1. One year of experience using multiplex
technology in Autoimmunity Lab
Marta Talise Astier, MD;MT

2. Introduction
Autoantibodies: are immunoglobulins that bind to antigens originating in the
same individual or species (autoantigens).
ANA:
Antinuclear antibodies (ANA) are autoantibodies that target nuclear constituents of
cells, such as the nuclear membrane, nucleoplasm, nucleoli, and nuclear
organelles.
Measurement of ANA is extensively used for diagnosing and monitoring various
autoimmune diseases such as systemic lupus erythematosus, Sjögren's syndrome,
scleroderma, mixed connective tissue disease, polymyositis and dermatomyositis

3. Screening Techniques
IIF (Indirect Immunofluorescence )
Manual
Semi-automatic
Ag Hep-2cell
ANA-Ab
Sample
PhD System (Bio-rad)
Fluorescein (FITC)- conjugated
anti-human immunoglobulin

4. Screening Techniques
IIF (Indirect Immunofluorescence )
Homogeneus Pattern Speckled Pattern
Nucleolar Pattern Centromere-B Pattern

5. Disadvantages of the IIF technique
Difficult to Standardize:
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.

6. Disadvantages of the IIF technique
Limited capacity to differentiate Antigens
• Very high titers of an autoantibody could mask the fluorescence
pattern of another.
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 laboratory’s information system.

7. Screening Techniques
ELISA ANA Screen
Ags DNA (dsDNA y nDNA), Histonas, Ro, La,
Sm, SmRNP, Scl 70 y Jo-1, Ags
Centroméricos
Limitation
Sensitivity Positive false

8. Screening Techniques
Western Blot

9. Screening Techniques
Inmunodot

10. Screening Techniques
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.
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.

11. ANA Screen Kit Bioplex 2200
ANA Screen Reagent Pack
use 16 microspheres: 13
Antigens and 3 quality
Control

12. Evaluation of ANA´s by Multiplex technology at the
University Hospital “12 de Octubre” (Madrid-Spain)
M. Talise1, M. Sevilla1, S. Lermo1, L. Borque2, E. Paz-Artal1, A. Serrano1
1Immunology Service, University Hospital 12 de Octubre, Madrid, 2San Pedro Hospital, Logroño, Spain
Methodology
• 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 BioPlex™2200 ANA screen
(by Bio-Rad Laboratories, Hercules, CA).
• IIFwas considered positive from the dilution 1 / 80.

13. 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

14. 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

15. 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

16. Evaluation of ANA´s by Multiplex technology at the
University Hospital “12 de Octubre” (Madrid-Spain)
Disagreement
ANA Positive by IIF - Negative ANA Negative by IIF- Positive by
by Bioplex 2200: 27 Bioplex 2200: 29
12 false positives for autoimmune 13 false negatives by IIF for
diseases: autoinmune diseases:
• 5 patients had rheumatoid arthritis, • 4 patients had rheumatoid arthritis
• 3 patients Lupus treated • 3 Lupus
• 2 autoimmune hepatitis type I, • 1 autoimmune hepatitis type I
• 1 Dermatomyositis, • 1 Bowel,
• 1 Behcet's disease, • 1 Systemic Esclerosis,
• 1 Sjögren Sx, • 1 Sweet sx,
• 1 Tumido lupus • 1 vasculitis

17. 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

18. Bioplex 2200 Vs Athena Luminex (500 samples)
Luminex Athena
Positive Negative
Bioplex Positive 35 49 84
Negative 18 398 416
Total 53 447 500
Global Agreement 86.6%
Agreement %: Positive Athena-Bioplex + Negative Athena-Bioplex/Total (500) x 100

19. Bioplex 2200 Vs ELISA SeroQuest (450 samples)
ELISA SeroQuest
Positive Negative
Bioplex Positive 65 15 80
Negative 90 280 370
Total 155 295 450
Global Agreement 76.7%
Agreement %: Positive Elisa-Bioplex + Negative Elisa-Bioplex/Total (450) x 100

20. Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex
technology at the University Hospital “12 de Octubre” (Madrid-Spain)
M. Talise1, M. Sevilla1, S. Lermo1, L. Borque2, E. Paz-Artal1, A. Serrano1
1Immunology Service, University Hospital 12 de Octubre, Madrid, 2San Pedro Hospital, Logroño, Spain
INTRODUCTION
•Autoantibodies to Ro/SS-A and La/SS-B ribonucleoproteins are found in autoimmune
diseases such as primary Sjögren´s 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).

21. Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex
technology at the University Hospital “12 de Octubre” (Madrid-Spain)
45 42 42
40 37
35 33
30 27
25
20 17
15 11
10
5
0

22. Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex
technology at the University Hospital “12 de Octubre” (Madrid-Spain)
Disagreement
Anti-SSA Negative Athena - Positive Anti-SSA Positive Athena- Negative
Bioplex 2200: 15 Bioplex 2200: 3
12 Positive by IIF 3 Negative by IIF= False
positive by Athena
•6 SLE
•2 Systemic Sclerosis
•2 Autoimmune Hepatitis
•1 SjÖgren Sx + Raynaud
•1 Dermatomyositis

23. Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex
technology at the University Hospital “12 de Octubre” (Madrid-Spain)
Disagreement
Anti-SSB Negative by Athena- Anti-SSB Positive by Athena-
Positive by Bioplex 2200: 8 Negative by Bioplex 2200: 2
8 Positive by IIF 1 Negative by IIF
•4 Patients SLE 1 Positive by IIF Rheumatoid A
•2 Rheumatoid arthritis
•2 Sjögren Sx

24. Conclusion
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.
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.
3.- Bioplex 2200 can be a good solution as a multiplex approach to diagnose
autoimmune diseases like Sjögren´s 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.

25. 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) 632–637

26. Gracias