13% greater for the common nomenclature [87%, 95% CI 82–90%] compared to the ICAP nomenclature [74%, 95% CI 68–79%] for all participants. Participants reporting all three main categories demonstrated better performances compared to those reporting 2 or less categorical patterns. The average accuracies varied between participant groups, however, with the lowest and most variable performances for cytoplasmic pattern specimens. The reported titers for all specimens varied, with the least variability for nuclear patterns and most titer variability associated with cytoplasmic patterns. Conclusions Our study demonstrated significant accuracy for all participants in identifying the categorical nuclear staining as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cytoplasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel.

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Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-1262 ◽

2020 ◽

Vol 58 (9) ◽

pp. 1489-1497 ◽

Cited By ~ 1

Author(s):

Lisa K. Peterson ◽

Anne E. Tebo ◽

Mark H. Wener ◽

Susan S. Copple ◽

Marvin J. Fritzler

Keyword(s):

Indirect Immunofluorescence ◽

Antinuclear Antibodies ◽

Current Practice ◽

Clinical Laboratory ◽

Immunofluorescence Assay ◽

Indirect Immunofluorescence Assay ◽

Medical Laboratory ◽

International Consensus ◽

Clinical Laboratories ◽

Reading Interpretation

AbstractBackgroundThe indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting.MethodsWe developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting.ResultsOne hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials.ConclusionsBased on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

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