1. Immunopathology / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTING
Guidelines for the Laboratory Use of Autoantibody Tests
in the Diagnosis and Monitoring of Autoimmune
Rheumatic Diseases
Renato Tozzoli, MD,1 Nicola Bizzaro, MD,2 Elio Tonutti, MD,3 Danilo Villalta, MD,4
Danila Bassetti, MD,5 Fabio Manoni, MD,6 Anna Piazza, MD,7 Marco Pradella, MD,8
and Paolo Rizzotti, MD9
Key Words: Antinuclear antibodies; Autoimmune rheumatic disease; Guidelines; Dermatopolymyositis; Systemic sclerosis; Systemic lupus
erythematosus; Sjögren syndrome
Abstract Guidelines in the clinical laboratory recommend the
The Italian Society of Laboratory Medicine Study most appropriate modalities for conducting a diagnostic
Group on the Diagnosis of Autoimmune Diseases has procedure, with the aims of improving the efficiency and effi-
generated a series of guidelines for the laboratory cacy of the diagnosis and monitoring the disease. As guide-
diagnosis and monitoring of systemic autoimmune lines are the result of a methodologic approach in which data
rheumatic diseases intended for the use of clinical based on scientific observation are screened by expert
pathologists and laboratory physicians. These opinion and discussion and then coordinated, they thus repre-
guidelines are based on a systematic review of sent a working tool and should be considered temporary steps
published works and expert panel discussion and in a continuous process of updating and formation.1 The
consist of 13 recommendations for antinuclear practice of evidence-based medicine2,3 has resulted in an
antibodies, anti–double-stranded native DNA, and important upsurge of interest in guidelines; international
antinuclear specific antibodies. To improve analytic experiences have confirmed that scientific-medical societies
performances and help select the most appropriate test have a central role in their elaboration and in defining indica-
for specific autoantibodies, as well as provide tors of efficiency in the provision of diagnostic services. This
education and guidance in the use of these tests, special issue is particularly important in the field of laboratory diag-
emphasis is placed on laboratory methods. nosis of autoimmune diseases, because methods for the assay
of organ-specific and non–organ-specific autoantibodies have
evolved considerably since the lupus erythematosus (LE) cell
phenomenon was reported more than 50 years ago.4 Requests
for these tests have risen remarkably, mainly owing to a
growing understanding of the nature of autoantibodies, as
well as the molecular characterization of the main target
autoantigens and the recent insights into the diagnostic and
prognostic significance of the presence and concentration of
some autoantibodies in the serum of patients with autoim-
mune diseases.
On the other hand, the parallel, almost chaotic prolifera-
tion of new methods and analytic systems in clinical
immunology has involved a constantly increasing expenditure
of economic resources for the assay of autoantibodies, esti-
mated in hundreds of millions of dollars throughout the world5;
this phenomenon thus demands the adoption of measures of
316 Am J Clin Pathol 2002;117:316-324 © American Society for Clinical Pathology

2. Immunopathology / REVIEW ARTICLE
standardization and verification of the quality of the methods, ❚Table 1❚
as well as the introduction and widespread use of guidelines Summary of Guidelines for the Laboratory Use of
Autoantibody Tests
for the diagnosis and monitoring of autoimmune diseases.
Autoantibodies directed against intracellular antigens • Test for antinuclear and anticytoplasmic antibodies (ANA) only in
(eg, antinuclear antibodies [ANAs], anti-DNA, antinuclear patients with symptoms of autoimmune rheumatic disease
(ARD), because weak ANA reactivity may be present in many
specific antibodies) and autoimmune rheumatic diseases nonrheumatic patients and even in “healthy” control subjects.
constitute a major interest of physicians working in the field • To diagnose ARD, screen for ANA using indirect
immunofluorescence (IIF) on HEp-2 cells, and specify
of clinical immunology (eg, rheumatologists, immunologists, immunohistochemical pattern (nuclear, cytoplasmic, mitotic) and
dermatologists) and in the laboratory.6 quantity (titer, concentration).
• Consider ANA titer (or concentration) of 1:40 (5 IU/mL) and 1:160
For about 10 years, progressive review of the cardinal (20 IU/mL) as decision-making levels: negative if <1:40, low
criteria for the diagnosis of the principal autoimmune positive from 1:40 to 1:80, and positive if 1:160 or more.
• Do not use ANA titer or concentration to monitor ARD.
rheumatic diseases has increasingly centralized the role of the • Use enzyme-linked immunosorbent assay (ELISA)-ANA screening
diagnostic laboratory since the presence of diagnostic criteria test only when your procedure has shown good clinical and
in the serum autoantibody profile of the patient represents an analytic correlation with the IIF method.
• Test for anti–double-stranded native DNA (anti-dsDNA) antibody
important prerequisite for the clinical diagnosis. Indeed, posi- only in ANA-positive patients in whom systemic lupus
tive results for ANA and the presence of anti–double-stranded erythematosus (SLE) is suspected clinically, using the IIF on
Crithidia luciliae or ELISA methods. Positive ELISA findings must
native DNA (anti-dsDNA) or anti-Sm antibodies constitute 2 be confirmed by IIF or the Farr technique.
of the 11 criteria for the diagnosis of systemic lupus erythe- • Use the ELISA or the Farr technique to monitor anti-
dsDNA–positive SLE patients.
matosus (SLE)7,8; positivity for ANA in high titer or the pres- • Test for antinuclear specific antibody only in IIF-ANA–positive
ence of anti-Ro/SSA or anti-La/SSB antibodies are diagnostic patients or IIF-ANA–negative patients who have clear symptoms
of ARD (especially Sjögren syndrome and polymyositis).
criteria for Sjögren syndrome9; the presence of anti–Jo-1 anti- • Use counterimmunoelectrophoresis, ELISA, line-immunoblot, or
bodies is a criterion for the diagnosis of dermatopoly- immunodiffusion to detect Ro/SSA, La/SSB, Sm, uridin-rich 1
myositis10; the presence of anticentromere or anti–topoiso- ribonucleoprotein, Scl70, Jo-1, CENP-B, rRNP and chromatin
,
antibodies; reserve Western blot for diagnostic workups.
merase I (anti-Scl70) antibodies is the criterion for classifying • In the case of isolated positivity for U1RNP report quantitative
,
subtypes of cutaneous systemic sclerosis as limited or results because a high antibody titer is diagnostic for mixed
connective tissue disease.
diffuse11; and a high titer of anti–uridin-rich 1 ribonucleopro-
tein (U1RNP) antibodies is the main diagnostic criterion for
mixed connective tissue disease (MCTD).12
Recommendations for ANA and Anticytoplasmic
Antibodies
The prevalence of autoimmune rheumatic diseases in
Guidelines for the Laboratory Diagnosis
the population is not high; with the exception of rheuma-
of Inflammatory Connective Tissue
toid arthritis, which affects about 1% of adults and for
Diseases
which at present there is no specific serologic autoanti-
Many literature reports describe the standardization of body marker, the other rheumatic diseases on the whole do
methods and analytic procedures for the detection of autoan- not affect more than 0.5% of the population.17,18 Most of
tibodies,13,14 with particular attention to the preparation of the affected subjects initially have modest, nonspecific
reference materials,15 and a recent article gives guidelines for clinical signs, generally with an indistinct and gradual
the clinical use of autoantibody testing16; however, none course; included among these is the possibility of a casual
treats the production of guidelines for the use of laboratory finding of abnormal laboratory test results in asympto-
methods to diagnose autoimmune rheumatic diseases. The matic subjects.
Italian Society of Laboratory Medicine Study Group on the Autoantibodies in low titer without clinical significance in
Diagnosis of Autoimmune Diseases has generated a series of healthy subjects or individuals with diseases other than
guidelines intended for the use of clinical pathologists and rheumatic diseases are a relatively frequent finding in the popu-
laboratory physicians ❚Table 1❚. These guidelines are based lation. On average, it is possible that 1 individual in 4 will show
on systematic review of published works (articles published positivity for autoantibodies with common laboratory tests, and
from 1966 through 2000 were identified by searching the this finding does not necessarily indicate the existence of an
National Library of Medicine MEDLINE and by examining autoimmune rheumatic disease19,20; for these reasons:
the bibliographies of retrieved original articles and review We recommend that tests to detect autoantibodies are
articles), as well as expert panel discussion, and consist of 13 performed only when a consistent clinical suspicion of autoim-
recommendations subdivided for ANA, anti-dsDNA, and mune rheumatic disease is present. ANA determination should
antinuclear specific antibodies. not be used to screen subjects without specific symptoms,
© American Society for Clinical Pathology Am J Clin Pathol 2002;117:316-324 317

3. Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTING
because weak ANA reactivity is present in many nonrheumatic Among the nuclear patterns, the most frequent consist
patients and even in “healthy” control subjects. of homogeneous/peripheral fluorescence (DNA, deoxyri-
Systemic autoimmune diseases are characterized by bonucleoprotein, histones) and speckled fluorescence (RNP,
the presence of high titers of serum autoantibodies to Sm, Ro/SSA, La/SSB); the relatively frequent patterns
intracellular proteins and nucleic acids.21 These autoanti- include the centromeric (CENP-A, CENP-B, CENP-C),
bodies are generally referred to as antinuclear antibodies nucleolar (PM/Scl, nucleolin, fibrillarin, RNA polymerase I,
owing to their predominant reactivity with nuclear anti- human upstream binding factor), and the diffuse grainy
gens, but they also include anticytoplasmic antibodies. (topoisomerase I or Scl70) patterns, while the rarer pictures
The test for ANAs has a very high diagnostic sensitivity, include the proliferating cell nuclear antigen (PCNA) or
such that it is considered the best test for the screening for cyclin, nuclear dots (coilin, Sp-100), and nuclear membrane
these diseases.7,22 (lamins A, B, and C; glycoprotein 210) patterns.
As most ANAs have an IgG isotype, and the detection Cytoplasmic patterns are relatively less frequent and
of IgM and IgA antibodies will not improve sensitivity but consist of speckled fluorescence (tRNA synthetase), mitochon-
may reduce specificity23-25: testing for IgG ANA will suffice drial fluorescence (proteins of the pyruvate-dehydrogenase
to detect serum samples that contain ANAs in patients with complex), ribosomal fluorescence (ribosomal ribonucleopro-
autoimmune rheumatic disease.24 tein), cytoskeletal filament fluorescence (eg, actin, vimentin),
Indirect immunofluorescence (IIF) is the most and fluorescence of the Golgi apparatus and lysosomes.
commonly used technique for ANA determination because it In reference to the rare mitotic patterns, these include
is highly sensitive with good specificity, easy to perform, and fluorescence of the spindle (tubulin), centrosomes (enolase),
inexpensive.26 Therefore: poles or NuMa (nuclear matrix protein), midbody, and the
IgG ANA determination using indirect immunofluores- chromosomal protein CENP-F (autoimmunity to which is
cence should be the initial step in the laboratory diagnosis of associated mainly with cancer and hepatitis B or C and not
autoimmune rheumatic diseases. with systemic autoimmune disease).30 For this reason:
In the IIF technique, tissue sections of murine liver and When performing IIF-ANA, use the epithelial cell line
kidney or epithelial cell lines obtained from human laryn- HEp-2 (American Type Culture Collection CCL 23) from
geal carcinoma may be used as substrate; the latter has human laryngeal carcinoma, provided that the expression
many advantages over murine tissues, including good visu- and integrity of the clinically significant antigens is
alization of all cellular structures; the presence of a homo- preserved.
geneous, monolayer cell population; capacity to express Specify the immunohistochemical patterns as follows,
antigens present in all phases of the cell cycle; and the and add a short interpretation of the significance:
possibility to identify antibodies restricted to human nuclear 1. Nuclear patterns: homogeneous/peripheral; speckled;
antigens.24,26,27 The use of increasingly optimized cellular centromeric; nucleolar; PCNA; nuclear dots; nuclear
substrates26,28 has increased the technique’s sensitivity while membrane; diffuse grainy
maintaining a high specificity for detecting a wide spectrum 2. Cytoplasmic patterns: speckled; mitochondrial-like;
of autoantibodies directed against intracellular antigens.25 ribosomal-like; Golgi apparatus; lysosomal-like;
However, there are some exceptions: anti-Ro/SSA and cytoskeletal filaments (actin, vimentin, cytokeratin)
anti–t-RNA synthetases are not always readily detected 3. Mitotic patterns: mitotic spindle; centrosomes; NuMA;
owing to scarcity of antigen in HEp-2 cells or other tissues midbody; CENP-F
and/or antigen leaching and denaturation secondary to fixa- The fluorescence intensity observed in IIF is expressed
tion procedures.24,29 To this end, acetone-fixed substrate in different ways in clinical laboratories. Expression with a
slides are preferable since ethanol and methanol fixations qualitative scale of values from + to ++++ has the advantage
may remove Ro/SSA antigen.16 of saving time and resources because titration of serum
The type of picture observed in IIF on HEp-2 cells in samples is not requested; however, the intensity of fluores-
interphase is correlated with antibody specificity, and the cence in the working dilution is not always proportional to
signals can originate from nuclear, mitotic, or cytoplasmic the antibody concentration. This is particularly true for
domains. Several immunohistochemical patterns are known centromeric and nucleolar patterns in which the reduced
for which an association with autoimmune and nonautoim- amount of antigen makes the definition of fluorescence
mune diseases has been demonstrated. At present, of the intensity highly subjective and, thus, poorly accurate.
approximately 40 types of fluoroscopic patterns identified, In the majority of laboratories, antibody concentration is
only 19 are reasonably associated with clinical pictures; expressed with a quantitative scale of values, using titers
moreover, for many autoantibodies, the target autoantigen (reciprocal of the last dilution of the reactive serum); this
has been identified with certainty. method better differentiates low- from high-positive titers
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4. Immunopathology / REVIEW ARTICLE
and enables the establishment of different cutoff levels, each would classify virtually all patients with SLE, systemic scle-
one with its own characteristics of sensitivity and specificity; rosis, and Sjögren syndrome as positive for ANA.20
however, this method also is bound to a subjective definition High autoantibody titers (>1:160) are present in sick
of the end point and, thus, is poorly reproducible, as subjects, but in only 5% of healthy people; this threshold is a
evidenced by the wide scattering of the titers obtained in little less sensitive but more specific.20 Indeed, at an end point
numerous collaborative studies.20,31,32 titer less than 1:160, the follow-up secondary tests will yield
The analytic imprecision or inaccuracy related to the use positive results in fewer than 5% of the cases.36,40 Intermediate
of dilutions makes it preferable to use calibrators adjusted titers may be present in about 20% of the healthy population
against an international reference standard (World Health and in a small percentage of sick subjects. For these reasons:
Organization [WHO] International Reference Preparation Titers of 1:40 and 1:160 (or concentrations of 5 and 20
[IRP] 66/233, homogeneous pattern) and to express the IU, respectively) are considered decision-making levels that
results in international units of concentration (IU/mL).23,33 require different operative algorithms:
Available since 1982 are a number of reference sera for the 1. Titers less than 1:40 (5 IU) should be considered as
standardization and verification of the quality of the various negative.
commercial preparations of HEp-2 cells34 and the different 2. Titers equal to or more than 1:40 (5 IU) and less than
types of nuclear fluorescence in the ANA test.31,35 However, 1:160 (20 IU) should be considered positive at low titer (in
the WHO standard is not easy to obtain on a regular basis, the absence of specific symptoms, further diagnostic study is
and secondary or tertiary standards should be prepared and not advised, but the patient should be clinically monitored).
eventually used.31 Consequently: 3. Titers equal to or higher than 1:160 (20 IU) should be
We recommend expressing ANA quantity in titer (recip- considered positive, and patients should undergo further
rocal of the last dilution of the reactive serum). If a reference diagnostic study because they are probably affected by an
standard is available, expression in antibody concentration autoimmune disease.
(IU/mL, WHO-IRP 66/233) is preferable. Each laboratory should verify the consistency of these
When ANA is expressed as a titer, which is the most used cutoff levels based on the characteristics of its own case
method, the recommended initial dilution of the sample is 1:40 material and possibly set cutoff levels at a dilution that gives
(corresponding approximately to 5 IU),36 and a titer equal to high diagnostic specificity as determined against inflamma-
or greater than 1:160 (20 IU) should be considered positive.20 tory diseases and healthy control subjects.
Low titers (1:40-1:80; 5-10 IU) of ANA may be present The variation in ANA titer in IIF seems to have a poor
in healthy subjects (in particular, pregnant women, women correlation with the course of the disease, with the sole
older than 40 years, and elderly persons) and as a phenom- exception of the anti-PCNA and antichromatin antibodies
enon associated with viral infections, paraneoplastic neuro- (homogeneous nuclear pattern) for which a titer decrease or
logic syndromes, liver disease, chronic fatigue syndrome, disappearance is associated with the efficacy of therapy.41
and cancers of various types.37 For the most part, these are This may be related to the fact that most assay systems are
natural autoantibodies with low avidity that in some cases not rigorously quantitative, and the use of variations in anti-
may be directed against microbial antigens and environ- body levels as a reflector of disease activity will need to
mental chemical haptens, with a repertoire of antigenic await the introduction of more quantitative assays; therefore:
specificity similar to that of autoantibodies present in In general, the use of variations in the ANA titer in IIF
autoimmune rheumatic diseases.38 to monitor the course and therapy of autoimmune rheumatic
The autoantibodies detected in serum samples of disease is not advised.
patients with autoimmune disease are most likely a heteroge- Despite recent advances in the standardization of the IIF
neous mixture of polyreactive, low-affinity natural autoanti- method (automation of the analytic procedure and recogni-
bodies of IgM isotypes and monoreactive, high-avidity path- tion of the immunohistochemical pattern by way of comput-
ogenic autoantibodies of IgG and IgA isotypes. 39 The erized systems), the technique still has some methodologic
methods commonly used in the clinical laboratory are not and interpretive limitations. A negative finding for ANA on
able to distinguish these two types of autoantibodies, so that IIF may occur in connective tissue diseases owing to the
the differential diagnosis must necessarily be conducted on effective absence of antinuclear autoantibodies,42 the pres-
the basis of the patient’s clinical history and symptoms and ences of antibodies to very soluble antigens (such as
the autoantibody levels present in serum. Ro/SSA), or the presence of autoantibodies directed against
Low autoantibody titers (1:40) are rarely present in scarce cytoplasmic antigens (such as Jo-1 and Ro/SSA).
autoimmune patients, but are observed in about 32% of To simplify and standardize the ANA test, solid phase
healthy subjects20; this threshold is highly sensitive and poorly immunoenzymatic methods (EIA-ANA) have been promoted
specific. However, it could have diagnostic value, since it in recent years as alternatives to the immunofluorescence test
© American Society for Clinical Pathology Am J Clin Pathol 2002;117:316-324 319

5. Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTING
for both screening and detection of total ANAs. While such became evident that the DNA molecule presented several
EIA-ANA tests are less labor intensive and can be used to epitopes and, therefore, that the anti-DNA antibodies
screen and detect a panel of common specific autoantibodies included a heterogeneous group of immunoglobulins with
usually observed in systemic rheumatic diseases, they are not different specificities. The most commonly identified anti-
100% sensitive when compared with IIF-ANA43-47; in addi- bodies are those directed against single-stranded DNA
tion, the EIA-ANA test may fail to detect certain ANAs with (ssDNA), whose antigenic determinants seem to be localized
atypical or rare immunofluorescent patterns.39 in the basic purine and pyrimidine sequences; the antibodies
Moreover, there are remarkable qualitative differences directed against dsDNA instead recognize epitopes localized
between the different reagents48; some kits contain only a along the deoxyribose-phosphate backbone.49
narrow assortment of antigens, whereas other include whole Compared with the lack of specificity of anti-ssDNA
cell extracts and others whole cell extracts spiked with scarcely antibodies, anti-dsDNA antibodies are highly specific for
represented antigens. Furthermore, some manufacturers use SLE and are present in affected subjects with a prevalence
IgG-specific conjugates, some use conjugates directed to the varying from 40% to 80%, such that they constitute the 10th
whole immunoglobulin molecule, and some to all of the diagnostic criteria for SLE as defined by the American
immunoglobulin classes. Therefore, to validate every single College of Rheumatology.8 Moreover, the presence of anti-
reagent or kit, it is necessary to study a sufficient number of dsDNA antibodies in an asymptomatic patient is highly
patients with well-established diagnoses, as well as a large suggestive of subclinical SLE, as these antibodies are nega-
sample number of IIF-positive serum samples having all the tive in SLE induced by drugs and positive in fewer than 2%
different staining patterns and fully representative of the of the cases with other autoimmune diseases.50
healthy population from the area (excluding hospital and labo- Anti-dsDNA and antichromatin antibodies are the main
ratory personnel), before substituting IIF with the enzyme- cause of the homogeneous/peripheral nuclear staining
linked immunosorbent assay (ELISA). In addition, in reporting pattern in IIF (although they also may be present less
negative EIA-ANA screening test results, it should be stated frequently with other staining patterns). Patients with SLE
that autoantibodies to specific antigens in this test (eg, Ro/SSA, and other autoimmune diseases may be initially recognized
La/SSB, RNP, Sm, Scl70-topoisomerase I, Jo-1, and dsDNA) by means of this test, which constitutes the 11th American
were not detected. This clarification will alert clinicians that a College of Rheumatology diagnostic criterion for SLE.8
negative EIA-ANA test result is not the equivalent of a negative Therefore:
finding for all nuclear antigens. Furthermore, a positive EIA- Determine anti-dsDNA autoantibodies only when clin-
ANA screen should always be confirmed by IIF-ANA on HEp- ical symptoms raise the suspicion of SLE and when ANA is
2 cells, reporting titer and pattern.43 For these reasons: positive by IIF.
At the present state of scientific knowledge and tech- The diagnostic and prognostic usefulness of the titer of
nology, do not screen for ANAs with immunoenzymatic anti-dsDNA antibodies has led to the development of
methods, unless the following conditions are present: numerous quantification techniques. Among these, the most
1. The method used must have at least 90% concordance commonly used are the techniques of radiobinding (among
with IIF, and prognostically important ANAs, such as those which is the original method of Farr51), IIF on Crithidia
directed to nucleoli or the nuclear membrane, must be recog- luciliae,52 and ELISA.53
nized as positive on the ELISA. The technique of Farr prevalently detects high-avidity
2. Positive results must be subsequently confirmed by IIF, anti-dsDNA antibodies and provides greater specificity for
specifying pattern and titer or reactivity. diagnosing SLE, as well as greater usefulness for monitoring
3. Discordant results (ELISA-positive, IIF-negative) the clinical course. Nevertheless, the need for radioactive
should be considered false-positive unless anti-Ro/SSA or isotopes limits its application in many clinical laboratories.
anti–Jo-1 antibodies are found. If it is strongly suspected that IIF using an anti-IgG conjugate54 is highly specific, has a
the patient has an autoimmune rheumatic disease, the good sensitivity and a relative accuracy, reveals antibodies
patient must be monitored over time. with high and intermediate avidity, and enables the identifica-
4. Negative results should specify which autoantibodies tion of the various antibody classes and their ability to fix
were studied and found negative, and patients with a clinical complement. This assay is easy to perform, very practical,
picture that raises suspicion of autoimmune rheumatic less technically demanding than the Farr technique, and the
disease must be evaluated successively over time. most widely used test for anti-dsDNA antibody detection, but
it does not provide an accurate quantitative determination.
Recommendations for Anti-DNA Antibodies The ELISA technique can be automated, it reveals
Since the 1950s it has been known that anti-DNA different immunoglobulin classes, provides quantitative find-
autoantibodies may exist in patients with SLE. It soon ings, and is more sensitive than the Farr and IIF techniques;
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6. Immunopathology / REVIEW ARTICLE
however, it may also detect antibodies with low avidity of classification has been demonstrated. At present, the
having uncertain clinical significance.55 Therefore, positive autoantibodies with these characteristics are anti-Ro/SSA,
results require subsequent confirmation by IIF, which has a anti-La/SSB, anti-Sm, anti-U1RNP, anti-Scl70, anti–Jo-1,
higher specificity. anti–CENP-B, anti-rRNP, and antinucleosome (chromatin);
Quantification of anti-dsDNA antibodies is useful for therefore:
the clinical management of the patient with SLE. Several In the diagnostic phase, extend the detection of antinu-
studies have documented a close relationship between anti- clear specific autoantibodies to the following autoantigens:
body titer and disease activity, particularly lupus nephritis56; Ro/SSA, La/SSB, Sm, U1RNP, Scl70 (topoisomerase I), Jo-1
an increase in the antibody concentration may precede flares (histidyl-tRNA synthetase), CENP-B, rRNP, and nucleosome
of disease by a few weeks.57,58 In general, for patients with (chromatin).
relatively active disease, anti-dsDNA can be tested every 6 to Serum antinuclear specific antibodies can be detected
12 weeks, whereas for those with less active disease, a with several techniques currently used in the laboratory:
frequency of every 6 to 12 months may suffice.16 Both the double immunodiffusion, counterimmunoelectrophoresis,
Farr and the ELISA technique will give an accurate quantita- ELISA, line-immunoblot, immunodot, and Western blot. The
tive result, but the Farr is not as cost-effective as ELISA. For strategy for choosing the techniques and their sequence of
these reasons: use depends on the clinical situation, the cost of the reagents,
In the diagnostic phase, use the highly specific radioim- and the time required for a result, as well as the organization
munologic method (Farr technique) or the IIF assay on of the laboratory and the experience of its personnel.
Crithidia luciliae at the initial serum dilution of 1:10 to The ideal method should fulfill criteria of clinical sensi-
determine anti-dsDNA antibodies. ELISA also may be used, tivity and high specificity,60 precision and accuracy, ease of
but positive results must be subsequently confirmed by IIF. execution, limited use of technology, quick availability, and
In monitoring the clinical course of SLE, perform a contained costs. At present, no method exists that fulfills all
quantitative determination of anti-dsDNA antibodies every 6 these requirements. Indeed, double immunodiffusion and
to 12 weeks, using ELISA or Farr assay, and express results counterimmunoelectrophoresis are not reliable for wide
in IU/mL (WHO/ISP Wo/80). routine application, as they are remarkably affected by the
method of preparation and execution, have a satisfactory
Recommendations for Antinuclear Specific Antibodies sensitivity only for some antinuclear specific antibodies
In the initial diagnostic phase in patients with clinical (anti-Ro/SSA),61,62 and are qualitative methods. ELISA or
symptoms that raise suspicion for autoimmune rheumatic other immunoenzymatic methods have excellent sensitivity
disease, the first test is the detection of ANAs by IIF; the and are quantitative, but they do not give reliable results for
pattern of nuclear or cytoplasmic fluorescence determines the entire antinuclear specific pattern; if screening methods
the subsequent test, represented by the search for autoanti- are not used, tests for each antigen are required.63 Western
bodies directed against one or more specific intracellular blot is a qualitative method that furnishes a complete pattern
autoantigens. 40 In some patients affected by rheumatic of the antinuclear specific antibodies and indicates the
diseases (in particular, Sjögren syndrome and composition and molecular weight of the different antigenic
dermatopolymyositis), the IIF-ANA test may be negative59; systems,64 but the proteins used as antigens are modified in
therefore, in patients in whom these diseases are suspected their conformation by sodium dodecyl sulfate–polyacryl-
clinically, antinuclear specific antibodies should be sought amide gel electrophoresis.65 This method is used mostly to
even if the ANA screening test is negative.40 For these confirm autoantibody specificity and to attempt recognizing
reasons: antigens different from those listed in the preceding guide-
In general, determine antinuclear specific antibodies line. Line-immunoblot is a qualitative method that furnishes
only when ANA screening is positive. In the case of a nega- limited information about the selected antigens and has the
tive ANA or positive cytoplasmic antibody finding, antinu- same technical limits as Western blot analysis.64 Immunodot
clear specific antibody testing is indicated only if the patient has reached the market and is a qualitative method that uses
has clear signs of an autoimmune rheumatic disease (espe- native proteins; it is easy to use and interpret, but at the
cially Sjögren syndrome or polymyositis). moment offers a limited panel of autoantigens.64
The variety of target autoantigens of the antinuclear Recent experiences of external quality assess-
autoantibodies is extremely wide; from a correct cost-benefit ment13,14,32,66-69 have shown significant variability in terms of
viewpoint, it is not reasonable to try to identify the specific analytic sensitivity among the methods and reagents
autoantibody in all patients with a known fluoroscopic commonly used in clinical laboratories for the detection of
pattern, but it is wise to limit the study to the autoantibodies antinuclear specific antibodies, none of which was found to
for which importance in the clinical diagnosis or as criteria be completely reliable. Therefore, a correct diagnostic
© American Society for Clinical Pathology Am J Clin Pathol 2002;117:316-324 321

7. Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTING
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