| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1
Bio-Rad Labs., 4000 Alfred Nobel Dr., Hercules, CA 94547;
2
Arthritis Center of Nebraska, Lincoln, NE 68506;
3
Natl. Inst. of Dental Res., NIH, Bethesda, MD 20892-1190;
a address for correspondence: fax 510-741-5824, e-mail rich_gniewek{at}bio-rad.com
Assessing the concentration of antinuclear antibodies (ANA) in a patient's serum is commonly done by the ANA-immunofluorescence assay (ANA-IFA) (1)(2)(3)(4)(5)(6), a very tedious and subjective procedure. By contrast, ANA enzyme immunoassays (ANA-EIA) are easy to perform, and the results are not subjective. Although the performance of ANA-EIA was judged to be less than satisfactory (7), five different ANA-EIA kits were recently proven to be comparable with ANA-IFA (8). Similarly, in a recent study involving one of us (9), a comparison of ROC curves for ANA-IFA and ANA-EIA showed equivalent performance. In this study we generated ROC curves with disease diagnosis as the gold standard and compared them with the ROC curves from the earlier study (10). We also tested 98 serum samples from patients with primary Sjögren syndrome (SS).
The patients' serum samples entered into this study from the Arthritis Center of Nebraska (ACN) were part of the routine medical care when ANA-IFA was ordered. A total of 283 serum samples were collected from April 4, 1995, to October 26, 1995. Diagnosis was made according to the criteria established by the American Rheumatism Association (11). All of these samples were part of the daily laboratory routine, and none of the samples processed by the laboratory during this period was excluded. The 98 serum samples from patients diagnosed with primary SS had been stored frozen at the National Institute of Dental Research of the National Institutes of Health until needed for testing by ANA-EIA. Diagnosis of primary SS was made according to established criteria (12). All information regarding ACN samples, which included ANA-IFA results, age, sex, diagnosis, and medications, was withheld until ANA-EIA results had been sent back to ACN. All information regarding primary SS samples was withheld until ANA-EIA results had been generated and sent back to NIH.
Samples from the ACN were tested by ANA-IFA (Kallestad Quantafluor; Sanofi Diagnostics Pasteur) according to the manufacturer's instructions. Samples from NIH were tested by ANA-IFA (ANA-HEp-2; ImmunoConcepts) according to the manufacturer's instructions. Samples were then stored frozen (-20 °C) and shipped to Bio-Rad Labs., where they were tested by ANA-EIA with the automated analyzer Radias (Bio-Rad). The Radias instrument was programmed by the manufacturer to interpret ANA-EIA results of <0.9 as negative, 0.9 to 1.1 as indeterminate, and >1.1 as positive. The ANA-EIA plates for the Radias are coated with a HEp-2 cell extract containing ANA antigens, which include double-stranded DNA, SS antigen A (SS-A/Ro), SS antigen B (SS-B/La), Sm, ribonucleoprotein (RNP), Jo-1, and Scl-70.
Diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and agreement were calculated with standard formulas (2). These values were calculated after defining those diagnoses for which ANA is commonly used as a part of connective tissue disease (CTD) diagnosis as disease-positive. Hence, systemic lupus erythematosus, discoid lupus erythematosus, scleroderma/CREST (calcinosis, Raynaud phenomenon, esophageal motility abnormalities, sclerodactyly, and telangiectasia), Raynaud syndrome, SS, MCTD, overlap CTD syndromes, polymyositis, and dermatomyositis were considered disease-positive (11) and all other diagnoses were considered disease-negative. The exact McNemar statistic (10) was used to compare sensitivities and specificities. The 95% confidence intervals (CI) and ROC curves were constructed, and the areas under the curves were compared (13).
Approximately 14% (39 of 283) of the ACN samples entered into this study were from patients diagnosed with a CTD. The most common diagnosis was systemic lupus erythematosus (n = 16), followed by Raynaud syndrome (n = 6), SS (n = 6), scleroderma/CREST (n = 5), MCTD (n = 3), discoid lupus erythematosus (n = 2), dermatomyositis/polymyositis (n = 2), and an undefined CTD (n = 9). Some of these patients were diagnosed with more than one CTD.
The area under the ROC curve for ANA-IFA (Fig. 1
A) was 0.804 (CI 0.758–0.850) and did not differ significantly
(P >0.05) from the area under the ROC curve for ANA-EIA,
which was 0.817 (CI 0.772–0.862). The ROC curve for the ANA-IFA from
this study did not differ significantly (P >0.05) from the
ROC curves for the ANA-IFA in the previous study (Fig. 1B
).
|
With a reference range of <1:80 for the ANA-IFA, the sensitivity, specificity, positive predictive value, and negative predictive value were 64.1%, 80.7%, 34.7%, and 93.4%, respectively. With a reference range of <1.2 for the ANA-EIA, the sensitivity, specificity, positive predictive value, and negative predictive value were 71.8%, 76.2%, 32.6%, and 94.4%, respectively. Comparison of the sensitivity and specificity between the ANA-IFA with a reference range of <1:80 and the ANA-EIA with a reference range of <1.2 showed no significant difference (P >0.05).
When we tested the 98 serum samples from patients diagnosed with primary SS with a reference range for ANA-IFA of <1:80 and a reference range of <0.9 for ANA-EIA, 6 samples were commonly called negative and 86 samples were commonly called positive. With these reference ranges, the difference between the sensitivity for ANA-IFA (88.8%) and ANA-EIA (92.9%) was not significant (P >0.05). The agreement between the two methods was 93.9%.
Some bias may have been introduced into this study because the ANA-IFA results were used by the physicians to establish diagnoses whereas the ANA-EIA results were not made available to the physicians. Although it may be reasonable to assume that this bias favored the ANA-IFA, it would be difficult to determine the extent of this bias because no definitive test for CTD exists.
Although information on patterns is lost with the ANA-EIA method, this may be a minor disadvantage. The ANA-IFA fluorescent patterns have been associated with certain clinical states (1)(2)(3)(4)(5)(6), but no matter how convincing such associations may seem, they are difficult to confirm when unselected patients are used (14). The earlier study (9) reported that only 3 of 8 patients with anticentromere antibody had a diagnosis of CREST syndrome, and only 1 of 19 patients with nucleolar antibody had a diagnosis of scleroderma. In this study, 2 of 4 patients with anticentromere antibody had a diagnosis of CREST, and none of the 4 patients with nucleolar antibody had a diagnosis of scleroderma. Although the association of certain patterns with certain clinical states has been reported, the incidence of these patterns in non-CTD clinical states has not been investigated thoroughly.
We conclude that the ANA-EIA method is equivalent to the ANA-IFA in screening for ANA in serum from patients with CTD. Differences in performance between the ANA-EIA and ANA-IFA may be insignificant when the appropriate cutoffs are selected. The appropriate cutoff may depend on many factors including the cost of false negatives and false positives as well as the prevalence of CTD. The ANA-EIA has the advantage of being easy to use, and the results are objective.
Acknowledgments
This study was supported by Bio-Rad Laboratories, the Arthritis Center of Nebraska, and the National Institutes of Health.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  E. Avaniss-Aghajani, S. Berzon, and A. Sarkissian Clinical Value of Multiplexed Bead-Based Immunoassays for Detection of Autoantibodies to Nuclear Antigens Clin. Vaccine Immunol., May 1, 2007; 14(5): 505 - 509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Hayashi, T. Kawamoto, M. Mukai, A. Morinobu, M. Koshiba, S. Kondo, S. Maekawa, and S. Kumagai 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., September 1, 2001; 47(9): 1649 - 1659. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
