EVALUATION OF ANA SCREENING BY BIOPLEX™ 2200 AT THE UNIVERSITY HOSPITAL “12 DE OCTUBRE” (MADRID-SPAIN)

M. Talise1, 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

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

dermatomyositis1. The traditional methods for detecting ANA are

indirect immunofluorescence (IIF) on HEp-2 cell and enzyme

immunoassay (ELISA). A positive ANA screen leads to further

laboratory investigations on the presence of markers useful for

diagnosis of autoimmune diseases such as anti-dsDNA antibodies or

anti-extractable nuclear antigens (anti-ENA) antibodies. These

techniques are time-consuming, laborious and characterized by

frequent discrepancies between laboratories . We compare the

BioPlex™2200 ANA screen (by Bio-Rad Laboratories, Hercules, CA)

with IIF, ELISA SeraQuest® ANA screening and AtheNA Multi-lyte.

METHODOLOGY

We selected 500 serum samples 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. All samples were tested by indirect

immunofluorescence (IIF) on Hep-2 cell line substrate Bio-Rad Lab; by

enzyme immunoassay ELISA SeraQuest® ANA screening; AtheNA

Multi-lyte® ANA test system a multiplex fluorescent microsphere

immunoassay for the semi-quantitative of Ig G class antibody to 8

separate analytes (SSA, SSB, Sm, RNP, Scl-70, Jo-1, Centromere B, and

Histone and finally by BioPlex™2200 ANA screen (by Bio-Rad

Laboratories, Hercules, CA) a fully automated Luminex-based system

developed for analysis of 13 antibodies simultaneously in a primary

tube {SS-A (52, 60 kDa), SS-B, Sm, Sm/RNP, RNP-A, 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.

Table 1. ANA IIF Vs ANA Bioplex 2200 (N=500)

ANA Positive IIF Negative IIF Total

Positive Bioplex 2200

51 29 80

Negative Bioplex 2200

27 393 420

Total 78 422 500

Table 2. Athena Multi-lyte Vs Bioplex 2200 (N=500)

ANA Positive Athena

Negative Athena

Total

Positive Bioplex 2200

35 49 84

Negative Bioplex 2200

18 398 416

Total 53 447 500

RESULTS Five hundred patients samples were evaluated for presence of

autoantibodies ANA’s by four different assays IIF, ELISA, Athena

Multilyte and BioPlex™2200 obtaining a global concordance of 88.8%,

76.7% and 86.6%, respectively.

We observed 27 discrepancies: ANA positive IIF but ANA negative

by Bioplex 2200, of which discrepancies, 12 were false positives

for autoimmune diseases, 5 patients had rheumatoid arthritis, 3

patients lupus treated with corticosteroids, 2 autoimmune

hepatitis type I, 1 dermatomyositis, 1 Behcet's disease, 1

Sjögren's Sx, 1 tumido lupus; on the other hand we observed 29

ANA negative discrepancies by IIF but ANA positive by Bioplex,

these were 13 false negatives by IIF for autoimmune diseases.

CONCLUSIONS

We conclude that BioPlexTM2200 has a good percentage of

concordance with the comparative method and represents a fast

solution, sensitive and specific for diagnosis of autoimmune

diseases.

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

The observed discrepancies 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.-