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Objective: Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria [anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III]. However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP), that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9), are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies
Methods: A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n=32) and Italy (Careggi Hospital, Florence, n=15) selected based on AC-9 HEp-2 IFA staining (>1:640, clumpy nucleolar pattern) and 102 non-SSc controls [inflammatory bowel disease (IBD) n=20, Sjögren’s syndrome (SjS) n=20, infectious disease (ID) n=7, systemic lupus erythematosus (SLE) n=17, rheumatoid arthritis (RA) n=17, and healthy individuals (HI) n=21]. All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples.
Results: The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0 %, Italy) of samples preselected based on the AC-9 IIF pattern. Collectively, the PMAT assay showed 91.5% [95% Confidence Interval (CI): 80.1-96.6%] sensitivity with 100.0% (95% CI: 96.4-100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman’s rho=0.89, 95% CI:0.77.9-0.95%, p<0.0001). Although most anti-fibrillarin positive samples were mono-specific (69.8%), some expressed additional antibodies (namely Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS70, and EJ).
Conclusion: The new PMAT assay had high level of agreement to FEIA for the detection of anti-fibrillarin antibodies. Further studies are warranted to investigate the clinical associations and performance of the new method in combination with other critical markers in the SSc panel.
Keywords
systemic sclerosis, autoantibodies, multi-analyte, immunoassay
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CURRENT STATUS: Under Review
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Posted 21 Dec, 2020
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Editorial decision: Minor revision
On 08 Feb, 2021
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Received 05 Feb, 2021
Reviewer #3 agreed
On 31 Jan, 2021
Review #1 received
Received 23 Dec, 2020
Reviewer #2 agreed
On 16 Dec, 2020
Reviewers invited
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Reviewer #1 agreed
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This preprint is under consideration at Autoimmunity Highlights. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Original Research
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 21 Dec, 2020
No community comments so far
Editorial decision: Minor revision
On 08 Feb, 2021
Review #2 received
Received 05 Feb, 2021
Reviewer #3 agreed
On 31 Jan, 2021
Review #1 received
Received 23 Dec, 2020
Reviewer #2 agreed
On 16 Dec, 2020
Reviewers invited
Invitations sent on 16 Dec, 2020
Reviewer #1 agreed
On 16 Dec, 2020
Editor assigned
On 14 Dec, 2020
Submission checks complete
On 14 Dec, 2020
Editor invited
On 14 Dec, 2020
First submitted
On 10 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Objective: Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria [anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III]. However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP), that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9), are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies
Methods: A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n=32) and Italy (Careggi Hospital, Florence, n=15) selected based on AC-9 HEp-2 IFA staining (>1:640, clumpy nucleolar pattern) and 102 non-SSc controls [inflammatory bowel disease (IBD) n=20, Sjögren’s syndrome (SjS) n=20, infectious disease (ID) n=7, systemic lupus erythematosus (SLE) n=17, rheumatoid arthritis (RA) n=17, and healthy individuals (HI) n=21]. All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples.
Results: The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0 %, Italy) of samples preselected based on the AC-9 IIF pattern. Collectively, the PMAT assay showed 91.5% [95% Confidence Interval (CI): 80.1-96.6%] sensitivity with 100.0% (95% CI: 96.4-100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman’s rho=0.89, 95% CI:0.77.9-0.95%, p<0.0001). Although most anti-fibrillarin positive samples were mono-specific (69.8%), some expressed additional antibodies (namely Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS70, and EJ).
Conclusion: The new PMAT assay had high level of agreement to FEIA for the detection of anti-fibrillarin antibodies. Further studies are warranted to investigate the clinical associations and performance of the new method in combination with other critical markers in the SSc panel.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
The full text of this article is available to read as a PDF.
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