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Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate
Published on: January 17, 2018
Reflex testing of speckled cytoplasmic patterns observed in routine ANA HEp-2 indirect immunofluorescence with a
Maria Infantino1, Boaz Palterer2, Roberta Biagiotti2
1SOS Immunology and Allergology Laboratory Unit, Department of Laboratory Medicine, Azienda Usl Toscana Centro, S. Giovanni di Dio Hospital, Florence, Italy. maria2.infantino@uslcentro.toscana.it.
Insights
Routine anti-nuclear antibody (ANA) testing can identify cytoplasmic speckled patterns, often linked to anti-synthetase antibodies. A multiplex dot-blot assay effectively detects these antibodies, aiding diagnosis in patients with atypical connective tissue disease presentations.
Area of Science:
- Immunology
- Rheumatology
- Clinical Chemistry
Background:
- Anti-nuclear antibody (ANA) immunofluorescence on HEp2-cells is standard for diagnosis.
- Cytoplasmic speckled patterns in ANA testing are associated with anti-synthetase antibodies, but traditional ENA testing often misses these.
- Anti-synthetase syndrome presents diversely, including isolated arthritis or interstitial lung disease.
Purpose of the Study:
- To evaluate a commercial synthetase profile dot-blot assay for detecting anti-cytoplasmic antibodies.
- To assess the utility of this assay in a cohort with cytoplasmic speckled patterns during routine ANA testing.
- To determine if this approach identifies patients with rare anti-synthetase antibodies and atypical clinical presentations.
Main Methods:
- Selected 493 sera: 254 with cytoplasmic speckled ANA patterns and 239 controls.
- Tested all samples using a commercial synthetase profile dot-blot (D TEK - Alphadia-Alifax).
- Retrospectively analyzed clinical data for positive cases.
Main Results:
- The dot-blot assay identified 7.1% positive sera in the cytoplasmic speckled group versus 1.7% in controls (p=0.0036).
- Blot intensity was significantly higher in samples with concordant cytoplasmic staining (p=0.0027).
- Of positive samples, 8/18 had compatible diagnoses (myositis, ILD, arthritis), 7/18 had uncharacterized CTD, and 3 had unrelated diagnoses.
Conclusions:
- A multiplex dot-blot assay is effective for identifying anti-cytoplasmic antibodies in patients with cytoplasmic speckled ANA patterns.
- This strategy aids in diagnosing rare anti-synthetase antibodies, especially in patients with incomplete or atypical clinical features.
- Implementing reflex testing for cytoplasmic speckled patterns with multiplex assays is recommended for routine ANA testing.
Abstract:
Immunofluorescence on HEp2-cells is the standard diagnostic assay for the detection of anti-nuclear antibodies (ANA). Cytoplasmic speckled patterns are a common finding, and are associated with various antibodies, including anti-synthetase antibodies. However, classic ENA testing generally identifies only anti-Jo-1. Moreover, anti-synthetase syndrome is increasingly recognized as a pleomorphic entity, possibly presenting as isolated arthritis or interstitial lung disease. Sera referred for routine ANA testing were selected on the basis of the presence of a fine dense speckled cytoplasmic pattern (254 samples) and compared to control sera with negative cytoplasm (239 samples). All 493 samples were tested with a commercial synthetase profile dot-blot (D TEK - Alphadia-Alifax) including anti-Jo1, anti-PL7, anti-PL12, anti-EJ, anti-OJ, anti-KS, anti-ZO, anti-HA, anti-SRP, and anti-Ribosome P0. Retrospective clinical data was searched for positive patients. Dot-blot identified 18/254 (7.1%) positive sera in the samples with a cytoplasmic fluorescence pattern and 4/239 (1.7%) in the control group (χ2 = 8.4627; p = 0.003625). Blot intensity was more intense in samples with concordant cytoplasmic staining (cytoplasmic negative 27 ± 12.4; cytoplasmic positive 53.9 27 ± 27.7; p = 0.0027). In the positive samples, 8/18 had a highly compatible diagnosis (myositis, interstitial lung disease, arthritis), 7/18 an uncharacterized connective tissue disease, and 3 a diagnosis not associated with the presence of anti-synthetase antibodies. We evaluated the performance of a dot-blot assay for anti-cytoplasmic antibodies in a serologic cohort presenting a cytoplasmic speckled pattern found during routine ANA testing. This algorithm enabled the identification of a significant quota of patients with rare anti-synthetase antibodies and an incomplete or atypical clinical picture. Reflex testing strategies of speckled cytoplasmic patterns with multiplex assays containing cytoplasm-specific antigens, as opposed to standard ENA testing, may yield important data and for this reason should be implemented in routine ANA testing.
