- Review Article
- Open Access
The universe of ANA testing: a case for point-of-care ANA testing
© The Author(s) 2017
- Received: 7 March 2017
- Accepted: 14 March 2017
- Published: 21 March 2017
Testing for total antinuclear antibodies (ANA) is a critical tool for diagnosis and management of autoimmune diseases at both the primary care and subspecialty settings. Repurposing of ANA from a test for lupus to a test for any autoimmune condition has driven the increase in ANA requests. Changes in ANA referral patterns include early or subclinical autoimmune disease detection in patients with low pre-test probability and use of negative ANA results to rule out underlying autoimmune disease. A positive result can lead to further diagnostic considerations. Currently, ANA tests are performed in centralized laboratories; an alternative would be ANA testing at the clinical point-of-care (POC). By virtue of its near real-time data collection capability, low cost, and ease of use, we believe the POC ANA has the potential to enable a new paradigm shift in autoimmune serology testing.
- Antinuclear autoantibodies (ANA)
- Point-of-care (POC) testing
- Electrochemical biosensor
Autoantibodies are essential serological markers that define and classify most autoimmune diseases. Testing for total antinuclear antibodies (ANA of undefined specificity and which includes anti-cytoplasmic autoantibodies) has become an invaluable tool at both the primary care and subspecialty settings as a window into further clinical investigation. The presence of total ANA triggers follow-up diagnostic studies for specific autoimmune disorders that are part of the diverse clinical landscape seen in rheumatology, neurology, oncology, and in infectious, pulmonary, and renal diseases among others. In many of these conditions ANA constitutes part of classification and diagnostic criteria for those diseases. The total and ANA sub-serology tests also facilitate differential diagnosis and its refinement , predict incipient disease , indicate disease severity or impending flares , serve as prognostic markers for further organ involvement , monitor efficacy of therapy , and asses induction of autoimmunity by drugs .
The present article attempts to summarize current trends in ANA serology testing by diverse physician groups and to provide ideas on how point-of-care delivery of ANA results may produce the framework, knowledge, and practices which may benefit patients, providers, and the health care industry as a whole. While methods for ANA detection have markedly evolved in recent years, newer methodologies generally require expensive instrumentation and/or central clinical laboratories. An inexpensive and reliable point-of-care device for ANA testing could be applicable not only in communities with modern health care infrastructures but also in more resource-poor settings that struggle with provision of medical care and high burden of disease.
Intent to diagnose (subspecialists)
Intent to refer (primary care physicians, PCP)
Intent to case find for early disease prevention (PCPs)
Intent to reassure patient (PCPs and subspecialists)
Testing with the intent of “reassurance” is not infrequent, despite the fact that when done for symptoms with low risk of serious illness, it is doing little to decrease anxiety or resolve complaints, but may reduce further primary care visits .
The total global autoimmune diagnostic tests’ market is predicted to reach $14.2 billion by 2020 . While market growth is due in part to the increased use of multiplex testing in which many analytes from single or multiple samples undergo high-throughput semi-automated screening, there remains an underlying need for only single sample testing for one analyte. For example, there are many circumstances in which a patient presents to primary care or emergency medicine physician with nonspecific symptoms in which the utilization of specific tests such as ANA is particularly informative [11, 17].
Current ANA testing is performed exclusively in centralized clinical laboratories. This is a protracted, labor-intensive, and expensive procedure that can slow the diagnostic process and restricts use in a large segment of the population. Also, the requirement for blood drawing, transport to the testing lab, blood processing, test execution, and communication of results is cumbersome, time consuming, error prone and costly, which detract from its diagnostic value. Primary care physicians recognize delays in test result review as a significant problem affecting quality of care and patient safety—“I wish I has seen this test result earlier”, even raising malpractice concerns .
Comparison between the standard indirect immunofluorescence method and electrochemical sensor for ANA measurement
Costly fluorescence microscope, infrastructure
Access to providers
Off-site clinical laboratory
Substantial training needed
Simple to operate
Readout and interpretation
Subjective signal intensity and pattern
Objective continuous digital scale output
Substantial processing time (~3 h)
Near real-time data (~20 min)
Semi-quantitative + pattern
Quantitative; no pattern
Usually run in sample batches
Single sample per run
Potential for improvement
Readily enhanced at additional cost
Clinical false positives due to DFS70
Control for non-specific binding
We have described development and application of an electrochemical biosensor for rapid quantitation of total ANA having performance characteristics well correlated with ANA titer determination by indirect immunofluorescence in a clinical laboratory . The immunoreactive surface consists of a native autoantigen-rich substrate largely derived from an inexpensive commercially available source that is bound to a porous membrane at high antigen density. By forced transport of the test sample through the membrane loaded with excess autoantigen, antibody–antigen complex formation is complete in less than 3 min. Detection of isotype-specific autoantibodies is achieved by transporting a high-affinity secondary antibody conjugated to peroxidase. Addition of enzyme–substrates results in production of a redox active intermediate transiently captured on the electrode, permitting its detection by amperometry under low voltage. The readout on a digital display is proportional to the amount of antigen-bound antibody. Of particular importance, the autoantigen substrate is devoid of DFS-70/LEDGF, a troublesome antibody-binding ligand of no diagnostic value that detracts from the utility of ANA testing for autoimmune disease . Unlike multiplex testing that is restricted to a limited number of selected autoantigens, the total ANA biosensor employs a complex mix of potential autoantigens and has the plasticity and capacity to accommodate additional antigenic species to produce a universal platform for ANA screening.
In 2012, the American Board of Internal Medicine Foundation (Philadelphia, PA) launched the “Choosing Wisely” campaign, aiming to advance a dialogue on avoiding wasteful and unnecessary medical tests, treatments, or procedures. Specialty society partners American College of Rheumatology  and Canadian Rheumatology Association  contributed recommendations for appropriate ANA utilization, stressing that duplicative ANA testing is not warranted if there is high pre-test probability for immune-mediated disease and/or active disease. In this situation, sub-serology testing for antigen-specific autoantibodies can enable stratification of patients into particular autoimmune diseases . However, in situations where there is minimal or even no suggestion of an immune-mediated disease other than vague symptoms, screening for total ANA could be most impactful . Even though the pre-test probability for an ANA-related disease may be low, testing for ANA offers the chance to case-find pre- or early autoimmune disease so that organ damage might be prevented (see ANA ordering pattern #3). To this end, the availability to primary care physicians of a convenient POC ANA testing platform could enhance the goal of improving patient outcomes and reducing health care costs.
Compliance with ethical standards
Conflict of interest
Human and animal rights
No animals were used for studies that are base of this review.
Blood from donors (used for the studies that are the base of this review) was obtained under auspices of a human subjects protocol approved by the Institutional Review Board.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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