A 15-year-old girl with a past medical history of asthma presented to our pediatric rheumatology outpatient clinic for evaluation of knee pain, which was associated with a positive antinuclear antibody (ANA). She developed bilateral knee pain 6 months prior to presentation. The pain occurs when she is doing squats and going up several flights of stairs in succession. She denies joint swelling or morning stiffness. The patient has remained physically active despite the pain, participating in Tae Kwon Do, Pilates, and yoga without limitation. She denies rashes, cough, abdominal pain, weight changes, vomiting, diarrhea, fatigue, dry eyes, dry mouth, or recent travel. Family history was noncontributory for autoimmune disease.
Her temperature was 37.1°C, blood pressure 95/66 mm Hg, heart rate 86, and resting rate 18. Her physical assessment was entirely unremarkable with the exception of the musculoskeletal examination. She had markedly hypermobile joints; she was able to hyperextend the bilateral knees and elbows by >10 degrees, touch both thumbs to the volar surface of the forearms, and place her palms flat on the floor with her knees straight. No restriction of any joints were noted, and there was no joint swelling. Stance was notable for bilateral foot hyperpronation. Her gait was normal.
A complete blood count demonstrated a white blood cell count of 6,300 K/uL (granulocytes 43%, lymphocytes 43%, monocytes 8%, eosinophils 5%), hemoglobin of 12.5 g/dL, platelet count of 284,000 K/uL, C-reactive protein (CRP) <0.29 mg/L, erythrocyte sedimentation rate (ESR) of 5 mm/h, a negative urinalysis, and ANA 1:320 (speckled).
This patient had arthralgias without arthritis. Evidence of joint inflammation, such as morning stiffness, swelling, or restricted range of motion, was absent. Articular hypermobility was noted during the assessment; young people with hypermobile joints are known to have increased complaints of musculoskeletal pain. The review of her systems and physical examination did not reveal any results suggestive of autoimmune disease. The blood count, inflammatory markers, and urinalysis were normal. Therefore, we concluded that the positive ANA was not of clinical significance. She was diagnosed with arthralgias, most likely secondary to hypermobility. Physical therapy was recommended for quadriceps strengthening, joint protection education, and establishment of a home-exercise program. Reassurance was provided regarding the positive ANA. No further laboratory evaluations were recommended at the time of evaluation. The family was instructed to return to the rheumatology clinic for re-evaluation of any new or concerning symptoms.
Physicians evaluate children often with complaints such as musculoskeletal pain, rash, or fatigue. It can be challenging to identify those children with a significant likelihood of a serious rheumatologic disease such as systemic lupus erythematosus (SLE) or juvenile idiopathic arthritis (JIA). Therefore, ANA assessment is frequently considered. However, clinicians often overestimate the utility of the ANA in screening for rheumatologic disease. This review clarifies when ANA testing is most likely to be useful, and elucidate guidelines for appropriate follow up for a positive result.1
Antinuclear Antibody Testing: History and Methods
In 1948, Hargraves et al.1 observed an unusual phenomenon in the bone marrow preparations of 25 patients with confirmed or suspected SLE. He described mature polymorphonuclear leukocytes that phagocytized and digested nuclei of other cells.2 As this cell was found exclusively in patients with SLE, he termed it the lupus erythematous (LE) cell, and the new laboratory evaluation became known as the LE preparation (or LE prep). However, it soon became apparent that the presence of this cell lacked both sensitivity and specificity for the diagnosis of SLE. The antinuclear autoimmune activity seen in the LE cell in association with the SLE phenotype led investigators to explore other methods of identifying antinuclear activity in SLE.
In 1950, Coons and Kaplan2 described an immunofluorescence (IF) method for the detection of antigen in tissue cells. Eight years later, Friou3 first used an indirect IF method for the detection of antinuclear antibodies. Compared with the LE cell preparation, the IF ANA test, which was performed on rodent tissues (mouse liver cells), was much more sensitive for the diagnosis of SLE. However, this increased sensitivity was associated with decreased specificity and significant numbers of patients with other diseases, and even healthy individuals were found to have a positive ANA. The human tumor cell line substrate (Hep-2 cell line) later replaced rodent tissues, and became the standard substrate for performing the ANA test. The human cell line is even more sensitive than the rodent line for the detection of ANAs, decreasing specificity still further. Some laboratories have attempted to adjust for this by using a higher titer of ANA as a cutoff for a positive result. For example, on rodent tissues, ANA titers of 1:20 or 1:40 or higher were called positive, whereas on the Hep-2 substrate titers of 1:80 or higher are typically considered positive.3
Currently, the ANA can be performed by IF or via the enzyme-linked immunosorbent method (ELISA). IF is more specific, and more false positives are noted with ELISA. Therefore, the American College of Rheumatology recommends that IF be used.4 This assay evaluates the reactivity of immunoglobulins from a patient’s serum with antigens expressed in human epithelioid cells (Hep-2 cells). To perform the ANA assay, patient serum is diluted with saline by a factor of 40 and is then layered on a slide with a fixed substrate of Hep-2 cells. After a brief incubation with diluted patient serum, the fixed cells are washed and a fluorescent-labeled antihuman immunoglobulin is added. Thus, patient antibodies bound to antigens within the fixed cells will acquire a fluorescent label. If nuclear fluorescence is observed, then the specimen is interpreted as positive. The highest serum dilution factor that still allows for the observation of fluorescence correlates with the titer of the ANA. For example, a positive ANA of 1:80 means that fluorescence is visible at a dilution factor of 80. Most assays can measure a positive titer as low as 1:40 and as high as 1:5,120. Patterns of fluorescence are described as homogeneous, speckled, nucleolar, centromere, atypical, or peripheral in appearance, and are more subjective than ANA titer values as these depend on laboratory technician interpretation. The most common and least specific patterns are homogeneous (also known as diffuse) and speckled. Nucleolar pattern is sometimes described in patients with mixed connective tissue disease or scleroderma, and centromere in those with CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) syndrome. However, ANA patterns are generally nonspecific and not very helpful in diagnosing the presence of a rheumatologic disease. They should be interpreted only in the context of the patient’s clinical and other laboratory findings.
After the development of the ANA IF test, several ANA subserologies were identified and associated with specific autoimmune diseases or presentations. Anti–double-stranded DNA (anti-dsDNA) and anti-Smith are found in many patients meeting criteria for diagnosis of SLE, and are considered to be specific for this disease. The presence of anti-ribonuclear protein (RNP) in the absence of anti-dsDNA and anti-Smith suggests the diagnosis of mixed connective tissue disease. SS-A (Ro) and SS-B (La) are often found in patients with dry eyes or mouth (sicca syndrome) and recurrent parotitis, which are symptoms suggestive of Sjögren’s syndrome. Ro and La are also implicated in neonatal lupus syndromes.
Clinical Utility of Antinuclear Antibody Testing
ANA is sometimes mistakenly characterized as the “lupus test.” Indeed, ANA is present in well over 90% of patients with SLE.5 However, SLE is a complex multisystem illness, and a positive ANA is just one piece of information used in evaluation for this disease. ANA is 1 of the 11 criteria for diagnosis of SLE; at least four criteria must be present to confirm the diagnosis (Table 1). On the other hand, a negative ANA is useful in excluding SLE, as a patient with a negative ANA is unlikely to have SLE. In addition to its association with SLE, ANA can be identified in patients with a variety of other conditions such as SLE JIA, infectious diseases (Epstein–Barr virus, hepatitis c) malignancy (acute lymphoblastic leukemia, inflammatory bowel disease), and medication induced (minocycline, hydralazine, quinidine, isoniazid, methyldopa, chlorpromazine) as well as in healthy people.
Diagnostic Criteria for Systemic Lupus Erythematosus
After SLE, the pediatric rheumatologic disease most associated with a positive ANA is JIA. However, ANA is neither sensitive nor specific in the diagnosis of JIA. In 2004, McGhee et al.6 demonstrated that the ANA was of no utility in discriminating children with JIA from children with other musculoskeletal complaints. The diagnosis of JIA is clinical, and based on the presence of joint swelling for 6 weeks or more in a patient younger than age 16 years, with exclusion of other etiologies. Persistent joint swelling in a child should prompt evaluation by a pediatric rheumatologist, regardless of the presence or absence of ANA. Conversely, in patients with noninflammatory musculoskeletal pain, a positive ANA may not have any clinical significance. Of note, although not useful in diagnosis, ANA positivity in a child with known JIA is associated with an increased risk of uveitis.
The majority of individuals with a positive ANA test do not have a diagnosable rheumatologic condition. Approximately 20% of the healthy adult population at some time during their lives, will have an ANA titer of 1:40 or higher, as measured with the Hep-2 substrate.4 As in adults, most children with a positive ANA do not have a rheumatologic disorder. Malleson et al.7 reviewed 445 positive ANA results in children, performed at a single laboratory (the indications for ANA testing were not included in the study). Of these children, 135 (or approximately 30%) were found to have a rheumatic disease (juvenile arthritis, SLE, mixed connective tissue disease, or juvenile dermatomyositis).7 The majority of children studied lacked convincing evidence of a rheumatic disease.
Given the poor specificity of the ANA, this test must be ordered thoughtfully rather than as part of a broad screen for autoimmune disease. In 2013, as part of the “Choosing Wisely” campaign,8 the American College of Rheumatology surveyed 1,052 rheumatologists to assess which laboratory studies or other practices were considered to be of low value in evaluating patients for a rheumatologic disease. ANA testing was first on the “Top 5 list of things physicians and patients should question.” The respondents overwhelmingly recommended against ANA screening for patients with only nonspecific symptoms, such as fatigue or myalgia. The respondents additionally expressed concerns about sending ANA sub-serologies (including anti-dsDNA, anti-Smith, RNP, SS-A, SS-B, Scl-70, and centromere antibodies, among others) as part of initial testing prior to confirming the ANA titer.1 This extensive testing is costly, and positive values are difficult to interpret. The specificity of a positive test depends on pretest probability of the particular disease tested being present, and therefore varies greatly depending on the clinical situation.
An ANA study may be appropriate when a patient presents with symptoms suggestive of a multisystem autoimmune disease. These can include recurrent fevers, mucocutaneous ulcerations, malar or vasculitic rashes, arthritis, arterial or venous thrombosis, alopecia, or seizures/psychosis, among many others. Laboratory and imaging results that suggest that an ANA study is indicated include cytopenias, proteinuria/hematuria, and polyserositis. As mentioned earlier, ANA status is also informative regarding the risk of uveitis in children with known JIA.
Management of a Positive Antinuclear Antibody
There are no definitive guidelines for follow up of a positive ANA result. If the clinician is concerned about the patient’s symptoms, the authors generally recommend performing a complete blood count, complete metabolic panel, ESR, CRP, and urinalysis. These studies should capture the most significant systemic processes requiring further evaluation or prompt treatment. “Reflex” ANA subtype panels are not recommended nor are the “rheumatology panels” offered by many commercial laboratories. These tests are expensive and are often difficult to interpret because of frequent false-positive results. If the initial ANA was performed via ELISA screen, a repeat test by IF may be helpful. IF is more specific and is reported with a titer. On the other hand, frequent reassessment of a positive ANA is likely to increase parental anxiety without yielding useful information. A transient positive ANA may be detected for 8 weeks or more after some viral illnesses. In other patients, ANA positivity may persist longer, perhaps indefinitely. In 1992, Cabral et al.9 analyzed the persistence of ANAs in children without an identifiable rheumatologic disease. They followed 24 children who had noninflammatory musculoskeletal pain and a positive ANA for between 1 and 11 years. The positive ANA persisted in 21 of the 24 children, but none of them developed an autoimmune or chronic inflammatory rheumatologic disease.8
The ANA titer is of moderate utility in assessing the importance of a positive ANA. The positive predictive value of a positive ANA for SLE or other rheumatologic diseases does rise with increasing titers. Even so, there is no reliable cutoff for a “significant” positive ANA titer. Various authors disagree regarding the importance of ANA titer. McGhee et al.6 found that titers <1:320, particularly in children younger than age 13 years, excluded SLE, although titers of 1:1,280 were highly predictive for lupus. In 2010, Malleson et al.10 concluded that ANA titers <1:640 should usually be ignored unless the child shows signs of SLE or another systemic disease. Nevertheless, patients with SLE with low-titer positive ANAs and healthy patients with persistent, very high-titer positive ANA are sometimes seen in clinical practice. The totality of history, physical examination, and other laboratory and diagnostic test results must be considered together with the ANA titer to accurately assess the implications of a positive ANA.
Patients with a positive ANA are often referred to pediatric rheumatologists for further evaluation. However, the presence or absence of antinuclear antibodies should not be the lone determinant as to whether or not to refer the patient for subspecialist evaluation. Instead, patients with persistent signs and symptoms concerning for rheumatologic disease should be appropriately referred regardless of laboratory results. Conversely, an isolated positive ANA does not mandate pediatric rheumatology evaluation.
Pertinent findings in the patient’s history that could be suggestive of rheumatologic disease include morning stiffness, rash, fatigue, and alopecia, among many others. Physical assessment results, such as joint swelling, malar rash, mouth ulcers, or Raynaud’s phenomenon, and laboratory abnormalities, including cytopenias, proteinuria, or hematuria, depressed complement levels, and elevated inflammatory markers are more worrisome than positive ANA alone.
Understanding when to order and how to interpret a positive ANA appropriately may reduce unnecessary referrals and costly evaluations. Many patients and parents also experience tremendous anxiety due to a misperception that a positive ANA test indicates the presence of a serious rheumatologic disease. ANA testing is an important, but limited piece of information within the broad clinical context, and cannot be interpreted in isolation from the complete history, physical examination, and other laboratory test results.
- Hargraves MM, Richmond H, Morton R. Presentation of two bone marrow elements; the ‘tart’ cell and the L.E. cell. Proc Staff Meet Mayo Clin. 1948;23(2):25–28.
- Coons A, Kaplan M. Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med. 1950;91(1):1–13. doi:10.1084/jem.91.1.1 [CrossRef]
- Friou GJ. Clinical application of lupus serum-nucleoprotein reaction using fluorescent antibody technique. J Clin Invest. 1957;36:890–898.
- Schur P. Know your labs. The Rheumatologist. http://www.the-rheumatologist.org/article/know-your-labs/. Accessed May 28, 2015.
- Silverman E, Eddy A. Systemic connective diseases. In Cassidy J, Petty R, eds. Textbook of Pediatric Rheumatology. 6th ed. Philadelphia, PA: Elsevier; 2011:315–343.
- McGhee JL, Kickingbird LM, Jarvis JN. Clinical utility of antinuclear antibody tests in children. BMC Pediatr. 2004;4:13. doi:10.1186/1471-2431-4-13 [CrossRef]
- Malleson PN, Sailer M, Mackinnon MJ. Usefulness of antinuclear antibody testing to screen for rheumatologic diseases. Arch Dis Child. 1997;77(4):299–304. doi:10.1136/adc.77.4.299 [CrossRef]
- Yazdany J, Schmajuk G, Robbins M, et al. Choosing wisely: the American College of Rheumatology’s Top 5 list of things physicians and patients should question. Arthritis Care Res (Hoboken). 2013;65(3):329–339. doi:10.1002/acr.21930 [CrossRef]
- Cabral DA, Petty RE, Fung M, Malleson PN. Persistent antinuclear antibodies in children without identifiable inflammatory rheumatic or autoimmune disease. Pediatrics. 1992; 89(3):441–444.
- Malleson PN, Mackinnon MJ, Sailer-Hoeck M, Spencer CH. Review for the generalist: the antinuclear antibody test in children - when to use it and what to do with a positive titer. Pediatr Rheumatol Online J. 2010;8:27. doi:10.1186/1546-0096-8-27 [CrossRef]
- Hochberg MC: Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40(9):1725. doi:10.1002/art.1780400928 [CrossRef]
Diagnostic Criteria for Systemic Lupus Erythematosusa
Positive antinuclear antibody
Polyserositis (pleuritis, pericarditis)
Hematologic involvement (leukopenia, lymphopenia, thrombocytopenia, autoimmune hemolytic anemia)
Neurologic phenomena (seizures, psychosis, and many others)
Immunologic phenomena (anti-double–stranded DNA, anti-Smith, or antiphospholipid antibodies)