Antinuclear Antibody (ANA) Testing

Effective Date: October 24, 2024

Recommendations and Topics

• Scope
• Key Recommendations
• Background
• Definitions
• Testing
• Indications for Referral
• Resources

Scope

This guideline describes the appropriate use of antinuclear antibody (ANA) testing in the diagnosis of autoimmune Connective Tissue Diseases (CTDs)* in adults ages ≥ 19 years. The guideline does not address ANA testing for the investigation of unexplained infertility, adverse pregnancy outcomes or other CTD investigations in pregnancy, liver disease or thrombotic disorders. 

Key Recommendations

• ANA testing is only indicated if the diagnosis of a CTD (i.e., systemic lupus erythematosus, scleroderma, polymyositis/dermatomyositis, Sjögren’s syndrome) is a significant clinical possibility
• ANA testing is not indicated:
  • as a screening test to evaluate fatigue, back pain, or other musculoskeletal pain without other clinical indications.
  • to confirm a diagnosis of rheumatoid arthritis or osteoarthritis.
  • to monitor lupus flares.
• ANA testing need only be ordered once in most cases.
• When the ANA test result is:
  • Positive: Repeat or serial testing is not indicated, as changes in ANA titres do not correlate with disease activity.
  • Negative: Repeat testing may be indicated only if the clinical presentation has changed.

Background

CTDs are a group of uncommon inflammatory conditions associated with autoimmune dysregulation, that can lead to disability, organ failure and premature mortality.¹ CTDs include systemic lupus erythematosus (SLE), systemic sclerosis, inflammatory myositis (i.e. polymyositis and dermatomyositis), and Sjögren’s syndrome.

As of 2021/22, only 0.56% of the B.C. population was diagnosed with CTD; the estimated incidence per million is 56 for SLE, 19 for scleroderma, and <10 for dermatomyositis and polymyositis. Although the incidence of CTDs is low, ANA testing is frequently ordered. In 2022/23 over 114,000 ANA tests were performed in B.C., at a total cost of $2.2 million. The number of ANA tests ordered greatly exceeds the small number of new cases of CTDs expected per annum. This volume of testing suggests that ANA tests are being ordered for patients with little probability of having ANA-associated CTD. Moreover, 31% of positive ANA tests were from repeat testing with the previous ANA test being positive.^† This represents improper ordering, as repeat testing is not indicated.

 

Definitions

ANA: Antinuclear Antibodies are a class of self-directed antibodies that bind to any cellular component of the nucleus, including proteins, DNA, RNA, and nucleic acid-protein complexes. ANAs are involved in disease pathogenesis, and the ANA test has been the foundation of diagnosis for autoimmune connective tissue diseases, including SLE, Sjögren’s syndrome, and polymyositis/dermatomyositis.^2,3 

ENA: Extractable Nuclear Antigens (ENA) are a subset of ANAs. ENA testing is performed to subclassify patients known to have a positive ANA screening test. 

ENA testing is a multicomponent panel test used to diagnostically distinguish the different CTDs. In B.C., the ENA panel is minimally comprised of dsDNA, anti-Ro (also called anti-SSA), anti-La (also called anti-SSB), anti-Sm (antiSmith antibody), anti-RNP (anti-ribonucleoprotein), anti-Jo-1, and anti-Scl70. Discussion of the disease correlates of these tests is beyond the scope of this document but the interested reader is directed to pertinent reviews. 

Testing

ANA testing is only indicated after clinical assessment reveals signs and/or symptoms suggestive of SLE, scleroderma, Sjögren’s syndrome or polymyositis/ dermatomyositis.⁴

Indications to order an ANA test requires the presence of at least two of the following clinical findings unexplained by other causes (See Table 1: Clinical Features of CTDs).

Table 1: Clinical Features of CTDs⁵

*In the context of a clinical presentation compatible with lupus.⁶

In the absence of two or more of the clinical signs and symptoms listed above, a positive ANA test only confounds the diagnostic process and causes unnecessary anxiety for patients. ANAs are found in up to 16% of the normal population, higher in females and increasing with age.^7,8 Positive ANA tests may also be seen in a wide range of diseases other than CTD where they have no diagnostic or prognostic value. For example, individuals with viral infections and conditions including malignancies, primary biliary cirrhosis, and other autoimmune disorders may have an elevated ANA. Additionally, certain medications [e.g., statins, ß-blockers, Angiotensin-converting enzyme (ACE) inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs) and biologics] can induce lupus-like symptoms, which are accompanied by positive ANA results.^9,10 

More selective ordering of ANA tests would reduce the volume of tests performed, improve the predictive value of the test, reduce avoidable misdiagnoses, reduce unnecessary referrals, and avoid inappropriate therapy.¹¹ However, atypical clinical presentations of CTD can occur, so clinical judgment should guide ANA testing in these cases.

ANA testing is indicated:

• if a CTD is clinically suspected based on the presentation of two clinical features (See Table 1: Clinical Features of CTDs).

ANA testing is NOT indicated:

• to investigate a possible diagnosis of rheumatoid arthritis or osteoarthritis
• to investigate chronic fatigue, back pain, or other musculoskeletal pain unless accompanied by two or more of the clinical findings listed above (See Table 1: Clinical Features of CTDs). 
• for serial monitoring, as changes in ANA titres do not correlate with disease activity (e.g., lupus flares). 
• as a screening test for disease in the general healthy population

Repeat ANA testing is rarely indicated:  Repeat testing after a prior negative test is only indicated when there is a change in the patient’s condition that now suggests the emergence of a CTD. Although higher ANA values are more specific for CTD, serial monitoring of ANAs does not contribute to improved clinical outcomes.^12,13

ENA testing

ENA testing may help characterize nuclear antibodies associated with specific CTD diagnoses and may help to further distinguish CTD diagnoses. It is not a standalone test and will be performed by laboratories in B.C. only following a positive ANA. It may be ordered by practitioners in the presence of a positive ANA or performed at laboratory discretion to help further characterize high ANA titres. In either case, the primary care practitioner may receive a quantitative report of tests in the ENA battery. ENA interpretation is challenging. Prior to making a clinical decision based on ENA results, a call to the RACE line or a discussion with a rheumatologist, internist, or laboratory consultant is recommended.

For patients with known CTD who are pregnant or planning pregnancy, repeat ANA and ENA testing is often indicated. Please consult with a rheumatologist, OB/GYN or appropriate specialist to understand what additional testing is required for these known CTD patients. The ordering physician should indicate “pregnant” or “planning pregnancy” status on the requisition.

Indications for Referral

If the patient has two clinical symptoms suggestive of CTD (See Table 1: Clinical Features of CTDs) and a positive ANA/ENA, a referral or a call to the RACE line is recommended. Based on positive ANA/ENA results alone, without clinical correlates, only a minority of patients need referral.

Resources

Abbreviations:

CTDs                     Connective Tissue Diseases

ANA                       Antinuclear Antibody

SLE                       Systemic Lupus Erythematosus

ENA                       Extractable Nuclear Antigens

dsDNA                  Double stranded DNA

ACE                      Angiotensin Converting Enzyme

NSAIDs                Non-Steroidal Anti-Inflammatory Drugs

OB/GYN               Obstetrics and Gynaecology

Practitioner Resources

• RACE Line: Rapid Access to Consultative Expertise Program: raceconnect.ca/. A phone consultation line for physicians, nurse practitioners and medical residents. If the relevant specialty area is available through your local RACE line, please contact them first.
• Real Time Virtual Support (RTVS):  Available to support practitioners in rural, remote, and First Nations communities in BC.
  • Rural Urgent Doctor in aid (RUDi) for instant emergency medicine support.
• Provincial Laboratory Medicine Services (PLMS):  Accountable for various functions related to the administration of lab services in B.C., and for implementing policies and processes on behalf of the Ministry of Health. To add a test to an existing order, change a test priority or to obtain a test result by phone, see PLMS Contact Us page, call 604-714-2800 or email plmsinfo@phsa.ca.
• PathwaysBC: An online resource that allows physicians, nurse practitioners and their office staff to quickly access current and accurate referral information. This includes specialists and specialty clinic wait times and areas of expertise. See: pathwaysbc.ca/login
• Health Data Coalition: An online, physician-led data sharing platform that can assist you in assessing your own practice in areas such as chronic disease management or medication prescribing. HDC data can graphically represent patients in your practice with chronic diseases in a clear and simple fashion, allowing for reflection on practice and tracking improvements over time. See: Health Data Coalition – Better Information. Better Care. Better Patient Outcomes. (hdcbc.ca)

Patient, Family and Caregiver Resources

• HealthLinkBC: Patients can call HealthLinkBC at 8-1-1 toll-free in B.C., or for the deaf and the hard of hearing, call 7-1-1. They will be connected with an English-speaking health-service navigator, who can provide health and health-service information and connect them with a registered dietitian, exercise physiologist, nurse, or pharmacist. See: healthlinkbc.ca/
  • HealthLinkBC ANA testing

Laboratory Fee Codes

90280 ($20.44 per test)

90281 ($16.24 per test)

Associated Documents

The following documents accompany this guideline:

• List of Contributors

References

1. Firestein GS, Budd RC, Gabriel SE, McInnes IB, O’Dell JR. Kelley’s Textbook of Rheumatology E-Book. Elsevier Health Sciences; 2012. 2345 p.
2. Kumar Y, Bhatia A, Minz RW. Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited. Diagn Pathol. 2009 Jan 2;4:1.
3. Nosal RS, Superville SS, Amraei R, Varacallo M. Biochemistry, Antinuclear Antibodies (ANA). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 [cited 2024 Feb 16]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK537071/
4. Abeles AM, Abeles M. The clinical utility of a positive antinuclear antibody test result. Am J Med. 2013 Apr;126(4):342–8.
5. Aringer M, Costenbader KH, Daikh DI, Brinks R, Mosca M, Ramsey-Goldman R, et al. 2019 EULAR/ACR Classification Criteria for Systemic Lupus Erythematosus. Arthritis Rheumatol Hoboken NJ. 2019 Sep;71(9):1400–12.
6. Abrol E, Coutinho E, Chou M, Hart M, Vincent A, Howard R, et al. Psychosis in systemic lupus erythematosus (SLE): 40-year experience of a specialist centre. Rheumatology. 2021 Dec 1;60(12):5620–9.
7. Dinse GE, Parks CG, Weinberg CR, Co CA, Wilkerson J, Zeldin DC, et al. Increasing Prevalence of Antinuclear Antibodies in the United States. Arthritis Rheumatol. 2022;74(12):2032–41.
8. Satoh M, Chan EKL, Ho LA, Rose KM, Parks CG, Cohn RD, et al. Prevalence and sociodemographic correlates of antinuclear antibodies in the United States. Arthritis Rheum. 2012 Jul;64(7):2319–27.
9. Kraev K, Hristov B, Uchikov P, Kraeva M, Basheva-Kraeva Y, Valova S, et al. Comprehensive Exploration of Antinuclear Antibodies (ANAs): Unveiling Clinical Significance, Associations with Cancer, and the Nuances of Differential Diagnosis in Positive ANA Patients. Diagnostics. 2024 Jan;14(3):320.
10. Solhjoo M, Goyal A, Chauhan K. Drug-Induced Lupus Erythematosus. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 [cited 2024 Apr 16]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK441889/
11. Man A, Shojania K, Phoon C, Pal J, de Badyn MH, Pi D, et al. An evaluation of autoimmune antibody testing patterns in a Canadian health region and an evaluation of a laboratory algorithm aimed at reducing unnecessary testing. Clin Rheumatol. 2013 May;32(5):601–8.
12. Alsaed OS, Alamlih LI, Al-Radideh O, Chandra P, Alemadi S, Al-Allaf AW. Clinical utility of ANA-ELISA vs ANA-immunofluorescence in connective tissue diseases. Sci Rep. 2021 Apr 15;11(1):8229.
13. Yeo AL, Leech M, Ojaimi S, Morand E. Utility of repeat extractable nuclear antigen antibody testing: a retrospective audit. Rheumatol Oxf Engl. 2023 Mar 1;62(3):1248–53.

* Systemic Autoimmune Rheumatic Diseases (SARDs) is the emerging term for autoimmune disease states that encompass the Connective Tissue Diseases (CTDs). However, for the purposes of this document, we will continue to use the term CTDs.

†  Antibody Testing Update on Test Utilization Fiscal Year 2022/23, provided by B.C. Provincial Laboratory Medical Services

BC Guidelines are developed for the Medical Services Commission by the Guidelines and Protocols Advisory Committee, a joint committee of Government and the Doctors of BC. BC Guidelines are adopted under the Medicare Protection Act and, where relevant, the Laboratory Services Act. Disclaimer: This guideline is based on best available scientific evidence and clinical expertise as of October 24, 2024. It is not intended as a substitute for the clinical or professional judgment of a health care practitioner.