Antinuclear Antibodies by Bio-Plex 2200

One year of experience using multiplex technology in Autoimmunity Lab

Marta Talise Astier, MD;MT

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

IIF (Indirect Immunofluorescence ) Manual Semi-automatic

PhD System (Bio-rad)

Ag Hep-2cell

ANA-Ab

Fluorescein (FITC)- conjugated anti-human immunoglobulin

Sample

Screening Techniques

Homogeneus Pattern Speckled Pattern

Nucleolar Pattern Centromere-B Pattern

IIF (Indirect Immunofluorescence ) Screening Techniques

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.

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.

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

Western Blot


Inmunodot


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.

ANA Screen Kit Bioplex 2200

ANA Screen Reagent Pack use 16 microspheres: 13 Antigens and 3 quality Control

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

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




Negative 27 393 420 Total 78 422 500





Bioplex Positive 51 29 80 Negative 27 393 420 Total 78 422 500

Global Agreement 88.8%


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

Bioplex 2200 Vs IIF (2150) samples)



Negative 30 1655 1685

Total 327 1823 2150



Luminex Athena

Positive Negative


Negative 18 398 416

Total 53 447 500

Bioplex 2200 Vs Athena Luminex (500 samples)


Agreement %: Positive Athena-Bioplex + Negative Athena-Bioplex/Total (500) x 100

ELISA SeroQuest

Positive Negative


Negative 90 280 370

Total 155 295 450

Bioplex 2200 Vs ELISA SeroQuest (450 samples)


Agreement %: Positive Elisa-Bioplex + Negative Elisa-Bioplex/Total (450) x 100

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

37

42

33

42

27

11

17

0 5

10 15 20 25 30 35 40 45


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


Disagreement

3 Negative by IIF= False positive by Athena

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


Disagreement

1 Negative by IIF

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.

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

Gracias

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Health & Medicine

Antinuclear Antibodies by Bio-Plex 2200