One year of experience using multiplex  technology in Autoimmunity Lab                     Marta Talise Astier, MD;MT
IntroductionAutoantibodies: are immunoglobulins that bind to antigens originating in thesame individual or species (autoan...
Screening Techniques                    IIF (Indirect Immunofluorescence )   Manual                                       ...
Screening Techniques                 IIF (Indirect Immunofluorescence )Homogeneus Pattern                            Speck...
Disadvantages of the IIF techniqueDifficult to Standardize: Large lab-to-lab variation in reporting results of IIF on HEp-...
Disadvantages of the IIF techniqueLimited capacity to differentiate Antigens • Very high titers of an autoantibody could m...
Screening Techniques  ELISA ANA Screen        Ags DNA (dsDNA y nDNA), Histonas, Ro, La,        Sm,   SmRNP,    Scl   70   ...
Screening Techniques   Western Blot
Screening Techniques    Inmunodot
Screening Techniques                                MultiplexThese systems utilize a new approach for the simultaneous mea...
ANA Screen Kit Bioplex 2200ANA Screen Reagent Pack use 16 microspheres: 13 Antigens and 3 quality Control
Evaluation of ANA´s by Multiplex technology at the    University Hospital “12 de Octubre” (Madrid-Spain)                  ...
Bioplex 2200 Vs IIF (500 samples)                                 IIF                  ANA         Positive      NegativeB...
Bioplex 2200 Vs IIF (500 samples)                                 IIF                  ANA         Positive      NegativeB...
Bioplex 2200 Vs IIF (500 samples)                                 IIF                  ANA         Positive      NegativeB...
Evaluation of ANA´s by Multiplex technology at the     University Hospital “12 de Octubre” (Madrid-Spain)                 ...
Bioplex 2200 Vs IIF (2150) samples)                                   IIF                   ANA          Positive      Neg...
Bioplex 2200 Vs Athena Luminex (500 samples)                                   Luminex       Athena                       ...
Bioplex 2200 Vs ELISA SeroQuest (450 samples)                                  ELISA       SeroQuest                      ...
Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex technology at the University Hospital “12 de Octubre...
Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex technology at the University Hospital “12 de Octubre...
Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex technology at the University Hospital “12 de Octubre...
Evaluation of Antinuclear Ro/SS-A and La/SS-B antibodies by Multiplex technology at the University Hospital “12 de Octubre...
Conclusion1.-We conclude that BioPlex 2200 has a good percentage of concordancewith the comparative method and represents ...
REFERENCES1. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes forcell biology. Adv. I...
Gracias
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Antinuclear Antibodies by Bio-Plex 2200

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Antinuclear Antibodies by Bio-Plex 2200

  1. 1. One year of experience using multiplex technology in Autoimmunity Lab Marta Talise Astier, MD;MT
  2. 2. IntroductionAutoantibodies: are immunoglobulins that bind to antigens originating in thesame individual or species (autoantigens).ANA:Antinuclear antibodies (ANA) are autoantibodies that target nuclear constituents ofcells, such as the nuclear membrane, nucleoplasm, nucleoli, and nuclearorganelles.Measurement of ANA is extensively used for diagnosing and monitoring variousautoimmune diseases such as systemic lupus erythematosus, Sjögrens syndrome,scleroderma, mixed connective tissue disease, polymyositis and dermatomyositis
  3. 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. 4. Screening Techniques IIF (Indirect Immunofluorescence )Homogeneus Pattern Speckled Pattern Nucleolar Pattern Centromere-B Pattern
  5. 5. Disadvantages of the IIF techniqueDifficult 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. 6. Disadvantages of the IIF techniqueLimited 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. 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. 8. Screening Techniques Western Blot
  9. 9. Screening Techniques Inmunodot
  10. 10. Screening Techniques MultiplexThese systems utilize a new approach for the simultaneous measurement ofautoantibodies based on multiple-dyed (fluorescent) beads coated with specificantigens and flow cytometry detection.The BioPlex 2200 ANA screen (by BioRad) is a fully automated Luminex-based systemdeveloped for high-throughput analysis of 13 autoimmune analytes simultaneously in asingle 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. 11. ANA Screen Kit Bioplex 2200ANA Screen Reagent Pack use 16 microspheres: 13 Antigens and 3 quality Control
  12. 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 sectionduring 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; AtheNAMulti-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. 13. Bioplex 2200 Vs IIF (500 samples) IIF ANA Positive NegativeBioplex 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. 14. Bioplex 2200 Vs IIF (500 samples) IIF ANA Positive NegativeBioplex 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. 15. Bioplex 2200 Vs IIF (500 samples) IIF ANA Positive NegativeBioplex 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. 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 Behcets disease, • 1 Systemic Esclerosis,• 1 Sjögren Sx, • 1 Sweet sx,• 1 Tumido lupus • 1 vasculitis
  17. 17. Bioplex 2200 Vs IIF (2150) samples) IIF ANA Positive NegativeBioplex 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. 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. 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. 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, SpainINTRODUCTION•Autoantibodies to Ro/SS-A and La/SS-B ribonucleoproteins are found in autoimmunediseases such as primary Sjögren´s syndrome, systemic lupus erythematousus (SLE) andrheumatoid 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 stablemacromolecular complex and that anti-Ro52 and anti-Ro60 (SS-A) antibodies have differentclinical associations and each specific to different antigens2, for this reason it is necessary tobe able to individually identify these antibodies in the autoimmunity laboratory and the BioplexTM 2200 automated system offers this advantage;•We compare this technique with the immunofluorescence (IIF) and AtheNA Multi-lyte® ANAtest system a multiplex fluorescent microsphere immunoassay for the semi-quantitative of IgG class antibody to 9 separate analytes (DNA, SSA, SSB, Sm, RNP, Scl-70, Jo-1,Centromere B, and Histone).
  21. 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. 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. 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. 24. Conclusion1.-We conclude that BioPlex 2200 has a good percentage of concordancewith the comparative method and represents a fast solution, sensitive andspecific for diagnosis of autoimmune diseases.2.-The observed disagreement may be due to different design and nature ofthe antigens used in each method and maybe with slight modifications in thecutoff point would ensure greater consistency.3.- Bioplex 2200 can be a good solution as a multiplex approach to diagnoseautoimmune diseases like Sjögren´s syndrome, systemic lupuserythematousus and rheumatoid arthritis because the separation of Ro /SSA 52 and 60 antigens conferred increased sensitivity, provides greaterspecificity with a lower rate of false negatives.
  25. 25. REFERENCES1. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes forcell biology. Adv. Immunology. 1989. 44:93-1512. Ulvestad E, Kanestrom A, Madland TM et al. Evaluation of diagnostic tests for antinuclearantibodies in rheumatological practice. Scand. J. Immunol. 2000. 52:3093. van Woerkom JM, Geertzema JG, Nikkels PG, et al. Expression of Ro/SS-A and La/SS-Bdetermined by immunohistochemistry in healthy, inflamed and autoimmune diseased humantissues: 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 autoantibodysystem. Autoimmunity Reviews 8 (2009) 632–637
  26. 26. Gracias

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