An ANA should be ordered whenever there is a strong clinical suspicion of autoimmune disease. In other words, a high pretest probability of the ANA being positive based on the clinician’s suspicion and differential diagnosis leads to quality utilization of the ANA.

It is inappropriate to randomly order an ANA in patients with symptoms not suggestive of autoimmune disease. A false-positive ANA result in such an individual can cause anxiety for both the patient and the provider.


Although a positive ANA is one component in the diagnosis of SLE, a positive ANA in and of itself is not diagnostic of SLE. As with all rheumatology serologies, established sensitivities (true positive rate) and specificities (true negative rate) for ANA exist. It is important for providers to recognize that false-positive ANA results occur. In fact, healthy individuals may have a positive ANA and never develop clinical manifestations suggestive of an autoimmune condition.5

In addition to SLE, a positive ANA can be associated with other rheumatic and non-rheumatic diseases (Table 2).


Table 2. Conditions associated with a positive ANA
Mixed connective tissue disease
Polymyositis/dermatomyositis
Rheumatoid arthritis
Sjögren’s syndrome
Drug-induced lupus
Discoid lupus
Pauciarticular juvenile chronic arthritis
Hashimoto thyroiditis
Graves’ disease
Autoimmune hepatitis
Primary autoimmune cholangitis
Idiopathic pulmonary arterial hypertension
Infection (mononucleosis, hepatitis C, subacute bacterial endocarditis, TB, HIV)
Malignancy (rarely)
Certain drugs (with or without drug-induced lupus)
Source: Schur PH. “Measurement and clinical significance of antinuclear antibodies. UpToDate. Accessed Feb. 13, 2014. Available at http://www.uptodate.com/contents/measurement-and-clinical-significance-of-antinuclear-antibodies

A helpful determinant (together with a thorough history and physical examination) as to whether an ANA is a true-positive representing autoimmune disease is the strength of the titer. In general, low titers (≤1:160) are more likely to be false-positive results.


Rheumatoid Factor

Waaler and Rose first described RF in 1940.6 RFs are immunoglobulin (Ig) antibodies directed against the Fc portion of IgG. There are various types immunoglobulin antibodies, but IgM is the type most commonly measured today. 


As with ANAs, positive RFs can be associated with other rheumatic and non-rheumatic conditions besides rheumatoid arthritis (Table 3). Every positive RF is not absolutely diagnostic of rheumatoid arthritis. False-negative RFs also occur, occasionally leading to a diagnosis of seronegative rheumatoid arthritis when the history and physical are supportive after appropriately ruling out other diagnostic considerations.


Table 3. Conditions associated with a positive RF
Sjögren’s syndrome
Mixed connective tissue disease
Mixed cryoglobulinemia (type II and III)
Systemic lupus erythematosus
Polymyositis/dermatomyositis
Infection (subacute bacterial endocarditis, hepatitis B, hepatitis C)
Sarcoidosis
Malignancy
Primary biliary cirrhosis
Source: Schmerling RH. “Origin and utility of measurement of rheumatoid factors. UpToDate. Accessed Feb. 13, 2014. Available at http://www.uptodate.com/contents/origin-and-utility-of-measurement-of-rheumatoid-factors

RFs should not be ordered in every person who presents with joint pain. Obtaining an appropriate history of the location of joints involved in a patient’s symptoms is vital (rheumatoid arthritis generally does not affect the low back or the distal interphalangeal joints). A high pretest probability should exist before a diagnosis of inflammatory polyarthritis is suspected (Table 4).


Table 4. Distinguishing between inflammatory and noninflammatory arthritis
History Inflammatory arthritis Noninflammatory arthritis
+++ +
Morning stiffness +++ +
Aggravating symptoms Rest Activity
Alleviating symptoms Activity Rest
Examination
Swelling, warmth, erythema, tenderness +++ +
Limited range of motion + +
Extra-articular manifestations +++

False-positive RFs do occur. Higher RF titers are less likely to be false-positive. Additionally, higher RF titers can be associated with greater disease severity, erosions, extra-articular manifestations, and disability.7,8 Positive RFs are noted in healthy individuals and become more common with age.



Anti-Citrullinated Peptide Antibody

ACPA is the newest of the three antibodies discussed in this article, having been used in clinical practice for just over a decade. The most commonly used ACPA assay measures antibodies against cyclic citrullinated peptides (CCPs). Some clinicians also measure ACPA antibodies against mutated citrullinated vimetin (MCV), but this is not as widely ordered as CCP. ACPAs are determined by enzyme-linked immunosorbent assay (ELISA) in the lab.


ACPA can be used in combination with RF when confirming a diagnosis of rheumatoid arthritis. ACPA has a higher specificicity for rheumatoid arthritis (95%) than does RF.9,10

As with ANA and RF, anti-CCP can be seen in other conditions. Notably, anti-CCP has been noted in 7% to 39% of patients with active TB,11-13 but less commonly in hepatitis C infection.14

Common Questions Regarding Rheumatology Serologies

1. I refer patients to labs that offer several techniques for obtaining ANAs. The results of these varying techniques are reported in ­different ways. Which technique should I choose?

There are various methods by which ANAs are tested in a laboratory, including the following: immunofluorescent microscopy (rodent liver or kidney, Hep-2 cell lines), immunodiffusion, hemagglutination, complement fixation, solid-phase immunoassay (ELISA or immunoblotting) and radioimmunoassay.

The American College of Rheumatology updated its position statement in 2011, recommending the immunofluorescent ANA assay as the gold standard based on a thorough review of the literature.15 A positive immunofluorescent ANA assay results in the reporting of a titer and pattern.

Other methods may yield either a positive or negative result or may numerically quantify the result obtained without a pattern reported. Many laboratories rely on other techniques for detecting ANAs because these techniques are less costly and less labor intensive to perform. The immunofluorescene ANA method is felt to be more sensitive for diagnosing SLE and other autoimmune conditions.15

2. Is the pattern of a positive ANA diagnostic of an autoimmune condition?

ANA patterns are provided when an ANA is ordered using the immunofluorescent method and the result is positive. Various staining patterns may be described (i.e., speckled, nucleolar, diffuse, centromere).

Except for centromere, the pattern is generally not helpful in the diagnosis. A centromere pattern can be associated with limited scleroderma, or CREST syndrome (Calcinosis, Raynaud phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia).


Occasionally a health-care provider may receive a report stating that the ANA result shows two patterns. Although not diagnostic, this should raise clinical suspicion for autoimmune disease and is felt to be less, likely a false-positive result. 


3. Should ANAs, RFs, or CCPs be ordered routinely once a diagnosis has been established?

ANAs, RFs, and CCPs are diagnostic laboratory tests and do not measure disease activity. Generally, once a level of certainty is obtained for a given diagnosis, these serologies do not need to be repeated serially, as the results would not be expected to change the course of treatment.

If there is uncertainty regarding a diagnosis or if new signs or symptoms suggest an additional or alternative diagnosis, repeat serologies may be indicated. 


Together with a comprehensive history and physical, other lab tests may be helpful when evaluating for disease activity. These include acute phase reactants (ESR, CRP) and system review labs to monitor organ function. When monitoring lupus patients for lupus nephritis, dsDNA, a specific antinuclear antibody, may be used.


4. I follow several patients who have a high titer ANA (or RF/CCP) but no signs of SLE or other autoimmune condition. This has been verified by a previous rheumatology referral. What should I do?

It is not uncommon to incidentally uncover a patient with positive serologies but no clinical suggestion of autoimmune disease or other explanation for the positive results. The development of autoantibodies may precede the development of clinically recognizable disease by a number of years.16-18

Patients who have positive serologies but no clinical disease should be monitored regularly (every six to 12 months) and instructed to contact a clinician between scheduled appointments if any additional symptoms develop. 


Conclusion

It is vital for health-care professionals to be able to obtain and interpret data when diagnosing and monitoring patients. Knowing which data is pertinent to a clinical investigation is a useful skill when caring for those with rheumatic disease.

Early diagnosis of disease can lead to better outcomes. When used appropriately, rheumatology serologies assist in making early diagnosis and improve patient care. 


Benjamin J. Smith, PA-C, is a rheumatology physician assistant at the McIntosh Clinic in Thomasville, Ga. He would like to thank Victor M. McMillan, MD, and James M. Hunt, DO, for their expert review in the preparation of this article. 


References

  1. Crowson CS, Matteson EL, Myasoedova E, et al. The lifetime risk of adult-onset rheumatoid arthritis and other inflammatory autoimmune rheumatic diseases. Arthritis Rheum. 2011;63:633-639. Available at www.ncbi.nlm.nih.gov/pmc/articles/PMC3078757/.

  2. American College of Rheumatology. Rheumatic diseases in America. Available at www.simpletasks.org/resources/ACR_Whitepaper_SinglePg.pdf. 

  3. Helmick CG, Felson DT, Lawrence RC, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part I. Arthritis Rheum. 2008;58:15-25. 

  4. Hargraves MM. Discovery of the LE cell and its morphology. Mayo Clin Proc. 1969;44:579-599.

  5. Tan EM, Feltkamp TE, Smolen JS, et al. Range of antinuclear antibodies in “healthy” individuals. Arthritis Rheum. 1997;40:1601-1611.

  6. Waaler E. On the occurrence of a factor in human serum activating the specific agglutintion of sheep blood corpuscles. APMIS. 2007;115:422-438. 

  7. van der Heijde DM, van Riel PL, van Rijswijk MH, van de Putte LB. Influence of prognostic features on the final outcome in rheumatoid arthritis: a review of the literature. Semin Arthritis Rheum. 1988;17:284-292.

  8. Cats A, Hazevoet HM. Significance of positive tests for rheumatoid factor in the prognosis of rheumatoid arthritis. A follow-up study. Ann Rheum Dis. 1970;29:254-260. Available at ard.bmj.com/content/29/3/254.long.

  9. Nishimura K, Sugiyama D, Kogata Y, et al. Meta-analysis: diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid factor for rheumatoid arthritis. Ann Intern Med. 2007;146:797-808.

  10. Avouac J, Gossec L, Dougados M. Diagnostic and predictive value of anti-cyclic citrullinated protein antibodies in rheumatoid arthritis: a systematic literature review. Ann Rheum Dis. 2006;65:845-851. Available at www.ncbi.nlm.nih.gov/pmc/articles/PMC1798205/.

  11. Elkayam O, Segal R, Lidgi M, Caspi D. Positive anti-cyclic citrullinated proteins and rheumatoid factor during active lung tuberculosis. Ann Rheum Dis. 2006;65:1110-1112. Available at www.ncbi.nlm.nih.gov/pmc/articles/PMC1798246/.

  12. Kakumanu P, Yamagata H, Sobel ES, et al. Patients with pulmonary tuberculosis are frequently positive for anti-cyclic citrullinated peptide antibodies, but their sera also react with unmodified arginine-containing peptide. Arthritis Rheum. 2008;58:1576-1581. Available at www.ncbi.nlm.nih.gov/pmc/articles/PMC3621955/.

  13. Mori S, Naito H, Ohtani S, et al. Diagnostic utility of anti-cyclic citrullinated peptide antibodies for rheumatoid arthritis in patients with active lung tuberculosis. Clin Rheumatol. 2009;28:277-283.

  14. Liu FC, Chao YC, Hou TY, et al. Usefulness of anti-CCP antibodies in patients with hepatitis C virus infection with or without arthritis, rheumatoid factor, or cryoglobulinemia. Clin Rheumatol. 2008;27:463-467. 

  15. American College of Rheumatology. Methodology of testing for antinuclear antibodies. Available at www.rheumatology.org/ACR/Practice/Clinical/Position/ana_position_stmt.pdf.

  16. Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. 2003;349:1526-1533. Available at www.nejm.org/doi/full/10.1056/NEJMoa021933.

  17. Heinlen LD, McClain MT, Merrill J, et al. Clinical criteria for systemic lupus erythematosus precede diagnosis, and associated autoantibodies are present before clinical symptoms. Arthritis Rheum. 2007;56:2344-2351. Available at onlinelibrary.wiley.com/doi/10.1002/art.22665/full.

  18. Nielen MM, van Schaardenburg D, Reesink HW, et al. Specific 
autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum. 2004;50:380-386. 


All electronic documents accessed February 15, 2014.


This article originally appeared on Clinical Advisor