The Clinical Utility of a Positive Antinuclear Antibody Test Result

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1 CLINICAL RESEARCH STUDY The Clinical Utility of a Positive Antinuclear Antibody Test Result Aryeh M. Abeles, MD, Micha Abeles, MD Division of Rheumatology, University of Connecticut Health Center, Farmington. ABSTRACT BACKGROUND: This retrospective study investigated the clinical utility of a positive antinuclear antibody (ANA) test performed outside of the rheumatology setting. Prior studies have investigated the frequency of ANA positivity within the general population. The purpose of this investigation was to evaluate the clinical utility of a positive ANA test result in a real-world setting by reviewing the final diagnoses of patients who were referred to a tertiary rheumatology clinic for evaluation of a positive ANA test result. METHODS: We reviewed the records of patients presenting to the authors between July 2007 and July Patients were included in the evaluation if they were referred for a positive ANA test result. All relevant descriptive and laboratory data were collated, as were the initial reasons for ordering ANA testing and the ultimate diagnoses reached. Positive predictive values for a positive ANA test result were calculated for all antinuclear antibody-associated rheumatic diseases and for lupus specifically. RESULTS: A total of 232 patients were referred for a positive ANA test result. The positive predictive value of a positive ANA test result in this cohort was 2.1% for lupus and 9.1% for any antinuclear antibodyassociated rheumatic disease. No antinuclear antibody-associated rheumatic disease was identified in patients with an ANA 1:160. The most common reason for ordering ANA testing was widespread pain (54/232, 23.2%). CONCLUSIONS: In this retrospective study, more than 90% of patients who were referred to a tertiary rheumatology clinic for a positive ANA test result had no evidence for an ANA-associated rheumatic disease. The poor predictive value of a positive ANA in this cohort was largely attributable to unnecessary testing in patients with low pretest probabilities for ANA-associated rheumatic disease Elsevier Inc. All rights reserved. The American Journal of Medicine (2013) 126, KEYWORDS: Antinuclear antibody; Lupus; Positive predictive value Spiraling healthcare costs have become an increasing problem in the United States, and one cause is unnecessary and inappropriate testing. 1 Screening tests that have high background positivity can prove particularly taxing to the healthcare system. When unnecessarily requested, tests that screen for uncommon diseases but have a high rate of background positivity can lead to multiple unfavorable consequences, including misdiagnosis, unnecessary or potentially harmful Funding: None. Conflict of Interest: None. Authorship: Both authors had access to the data and played a role in writing this manuscript. Requests for reprints should be addressed to Aryeh M. Abeles, MD, Division of Rheumatology, Medical Arts and Research Building, UCHC 263 Farmington Ave, Farmington, CT address: aabeles@uchc.edu follow-up testing, and even inappropriate treatment. The antinuclear antibody (ANA) test is one such example. Antinuclear antibodies were discovered in 1957 in the sera of patients with systemic lupus erythematosus. 2,3 These antibodies were recognized to react with then-unspecified antigens from human nuclei (now appreciated to include multiple possible antigens, including the Smith, SSA and SSB antigens, and double-stranded DNA). Although antinuclear antibodies were discovered in association with systemic lupus erythematosus, a number of other autoimmune diseases were found subsequently to be associated with them, most of which fall under the rubric of rheumatology, including systemic sclerosis (scleroderma), Sjögren s syndrome, and mixed connective tissue disease. The most commonly used method for testing is the indirect fluorescent assay. 4 The indirect fluorescent assay is performed by in /$ -see front matter 2013 Elsevier Inc. All rights reserved.

2 Abeles and Abeles Clinical Utility of Positive Antinuclear Antibody 343 cubating patient serum on a cellular antigen substrate (typically HEp-2 cells, which are clonal cancer cells useful in this assay for their large nuclei), and then, after a washing step, detecting patient antibodies bound to nuclear antigens through the addition of fluorescence-tagged anti-immunoglobulin G antibodies. Although an American College of Rheumatology expert panel has recognized indirect fluorescent assay as the gold standard for ANA testing, 5 the method has a number of acknowledged shortcomings, including interobserver variability and differences in sensitivity and specificity of the test depending on the substrate used (eg, different HEp-2 kits). Although a positive ANA test result can reflect an interaction between serum antibodies and as many as 150 different nuclear antigens, only a minority of these antigens are well described and have established associations with disease, whereas many of the remaining antigenantibody reactions may not be clinically relevant. Other widely CLINICAL SIGNIFICANCE used, commercially available ANA kits use different techniques, including enzyme-linked immunosorbent assay and the multiplex bead assay, and studies have demonstrated that ANA test results depend partly on the assay used. 6,7 Slater et al 8 determined the positive predictive value of all consecutive ANA testing run at a single medical center and concluded that positive ANA test results are poorly predictive of connective tissue disease. That study included all testing ordered within a single academic healthcare system, by all providers, on both hospitalized and outpatients. However, to date, the strengths and limitations of ANA testing are less well studied in real-world outpatient settings. Our goal was to evaluate the clinical utility of the positive ANA test result in a real-world setting, in a purely outpatient population referred by a heterogeneous group of medical providers (who used a heterogeneous group of testing centers). To do so, we determined the final diagnoses of patients who were referred to a tertiary rheumatology clinic for evaluation of a positive ANA test result and reviewed these outcomes in the context of descriptive data as to why testing was ordered by the referring physicians. To our knowledge, ours is the first study to investigate the positive predictive value of ANA testing in a cohort of adult patients referred to a rheumatology center specifically for a positive ANA test result. Our results indicate that ANA testing outside the rheumatology setting is often inappropriately ordered and interpreted, and that in clinical practice, the test has a poor positive predictive value for diagnosing connective tissue disease in general, and lupus in particular. A positive antinuclear antibody (ANA) test result was rarely clinically meaningful. The positive predictive value of an ANA test was less than 10% for any connective tissue disease and less than 3% for lupus. Most patients were sent for ANA testing despite low pretest likelihood of connective tissue disease. ANA testing often was ordered indiscriminately, which can lead to adverse consequences. MATERIALS AND METHODS The University of Connecticut Health Center Institutional Review Board granted full approval for this study. We retrospectively reviewed the records for all consultations presenting to the authors at the University of Connecticut Health Center rheumatology clinic between July 2007 and July Patients were included in the evaluation if they had been referred for subspecialty evaluation of a recently ordered positive ANA test result. Patients presenting with a previously diagnosed antinuclear antibody-associated rheumatic disease and sent for a second opinion or transfer of care from an outside rheumatologist were excluded. Positive predictive values were determined as the number of patients within the cohort (or subcohort, as defined below) with an antinuclear antibody-associated rheumatic disease divided by the number of total patients in the study (or total patients in the subcohort). The following were considered to be antinuclear antibodyassociated rheumatic diseases: systemic lupus erythematosus (diagnosed according to American College of Rheumatology criteria), 9,10 Sjögren s syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, and undifferentiated connective tissue disease. Although antinuclear antibody positivity is frequently seen in rheumatoid arthritis, the disease is not considered to be an antinuclear antibody-associated illness, and thus was not counted as such in this study. The medical chart for each patient was reviewed, and all relevant data were extracted by the first author (AMA), including the reason for ANA testing, results of laboratory tests ordered at the time of the visit (and all laboratory results before and after the visit), and all other pertinent objective data, including examination findings and the patient s ultimate diagnosis. Additional laboratory testing was done at the discretion of the evaluating rheumatologist at the time of consultation as clinically appropriate. Once data, including patient diagnoses, were recorded, the second investigator independently reviewed the electronic medical records, the extracted data, and final diagnoses to confirm agreement. Discussion took place wherever questions arose, such that there was agreement on all patients diagnoses by the end of the study. Positive predictive values for a positive ANA test result were further evaluated after stratification according to titer, as well as according to clinically relevant key patient characteristics, including age (younger [ 65 years] vs older

3 344 The American Journal of Medicine, Vol 126, No 4, April 2013 Table 1 Positive Predictive Value of Antinuclear Antibody Screen, by Titer ANA Titer No. of Patients (Total 232; Total With Available Titer at Time of Evaluation 227)* No. of Patients with ANA-Associated Rheumatic Diseases (and No. With SLE) PPV (for any AARD) of ANA Test Using This Titer as Cutoff Value 1: (5) 8.8% 2.2% 1: (5) 10% 2.5% 1: (5) 11.6% 2.9% 1: (4) 18.9% 4% 1: (4) 26.9% 6% 1: (2) 38.9% 5.6% 1: (0) 46.2% N/A 1: (0) 57.1% N/A No titer 5 1 (0) N/A N/A PPV of ANA Test for SLE ANA antinuclear antibody; AARD antinuclear antibody associated rheumatic disease; PPV positive predictive value; SLE systemic lupus erythematosus. *Number of all patients in cohort with ANA positivity of at least this titer (ie, in this table, all patients with a titer 1:40, including those with titers up to 1:5120, are included in the first row, all those with a titer of 1:80 are included in the second row, and so forth). patients), gender, setting where the ANA test was drawn (categorized as those who had ANA testing performed within our academic medical center vs testing elsewhere), and characteristics of the referring provider (academic vs community-based, primary care physician vs specialist). Subsequently, sensitivity analysis was performed using different cutoff values to define a positive test. RESULTS Of 1306 consultation visits over the 2-year period, 227 patients (17.4%) had been referred for a positive ANA of 1:40 performed via indirect fluorescent assay, and an additional 5 patients had been referred for a positive antinuclear antibody screening test result without an associated titer performed. All ANA tests and referrals were ordered by nonrheumatologists. There were a total of 138 referring providers, of whom more than 80% (112/138) were primary care practitioners (internal medicine, family practice, pediatrics, and obstetrics-gynecology). Correspondingly, 85% of the referrals came from primary care providers (197/232). The breakdown of specialists in the referral base was as follows: dermatology (3), orthopedic surgery (4), pulmonary (4), physiatry (2), neurology (4), ophthalmology (2), gastroenterology (3), hematology (2), infectious disease (1), and endocrinology (1). Among the 232 patients with a positive ANA test result, extensive rheumatologic evaluation revealed an antinuclear antibody-associated rheumatic disease in 21 individuals (including 1 of the 5 who had a positive ANA screen without a reported titer). By using a cutoff titer for test positivity of 1:40 (and excluding the patient with no titer performed in this analysis), the positive predictive value of an antinuclear antibody so obtained was 8.8% for any antinuclear antibody-associated rheumatic disease (Table 1). Using higher-titer cutoffs yielded slightly higher positive predictive values. For example, setting the threshold value for positivity at 1:160 (as our university laboratory recommends) yielded a positive predictive value of 11.6%. No patients with an ANA titer 1:160 had an antinuclear antibody-associated rheumatic disease (0% positive predictive value), and the overwhelming majority (18/21, 85.7%) of patients presenting with an antinuclear antibody-associated rheumatic disease in this study had an ANA of 1:640. Nonetheless, even when using a titer of 1:640 as a cutoff, the positive predictive value for antinuclear antibody positivity was still fairly low (26.9% for any antinuclear antibody-associated rheumatic disease and 6% for systemic lupus erythematosus). The specific breakdown of antinuclear antibody-associated rheumatic diseases diagnosed in this cohort is listed in Table 2 by ANA titer. Only 5 patients were diagnosed with lupus, the disease that was generally being screened for when an ANA was ordered; the positive predictive value for any positive ANA with regard to sys- Table 2 Antinuclear Antibody-Associated Rheumatic Diseases Diagnosed in Patients, by Antinuclear Antibody Titer Reported ANA Titer No. of Patients No. of Patients with AARD (and Specific Diagnoses) 1:40 (and 1:80) :80 (and 1:160) :160 (and 1:320) 71 1 (SLE) 1:320 (and 1:640) 34 1 (SjS) 1:640 (and 1:1280) 31 4 (2 SLE, 2 SjS) 1:1280 (and 1:2560) 23 8 (2 SLE, 4 SjS, 1 SSc, 1 UCTD) 1:2560 (and 1:5120) 6 2 (1 SSc, 1 SjS) 1: (1 MCTD, 1 SSc, 2 SjS) No titer 5 1 (UCTD) AARD antinuclear antibody-associated rheumatic disease; ANA antinuclear antibody; MCTD mixed connective tissue disease; SjS Sjögren s syndrome; SLE systemic lupus erythematosus; SSc systemic sclerosis; UCTD undifferentiated connective tissue disease.

4 Abeles and Abeles Clinical Utility of Positive Antinuclear Antibody 345 Table 3 Disease Key Characteristics of Antinuclear Antibody-Positive Patients Stratified by Presence of Variable With Disease Without Disease Gender Female (%) 18 (9.2) 177 (90.8) 1.00 (NS) Male (%) 3 (8.1) 34 (91.9) Age, y 65 (%) 17 (8.9) 175 (91.1) 0.77 (NS) 65 (%) 4 (10) 36 (90) Referring provider Academic (%) 12 (11.3) 94 (88.7) 0.36 (NS) Community-based (%) 9 (7.1) 117 (92.9) Referring provider Primary care (%) 16 (8.1) 181 (91.9) 0.33 (NS) Specialist (%) 5 (14.3) 30 (85.7) Primary care Internal medicine (%) 14 (8.8) 146 (91.2) 1.00 (NS) Family practice (%) 2 (6.3) 30 (93.4) Site where ANA screen was performed UCHC laboratory (%) 14 (10.9) 115 (89.1) 0.36 (NS) Outside laboratory (%) 7 (6.8) 96 (93.2) P value (Fisher Exact Test, 2-Tailed) ANA antinuclear antibody; NS not significant; UCHC University of Connecticut Health Center. temic lupus erythematosus only was 2.1% for the entire cohort (positive predictive value 2.9% for ANA 1:160). Although the prevalence of clinically unimportant positive ANA test results reportedly is increased in older populations, the positive predictive value of a positive ANA test result was not greater among the younger patients in our cohort. By using 65 years of age as a cutoff separating younger from older subjects (Table 3), 17 of 192 patients in the younger subgroup had an antinuclear antibody-associated rheumatic disease (positive predictive value 8.9%) versus 4 of 40 subjects in the older group (positive predictive value 10%). A majority of subjects were female (195 women vs 37 men), but we found no gender difference in the rate of true disease (8.1% [3/37] vs 9.2% [18/195]). ANA tests performed at our academic medical center s laboratory (n 129) had a positive predictive value of 10.9% (14/129 patients), whereas the positive predictive value was 6.8% for the subgroup of patients who had laboratory testing performed elsewhere (7/103). Likewise, the positive predictive value for those referred by practitioners within the University of Connecticut was 11.3% (12/106), whereas the positive predictive value for the subcohort referred by outside physicians was 7.1% (9/126). Broken down by specialty, the positive predictive value was 8.1% (16/197) for primary care providers and 14.3% (5/35) for specialists. The difference in positive predictive values between each of the paired groups above did not achieve statistical significance. A total of 23 of 232 patients had a non-antinuclear antibody-associated inflammatory or autoimmune rheumatic disease (9.9%), including 8 with rheumatoid arthritis. Of the remaining 15 patients with a non-antinuclear antibody-associated rheumatic disease, 3 had gout; 6 had polymyalgia rheumatica or giant cell arteritis; 4 had psoriatic arthritis or ankylosing spondylitis; 1 had antineutrophil cytoplasmic antibody-associated vasculitis; and 1 had pseudogout. A total of 28 patients were found to have antithyroid antibodies (microsomal or thyroglobulin antibodies); of these, 4 were previously known to have clinical hypothyroidism, and 1 was diagnosed with hypothyroidism at the time of consultation. Of the 232 patients in this cohort, only 91 patients were tested for antithyroid antibodies, and of these, 28 patients (30.8%) had positive test results. The most common reason for ANA testing was widespread pain (Table 4). Fifty-four of 232 patients (23%) had ANA testing performed for this chief symptom, none of whom had a rheumatologic disease. Of these 54 patients, 46 were subsequently diagnosed with fibromyalgia. Other common reasons for obtaining an ANA and consequently being referred are noted in Table 4 and included knee pain, chronic lower back pain, chronic headaches, and unilateral hip pain. Table 4 includes both the original reasons for ANA testing and the ultimate diagnoses made in the course of consultation. DISCUSSION Our study suggest that a positive ANA test result obtained outside of the rheumatologic setting is poorly predictive of an antinuclear antibody-associated rheumatic disease, even when using a high-titer value ( 640) as a cutoff. Moreover, these data demonstrate that ANA testing by nonrheumatologists is commonly performed in clinical scenarios neither suggestive

5 346 The American Journal of Medicine, Vol 126, No 4, April 2013 Table 4 Common Reasons for Antinuclear Antibody Test (and Ultimate Diagnoses) Reason for ANA Test No. of Patients (% of Cohort) Ultimate Diagnosis (or Diagnoses) Widespread pain 54 (23.2%) Fibromyalgia in 46 (8 with other diagnoses) Hand pain 21 (9.1%) Osteoarthritis (7), carpal tunnel syndrome (6) (1 patient with osteoarthritis and carpal tunnel syndrome), rheumatoid arthritis (2), psoriatic arthritis (2), pseudogout (1), trigger finger (1), post-fracture pain (1), mixed connective tissue disease (1), congenital dystonia (1) Knee pain 9 (3.9%) Osteoarthritis Chronic lower back pain 8 (3.4%) Osteoarthritis, regionalized pain syndrome, ankylosing spondylitis Chronic headache 5 (2.2%) Headache syndrome Unilateral hip pain 4 (1.7%) Trochanteric bursitis (3), hip osteoarthritis (1) Unilateral shoulder pain 3 (1.3%) Mechanical shoulder pain (tendinopathy/bursitis) Fatigue 3 (1.3%) Chronic fatigue (no clear cause) Thinning hair 2 (0.9%) Age-related hair loss ANA antinuclear antibody. Miscellaneous (n 1) reasons for an ANA test (and ultimate diagnoses) included chronic rhinorrhea (chronic rhinitis), intermittent toe pain and swelling (podagra/gout), right-sided jaw pain (temporomandibular joint syndrome), groin rash (tinea cruris), unilateral foot pain (immediately after foot surgery), 3 days of generalized arthralgias (immediately after zoledronic acid infusion), loss of motion in the left fourth digit (Dupuytren s contracture), left hip and left shoulder pain (that began after falling from a ladder), and elevated erythrocyte sedimentation rate (active osteomyelitis in a diabetic foot). of nor suspicious for connective tissue disease (Table 3). To our knowledge, studies have not addressed whether the antinuclear antibody test performs any better in the hands of rheumatologists, a question of significant clinical importance. Multiple studies show ANA positivity to be highly prevalent in both the general and various patient populations. In a Belgian study by Verstegen et al, % of 6422 consecutively tested patients at a large community hospital were ANA-positive ( 1:80), and Marin et al 15 demonstrated the prevalence of ANA positivity ( 1:40) in a group of 304 healthy individuals to be 54.3%. Fernandez et al 16 demonstrated ANA positivity in 22.6% of normal blood donors (antinuclear antibody 1:40), and Peene et al 17 found that 23.5% of 10,550 consecutive patients referred for ANA testing at a single medical center had a positive result (in a titer of 1:40). Of note, the similar rates in these latter 2 seemingly disparate populations suggest that ANA positivity rates may be virtually identical between healthy controls and patients who undergo ANA testing according to clinical (nonexpert) judgment. Even children have been found to have a high background ANA positivity rate. Of 1369 consecutive samples assessed at 1 academic hospital, 36% were ANA positive with a titer of 1:40 (only 8.2% of whom were determined to have systemic lupus erythematosus or an overlap syndrome). 18 Of note, even when the cutoff for ANA positivity is defined more rigorously (as a dilution 1:160), the rate of ANA positivity in the general population remains fairly high, at approximately 9.5%. 19 With such a high positivity rate in the general population, our study confirms that the ANA test can be problematic when not appropriately applied. As basic statistical principles would predict, a test that is positive in approximately half of the population results in an unacceptably high rate of false positivity and a poor positive predictive value when ordered in the face of relatively low pretest probability. Our results are in agreement with previous studies indicating that the antinuclear antibody test is most appropriately ordered in settings where the pretest probability of antinuclear antibody-associated disease is high. 15,20 However, our study further shows that antinuclear antibody testing is frequently ordered in settings where the pretest likelihood is low. Moreover, overuse of the ANA test and the consequent false-positives that result often fuel further follow-up testing, patient anxiety, and even misdiagnosis and inappropriate treatment. 20 In this regard, Narain et al 20 reported that of 137 patients with a positive ANA but without systemic illness, 39 had been treated with prednisone at doses as high as 60 mg per day. Thus, an incomplete understanding of when to order an ANA test and how to interpret it both harms patients and strains the resources of an already burdened healthcare system. We were particularly struck by the frequency with which patients with widespread pain underwent ANA testing, an observation concordant with at least 1 prior study. 21 Considering the intrinsically high rate of ANA positivity in the general population, and the fact that both ANA positivity and fibromyalgia occur at higher rates in women, 22 the potential for unnecessary ANA testing and misdiagnosis is apparently enhanced in the patient population presenting with chronic widespread pain. Epidemiologically, population studies suggest that 10% to 13% of the population has widespread pain, 22,23 whereas between 0.04% and 0.15% of the population of the United States is estimated to have systemic lupus erythematosus Thus, the potential for incorrectly diagnosing fibromyalgia as lupus or a lupus-like illness, on the basis of widespread pain and a positive ANA, is significant. In one study of 149 patients referred for lupus to an academic center, 24.8% were determined

6 Abeles and Abeles Clinical Utility of Positive Antinuclear Antibody 347 not to have lupus, but rather, fibromyalgia-like symptoms with a positive ANA. 27 Of note, a long-term follow-up study of patients with fibromyalgia demonstrated no increased risk of developing connective tissue disease compared with the general population, and no increased incidence of ANA positivity. 28 STUDY LIMITATIONS AND STRENGTHS Our study had a number of limitations. Because our data were collected retrospectively, the tests obtained by the referring clinicians at the time of clinical evaluation varied from patient to patient. However, most patients with highertiter ANAs (91% of subjects with an ANA 1:320 and 97% of patients with an ANA 1:640) had an extensive laboratory evaluation performed in the course of consultation (usually including at least the following: anti-smith, ribonucleoprotein, SSA, SSB, and double-stranded DNA antibodies, urinalysis, C3 and C4 complement levels, complete blood count, and erythrocyte sedimentation rate [Westergren]), so it is unlikely that antinuclear antibody-associated autoimmune disease went unrecognized. Of note, we do not have long-term follow-up data on our study patients. Because patients with systemic lupus erythematosus develop ANA positivity years before the onset of clinical disease, 29 it is possible that some of our patients without an antinuclear antibody-associated rheumatic disease may go on to develop one in the future. However, prior studies have shown that most patients presenting even with a high-titer ANA ( 1:640), but lacking a clinically diagnosable antinuclear antibody-associated rheumatic disease at the time of evaluation, do not develop connective tissue disease over a 10-year follow-up period. 30 Although the study was performed in a single center by 2 practitioners, we believe that the study findings are generalizable. The study investigated the performance of a clinical test in the hands of a large and representative medical community (with 138 referring physicians from both outside and within a university system), and disease diagnosis was confirmed or ruled out by 2 academically based full-time clinicians using accepted disease criteria. Our findings concur with prior studies. This study also has several noteworthy strengths. Although the utility of ANA testing has been studied in other settings, ours is the first to investigate a cohort of patients sequentially referred for a positive ANA test result. Another strength of our study is that this approach reflects real-world practice. In addition, our referring providers were a heterogeneous group, composed of both community and university-based practitioners, and the ANA test results leading to rheumatology consultation were performed at a number of different laboratories. CONCLUSIONS We showed that a positive ANA only occasionally implies the presence of an antinuclear antibody-associated rheumatic disease in clinical practice. Our study suggests that the ANA test is overused as a screening tool, often drawn in clinical scenarios that do not suggest connective tissue disease (eg, lower back pain), and potentially leading to adverse consequences. Although we do not discourage primary care physicians from ordering the ANA test, we would encourage careful consideration before ordering the screen as to whether an antinuclear antibody-associated disease seems likely. For cases that do not suggest an antinuclear antibody-associated disease (eg, chronic widespread pain), the test should not be ordered. In cases in which the ANA screen comes back as positive, it also may be important for providers to counsel patients appropriately, explaining that serologic testing is but one aspect of a diagnostic evaluation, and that ANA positivity should not be equated with definite, or even likely, disease. It also would be helpful for laboratories to include their in-house data on the background rate of ANA positivity by titer when reporting results, because many practicing clinicians are not aware of the high background rate of ANA positivity in the general population (and rates of ANA positivity vary from laboratory to laboratory). 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