Enhanced Detection of Autoimmune Disease: Implementation of a Fully Automated, Random Access Multiplexing Platform for Antinuclear Antibody (ANA) Screening

Scott Bainbridge, CLS, Special Chemistry/Toxicology Supervisor
Nam K. Tran, PhD, MS, FACB, Director of Clinical Chemistry and POCT

Background: Autoimmune diseases are often characterized as immune responses directed toward self. These diseases can be localized or systemic and can involve a specific antigen or a variety of antigens. Because these antigens are “self” they are continuously present and diseases involving autoimmune response are nearly impossible to cure, leading to a chronic condition of inflammation. This presents a strain on the resources of the medical community and has a negative effect on the quality of life of the patient.1,2

As with many diseases, laboratory testing is highly valuable in diagnosing autoimmune diseases, and the detection of autoantibodies is considered the most valuable both in diagnosis and in monitoring disease activity and response to treatment. In the laboratory, autoantibodies are detected using an anti-nuclear antibody (ANA) screen. Indirect Fluorescent Antibody (IFA) is traditionally used for screening. This method involves incubating patient serum on a slide containing tissue with a variety of antigens, labelling the bound antibodies with fluorescein bound antibody markers and viewing the slide under a fluorescence microscope to determine patterns and titers. This method is clinically sensitive, but has a high false-positive rate (lower clinical specificity), is time consuming for the laboratory, and due to the subjective nature of pattern interpretation, it is often plagued with inconsistencies between laboratories and even between technologists.2,3

Laboratory Best Practice: Multiplex flow immunoassay (Bioplex, Bio-Rad, Hercules, CA) is an emerging as a high performance technique for ANA screening. This new method is similar to enzyme immunoassay (EIA), however, the multiplex flow immunoassay has the advantage of greater clinical sensitivity and specificity. Positive results from Bioplex ANA screens are more likely to be associated with rheumatic disease than IFA results, according to clinical studies.3-4 This method also has the advantage of being automated, having a much shorter time to result, and, due to the standardized output of results, very little subjectivity between individual technologists and laboratories.

Bioplex ANA testing generates a positive result by simultaneously detecting levels of 11 antibodies. If one or more of the individual antibodies is present at an abnormal level, the ANA Screen will be positive. If levels of all 11 antibodies are negative then the ANA Screen will be negative. In addition, individual antibody results are available if ordered by a physician or added on after the ANA Screen is resulted. The individual antibodies include: anti-dsDNA, anti-Chromatin, anti-Ribosomal P, anti-SSA, anti-SSB, anti-Smith, anti-SmRNP, anti-RNP, anti-Scl-70, anti-Jo-1 and anti-Centromere.

Table 1: Suggested Follow-up testing for Positive ANA:5,6,7

Clinical Suspicion Follow up testing
Systemic Lupus Erythematosus (SLE) anti-dsDNA, anti-Smith, anti-Ribosomal P, anti-Chromatin, anti-SSA
Scleroderma anti-Scl-70, anti-Centromere
Sjogren’s syndrome anti-SSA, anti-SSB
Polymyositis anti-Jo-1
Drug-induced Lupus anti-Chromatin
Mixed connective tissue disease (MCTD) anti-Sm/RNP, anti-RNP, anti-Chromatin

 

It is not possible to predict the result of the Bioplex ANA Screen based on the IFA titer for a patient because there is no direct correlation between IFA titers and the testing of specific autoantibodies. However, in general, as the IFA titer increases, the likelihood of a positive Bioplex ANA Screen also increases. For patients that have previously been monitored by IFA, the following may help identify appropriate follow-up testing:

Table 2: Common IFA patterns and associated follow-up testing:1

Pattern Follow up testing
Speckled anti-RNP, anti-Smith, anti-SmRNP, anti-SSA, anti-SSB
Homogeneous anti-Chromatin, anti-dsDNA
Centromere anti-Centromere
Cytoplasmic anti-Jo-1, anti-Ribosomal P
Mixed Anti-Scl-70

 

Some samples that are positive by IFA may be negative by the Bioplex ANA screen. One of the IFA patterns, the nucleolar pattern, includes antibodies that are not measured by the Bioplex ANA screen. Studies have shown that this pattern has a poor positive predictive value for its associated diseases (i.e., polymyositis, systemic lupus erythematosus, and scleroderma).7 Additionally, several antibodies that are detected by the Bioplex (anti-Scl-70, anti-Centromere, anti-RNP, anti-SSA) may be positive in these patients.4 Patients who are currently being monitored with IFA and exhibit the nucleolar pattern should be interpreted with caution when screened using the Bioplex method.

The inclusion of anti-chromatin is a new feature. Studies have found that anti-chromatin antibodies are more often positive in SLE patients than anti-dsDNA,4 correlate better with kidney involvement than anti-dsDNA,6 and are often detected in patients with Drug Induced Lupus.9 Unfortunately anti-Chromatin is not standardized and has variable performance across manufacturers. Therefore results using assays other than the Bioplex should not be used interchangeably. Figure 1 illustrates the recommend testing process using multiplex flow immunoassay-based ANA screening.

Acknowledgements: We thank the UC Davis Division of Rheumatology, Allergy, and Clinical Immunology for their feedback.

 Figure 1. Autoimmune Testing Algorithm

Autoimmune Testing Algorithm

References:

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  7. Reveille JD, Solomon DH; American College of Rheumatology Ad Hoc Committee of Immunologic Testing Guidelines. Evidence-based guidelines for the use of immunologic tests: anticentromere, Scl-70, and nucleolar antibodies. Arthritis & Rheumatism (Arthritis Care & Research) 2003; 49:399-412.
  8. Mahler M, Kessenbrock K, Szmyrka M, Takasaki Y, Garcia-De La Torre I, Shoenfeld Y, Hiepe F, Shun-le C, von Mühlen CA, Locht H, Höpfl P, Wiik A, Reeves W, Fritzler MJ. International multicenter evaluation of autoantibodies to ribosomal P proteins. Clin Vaccine Immunol. 2006; 13:77-83.
  9. Burlingame RW, Cervera R. Anti-chromatin (anti-nucleosome) autoantibodies. Autoimmunity Reviews 2002; 1:321-328.
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