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● The following guideline presents a series of recommen- that are pathognomonic for particular diseases. For these
dations based on published medical literature for use of reasons, test results for autoantibodies alone are insuffi-
the antinuclear antibody (ANA) test and tests for specific cient to establish the diagnosis of a systemic rheumatic
autoantibodies to nuclear antigens in the diagnostic eval- disease; they must always be interpreted in the clinical
uation, prognostic assessment, and monitoring of patients context. Positive results for tests such as the ANA test are
with systemic rheumatic diseases. The guideline emphasiz- seen quite commonly in patients with nonrheumatic dis-
es the need for clinical evaluation to improve the useful- eases and even among normal, healthy persons. Moreover,
ness of test results in patient management. Consideration technical considerations are critical to the accurate inter-
is given to appropriate use of the generic ANA test in the pretation of results of autoantibody tests. Improper use of
initial evaluation of patients with signs and symptoms of a immunologic tests can result in misdiagnosis, inappropri-
systemic rheumatic disease, the evaluation of patients sus- ate therapy, and wasted health care resources.
pected of having lupus erythematosus, use in clinical sit- In an effort to help define the optimal clinical use of the
uations in which the ANA test is required to establish a ANA and related tests for specific autoantibodies (anti-
disease diagnosis, and identification of clinical situations dsDNA, anti–Ro [SS-A], anti–La [SS-B], anti-Sm, anti-
in which the ANA test has little value. Sections are also nRNP, antihistone, and anti–Scl-70 antibodies), the College
devoted to recommendations aimed at improving the an- of American Pathologists (CAP) assembled a panel of rep-
alytic methods used to detect and measure ANA and spe- resentatives from the CAP (Drs Homburger and Tomar),
cific autoantibodies to nuclear antigens and to the appro- the American College of Rheumatology (Drs Kavanaugh
priate use of tests for specific autoantibodies in several dis- and Reveille), and the Clinical Immunology Society and
ease situations that commonly occur in patients with sus- Clinical Center of the National Institutes of Health (Tho-
pected or documented systemic rheumatic diseases. mas Fleisher, MD). The guidelines presented herein rep-
Emphasis is placed on the use of these tests only in situa- resent the work of members of the panel and other invited
tions in which the test results can be expected to provide contributors (Robert Lahita, MD; Peter Schur, MD; Yvonne
information necessary for clinical decision making. Those Sherrer, MD; and Dr Solomon).
tests of limited medical usefulness and situations in which Development of these recommendations began with a
test results are likely to be misleading are also identified. thorough search of the relevant medical literature. More
(Arch Pathol Lab Med. 2000;124:71–81) than 800 published articles were retrieved and critiqued
using criteria published by the Evidence-Based Medicine
Working Group.1,2 Individual recommendations in the
R esults of serologic tests for autoantibodies, including
tests for antinuclear antibodies (ANAs) and antibod-
ies to specific nuclear antigens such as double-stranded
guideline are based on the best relevant literature.
This guideline is intended to serve as an aid to the clin-
ical use and interpretation of tests for autoantibodies.
DNA (dsDNA), play an important role in the diagnosis of
These recommendations are not designed to be a substi-
systemic rheumatic diseases. Although the results are of-
tute for clinical judgment. In atypical cases, clinical judg-
ten useful, they can be misleading. Few tests yield results
ment may require deviation from the recommendations.
Also, many of the rheumatologic diseases discussed here-
in follow a variable or evolving course, and changes in
Accepted for publication August 31, 1999. clinical status may require reevaluation and repeat testing.
From the University of Texas Southwest Medical School, Dallas (Dr
Kavanaugh); University of Wisconsin Hospital and Clinic, Madison (Dr Finally, the recommendations in this guideline may evolve
Tomar); University of Texas Health Science Center, Houston (Dr Rev- over time, as newer analytic methods and additional clin-
eille); Brigham & Woman’s Hospital, Boston, Mass (Dr Solomon); and ical research yield important results.
Mayo Clinic, Rochester, Minn (Dr Homburger). The recommendations contained herein are presented in
Presented at the College of American Pathologists Conference XXXII, several parts. The first section reviews the history of de-
Guidelines for Laboratory Evaluation and Use of Antinuclear Antibod-
velopment of tests for autoantibodies to nuclear antigens.
ies and Laboratory Diagnosis and Monitoring of Monoclonal Gam-
mopathies, Chicago, Ill, May 29–31, 1998. The second section discusses technical considerations im-
Reprints: Henry A. Homburger, MD, Mayo Clinic, Hilton 920, 200 portant to the performance of these laboratory tests. The
First St SW, Rochester, MN 55905. third section presents guidelines for the use of autoanti-
Arch Pathol Lab Med—Vol 124, January 2000 Antinuclear Antibody and Autoantibody Tests—Kavanaugh et al 71
body tests to nuclear antigens in the evaluation of patients Table 1. American College of Rheumatology
suspected of having a systemic rheumatic disease or for Classification Criteria for Systemic Lupus
prognostic assessment of the disease. The final 2 sections Erythematosus*
are devoted to frequently asked questions about the clin- Malar rash: fixed malar erythema, flat or raised
ical application of autoantibody tests and additional in- Discoid rash: erythematous raised patches with keratotic scaling
formation about individual tests for autoantibodies. and follicular plugging; possible atrophic scarring
Photosensitivity: rash as an unusual reaction to sunlight
HISTORY Oral ulcers: oral or nasal ulcers, usually painless
Arthritis: inflammatory arthritis of 2 or more peripheral joints
Observation of the ‘‘LE cell’’ by Hargraves et al3 in 1948
Serositis: documented pleuritis or pericarditis
led to the first laboratory test for ANA. This was an im- Renal disease: persistent proteinuria (.0.5 g/d or .31) or cellular
portant discovery, as it provided the clinician with a test casts
that could be used to support the diagnosis of systemic Neurologic disorder: unexplained seizures or psychosis
lupus erythematosus (SLE). Previously, the diagnosis of Hematologic disorder: hemolytic anemia; or leukopenia (white
SLE often could not be established until tissue specimens blood cell count ,4.0 3 109/L) or lymphopenia (white blood
cell count , 1.5 3 109/L) on 2 occassions
were obtained. Even though the lupus erythematosus (LE)
Immunologic disorder: anti-dsDNA antibodies; or anti-Sm
cell preparation was recognized as a useful laboratory test, antibodies; or antiphospholipid antibodies
it soon became apparent that it was neither absolutely sen- Antinuclear antibody: positive antinuclear antibody test result
sitive nor specific for the diagnosis of SLE. In an effort to * Additional signs and symptoms suggestive of systemic lupus ery-
further explain and refine this test, work conducted in a thematosus include, Raynaud phenomenon, excessive hair loss, un-
number of laboratories led to the recognition that the fac- explained fever, unexplained lymphadenopathy or splenomegaly, and
tors responsible for the LE cell phenomenon were a family unexplained thromboembolic phenomena.
of antibodies to various nuclear constituents.4 By the late
1950s, tests were developed to detect these ANAs using
immunofluorescence (IF) techniques on animal tissue sub- nucleoproteins, including Ro (SS-A), La (SS-B), nRNP, and
strates. An assortment of tissue types were used to detect Sm; enzymes such as topoisomerase-1 (Scl-70); and his-
ANA, and many of the early studies assessing clinical util- tone proteins. Reactivity with these antigens is more dis-
ity of the ANA test in SLE and other diseases used kidney ease specific than the above-mentioned patterns and may
or liver sections from rats or mice as substrates. Compared also provide clinically useful prognostic information (see
with the LE cell preparation, the immunofluorescent ANA below).
test on rodent tissues was more sensitive for the diagnosis Over the past decade, most laboratories worldwide have
of SLE. However, this increased sensitivity was associated come to use a human tumor cell line substrate (the HEp-
with reduced specificity, and substantial numbers of pa- 2 cell line) for routine ANA testing.5 The HEp-2 substrate
tients with other diseases and even healthy persons were has largely replaced rodent tissues and has become the
found to have a positive ANA test result.5 In an effort to standard substrate for performing the ANA test. Most rel-
reduce the numbers of false-positive or clinically irrele- evant literature in recent years is based on results ob-
vant results, laboratories began to report titers; that is, the tained with HEp-2 cells. There are several important dif-
highest dilution of test serum that would still show im- ferences between these 2 methods of ANA testing. The
munofluorescent nuclear staining. In general, patients human cell line is more sensitive than the rodent line for
with SLE had higher titers of ANA than normal persons the detection of ANAs. As a result, virtually all SLE pa-
or persons with other diseases. Although testing often be- tients have a positive ANA finding with use of the HEp-
gan at 1:10 or 1:20 dilutions, a positive test result typically 2 substrate. Increased sensitivity results from the expres-
was reported only when immunofluorescent staining per- sion of more relevant nuclear antigens in the human tu-
sisted at dilutions of 1:40 or higher. While this practice mor cells. For example, rodents do not express Ro (SS-A)
reduced the problem of spurious results, large numbers antigen. Also, centromeres, nucleoli, and other cellular or-
of patients had positive ANA test results but did not have ganelles are more readily seen in transformed tumor cells
SLE, and occasional SLE patients had negative ANA test like HEp-2. A sizable number of patients, for whom the
results. Despite these limitations, a positive ANA finding term ‘‘ANA-negative lupus’’ was coined, have reactivity
was incorporated as a diagnostic criterion for SLE (Table predominantly with Ro (SS-A).8 Others have nucleolar
1).6,7 reactivity that is not readily detected on rodent tissue sub-
Analysis of the results of ANA tests on animal tissue strates. While such patients may have had negative ANA
substrates revealed different appearances or patterns of test results on rodent tissue substrates, they are almost
immunofluorescent staining. It became standard practice always positive when the HEp-2 substrate was used.9
for laboratories to report the patterns observed (eg, speck- The increased sensitivity of the HEp-2 ANA test com-
led, homogeneous/diffuse, rim/peripheral, nucleolar, or pared with the ANA test performed on rodent tissue is
centromere) in addition to titers for positive ANA test re- associated with a lower specificity. Thus, more patients
sults. In some cases, there appeared to be some clinical with diseases other than SLE as well as normal healthy
correlation with particular staining patterns. However, the persons have positive ANA test results. Some laboratories
relevance of these associations has been largely supplant- have attempted to adjust for this by using a higher titer
ed by the ability to categorize positive ANA test results of ANA as a cutoff for a positive result. For example, on
on the basis of the antigen specificities of the autoantibod- rodent tissues, ANA titers of 1:20 or 1:40 or higher have
ies. Using laboratory techniques such as immunodiffusion, been called positive, whereas on the HEp-2 substrate, ti-
immunoprecipitation, radioimmunoassay (RIA), hemag- ters of 1:80 or higher are usually called positive.
glutination, and enzyme immunoassay (EIA), it has been Another difference between the types of ANA tests is
shown that ANA-positive sera react with several different that the staining patterns previously described with ro-
nuclear antigens, including dsDNA; small nuclear ribo- dent tissue are not the same on HEp-2 cells. For example,
72 Arch Pathol Lab Med—Vol 124, January 2000 Antinuclear Antibody and Autoantibody Tests—Kavanaugh et al
a rim pattern is rarely seen on HEp-2 cells. Because of this Table 2. Regulatory Requirements for IF-ANA and
and because the more relevant tests for autoantibodies to EIA-ANA Test Methods*
specific nuclear antigens are now widely available, inter-
Personnel: federal regulations define the minimum
pretation of patterns has become much less important clin- qualifications required for laboratory directors (doctoral
ically. degree), technical supervisors (doctoral degree, master’s
More recently, EIA techniques have been used for ANA degree, or bachelor’s degree plus experience), clinical
testing. In a typical EIA, nuclear antigens from cell prep- consultants, general supervisors, and testing personnel
arations or mixtures of purified or recombinant antigens (associate degree) engaged in ‘‘high-complexity’’ testing
Competency assessment: required yearly for individuals that
are adsorbed to microtiter plates. The EIA has several ad-
perform tests and includes direct observation of testing and
vantages over IF, including ease of performance and lower reporting of results
cost of the test. The EIA is now widely used to test for Quality control: laboratories must have an ‘‘ongoing
specific autoantibodies to nuclear antigens such as ds- mechanism’’ to identify problems and produce corrective
DNA, Ro (SS-A), and La (SS-B). Nevertheless, at present, actions
use of the EIA for the generic ANA test has not been sub- Proficiency testing: ANA is a ‘‘regulated analyte’’; acceptable
performance on proficiency testing is defined by a result
ject to widespread population testing. Until comparative
equal to the target value 6 2 dilutions; acceptable results
studies are available that contrast the operational charac- must be obtained on 4 of 5 challenges in each mailing;
teristics of the EIA with those of the established immu- specific ANAs are not ‘‘regulated analytes’’ and acceptable
nofluorescent methods, it is difficult to make recommen- performance is defined by the proficiency provider
dations concerning the utility of the EIA technique for de- * IF indicates immunofluorescent; ANA, antinuclear antibody; and
termining ANA. EIA, enzyme immunoassay.
Arch Pathol Lab Med—Vol 124, January 2000 Antinuclear Antibody and Autoantibody Tests—Kavanaugh et al 81