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Clinical Features of 39 Patients With Antibodies to Extractable Nuclear Antigens Despite Negative Antinuclear Antibodies: Evidence for Autoimmunity Including Neurologic and Connective Tissue Diseases

Davis, John M. III MD; Moder, Kevin G. MD; Homburger, Henry A. MD; Ytterberg, Steven R. MD

doi: 10.1097/01.md.0000173181.87969.eb
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Abstract: Systemic lupus erythematosus (SLE) rarely presents with a negative antinuclear antibody (ANA). Although antibodies to extractable nuclear antigens (ENA) are sometimes ordered despite a negative ANA, it is unclear if this contributes to the diagnosis of SLE or other forms of connective tissue disease (CTD). We reviewed 39 patients with anti-ENA antibodies despite a negative ANA during a 1-year period to determine the presence of SLE or other CTD. Several patients had clinical features suggestive of CTD, including 1 with possible SLE. A number of patients had neurologic disorders, especially peripheral neuropathy. In this study, the finding of anti-ENA despite negative ANA was associated with neurologic disorders and CTD. This may represent test bias or false-positive anti-ENA assays or false-negative ANA assays, or may imply immune-related mechanisms not previously described.

Abbreviations: ANA = antinuclear antibody, CTD = connective tissue disease, EIA = enzyme immunoassay, ENA = extractable nuclear antigens, ESR = erythrocyte sedimentation rate, Hep = human epithelial, IIF = indirect immunofluorescence, LIA = line immunoassay, SLE = systemic lupus erythematosus.

From Division of Rheumatology (JMD, KGM, SRY), Department of Medicine; Division of Clinical Biochemistry and Immunology (HAH), Department of Laboratory Medicine and Pathology; Mayo Clinic College of Medicine and Mayo Clinic, Rochester, Minnesota.

Address reprint requests to: John M. Davis III, MD, Division of Rheumatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Fax: 507-284-0564; e-mail: davis.john4@mayo.edu.

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INTRODUCTION

Systemic lupus erythematosus (SLE) rarely presents with a negative antinuclear antibody (ANA). The incidence of ANA-negative SLE has been estimated to be 1%-5%22. The increasing use of human epithelial (HEp-2) substrate has increased the sensitivity of ANA assays, and the perceived incidence of ANA-negative SLE has decreased22,40. In the past, ANA-negative SLE was thought to manifest primarily with dermatologic disease40 including subacute cutaneous lupus erythematosus33. In 1 study, sera of patients with SLE or subacute cutaneous lupus erythematosus previously ANA-negative using mouse liver substrate were found to be anti-SSA/Ro positive by enzyme-linked immunosorbent assays (ELISA)33. Other investigators have debated whether or not ANA-negative SLE exists or represents falsely negative testing9.

The utility of testing for antibodies to specific nuclear antigens including ENA has been reviewed22,23,28,35,37. Specific autoantibodies are associated with several connective tissue diseases (CTDs), including anti-SSA/Ro and anti-SSB/La with Sjögren syndrome, anti-Sm and anti-dsDNA with SLE, anti-U1RNP with mixed CTD, anti-Jo-1 with polymyositis/dermatomyositis, anti-centromere with limited cutaneous systemic sclerosis/CREST syndrome, and anti-Scl-70 with diffuse cutaneous systemic sclerosis.

New methods of detecting ANA and anti-ENA antibodies have been developed over the last decade. Several studies have compared indirect immunofluorescence (IIF) assays to enzyme immunoassays (EIA) and ELISA for the detection of ANA. The sensitivity and specificity of EIA-based commercial kits for detecting ANA using HEp-2 substrate compared with IIF vary from 62% to 97.7% and from 50% to 97.9%, respectively10,20,21,34.

EIA is becoming more widely used for the detection of ANA, but few published reports have evaluated the characteristics of EIA-based testing37. False-positive EIA tests have been reported to be a problem25,37. Agreement between commercial EIA kits and IIF for ANA detection was marginal with a kappa score < 0.4 in 1 study10. Another study raised the question of false-negative results with EIA ANA tests, as an ELISA was found to miss anti-SSA/Ro even when using Centers for Disease Control and Prevention (CDC) reference sera25.

One author of the current study (HAH) has previously recommended a cascade approach to ANA testing, largely based on the finding that patients with a negative ANA will test positive for anti-ENA antibodies in less than 5% of cases19. Of patients who have anti-ENA antibodies, 92% will have an ANA by EIA >3 units or an IIF titer >1:16020. In this approach, second-order testing for antibodies to ENA is performed only after an initial positive ANA screening test. However, in clinical practice, both ANA and ENA antibody assays are often ordered simultaneously, presumably for expedience and to increase the sensitivity in detecting SLE or other CTD. Uncommonly, patients have been seen with a negative ANA and positive ENA, but the significance of this is not clear. We reviewed the medical records of patients with a positive anti-ENA antibody assay despite a negative ANA to determine the presence of SLE or other CTD.

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PATIENTS AND METHODS

ANA and ENA assays were performed by EIA using a commercial kit (Helix Diagnostics, West Sacramento, CA) that uses antigens purified from HEp-2 cell nuclei. A negative ANA was defined as EIA units less than 1, and a positive ENA was defined as EIA units greater than 20. ENA included a screening EIA and, for positive sera, specific EIA for anti-SSA/Ro, anti-SSB/La, anti-Sm, anti-U1RNP, anti-Jo-1, and anti-Scl-70.

Using records of an internal laboratory database from January 1, 2000, to January 2, 2001, we identified 47 patients who simultaneously had a negative ANA yet positive ENA. Of these, 8 patients were excluded because their medical records were located at Mayo Clinic Scottsdale. The final sample size was 39.

A single reviewer (JMD) examined the medical records using a structured data collection form and recorded demographic data, reasons for testing, ordering physician specialty, specific SLE classification criteria and related clinical features, and final diagnoses. To assess for SLE, we used the American College of Rheumatology (ACR) revised criteria for the classification of SLE as updated in 199717,39. Presence of peripheral neuropathy was based on report of clinical electrodiagnostic testing, including electromyography, thermoregulatory sweat testing, and/or autonomic reflex screen. The Mayo institutional review board approved this study.

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RESULTS

In 2000, 24,453 ENA screens were performed at our institution. Of 918 patients who had a positive ENA, 47 (5.1%) had a simultaneously negative ANA. Eight charts located at Mayo Clinic Scottsdale were not reviewed. Of the 39 cases reviewed, there were 24 female and 15 male patients (female:male ratio 1.6). The mean age was 53.8 ± 14.8 years. One patient was deceased at the time of review. The autoantibody testing was requested by multiple departments and divisions, including Neurology, 15; General Internal Medicine, 13; Rheumatology, 7; and other internal medicine specialties, 4.

Table 1 depicts the demographic data, ENA results, specific positive ENA antibodies, indications for testing, and diagnoses. Several patients had specific ACR classification criteria for SLE: 2 patients with 2, and 11 with 1; 26 had no criteria. For all patients, there were a total of 96 criteria not adequately assessed. The SLE criteria that were positive are depicted in Figure 1.

TABLE 1

TABLE 1

TABLE 1

TABLE 1

FIGURE 1

FIGURE 1

Table 2 contains ANA and ENA assay results for patients who were tested on at least 1 occasion before or after the index date in 2000. Four patients had a positive ANA before the index date (Patients 7, 17, 21, 29). These patients satisfied the SLE criterion for "ever" having a positive ANA. Patients 7, 21, and 29 had a history of a positive ANA by IIF, each at low titers, 2 of which were at other institutions, but these patients subsequently tested negative by EIA at our institution. Patient 17 had a weakly positive ANA by EIA at our institution in October 1999 but had negative ANA assays on 3 other occasions by the same method.

TABLE 2

TABLE 2

Several patients had results from more than 1 negative ANA in their records. Patient 25 had a negative ANA by IIF in 1995, then tested negative by EIA in 2000. Patients 25, 27, 29, and 30 had repeatedly negative ANA assays by EIA.

Multiple ENA results were documented in the records of 5 patients (Patients 4, 11, 17, 30, 31) (see Table 2). In 3 of 5 cases, the ENA testing occurred before the index date in 2000-these prior ENA results were positive. The EIA values for these patients remained stable over the course of 1-4 years. Two patients had repeat ENA assays after the index date testing in 2000, and these results were negative. Patient 4 had a repeat ENA screen in February 2001 that was negative. Likewise, Patient 31 had a repeat ENA screen in January 2002 that was negative at 16.9 units.

Patients 17, 30, and 31 had multiple occasions of simultaneous ANA/ENA testing (see Table 2). Patient 17 had a single weakly positive ANA out of 4 testing episodes spanning 4 years; the SSB/La antibody was persistently positive. Patient 30 had a persistently negative ANA yet tested positive for SSA/Ro and SSB/La antibodies on 2 occasions. Patient 31 had negative ANA results and had a positive ENA screen with positives for SSA/Ro and U1RNP in 2000; a repeat ENA screen in 2002 was negative.

Combinations of specific ENA antibodies were present (see Table 1). Most patients had a single positive ENA antibody, including 12 patients with anti-SSB/La, 9 with anti-SSA/Ro, 3 with anti-U1RNP, and 5 with anti-Scl-70. A few patients had 2 positive ENA antibodies, including 2 with anti-Scl-70 and anti-U1RNP, 1 with anti-SSA/Ro and anti-SSB/La, and 1 with anti-SSA/Ro and anti-U1RNP. No patient had a positive anti-Sm or anti-Jo-1, and 6 patients had no specific positive ENA antibody despite a positive screening assay. One of these, Patient 37, was not tested for specific ENA antibodies.

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Connective Tissue Diseases

Several patients were diagnosed with CTD including 2 with Sjögren syndrome. Patient 11 developed keratoconjunctivitis sicca 6 weeks postpartum and had a positive Schirmer test and a lip biopsy with 50 lymphocytes per high-powered field. She had good control of symptoms with hydroxychloroquine. She later developed hypothyroidism due to Hashimoto thyroiditis. Patient 15 presented with low back pain and tested positive for anti-SSB/La. She had symptoms of keratoconjunctivitis sicca, with a history of a corneal ulcer and dysphagia.

Two patients were diagnosed with undifferentiated CTD. Patient 30 had recurrent pericarditis on 3 occasions. A rheumatologist was not able to identify any other signs of SLE. She tested negative for ANA, anti-dsDNA, antiphospholipid antibodies, lupus anticoagulant, and human immunodeficiency virus (HIV)-1, but assays for anti-SSA/Ro and anti-SSB/La were positive on 2 occasions. A diagnosis of undifferentiated CTD was made. She responded to hydroxychloroquine 400 mg daily, but had another episode of pericarditis after the dose was tapered to 200 mg daily. Patient 1 presented with long-standing polyarthralgia and fatigue. Previously, she had documented inflammatory synovitis. At the time of testing, there were no symptoms of joint swelling, morning stiffness, or sicca symptoms; however, she was managed with a combination of methotrexate, celecoxib, and etanercept. Rheumatoid factor and ANA were negative, but she had high-titer anti-SSA/Ro antibodies.

Other patients had clinical features suggestive of possible CTD but had no specific diagnosis. Patient 26 presented at age 63 years with fatigue, diffuse joint pain, weakness, headaches, and orthostatic hypotension. She had a persistently elevated Westergren erythrocyte sedimentation rate (ESR), and previously had been diagnosed with polymyalgia rheumatica. She had a positive anti-Scl-70 assay with negative ANA. Her ESR was 50 mm/h. Pulmonary function testing revealed mild restriction with total lung capacity at 74% predicted and slightly reduced diffusing capacity. Serum creatinine was 1.8 mg/dL. Systemic sclerosis was suspected, but there were no characteristic skin findings. The patient declined renal biopsy. Patient 25 had chronic intermittent back and hip girdle pain, pleuritic chest pain, and Raynaud phenomenon. Her examination documented fingertip hyperemia and livedo reticularis, but no synovitis, rash, or skin tightening. Vascular studies demonstrated bilateral digital artery vasospasm with moderate improvement on rewarming. Anti-Scl-70 was strikingly positive. The final diagnosis was idiopathic Raynaud phenomenon. Patient 31 had a history of carpal tunnel syndrome, bilateral hand swelling, and hand joint stiffness and gel phenomenon that was steroid-responsive. Rheumatology evaluation revealed bilateral flexor tenosynovitis and metatarsophalangeal synovitis. ANA and RF were negative but anti-SSA/Ro and anti-U1RNP antibodies were detected. Although possible CTD or the syndrome of relapsing symmetrical seronegative synovitis and pitting edema (RS3PE) was suspected, he had no other features of these disorders and remained without a specific diagnosis.

In a noteworthy case, Patient 7 was a 67-year-old woman with a history of long-standing polyarthralgia and myalgia of the upper and lower extremities. In 1993, she developed skin lesions on her chest that on biopsy were consistent with discoid lupus erythematosus. On careful review of her records, it was noted that she had a history of a "weakly positive" ANA in 1992 at another institution, and she had a prolonged dilute Russell viper venom time that partially corrected in the mixing study with a normal activated partial thromboplastin time. The hematologist suggested this might represent a lupus anticoagulant. Her symptoms were managed with steroids and hydroxychloroquine 400 mg daily for a number of years before the clinical episode leading to repeat autoantibody testing at our institution. In 2000, she was seen for a lip lesion that proved to be a squamous cell carcinoma and was found to be ANA negative yet positive for SSA/Ro antibodies. She definitely met 2 (and possibly 3) SLE criteria, consistent with "possible" SLE.

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Neurologic Diseases

Twenty-three patients were diagnosed with a primary neurologic disorder, including 11 patients with various forms of peripheral neuropathy. Table 3 depicts the neurologic diagnoses along with ENA specificities, and Table 4 shows the types of peripheral neuropathies along with ENA specificities, electrodiagnostic testing, additional pertinent evaluation, clinical symptoms, and etiology if known.

TABLE 3

TABLE 3

TABLE 4

TABLE 4

Patient 18 had Devic neuromyelitis optica, an entity related to multiple sclerosis with optic neuritis and features of spinal demyelination. Two patients had amyotrophic lateral sclerosis. Another patient had a chronic neuronal hyperexcitability syndrome. As depicted in Table 3, a variety of other neurologic diagnoses was present.

As presented in Table 4, several forms of peripheral neuropathy were observed: 3 patients had small fiber neuropathy; 4 had large fiber, length-dependent peripheral neuropathy; and 2 had combined small and large fiber neuropathy. Patient 10 initially presented with sensory ataxia, flaccid dysarthria, lancinating pains in his extremities, and keratoconjunctivitis sicca. A sural nerve biopsy revealed decreased density of myelinated fibers, early "onion bulb" formation, and epineural mononuclear cell infiltrates, suggesting a chronic inflammatory process. Laboratory evaluation revealed type II cryoglobulinemia, hepatitis C with evidence of active viral replication, positive rheumatoid factor, and high-titer anti-SSB/La antibodies. Clinical examination and electromyography resulted in a diagnosis of pure sensory neuropathy, questionably due to cryoglobulinemia or possible Sjögren syndrome. Patient 26 suffered from poorly controlled diabetes, and this was attributed as the cause of her small fiber autonomic neuropathy. Patient 33 had multifocal motor neuropathy with conduction block associated with elevated anti-myelin-associated glycoprotein antibodies. After extensive testing (see Table 4), no cause of neuropathy was identified in the 8 remaining patients with neuropathy.

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Other Diseases

A significant number of patients presented with immunologic, infectious, and hematologic diseases. As examples of immunologic diseases, Patient 24 had idiopathic cutaneous leukocytoclastic vasculitis without systemic manifestations, associated with a positive U1RNP. Patient 23 developed intermittent and recurrent purpuric lower extremity skin lesions, abdominal pain, diarrhea, oral aphthous ulcers, and was partially responsive to steroids, methotrexate, and indomethacin. He had an elevated ESR, anemia, and episodic leukopenia. Previous biopsies showed lobular panniculitis, but repeat biopsies at our institution were not remarkable, presumably due to his treatment. There was suspicion of chronic erythema nodosum.

As an example of an infectious diagnosis, Patient 29, from Oklahoma, presented with fever of unknown origin, a history of tick exposure, and serologic evidence of chronic Q fever due to Coxiella burnetii infection. He had a marginally positive anti-Scl-70 antibody. Patient 14 was diagnosed with solar urticaria, but also had evidence of chronic sinusitis and allergic bronchopulmonary aspergillosis; she had antibody to SSB/La.

There was a myriad of hematologic diagnoses. Two patients suffered from non-Hodgkin lymphoma: Patient 21 with diffuse large B-cell lymphoma, and Patient 27 with celiac disease and enteropathy-associated T-cell lymphoma. Five patients had dysproteinemias, including Patient 3 with monoclonal plasma cell proliferative disorder and Patient 10 with IgA monoclonal gammopathy. Patients 10, 24, and 25 had polyclonal gammopathies on serum protein electrophoresis.

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DISCUSSION

In the current study, we reviewed the records of patients who had ENA antibodies despite a negative ANA screen to identify SLE or other CTD. Our first main finding was that no patient had definite SLE, but 1 patient had a syndrome suspicious for SLE and arguably met criteria for "possible" SLE. Several patients did have CTD diagnoses including Sjögren syndrome and undifferentiated CTD. Sjögren syndrome is known to occur with a negative ANA. In contrast, nearly all patients with undifferentiated CTD are ANA positive4,29, although 1 report found that only 58% of patients with undifferentiated CTD were ANA positive7.

There are several possible explanations for these findings. The ANA in this cohort may be falsely negative. The process of protein purification may denature nuclear antigens, causing unreliable detection of certain "conformational epitopes," thereby resulting in a negative ANA result25. Alternatively, nuclear antigens extracted from HEp-2 cells may not be coated to assay plates uniformly, leading to different affinities for the solid phase and a false-negative assay16. At our institution's laboratory, several centralized stations perform each step of the ANA and ENA assays. If the pipetting station would fail to aspirate the patient's specimen and add it to the microtiter plate, then the assay would be falsely negative. In our cohort, a few patients were positive for ANA by IIF and then negative by EIA, suggesting methodologic differences may account for some of the negative ANA results. Another potential explanation is that the ENA results in this cohort may be false positives, and the enrichment for CTD may be occurring through referral or selection bias. However, as we have shown, several patients in our cohort with anti-ENA antibodies repeatedly tested negative for ANA. Of 3 patients who had assays for both ANA and ENA simultaneously on more than 1 occasion, 2 were persistently negative for ANA yet had persistently positive anti-ENA antibodies. Therefore, technical factors are unlikely to be the sole explanation for our findings.

Our second main finding was that a significant number of patients had neurologic diseases, a few of which have a known autoimmune basis. One patient in the study had multiple sclerosis, an autoimmune disorder characterized by immune-mediated demyelinating lesions of the central nervous system. Patients with multiple sclerosis are frequently ANA positive and often have other signs of autoimmune disease3,38,41, and there is evidence that ANA are correlated with a chronic progressive disease course6,8,38. Devic neuromyelitis optica, seen in Patient 18, is an idiopathic inflammatory disease of the central nervous system related to multiple sclerosis and characterized by optic neuritis, myelitis, and absence of lesions seen on magnetic resonance imaging of the brain14,26. Devic disease has been reported with SLE13, and humoral immune-mediation has been demonstrated by immunopathologic analysis26. Evidence for immune-mediated pathogenesis with calcium channel antibodies has been described for amyotrophic lateral sclerosis, although this has been controversial5,30. Neuronal hyperexcitability syndromes including neuromyotonia, cramp-fasciculation syndrome, and rippling muscle syndrome have been demonstrated to be caused by voltage-gated potassium channel antibodies and are responsive to plasma exchange and corticosteroids15,42.

Additionally, there was a significant prevalence of peripheral neuropathy in our cohort. Many neuropathies have a known association with autoantibodies such as anti-myelin-associated glycoprotein31 or anti-GM1 ganglioside32, as was the case for Patient 33 who had multifocal motor neuropathy. Patient 10, 1 patient among 5 with dysproteinemia, was diagnosed with a pure sensory neuropathy. It is relevant that dysproteinemias have been associated with various forms of neuropathy. For example, 7%-15% of patients with cryoglobulinemia in 1 study had peripheral neuropathy24. Relevant to Patient 10, subacute severe neuropathy associated with a monoclonal IgM-kappa cryoglobulinemia has been reported24. Additionally, a plausible mechanism for a severe neuropathy caused by monoclonal anti-DNA in a patient with a monoclonal gammopathy has been reported12. In 1 series of patients with peripheral neuropathy, 5% were ANA positive, and 4 of these 5 patients had a CTD27.

Patients with Sjögren syndrome can also present with peripheral neuropathy. One study described 5 patients with pure sensory neuropathy of insidious onset who were later diagnosed with Sjögren syndrome11. While only 2 of 5 were ANA positive, 4 of 5 were anti-SSA/Ro positive, and 3 of 5 were anti-SSB/La positive. Another study1 identified neurologic disease, mainly peripheral neuropathy, in 32 of 63 patients with Sjögren syndrome.

A wide variety of neurologic diseases was present in our cohort, and many of these are either relatively common, such as stroke, or have no association with autoantibodies, such as Alzheimer disease. In these cases, it is unlikely that the anti-ENA antibodies are related to the neurologic diseases. However, a few patients in our series had neurologic diseases with known autoantibody associations and/or autoimmune pathophysiology as discussed above. It is possible that the anti-ENA antibodies seen in these patients are markers of immune dysregulation, which is involved in the pathogenesis of neurologic dysfunction. This association is novel and noteworthy, but our data are strictly hypothesis generating and should be interpreted as such. Whether the association between anti-ENA antibodies and autoimmune neurologic diseases in this cohort relates to undiagnosed Sjögren syndrome or sicca complex, expansion of clones of B cells producing autoantibodies, or some other mechanism will require further study.

Previous studies have addressed similar questions as those in the current study. One group tested 468 patient sera over 12 months for ANA and anti-ENA40. The methods of ANA and anti-ENA detection other than the use of HEp-2 substrate were not specified. Only 9 patients had a negative ANA yet positive anti-ENA, and 6 of these were previously ANA positive. The remaining 3 patients were anti-SSA/Ro positive, representing 0.64% of all ANA tested. In 1995, a study compared the accuracy, sensitivity, and specificity of ANA detection for various EIA-based commercial kits compared to IIF on HEp-2 cells21. Nine patients had insignificant IIF-ANA titers (4 patients <1:40, 4 patients <1:160, and 1 patient <1:640) associated with 1 or more positive EIA-ANA. Specific EIAs in 4 of these patients were positive for anti-dsDNA but not for other ENA antibodies. Some of these patients had anti-histone and anti-ssDNA antibodies, none considered significant. Only 1 of the anti-dsDNA antibodies was considered clinically significant although the titer was not much higher than the others21. In 2002, a study reported 494 sera tested for ANA by IIF on HEp-2 and HEp-2000 substrate as well as line immunoassay (LIA)18. Of the ANA-positive sera by LIA, only 72% and 75% were positive by IIF on HEp-2 or HEp-2000 substrates, respectively. Moreover, of 291 patients ANA negative on both IIF assays, 12 patients were ANA-LIA positive, including 3 with suspected CTD and 4 with definite CTD. In the definite CTD group were SLE, polymyositis, and Sjögren syndrome. These findings suggest that there can be significant disagreement between different ANA assays, and they also are consistent with the finding of CTD associated with a negative ANA screen yet with positive results on specific autoantibody tests.

Regarding the patients in this cohort, it is not clear if these results were seen as clinically relevant or important to the ordering physicians. It was beyond the scope of the current retrospective study to determine if the testing affected the diagnosis or clinical management. In each case, it appears that simultaneous ANA and ENA testing proceeded based on high pretest suspicion of CTD. Based on results of a study discussed above18, some investigators recommend that both ANA and specific autoantibodies be checked when the pretest probability of CTD is high.

It has been suggested that certain autoantibodies identified in this study can have prognostic significance, so their identification would seem important36. One report demonstrated progressive accumulation of autoantibodies over time in a predictable fashion before the onset of overt SLE2. It is possible that anti-ENA antibodies may precede development of positive ANA in some patients moving along a continuum of autoimmunity.

Several limitations of this study deserve mention. The retrospective nature of the study may introduce bias. In several cases, documentation of examination or testing for specific SLE criteria was not available. There was no feasible way to identify a control group retrospectively, which precluded any statistical analysis of the data. In the case of the neurologic diseases, it is possible that selection or referral bias may explain the positive ENA tests. Further prospective controlled studies will be necessary to answer these concerns.

Previous investigators, including 1 of the authors of the current study, have recommended a cascade approach to autoantibody testing. Using this strategy, the ANA is the initial screening test. If positive, then a screening ENA is performed, and finally, specific EIA to each ENA performed. In the sense that no patient in this study with a negative ANA and positive anti-ENA antibodies had definite SLE, the findings support this approach. However, our findings suggest that anti-ENA do uncommonly occur with negative ANA in patients with true CTD or other autoimmune diseases. At this time, it would seem reasonable to obtain both ANA and ENA screens when the pretest probability of CTD is high.

In summary, we report a cohort of consecutive patients in the year 2000 with positive anti-ENA antibodies despite negative ANA. This cohort was enriched for connective tissue disorders and possible SLE as well as neurologic diseases, many of which have a known autoimmune pathophysiology. Further studies are required to verify this finding with a control group and to clarify the significance of these findings.

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REFERENCES

1. Andonopoulos AP, Lagos G, Drosos AA, Moutsopoulos HM. The spectrum of neurological involvement in Sjogren's syndrome. Br J Rheumatol. 1990;29:21-23.
2. Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, Harley JB. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. New Engl J Med. 2003;349:1526-1533.
3. Barned S, Goodman AD, Mattson DH. Frequency of anti-nuclear antibodies in multiple sclerosis. Neurology. 1995;45:384-385.
4. Belfiore N, Rossi S, Bobbio-Pallavicini F, Epis O, Caporali R, Montecucco C. Anti-Ro(SS-A) 52 kDa and 60 kDa specificities in undifferentiated connective tissue disease. Joint Bone Spine. 2000;67:183-187.
5. Benatar M. Antibodies from ALS patients inhibit dopamine release mediated by L-type calcium channels. Neurology. 1999;52:1520-1521.
6. Collard RC, Koehler RP, Mattson DH. Frequency and significance of antinuclear antibodies in multiple sclerosis. Neurology. 1997;49:857-861.
7. Danieli M, Fraticelli P, Franceschini F, Cattaneo R, Farsi A, Passaleva A, Pietrogrande M, Invernizzi F, Vanoli M, Scorza R, Sabbadini M, Gerli R, Corvetta A, Farina G, Salsano F, Priori R, Valesini G, Danieli G. Five-year follow-up of 165 Italian patients with undifferentiated connective tissue diseases. Clin Exp Rheumatol. 1999;17:585-591.
8. de Andres C, Guillem A, Rodriguez-Mahou M, Lopez Longo FJ. Frequency and significance of anti-Ro (SS-A) antibodies in multiple sclerosis patients. Acta Neurol Scand. 2001;104:83-87.
9. Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol. 2000;53:424-432.
10. Emlen W, O'Neill L. Clinical significance of antinuclear antibodies: comparison of detection with immunofluorescence and enzyme-linked immunosorbent assays. Arthritis Rheum. 1997;40:1612-1618.
11. Font J, Valls J, Cervera R, Pou A, Ingelmo M, Graus F. Pure sensory neuropathy in patients with primary Sjogren's syndrome: clinical, immunological, and electromyographic findings. Ann Rheum Dis. 1990; 49:775-778.
12. Freddo L, Hays AP, Nickerson KG, Spatz L, McGinnis S, Lieberson R, Vedeler CA, Shy ME, Autilio-Gambetti L, Grauss FC, Petito F, Chess L, Latov N. Monoclonal anti-DNA IgM kappa in neuropathy binds to myelin and to a conformational epitope formed by phosphatidic acid and gangliosides. J Immunol. 1986;137:3821-3825.
13. Gibbs AN, Moroney J, Foley-Nolan D, O'Connell PG. Neuromyelitis optica (Devic's syndrome) in systemic lupus erythematosus: a case report. Rheumatology (Oxford). 2002;41:470-471.
14. Gold R, Linington C. Devic's disease: bridging the gap between laboratory and clinic. Brain. 2002;125(pt. 7):1425-1427.
15. Hart IK. Acquired neuromyotonia: a new autoantibody-mediated neuronal potassium channelopathy. Am J Med Sci. 2000;319:209-216.
16. Hayashi N, Kawamoto T, Mukai M, Morinobu A, Koshiba M, Kondo S, Maekawa S, Kumagai S. Detection of antinuclear antibodies by use of an enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: comparison with immunofluorescence assay in 307 patients. Clin Chem. 2001;47:1649-1659.
17. Hochberg M. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40:1725.
18. Hoffman IE, Peene I, Veys EM, De Keyser F. Detection of specific antinuclear reactivities in patients with negative anti-nuclear antibody immunofluorescence screening tests. Clin Chem. 2002;48:2171-2176.
19. Homburger HA. Cascade testing for autoantibodies in connective tissue diseases. Mayo Clin Proc. 1995;70:183-184.
20. Homburger HA, Cahen YD, Griffiths J, Jacob GL. Detection of antinuclear antibodies: comparative evaluation of enzyme immunoassay and indirect immunofluorescence methods. Arch Pathol Lab Med. 1998; 122:993-999.
21. Jaskowski TD, Schroder C, Martins TB, Mouritsen CL, Litwin CM, Hill HR. Screening for antinuclear antibodies by enzyme immunoassay. Am J Clin Pathol. 1996;105:468-473.
22. Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. American College of Pathologists. Arch Pathol Lab Med. 2000;124:71-81.
23. Keren DF. Antinuclear antibody testing. Clin Lab Med. 2002;22:447-474.
24. Lippa CF, Chad DA, Smith TW, Kaplan MH, Hammer K. Neuropathy associated with cryoglobulinemia. Muscle Nerve. 1986;9:626-631.
25. Lock RJ, Unsworth DJ. Antibodies to extractable nuclear antigens. Has technological drift affected clinical interpretation? J Clin Pathol. 2001; 54:187-190.
26. Lucchinetti CF, Mandler RN, McGavern D, Bruck W, Gleich G, Ransohoff RM, Trebst C, Weinshenker B, Wingerchuk D, Parisi JE, Lassmann H. A role for humoral mechanisms in the pathogenesis of Devic's neuromyelitis optica. Brain. 2002;125(pt 7):1450-1461.
27. Michielsens B, Walravens M, Vermylen J, Carton H. Diagnostic significance of antinuclear antibodies in neurologic patients. Acta Neurol Scand. 1991;84:102-106.
28. Moder KG. Immunologic tests in rheumatology. Ann Allergy Asthma Immunol. 1998;81:539-544, 547.
29. Mosca M, Tavoni A, Neri R, Bencivelli W, Bombardieri S. Undifferentiated connective tissue diseases: the clinical and serological profiles of 91 patients followed for at least 1 year. Lupus. 1998;7:95-100.
30. Offen D, Halevi S, Orion D, Mosberg R, Stern-Goldberg H, Melamed E, Atlas D. Antibodies from ALS patients inhibit dopamine release mediated by L-type calcium channels. Neurology. 1998;51:1100-1103.
31. O'Hanlon GM, Veitch J, Gallardo E, Illa I, Chancellor AM, Willison HJ. Peripheral neuropathy associated with anti-GM2 ganglioside antibodies: clinical and immunopathological studies. Autoimmunity. 2000; 32:133-144.
32. Pestronk A, Choksi R. Multifocal motor neuropathy. Serum IgM anti-GM1 ganglioside antibodies in most patients detected using covalent linkage of GM1 to ELISA plates. Neurology. 1997;49:1289-1292.
33. Reichlin M. ANA negative systemic lupus erythematosus sera revisited serologically. Lupus. 2000;9:116-119.
34. Reisner BS, DiBlasi J, Goel N. Comparison of an enzyme immunoassay to an indirect fluorescent immunoassay for the detection of antinuclear antibodies. Am J Clin Pathol. 1999;111:503-506.
35. Sanchez-Guerrero J, Lew RA, Fossel AH, Schur PH. Utility of anti-Sm, anti-RNP, anti-Ro/SS-A, and anti-La/SS-B (extractable nuclear antigens) detected by enzyme-linked immunosorbent assay for the diagnosis of systemic lupus erythematosus. Arthritis Rheum. 1996;39:1055-1061.
36. Simmons-O'Brien E, Chen S, Watson R, Antoni C, Petri M, Hochberg M, Stevens MB, Provost TT. One hundred anti-Ro (SS-A) antibody positive patients: a 10-year follow-up. Medicine (Baltimore). 1995;74: 109-130.
37. Solomon DH, Kavanaugh AJ, Schur PH. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum. 2002;47:434-444.
38. Spadaro M, Amendolea MA, Mazzucconi MG, Fantozzi R, Di Lello R, Zangari P, Masala G. Autoimmunity in multiple sclerosis: study of a wide spectrum of autoantibodies. Mult Scler. 1999;5:121-125.
39. Tan E, Cohen A, Fries J, Masi A, McShane D, Rothfield N, Schaller J, Talal N, Winchester R. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271-1277.
40. Thomson K, Murphy A, Goodfield M, Misbah S. Is it useful to test for antibodies to extractable nuclear antigens in the presence of a negative antinuclear antibody on Hep-2 cells? J Clin Pathol. 2001;54:413.
41. Tourbah A, Clapin A, Gout O, Fontaine B, Liblau R, Batteux F, Stievenart JL, Weill B, Lubetzki C, Lyon-Caen O. Systemic autoimmune features and multiple sclerosis: a 5-year follow-up study. Arch Neurol. 1998;55:517-521.
42. Vernino S, Auger RG, Emslie-Smith AM, Harper CM, Lennon VA. Myasthenia, thymoma, presynaptic antibodies, and a continuum of neuromuscular hyperexcitability. Neurology. 1999;53:1233-1239.
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