Comparison of the analytical and clinical performances of two different routine testing protocols for antinuclear antibody screening

J Clin Lab Anal. 2021;35:e23914.   | 1 of 8 https://doi.org/10.1002/jcla.23914 wileyonlinelibrary.com/journal/jcla Received:1June2021 | Revised:6July2021 | Accepted:11July2021 DOI: 10.1002/jcla.23914 RESEARCH ARTICLE Comparison of the analytical and clinical performances of two different routine testing protocols for antinuclear antibody screening Concepción González Rodríguez | Sandra Fuentes Cantero | Antonio Pérez Pérez | Francisco Javier Vázquez Barbero | Antonio León Justel ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium, provided the original work is properly cited. ©2021TheAuthors.Journal of Clinical Laboratory AnalysispublishedbyWileyPeriodicalsLLC DepartmentofBiochemistry,Hospital UniversitarioVirgenMacarena,Seville, Spain Correspondence ConcepciónGonzálezRodríguez, DepartmentofBiochemistry,Hospital UniversitarioVirgenMacarena,Seville, Spain. Email: concepcion.gonzalez.r.sspa@ juntadeandalucia.es Funding information VirgenMacarenaUniversityHospital Abstract Background: The diagnosis of systemic autoimmune rheumatic diseases (SARD) is basedonthedetectionofserumantinuclearantibodies(ANA)forwhichindirectimmunofluorescence(IIF)isthegoldenstandard.Newsolid-phaseimmunoassayshave been developed to be used alone or in combination with the detection of extractable antinuclearantibodies(ENA)toimproveSARDdiagnosis.Thepurposeofthisstudy wastocomparetheclinicalperformancesofdifferentANAscreeningmethodsalone orincombinationwithENAscreeningmethodsforSARDdiagnosis. Methods: Atotalof323patientswerescreenedforANAbyIIF,EliA™CTDScreen, andELISAmethods.Agreementswerecalculatedbetweenthemethods.Then,EliA™ CTDScreenpositivesampleswerescreenedforENAbylineimmunoassay(LIA)and fluorescenceenzymeimmunoassay(FEIA). Results: ThediagnosticaccuracyofEliA™CTDScreen(79%sensitivityand91%specificity)wasbetterthanthatofELISAorIIF.ThecombinationofEliA™CTDplusIIFhad thehighestsensitivity(93%).ENAdeterminationrevealedthatRo52andRo60were the most prevalent specificities. The use of IIF alone was not able of detecting up to 36%ofsamplespositiveforRo52,and41%forRo60. Conclusions: EliA™CTDScreenhasabetterdiagnosticperformancewhencomparedto IIFandELISA.ThecombineduseofEliA™CTDScreenandIIFclearlyimprovestherate andaccuracyofSARDdiagnosis.TheuseofEliA™CTDScreenasfirst-linescreening techniqueallowsthedetectionofantibodies,whichcouldnotbedetectedbyIIFalone. KEYWORDS antinuclearantibodies,diagnosis,EliA,enzyme-linkedimmunosorbentassay,Indirect immunofluorescence,methodcomparison,systemicautoimmunerheumaticdiseases 1 | INTRODUCTION Systemicautoimmunerheumaticdiseases(SARD),alsoknownas connectivetissuediseases(CTD),includingalldiseasestriggered bytheformationofimmunecomplexesthatenterthecirculation, are then deposited in different tissues and organs, and cause damage.1Thedetectionofantinuclearantibodies(ANA)andof antibodies to extractable nuclear antigens (ENA) is used in the 10982825, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcla.23914 by Universidad De Sevilla, Wiley Online Library on [28/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2 of 8 | GONZÁLEZ RODRÍGUEZ Et aL. diagnosis of SARD and in the prediction of an early onset of disease.2 Indirect immunofluorescence (IIF) on HEp-2 cells (human epidermoid laryngeal carcinoma cells) has been defined as the referencescreeningmethodforANAintheclinicallaboratoryroutine.3 However,IIFisalabor-intensive,time-consumingprocedureandhas poor reproducibility due to the subjective interpretation of results.4 Therefore, in 2014, an international workgroup of experts representing 15 European countries developed a set of recommendations fortheappropriateassessmentandinterpretationofANAdetected by different methods. According to this expert panel, alternative assaysmightbepreferredforANAscreeningwhenclinicalsuspicion is strong; and when the results of these alternative methods are negative, IIF should be used for the definitive diagnosis.3 In thisregard,variousautomatedsolid-phaseimmunoassays(suchas enzyme-linkedimmunosorbentassay(ELISA)andfluorescenceenzymeimmunoassay-based(FEIA)assays)havebeendevelopedtobe usedasfirst-linescreeningmethodsinSARDdiagnosis.2ELISAisa plate-basedassaytechniqueinwhichantigensofHEp-2cellextracts areimmobilizedonasolidsurface,whereasFEIAisdesignedasa sandwich assay where a mix of antigens is coated to the solid phase. Antinuclearautoantibodiesbindtotheseantigens,whicharecoupledtoanenzyme-linkedantibody,producingafluorescentsignal upon binding.2 SuchsystemsareattractivealternativestoIIF,notonlybecause of the automated process but also because of the improved specificitycomparedtoIIF.Moreover,ithasbeenwidelydemonstratedthat a combination of these two techniques may improve the diagnostic accuracyofANAscreening.5- 8 Inroutinelaboratorytesting,afterapositiveresultisobtainedon ascreeningplatform,itisusefultodeterminethespecificityofthe antibodyusing differentENAtestingplatforms duetotheir prognosticanddiagnosticpower.Takingintoaccount,theavailabilityof differentmethodsforSARDdiagnosis,itisimportanttoevaluatethe performanceofdifferenttestsinordertodeterminewhichANAand ENAcombinationperformsbestandismoresustainable. The aim of this study was the comparative analysis of three differentANAscreeningmethods(EliA™CTDScreen,IIF,andELISA). Ananalysiswascarriedoutconsideringsingleorcombinationtests (EliA™ CTD Screen plus IIF vs. ELISA plus IIF). We also evaluated thepotentialANAplusENAcombinationtestinginSARDdiagnosis usingtwodifferentENAscreeningmethods(LIAandFEIA). 2 | MATERIALS AND METHODS 2.1 | Patients Cross-sectional study including samples referred for ANA testing fromApriltoAugust2019.Atotalof323patientsfromtheprimary care, rheumatology, nephrology, and internal medicine services of the Virgen Macarena University Hospital (Seville, Spain) were evaluated.Thesampleswerecollectedrandomly,includingjustone sampleperpatient.Thesampleswereretrospectivelyclassified,accordingtopatientsfinalclinicaldiagnosis,intothefollowinggroups: SARD group, N = 147 (including systemic lupus erythematosus, Sjögren'ssyndrome,mixedconnectivetissuedisease,polymyositis/ dermatomyositis, systemic sclerosis, undifferentiated connective tissuediseases,rheumatoidarthritis,andvasculitis);Organ-specific autoimmunediseasegroup,N =31;Malignanciesgroup,N = 12 and Non-autoimmunediseases,N =147).Thisstudywasapprovedbythe localEthicsCommitteeoftheVirgenMacarenaUniversityHospital. 2.2 | Antinuclear antibodies (ANA) Screening ANAscreeningwasperformedwiththefollowingthreetechniques: IIF:ANAwasperformedusing HEp-2®cells(Immunoconcepts) (screeningdilution1:80)using≥1:80cut-offwhichallowsthedetection of antibodies against a wide variety of nuclear molecules and antigens locatedinthecytoplasm,includingthoseinmitoticcells. ANAIIFwerecarriedoutbyDr.GonzálezandDr.Pérez,whohave 30 and 7 years of experience, respectively; using an automated microscope(ImageNavigator,Nikon)andSIExpertsoftware(Palex Medical,SantCugatdelVallés). FEIA: The EliA™ CTD Screen (Thermo Fisher Scientific) was performed using the Phadia 250 instrument. This assay contains a mixture of the following antigens: dsDNA, Ro52, Ro60, SSB, Sm, U1RNP(RNP-70,A,C),Jo-1,Scl-70,CENP-B,Mi-2,RNAPolIII,PM- Scl,PCNA,RibosomalP,andFibrillarin.Theratiosoftestsampleto calibrator (cut-off)recommendedbythemanufacturerwereused: <0.7,negative;0.7–1,equivocal;>1,positive. ELISA:RELISA(Immunoconcepts)aqualitativeindirect enzyme immunoassay was used. Stabilized antigens (dsDNA, histones, SSA/Ro60, SSB/la, Sm, Sm/RNP, Scl70, Centromere, Jo-1, PCNA, RibosomalP,Mi-2mitochondrial)andotherantigensfromHEp-2nucleus and cytoplasm are coated onto the surface of the microwells to serve as antigenic substrates. Cutoff values recommended by the manufacturer were used: <10U/ml,negative;10–15U/ml,borderline; >15U/ml,positive. For combined testing (ie, when more than one of these tests wereused),patientswithapositiveresultinoneofthetestswere consideredpositive,andpatientswithnegativeresultsinbothtests were considered negative. 2.3 | Extractable nuclear antigens (ENA) Screening EliA™CTDScreenpositivesamples(n =123)wereanalyzedforthe followingENAspecificitiesusingtwodifferentmethods: LIA:Euroline(Euroimmun),includingRNP-U1(70kDa+A+C),Sm, SSA/Ro60,SSA/Ro52,SSB,Scl70,Jo-1,CENP-B,PCNA,Histones, Ribosomal P proteins, PM-Scl, Mitochondrial, and Nucleosomes. The intensity of the resulting staining is proportional to the antibody concentration in the sample. Therefore, semi-quantitative cut-off values recommended by the manufacturer were used as follows: 10982825, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcla.23914 by Universidad De Sevilla, Wiley Online Library on [28/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 3 of 8 GONZÁLEZ RODRÍGUEZ Et aL. negative(0–10intensityoftheresultingstaining);weakpositive(11– 25);moderatepositive(26–50),andstrongpositive(>50). FEIA:EliAspecificities(ThermoFisherScientific),includingSmDP, RibP,PCNA,U1RNP,Ro,Ro52,Ro60,La,CENP,Scl-70s,Fibrillarin, RNAPolIII,PM-Scl,Jo-1,andMi-1.Cut-offvaluesrecommendedby themanufacturerwereused:≤1.0U/ml,negative;>1U/ml,positive. 2.4 | Statistical analysis The differences between the results obtained from different methodswereanalyzedbychi-square,phicoefficient,andcontingency coefficienttests.Cohen'skappacoefficientswereusedtoestimate themeasuringagreementamongmethods.Statisticalanalyseswere performedusingthesoftwareSPSSStatisticsv25. 3 | RESULTS 3.1 | Demographics Inthispatientcohort,theprevalenceofSARDwas41.2%(116of 246women,and17of77men).Themedianageofthepatientswas 56 ± 16 years. Table 1 describes the demographic data for each group. 3.2 | Comparison of ANA Screening platforms TheresultsofANAscreeningwithIIF,ELISA,andEliA™CTDScreen wereobtainedandcompared.AsshowninTable2183oftheresults wereconsistent(56.7%)and140results(43.3%)werecontradictory betweenIIFandEliA™CTDScreentechniques,whereas251ofthe resultswereconsistent(77.7%)and72results(22.3%)werecontradictorybetweenELISAScreenandEliA™CTDScreentechniques. Bivariate analysis between EliA™ CTD Screen, IIF, and ELISA techniquesmeasuredbyPearson'schi-squaretestrevealedstatistically significant differences between each test (p <0.05)(Table3). Phi coefficient and contingency coefficient were also calculated and presentedinTable3.Methodagreementanalysis(Kappacoefficient) revealedthattheagreementbetweenEliA™CTDScreenandELISA (moderate,0.540)washigherthantheagreementbetweenEliA™ CTDandIIF(weak,0.150)orbetweenELISAandIIF(weak,0.203). Importantly, the agreement between combined tests (EliA™ CTD plusIIFvsELISAplusIIF)wasstronger(strong,0.814).Discrepant results are shown in Table4. Next,SARDdiagnosisperformancesofthreescreeningplatforms werecompared(Table5).ForEliA™CTDScreen,sensitivitywas79% (95%CI:72%–86%),specificitywas91%(95%CI:86–95%),andpositiveandnegativelikelihoodratios(LR)valueswere8.33(2.05–3.50) and0.23(0.32–0.55),respectively.IIFhadthefollowingvalues:sensitivity69%(95%CI:61%-77%),specificity56%(95%CI:49%-63%), positiveLR1.58(1.30–1.93),andnegativeLR0.55(0.41–0.73);while ELISAshowedasensitivityof69%(95%CI:61%–77%),aspecificity of74%(95%CI:68%-80%),positiveLR2.68(2.05–3.50),andnegativeLR0.42(0.32–0.55).Therefore,itcanbeconcludedthatEliA™ CTDScreenhadhighersensitivity,specificity,andpositiveLR,and betternegativeLRcomparedtobothIIFandELISA. Moreover,whentheperformanceofEliA™CTDScreenplusIIF wascomparedtoELISAplusIIFscreening,EliA™CTDScreenplus IIFscreeningshowedhighersensitivity(93%vs.89%)andspecificity (50%vs45%),andhigherpositiveLR(1.86vs.1.63)andlowernegativeLR(0.14vs.0.23)values. 3.3 | Specific ENA profile Theantigenicspecificitieswereassessedusingtwomethods:EliA™ and LIA. Both platforms displayed the highest positivity rates for Ro60andRo52antibodiesthataremostcommonlyreportedinthe literature for SARD diagnosis. Therefore, the percentage of these antigens (as calculated from the total positive specificities of each method)wasassessedinsystemicautoimmunediseases(Table6). Forthetwomostprevalentsystemicautoimmunediseases,systemic lupuserythematosus,and Sjögren's syndrome, thefollowing positivitypercentagesweremeasured:SystemicLupusErythematosus, E l i A ™ - R o 5 2  =3.42%andRo60=8.12%,LIA-Ro52=6.59%and Ro60 =9.89%; andSjögren's Syndrome,EliA™-Ro52= 8.55%and Ro60=10.26%,LIA-Ro52=12.64%,andRo60=11.5%. Finally, diagnostic performances of EliA™ and LIA were comparedforsamplespositiveforRo52orRo60(CTD+)andpositivefor SARD(IFF+,andIFF−).AsshowninTable7,25–36%ofthesamples positiveforRo52and37–41%ofthesamplespositiveforRo60could not be detected by IIF only. 4 | DISCUSSION ThisstudyincludedpatientswithsuspicionofSARDreferredfrom primarycare,rheumatology,nephrology,andinternalmedicinedepartments. It is important to distinguish these different groups of patientswhichgenerallyarereferredtoasSARDorANA-associated rheumaticdiseases(AARD)asthereisconsiderablevariabilityamong them.9- 11Inourstudycohort,thepercentagesofpatientsdetected byIIF(55%,n =175)weresimilartothoseobtainedintheroutine work of our laboratory and in other studies such as Bizzaro et al.5 and Dellavance et al.12which reported prevalence ratesof 46.7% and44.3%,respectively,whichalsouseda1/80dilutionasacut-off point as we used in this study. The comparative analysis of the three screening methods revealedthatthesensitivitiesandspecificitiesoftheIIFandELISA methodsweremoresimilartoeachother(69%sensitivityinboth methods,and56%and74%specificityinIIFandELISA,respectively),whileEliA™CTDScreenhadhighersensitivityandspecificitylevelsforSARD(79%sensitivity,91%specificity).Positive and negative LRs(8.33and 0.23, respectively) werealsobetter 10982825, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcla.23914 by Universidad De Sevilla, Wiley Online Library on [28/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 8 | GONZÁLEZ RODRÍGUEZ Et aL. N%Ratio W/M Age (average) Non-autoimmunedisease(n =147) 2/1 59 Arthrosis 23 7.12 Arthralgias 16 4.95 Neuropathies 11 3.40 Psoriasis 61.86 Fibromyalgia 17 5.26 Nephropathies 25 7.74 Skinlesions 72.17 Thrombopenia 11 3.40 Synovitis 51.55 Raynaud'sandvasculardisorders 7 2.17 Pulmonary thromboembolism 30.92 Infections 20.62 Others 14 4.33 Malignancies (n =12) 3/1 62 Malignancies 12 3.71 Organspecific autoimmune diseases (n =31) 3.3/1 56 Autoimmunethyroiddiseases 82.48 Autoimmuneliverdiseasesandcholangitis 11 3.40 Diabetes 10.31 Otherarthritis(undifferentiated,psoriasis) 7 2.17 Crohn´s disease 20.31 Celiac disease 20.62 Myasthenia gravis 10.31 SARD(n =133) 8/1 54 Rheumatoid arthritis 22 6.81 Mixed connective tissue disease 51.55 Systemicsclerosis 11 3.40 Systemiclupuserythematosus 38 11.76 Dermatomyositis/polymyositis 41.24 Antiphospholipidsyndrome Primary 41.24 Secondary 4* 1.24* Sjögrensyndrome 39 12.07 Undifferentiated connective tissue diseases 61.86 Systemicvasculitis ANCA+11.24 ANCA− 3 TOTAL 323 100% Abbreviations:M,men;W,women. TABLE 1 Demographicsandpatients’ clinical profile description 10982825, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcla.23914 by Universidad De Sevilla, Wiley Online Library on [28/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 5 of 8 GONZÁLEZ RODRÍGUEZ Et aL. thanIIFandELISA.ThesensitivityandspecificityvaluesforIIF were lower than reported by Orme et al.2 at 1/80 titer (sensitivity between84%and93%,andspecificitybetween62%and81%)in a metaanalysis performed to compare the diagnostic accuracy of IIFandEliA™CTDScreenmethods.However,inthesamestudy, sensitivityandspecificityvaluesforEliA™CTDScreen(sensitivity Pearson's chisquare test Phi Coefficient Φ Contingency Coefficient Kappa Coefficient EliA™CTDvsIIF 8.079(p <0.004) 0.158 0.156 0.150 EliA™CTDvs ELISA 95.54 (p <0.0001) 0.544 0.478 0.540 ELISAvsIIF 13.98 (p <0.0001) 0.208 0.204 0.203 EliA™CTD&IIF vsELISA&IIF 214.1 (p <0.0001) 0.814 0.631 0.814 Abbreviations:IIF,indirectimmunofluorescence. TABLE 3 Comparativestatisticaltests and agreement coefficients between differentANAscreeningplatformsalone or combined TABLE 4 (A)Discrepantresultsforthethreemethodscompared.(B)DescriptionoftiterandpatternfordiscrepantresultsbetweenIIF andEliAorELISA (A) EliA +ELISAELIA - ELISA+IIF- EliA+IIFELISA+IIF+EliA- IIF+ELISA- N27 45 44 48 96 82 (B) IIF+EliA- IIF+ELISAIIF Pattern 29.0%NuclearHomogeneous(AC−1) 29.3%NuclearHomogeneous(AC−1) 29.0%NuclearFineSpeckled(AC−4) 30.5%NuclearFineSpeckled(AC−4) 8.3%NuclearDenseFineSpeckled(AC−2) 9,8%NuclearDenseFineSpeckled(AC−2) 2.0%NuclearCoarseSpeckled(AC−5) 2.4%NuclearCoarseSpeckled(AC−5) 12.5%Nucleolar(AC−8,9,10) 9.8%Nucleolar(AC−8,9,10) 5,2%Reticular/AMA(AC−21) 6.0%Reticular/AMA(AC−21) 4.0%Discretenucleardots(AC−6,7) 3.6%Discretenucleardots(AC−6,7) 2.0%Centromere(AC−3) 1.2%Centromere(AC−3) 2.0%FibrillarCytoplasmic(AC−15,16,17) 2.4%FibrillarCytoplasmic(AC−15,16,17) 3.0%CytoplasmicSpeckled(AC−18,19,20) 3.6%CytoplasmicSpeckled(AC−18,19,20) 2.0%Other 24.4%Other IIF titer 1/80:66.7% 1/80:70.7% 1/160:20.8% 1/160:17.1% 1/320;10.4% 1/320:11.0% >1/640:2.1%a >1/640:1.2% EliA™CTD SCREEN TotalNegative Positive IIF Negative 104(32.2%) 44(13.6%) 148(45.8%) Positive 96(29.7%) 79(24.4%) 175(54.2%) Total 200(61.9%) 123(38%) 323(100%) ELISAScreen Negative 155(48%) 27(8.3%) 182(56.3%) Positive 45(13.9%) 96(29.7%) 141(43.6%) Total 200(61.9%) 123(38%) 323(100%) TABLE 2 ComparisonofANA screeningresultsbyEliA™CTD,IIFand ELISAscreen 10982825, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcla.23914 by Universidad De Sevilla, Wiley Online Library on [28/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 8 | GONZÁLEZ RODRÍGUEZ Et aL. between 71% and 84%, and specificity between 90% and 96%) were similar to the values demonstrated in this publication. This has been also reported by Bizzaro et al.13Intheirstudy,thediagnosticefficiencyofEliA™CTDScreenandIIFobtainedbyseven differentstudieswascompared,obtaininganaverageefficiency of87.1%forEliA™CTDScreenand77.1%forIIF(rangesbetween 77%-96%and70–87%,respectively).Theperformanceobtained inthepresentstudyforEliA™CTDScreenisconsistentwiththe numbers reported by Bizzaro; while for IIF, we have obtained slightly lower values. It is important to mention that the results ofIIFandELISAmethodshavebeenshowntobehighlydependent on the equipment used and also between equipment from the same manufacturer.14 In a study that evaluated the sensitivity ofautomatedIIFandELISA,theauthorsreportedvaluesbetween differentmanufacturersrangingfrom77.7%and95.5%forIIF,and ImmunoconceptsHEp-2 cellswere the ones displaying thelowestvalue(77.7%).ValueswerehigherforELISA,88.3%and86.4% for the two studied methods.15 It should also be noted that the performanceofIIFmayvary amonglaboratories,and itis more consistent in fully automated tests.16 When the diagnostic efficiency of combined tests was evaluated (EliA™CTDScreenplusIIF,andELISAplusIIF),sensitivityandnegativeLRvalueswereobservedtobeincreasedcomparedtoEliA™CTD ScreenorELISAalonemeasurements.Inparticular,EliA™CTDScreen plus IIF combination results were highly promising with high sensitivityandlownegativeLR(sensitivity=93%andnegativeLR=0.14). Although combination tests had lower specificity and positive LR valuescomparedtoindividualtests,sensitivitywashigherandnegativeLRwasbelowthelimitsconsideredclinicallysignificant.4 These results are in line with published studies and current recommendations which advocate for the joint performances of IIF and a screening testwith alargenumber of solid-phasefixed antigens,suchas the EliA™CTDScreen(FEIA),orchemiluminescenceassays.5,16Second- generationANAscreeningapproacheshavebeendevelopedcombining IIF with antigenic specificity assays in a single test consisting of HEp-2cellsimmobilizedinacentralcompartmentofaglass holder and surrounded by microparticles with specific antigens.17 HighermeasuringagreementhasbeenobservedbetweenEliA™ CTDScreenandELISAcomparedtoEliA™CTDScreenandIIF,most likelyduetothefactthatEliAincludesalltheantigenswithenhanced expressioninELISA.InEliA™CTDScreenamixtureof17ANAsassociatedwithSARDwaspresent,whileinELISAtherewere12.On theotherhand,IIFusesamuchwiderrangeofantigens.Asassessed by Pearson's chi-square test, although there were statistically significant differences between the results of the methods (alone or in combination),whenEliA™CTDScreenorELISAwerecombinedwith IIF, sensitivity, specificity, and measure agreement improved. The TABLE 7 DiagnosticperformanceofANAplusENAscreenings SARD and Ro52/ Ro60 positive EliA™ CTD LIA Ro52 Ro60 Ro52 Ro60 IIF+&CTD+66 54 51 51 IIF−&CTD+24 22 13 19 LossRatewithIIF 36% 41% 25% 37% Abbreviations:CTD,EliA™CTDScreen;IIF,indirect immunofluorescence. EliA™ CTD LIA Ro52 Ro60 Ro52 Ro60 Systemiclupuserythematosus 8(3.42%) 19(8.12%) 12(6.59%) 18(9.89%) Sjögren'ssyndrome 20(8.55%) 24(10.26%) 23(12.64%) 21(11.53%) Systemicsclerosis 1 1 1 3 MCTD 1 0 3 0 DMPM 2 1 1 1 Undifferentiated CTD 1 1 1 1 Other 5 9 8 8 Total 38 55 49 52 Abbreviations:CTD,connectivetissuedisease;DM-PM,dermatomyositis–polymyositis;MCTD, Mixedconnectivetissuedisease;Others,rheumatoidarthritisandvasculitis. TABLE 6 FrequencyofRo52and Ro60antibodiesperautoimmunedisease assessedbyEliA™andLIA TABLE 5 DiagnosticperformancesANAscreenplatforms(aloneorcombined) EliA™ CTD IIF ELISA EliA™ CTD +IIF ELISA +IIF Sensitivity(%) 79% 69% 69% 93% 89% Specificity(%) 91% 56% 74% 50% 45% LR+8.33 1.58 2.68 1.86 1.63 LR− 0.23 0.55 0.42 0.14 0.23 Abbreviations:LR,Likelihoodratio;IIF,indirectimmunofluorescence. 10982825, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcla.23914 by Universidad De Sevilla, Wiley Online Library on [28/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 7 of 8 GONZÁLEZ RODRÍGUEZ Et aL. metaanalysis by Orme et al.2 has also reported significant differences betweenthediagnosticoddsratiosofEliA™CTDScreenandIIF. ForEliA™CTDScreenpositivesamples,specificantibodiesassociatedwithSARDhavebeentested,usingLIAandEliA™(FEIA). However, dsDNA has not been considered for the study, since it wouldnotbeusedforcomparingLIAandFEIA,asitisnotrecommended to analyze anti-dsDNA autoantibodies by LIA.18 One or moreantibodiesweredetectedbyLIAin84%ofSARDsamplesin our population, while in 89% of the samples antibodies were detectedbyFEIA.Itisremarkablethat,inthe18EliA™-CTDpositive SARDnegativesamples,43%and38%werepositiveinLIAandFEIA tests,respectively;amongthese,anti-SSA/Ro(60kD)autoantibodiesweredetectedin82%ofthecasesandanti-centromereautoantibodiesin18%.Anti-SSA/Ro(60kD)antibodieshavebeenreported to be among the ANA most frequently detected in routine SARD testing.19 They are mainly detected in patients with systemic lupus erythematosusandSjögren'sdisease.20Inourcohort,anti-Ro60autoantibodiesweremoreprevalentthananti-Ro52.Interestingly,up to41%ofthesamplespositiveforRo60couldnotbedetectedwhen IIF was used as the only screening method. The study has three main limitations. The first is sample size. The cohort size (n =323)waslimited;however,itcanbeconsidered representative of the routine work of a laboratory specialized in the diagnosisofSARD.Thesecondreliesonthefactthattheanalysis of antibodies against the different antigenic specificities associated withSARDwasperformedexclusivelyinpatientswithpositiveEliA™ CTDScreenfollowingtheusualprotocolofthelaboratory.Finally, neither disease duration nor concurrent therapies and disease activitywereconsideredfortheanalysis,soitsimpactontheresultshas not been evaluated. Insummary,EliA™CTDScreenpresentsbetterdiagnosticperformanceresultsversusIIFandELISAindividualmeasurements. The combined use of IIF with a screening test based on the use ofsolid-phasefixedantigens,suchasEliA™CTDScreen,clearly improvesthedetectioncapacityofSARD(sensitivity=93%and LR− =0.14).Furthermore,theuseofEliA™CTDScreenasfirst- linescreeningtechniqueallowsthedetectionofRo52andRo60 antibodies (considered two of the most prevalent antibodies in SARD)whichwouldnotbedetectedbyIIFalone,thusincreasing the falsenegative rate. ACKNOWLEDGEMENT TheauthorsgreatlyacknowledgeThermoFisherScientificforproviding EliA™ reagents, and also the Virgen Macarena University Hospitalforfunding. CONFLICT OF INTEREST Dr. González has received a speaker honorarium from Thermo Fisher Scientific. AUTHOR CONTRIBUTIONS Allauthorsconfirmedtheyhavecontributedtotheintellectualcontent of this paper and have met the following 3 requirements: (a) significantcontributionstotheconceptionand design, acquisition ofdata,oranalysisandinterpretationofdata;(b)draftingorrevisingthearticleforintellectualcontent;and(c)finalapprovalofthe published article. ETHICS APPROVAL This study was approved by the Ethics Committee of the University of Virgen Macarena Hospital (Approval reference number: 201932115312).Authorsdeclarethatthemanuscripthasnotbeen submittedtoanyotherjournalforsimultaneousconsideration,has not been published previously and data have not been manipulated or copied from other authors. DATA AVAILABILITY STATEMENT The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 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J Clin Lab Anal. 2021;35:e23914. https://doi. org/10.1002/jcla.23914 10982825, 2021, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcla.23914 by Universidad De Sevilla, Wiley Online Library on [28/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License