Article
Are Gold Standards Also Gold Handcuffs?
The 2009 position statement of the American College of Rheumatology (ACR) regarding ANA screening methods states:
“1) The ACR supports the immunofluorescence antinuclear antibody (ANA) test using Human Epithelial type 2 (HEp-2) substrate, as the gold standard for ANA testing.
2) Hospital and commercial laboratories using alternative bead-based multiplex platforms or other solid phase assays for detecting ANAs must provide data to ordering healthcare providers on request that the alternative assay has the same or improved sensitivity compared to IF ANA.” 4
This position on ANA testing methods was updated in 2019 in a joint recommendation of the ACR and the European League Against Rheumatism (EULAR) on systemic lupus erythematosus (SLE) classification:
“Since some SLE centers do not have access to HEp-2 ANA, and in view of ongoing work on the standardization of serology and potential future advances in the field … testing by immunofluorescence on HEp-2 cells or a solid phase ANA screening immunoassay with at least equivalent performance is highly recommended.” 5
In other words, the ACR and EULAR recognize the need to consider newer and potentially advantageous technologies beyond the traditional HEp-2 immunofluorescence assay (IFA). The guidelines do not preclude the adoption of new methods; they merely recommend that clinicians be provided with comparative data that demonstrate performance equivalence.
Comparisons of Autoantibody Assay Performance in the Literature
Many studies have been conducted in the last two decades to compare the performance of different autoantibody detection assays, and four key themes have emerged that influence the decision and expectations around updating autoantibody test methods.
-
IFA Challenges
The traditional IFA method for ANA detection is labor-intensive, inefficient, and requires specialized expertise to use.1-3 It is also difficult to achieve reproducibility and consistency within and between laboratories. When performed using the most sensitive protocol with a 1:40 dilution, specificity tends to drop below 50%.2,6
-
Balancing Sensitivity and Specificity
No single ANA detection assay can identify more than 90% of systemic autoimmune cases with more than 50% specificity.2,6 However, using two or more assays for screening and confirmation can increase sensitivity and specificity beyond these levels.3,7
-
Solid Phase Assay Advantages
The clinical performance of solid phase ANA assays (SPAs) can be equivalent to that of IFA assays.2,6 A multiplex bead-based immunoassay (MBIA) has also been reported to be more sensitive than IFA assays when performed with equivalent specificity cutoffs.8 SPAs have significant efficiency and reproducibility advantages over IFAs, including full automation, simple objective read-outs, and screen/confirmation reflex options.
-
Discordance Is Normal
No two autoantibody assays produce identical results, even in qualitative terms. Even if the overall clinical performance of two assays demonstrates equivalent sensitivity, specificity, and predictive value, there may be significant discordance between results obtained with the assays on a sample-by-sample basis. In fact, qualitative concordance is often below 90%.
No Perfect Assays
Updating an autoantibody assay is not simple. The lab needs to balance not only clinical sensitivity and specificity but also operational factors such as efficiency and reproducibility. While there are no perfect assays that meet every lab need completely, replacement or addition of a complementary assay system can have significant benefits.
Key Contributors to Discordance Between Autoantibody Assays
As a result of these comparative studies, many laboratories have integrated solid phase assays such as enzyme-linked immunoassays (EIAs) and MBIAs into their autoimmunity testing algorithms. While this has provided significant benefits for laboratory operations and patient care, the assay discordance rates make some clinicians hesitant to accept results obtained with new assays. It is thus critical to understand the inherent sources of discordance between autoantibody assays when communicating with clinicians about the use of new tests. Five key sources of discordance are summarized below.
Assay Technology
The wide variety of available methods for autoantibody detection includes IFA assays, EIAs, chemiluminescence assays, and MBIAs. While each technology has unique advantages and disadvantages in terms of workflow, cost, and performance, differences between methods contribute to the inherent variability of assay performance and discordance between results. For example, the substrates for IFA autoantibody detection assays are whole cells affixed to slides, whereas MBIAs utilize purified proteins attached to beads.9
Assay Design Specifications
Assay design specifications can affect relative performance, even when equivalent materials and methods are used to measure the same analyte. For example, the choice of buffer can influence the binding efficiencies of target-specific autoantibodies. Assay cutoffs can impact relative performance as well, because the cutoffs for discerning positive from negative samples are highly dependent on the sample sets manufacturers used to develop their assays. Cutoffs are also influenced by the intended uses of assays and the balance of sensitivity and specificity desired by the assay manufacturers. Consequently, samples that test positive at the low end of the range in one assay might test negative with a different assay.9
Antigen Target Production
There is no industry or scientific consensus on standardized antigen target production methods. Manufacturers individually interpret the scientific literature to determine an optimal approach and must establish their own internal metrics to control assay quality and reliability. Assays designed to detect the same analytes may employ versions of the target molecules or cells that differ extensively; examples include synthetic oligos vs. plasmids for dsDNA, or recombinant protein vs. peptides. Even Hep2 cell IFA slide manufacturing is not standardized. This variety of production methods in turn contributes to discordance between results.9
Lack of Reference Autoantibody Standards
Much of the variability in autoimmunity assay performance arises from a lack of internationally agreed-upon reference standards apart from the anti-dsDNA Wo/80 standard. This means units of measurement are arbitrary because there is no common index. Consequently, one cannot compare numerical values across assays made by different manufacturers.9 Although anti-dsDNA assays share a standard unit of measurement, the factors described above still contribute to discordance.
Autoantibody Diversity
The autoimmune response to autoantigens is extremely heterogenous. This makes it difficult to prepare reference standards and contributes to the inherent variability of assay performance.
Method discordance is an inevitable consequence of the many sources of variability inherent to autoantibody assays. Discordant results do not necessarily mean that one test method is better or more accurate than another.
Key Takeaways
- Current guidelines allow the use of alternatives to traditional "gold standard" ANA IFA assays and recommend demonstration of performance equivalence
- Performance equivalence between ANA IFA and solid phase assays has been shown in multiple published studies6,10
- No single autoantibody assay is perfect from a performance perspective
- Some degree of discordance between autoantibody detection methods is inevitable
Why Is This Important?
Staying informed about the performance of different assays and the reasons for discordance is critical for implementing test methods that optimize laboratory operations. Having this information is also important for the lab to discuss assay transitions from IFAs to EIAs and MBIAs with clinicians. Laboratories can improve patient care and optimize operations by utilizing newer autoantibody testing technologies that produce equivalent or better results more effectively and objectively than traditional methods.References
- Retzlaff K (2018). New study reveals limitations in ANA test kits for lupus. https://www.the-rheumatologist.org/article/new-study-reveals-limitations-in-ana-test-kits-for-lupus/, accessed August 4, 2023.
- Snyder MR (2019). A basic guide to ANA testing. https://www.aacc.org/cln/articles/2019/april/a-basic-guide-to-antinuclear-antibody-ana-testing, accessed August 4, 2023.
- Albert C (2020). Addressing the shortcomings of ANA testing by IFA. https://www.captodayonline.com/addressing-the-shortcomings-of-ana-testing-by-ifa/, accessed August 4, 2023.
- American College of Rheumatology (2019). American College of Rheumatology position statement: methodology of testing for antinuclear antibodies.https://rheumatology.org/api/asset/blta48818378bc89445, accessed August 4, 2023.
- Aringer M et al. (2019). 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Arthritis Rheumatol 71, 1400–1412.
- Deng X et al. (2016). Utility of antinuclear antibody screening by various methods in a clinical laboratory patient cohort. J Appl Lab Med 1, 36–46.
- Bizzaro N et al. (2018). The association of solid-phase assays to immunofluorescence increases the diagnostic accuracy for ANA screening in patients with autoimmune rheumatic diseases. Autoimmun Rev 17, 541–547.
- Pérez D et al. (2018). Predictive autoimmunity using autoantibodies: screening for antinuclear antibodies. Clin Chem Lab Med 56, 1771–1777.
- Meroni PL et al. (2014). Standardization of autoantibody testing: a paradigm for serology in rheumatic diseases. Nat Rev Rheumatol 10, 35–43.
- Alsaed OS et al. (2021). Clinical utility of ANA-ELISA vs ANA-immunofluorescence in connective tissue diseases. https://www.nature.com/articles/s41598-021-87366-w, accessed July 24, 2023.
