Immunopathology / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTINGGuidelines for the Laboratory Use of Autoantibody Testsin ...
Immunopathology / REVIEW ARTICLEstandardization and verification of the quality of the methods,     ❚Table 1❚as well as th...
Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTINGbecause weak ANA reactivity is present in many nonrheumatic ...
Immunopathology / REVIEW ARTICLEand enables the establishment of different cutoff levels, each      would classify virtual...
Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTINGfor both screening and detection of total ANAs. While such  ...
Immunopathology / REVIEW ARTICLEhowever, it may also detect antibodies with low avidity            of classification has b...
Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTINGstrategy for the search for antinuclear specific antibodies ...
Immunopathology / REVIEW ARTICLE 19. Fritzler MJ, Pauls JD, Douglas-Kinsella T, et al. Antinuclear,     37. Casiano CA, Ta...
Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTING 56. Bootsma H, Spronk PE, Derksen R, et al. Prevention of  ...
Upcoming SlideShare
Loading in …5

Guidelines for the Laboratory Use of Autoantibody Tests in the Diagnosis and Monitoring of Autoimmune Rheumatic Diseases


Published on

  • Be the first to comment

  • Be the first to like this

No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide

Guidelines for the Laboratory Use of Autoantibody Tests in the Diagnosis and Monitoring of Autoimmune Rheumatic Diseases

  1. 1. Immunopathology / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTINGGuidelines for the Laboratory Use of Autoantibody Testsin the Diagnosis and Monitoring of AutoimmuneRheumatic DiseasesRenato Tozzoli, MD,1 Nicola Bizzaro, MD,2 Elio Tonutti, MD,3 Danilo Villalta, MD,4Danila Bassetti, MD,5 Fabio Manoni, MD,6 Anna Piazza, MD,7 Marco Pradella, MD,8and Paolo Rizzotti, MD9Key Words: Antinuclear antibodies; Autoimmune rheumatic disease; Guidelines; Dermatopolymyositis; Systemic sclerosis; Systemic lupuserythematosus; Sjögren syndromeAbstract Guidelines in the clinical laboratory recommend the The Italian Society of Laboratory Medicine Study most appropriate modalities for conducting a diagnosticGroup 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 datarheumatic diseases intended for the use of clinical based on scientific observation are screened by expertpathologists 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 stepspublished works and expert panel discussion and in a continuous process of updating and formation.1 Theconsist of 13 recommendations for antinuclear practice of evidence-based medicine2,3 has resulted in anantibodies, anti–double-stranded native DNA, and important upsurge of interest in guidelines; internationalantinuclear specific antibodies. To improve analytic experiences have confirmed that scientific-medical societiesperformances 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. Thiseducation 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 of316 Am J Clin Pathol 2002;117:316-324 © American Society for Clinical Pathology
  2. 2. Immunopathology / REVIEW ARTICLEstandardization 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 Testsfor 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 manyspecific 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 specifyof clinical immunology (eg, rheumatologists, immunologists, immunohistochemical pattern (nuclear, cytoplasmic, mitotic) anddermatologists) 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, lowcriteria 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 screeningdiagnostic laboratory since the presence of diagnostic criteria test only when your procedure has shown good clinical andin the serum autoantibody profile of the patient represents an analytic correlation with the IIF method. • Test for anti–double-stranded native DNA (anti-dsDNA) antibodyimportant prerequisite for the clinical diagnosis. Indeed, posi- only in ANA-positive patients in whom systemic lupustive 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 mustnative 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–positiveence 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, orbodies is a criterion for the diagnosis of dermatopoly- immunodiffusion to detect Ro/SSA, La/SSB, Sm, uridin-rich 1myositis10; 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 formixed connective tissue disease (MCTD).12 Recommendations for ANA and Anticytoplasmic Antibodies The prevalence of autoimmune rheumatic diseases inGuidelines 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 forDiseases 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 domethods and analytic procedures for the detection of autoan- not affect more than 0.5% of the population.17,18 Most oftibodies,13,14 with particular attention to the preparation of the affected subjects initially have modest, nonspecificreference materials,15 and a recent article gives guidelines for clinical signs, generally with an indistinct and gradualthe clinical use of autoantibody testing16; however, none course; included among these is the possibility of a casualtreats 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 inDiagnosis of Autoimmune Diseases has generated a series of healthy subjects or individuals with diseases other thanguidelines 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 showon systematic review of published works (articles published positivity for autoantibodies with common laboratory tests, andfrom 1966 through 2000 were identified by searching the this finding does not necessarily indicate the existence of anNational 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 arearticles), 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 shouldantinuclear 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. 3. Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTINGbecause weak ANA reactivity is present in many nonrheumatic Among the nuclear patterns, the most frequent consistpatients 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 patternsintracellular 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 grainygens, but they also include anticytoplasmic antibodies. (topoisomerase I or Scl70) patterns, while the rarer picturesThe test for ANAs has a very high diagnostic sensitivity, include the proliferating cell nuclear antigen (PCNA) orsuch that it is considered the best test for the screening for cyclin, nuclear dots (coilin, Sp-100), and nuclear membranethese 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 andof 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-dehydrogenaseto 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 includeis 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 iscence should be the initial step in the laboratory diagnosis of associated mainly with cancer and hepatitis B or C and notautoimmune 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 linekidney or epithelial cell lines obtained from human laryn- HEp-2 (American Type Culture Collection CCL 23) fromgeal carcinoma may be used as substrate; the latter has human laryngeal carcinoma, provided that the expressionmany advantages over murine tissues, including good visu- and integrity of the clinically significant antigens isalization 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; nuclearsubstrates26,28 has increased the technique’s sensitivity while membrane; diffuse grainymaintaining 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-Fand/or antigen leaching and denaturation secondary to fixa- The fluorescence intensity observed in IIF is expressedtion procedures.24,29 To this end, acetone-fixed substrate in different ways in clinical laboratories. Expression with aslides are preferable since ethanol and methanol fixations qualitative scale of values from + to ++++ has the advantagemay 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 tosignals can originate from nuclear, mitotic, or cytoplasmic the antibody concentration. This is particularly true fordomains. Several immunohistochemical patterns are known centromeric and nucleolar patterns in which the reducedfor which an association with autoimmune and nonautoim- amount of antigen makes the definition of fluorescencemune 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 isonly 19 are reasonably associated with clinical pictures; expressed with a quantitative scale of values, using titersmoreover, for many autoantibodies, the target autoantigen (reciprocal of the last dilution of the reactive serum); thishas been identified with certainty. method better differentiates low- from high-positive titers318 Am J Clin Pathol 2002;117:316-324 © American Society for Clinical Pathology
  4. 4. Immunopathology / REVIEW ARTICLEand 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.20however, this method also is bound to a subjective definition High autoantibody titers (>1:160) are present in sickof the end point and, thus, is poorly reproducible, as subjects, but in only 5% of healthy people; this threshold is aevidenced by the wide scattering of the titers obtained in little less sensitive but more specific.20 Indeed, at an end pointnumerous 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 Intermediateof dilutions makes it preferable to use calibrators adjusted titers may be present in about 20% of the healthy populationagainst 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 thatresults 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 asstandardization 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 thantypes of nuclear fluorescence in the ANA test.31,35 However, 1:160 (20 IU) should be considered positive at low titer (inthe WHO standard is not easy to obtain on a regular basis, the absence of specific symptoms, further diagnostic study isand 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 furtherrocal of the last dilution of the reactive serum). If a reference diagnostic study because they are probably affected by anstandard 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 casemethod, 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 poorin healthy subjects (in particular, pregnant women, women correlation with the course of the disease, with the soleolder than 40 years, and elderly persons) and as a phenom- exception of the anti-PCNA and antichromatin antibodiesenon associated with viral infections, paraneoplastic neuro- (homogeneous nuclear pattern) for which a titer decrease orlogic syndromes, liver disease, chronic fatigue syndrome, disappearance is associated with the efficacy of therapy.41and cancers of various types.37 For the most part, these are This may be related to the fact that most assay systems arenatural 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 tomental 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 IIFautoimmune 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 IIFneous 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 methodologicmethods commonly used in the clinical laboratory are not and interpretive limitations. A negative finding for ANA onable to distinguish these two types of autoantibodies, so that IIF may occur in connective tissue diseases owing to thethe 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 asthe 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 phasehealthy subjects20; this threshold is highly sensitive and poorly immunoenzymatic methods (EIA-ANA) have been promotedspecific. 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. 5. Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTINGfor both screening and detection of total ANAs. While such became evident that the DNA molecule presented severalEIA-ANA tests are less labor intensive and can be used to epitopes and, therefore, that the anti-DNA antibodiesscreen and detect a panel of common specific autoantibodies included a heterogeneous group of immunoglobulins withusually 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 DNAtion, the EIA-ANA test may fail to detect certain ANAs with (ssDNA), whose antigenic determinants seem to be localizedatypical 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 localizedbetween the different reagents48; some kits contain only a along the deoxyribose-phosphate backbone.49narrow assortment of antigens, whereas other include whole Compared with the lack of specificity of anti-ssDNAcell extracts and others whole cell extracts spiked with scarcely antibodies, anti-dsDNA antibodies are highly specific forrepresented antigens. Furthermore, some manufacturers use SLE and are present in affected subjects with a prevalenceIgG-specific conjugates, some use conjugates directed to the varying from 40% to 80%, such that they constitute the 10thwhole immunoglobulin molecule, and some to all of the diagnostic criteria for SLE as defined by the Americanimmunoglobulin 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 highlypatients 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.50healthy population from the area (excluding hospital and labo- Anti-dsDNA and antichromatin antibodies are the mainratory personnel), before substituting IIF with the enzyme- cause of the homogeneous/peripheral nuclear staininglinked immunosorbent assay (ELISA). In addition, in reporting pattern in IIF (although they also may be present lessnegative EIA-ANA screening test results, it should be stated frequently with other staining patterns). Patients with SLEthat autoantibodies to specific antigens in this test (eg, Ro/SSA, and other autoimmune diseases may be initially recognizedLa/SSB, RNP, Sm, Scl70-topoisomerase I, Jo-1, and dsDNA) by means of this test, which constitutes the 11th Americanwere not detected. This clarification will alert clinicians that a College of Rheumatology diagnostic criterion for SLE.8negative 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 is2 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 ofnology, do not screen for ANAs with immunoenzymatic anti-dsDNA antibodies has led to the development ofmethods, 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 (amongwith IIF, and prognostically important ANAs, such as those which is the original method of Farr51), IIF on Crithidiadirected to nucleoli or the nuclear membrane, must be recog- luciliae,52 and ELISA.53nized 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 forspecifying 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 radioactiveshould 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 athe patient has an autoimmune rheumatic disease, the good sensitivity and a relative accuracy, reveals antibodiespatient 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 fixwere 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 thedisease 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;320 Am J Clin Pathol 2002;117:316-324 © American Society for Clinical Pathology
  6. 6. Immunopathology / REVIEW ARTICLEhowever, it may also detect antibodies with low avidity of classification has been demonstrated. At present, thehaving 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-1an increase in the antibody concentration may precede flares (histidyl-tRNA synthetase), CENP-B, rRNP, and nucleosomeof 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 detected12 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. Thetive result, but the Farr is not as cost-effective as ELISA. For strategy for choosing the techniques and their sequence ofthese 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 organizationmunologic 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 ofbut 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 allquantitative determination of anti-dsDNA antibodies every 6 these requirements. Indeed, double immunodiffusion andto 12 weeks, using ELISA or Farr assay, and express results counterimmunoelectrophoresis are not reliable for widein IU/mL (WHO/ISP Wo/80). routine application, as they are remarkably affected by the method of preparation and execution, have a satisfactoryRecommendations 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 orsymptoms that raise suspicion for autoimmune rheumatic other immunoenzymatic methods have excellent sensitivitydisease, the first test is the detection of ANAs by IIF; the and are quantitative, but they do not give reliable results forpattern of nuclear or cytoplasmic fluorescence determines the entire antinuclear specific pattern; if screening methodsthe subsequent test, represented by the search for autoanti- are not used, tests for each antigen are required.63 Westernbodies directed against one or more specific intracellular blot is a qualitative method that furnishes a complete patternautoantigens. 40 In some patients affected by rheumatic of the antinuclear specific antibodies and indicates thediseases (in particular, Sjögren syndrome and composition and molecular weight of the different antigenicdermatopolymyositis), the IIF-ANA test may be negative59; systems,64 but the proteins used as antigens are modified intherefore, 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 toeven if the ANA screening test is negative.40 For these confirm autoantibody specificity and to attempt recognizingreasons: antigens different from those listed in the preceding guide- In general, determine antinuclear specific antibodies line. Line-immunoblot is a qualitative method that furnishesonly when ANA screening is positive. In the case of a nega- limited information about the selected antigens and has thetive ANA or positive cytoplasmic antibody finding, antinu- same technical limits as Western blot analysis.64 Immunodotclear specific antibody testing is indicated only if the patient has reached the market and is a qualitative method that useshas clear signs of an autoimmune rheumatic disease (espe- native proteins; it is easy to use and interpret, but at thecially 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 ofviewpoint, it is not reasonable to try to identify the specific analytic sensitivity among the methods and reagentsautoantibody in all patients with a known fluoroscopic commonly used in clinical laboratories for the detection ofpattern, but it is wise to limit the study to the autoantibodies antinuclear specific antibodies, none of which was found tofor 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. 7. Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTINGstrategy for the search for antinuclear specific antibodies must Referencesuse several methodologic approaches. For these reasons: 1. Dans PE. Credibility, cookbook medicine and common sense: In the diagnostic phase, determine antinuclear specific guidelines and the college [editorial]. Ann Intern Med. 1994;120:966-968.antibodies with counterimmunoelectrophoresis, immunoen- 2. Sackett DL, Rosenberg WMC, Gray JAM, et al. Evidence-zymatic, immunoblot, or immunodot methods. based medicine: what it is and what it isn’t [editorial]. BMJ. The use of one of the aforementioned methods suffices to 1996;312:71-72.identify anti-Ro/SSA, anti-La/SSB, anti-U1RNP, anti-Sm, 3. Moore RA. Evidence-based clinical biochemistry. Ann Clinanti–Jo-1, anti-Scl70, anti-rRNP, anti–CENP-B, and antinu- Biochem. 1997;34:3-7.cleosome (chromatin) antibodies; however, the finding of a 4. Hargreaves MM, Richmond H, Morton R. Presentation of two bone marrow elements: the “tart” and “L.E.” cell. Mayolow or negative antibody concentration in a subject for Clin Proc. 1948;23:25-28.whom clinical suspicion of autoimmune rheumatic disease is 5. Smolen JS, Steiner G, Tan EM. Standards of care: the valuestrong imposes the use of 2 of the above methods. Use and importance of standardization [editorial]. Arthritis Rheum.Western blot analysis for confirmation of autoantibody speci- 1997;40:410-412.ficity and possible identification of antibodies that recognize 6. Humbel RL. Auto-immunité, auto-anticorps et maladie auto- immunes. In: Humbel RL, ed. Autoanticorps et Maladiesantigens other than those listed. Autoimmunes. Paris, France: Edition Scientifiques Elsevier; In all of the rheumatic diseases, detection of the concen- 1997:17-20.tration of the antinuclear specific autoantibodies does not 7. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteriaseem to provide additional information for the study of the for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271-1277.disease course and prognosis or the efficacy of therapy, with 8. Hochberg M. Updating the American College ofthe single exception of MCTD, in which the finding of a Rheumatology revised criteria for the classification of systemichigh anti-U1RNP autoantibody titer represents the main diag- lupus erythematosus. Arthritis Rheum. 1997;40:1725-1734.nostic criterion; therefore: 9. Vitali C, Bombardieri S, Moutsopoulos HM, et al. Preliminary Report quantitative results (ELISA method) only in the criteria for the classification of Sjögren’s syndrome: results of a prospective concerted action supported by the Europeancase of an isolated positivity for U1RNP, because a high anti- Community. Arthritis Rheum. 1993;36:340-347.body concentration is a diagnostic criterion for MCTD. 10. Tanimoto K, Nakano K, Kano S, et al. Classification criteria for polymyositis and dermatomyositis. J Rheumatol. 1995;22:668-674. 11. Leroy EC, Black C, Fleischmajer R, et al. Scleroderma (sys-Conclusions temic sclerosis): classification, subsets, and pathogenesis. J Rheumatol. 1988;15:202-205. These recommendations are subject to further modifica- 12. Amigues JM, Cantagrel A, Abbal M, et al. Comparative studytions and additions as new scientific acquisitions become of 4 diagnosis criteria sets for mixed connective tissue diseasesavailable; they are offered to the attention of all operators in in patients with anti-RNP antibodies. J Rheumatol. 1996;23:2055-2062.the field, both in the clinic and the laboratory, as a working 13. van Venrooij WJ, Charles P, Maini RN. The consensustool; they do not represent definitive documents, but tempo- workshop for the detection of autoantibodies to intracellularrary steps of a continuous process of updating and formation. antigens in rheumatic diseases. J Immunol Methods. 1991;140:181-189. 14. Charles PJ, van Venrooij WJ, Maini RN, and the ConsensusFrom the 1Department of Laboratory Medicine, Civic Hospital, Finding Group for Autoantibodies. The consensus workshopLatisana (UD); 2Laboratory of Clinical Pathology, Civic Hospital, for the detection of autoantibodies to intracellular antigens inS Donà di Piave (VE); 3Institute of Clinical Chemistry, Hospital S rheumatic diseases: 1989-1992. Clin Exp Rheumatol.Maria della Misericordia, Udine; 4Immunology and Microbiology 1992;10:507-511.Unit, Hospital S Maria degli Angeli, Pordenone; 5Laboratory of 15. Tan EM. International cooperative activities in standardi-Clinical Pathology II, Hospital S Chiara, Trento; 6Department of zation of antinuclear antibodies. In: van Venrooij WJ, MainiLaboratory Medicine, Civic Hospital, Chioggia (VE); 7Laboratory RN, eds. Manual of Biological Markers of Disease. Dordrecht,of Clinical Chemistry and Microbiology, Geriatric Hospital, the Netherlands: Kluwer; 1993:A1:1-4.Padova; 8Laboratory of Clinical Chemistry and Microbiology, 16. Kavanaugh A, Tomar R, Reveille J, et al. Guidelines forCivic Hospital, Castelfranco Veneto (TV); and 9Laboratory of clinical use of the antinuclear antibody test and tests forClinical Chemistry and Hematology, Borgo Trento Hospital, specific autoantibodies to nuclear antigens. Arch Pathol LabVerona, Italy. Med. 2000;124:71-81. 17. Morrow J, Isenberg D. Setting the scene. In: Morrow J, Address reprint requests to Dr Bizzaro: Laboratorio di Patologia Isenberg D, eds. Autoimmune Rheumatic Disease. Oxford,Clinica, Ospedale Civile, 30027 S Donà di Piave (VE), Italy. England: Blackwell Scientific; 1987:1-23. Acknowledgments: We thank Allan Wiik, Staten Serum 18. Jacobson DL, Gange SJ, Rose NR, et al. Epidemiology andInstitute, Copenhagen, Denmark, and Eng M. Tan, Scripps estimated population burden of selected autoimmune diseasesResearch Institute, La Jolla, CA, for valuable suggestions and in the United States. Clin Immunol Immunopathol.critical reading of the manuscript. 1997;84:223-243.322 Am J Clin Pathol 2002;117:316-324 © American Society for Clinical Pathology
  8. 8. Immunopathology / REVIEW ARTICLE 19. Fritzler MJ, Pauls JD, Douglas-Kinsella T, et al. Antinuclear, 37. Casiano CA, Tan EM. Autoantibodies to centromere protein anticytoplasmic and anti-Sjögren’s syndrome antigen A (SS- F. In: Conrad K, Humbel RL, Meurer M, et al, eds. Pathogenic A/Ro) antibodies in female blood donors. Clin Immunol and Diagnostic Relevance of Autoantibodies. Berlin, Germany: Immunopathol. 1985;36:120-128. PABST; 1998:395-411. 20. Tan EM, Feltkamp TEW, Smolen JS, et al. Range of 38. Abu-Shakra M, Schoenfeld Y. What are natural antinuclear antibodies in “healthy” individuals. Arthritis autoantibodies? In: Schoenfeld Y, Isenberg DA, eds. Natural Rheum. 1997;40:1601-1611. Autoantibodies: Their Physiological Role and Regulatory 21. von Mühlen CA, Tan EM. Autoantibodies in the diagnosis of Significance. Boca Raton, FL: CRC Press; 1993:15-33. systemic rheumatic diseases. Semin Arthritis Rheum. 39. LeMing H, Nakamura RM. Current concepts and advances in 1995;24:323-358. clinical laboratory testing for autoimmune diseases. Crit Rev 22. Slater CA, Davis RB, Shmerling RH. Antinuclear antibody Clin Lab Sci. 1997;34:275-311. testing; a study of clinical utility. Arch Intern Med. 40. Homburger HA. Cascade testing for autoantibodies in 1996;156:1421-1425. connective tissue diseases. Mayo Clin Proc. 1995;70:183-184. 23. Molden DP, Nakamura RM, Tan EM. Standardization of the 41. Takasaki Y, Tan EM. Clinical significance of autoantibodies to immunofluorescence test for autoantibody to nuclear antigens PCNA. In: van Venrooij WJ, Maini RN, eds. Manual of (ANA): use of reference sera of defined antibody specificity. Biological Markers of Disease. Dordrecht, the Netherlands: Am J Clin Pathol. 1984;82:57-66. Kluwer; 1993:C2-4:1-13. 24. Hollingsworth PN, Pummer SC, Dawkins RL. Antinuclear 42. Maddison P, Provost T, Reichlin M. Serological findings in antibodies. In: Peter JB, Shoenfeld Y, eds. Autoantibodies. patients with “ANA” negative lupus. Medicine. 1981;60:87-94. Amsterdam, the Netherlands: Elsevier Science BV; 1996:74-90. 43. Jaskowski TD, Schroder C, Martins TB, et al. Screening for 25. Humbel RL. Detection of antinuclear antibodies by antinuclear antibodies by enzyme immunoassay. Am J Clin immunofluorescence. In: van Venrooij WJ, Maini RN, eds. Pathol. 1996;105:468-473. Manual of Biological Markers of Disease. Dordrecht, the Netherlands: Kluwer; 1993:A2:1-16. 44. Emlen W, O’Neill L. Clinical significance of antinuclear antibodies: comparison of detection with immunofluorescence 26. NCCLS. Quality Assurance for the Indirect Immunofluorescence and enzyme-linked immunosorbent assay. Arthritis Rheum. Test for Autoantibodies to Nuclear Antigen (IF-ANA); Approved 1997;40:1612-1618. Guideline I/LA2-A. Wayne, PA: NCCLS; 1996. Vol 16. 45. Monce NM, Bogusky RT, Cappel NN. An enzyme 27. Humbel RL, Kadusch P. Use of HEp-2 cells in the IF test for immunoassay screening test for the detection of total detection of antinuclear antibodies. In: Peeters H, ed. Protides antinuclear antibodies. J Clin Lab Anal. 1991;5:439-442. of the Biological Fluids. New York, NY: Pergamon Press; 1985:385-387. 46. Jitsukawa T, Nakajima S, Usui J, et al. Detection of anti- nuclear antibodies from patients with systemic rheumatic 28. Fritzler MJ, Miller BJ. Detection of autoantibodies to SSA/Ro diseases by ELISA using HEp-2 cell nuclei. J Clin Lab Anal. by indirect immunofluorescence using a transfected and 1991;5:49-53. overexpressed human 60 kd Ro autoantigen in HEp-2 cells. J Clin Lab Anal. 1995;9:218-224. 47. Gniewek RA, Stites DP, McHugh TM, et al. Comparison of antinuclear antibody testing methods: immunofluorescence 29. Chan EKL, Buyon JP. The SS-A/Ro antigen. In: van Venrooij assay versus enzyme immunoassay. Clin Diagn Lab Immunol. WJ, Maini RN, eds. Manual of Biological Markers of Disease. 1997;4:185-188. Dordrecht, the Netherlands: Kluwer; 1994:B4.1:1-18. 48. Jen SN. Comparison of EIA methods against IFA method for 30. Casiano CA, Humbel RL, Peebles C, et al. Autoimmunity to ANA qualitative screening [abstract]. Clin Chem. the cell cycle–dependent centromere protein p330d/CENP-F 1999;45(S6):A152. in disorders associated with cell proliferation. J Autoimmun. 1995;8:575-586. 49. Reeves WH, Satoh M, Wang J, et al. Systemic lupus 31. Feltkamp TEW. Standards and reference preparations. In: van erythematosus: antibodies to DNA, DNA binding proteins, Venrooij WJ, Maini RN, eds. Manual of Biological Markers of and histones. Rheum Dis Clin North Am. 1994;20:1-28. Disease. Dordrecht, the Netherlands: Kluwer; 1993:A11:1-12. 50. Ballou SP, Kushner I. Anti-native DNA detection by the 32. Bizzaro N, Tozzoli R, Tonutti E, et al. Variability between Crithidia luciliae method: an improved guide to the diagnosis methods to determine ANA, anti-dsDNA and anti-ENA and clinical management of systemic lupus erythematosus. autoantibodies: a collaborative study with the biomedical Arthritis Rheum. 1979;22:321-327. industry. J Immunol Methods. 1998;219:99-107. 51. Wold ET, Young FE, Tan EM, et al. Deoxyribonucleic acid 33. Hollingsworth PN, Bonifacio E, Dawkins RL. Use of a antibody: a method to detect primary interaction with standard curve improves precision and concordance of deoxyribonucleic acid. Science. 1968;161:806-807. antinuclear antibody measurements. J Clin Lab Immunol. 52. Crowe W, Kushner I. An immunofluorescent method using 1987;22:197-200. Crithidia luciliae to detect antibodies to double stranded DNA. 34. Tan EM, Feltkamp TEW, Alarcon-Segovia D. Reference Arthritis Rheum. 1977;20:811-814. reagents for antinuclear antibodies. Arthritis Rheum. 53. Smeenk RJ. Measurement of antibodies to DNA. In: van 1988;31:1331-1333. Venrooij WJ, Maini RN, eds. Manual of Biological Markers of 35. Feltkamp TE, Klein F, Janssens MB. Standardisation of the Disease. Dordrecht, the Netherlands: Kluwer; 1993:A8:1-12. quantitative determination of antinuclear antibodies (ANAs) 54. Smeenk RJT, Berden JHM, Swaak AJG. dsDNA autoanti- with a homogeneous pattern. Ann Rheum Dis. 1988;47:906-909. bodies. In: Peter JB, Shoenfeld Y, eds. Autoantibodies. 36. Dawkins RL, Martinez OP, Freitas EM, et al. Diagnosis of Amsterdam, the Netherlands: Elsevier Science BV; 1996:227- autoimmune disease. In Rose NR, Mackay IR, eds. The 236. Autoimmune Diseases. 3rd ed. San Diego, CA: Academic 55. Swaak AJ, Groenwold J, Aarden LA, et al. Prognostic value Press; 1998:821-831. of anti-dsDNA in SLE. Ann Rheum Dis. 1982;41:388-395.© American Society for Clinical Pathology Am J Clin Pathol 2002;117:316-324 323
  9. 9. Tozzoli et al / LABORATORY GUIDELINES FOR AUTOANTIBODY TESTING 56. Bootsma H, Spronk PE, Derksen R, et al. Prevention of 63. van Venrooij WJ. Autoantigens in connective tissue disease. relapses in systemic lupus erythematosus. Lancet. In: Panayi GS, ed. Immunology of the Connective Tissue 1995;345:1595-1599. Diseases. Dordrecht, the Netherlands: Kluwer; 1994:305-334. 57. ter Borg EJ, Horst G, Hummel EJ, et al. Measurement of 64. Stott DI. Immunoblotting and dot blotting. J Immunol increases in anti–double-stranded DNA antibody levels as a Methods. 1991;119:153-187. predictor of disease exacerbation in systemic lupus 65. Boire G, Lopez-Longo FJ, Lapointe S, et al. Sera from patients erythematosus. Arthritis Rheum. 1990;33:634-643. with autoimmune disease recognize conformational 58. Bootsma H, Spronk PE, Hummel EJ, et al. Anti–double determinants on the 60-kd Ro/SS-A protein. Arthritis Rheum. stranded DNA antibodies in systemic lupus erythematosus: 1991;34:722-730. detection and clinical relevance of IgM-class antibodies. 66. Bizzaro N, Tozzoli R, Tonutti E, et al. Variabilità analitica nella Scand J Rheumatol. 1996;25:352-359. determinazione degli autoanticorpi anti-ENA: risultati 59. Bylund DJ, Nakamura RM. Importance of detection of SS- preliminari di un’esperienza di VEQ. Med Lab. 1998;2:161-165. A/Ro autoantibody screening immunofluorescence tests for 67. Bizzaro N, Tonutti E, Villalta D, et al. Sensitivity and autoantibodies to nuclear antigens. J Clin Lab Anal. specificity of immunological methods for the detection of 1991;5:212-217. anti–topoisomerase I (Scl70) autoantibodies: results of a 60. Zweig MH, Campbell G. Receiver-operating characteristics multicenter study. Clin Chem. 2000;46:1681-1685. (ROC) plots: a fundamental valuation tool in clinical 68. Tan EM, Smolen JS, McDougal JS, et al. A critical evaluation medicine. Clin Chem. 1992;39:561-577. of enzyme immunoassays for detection of antinuclear 61. Reichlin M, Scofield H. SS-A (Ro) autoantibodies. In: Peter autoantibodies of defined specificity, I: precision, sensitivity, JB, Shoenfeld Y, eds. Autoantibodies. Amsterdam, the and specificity. Arthritis Rheum. 1999;42:455-464. Netherlands: Elsevier Science BV; 1996:783-788. 69. Villalta D, Bizzaro N, Tonutti E, et al. Detection of anti-ENA 62. Manoussakis MN, Kistis KG, Liu X, et al. Detection of anti- autoantibodies in patients with systemic connective tissue Ro(SSA) antibodies in autoimmune diseases: comparison of diseases: analytical variability and diagnostic sensitivity of 4 five methods. Br J Rheumatol. 1993;32:449-455. methods [in Italian]. Recenti Prog Med. 1999;90:579-584.324 Am J Clin Pathol 2002;117:316-324 © American Society for Clinical Pathology