Traditional ANA Screening
Antinuclear antibodies have been shown to be associated with systemic autoimmune rheumatic diseases (SARDs), also known as connective tissue diseases (CTDs); tests for such antibodies are used as an aid in diagnosis of CTDs. In 2009, the American College of Rheumatology (ACR) released a position statement supporting the HEp-2 immunofluorescence assay (IFA) method as the “ gold standard for ANA testing.”2 The position statement had a significant effect on the clinical laboratory community. Many labs remained loyal to HEp -2 IFA. Other labs, which had previously switched to solid phase methods, switched back to HEp-2 IFA or added HEp-2 IFA as an option following the release of the position statement.

In 2019, the ACR and the European Alliance of Associations for Rheumatology (EULAR) released classification criteria for systemic lupus erythematosus (the most commonly diagnosed CTD).3 For ANA screening, “testing by immunofluorescence on HEp-2 cells or a solid phase ANA screening immunoassay with at least equivalent performance is highly recommended.” Though the term “gold standard” does not appear in the 2019 classification criteria, many in the clinical laboratory community today continue to reference HEp-2 IFA as the “gold standard” for ANA screening.
Using the HEp-2 IFA method requires in-depth technical expertise, as the test results are reported as fluorescent staining patterns that require advanced training to identify. In addition, for positive HEp-2 IFA test results, clinical laboratories typically dilute the patient sample further to determine an endpoint titer, or the dilution at which the patient sample appears negative. This also requires the expertise of an MLS, as the distinction between a weak positive and a negative is very small.
Automated IFA slide readers with pattern recognition capabilities have been introduced in the market. These readers have helped minimize transcription errors. However, the pattern recognition capabilities are limited, and an experienced MLS is still required to review the images.
Loss of Expertise
What should clinical laboratories do when they lose their MLSs with the unique expertise of identifying HEp-2 IFA patterns? Is the only option to send samples out to a reference lab for HEp-2 IFA testing?
At first glance, send-out testing can appear to be an attractive solution. The lab does not have to spend time and money to identify and recruit MLSs with HEp-2 IFA slide reading expertise, and the cost of implementing send-out testing is relatively low. However, the cost-per-test may be quite expensive, and there are hidden costs incurred due to the additional handling and administrative tasks required to manage the process. In addition, send-out testing extends the time between sample receipt and delivery of the test result to the clinician, thereby delaying patient care.
Solid Phase Alternatives for ANA Screening
Instead of resorting to send-out testing, laboratories can consider moving away from HEp-2 IFA testing and adopting solid phase alternatives for ANA screening in-house.

Over the last 20+ years, many solid phase assays (SPAs) have been introduced as alternatives to the HEp-2 IFA method for ANA screening. These include screening through enzyme-linked immunosorbent assays (ELISAs), line blots, chemiluminescent assays, and multiplex bead-based assays (MBIAs).
A major advantage of solid phase methods for ANA screening is that they use a predetermined cut-off value as provided by the manufacturer (except for line blots, in which interpretation of results can be subjective when read manually). This provides the clinical laboratory and clinician with an objective test result without requiring an MLS with specialized expertise.
MBIAs provide a further advantage in that multiple autoantibodies can be detected simultaneously in a single test. A composite ANA result is provided and the results for the individual autoantibodies assays can be revealed through the instrumentation software, requiring no additional follow-up testing. Therefore, MBIAs introduce an added level of efficiency for the clinical laboratory over other SPAs and can improve turnaround time.
Compromising on Quality
Are clinical laboratories compromising on the quality of results by switching from HEp-2 IFA to solid phase methods for ANA screening?
Limitations of HEp-2 IFA
In recent years, the rheumatology and clinical laboratory communities have recognized and actively discussed the limitations of HEp-2 IFA.4 Variability in HEp-2 IFA testing exists due to several factors including differences in the substrate, screening dilution, conjugate, microscope characteristics, and the experience level of the MLSs reading the slides.
Although there has been some success in harmonization of HEp-2 IFA pattern naming and reporting through the International Consensus on ANA Patterns (ICAP), substantial differences between laboratories remain.5 There will always be variability due to the inherent differences in slide manufacturing as well as the subjectivity of the MLS reading the slides. There are still labs that choose not to report cytoplasmic patterns associated with clinically relevant autoantibodies because they are not technically “antinuclear antibodies.”6 In addition, harmonization of HEp-2 IFA patterns and reporting do not address the significant variability seen in endpoint titer determination (even when slides are from the same manufacturer).7
Effectiveness of Solid Phase Methods
While limitations of HEp-2 IFA have been acknowledged, solid phase methods have been recognized to be just as effective as HEp-2 IFA for ANA screening. Most notably, Deng et al. demonstrated in 2016 that the overall diagnostic performance of IFA, ELISA, and MBIA are not statistically different.8 Instead, the clinical sensitivity and specificity observed varied depending on the cut-off used.
A three-year follow-up study of BioPlex 2200 ANA Screen positive/HEp-2 IFA negative patients suggested that the BioPlex 2200 ANA Screen may have greater sensitivity than HEp-2 IFA for autoantibodies detection and that early detection of these antibodies may predict possible development of connective tissue diseases.9 During the three-year follow-up period, more than 75% of patients tested positive for HEp-2 IFA and more than 85% of patients were diagnosed with a connective tissue disease. This study, along with the Deng et al. study mentioned earlier, suggests that, since SPAs have shown equivalent or better performance than HEp-2 IFAs, they can be adopted without compromising quality.
Taken together, the demonstration of the effects of HEp-2 IFA limitations, alongside the performance of solid phase methods for ANA screening, should reassure clinical laboratories that they are not compromising on quality by bringing a solid phase method in-house. Additional studies have shown that there is a place for both solid phase methods and HEp-2 IFA and that these methods can be complementary to each other.10-13 For instance, laboratories may consider offering HEp-2 IFA testing as a send-out option for rheumatologists when clinical suspicion of a connective tissue disease remains after a negative solid phase ANA screen result.
Conclusion
When HEp-2 IFA expertise is lost in the clinical laboratory, send-out testing for ANA screening is not the only option. Solid phase assays offer an opportunity for laboratories to maintain ANA screening in-house without requiring an MLS with specialized expertise, while also minimizing some of the limitations of HEp-2 IFA. Multiplex bead immunoassays offer additional workflow advantages by reducing the need for further testing after a positive composite ANA screen result is received. In these ways, clinical laboratories can accommodate loss of expertise without compromising on quality of results.
References
- Robinson AT (2023). Healthcare-Severe Shortages of Qualified Medical Laboratory Professionals. Biomed J Sci & Tech Res 48, 39710-39713.
- American College of Rheumatology (2009). American College of Rheumatology position statement: methodology of testing for antinuclear antibodies. https://rheumatology.org/api/asset/blta48818378bc89445, accessed August 22, 2023.
- Aringer M et al. (2019). European League Against Rheumatism/American College of Rheumatology Classification Criteria for Systemic Lupus Erythematosus. Arthritis Rheumatol 71(9), 1400-1412.
- 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 31, 2023
- Vercammen M et al. (2023). Analytical aspects of the antinuclear antibody test by HEp-2 indirect immunofluorescence: EFLM report on an international survey. Clin Chem Lab Med 61, 1199–1208.
- Tebo AE et al. (2021). The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory performance, pattern recognition and interpretation. Auto Immun Highlights 12, 1-10.
- Wener MH et al. (2021). Long-term variability in immunofluorescence titer of antibodies to nuclear antigens observed in clinical laboratory proficiency testing surveys. Archives of Pathology & Laboratory Medicine 145, 937-942.
- Deng X et al. (2016). Utility of antinuclear antibody screening by various methods in a clinical laboratory patient cohort. Journal of Applied Laboratory Medicine 1, 36-46.
- Pérez D et al. (2018). Predictive autoimmunity using autoantibodies: screening for anti-nuclear antibodies. Clinical Chemistry and Laboratory Medicine (CCLM), 56, 1771-1777.
- Tacker DH and Perrotta PL (2017). Quality monitoring approach for optimizing antinuclear antibody screening cutoffs and testing work flow. The Journal of Applied Laboratory Medicine, 1, 678-689.
- Claessens J et al. (2018). Solid phase assays versus automated indirect immunofluorescence for detection of antinuclear antibodies. Autoimmunity Reviews, 17, 533-540.
- Bizzaro N (2020). Can solid-phase assays replace immunofluorescence for ANA screening? Annals of the Rheumatic Diseases, 79, e32-e32.
- Naranjo L et al. (2020). Algorithm for antinuclear antibodies in subjects with clinical suspicion of autoimmune diseases. Clin. & Exp. Rheum 38, 633-639.