A Better Way to Manage Antinuclear Autoantibody Testing

EULAR/ACR ANA Testing Guidelines for Systemic Lupus Erythematosus (SLE)

The EULAR and the ACR jointly updated their classification criteria for SLE and published their recommendations in 2019. A key recommendation in their report is the use of an ANA titer of ≥1:80 with a HEp-2 IFA assay as an entry criterion for SLE. The steering committee added a provision stating that the use of a solid phase ANA screening immunoassay that performed as well as or better than an IFA assay was acceptable "in view of ongoing work on the standardization of serology and potential future advances in the field.”²

IFAs and the Ongoing Debate about ANA Testing Methodology

ANA screening by IFA is performed with cells derived from a human epithelial cell tumor line (HEp-2) affixed to a substrate such as a microscope slide. A fluorescently labeled secondary antibody is used to detect ANAs present in the patient sample. As such, ANAs will bind to nuclear antigens in the fixed cells. A sample that tests positive at a screening dilution ranging from 1:40 to 1:160 (depending on the laboratory) is typically diluted further, and the final “endpoint titer” dilution is reported with a staining pattern.³ There are six "basic level" staining patterns that are typically recognized by medical laboratory scientists with IFA expertise but numerous others that require an advanced level of expertise to interpret.⁴ A lack of standardization in the ANA IFA screening methodology and manufacturing of HEp-2 cell substrates, combined with varying levels of individual experience and training, can make interpretation of results inconsistent and somewhat subjective, even for experts.^{5, 6}

Historically, only rheumatologists and clinical immunologists ordered ANA screens to aid in the diagnosis of CTD, primarily SLE.⁷ But as awareness of such diseases grew, clinicians outside these specialties started ordering ANA screens. Today, even general practitioners order ANA screens, sometimes in the absence of classical indications of CTD. ANA testing is now applied broadly to a population with a lower pre-test probability, and the limitations of HEp-2 IFA for ANA screening are increasingly being recognized. A growing body of evidence also indicates that, despite its high sensitivity, the traditional approach of ANA screening by IFA is not effective for detecting some non-SLE CTDs.^{8, 9}

Rheumatologists and clinical laboratory professionals are actively discussing the use of newer and automated technologies for ANA screening.^10–12 These discussions stem from the limitations of HEp-2 IFA assays, which lack manufacturing standardization and include a labor-intensive workflow involving pattern recognition that is not easily automated. Current IFA slide readers offer imperfect and incomplete pattern interpretation, requiring a medical laboratory scientist with expertise to read or review images. There is clearly a need for alternative immunoassays that can complement or replace HEp-2 IFA screening for ANA. Many studies have shown that solid phase assays (SPAs) for ANA detection can provide comparable results with similar clinical utility.^{8, 9, 11, 13} The most commonly used solid phase alternatives for ANA screening, EIAs and MBIAs, are described in the following sections.

EIAs: A First Step Towards Efficiency

Enzyme-linked immunoassays (EIAs) are a more efficient alternative to IFAs and offer several key advantages. With an EIA, an enzyme-labeled anti-human immunoglobulin (Ig) is used to detect antinuclear antibodies that react with antigen bound to a solid surface. The enzyme linked to the antibody converts its substrate to a light-absorbing substance. Sample light absorbance is measured against a standard and the result is reported as a numeric value. A cutoff is specified by the assay manufacturer for interpretation of the numeric value as positive or negative.

EIAs can be used for ANA screening and testing for specific antigens associated with CTDs. EIA workflows can be automated but require batched testing, which is inefficient since it increases turnaround time. Because each EIA only measures one analyte, follow-up testing involving multiple analytes after an ANA screen positive result can be labor intensive, time consuming, and expensive. However, interpreting the data is much more straightforward than interpreting HEp-2 IFA staining patterns.

• EIAs Advantages

  • Both screen and antigen-specific options available
  • Can be fully automated or semi-automated
  • Simple, objective result read-out
  • Cut-offs generally set to maximize sensitivity
  • Can be performed in large batches (beneficial for high-volume laboratories)

• EIAs Limitations

  • Batched testing required, slowing turnaround time
  • Controls/calibrations required for every run
  • Expensive and inefficient to run single-analyte assays if multiple targets are needed
  • Education of clinicians may be needed when switching from or complementing ANA IFA

Driving Efficiency and Patient Care with the Power of Multiplexing

Multiplex bead-based immunoassays (MBIAs) can provide high result throughput while helping clinical laboratories expedite turnaround time and increase testing capacity. MBIAs, run on platforms like the Bio-Rad BioPlex 2200 System, employ polystyrene beads coated with specific ANA antigens. Each antigen-specific bead set has a unique fluorescent label, so different antigen-specific bead sets can be combined into a multi-analyte detection assay. The assay beads are mixed with a patient sample, and ANAs bound to the target antigens on the beads are detected and measured with a fluorescent-labeled anti-human detection reagent. Results are reported semi-quantitatively or quantitatively with unique ranges and cutoffs for the individual analytes.

• MBIAs Advantages

  • Good balance of sensitivity and specificity
  • High result throughput (simultaneous detection of multiple analytes per test)
  • Can be fully automated or semi-automated
  • Random access (enables faster turnaround time)
  • Results linked to specific antibodies of interest
  • Simple, objective result read-out
  • Screening and specific autoantibody results available from one test; no follow-up testing required

• MBIAs Limitations

  • Screening option is based on detection of specific antibodies (may reduce sensitivity for some diseases)
  • Relatively large laboratory footprint
  • Education of clinicians may be needed when switching from or complementing ANA IFA

* SS-A 52 and SS-A 60 are a composite result in the U.S.
† RNP A and RNP 68 are a composite result in the U.S.

Simultaneous Autoantibody Identification

Fully automated multiplex analysis on the BioPlex 2200 System can significantly increase workflow efficiency in clinical laboratories that perform autoantibody testing. The BioPlex ANA Screen panel can simultaneously identify many of the most clinically relevant autoantibodies. Autoantibodies that target double-stranded DNA (dsDNA), extractable nuclear antigens (SS-A 52 and 60, SS-B, Sm, Scl-70, Jo-1, RNP A, RNP 68), Sm/RNP, chromatin, ribosome P, and centromere B can be detected simultaneously in a single test.*

Testing Workflow Optimization

With this broad analytical capability, an ANA test run on the BioPlex 2200 System can be used as an initial screening assay, a confirmatory test, or both. Results for up to 13 individual analytes* can be available immediately if ordered by the clinician without further testing through the add-on feature on the instrument software. In this way, multiplexing on the BioPlex 2200 System can save laboratories a significant amount of time. Full automation, track line compatibility, and continuous (non-batch) testing can save additional time and labor.

Performance

Several comparative studies of SPAs and IFA assays have demonstrated similar clinical performance, and it has been shown that a positive HEp-2 IFA or EIA ANA screening results confirmed with the BioPlex 2200 ANA Screen can have greater predictive value for clinicians.^{8, 9, 11, 13} Another study suggests that results obtained with the BioPlex ANA Screen can be more predictive of a future CTD diagnosis than results from an ANA IFA, when the tests are run with similar specificity.¹⁴ Integrating multiplex ANA testing into existing ANA testing algorithms can thus have potential benefits for patient care.

Integrating Multiplex Immunoassays with Existing ANA Testing Algorithms

Before updating test menus, laboratories should analyze their unique operational and testing needs and carefully evaluate new ANA assay methods. The ACR and EULAR recommend that laboratories provide comparative data demonstrating the efficacy of new ANA testing methods to their clinical clients on request.^1,2

There are multiple ways in which MBIAs can be integrated into ANA workflows. Some laboratories use an MBIA assay such as the BioPlex 2200 ANA panel to identify specific autoantibodies following a positive EIA or IFA ANA screen.^15–20 Other laboratories seek further workflow streamlining by using the BioPlex 2200 ANA Screen panel for both screening and confirmation.²¹ With respect to confirmation, some laboratories may offer test results for all of the analytes included in the panel at one time while others will take a cascade approach.

A common concern in clinical laboratories is how best to communicate test method changes.²² For example, rheumatologists who are used to IFAs could be reluctant to move away from results based on patterns and titers. Many laboratories that move from IFA to a solid phase method will engage early with healthcare stakeholders and share the advantages of adopting the new method in advance. Manufacturers can also typically help the laboratory with the transition. Manufacturers usually have Field Application Specialists who can advise on the performance of the new method, including interpretation of results.

Key Takeaways

Why is This Important?

Clinical labs are experiencing significant growth in autoimmune testing volumes while facing operational challenges including loss of legacy expertise and the need for greater workflow efficiencies. Adding ANA testing on an MBIA system such as the BioPlex 2200 System can help laboratories boost their testing efficiency to meet these challenges.