Rheumatology Advance Access originally published online on December 20, 2005
Rheumatology 2006 45(5):629-635; doi:10.1093/rheumatology/kei260
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cost-effective detection of non-antidouble-stranded DNA antinuclear antibody specificities in daily clinical practice
Department of Rheumatology and Clinical Immunology and 1 Laboratory of Immunology, University Medical Centre, Utrecht, The Netherlands.
Correspondence to: P. A. J. M. Vos, Department of Rheumatology and Clinical Immunology (F02.127), University Medical Centre, PO Box 85500, 3508GA Utrecht, The Netherlands. E-mail: p.a.j.m.vos{at}azu.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Objectives. To compare the utility of indirect immunofluorescence for the detection of antinuclear antibodies (ANA-IIF) and a fully automated test (ELiA SymphonyTM) that detects antibodies against a mixture of nuclear and cytoplasmic antigens (ENA), to select sera that should be tested for non-antidouble-stranded DNA (dsDNA) antinuclear antibodies in a relatively expensive automated line immunoassay (INNO-LIATMANA update, Lineblot).
Methods. All 328 sera sent to the laboratory for ANA or anti-ENA tests, over a 4 month period were evaluated in all three assays. Results were related to signs and symptoms of systemic autoimmune disease (AID) that patients had before or at the time of blood sampling.
Results. Overall, 72 (22%) sera were Lineblot positive. Of 198 patients without clinical manifestations of AID, 7% were Lineblot positive. Limiting Lineblot to sera positive in either ANA-IIF or Symphony tests failed to detect 26 (ANA-IIF) and 22 (Symphony) Lineblot-reactive sera, with 15 sera being negative in both assays. From a clinical point of view, failure to detect these reactivities was not important in most cases.
Conclusions. Restriction of performance of Lineblot to patients with at least one criterion for AID is an ideal and cost-effective strategy. In ignorance of clinical signs and symptoms, screening of sera by ANA-IIF or Symphony strongly reduces the costs of anti-ENA detection, with minimal loss in diagnostic capacity. Based on small differences, including the fact that anti-dsDNA antibodies give a positive ANA-IIF, we prefer screening with ANA-IIF over Symphony.
KEY WORDS: Antinuclear antibodies, ENA, Health economics
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The presence of antinuclear antibodies (ANA) is a hallmark of many systemic autoimmune diseases (AID). The technique commonly used for their detection is indirect immunofluorescence (IIF) with fixed tissue or cultured cell lines as the substrate. Apart from antidouble-stranded DNA (anti-dsDNA) antibodies, a positive ANA-IIF can be caused by antibodies against a variety of nuclear antigens. Although some IIF patterns strongly suggest distinct specificities, additional tests are required to demonstrate antibody reactivities against specific nuclear and cytoplasmic antigens (ENA).
Clinicians have different reasons for ordering anti-ENA tests. In patients with sufficient signs and symptoms to make the clinical diagnosis of an AID, anti-ENA tests are used to support the diagnosis (disease specificity) or to identify subsets of patients that are prone to particular disease manifestations (prognostic marker). At the other end of the spectrum, requests for anti-ENA are made in patients with aspecific signs and symptoms that have a wide differential diagnosis, including AID. Here, the clinician (rightly or wrongly) uses the results of the tests to exclude systemic autoimmunity (negative predictive value). A third group of patients have signs and symptoms that fit into a diagnosis of AID but insufficient in number to meet the diagnostic criteria of a particular AID. Here, clinicians hope that the results of anti-ENA tests will assist the diagnostic process (disease specificity).
Over the years, laboratory scientists and the diagnostic industry have developed a variety of techniques for the detection of anti-ENA antibodies. Traditional assays based on haemagglutination reactions and immunodiffusion are increasingly being replaced by less cumbersome tests based on enzyme linked immunosorbent assays (ELISAs) and immunoblotting techniques. The antigens used in the assays evolved from crude extracts from organs (frequently the thymus) to purified natural antigens and recombinant nuclear antigens. In general, the new technologies focus on short hands-on times and lower costs. However, as the generally accepted disease specificities of autoantibodies against certain nuclear and cytoplasmic antigens were established many years ago in studies that used older technologies, and because diagnostic specificity usually decreases with the use of more sensitive assays, it is important that new techniques are evaluated for their diagnostic value in daily practice and that studies are done to evaluate how these new techniques are best used from a health economics point of view. The use of very sensitive tests with low disease specificity increases the chance of finding a reactivity against ENA that does not fit into the clinical picture. This can confuse clinicians and may result in unnecessary additional tests and follow-up.
Based on the principles of health economics, we have evaluated in what way ANA, as detected by IIF and a fully automated screening test for antibodies against a mixture of specific nuclear and cytoplasmic antigens (Symphony; EliA SymphonyTM system, Pharmacia Diagnostics, Uppsala, Sweden), can be optimally used to select sera that should be tested for specific reactivities in an automated line immunoassay that identifies fine antibody specificities (Lineblot; INNO-LIATMANA update, Innogenetics, Ghent, Belgium). We performed these three tests in all samples that were sent to the laboratory by any department of our hospital over a 4-month period with a request for ANA and/or anti-ENA testing. Test results were related to the number of signs and symptoms of AID that patients had before or at the time the samples were taken.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Samples and patients
All 328 serum samples that were sent to the Laboratory of Immunology of the University Hospital Utrecht by any hospital department between 11 December 2003 and 6 April 2004 with a request to test for ANA, anti-ENA or both, were included in the study. The blood samples used were collected in daily clinical practice. Patient treatments were not influenced, nor were extra investigations performed, so ethical approval was not necessary. One of us (PV) performed a chart review to analyse the clinical signs and symptoms as well as laboratory and histological data that were present in the patients before and at the time blood samples were taken. A composite list of clinical and laboratory findings (excluding ANA and anti-ENA results obtained with test samples) that characterize AID was derived from accepted diagnostic and classification criteria for specific AIDs [1, 2] (Table 1). Patients were classified into one of three groups according to the number of AID criteria that were fulfilled (group A, meeting no criteria; group B, meeting one criterion; group C, meeting two or more criteria).
|
Laboratory tests
All samples were tested by ANA-IIF, Symphony and Lineblot. ANA-IIF was performed with samples diluted 1:40 and 1:400 with HEp 2000 cells (ImmunoConcepts, Sacramento, CA, USA) as substrate. HEp-2000 cells are Hep-2 cells (a human larynx epithelioma cell line) transfected with Ro/SSA60 complementary DNA (cDNA) to improve its sensitivity for detecting anti-Ro/SSA antibodies [3]. In the Symphony test sera were incubated according to the manufacturer's instruction in wells coated with a mixture of recombinant antigens [U1 ribonucleoprotein (RNP)-A, -C, -70 kDa, Ro/SSA 52 and 60 kDa, La/SSB, Scl-70/topoisomerase I, CENP-B and Jo-1] and the natural purified Sm antigen. After incubation with a fluorogenic antihuman immunoglobulin (Ig) G conjugate, the amount of bound antibody is measured by fluorescence and directly compared with the response obtained for the calibrator. The ratio of test sample to calibrator is calculated and reported as negative (ratio <0.7), equivocal (ratio 0.7–1.0) or positive (ratio >1).
The Lineblot identifies reactivity against specific antigens. The test is based on the principle of a line immunoassay in which recombinant antigens (RNP-A, -C, -70 kDa, Ro/SSA 52 kDa, La/SSB, Scl70, CENP-B, Jo-1 and SmB), synthetic peptides (SmD and ribosomal P) and natural proteins (Ro/SSA 60 kDa and histones) are coated as discrete lines on a nylon membrane with a plastic backing. These strips are incubated sequentially with sera, alkaline phosphatase-labelled antihuman IgG antibody and substrate. Readings are made by comparing the intensity of the reaction with control lines and measured by image analysis. Results are reported as negative (<67% of control line), equivocal (between 67% and 100% of control line) or positive (>100% of control line).
Given the fact that Symphony is a screening test and Lineblot a confirmatory test, we decided to consider equivocal results with Symphony as positive and equivocal results with Lineblot as negative.
Sera with a positive ANA-IIF were tested for anti-dsDNA antibodies with the Farr assay (Diagnostic Products Corporation, Los Angeles, CA, USA) according to the manufacturer's instructions with 125I-labelled recombinant dsDNA. As advised by the manufacturer >10 IU/ml was interpreted as a positive result.
Costs
According to current Dutch financial regulations (imposed by the ‘Centraal Orgaan Tarieven Gezondheidszorg’, COTG), the Lineblot costs 50 points, the Symphony 15 points, an untitrated ANA-IIF 10 points and a titrated ANA-IIF 15 points. Points are paid according to local rules. In our hospital, 1 point equals 70 Euro cents.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Clinical data
The mean age of the 328 patients in the study was 44 ± 20 yr (range 3 months to 91 yr); 224 (68%) patients were female. Based upon the number of AID criteria these patients fulfilled, 198 patients were classified in group A (no criteria), 75 in group B (one criterion) and 55 in group C (two or more criteria) (Tables 1 and 2).
|
The most frequent diagnoses in patients from group A were arthralgia (31%) and polyneuropathy (25%) (data not shown). Arthritis, discoid rash, oral or ocular dryness and Raynaud's phenomenon were the most frequent signs and symptoms in patients from group B (Table 1). The diagnosis in patients with two or more criteria (group C) were systemic lupus erythematosus (SLE), lupus-like disease, primary Sjögren's syndrome (pSS), definite or possible polymyositis (PM), scleroderma and mixed connective tissue disease (Table 2).
Laboratory results
Of the 328 samples, 69 (21%) tested positive for ANA in a 1:40 serum dilution and 20 (6%) in a dilution of 1:400. Sixty (18%) samples were positive in the Symphony, including six with equivocal results. Seventy-two (22%) sera were Lineblot-positive (including three with equivocal results in the Symphony). Figure 1 shows the results with the three assays in relation to the clinical category to which the patients from whom the samples were taken had been assigned. Of the 198 patients of group A (no AID criterion), 15 (7.5%) were positive for ANA (1:40), 9 (5%) in the Symphony and 14 (7%) in the Lineblot. Of the 75 patients with one AID criterion (group B) 17 (23%) were ANA positive, 18 (24%) Symphony-positive and 24 (32%) positive in the Lineblot. Of 55 patients with two or more AID criteria (group C) 37 (67%) were ANA positive, 33 (60%) positive with Symphony and 34 (62%) with Lineblot. Sixty per cent of samples in the study were from patients without any AID criteria (group A) and, of these, 93% were negative in the Lineblot.
|
Within the total of 328 samples, a positive ANA-IIF at 1:400 dilution was present in 20 (6%). All but one sample came from patients with at least one criterion of AID [group B (n = 3) or C (n = 16)]. Lineblot was positive in 15/20 samples and anti-dsDNA antibodies were found in 8/15. Anti-dsDNA antibodies were also present in two of the five strongly ANA-positive and Lineblot-negative samples.
Clinico-serological correlations
Most (87%) of the 39 sera that were positive in all three assays (ANA-IIF, Symphony and Lineblot) came from patients with at least one criterion for AID (groups B and C, Fig. 1). Table 3 relates the specificities with Lineblot in relation to clinical details. Most samples had reactivities for more than one antigen. Antibodies against Ro/SSA and/or La/SSB antigens were found in four of the five patients from group A (no criterion for AID) with positive results in all three assays.
|
In seven samples with a positive result in all three assays, reactivity against CENP-B was found. In five of these seven samples anti-CENP-B was the only detectable reactivity and three of these five samples had a centromeric fluorescence pattern with ANA-IIF. In two other samples (both with anticentromeric ANA-IIF), anti-CENP-B plus anti-Ro/SSA52 reactivity was found. In two of the seven samples (one from a patient with scleroderma and one with Raynauds’ phenomenon) the presence of CENP-B antibodies was compatible with clinical findings. This also holds for a patient with achalasia (group A), assuming that the diagnosis of achalasia may be a scleroderma-related oesophageal problem. In the patients with lupus-like disease (LLD), discoid lupus (CDLE), sicca and pSS the finding of anti-CENP-B antibodies was unexpected. None of the patients had primary biliary cirrhosis, another clinical condition with which anticentromere antibodies are associated [4].
All seven samples that were positive for ANA-IIF and Lineblot but negative in the Symphony test came from patients with at least one criterion for AID (groups B and C) (Fig. 1, Table 4). One sample had monoreactivity against histones. As histones are absent in the antigen mixture used in the Symphony test, the negative Symphony result is explained. Excluding reactivity against histones, all but one of the sera had reactivity against single antigens. None of the reactivities found were ‘incompatible’ with the clinical diagnosis.
|
Details on reactivities in the Lineblot of 11 samples with a positive Symphony and a negative ANA-IIF test are given in Table 5. All but two samples came from patients with at least one criterion for AID (groups B and C). Seven of 11 samples had reactivity against Ro/SSA or La/SSB. One sample from a patient with PM had anti-Jo-1 reactivity. Failure of ANA-IIF to detect this reactivity is clinically relevant.
|
Table 6 presents details of 15 samples that were positive in the Lineblot but negative with ANA-IIF and Symphony. For one sample a negative Symphony was explained by monoreactivity against histones, an antigen not present in the mixture used in the Symphony test. Seven out of 15 samples came from patients in group A (no criteria for AID). Disregarding reactivity against histones, all samples had reactivity against single antigens. A remarkable finding is that none of the three samples with anti-topoisomerase I antibodies was detected with either ANA or Symphony. All three corresponding patients had an AID criterion (Raynaud's phenomenon, arthritis and sicca, respectively) that can occur in scleroderma.
|
Out of the 10 samples that were negative for ANA-IIF and Lineblot but positive with Symphony (Figure 1), three (33%) had an equivocal result in Symphony. A total of six samples had an equivocal result with Symphony. Of these, three (50%) had a positive Lineblot and only one (2%) a positive ANA-IIF.
In the total of 69 sera with a positive ANA-IIF test, 22 (32%) had anti-dsDNA antibodies as detected by Farr assay. Of the 23 samples with a positive ANA-IIF test, but negative results with both Symphony and Lineblot, anti-dsDNA antibodies were found in four (17%).
Costs
Table 7 shows the costs in relation to the number of Lineblot-positive samples found for different strategies in anti-ENA testing. When the Lineblot is performed only in patients with at least one criterion for AID, cost reduction is optimal. In ignorance of clinical signs and symptoms, limitation of the Lineblot to samples with either a positive result in ANA-IIF or Symphony strongly reduced costs, but failed to detect 26 and 22 samples, respectively, that were positive with Lineblot. All other algorithms with ANA-IIF and Symphony were more expensive and always failed to detect 15 Lineblot-positive samples that were negative for both ANA-IIF and Symphony.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Our study demonstrates that in daily clinical practice over half of the samples that came to the laboratory with a request to test for ANA/anti-ENA were from patients with a low pre-test probability for AID (group A) (Fig. 1). Only 7% of these group A sera had a positive Lineblot, and in more than half of cases reactivity concerned Ro/SSA and/or La/SSB antigens (Tables 3–6
). Knowledge of the presence of such reactivities in the absence of at least one criterion for AID (notably sicca complaints in this example) is not clinically helpful. The same holds for the other monoreactivities that were found in sera from group A, with the finding of CENP-B in a patient with achalasia as a possible exception. Here, the serological finding may lead to consideration of scleroderma-related oesophageal problems as an alternative diagnosis. Taken together, our data imply that in the ideal situation the laboratory should perform anti-ENA tests only in those patients with at least one criterion for AID, as has been reported previously for the ANA-IIF test [5]. Such a policy is cost-effective and makes pre-screening of sera before a Lineblot is performed unnecessary. Accepting that in daily life many tests are ordered in patients without manifestations of AID and that laboratory management should be price-conscious, the laboratory needs adequate screening assays that are relatively cheap in comparison to the Lineblot, the assay we used as gold standard in this study. In head-to-head comparison the Lineblot equals or surpasses counterimmunoelectrophoresis, ELISA and immunoblotting techniques in the identification of antibodies against RNP, Sm, SSB, SSA, topoisomerase I and Jo-1 antigens [6]. Whereas the Symphony test uses recombinant Ro/SSA60 antigen in the antigenic mixture, the Lineblot contains native Ro/SSA60 antigen to increase the chance that antibodies reactive with conformational, instead of continuous, epitopes are also detected [6]. Indeed we found that two of the 23 sera containing only antibodies against SSA or SSB antigens were positive for Lineblot and negative for Symphony.
Early studies with haemagglutination and counterimmunoelectrophoresis that used crude tissue extracts as antigens established a high correlation between the presence of Sm antibodies and SLE, and between U1 RNP antibodies and mixed connective tissue disease (MCTD) (especially at high titres) [7]. However, as immunoblotting and, in particular, the Lineblot detects reactivity against the individual components of Sm (notably SmB and SmD) and U1 RNP (notably RNP-A, -C and -70 kDa) complexes, the question arises as to which of the reactivities against individual Sm and U1 RNP components are to be considered disease specific and therefore clinically relevant. Guidelines published by a European consensus workshop suggest that SmD antibodies should primarily be taken into account for assigning anti-Sm (and disease specificity for SLE) because SmB antibodies ‘are often found in non-Sm sera (mostly via crossreaction)’ [8]. That anti-Sm determination should be based on anti-SmD reactivity was adopted in several studies [6, 9–11]. In a multicentre validation study of the Lineblot, SmD reactivity was found in 5% of SLE (n = 40), 12.5% of MCTD (n = 39) and 1% of patients with Sjögren's syndrome (n = 93). SmB was found in SLE (17.5%), MCTD (43%), scleroderma (3%) and rheumatoid arthritis (RA) (2.5%) [9]. The statement that ‘at least anti-SmD reactivity resulted in 100% disease specificity for SLE’ [9] did not hold in our cohort (Tables 3, 4 and 6).
Because a positive signal for at least two (out of three) antigenic determinants of RNP showed the highest statistical correlation with the results obtained with immunodiffusion (the technique used as the standard criterion for RNP reactivity) [9] this has been accepted as the definition of RNP positivity in the Lineblot [9–11]. Reactivity against two out of three RNP proteins is rare in healthy and diseased controls and occurs in 18–20% of SLE patients [10, 11], a high percentage of MCTD, 3% of scleroderma and 2.5% of RA patients [10]. In our cohort 11/328 (3%) of the sera had reactivity against two or three RNP proteins. The diagnosis in these patients was SLE (n = 4), MCTD (n = 3), LLD (n = 2), pSS (n = 1) and CDLE (n = 1), confirming that such reactivity is not 100% specific for MCTD and SLE.
We evaluated the Symphony and ANA-IIF as tools for selecting sera that should be evaluated further with Lineblot. Restriction of the Lineblot to samples with positive results in either ANA-IIF or Symphony strongly reduces the costs of anti-ENA testing compared with performance of the Lineblot with all samples (Table 7). However, in doing so, a number of sera with anti-ENA antibodies (26 and 22, when ANA-IIF and Symphony are used for screening, respectively) remained undetected (Fig. 1). Fifteen Lineblot-positive sera were missed, irrespective of whether screening was done with ANA-IIF or Symphony (Fig. 1, Table 6). Remarkably, 7/15 samples were from patients without manifestations of AID and, disregarding reactivity against histones (an antigen not present in the Symphony), all samples had reactivities against single antigens. The key question is whether ignorance of the presence of these reactivities is clinically important. The answer is positive with respect to topoisomerase I (Scl70) antibodies. All three samples with this reactivity in our cohort came from patients with manifestations that can occur in scleroderma and knowledge of their presence is therefore clinically relevant. Although our sample size of only three sera with topoisomerase I antibodies is small, our finding suggests that both ANA-IIF and Symphony are insensitive for their detection. This implies that when ANA-IIF or Symphony are used for screening, clinicians should be alerted that in case a specific search for topoisomerase I antibodies is indicated, a direct request for a specific test such as Lineblot should be made to the laboratory. Table 6 also shows that no other clinically important reactivities with Lineblot are presenting in sera being negative in both ANA-IIF and Symphony. However, presence of SmD in a patient with Raynauds’ phenomenon or arthralgia might be interpreted by clinicians as a sign of evolving SLE, although no data on the positive predictive value of SmD as detected by Lineblot are available.
Apart from the 15 sera missed by both ANA-IIF and Symphony, screening with ANA-IIF failed to detect another 11 samples and screening with Symphony another seven. All but two of the additional 11 Lineblot-reactive sera that were missed with screening by ANA-IIF, and all of the additional seven Lineblot-reactive sera that were missed when screening was done with Symphony, came from patients with at least one criterion for AID (Fig. 1). This increases the chance that ignorance of the presence of these reactivities is clinically relevant. In the 11 sera missed by screening with ANA-IIF the reactivity in the Lineblot was often against Ro/SSA and La/SSB antigens (Table 5). Several of the patients had sicca, a condition in which their presence provides a criterion for pSS. That the Lineblot is more sensitive for the detection of Ro/SSA and La/SSB than ANA-IIF, even when HEp-2000 cells are used, has been shown before [12]. Screening with ANA-IIF also missed Jo-1 antibodies in a patient with PM. This is a clinically relevant omission as anti-Jo-1 antibodies are associated with pulmonary complications in PM [1]. That anti-Jo-1 reactivity goes undetected or unreported with ANA-IIF is well recognized because these antibodies give a cytoplasmic, rather than nuclear, staining pattern on IIF [12].
A positive result with ANA-IIF together with negative results in both Symphony and Lineblot may indicate the presence of anti-dsDNA antibodies. Indeed, a positive test for anti-dsDNA antibodies (Farr assay) was found in three out of 23 ANA-positive samples with a negative result in Symphony and Lineblot.
Of the reactivities in seven sera that were missed when the Symphony is used for screening, almost all were against components of Sm and/or RNP complexes. Ignoring antihistone antibodies and accepting that reactivities against SmB and single determinants of RNP are clinically unimportant, relevant reactivities were found in three out of six patients (reactivity against two RNP proteins in a patient with CDLE, SmD in a patient with arthritis and SmD in a patient who met the ACR criteria for SLE).
In conclusion, the most cost-effective method that does not harm patients’ interests is a policy in which the performance of the relatively expensive Lineblot is restricted to sera from patients with at least one criterion for AID. In daily clinical practice, however, many requests for ANA or anti-ENA testing that reach the laboratory involve samples from patients without any clinical criteria for AID. In such a situation pre-screening with either ANA-IIF (1:40) or Symphony and restriction of the performance of Lineblot to sera with a positive result in one of the screening tests strongly reduces the costs of the detection of anti-ENA antibodies, with minimal loss of diagnostic capacity.
It is hard to conclude from our data whether ANA-IIF or Symphony is preferable as a tool for screening. Differences are small. Both ANA-IIF and Symphony were insensitive for the detection of topoisomerase-I antibodies and ANA-IIF was insensitive for the detection of anti-Jo-1 and some SSA/SSB antibodies. However, as a positive ANA-IIF test may, in contrast to the Symphony, be indicative of the presence of anti-dsDNA antibodies, we would prefer the ANA-IIF test for screening purposes in daily clinical practice.
|
Acknowledgments |
|---|
The authors are grateful to Coby van der Velde and Hanny Wolters for their technical assistance.
The authors have declared no conflicts of interest.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
- Klippel JH, Crofford LJ, Stone JH, Weynand CM. Primer on the rheumatic diseases, 12th edn. Atlanta: Arthritis Foundation, 2001.
- Alarcon-Segovia D, Cardiel MH. Comparison between 3 diagnostic criteria for mixed connective tissue disease. Study of 593 patients. J Rheumatol 1989;16:328–34.[Web of Science][Medline]
- Keech CL, Howarth S, Coates T, Rischmueller M, McCluskey J, Gordon TP. Rapid and sensitive detection of anti-Ro (SS-A) antibodies by indirect immunofluorescence of 60kDa Ro HEp-2 transfectants. Pathology 1996;28:54–7.[CrossRef][Web of Science][Medline]
- Powell FC, Winkelmann RK, Venencie-Lemarchand F, Spurbeck JL, Schroeter AL. The anticentromere antibody: disease specificity and clinical significance. Mayo Clin Proc 1984;59:700–6.[Medline]
- Slater CA, Davis RB, Shmerling RH. Antinuclear antibody testing. A study of clinical utility. Arch Intern Med 1996;156:1421–5.
[Abstract/Free Full Text] - Lopez-Longo FJ, Rodriguez-Mahou M, Escalona-Monge M, Gonzalez CM, Monteagudo I, Carreno-Perez L. Simultaneous identification of various antinuclear antibodies using an automated multiparameter line immunoassay system. Lupus 2003;12:623–9.
[Abstract/Free Full Text] - Sharp GC, Irvin WS, Tan EM, Gould RG, Holman HR. Mixed connective tissue disease–an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 1972;52:148–59.[CrossRef][Web of Science][Medline]
- van Venrooij WJ, Charles P, Maini RN. The consensus workshops for the detection of autoantibodies to intracellular antigens in rheumatic diseases. J Immunol Methods 1991;140:181–9.[CrossRef][Web of Science][Medline]
- Meheus L, van Venrooij WJ, Wiik A et al. Multicenter validation of recombinant, natural and synthetic antigens used in a single multiparameter assay for the detection of specific anti-nuclear autoantibodies in connective tissue disorders. Clin Exp Rheumatol 1999;17:205–14.[Web of Science][Medline]
- Pottel H, Wiik A, Locht H et al. Clinical optimization and multicenter validation of antigen-specific cut-off values on the INNO-LIA ANA update for the detection of autoantibodies in connective tissue disorders. Clin Exp Rheumatol 2004;22:579–88.[Web of Science][Medline]
- Hoffman IE, Peene I, Cebecauer L et al. Presence of rheumatoid factor and antibodies to citrullinated peptides in systemic lupus erythematosus. Ann Rheum Dis 2005;64:330–2.
[Free Full Text] - 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;48:2171–6.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||