Antinuclear Antibodies by Bio-Plex 2200
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Transcript of Antinuclear Antibodies by Bio-Plex 2200
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One year of experience using multiplex technology in Autoimmunity Lab
Marta Talise Astier, MD;MT
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Introduction
ANA: Antinuclear antibodies (ANA) are autoantibodies that target nuclear constituents of
cells, such as the nuclear membrane, nucleoplasm, nucleoli, and nuclear
organelles.
Autoantibodies: are immunoglobulins that bind to antigens originating in the same individual or species (autoantigens).
Measurement of ANA is extensively used for diagnosing and monitoring various
autoimmune diseases such as systemic lupus erythematosus, Sjgren's syndrome,
scleroderma, mixed connective tissue disease, polymyositis and dermatomyositis
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IIF (Indirect Immunofluorescence ) Manual Semi-automatic
PhD System (Bio-rad)
Ag Hep-2cell
ANA-Ab
Fluorescein (FITC)- conjugated anti-human immunoglobulin
Sample
Screening Techniques
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Homogeneus Pattern Speckled Pattern
Nucleolar Pattern Centromere-B Pattern
IIF (Indirect Immunofluorescence ) Screening Techniques
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Difficult to Standardize:
Disadvantages of the IIF technique
Large lab-to-lab variation in reporting results of IIF on HEp-2 cells due to:
Differences in microscopes such as:
- Power of the objectives
- Strength of the fluorescent light
Differences in technicians interpretation of the IIF patterns.
Differences in the conjugates used to detect the bound autoantibodies
(IgG-specific compared to IgG-, IgA-, IgM-, or polyspecific),
Differences of the starting dilution of serum (1:40, 1:80, 1:160) among
laboratories.
Differences in the ways that HEp-2 cells are fixed by various
manufacturers.
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Very high titers of an autoantibody could mask the fluorescence
pattern of another.
Limited capacity to differentiate Antigens
Disadvantages of the IIF technique
Laborious Technique
Relatively high amount of skilled labor is required to perform the test.
Addition of sample and detecting reagent, and wash steps, can be easily
automated.
However, a Trained Technologist must read and interpret the slide by
examining it under a fluorescent microscope and must manually enter
the resulting data into the laboratorys information system.
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Screening Techniques ELISA ANA Screen
Sensitivity Positive false
Limitation
Ags DNA (dsDNA y nDNA), Histonas, Ro, La,
Sm, SmRNP, Scl 70 y Jo-1, Ags
Centromricos
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Western Blot
Screening Techniques
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Inmunodot
Screening Techniques
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Multiplex These systems utilize a new approach for the simultaneous measurement of autoantibodies based on multiple-dyed (fluorescent) beads coated with specific antigens and flow cytometry detection.
Screening Techniques
The BioPlex 2200 ANA screen (by BioRad) is a fully automated Luminex-based system developed for high-throughput analysis of 13 autoimmune analytes simultaneously in a single tube.
Reacting with SSA (52 and 60 kDa), SSB, Sm, Sm/RNP, RNP-A, RNP-68 kDa, Scl70, centromere B, dsDNA, chromatin, Jo1, ribosomal P proteins.
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ANA Screen Kit Bioplex 2200
ANA Screen Reagent Pack use 16 microspheres: 13 Antigens and 3 quality Control
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Evaluation of ANAs by Multiplex technology at the University Hospital 12 de Octubre (Madrid-Spain)
500 serum samples randomly admitted to the autoimmunity section
during May and June 2009.
Samples were tested by IIF on Hep-2 cell line substrate Bio-Rad Lab;
by enzyme immunoassay ELISA SeraQuest ANA screening; AtheNA
Multi-lyte ANA test system and finally by BioPlex2200 ANA screen
(by Bio-Rad Laboratories, Hercules, CA).
IIFwas considered positive from the dilution 1 / 80.
Methodology
M. Talise1, M. Sevilla1, S. Lermo1, L. Borque2, E. Paz-Artal1, A. Serrano1
1Immunology Service, University Hospital 12 de Octubre, Madrid, 2San Pedro Hospital, Logroo, Spain
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Bioplex 2200 Vs IIF (500 samples)
IIF ANA Positive Negative
Bioplex Positive 51 29 80
Negative 27 393 420
Total 78 422 500
Global Agreement of 88.8%
Agreement %: Positive IIF-Bioplex + Negative IIF-Bioplex/Total (500) x 100
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Bioplex 2200 Vs IIF (500 samples)
IIF ANA Positive Negative
Bioplex Positive 51 29 80
Negative 27 393 420 Total 78 422 500
Global Agreement of 88.8%
Agreement %: Positive IIF-Bioplex + Negative IIF-Bioplex/Total (500) x 100
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Bioplex 2200 Vs IIF (500 samples)
IIF ANA Positive Negative
Bioplex Positive 51 29 80 Negative 27 393 420 Total 78 422 500
Global Agreement 88.8%
Agreement %: Positive IIF-Bioplex + Negative IIF-Bioplex/Total (500) x 100
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Evaluation of ANAs by Multiplex technology at the University Hospital 12 de Octubre (Madrid-Spain)
13 false negatives by IIF for autoinmune diseases:
4 patients had rheumatoid arthritis 3 Lupus 1 autoimmune hepatitis type I 1 Bowel, 1 Systemic Esclerosis, 1 Sweet sx, 1 vasculitis
Disagreement
12 false positives for autoimmune diseases:
5 patients had rheumatoid arthritis, 3 patients Lupus treated 2 autoimmune hepatitis type I, 1 Dermatomyositis, 1 Behcet's disease, 1 Sjgren Sx, 1 Tumido lupus
ANA Positive by IIF - Negative by Bioplex 2200: 27
ANA Negative by IIF- Positive by Bioplex 2200: 29
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Bioplex 2200 Vs IIF (2150) samples)
IIF ANA Positive Negative
Bioplex Positive 297 168 465
Negative 30 1655 1685
Total 327 1823 2150
Global Agreement of 90.8%
Agreement %: Positive IIF-Bioplex + Negative IIF-Bioplex/Total (2150) x 100
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Luminex Athena
Positive Negative
Bioplex Positive 35 49 84
Negative 18 398 416
Total 53 447 500
Bioplex 2200 Vs Athena Luminex (500 samples)
Global Agreement 86.6%
Agreement %: Positive Athena-Bioplex + Negative Athena-Bioplex/Total (500) x 100
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ELISA SeroQuest
Positive Negative
Bioplex Positive 65 15 80
Negative 90 280 370
Total 155 295 450
Bioplex 2200 Vs ELISA SeroQuest (450 samples)
Global Agreement 76.7%
Agreement %: Positive Elisa-Bioplex + Negative Elisa-Bioplex/Total (450) x 100
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Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex technology at the University Hospital 12 de Octubre (Madrid-Spain)
INTRODUCTION Autoantibodies to Ro/SS-A and La/SS-B ribonucleoproteins are found in autoimmune diseases such as primary Sjgrens syndrome, systemic lupus erythematousus (SLE) and rheumatoid arthritis1 (RA). Two types of anti-Ro/SSA antibodies have been described, anti-SSA-52 kDa and anti-SSA-60kDa, historically, these autoantibodies were considered as a uniform autoantibody-system. However, recent studies provided evidence that Ro60 and Ro52 are not part of a stable macromolecular complex and that anti-Ro52 and anti-Ro60 (SS-A) antibodies have different clinical associations and each specific to different antigens2, for this reason it is necessary to be able to individually identify these antibodies in the autoimmunity laboratory and the Bioplex TM 2200 automated system offers this advantage; We compare this technique with the immunofluorescence (IIF) and AtheNA Multi-lyte ANA test system a multiplex fluorescent microsphere immunoassay for the semi-quantitative of Ig G class antibody to 9 separate analytes (DNA, SSA, SSB, Sm, RNP, Scl-70, Jo-1, Centromere B, and Histone).
M. Talise1, M. Sevilla1, S. Lermo1, L. Borque2, E. Paz-Artal1, A. Serrano1
1Immunology Service, University Hospital 12 de Octubre, Madrid, 2San Pedro Hospital, Logroo, Spain
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37
42
33
42
27
11
17
0 5
10 15 20 25 30 35 40 45
Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex technology at the University Hospital 12 de Octubre (Madrid-Spain)
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Anti-SSA Negative Athena - Positive Bioplex 2200: 15
12 Positive by IIF
6 SLE
2 Systemic Sclerosis
2 Autoimmune Hepatitis
1 Sjgren Sx + Raynaud
1 Dermatomyositis
Anti-SSA Positive Athena- Negative Bioplex 2200: 3
Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex technology at the University Hospital 12 de Octubre (Madrid-Spain)
Disagreement
3 Negative by IIF= False positive by Athena
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Anti-SSB Negative by Athena- Positive by Bioplex 2200: 8
8 Positive by IIF
4 Patients SLE
2 Rheumatoid arthritis
2 Sjgren Sx
1 Positive by IIF Rheumatoid A
Anti-SSB Positive by Athena- Negative by Bioplex 2200: 2
Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex technology at the University Hospital 12 de Octubre (Madrid-Spain)
Disagreement
1 Negative by IIF
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Conclusion
2.-The observed disagreement may be due to different design and nature of the antigens used in each method and maybe with slight modifications in the cutoff point would ensure greater consistency.
1.-We conclude that BioPlex 2200 has a good percentage of concordance with the comparative method and represents a fast solution, sensitive and specific for diagnosis of autoimmune diseases.
3.- Bioplex 2200 can be a good solution as a multiplex approach to diagnose autoimmune diseases like Sjgrens syndrome, systemic lupus erythematousus and rheumatoid arthritis because the separation of Ro / SSA 52 and 60 antigens conferred increased sensitivity, provides greater specificity with a lower rate of false negatives.
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REFERENCES
1. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for
cell biology. Adv. Immunology. 1989. 44:93-151
2. Ulvestad E, Kanestrom A, Madland TM et al. Evaluation of diagnostic tests for antinuclear
antibodies in rheumatological practice. Scand. J. Immunol. 2000. 52:309
3. van Woerkom JM, Geertzema JG, Nikkels PG, et al. Expression of Ro/SS-A and La/SS-B
determined by immunohistochemistry in healthy, inflamed and autoimmune diseased human
tissues: a generalized phenomenon. Clin Exp Rheumatol. 2004 May-Jun;22(3):285-92.
4. J. Schulte-Pelkum a,M. Fritzler , M. Mahler . Latest update on the Ro/SS-A autoantibody
system. Autoimmunity Reviews 8 (2009) 632637
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Gracias
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