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, V. Peréz1, M. Sevilla1, S. Lermo1, G. Badra2, L. Borque3, A. Serrano1 1Immunology Service, University Hospital 12 de Octubre, 2University Rey Juan Carlos, Madrid, 3San 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).

METHODOLOGY

We studied sera from 600 patients randomly admitted to the

autoimmunity section during May and June 2009, these sera were

obtained from patients referred from primary care physicians, general

internists, rheumatologists and other subspecialties such as

nephrology, dermatology, etc . Antinuclear antibodies (ANA´s ) were

determined by immunofluorescence (IIF) on HEp-2 cell, AtheNA Multi-

lyte® multiplex technique and BioPlex™2200 (by Bio-Rad Laboratories,

Hercules, CA) a fully automated Luminex based system developed for

analysis of 13 antibodies including anti-SSA-52 kDa and anti-SSA-60

kDa in a primary tube and SS-B, Sm, Sm/RNP, RNPA, RNP-68, Scl70,

C e n t r o m e r e - B , d s D N A , c h r o m a t i n , J o 1 , r i b o s o m a l P.

Immunofluorescence was considered positive from the dilution 1/ 80.

RESULTS Six hundred sera were evaluated, 33 sera were Anti-SSA/Ro52, 42 Anti-

SSA/Ro60, and 17 Anti-La/SS-B positive by Bioplex 2200; 37 sera were

positive to Anti SS-A/Ro and 11 AntiSS-B/La by AtheNA Multi-lyte® with a

global concordance of 96.5% and 98.33% respectively; by IIF we obtained

48 positive sera, 25 (52.08%) with coarse and fine speckled pattern, 18

(37.50%) homogeneous, 2 (4.20%) Centromere-B, 2 (4,20%) Nuclear dots.

Finally, 80.70% of the patients had at least one diagnosis of autoimmune

disease.

0

5

10

15

20

25

30

35

40

45

Anti-SSA Athena Anti-SSA Bioplex 2200

Anti-SSA/Ro52 Bioplex

Anti-SSA/Ro60 Bioplex

Anti-SSA Ro52 and Ro60 Bioplex

Anti-SSB/La Athena

Anti-SSB/La Bioplex 2200

We observed 15 discrepancies: Anti-SSA negative by Athena but

positive Anti-SSA/Ro52 and/or Anti-SSA/Ro60 by Bioplex 2200; of

which 12 were IIF positive and had some autoimmunity disease: 6

SLE; 2 Systemic Sclerosis, 2 autoimmune Hepatitis, 1 Sjögren´s

syndrome + Raynaud , 1 primary biliary cirrhosis, 1

dermatomyositis. Only three discrepancies were Anti-SSA Athena

positive, Anti-SSA Bioplex Negative and IIF negative, one had

rheumatoid arthritis.

On the other hand other 8 discrepancies were observed: Anti-

SSB Athena’s Negative but Bioplex 2200 Positive, of which 8 were

IIF positive and four had diagnosis of SLE, 2 RA, 2 Sjögren´s Sx.

Only two sera were Anti-SSB Athena positive but Bioplex 2200

negative, of which 1 was IIF positive and 1 had RA.

CONCLUSIONS

We conclude that BioPlex2200 offers a good percentage of

concordance with other methods, and it 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.

REFERENCES 1. 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.

2. J. Schulte-Pelkum a,M. Fritzler , M. Mahler . Latest update on the Ro/SS-A

autoantibody system. Autoimmunity Reviews 8 (2009) 632–637