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Slide 1:

Hello, my name is Danyel Tacker. I am an Associate Clinical Professor in the Department of Pathology, Anatomy, and Laboratory Medicine at West Virginia University. Welcome to this Pearl of Laboratory Medicine on “Antinuclear Antibody Testing.”

Slide 2:

In this Pearl, we will start with a definition of antinuclear antibodies – or, ANA – and clinical disease states with strongest association to ANA. We’ll look at different techniques employed in ANA testing – with emphasis on the gold-standard testing technique – as well as benefits and limitations of these techniques. Finally, we’ll look at current clinical recommendations for ANA testing and reporting practices.

Slide 3:

ANA are defined as autoimmune antibodies that bind to epitopes in the cell nucleus. In most healthy individuals, these components are viewed by the immune system as “self” and evade immune attack. However, in patients with a significant derangement of immune self-recognition, production of autoantibodies can result in clinical illness. Listed here are some of the major classes of nuclear components that are targeted by ANA. Keep in mind that over decades of research, over 150 discrete epitopes have been identified, making ANA a diverse group of antibodies.

Slide 4:

The American College of Rheumatology – or, ACR – strongly recommends testing for ANA in a patient only after a clearly established suspicion of an associated disease state is developed clinically. Thus, ANA should support diagnosis largely for the conditions and indications listed in the table shown, rather than serve as a screening tool for the general population. Note that some of the conditions listed deem ANA results useful for diagnosis, whereas others have ANA positivity listed as a criterion for diagnosis. Several conditions have weaker associations to ANA positivity in terms of diagnosis, but employ ANA testing for monitoring disease status and assessing prognosis.

Slide 5:

The first of the modern ANA testing methods is the indirect immunofluorescence assay, or IFA. Briefly, ANA in the patient serum are allowed to bind to corresponding nuclear epitopes in human epithelium-derived HEp-2 cells on microscope slides. Fluorophore-conjugated detection antibodies bind to the ANA that are bound to the cells; the slide is then viewed with a fluorescence microscope. Typically, testing employs a dilution series, starting with a 1:40 or 1:80 minimal dilution that doubles with each additional dilution prepared. When positive, the staining pattern is reported along with the highest reportable dilution giving a positive fluorescence signal.

Slide 6:

Over the years, variability in IIFA testing and reporting practices pointed out a need for standardization. The International Consensus on ANA Pattern – or, ICAP – answered the call and has published several consensus documents. The ICAP initially gathered expert consensus regarding 28 distinct IIFA patterns found in ANA testing, categorizing them first by nuclear, cytoplasmic, and mitotic findings, and then by general pattern and complexity.

Since the initial release of the classification system in 2016, additional consensus documents released by ICAP have given negative results a classification number (AC-0), addressed the reporting of as-yet unidentified patterns, and named a classification for the DNA topoisomerase I pattern (AC-29).

Slide 7:

With this background in mind, here are some relatively common examples of IIFA staining patterns for ANA, with their associated ICAP classifications. ICAP’s website,, offers free registration and excellent educational resources, as well as access to images, for HEp-2 ANA patterns. It is a long-overdue training tool, available in several languages, and created and maintained by ICAP members.

Slide 8:

Standardization of pattern reporting for ANA is important clinically. Universal language about ANA results can point providers to targeted sub-serology testing, to determine which antigens may be associated with the pattern. This in turn can point to the likely clinical disease and aid in diagnosis, evaluating prognosis, and/or monitoring patients with known disease. Shown here are the first five ICAP classification numbers, with HEp-2 IIFA pattern, likely antigens targeted, and likely disease associations.

Slide 9:

The benefits of IIFA include clinically relevant information about pattern, and relatively good clinical sensitivity.

The limitations of the IIFA technique involve specificity for disease, particularly for weakly- positive test results. Preparation is still manual at most testing sites, with specimens typically batched for cost and workflow reasons; this can prolong turn-around time and delay result reporting. IIFA automation is available, but expensive. Also, the IIFA method requires relatively advanced local technical expertise for testing and interpretation and a dark or light-shielded space for optimal visibility of fluorescence patterns.

Slide 10:

ANA’s first modern evolutionary shift came with ELISA. When testing ANA with ELISA, epitopes are either cloned or extracted from source material – such as HEp-2 cells – and coated on solid- phase wells. ANA in the patient serum are captured when they bind corresponding epitopes, and an enzyme-conjugated detection antibody binds the ANA. Signal is generated from a reaction driven by the enzyme on the detection antibody, and is captured and measured with a colorimetric detector. Signal obtained from a patient specimen is compared to a decision point determined by the analytic calibration, and an index – or, cutoff – is used to determine if ANA are present or not.

EIA-based ELISA can take two different forms. The first form of ANA testing with ELISA can serve as a general screening assay; this is made possible by presenting HEp-2 cell or nuclear homogenates, or a blend of purified common antigens, in the assay wells.

The second form of ANA testing with ELISA serves to target specific antigens associated with IIFA staining patterns; this approach is called sub-serology testing and it commonly serves as the second-line test after a positive ANA pattern is detected.

Slide 11:

The benefits of ANA testing with ELISA methods include the ability to automate, avoidance of dilution series if using the test for screening, and reagents that are relatively inexpensive. Also, the level of technical expertise required to perform testing and result reporting is lower. Assay cutoffs are typically engineered for maximal clinical sensitivity, and high plate capacities allow for large batches, making for efficient work in high-volume laboratories.

The limitations of ELISA-based ANA testing include widely variable clinical performance due to lacking test standardization and differences in solid phase preparation. Also, due to the engineering of these methods to seek high clinical sensitivity, the specificity and overall accuracy of ELISA can be poor. ELISA-based ANA results yield limited clinical information beyond binary “positive or negative” outputs, so they require further testing to determine the clinical relevance of the result. Finally, ELISA performs best when automated, and the available platforms are relatively expensive.

Slide 12:

ANA testing evolved again in the early 2000’s with multiplexed immunoassay – or, MIA – approaches. MIA uses all of the same ideology as ELISA, but advances the technique by presenting epitopes on beads that are individually detectable on the analytic system.

For example, if a positive IIFA points to a need for sub-serology testing of several epitopes, ELISA-based testing would require a separate ELISA batch for each epitope, but MIA-based testing could cover most or all of these epitopes in a single test batch.

Most MIA for ANA include ten to twelve of the most commonly-detected sub-serology antigens. Fluorescence signal from one or more beads representing these markers can enable reporting for the specific antigen yielding signal.

As for ELISA-based ANA testing, MIA reports are typically qualitative, with index-based cutoffs driving the interpretation.

Slide 13:

The benefits of ANA testing with MIA are derived from their intrinsically automated nature and placement on random-access analyzers, giving them rapid cycle times. Also, linking signal to specific epitopes can allow for more specific, pattern-like reporting of results.

The limitations of ANA testing with MIA are also derived from the way the modern systems are designed. Primarily, only limited numbers of epitopes are currently included in MIA-based ANA methods, creating problems for clinical performance in terms of sensitivity and specificity when compared to IIFA and ELISA techniques that use HEp-2 nuclear homogenate as substrate.
Consequently, MIA are not recommended as screening tests for ANA. Another feature of MIA which limits its use in ANA testing is the expense of the testing systems on the market, which are currently considered high.

Slide 14:

With all of these testing options, it would seem difficult to determine which method is the right fit for a laboratory. Something one must consider is the ACR’s position statement about testing methods for ANA. This position statement, most recently reviewed and re-approved in 2015, maintains that IIFA is the gold-standard method for ANA testing. Consequently, regardless of the first-line testing involved at an institution, IIFA is likely to remain the definitive method in the eyes of specialist providers.

Also, due to the known limitations of ELISA- and MIA-based ANA methods, laboratories using these technologies in particular need to demonstrate the same or improved clinical sensitivity compared to IIFA and have data available for provider review.

In addition to these points, the ACR also calls for standardization of ANA testing methodology, reporting practices for ANA and sub-serology testing, and naming the method used for testing in result reports.

Slide 15:

In conclusion, we have discussed ANA testing in the framework of confirming the presence of clinical disease, and assisting clinical specialists in this task.

We have explored various testing modalities in-use currently for detecting ANA – with the greatest emphasis placed on IIFA – and listed the strengths and limitations of each. We are reminded that though many methods currently exist for ANA testing, IIFA remains the gold standard for clinical utility, and that ICAP is leading the effort to standardize reporting for this method.

Finally we have reviewed the American College of Rheumatology’s call for improved laboratory transparency regarding ANA testing methods used. The most important elements to keep in focus are how the tests are used and perform clinically, since both of these have large impacts in the clinical spectrum.

Slide 16: References

Slide 17: Disclosures

Slide 18: Thank You from

Thank you for joining me on this Pearl of Laboratory Medicine, “Antinuclear Antibody Testing.”