Review Article Current Concepts and Future Directions for the...

Review ArticleCurrent Concepts and Future Directions forthe Assessment of Autoantibodies to Cellular AntigensReferred to as Anti-Nuclear Antibodies

Michael Mahler1 Pier-Luigi Meroni2 Xavier Bossuyt3 and Marvin J Fritzler4

1 INOVA Diagnostics Inc 9900 Old Grove Road San Diego CA 92131-1638 USA2 Rheumatology amp Experimental Laboratory of Immuno-rheumatology University of Milan Istituto Auxologico ItalianoVia G Zucchi 18 20095 Cusano Milanino Milan Italy

3 Department of Microbiology and Immunology Laboratory Medicine University Hospitals Leuven KU Leuven Belgium4Faculty of Medicine University of Calgary Calgary Alberta Canada T2N 4N1

Correspondence should be addressed to Michael Mahler mmahlerinovadxcom

Received 13 December 2013 Accepted 27 January 2014 Published 27 April 2014

Academic Editor Ghislain Opdenakker

Copyright copy 2014 Michael Mahler et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The detection of autoantibodies that target intracellular antigens commonly termed anti-nuclear antibodies (ANA) is a serologicalhallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD) Different methods are available for detection ofANA and all bearing their own advantages and limitations Most laboratories use the indirect immunofluorescence (IIF) assaybased on HEp-2 cell substrates Due to the subjectivity of this diagnostic platform automated digital reading systems have beendeveloped during the last decade In addition solid phase immunoassays using well characterized antigens have gained widespreadadoption in high throughput laboratories due to their ease of use and open automation Despite all the advances in the field ofANA detection and its contribution to the diagnosis of SARD significant challenges persist This review provides a comprehensiveoverview of the current status on ANA testing including automated IIF reading systems and solid phase assays and suggests anapproach to interpretation of results and discusses meeting the problems of assay standardization and other persistent challenges

1 Introduction

In 1950 Coons and Kaplan described the improvement of animmunofluorescence method for the detection of antigens intissue cells [1] Eight years later Friou et al first describedan indirect immunofluorescence (IIF) assay for the detectionof anti-nuclear antibodies (ANA) [2 3] Along with theearlier discovery of the lupus erythematosus (LE) cell andthe development of the LE cell test [4 5] this ushered in along and productive age of ANA testing The ANA IIF testinitially relied on rodent tissue substrates but contemporarytests use HEp-2 cells a cell line established in 1952 by Mooreand her colleagues from tumors that had been producedin weanling rats exposed to irradiation and corticosteroidsinjected with epidermoid carcinoma tissue from the larynxof a 56-year-old male [6] In the following decades ANAtests using HEp-2 cells revolutionized the diagnosis of ANA

associated rheumatic diseases (AARD) including systemiclupus erythematosus (SLE) systemic sclerosis (SSc) Sjogrenrsquossyndrome (SjS) mixed connective tissue disease (MCTD)and idiopathic inflammatory myopathies (IIM) [7 8]

The IIF assay on HEp-2 cells has been replaced inmany laboratories since the development of ANA screeningassays based on ELISA and automated high throughputmultiplex assays using addressable laser bead and otherarray technologies for the detection of specific ANA [910] Due to a significant prevalence of ldquofalse negativerdquoANA results on these newer platforms and an insufficientcommunication between laboratorians and clinicians therehave been growing concerns about unilateral adoption ofthese newer screening and high throughput assays [11]Questions about which method should be used and thelack of standardization of the novel test algorithms ledthe American College of Rheumatology (ACR) to form

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2014 Article ID 315179 18 pageshttpdxdoiorg1011552014315179

2 Journal of Immunology Research

Table 1 Statistical terms relevant for ANA testing

Statistical measure General explanation Implication for ANA

Sensitivity Statistical measure of how accurately a testcorrectly identifies diseased individuals

ANA is used as screening test High sensitivity isimportant The sensitivity for different AARDvaries (ie higher in systemic lupuserythematosus versus myositis)

Specificity Statistical measure of how well a test correctlyidentifies absence of the disease in question

Importance of specificity depends on pretestprobability In settings with low pretestprobability high specificity is required

Diagnostic efficiency Combination of sensitivity and specificity Not commonly used

False negative (clinically) Negative test result of a diseased individual

ANA is used as screening test False negativeresults are undesirable However in all AARDpatients without a positive ANA test existTherefore a negative result should never be usedto rule out AARD

False positive (clinically) Positive test result of an individual without thedisease in question

In case of low pretest probability false positiveresults significantly impact the posttest probability

False negative (analytically) Negative test result in the presence of therespective analyte See negative positive (clinically)

False positive (analytically) Positive test result in the absence of therespective analyte See false positive (clinically)

Positive predictive value Ratio of true positive to combined true andfalse positives Depends on the prevalence (pretest probability)

Negative predictive value Ratio of true negatives to combined true andfalse negatives Depends on the prevalence (pretest probability)

Positive likelihood ratio

The probability of a positive test results inpatients with the disease divided by theprobability of a positive test result inindividuals without the disease Independentfrom prevalence

Important information for clinicians Should beincluded in the laboratory report together with anexplanation of its significance in the context of thetest result

Negative likelihood ratio

The probability of a negative test result inpatients with the disease divided by theprobability of a negative test result inindividuals without the disease Independentfrom prevalence


The importance of the likelihood ratio in the laboratory report is controversially discussed but might improve use of ANA test results in the future

a task force who recommended the use of the conventionalIIF HEp-2 platform for ANA detection [12] This recom-mendation was in part based on evidence that the HEp-2 cell substrates are essentially an ldquoarrayrdquo presenting gt100autoantibody targets whereas most high throughput screen-ing arrays are much more limited in autoantibody targetcomposition This has prompted a reevaluation of the ANAIIF method which was reflected by entire sessions dedicatedto HEp-2 ANA testing at international clinical and scientificmeetings

In recent years the first digital imaging systems for ANAIIF have been developed which eliminate some major draw-backs of the method namely the subjectivity of observersreading the slides and the lack of an automated proce-dure [15ndash17] Nevertheless several drawbacks of the HEp-2 IIF methods persist and other technologies for ANAdetection continue to emerge and evolve In this reviewnovel insights and updates on ANA detection are pre-sented and the pros and cons of different methods arediscussed

2 Statistical Considerations

21 Sensitivity and Specificity For diagnostic applicationsit is important to differentiate between analytical sensitiv-ityspecificity and clinical (diagnostic) sensitivityspecificityTherefore the terms clinical sensitivity and specificity falsenegative false positive and predictive values are describedin Table 1 In addition it is widely known and extensivelydocumented that certain autoantibodies can precede thediagnosis or full clinical expression of an underlying diseasefor many years and thus false positive results at a given pointin time might over a subsequent time period become a truepositive [18 19] Consequently the term ldquofalse positiverdquo forautoantibodies needs to be used carefully

22 ROC Analysis and Cut-Off Selection The receiver oper-ating characteristic (ROC) analysis has a broad range ofapplications and was first used for military purposes duringWorld War II [20] In medicine ROC analysis has been

Journal of Immunology Research 3

0

02

04

06

08

1

0 02 04 06 08 1

Postt

est p

roba

bilit

y

Pretest probability

IIF(+)IIF(minus)

Posttest probability

50 a young woman with photosensitivitymalar rash and symmetrical polyarthritis

6

080

008 19 020

42 72 3

87 7 96 21

Pretest probability

1 a young woman with hair loss andpolyarthralgias

10 a young woman with photosensitivity

IIF(+) IIF(minus)

and mild leucopenia (3000ndash3500WBCmm3)

SPA(+) SPA(minus)

SPA(+)SPA(minus)

Figure 1 Illustration of pretest and posttest probability Posttest probability (predictive value) for systemic lupus erythematosus as a functionof pretest probability and as a function of indirect immunofluorescence (IIF) and solid phase assay (SPA) (EliA CTD screenThermo Fisher)test result Values for likelihood ratios are from Bossuyt and Fieuws [31] WBC = white blood cell

extensively used for diagnostic testing to evaluate the effec-tiveness of a novel diagnostic method as compared to analready established one or so called ldquogold standardrdquo methodSeveral statistical methods can be applied using the ROCanalysis including the most commonly used area under thecurve (AUC)TheAUC is equivalent to theMann-Whitney119880which tests for themedian difference between scores obtainedin the two continuous data sets However any attempt tosummarize the ROC curve into a single number fails asinformation about the pattern of tradeoffs of the particulardiscriminator algorithm is not expressed

The manner by which immunoassay cut-offs are estab-lished varies significantly among researchers and scientists indiagnostic companies A common approach to define the cut-off value for certain assays is to test specimens from patientswith the respective disease and compare them to a broadrange of controls including related and unrelated diseasesas well as age and gender matched (apparently) healthyindividuals The mean value plus 3-fold standard deviationthe 95 or the 99 percentile of the controls are then oftenused to define the cut-off value Another popular approachfor definition of the cut-off value makes use of ROC analysisDespite broad application most references do not specifyhow to use the ROCanalysis to define the cut-off value [21] In

the majority of cases a visual approach is used to identify anappropriate point on the ROC curve which provides a goodcombination of sensitivity and specificity Following this it isimportant that the cohort used to define the cut-off (trainingset) is large enough to achieve statistical power and that thecut-off is validated using an independent cohort of patients(validation set)

The method used for cut-off definition strongly dependson the assay and how it is intended to be used in a routinesetting For a screening assay a high degree of sensitivity ismandatory to ensure that the number of patients that aremissed by the assay is kept as low as possible In contrastconfirmation tests need high specificity In general low cut-off values increase the sensitivity at the expense of decreasingspecificity and vice versa when a higher cut-off is defined[9 10]

The interpretation of the ANA test results depends on thepretest probability of having the disease or whether fulminantdisease is present In a setting of high index of suspicion evenlow titers of ANA-IIF can be interpreted as significant [22]In 1997 it was suggested by Tan and colleagues [23] that thetest should be performed and reported at two dilutions 1 40and 1 160 in order to preserve the appropriate sensitivity andspecificity A more recent study recommended using a more


economical single screening dilution of 1 160 [24] which wasconfirmed by a recommendation paper that used a Delphiapproach for assessment of autoantibodies to intracellularantigens [21] ANA-IIF testing on HEp-2 cells is currentlywidely accepted as the procedure of choice for the detectionof ANA but not as a corollary of disease activity or relapse[21]

23 Likelihood Ratio In clinical practice an important andrelevant question is ldquoWhat is the probability of a patienthaving a particular disease when the laboratory test ispositive or not having the disease when the laboratory test isnegativerdquo [25] Clinicians and laboratory professionals havedifficulties in estimating the posttest probability for a diseasebased on sensitivity and specificity (2) Likelihood ratio (LR)is an alternative and probably more easily understood wayto convey diagnostic accuracy data in a clinical setting [26]The LR for a disease is the probability of the test resultin patients with the disease divided by the probability ofthe same test result in individuals without the disease Theposttest probability for disease associated with a particulartest result can be estimated based on the pretest probabilityand the LR for that particular test result [25 27]

Traditionally a single cut-off is used for the interpretationof a laboratory test and all values above or below the cut-off value are given the same interpretation (positive ornegative resp) For many AARD the likelihood for diseaseincreases with increasing antibody concentration [28ndash30]This information is lacking when a single cut-off is used LRcan be assigned to a particular test result or to a test resultinterval (eg the antibody titer of ANA) It has been shownthat the LR for an AARD increases with increasing antibodylevels [28] Knowledge of test result (interval) specific LRimproves the clinical interpretation of a particular test resultcompared to knowledge related to a single cut-off value

The LR gives an estimation of whether there will be asignificant change in pretest to posttest probability of diseasegiven the test result [25] A LR of 1 implies that there will beno difference between pretest and posttest probability [25]LRs gt10 or lt01 indicate large often clinically significantdifferences LRs between 5 and 10 and between 01 and 02indicate modest clinical differences [25]

The LR for SLE for ANA by IIF has been estimated tobe 7 for a positive test result and 003 for a negative testresult whereas the LR for SLE based on solid phase assays(SPA) [in this case Fluoro enzyme immunoassay (FEIA)EliA CTD screen] has been estimated to be 24 for a positivetest result and 027 for a negative test result [31] Using LRsone can calculate the posttest probability for any given pretestprobability [25] Figure 1 illustrates a graphical representationof the posttest probability (predictive value) for SLE as afunction of the pretest probability for IIF as well as forSPA Such graphical representation has been shown to be aconvenient way to convey diagnostic information [26]

To illustrate the impact of pretest probability and assayperformance on the posttest probability clinical examples areprovided (unpublished data based on expert experience ofPier-Luigi Meroni)

Table 2 Anti-nuclear antibodies (ANA) in different ANA associ-ated autoimmune rheumatic diseases and healthy individuals

Antibody AARDSLE SSc SjS IIM MCTD HI

dsDNA 40ndash70 lt3 lt3 lt3 lt3 lt3Chromatin 40ndash70 lt3 lt3 lt3 5ndash18 lt3RNP 10ndash40 5ndash15 lt3 5ndash15 1005

lt3Sm 5ndash20 lt2 lt1 lt1 lt22

lt1SS-ARo60 40ndash70 3ndash10 60ndash90 lt3 lt3 lt3Ro52TRIM21 40ndash70 15ndash30 70ndash90 25ndash50 lt3 lt3

SS-BLa 15ndash30 1ndash5 60ndash80 5ndash15 lt3 lt3Scl-70 (topo I) 0ndash5 20ndash404

lt3 lt3 lt3 lt1Jo-1 1ndash3 1ndash3 lt2 15ndash30 lt2 lt1Centromere 2ndash5 20ndash404 5ndash10 1ndash3 2ndash5 lt3RNA Pol III lt1 5ndash254

lt1 lt1 lt1 lt1Ribosomal P 10ndash30 lt2 lt2 lt2 lt2 lt1PMScl 1ndash3 5ndash10 lt2 5ndash10 lt2 lt3Mi-2 lt1 3ndash8 lt1 5ndash151

lt1 lt3Ku 5ndash20 3ndash8 lt3 3ndash103

lt3 lt3PCNA lt5 lt1 lt1 lt1 lt1 lt3ThTo lt1 3ndash10 lt1 lt1 lt1 lt11Rare in PM higher prevalence in DM mild form of disease early duringdevelopment2Prevalence depends if antigen contains SmBB1015840 (cross-reactive with RNP)3Very high titer in PM4Anti-Scl-70 anti-centromere anti-RNA Pol III antibodies tend to bemutually exclusive5Part of the classification criteria therefore should be 100 howeverdepending on assay used some patients might be negativeNote Prevalence values were established based on literature and consensusof authorsAbbreviations DM dermatomyositis IIM idiopathic inflammatory myopa-thy (polymyositisdermatomyositis) MCTD mixed connective tissue dis-ease PCNA proliferating cell nuclear antigen PM polymyositis RArheumatoid arthritis RNA pol III RNA polymerase III RNP ribonu-cleoprotein Sm Smith antigens (U2-U6 RNP) SjS Sjogrenrsquos syndromeSLE systemic lupus erythematosus SPA Solid phase assay SSc systemicsclerosis TRIM tripartite motif

For example a young woman with hair loss and pol-yarthralgias which are very nonspecific signs of SLE isestimated to have a pretest probability for SLE of 1 If ANAby IIF turns out to be positive in this patient the probabilityfor SLE increases from 1 to 6 which is still low If the SPACTD screen reveals an autoantibody directed to a specificnuclear antigen (ENA or dsDNA) then the probability forSLE is higher (19) A second example is a young womanwho presents to her physician with photosensitivity and mildleucopenia (3000ndash3500WBCmm3) Based on this clinicalpresentation the probability for SLE is estimated to be 10A positive ANA by IIF increases the probability for SLEto 42 whereas a positive SPA CTD screen increases theprobability for SLE to 72 which makes the diagnosis forSLE likely However a woman positive for anti-SSARo coulddisplay the same clinical manifestations not necessarily hav-ing SLE (sometimes the sicca syndrome is clinically silent at


Table3Com

merciallyavailableimmun

oassaysfor

ANAtesting

Antigen

Automated

screeningassays

Multip

lexassays

Line

immun

oassaysd

otblots

EliA

CTD

EliA

Symph

ony

QUA

NTA

FlashEN

A7

QUA

NTA

Flash

CTDScreen

Plus

Alegria

ANA

Detect

Bio-Plex

2200

ANAALB

IA

AtheNAMulti-Lyte

Anti-N

uclear

Antibod

ies(ANA)

FIDIS

Con

nective

10ANAProfi

leIm

mcoStrip

eANA

Advanced

ANA12

IgG

BlueDot

ANA-

12Pro

(BLO

T)

recomLine

ANAENA

IgG

ANALia

MAX

Thermo

Scientific

Thermo

Scientific

INOVA

Diagn

ostic

sIN

OVA

Diagn

ostic

sOrgentec

Bio-Ra

dZe

usTh

eradiag

Euroim

mun

IMMCO

Diagn

ostic

sD-Tek

AES

KUDiagn

ostic

sMIK

ROGEN

Diagn

ostic

sHUMAN

Diagn

ostic

sU1-R

NP

XX

XX

XX

XX

XX

U1-R

NPSm

XX

XX

XX

U1-R

NP68

kDa

XX

XU1-R

NPA

XX

XU1-R

NPC

XX

XSS-ARo6

0X

XX

XX

X(i)

XX

XX

XX

XX

Ro52TRIM21

XX

XX

XX(

i)X

XX

XX

SS-BLa

XX

XX

XX

XX

XX

XX

XX

Centro

mere

XX

XX

XX

XX

XX

XX

Scl-7

0topo

IX

XX

XX

XX

XX

XX

XX

Jo-1

XX

XX

XX

XX

XX

XX

XX

FibrillarinU

3RNP

XRN

A-Po

lIII

XX

Ribo

somalP

XX

XX

XX

XX

XX

PMScl

XX

XX

PMScl-

75X

PMScl-

100

XX

PCNA

XX

XX

XX

XMi-2

XX

XX

XSm

XX

XX

XX

XX

XX

XdsDNA

XX

XX

XX

XX

XX

Nucleosom

eX

XX

XX

Histon

eX

XX

XX

XX

KuX

XX

XX

SRP5

4X

AMA-

M2

XX

X

Notesomec

ompanies

offer

severallineimmun

oassaysfor

ANAdetection

Mostcom

prehensiv

eassaysfrom

different

companies

ares

hown

AMAA

nti-m

itochon

drialantibod

iesPC

NAP

roliferatingcellnu

cleara

ntigenSRP

signalrecognitio

nparticle

NOTE

RNPandRN

PSm

containthes

ubun

itsRN

P-AR

NP-CandRN

P-68

kDa

(i)Re

ported

outsidethe

UnitedStates

asRo

60andRo

52andas

SS-A

intheU

nitedStates

Con

tainsC

ENP-AandCE

NP-B


the beginning of a primary SjS) A negative ANA by IIF resultwould reduce the probability for SLE from 10 to lt1 Thethird example is a young woman with photosensitivity malarrash and symmetrical polyarthritis This clinical picture issuggestive of SLE (50 probability) A positive IIF resultincreases the probability for SLE from 50 to 87 whereas apositive SPA CTD screen increases it to 96 A negative IIFresult would reduce the probability for SLE to 7 whereas anegative SPA test result would reduce the probability of SLEto 21 illustrating the NPV of SPA is lower than the NPV ofIIF

3 Nomenclature of Antibodies toCellular Antigens Commonly Referred toas Anti-Nuclear Antibodies (ANA)

Historically only antibodies targeting antigens present in thenuclear compartment of the cells (nuclear antigens) werecalled ANA Similarly the term extractable nuclear antigen(ENA) described in 1959 by Holman and Robbins was usedfor a group of nuclear antigens extractable by saline solutions[14] Nowadays with the identification of a variety of newautoantigens within various compartments of the cell thenomenclature has become rather imprecise and misleading[21] As an oversimplification even serum samples with anti-cytoplasmic but without ANA reactivity are sometimes con-sidered as ANA positive [32ndash34]This confusing terminologyhas even been adopted in the nomenclature of commercialautoantibody assays and kits Autoantibody arrays on varioustechnology platforms are often termed as ANA or ENAprofiles even though they contain relatively insoluble nuclearantigens such as dsDNA andor cytoplasmic targets such asribosomal P or Jo-1 antigens Therefore standardization ofthis nomenclature is highly desirable

4 Anti-Nuclear Antibodies inDifferent Conditions

To date more than 160 autoantigens many of them localizedto the cell nucleus have been described in sera of SLE patients[35] Therefore the spectrum of SLE associated autoantigenscontained in most ANA screening SPA includes only a smallproportion of antigens targeted by SLE autoantibodies [3536] However most SLE associated autoantigens apart fromthe standard ENAs are rarely the target of individual SLEsera and even more uncommon without reactivity to anyof the standard ENAs For example a recent study found asensitivity of a SPA for SLE of 79 (in diagnostic samples)compared to a sensitivity of IIF at cut-off 1 160 of 90[29] Thus the number of SLE patients having at least oneclinically meaningful autoantibody that are missed by ANASPA appears to be approximately 10 In contrast an evenlarger proportion of SSc patients have a negative test resultswhen an ANA SPA is used [37] Consequently the clinicalutility of novel assays for different AARD can be different andeach new assay has to be validated in all AARD subgroups(SLE SSc MCTD SjS and IIM)

In addition to SLE and SSc ANA can be found invarious other SARD including but not limited to IIM SjSand MCTD (Table 2) The appreciation that ANA are usefuldiagnostic biomarkers in a broad spectrum of autoimmuneconditions has led to a significant change in the referralpattern of ANA tests to diagnostic laboratories (see Figure 1)Historically primarily rheumatologists and clinical immu-nologists ordered ANA testing as an aid to the diagnosis ofSLE Much of this was due to the embedding of ANA andcertain ENA in the older and now more recent classificationcriteria for SLE [38 39] Nowadays a wider spectrumof clinicians order the ANA test (see Figure 1) includingbut not limited to internists dermatologists nephrologistsoncologists cardiologists neurologists gastroenterologistsotolaryngologists ophthalmologists gynecologists and evenprimary care physicians (Figure 2) This can be attributed tothe broadening spectrum of ANAs in rheumatoid arthritis(RA) antiphospholipid syndrome (APS) [40] autoimmuneliver diseases such as autoimmune hepatitis and primarybiliary cirrhosis (PBC) [41ndash46] vasculitis [47] inflammatorybowel disease (IBD) [48ndash51] and cancer [52ndash57] (Table 4)

5 Differential Diagnosis ofAutoimmune Diseases

The early and accurate diagnosis of autoimmune diseasescan be very challenging because the spectrum of signs andsymptoms are verywide and often overlap Initially anAARDhas to be differentiated from a wide spectrum disorders(ie infections malignancies allergic and adverse drugreactions) presenting with similar signs and symptoms Forexample a patient suspected to have SLE can first presentwith skin manifestations which need to be differentiatedfrom discoid lupus polymorphous light eruption rosaceadrug eruptions and other dermatoses If other organs areinvolved (ie kidney lung musculoskeletal cardiovascularor neuropsychiatric [58]) the differential diagnosis must takeother diagnostic possibilities into consideration Secondlyafter the presence of an AARD is confirmed on the basisof signs symptoms and physical examination the differentAARD need to be differentiated from each other so as toassist the clinician with decisions about appropriate ther-apeutic interventions This can be further complicated bythe evolution of autoimmune diseases from one conditionto another Many AARD especially SLE can present witharthritis but during follow-up a diagnosis of RA or ldquorupusrdquomight be established [59 60] Similarly MCTD can evolveinto SSc or RA The appropriate interpretation of a positiveor negative ANA can help enlighten the diagnostic andprognostic accuracy of AARD although very little is knownabout the LR to differentiate the different diseases

6 Screening and Profile Assays forANA Detection

61 ANA by Indirect Immunofluorescence on HEp-2 Cells Forwell over the last decade the IIF HEp-2 assay was beingreplaced by newer technologies for the detection of ANA [61]


RheumatologyClinical immunology

Internal medicineDermatology

Primary careHematologyNephrologyOncologyCardiologyObstetrics and

gynecologyNeurologyGastroenterology

hellip1960 Todayhellip

Pretest probability decreasedrarr significant consequences on posttest probability

Figure 2 Change in referral patterns Historically when the ANA HEp-2 test became available in around 1960 exclusively rheumatologistand clinical immunologists ordered the ANA test With the emerging recognition that many other diseases are associated with ANAs abroad range of clinical disciplines order the ANA test With changes in the ANA referral pattern and the associated decrease in the pretestprobability the posttest probability significantly decreases (indicated by the triangle)

Table 4 Clinical utility of ANA testing in different diseases

Diagnosis Clinical utility ANA prevalence Monitoringprognosis CommentsSLE Very useful 90ndash95 Not useful ANA IIF superior to ANA solid phase assaysSSc Very useful 85ndash95 Not useful ANA IIF superior to ANA solid phase assays

SjS Useful 50ndash60 Not useful ANA solid phase assays superior to ANA IIF SS-Areactivity can be missed by ANA HEp-2

AIM Somewhatuseful 50ndash60 Not useful ANA solid phase assays superior to ANA IIF Jo-1

reactivity can be missed by ANA HEp-2

MCTD Very useful 90ndash100 Not useful High titer anti-U1-RNP are highly indicative forMCTD

JCAJIA Somewhatuseful 50ndash60 Very useful Useful for subset that are at risk of developing uveitis

PBC Very useful 50ndash80 Not proven

ANA IIF superior to solid phase assays Antibodiesto SP100 gp210 nucleoporin p62 lamin B receptorand Ro52 TRIM21 Anti-gp210 reported associationwith poor prognosis

RA Not useful 15ndash20 Not useful Homogeneous and speckled staining are the mostcommon patterns

APS Not useful 40ndash70 Not useful Might indicate systemic autoimmunity in primaryAPS patients

AT Not useful 10ndash20 Not useful Higher in Graversquos disease as compared toHashimotolsquos thyroiditis

Cancer andparaneoplasticsyndromes

Not useful orutility notestablished

20ndash50 Not usefulAntibodies to CENP-F and to other proteins mightbe useful to help in the diagnosis of cancer p53 hasbeen discussed not many systematic studies onANA in cancer

AIH Useful 40ndash80 Not useful Prevalence depends on phase of the diseaseAbbreviations AIH autoimmune hepatitis AIM autoimmune inflammatory myopathy (polymyositis dermatomyositis) APS anti-phospholipid syndromeAT autoimmune thyroiditis JCAJIA juvenile chronic arthritisjuvenile inflammatory arthritis MCTDmixed connective tissue disease PBC primary biliarycirrhosis RA rheumatoid arthritis SjS Sjogrenrsquos syndrome SLE systemic lupus erythematosus SSc systemic sclerosis NOTE Prevalence values are based ondiagnostic samples (not treated patients)


Table 5 Advantages and disadvantages of the HEp-2 ANA test

Advantages DisadvantagesVariety of different target autoantigens (gt100) Subjectivity

Some autoantibodies can be identified withoutconfirmatory testing (ie anti-centromere)

Poorly standardized across manufacturers

Discovery tool for novel autoantibodies Requires training and expertise

Useful for a spectrum autoimmune diseases (ieautoimmune hepatitis)

Low sensitivity for certain clinically importantautoantibodies (ie Jo-1 ribosomal P SS-ARo60Ro52TRIM21)Low specificity (high false positive rate)

Table 6 Overview of defined ANA patterns (modified from Wiiket al 2010 [13])

Pattern group PatternNuclear envelope(membrane)

Smooth nuclear envelopePunctate nuclear envelope

Nuclear

Homogeneous patternLarge speckledCoarse speckledFine speckledFine grainy Scl-70-likePleomorphic speckled (ie PCNA)CentromereMultiple nuclear dotsCoiled bodies (few nuclear dots)Dense fine speckledIsolated metaphase chromosomes

NucleolarHomogeneous nucleolarClumpy nucleolarPunctate nucleolar

Mitotic spindleapparatus

Centriole (centrosome)Spindle pole (NuMa) (MSA-1) (HSeg5)Spindle fiberMidbody (MSA-2)CENP-F (MSA-3)

Cytoplasmic

DiffuseFine speckledMitochondrialDiscrete dots GW bodiesendosomes lysosomesGolgi complexIntercellular contact proteinsFibers and cytoskeletonRods and rings

Negative

and several larger laboratories switched to automated high-throughput immunoassay platforms [61] However in 2010 aposition paper was published indicating that IIF on HEp-2cells should remain the ldquogold standardrdquo for the detection

of ANA [12] triggering a renaissance of the IIF ANA testNevertheless in some cases an ANA result based on IIFANA on HEp-2 substrates may mislead the clinician and hasto be interpreted within the clinical context [62] (Table 5)In addition standardization of this assay is difficult due tointermanufacturer variations in the substrate and the fixationprocess characteristics of the secondary antibody used [63]interlaboratory variations in microscopy apparatus andespecially the subjective interpretation of the results [64]Detection of ANA by IIF may also yield false negative resultseven in the presence of high titers of antibodies such as thosedirected to SS-ARo60 Ro52TRIM21 Jo-1 (histidyl tRNAsynthetase) and others [65ndash67] Additionally the challengeof significant variation of staining patterns on the ANAHEp-2 IIF substrates obtained with slides from differentmanufacturers [63] has led to a proposed nomenclature forIIF patterns [13] (Table 6) For these reasons considerableeffort has been dedicated to the development of standardizedSPA for routine use such as ELISA [68] which are attendedby guidelines for the detection of ANA [21 62 69]

611 Automated Pattern Recognition of the ANA HEp-2 TestComputer assisted pattern recognition for ANA testing onHEp-2 cells has been described more than ten years ago [70]Automated hardware and software-based pattern recognitionplatforms that allow for the identification and archiving ofIIF patterns obtained on HEp-2 cell substrates however theyhave only become available during the last few years [1516 71ndash74] The operating principle of these new automatedsystems is acquiring storing and analyzing of digital imagesof stained IIF slides and displaying them on high resolutioncomputer monitors The inherent technical difficulties ofprocessing and reading IIF slides (manual reading real-timeinterpretation need for dark room and handwritten resultstranscription) make traditional IIF methods difficult to fit inthe workflow of modern automated laboratories The newautomated systems are powerful workflow and operationaltools that can eliminate the need for a darkroom and separateimage acquisition from image interpretation and have thepotential to improve the quality and utility of the ANAHEp-2assay

The currently available automated ANA IIF image ana-lyzing systems include NOVA View (INOVA DiagnosticsSan Diego US) [30] Aklides (Medipan Berlin Germany)[15 16 75] G-Sight (Menarini Florence Italy) [76 77]

Journal of Immunology Research 9Ta

ble7Au

tomated

digitalA

NAreadingsyste

ms

Instrument

NOVA

View

AKL

IDES

EURO

Patte

rnIm

ageN

avigator

Helios

ZENIT

GSight

Manufacturer

INOVA

diagno

stics

Medipan

Euroim

mun

Immun

ocon

cepts

Aesku

Menarini

LIMSconn

ectio

n(soft

ware)

Yes

(QUA

NTA

Link

)

Yes(syste

mindepend

ent

stand

ardXM

Linterfa

ce)

Yes

(EURO

LabO

ffice)

Yes(direct)

Optionallab

traffi

ccon

trol

Yes(direct)

Optional

AeskuLab

middlew

are

Yes(Ze

nIT)

Slideidentificatio

nviab

arcode

Yesb

yhand

held

scanner

Yesb

yhand

held

scanner

Yesb

yintegrated

scanner

Yes

Yesb

yintegrated

scanner

Yesb

yintegrated

scanner

Loadingcapacity

5slides

(upto

60wells)

5slides

(upto

60wells)

50slides

(upto

500wells)

4slides

(upto

84wells)

20slides

(upto

240wells)

5slides

(upto

70wells)

Imagea

cquisitionspeed

sim45

swellfor

3im

ages

sim40

swell

lt20

swell

sim25

swellfor

4im

ages

10spicture

Custo

mizable

from

1to10

images

gt60

swell

numbero

fpictures5

(smallscan)50

(medium

scan)or

220(fu

llscan)

100

QCforsub

strateandprocess

integrity

cou

ntersta

ining

YesDAPI

YesDAPI

YesProp

idium

iodide

Non

eNon

eNon

eNon

eNon

eNon

e

Automaticpo

snegdiscrim

inationincl

presortin

gof

images

Yes

Yes

Yes

Yes

Yes

Yes

Batchw

iseverifi

catio

nof

negativ

esam

ples

Yes

Yes

Yes

Yes

Yes

Yes

Automaticpatte

rnrecogn

ition

Yes

Yes

Yes

No

No

Yes

Patte

rnAnalysis

metho

dPatte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Nopatte

rnmatching

capabilities

Nopatte

rnmatching

capabilities

Patte

rnrecogn

ition

bymathematical

algorithm

Num

bero

frecognizableA

NAsta

iningpatte

rnlisto

ut

6Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

10Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

8Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Nuclear

rimMixed

patte

rnmito

tic

Non

eNon

e

5Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Cytoplasm

Negative

Analysis

ofmixed

staining

patte

rnLimited

(hom

ogeneous

nucle

olar)

Yes

Yes

No

No

No

Mergedresults

perp

atient

(differentd

ilutio

ns)

Yes

Yes

Yes

Yes

Yes

Yes

Finalresultvalidationpo

ssiblewhilesyste

mprocessesrem

aining

samples

Yes

No

Yes

Yes

Yes

No

Instrumentcalibratio

nto

minim

izev

ariability

Yes

Yes

Yes

Yes

Yes

No

Integrationwith

slide

processin

gin

1instrum

ent

No

No

No

No

Yes

No


EuroPattern (Euroimmun Lubeck Germany) [73] ImageNavigator (ImmunoConcepts Sacramento US) and Helios(Aesku Wendelsheim Germany) (Table 7) The systems dif-fer from each other with respect to the use of DNA-bindingcounterstains such asDAPI the cell substrate used (egmostsystems are restricted to using the respective manufacturerrsquosslides) the throughput the number of patterns that can beidentified and user-friendly features of the software [76 77]

Generally these automated systems are based on amicro-scope fitted with an automated stage a CCD digital cameraa LED light source and software that controls the movingparts and directs image acquisition All systems performsome kind of fluorescent light intensitymeasurement and usethe results for preliminarily categorization of the samples aspositive or negative and for pattern analysis The automatedreading is followed by human visual interpretation of thedigital images that are displayed on a computer monitorallowing user confirmation or revision of the automatedresults By providing good quality digital images and otherobjective information (such as preliminary classification andpattern interpretation) these automated systems supportthe operatorsrsquo decision making and increase the consistencybetween readers and readings In addition the digital imagescan be stored for training documentation follow-up andsecond opinion purposes In the future these digital imagesmight also become part of the patientrsquos electronic medicalrecord (EMR) At present the systems are highly reliable intheir ability to discriminate positive from negative reactionsand to estimate fluorescence intensity but the accuracy androbustness of pattern recognition does not reach the accuracyof human interpretation [77 78] An important feature is thequality of the digital images enabling the operators to makethe same clinically relevant interpretation as theywouldmakeusing a conventional microscope

The final result interpretation is made by the opera-tor therefore subjectivity cannot be completely removedMoreover the characteristics of the HEp-2 substrates andconjugates influence the appearance of certainANA specifici-ties and the automated pattern identification of the varioussystems is likely based on somewhat different programmingprincipals Nevertheless automated systems for ANA HEp-2 analysis are a significant step forward to reduce variabilityand offer opportunities to increase harmonization of ANAinterpretation [79]

Some systems offer automated assessment of ANA end-point titers on a single serum dilution thereby eliminatingthe need for serial dilutions [75]Moreover the potential inte-gration of the automated digital IIF systems with laboratoryinformation systems (LIS) provides sample traceability andeliminates manual transcription and associated transmittalerrors thereby improving patient safety The systems alsohold the promise to reduce hands on time Work flow studiesusing different systems are required to analyze the efficiencybenefits of those systems

612 Limited Sensitivity and Specificity of ANAHEp-2 IIF TestSeveral studies have demonstrated limited analyticalclinicalsensitivity [61 63 66 80 81] and clinical specificity [82ndash85]

of IIF on HEp-2 cells In particular for anti-Rib-P anti-SSARo60 and anti-Jo-1 autoantibodies the ANA HEp-2 IIFtest has been reported to lack analytical sensitivity whichtranslates to the clinical sensitivity for AARD [63 65 66 8186 87] In a comparative analysis of an ANAELISA andANAIIF equivalent sensitivity but significantly higher specificityof the ELISA was observed [84] When using the cut-offrecommended by the manufacturer the clinical specificity ofIIF for AARD was as low as 623 [84]

Based on these observations over three decades ago atransfected HEp-2 cell line overexpressing SS-ARo60 wasdeveloped and this cell-based IIF assay is marketed as HEp-2000 cells [81 88ndash90] A similar approach has recently beenused for SmD1 antigen in a research HEp-2 cell line [91]Thistechnological approach of overexpressing target antigens in avariety of cell lines has more recently become a productiveapproach to cell based IIF assays where structural or con-formational epitopes are important for human autoantibodydetection It was found that adding antigen specific assaysto IIF on HEp-2 cells significantly improved the diagnosticalgorithm for the diagnosis of SARD [92] However it wasconcluded in this study that changing from IIF to othermethods for ANA detection also requiredmodification of thedisease criteria In addition it was highly recommended touse anti-SS-A antibody assays in addition to ANAHEp-2 test[92]

62 Different Staining Patterns Have Different SpecificitiesDisease classification criteria such as the SLE criteria [3839] do not distinguish between different ANA IIF patternsAlthough certain ANA patterns on HEp-2 cells have a signif-icant disease associations in clinical practice differentiationof IIF patterns is rarely used as an aid in establishing theclinical diagnosis [21] Nevertheless it is well establishedthat the centromere staining pattern is primarily associatedwith the limited cutaneous form of SSc (also referred toas the CREST syndrome) [8 93] Additional examples arethe association of the homogenous IIF pattern with SLEand nucleolar IIF pattern with SSc [37] although thesegeneralizations have not been observed in all studies [94](most likely attributed to different pretest probabilities)Interestingly antibodies to dense fine speckled 70 (DFS70)also known as LEDGF (lens epithelium-derived growthfactor) which generate a DFS IIF pattern on HEp-2 cellswere not commonly observed in AARDs [82 95ndash102] Theseantibodies decorate interphase nucleoplasm outside of thenucleolus but in contrast to the anti-SSASSB and anti-Mi-2 antibodies the anti-DFS70LEDGF antibodies stainthe metaphase and telophase chromosomal cell plates [13](Figure 3) Just recently it was confirmed that theDFS patternwas not associated with AARD and was primarily found inapparently healthy individuals [103] In rare cases this patterncan also be observed in patients with AARD but these casesanti-DFS70 antibodies are commonly accompanied by otherautoantibodies [98 102] Follow-up of individuals with hightiters of anti-DFS70 antibodies revealed that they retain ANAreactivity even after four years but do not develop SARD[103 104] Hence it has been suggested that within some


(a)

(b)

(c)

Figure 3 Characteristic staining pattern of anti-DFS70 antibodiesThe characteristic dense fine speckled (DFS) staining pattern of interphaseHEp-2 cells is indicated by the blue arrow and the strong chromatin staining of mitotic cells by the red arrow (a) Wide field view using 40xmagnification (b) dense fine speckled pattern of an interphase nucleus and (c) of the metaphase chromatin of a mitotic cell

limits anti-DFS70 antibodies can be used to exclude thediagnosis of AARD [82] The major epitope of the moleculeis conformation dependent and is located in the C-terminalpart of the molecule [105] Although the immunoreactiveregion has been shown to be located within a stretch of 22amino acids (407ndash435) the use of 12 mer peptides failed toestablish reactivity with the presumed linear epitope [105]

63 ANA Screening ELISA During the last decade differ-ent strategies have been utilized to develop evaluate andcommercialize several ANA screening ELISAs [106ndash110]Themajority of ANA screening ELISAs make use of mixtures orldquoblendsrdquo of purified autoantigens from native sources andorrecombinant technologies [106 111]The composition of theseantigen preparations is quite diverse and is dependent onseveral factors including the availability of pure antigens andthe technical feasibility of combining all different antigensin a single assay Most available immunoassays contain SS-ARo60 [112] SS-BLa Scl-70topoisomerase I [113] CENP-B [93] Jo-1 U1-RNP Sm and dsDNA [114] ANA screen-ing ELISAs from some manufacturers also contain otherautoantigens such as PMScl or ribosomal P [36] Howeverbased on comparison to immunoprecipitation of radiola-belled native proteins and other techniques reactivity can bemissed by these ELISAs even if the autoantigens are containedin the mixture [36] This can be attributed to the notionthat individual autoantigens exhibit different biochemicalproperties and therefore display different binding behaviorswhen solid phase matrices are used to bind the targetautoantigens For example some antigensmight bind to othertargets in the same mixture resulting in a masking effect thatgives the impression that insufficient epitopes are available forhuman autoantibody binding Some antigens such as PCNA[115 116] RNAPol III [117] orThTo [118] are rarely includedas purified antigen in screening assays

64 Line Immunoassays Line immunoassays (LIA) can basi-cally be considered second generation dot-blot assays Abroad range of LIAs are available and they are typically used toconfirm autoantibodies previously identified by HEp-2 ANAIIF or other screening immunoassays [119] However in somelaboratories these LIAs have also been used as a screening testfor disease specific autoantibodies that are seen in SLE SScIIM paraneoplastic and autoimmune liver diseases [120]There have been recent advances in partially automatingthesemultiplex LIAsmaking them somewhatmore appealingto high throughput laboratory testing [121] Despite theirease of use LIAs have some drawbacks including the lackof sensitivity and specificity for certain autoantibodies [120122] The antigen compositions of several are shown inTable 3

65 Multiplex Bead-Based Assays Multiplex assays based onthe Luminex technology (Austin Texas USA) use address-able laser beads and are therefore often referred to as ALBIA(addressable laser bead immunoassays) [9] Today severalcommercial ALBIA kits are available for the detection ofautoantibodies to a variety of autoantigens [83 85 123ndash130]First generation ALBIAs showed polyreactivity which wascaused by nonspecific binding to the beads [131] Secondgeneration assays showed significant reduced polyreactivityand thus higher specificity [131] In 2007 a multiplex test forthe detection of ANAwas compared to ANA IIF and differentELISA assays 787 (74) of healthy donors were positive67 showed a speckled and 17 a nucleolar staining pattern[85] Similar to LIAs the number of antigens and the antigencompositions of these bead-based arrays significantly varyand are shown in Table 3

66 Other ANA Tests Additional methods have been devel-oped for automated ANA detection [84 108 132ndash139] In


1999 a fully automated ANA screening assay (COBAS CoreHEp2 ANA EIA Roche Diagnostics Mannheim Germany)was developed and evaluated The performance evaluationstudies showed promising results but were inclusive in theconclusion One study shows that the new assay is superiorto the ANA IIF as analyzed by ROC analysis [138] Howeverthis finding could not be confirmed in a second independentanalysis [133] Six years later the first-automated chemilu-minescent immunoassay (CIA) for the detection of ANA(LIAISON ANA screen DiaSorin) was evaluated in two cen-ters yielding a good positive (795) and negative agreement(912) when the LIAISONANA screen was compared to theANA IIF test (Bio-Rad) [140] A recent study using this assayshowedANAprevalence compatible with the expected valuesof theANA IIF test [42]Onlymoderate agreementwas foundbetween IIF and a multiplex assay based on the ALBIA Themajority of ANA IIF positive and multiplex ANA negativesera were also positive by an ANA ELISA utilizing nuclearextracts [129] In addition several other methods have beendeveloped for ANA testing but are not widely used in clinicalpractice In 2009 a novel method for quantitative ANAmeasurement using near-infrared imagingwas also described[141] Furthermore a novel microbead-based ELISA systemusing fluorescence-coded immobilized microbeads on theAKLIDIS system has been described [142] Similar to theALBIA but using nanobarcodes for the bead identificationthe Ultraplex system [143] was used to screen simultaneouslyfor nine ANA autoantibodies requiring significantly lesslabor and fewer reagents with performance equivalent toexisting gold-standard methods

Several publications have described protein arrays onplanar solid phase surfaces for the detection of autoanti-bodies to a wide range of viral proteins and autoantigens[144 145] bound to a variety of surfaces [146] Howeverthese immunoassays are still not used in routine diagnosticlaboratories

More recently novel ldquoANArdquo screening assays have beendeveloped on fully automated closed systems such as thePhadia (Thermo Fisher Freiburg Germany) or the BIO-FLASH System (INOVA San Diego USA) [147] The EliACTD Screen (Thermo Fisher) has been evaluated in severalstudies two of which have been published in peer-reviewedjournals [28 148] The first study showed satisfactory resultsfor anti-ribosomal P anti-PMScl anti-Mi-2 and anti-PCNAantibodies However the sensitivity for anti-fibrillarin andanti-RNA Pol III antibodies was rather limited [148] Inthe second study the CTD screen was compared to theANA HEp-2000 method [29] Additionally the QUANTAFlash CTD Screen Plus (on BIO-FLASH) was evaluated andshown to exhibit good sensitivity in different SARD [149]However further studies are needed to establish the clinicalperformance characteristics of the novel assays

7 Quality Aspects Standardization andReference Sera

For the development and quality assurance of autoantibodyassays a broad range of international reference samples are

mandatory [150 151] Consequently an ANA and relatedautoantibody reference serum panel was established andis now available through the Center for Disease Controland Prevention in Atlanta USA [152ndash155] Initially thispanel of sera was used to standardize ANA IIF tests andto define staining patterns but eventually this panel wasused to evaluate the performance of different autoantibodyimmunoassays [152] In 2000 another ANA reference serumpanel became available through the Association of MedicalLaboratory Immunologists (AMLI) [156] However only afew studies used these samples for their investigations [157]The first international standard for ANA became available in1990 [158] However the number of international referencesera is limited and not all autoantibodies are represented bythe available serum panels [150] Furthermore for screeningassays monospecific samples for each antigen are required toensure the presence of all antigens in sufficient quantity andquality

Different committees and organizations were formedwho worked to achieve a better standardized approach toautoantibody testing [150 159 160] including the Interna-tionalUnion of Immunology Specialties (IUIS)AutoantibodyStandardization Committee (httpascdentalufleduhomehtmltext) the European Autoimmunity Stand-ardisationInitiative (EASI) [httpwwweasi-networkcom] and theWorking Group on Harmonization of Autoantibody Tests(WG-HAT) in the framework of the International Federationof Clinical Chemistry and Laboratory Medicine [httpwwwifccorgifcc-scientific-divisionsd-working-groupsharmo-onisation-of-autoantibody-tests-wg-hat] Despite signifi-cant efforts to standardize autoantibody tests [152 161] andevidence that these groups are now working more closelytogether [21] significant variations still exist

8 Conclusions

(i) Performance data (including LRs) of themethod usedto detect ANA and appropriate explanation should bemade available to the clinician

(ii) ANA test results are only a portion of the informationthat aids in the diagnosis of systemic autoimmune dis-eases and are an adjunct to the clinicianrsquos diagnosticrepertoire

(iii) Both IIF on HEp-2 cells and solid phase immunoas-says have their individual advantages and limitations

(iv) Standardized nomenclature of diseases and associatedautoantibodies is an important goal for immediateconsideration by advisory groups

9 Future Perspectives

Despite significant evolution and improvements in ANAand related autoantibody testing including the arrival ofnovel and promising technologies several limitations stillpersist and need to be addressed First the terminologyand nomenclature used to identify and refer to variousautoantibodies need to be standardized Second the clas-sification criteria and nomenclature of individual SARD


and related autoimmune diseases must continue to evolveand keep abreast of biomarker identification Third thecorresponding immunoassays and diagnostic platforms usedfor the various clinical applications need to be based onstandardized reference samples of defined specificities Thispossibility could include the development and validation ofdisease specific screening assays (ie SLE Screen SSc Screen)on solid phase technologies Fourth a clearly defined strategyneeds to be developed to facilitate clinicians and laboratoryscientists alike becoming more familiar with and be ableto intelligently use objective interpretation of autoantibodyresults through an understanding of ROCs and LRs Lastlydiagnostic algorithms need to be adjusted to the clinicaland laboratory setting considering the referral pattern thesample testing volume and health economic aspects (iereimbursement)

Conflict of Interests

Michael Mahler is employed at INOVA Diagnostics a com-pany that manufactures and markets autoantibody assaysPier-Luigi Meroni received fees as consultant for INOVADiagnostics and from BioRad Xavier Bossuyt has been aconsultant to INOVA Diagnostics and has received lec-ture fees from Instrumentation Laboratory Thermo Fisherand Menarini Marvin J Fritzler is the Director of Mito-gen Advanced Diagnostics Laboratory (Calgary AlbertaCanada) which performs autoantibody testing and is also aconsultant to INOVA Diagnostics and has received gifts inkind from Euroimmun GmbH

References

[1] AHCoons andMHKaplan ldquoLocalization of antigen in tissuecells improvements in a method for the detection of antigenby means of fluorescent antibodyrdquoThe Journal of ExperimentalMedicine vol 91 no 1 pp 1ndash13 1950

[2] G J Friou S C Finch and K D Detre ldquoInteraction of nucleiand globulin from lupus erythematosis serum demonstratedwith fluorescent antibodyrdquo Journal of Immunology vol 80 no4 pp 324ndash329 1958

[3] G J Friou ldquoSetting the scene a historical and personal view ofimmunologic diseases autoimmunity and ANArdquo Clinical andExperimental Rheumatology vol 12 supplement 11 pp S23ndashS25 1994

[4] M M Hargraves H Richmond and R Morton ldquoPresentationof two bone marrow elements the tart cell and the LE cellrdquoProceedings of the Staff Meetings of the Mayo Clinic vol 23 pp25ndash28 1948

[5] H R Holman H R Deicher and H G Kunkel ldquoThe L E celland the L E serum factorsrdquo Bulletin of the New York Academyof Medicine vol 35 no 7 pp 409ndash418 1959

[6] A E Moore L Sabachewsky and H W Toolan ldquoCulturecharacteristics of four permanent lines of human cancer cellsrdquoCancer Research vol 15 pp 598ndash602 1955

[7] E M Tan ldquoAntinuclear antibodies in diagnosis and manage-mentrdquo Hospital Practice vol 18 no 1 pp 79ndash84 1983

[8] M Mahler and M J Fritzler ldquoEpitope specificity and signifi-cance in systemic autoimmune diseasesrdquoAnnals of theNewYorkAcademy of Sciences vol 1183 pp 267ndash287 2010

[9] M J Fritzler and M L Fritzler ldquoThe emergence of multiplexedtechnologies as diagnostic platforms in systemic autoimmunediseasesrdquoCurrentMedicinal Chemistry vol 13 no 21 pp 2503ndash2512 2006

[10] M J Fritzler ldquoAdvances and applications of multiplexed diag-nostic technologies in autoimmune diseasesrdquo Lupus vol 15 no7 pp 422ndash427 2006

[11] A S Wiik T P Gordon A F Kavanaugh et al Eds ldquoCut-ting edge diagnostics in rheumatology the role of patientsclinicians and laboratory scientists in optimizing the use ofautoimmune serologyrdquo Arthritis Care and Research vol 51 no2 pp 291ndash298 2004

[12] P L Meroni and P H Schur ldquoANA screening an old test withnew recommendationsrdquo Annals of the Rheumatic Diseases vol69 no 8 pp 1420ndash1422 2010

[13] A S Wiik M Hoslashier-Madsen J Forslid P Charles and JMeyrowitsch ldquoAntinuclear antibodies a contemporary nomen-clature using HEp-2 cellsrdquo Journal of Autoimmunity vol 35 no3 pp 276ndash290 2010

[14] HHolman andWRobbins ldquoAntinuclear antibodies in systemiclupus erythematosusrdquo Arthritis amp Rheumatology vol 2 pp468ndash471 1959

[15] K Egerer D Roggenbuck R Hiemann et al ldquoAutomatedevaluation of autoantibodies on human epithelial-2 cells as anapproach to standardize cell-based immunofluorescence testsrdquoArthritis Research andTherapy vol 12 no 2 article R40 2010

[16] S Kivity B Gilburd N Agmon-Levin et al ldquoA novel automatedindirect immunofluorescence autoantibody evaluationrdquo Clini-cal Rheumatology vol 31 no 3 pp 503ndash509 2012

[17] A Melegari C Bonaguri A Russo B Luisita T Trentiand G Lippi ldquoA comparative study on the reliability of anautomated system for the evaluation of cell-based indirectimmunofluorescencerdquo Autoimmunity Reviews vol 11 pp 713ndash716 2012

[18] M R Arbuckle M T McClain M V Rubertone et alldquoDevelopment of autoantibodies before the clinical onset ofsystemic lupus erythematosusrdquo The New England Journal ofMedicine vol 349 no 16 pp 1526ndash1533 2003

[19] C Eriksson H Kokkonen M Johansson G Hallmans GWadell and S Rantapaa-Dahlqvist ldquoAutoantibodies predate theonset of systemic lupus erythematosus in northern SwedenrdquoArthritis Research andTherapy vol 13 no 1 article R30 2011

[20] TKampfrath and S S Levinson ldquoBrief critical review statisticalassessment of biomarker performancerdquo Clinica Chimica Actavol 419 pp 102ndash107 2013

[21] N Agmon-Levin J Damoiseaux C Kallenberg et al ldquoInterna-tional recommendations for the assessment of autoantibodies tocellular antigens referred to as anti-nuclear antibodiesrdquo Annalsof Rheumatic Diseases vol 73 pp 17ndash23 2014

[22] A M Abeles and M Abeles ldquoThe clinical utility of a positiveantinuclear antibody test resultrdquo American Journal of Medicinevol 126 pp 342ndash348 2013

[23] E M Tan T E W Feltkamp J S Smolen et al ldquoRange ofantinuclear antibodies in ldquohealthyrdquo individualsrdquo Arthritis ampRheumatism vol 40 no 9 pp 1601ndash1611 1997

[24] D A Gonzalez A C D Leon A R Varela et al ldquoAutoanti-body detection with indirect immunofluorescence on HEp-2cells starting serum dilutions for systemic rheumatic diseasesrdquoImmunology Letters vol 140 no 1-2 pp 30ndash35 2011

[25] X Bossuyt ldquoClinical performance characteristics of a labora-tory test A practical approach in the autoimmune laboratoryrdquoAutoimmunity Reviews vol 8 no 7 pp 543ndash548 2009


[26] P Vermeersch and X Bossuyt ldquoComparative analysis of dif-ferent approaches to report diagnostic accuracyrdquo Archives ofInternal Medicine vol 170 no 8 pp 734ndash735 2010

[27] A F Kavanaugh D H Solomon P Schur J D Reveille Y R SSherrer and R Lahita ldquoGuidelines for immunologic laboratorytesting in the rheumatic diseases an introductionrdquo ArthritisCare and Research vol 47 no 4 pp 429ndash433 2002

[28] KOpDeBeeck PVermeersch PVerschueren et al ldquoDetectionof antinuclear antibodies by indirect immunofluorescence andby solid phase assayrdquo Autoimmunity Reviews vol 10 no 12 pp801ndash808 2011

[29] K Op De Beeck P Vermeersch P Verschueren et al ldquoAntinu-clear antibody detection by automated multiplex immunoassayin untreated patients at the time of diagnosisrdquo AutoimmunityReviews vol 12 pp 137ndash143 2012

[30] S Schouwers M Bonnet P Verschueren et al ldquoValue-addedreporting of antinuclear antibody testing by automated indirectimmunofluorescence analysisrdquo Clinical Chemistry and Labora-tory Medicine vol 52 no 4 pp 547ndash551 2014

[31] X Bossuyt and S Fieuws ldquoDetection of antinuclear antibodiesadded value of solid phase assayrdquo Annals of the RheumaticDiseases vol 73 article e10 2014

[32] M Vercammen P Meirlaen J Sennesael et al ldquoDiagnos-tic accuracy of the FIDIS multiplex fluorescent microsphereimmunodetection system for anti-extractable nuclear antigen(ENA) antibodies in connective tissue diseasesrdquo Clinical Chem-istry and Laboratory Medicine vol 45 no 4 pp 505ndash512 2007

[33] J Wenzel R Gerdsen M Uerlich R Bauer T Bieber andI Boehm ldquoAntibodies targeting extractable nuclear antigenshistorical development and current knowledgerdquo British Journalof Dermatology vol 145 no 6 pp 859ndash867 2001

[34] J G M C Damoiseaux and J W Cohen Tervaert ldquoFrom ANAto ENA how to proceedrdquo Autoimmunity Reviews vol 5 no 1pp 10ndash17 2006

[35] Y Sherer AGorsteinM J Fritzler andY Shoenfeld ldquoAutoanti-body explosion in systemic lupus erythematosus more than 100different antibodies found in SLE patientsrdquo Seminars inArthritisand Rheumatism vol 34 no 2 pp 501ndash537 2004

[36] S S Copple A D Sawitzke A M Wilson A E Tebo andH R Hill ldquoEnzyme-linked immunosorbent assay screeningthen indirect immunofluorescence confirmation of antinuclearantibodiesrdquo American Journal of Clinical Pathology vol 135 no5 pp 678ndash684 2011

[37] V K Shanmugam D R Swistowski N Saddic H Wang andV D Steen ldquoComparison of indirect immunofluorescence andmultiplex antinuclear antibody screening in systemic sclerosisrdquoClinical Rheumatology vol 30 no 10 pp 1363ndash1368 2011

[38] E M Tan A S Cohen and J F Fries ldquoThe 1982 revisedcriteria for the classification of systemic lupus erythrematosusrdquoArthritis amp Rheumatism vol 25 no 11 pp 1271ndash1277 1982

[39] M Petri A M Orbai G S Alarcon et al ldquoDerivation andvalidation of the systemic lupus international collaboratingclinics classification criteria for systemic lupus erythematosusrdquoArthritis amp Rheumatology vol 64 pp 2677ndash2686 2012

[40] L Andreoli F Pregnolato RW Burlingame et al ldquoAntinucleo-some antibodies in primary antiphospholipid syndrome a hintat systemic autoimmunityrdquo Journal of Autoimmunity vol 30no 1-2 pp 51ndash57 2008

[41] S Assassi M J Fritzler F C Arnett et al ldquoPrimary biliarycirrhosis (PBC) PBC autoantibodies and hepatic parame-ter abnormalities in a large population of systemic sclerosis

patientsrdquo Journal of Rheumatology vol 36 no 10 pp 2250ndash2256 2009

[42] Y Zafrir B Gilburd M G Carrasco et al ldquoEvaluation of anautomated chemiluminescent immunoassay kit for antinuclearantibodies in autoimmune diseasesrdquo Immunologic Research vol56 pp 451ndash456 2013

[43] A Granito P Muratori C Quarneti G Pappas R Cicola andLMuratori ldquoAntinuclear antibodies as ancillarymarkers in pri-mary biliary cirrhosisrdquo Expert Review of Molecular Diagnosticsvol 12 no 1 pp 65ndash74 2012

[44] PMuratori A Granito G Pappas LMuratori M Lenzi and FB Bianchi ldquoAutoimmune liver disease 2007rdquoMolecular Aspectsof Medicine vol 29 no 1-2 pp 96ndash102 2008

[45] D P Bogdanos and L Komorowski ldquoDisease-specific autoanti-bodies in primary biliary cirrhosisrdquo Clinica Chimica Acta vol412 no 7-8 pp 502ndash512 2011

[46] N Agmon-Levin Y Shapira C Selmi et al ldquoA comprehensiveevaluation of serum autoantibodies in primary biliary cirrho-sisrdquo Journal of Autoimmunity vol 34 no 1 pp 55ndash58 2010

[47] J A Savige M Gallicchio L Chang and J D Parkin ldquoAutoan-tibodies in systemic vasculitisrdquo Australian and New ZealandJournal of Medicine vol 21 no 4 pp 433ndash437 1991

[48] F Beigel F Schnitzler R Paul Laubender et al ldquoFormation ofantinuclear and double-strand DNA antibodies and frequencyof lupus-like syndrome in anti-TNF-120572 antibody-treated patientswith inflammatory bowel diseaserdquo Inflammatory Bowel Dis-eases vol 17 no 1 pp 91ndash98 2011

[49] C Mancho A Sainz M Garcıa-Sancho A Villaescusa M ATesouro and F Rodrıguez-Franco ldquoDetection of perinuclearantineutrophil cytoplasmic antibodies and antinuclear antibod-ies in the diagnosis of canine inflammatory bowel diseaserdquoJournal of Veterinary Diagnostic Investigation vol 22 no 4 pp553ndash558 2010

[50] H Nielsen A Wiik and J Elmgreen ldquoGranulocyte specificantinuclear antibodies in ulcerative colitis Aid in differentialdiagnosis of inflammatory bowel diseaserdquo Acta PathologicaMicrobiologica et Immunologica Scandinavica C Immunologyvol 91 no 1 pp 23ndash26 1983

[51] D Zauli C Crespi and P DallrsquoAmore ldquoAntibodies to thecytoskeleton components and other autoantibodies in inflam-matory bowel diseaserdquo Digestion vol 32 no 2 pp 140ndash1441985

[52] A K Al-Shukaili A A Al-Jabri and M S Al-MoundharildquoPrognostic value of auto-antibodies in the serum of Omanipatients with gastric cancerrdquo Saudi Medical Journal vol 27 no12 pp 1873ndash1877 2006

[53] J B Armas J Dantas D Mendonca et al ldquoAnticardiolipin andantinuclear antibodies in cancer patientsmdasha case control studyrdquoClinical and Experimental Rheumatology vol 18 no 2 pp 227ndash232 2000

[54] F Blaes M Klotz H Huwer et al ldquoAntineural and antinuclearautoantibodies are of prognostic relevance in non-small celllung cancerrdquo Annals of Thoracic Surgery vol 69 no 1 pp 254ndash258 2000

[55] T Daniels J Zhang I Gutierrez et al ldquoAntinuclear autoanti-bodies in prostate cancer immunity to LEDGFp75 a survivalprotein highly expressed in prostate tumors and cleaved duringapoptosisrdquo Prostate vol 62 no 1 pp 14ndash26 2005

[56] H Imai Y Nakano K Kiyosawa and E M Tan ldquoIncreasingtiters and changing specificities of antinuclear antibodies inpatients with chronic liver disease who develop hepatocellularcarcinomardquo Cancer vol 71 pp 26ndash35 1993


[57] A Imran F Neelam and M Tariq ldquoIncidence of circulatingantinuclear antibodies in cancer patientsrdquo Indian journal ofmedical sciences vol 57 no 3 pp 113ndash116 2003

[58] AM Borowoy J E Pope E Silverman et al ldquoNeuropsychiatriclupus the prevalence and autoantibody associations dependon the definition results from the 1000 faces of lupus cohortrdquoSeminars inArthritis andRheumatism vol 42 pp 179ndash185 2012

[59] A Fernandez G Quintana E L Matteson et al ldquoLupusarthropathy historical evolution from deforming arthritis torhupusrdquo Clinical Rheumatology vol 23 no 6 pp 523ndash5262004

[60] C Tani D DrsquoAniello S A Delle et al ldquoRhupus syndromeassessment of its prevalence and its clinical and instrumentalcharacteristics in a prospective cohort of 103 SLE patientsrdquoAutoimmunity Reviews vol 12 pp 537ndash541 2013

[61] H Nossent and O P Rekvig ldquoAntinuclear antibody screeningin this newmillennium farewell to the microscoperdquo Scandina-vian Journal of Rheumatology vol 30 no 3 pp 123ndash126 2001

[62] A Kavanaugh R Tomar J Reveille D H Solomon and HA Homburger ldquoGuidelines for clinical use of the antinuclearantibody test and tests for specific autoantibodies to nuclearantigensrdquo Archives of Pathology and Laboratory Medicine vol124 no 1 pp 71ndash81 2000

[63] M Mahler J T Ngo J Schulte-Pelkum T Luettich and M JFritzler ldquoLimited reliability of the indirect immunofluorescencetechnique for the detection of anti-Rib-P antibodiesrdquo ArthritisResearch andTherapy vol 10 no 6 article R131 2008

[64] I Kang R Siperstein T Quan and M L Breitenstein ldquoUtilityof age gender ANA titer and pattern as predictors of anti-ENAand -dsDNA antibodiesrdquo Clinical Rheumatology vol 23 no 6pp 509ndash515 2004

[65] X Bossuyt and A Luyckx ldquoAntibodies to extractable nuclearantigens in antinuclear antibody-negative samplesrdquo ClinicalChemistry vol 51 no 12 pp 2426ndash2427 2005

[66] I E A Hoffman I Peene E M Veys and F De KeyserldquoDetection of specific antinuclear reactivities in patients withnegative anti-nuclear antibody immunofluorescence screeningtestsrdquo Clinical Chemistry vol 48 no 12 pp 2171ndash2176 2002

[67] X Bossuyt G Marien and S Vanderschueren ldquoA 67-year-oldwoman with a systemic inflammatory syndrome and siccardquoClinical Chemistry vol 56 no 9 pp 1508ndash1509 2010

[68] G P Tri R C W Wong and S Adelstein ldquoAutoantibodies toextractable nuclear antigens making detection and interpre-tation more meaningfulrdquo Clinical and Diagnostic LaboratoryImmunology vol 9 no 1 pp 1ndash7 2002

[69] D H Solomon A J Kavanaugh P H Schur et al ldquoEvidence-based guidelines for the use of immunologic tests antinuclearantibody testingrdquo Arthritis Care and Research vol 47 no 4 pp434ndash444 2002

[70] P Perner H Perner and B Muller ldquoMining knowledgefor HEp-2 cell image classificationrdquo Artificial Intelligence inMedicine vol 26 no 1-2 pp 161ndash173 2002

[71] R Hiemann T Buttner T Krieger D Roggenbuck U Sackand K Conrad ldquoChallenges of automated screening and differ-entiation of non-organ specific autoantibodies on HEp-2 cellsrdquoAutoimmunity Reviews vol 9 no 1 pp 17ndash22 2009

[72] A Willitzki R Hiemann V Peters et al ldquoNew platform tech-nology for comprehensive serological diagnostics of autoim-mune diseasesrdquo Clinical and Developmental Immunology vol2012 Article ID 284740 8 pages 2012

[73] J Voigt C Krause E Rohwader et al ldquoAutomated indirectimmunofluorescence evaluation of antinuclear autoantibodieson HEp-2 cellsrdquo Clinical and Developmental Immunology vol2012 Article ID 651058 7 pages 2012

[74] D Bertin N Jourde-Chiche P Bongrand and N BardinldquoOriginal approach for automated quantification of antinuclearautoantibodies by indirect immunofluorescencerdquo Clinical andDevelopmental Immunology vol 2013 Article ID 182172 8pages 2013

[75] D Roggenbuck R Hiemann P Schierack D Reinhold andK Conrad ldquoDigital immunofluorescence enables automateddetection of antinuclear antibody endpoint titers avoiding serialdilutionrdquo Clinical Chemistry and Laboratory Medicine pp 1ndash32013

[76] C Bonroy C Verfaillie V Smith et al ldquoAutomated indi-rect immunofluorescence antinuclear antibody analysis is astandardized alternative for visual microscope interpretationrdquoClinical Chemistry and Laboratory Medicine vol 51 pp 1771ndash1779 2013

[77] X Bossuyt S Cooreman B H De et al ldquoDetection of antin-uclear antibodies by automated indirect immunofluorescenceanalysisrdquo Clinica Chimica Acta vol 415 pp 101ndash106 2013

[78] P Foggia G Percannella P Soda and M Vento ldquoBenchmark-ing HEp-2 cells classification methodsrdquo IEEE Transactions onMedical Imaging vol 32 pp 1878ndash1889 2013

[79] D Roggenbuck R Hiemann D Bogdanos D Reinhold andK Conrad ldquoStandardization of automated interpretation ofimmunofluorescence testsrdquo Clinica Chimica Acta vol 421 pp168ndash169 2013

[80] K Kidd K Cusi R Mueller M Goodner B Boyes and E HoyldquoDetection and identification of significant ANAs in previouslydetermined ANAnegative samplesrdquoClinical Laboratory vol 51no 9-10 pp 517ndash521 2005

[81] X Bossuyt J Frans A Hendrickx G Godefridis R West-hovens and G Marien ldquoDetection of anti-SSA antibodies byindirect immunofluorescencerdquo Clinical Chemistry vol 50 no12 pp 2361ndash2369 2004

[82] A Watanabe M Kodera K Sugiura et al ldquoAnti-DFS70 anti-bodies in 597 healthy hospital workersrdquo Arthritis amp Rheuma-tism vol 50 no 3 pp 892ndash900 2004

[83] O Shovman B Gilburd O Barzilai et al ldquoEvaluation of theBioPlex 2200 ANA screen Analysis of 510 healthy subjectsincidence of naturalpredictive autoantibodiesrdquo Annals of theNew York Academy of Sciences vol 1050 pp 380ndash388 2005

[84] R A Gniewek D P Stites T M McHugh J F Hilton and MNakagawa ldquoComparison of antinuclear antibody testing meth-ods immunofluorescence assay versus enzyme immunoassayrdquoClinical andDiagnostic Laboratory Immunology vol 4 no 2 pp185ndash188 1997

[85] E Avaniss-Aghajani S Berzon and A Sarkissian ldquoClinicalvalue of multiplexed bead-based immunoassays for detectionof autoantibodies to nuclear antigensrdquo Clinical and VaccineImmunology vol 14 no 5 pp 505ndash509 2007

[86] K Sugisaki I Takeda T Kanno et al ldquoAn anti-nuclearantibody-negative patient with systemic lupus erythematosus(SLE) accompanied with anti-ribosomal P antibody (anti-P)rdquoInternal Medicine vol 41 no 11 pp 1047ndash1051 2002

[87] Y Muro K Sugiura Y Morita and Y Tomita ldquoEvaluationof anti-ribosomal P protein immunoassay in Japanese patientswith connective tissue diseases comparison with an indirectimmunofluorescence assayrdquo Scandinavian Journal of Rheuma-tology vol 38 no 6 pp 460ndash463 2009


[88] I Peene W Van Ael M Vandenbossche T Vervaet E Veysand F De Keyser ldquoSensitivity of the HEp-2000 substrate for thedetection of anti-SSARo60 antibodiesrdquoClinical Rheumatologyvol 19 no 4 pp 291ndash295 2000

[89] M J Fritzler C Hanson J Miller and T Eystathioy ldquoSpecificityof autoantibodies to SS-ARo on a transfected and overex-pressed human 60 kDa Ro autoantigen substraterdquo Journal ofClinical Laboratory Analysis vol 16 no 2 pp 103ndash108 2002

[90] N Tanaka Y Muro K Sugiura and Y Tomita ldquoAnti-SS-ARo antibody determination by indirect immunofluorescenceand comparison of different methods of anti-nuclear antibodyscreeningrdquo Modern Rheumatology vol 18 no 6 pp 585ndash5922008

[91] S LWang F FWang S L Chen et al ldquoExpression localizationand clinical application of exogenous Smith proteins D1 in genetransfected HEp-2 cellsrdquo International Journal of RheumaticDiseases vol 16 pp 303ndash309 2013

[92] CDahle T Skogh A K Aberg A Jalal and POlcen ldquoMethodsof choice for diagnostic antinuclear antibody (ANA) screeningbenefit of adding antigen-specific assays to immunofluores-cence microscopyrdquo Journal of Autoimmunity vol 22 no 3 pp241ndash248 2004

[93] M J Fritzler J B Rattner L M Luft et al ldquoHistoricalperspectives on the discovery and elucidation of autoantibodiesto centromere proteins (CENP) and the emerging importanceof antibodies to CENP-Frdquo Autoimmunity Reviews vol 10 no 4pp 194ndash200 2011

[94] S Khan A Alvi S Holding et al ldquoThe clinical significance ofantinucleolar antibodiesrdquo Journal of Clinical Pathology vol 61no 3 pp 283ndash286 2008

[95] N Bizzaro E Tonutti D Visentini et al ldquoAntibodies to the lensand cornea in anti-DFS70-positive subjectsrdquo Annals of the NewYork Academy of Sciences vol 1107 pp 174ndash183 2007

[96] M Okamoto Y Ogawa A Watanabe et al ldquoAutoantibodiesto DFS70LEDGF are increased in alopecia areata patientsrdquoJournal of Autoimmunity vol 23 no 3 pp 257ndash266 2004

[97] N Kuwabara Y Itoh T Igarshi and Y Fukunaga ldquoAutoanti-bodies to lens epithelium-derived growth factortranscriptionco-activator P75 (LEDGFP75) in children with chronic non-specific complaints and with positive antinuclear antibodiesrdquoAutoimmunity vol 42 no 6 pp 492ndash496 2009

[98] Y Muro K Sugiura Y Morita and Y Tomita ldquoHigh concomi-tance of disease marker autoantibodies in anti-DFS70LEDGFautoantibody-positive patients with autoimmune rheumaticdiseaserdquo Lupus vol 17 no 3 pp 171ndash176 2008

[99] V Ganapathy and C A Casiano ldquoAutoimmunity to the nuclearautoantigenDFS70 (LEDGF) what exactly are the autoantibod-ies trying to tell usrdquo Arthritis amp Rheumatism vol 50 no 3 pp684ndash688 2004

[100] V Ganapathy T Daniels and C A Casiano ldquoLEDGFp75 anovel nuclear autoantigen at the crossroads of cell survival andapoptosisrdquo Autoimmunity Reviews vol 2 no 5 pp 290ndash2972003

[101] M Mahler J G Hanly and M J Fritzler ldquoImportance of thedense fine speckled pattern on HEp-2 cells and anti-DFS70antibodies for the diagnosis of systemic autoimmune diseasesrdquoAutoimmunity Reviews vol 11 no 9 pp 642ndash645 2012

[102] M Mahler and M J Fritzler ldquoThe clinical significance ofthe dense fine speckled immunofluorescence pattern on HEp-2 cells for the diagnosis of systemic autoimmune diseasesrdquoClinical and Developmental Immunology vol 2012 Article ID494356 6 pages 2012

[103] H A Mariz E I Sato S H Barbosa S H Rodrigues ADellavance and L E Andrade ldquoAna HEp-2 pattern is a criticalparameter for discriminating ana-positive healthy individualsand patients with autoimmune rheumatic diseasesrdquo Arthritis ampRheumatology Arthritis Rheum 201163191-200

[104] M J Fritzler ldquoThe antinuclear antibody test last or lastinggasprdquo Arthritis amp Rheumatism vol 63 no 1 pp 19ndash22 2011

[105] Y Ogawa K Sugiura A Watanabe et al ldquoAutoantigenicity ofDFS70 is restricted to the conformational epitope of C-terminalalpha-helical domainrdquo Journal of Autoimmunity vol 23 no 3pp 221ndash231 2004

[106] M Fenger A Wiik M Hoslashier-Madsen et al ldquoDetectionof antinuclear antibodies by solid-phase immunoassays andimmunofluorescence analysisrdquo Clinical Chemistry vol 50 no11 pp 2141ndash2147 2004

[107] E Tonutti D Bassetti A Piazza et al ldquoDiagnostic accuracyof Elisa methods as an alternative screening test to indirectimmunofluorescence for the detection of antinuclear antibod-ies Evaluation of five commercial kitsrdquo Autoimmunity vol 37no 2 pp 171ndash176 2004

[108] P Kern M Kron and K Hiesche ldquoMeasurement of antinuclearantibodies assessment of different test systemsrdquo Clinical andDiagnostic Laboratory Immunology vol 7 no 1 pp 72ndash78 2000

[109] M Lopez-Hoyos V Rodrıguez-Valverde and V Martinez-Taboada ldquoPerformance of antinuclear antibody connectivetissue disease screenrdquo Annals of the New York Academy ofSciences vol 1109 pp 322ndash329 2007

[110] D Sinclair M Saas D Williams M Hart and R GoswamildquoCan an ELISA replace immunofluorescence for the detectionof anti-nuclear antibodiesmdashthe routine use of anti-nuclearantibody screening ELISAsrdquo Clinical Laboratory vol 53 no 3-4 pp 183ndash191 2007

[111] J Schmitt and W Papisch ldquoRecombinant autoantigensrdquoAutoimmunity Reviews vol 1 no 1-2 pp 79ndash88 2002

[112] J Schulte-PelkumM Fritzler andMMahler ldquoLatest update onthe RoSS-A autoantibody systemrdquo Autoimmunity Reviews vol8 no 7 pp 632ndash637 2009

[113] MMahler ED Silverman J Schulte-Pelkum andM J FritzlerldquoAnti-Scl-70 (topo-I) antibodies in SLE myth or realityrdquoAutoimmunity Reviews vol 9 no 11 pp 756ndash760 2010

[114] M Mahler ldquoSm peptides in differentiation of autoimmunediseasesrdquo Advances in Clinical Chemistry vol 54 pp 109ndash1282011

[115] MMahler E D Silverman andM J Fritzler ldquoNovel diagnosticand clinical aspects of anti-PCNA antibodies detected by noveldetection methodsrdquo Lupus vol 19 no 13 pp 1527ndash1533 2010

[116] M Mahler K Miyachi C Peebles and M J Fritzler ldquoTheclinical significance of autoantibodies to the proliferating cellnuclear antigen (PCNA)rdquo Autoimmunity Reviews vol 11 pp771ndash775 2012

[117] L Maes D Blockmans P Verschueren et al ldquoAnti-PMScl-100 and anti-RNA-polymerase III antibodies in sclerodermardquoClinica Chimica Acta vol 411 no 13-14 pp 965ndash971 2010

[118] M Mahler C Gascon S Patel et al ldquoRpp25 is a major targetof autoantibodies to theThTo complex as measured by a novelchemiluminescent assayrdquo Arthritis Research amp Therapy vol 15article R50 2013

[119] C Bordet ldquoPerformance of the Inno-Lia Ana test in thedetection of the main autoantibodies involved in connectivetissue diseasesrdquo Annales de Biologie Clinique vol 59 no 6 pp767ndash768 2001


[120] S A Lee J Kahng Y Kim et al ldquoComparative studyof immunofluorescent antinuclear antibody test and lineimmunoassay detecting 15 specific autoantibodies in patientswith systemic rheumatic diseaserdquo Journal of Clinical LaboratoryAnalysis vol 26 pp 307ndash314 2012

[121] F J Lopez-Longo M Rodrıguez-Mahou M Escalona-MongeC M Gonzalez I Monteagudo and L Carreno-Perez ldquoSimul-taneous identification of various antinuclear antibodies usingan automated multiparameter line immunoassay systemrdquoLupus vol 12 no 8 pp 623ndash629 2003

[122] D Almeida Gonzalez A Cabrera de Leon M D C RodrıguezPerez et al ldquoEfficiency of different strategies to detect autoanti-bodies to extractable nuclear antigensrdquo Journal of Immunologi-cal Methods vol 360 no 1-2 pp 89ndash95 2010

[123] O Shovman B Gilburd G Zandman-Goddard A YehielyP Langevitz and Y Shoenfeld ldquoMultiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseasesrdquoAutoimmunity vol 38 no 1 pp 105ndash109 2005

[124] J G Hanly L Su V Farewell and M J Fritzler ldquoComparisonbetween multiplex assays for autoantibody detection in sys-temic lupus erythematosusrdquo Journal of Immunological Methodsvol 358 no 1-2 pp 75ndash80 2010

[125] J GHanly KThompson GMcCurdy L Fougere CTheriaultand K Wilton ldquoMeasurement of autoantibodies using multi-plex methodology in patients with systemic lupus erythemato-susrdquo Journal of ImmunologicalMethods vol 352 no 1-2 pp 147ndash152 2010

[126] P Caramaschi O Ruzzenente S Pieropan et al ldquoDeter-mination of ANA specificity using multiplexed fluorescentmicrosphere immunoassay in patients with ANA positivityat high titres after infliximab treatment preliminary resultsrdquoRheumatology International vol 27 no 7 pp 649ndash654 2007

[127] S S Copple T B Martins C Masterson E Joly and H R HillldquoComparison of three multiplex immunoassays for detectionof antibodies to extractable nuclear antibodies using clinicallydefined serardquo Annals of the New York Academy of Sciences vol1109 pp 464ndash472 2007

[128] B Gilburd M Abu-Shakra Y Shoenfeld et al ldquoAutoantibodiesprofile in the sera of patients with sjogrenrsquos syndrome the ANAevaluationmdasha homogeneous multiplexed systemrdquo Clinical andDevelopmental Immunology vol 11 no 1 pp 53ndash56 2004

[129] A-P Nifli G Notas M Mamoulaki et al ldquoComparison of amultiplex bead-based fluorescent assay and immunofluores-cence methods for the detection of ANA and ANCA autoan-tibodies in human serumrdquo Journal of Immunological Methodsvol 311 no 1-2 pp 189ndash197 2006

[130] K Uto N Hayashi S Kinoshita S Kawano and S KumagaildquoEvaluation of simultaneous detection of specific antinuclearantibodies using multiplexed technologyrdquo Rinsho Byori vol 57no 10 pp 941ndash953 2009

[131] M J Fritzler F Behmanesh and M L Fritzler ldquoAnalysis ofhuman sera that are polyreactive in an addressable laser beadimmunoassayrdquo Clinical Immunology vol 120 no 3 pp 349ndash356 2006

[132] S Bernardini M Infantino L Bellincampi et al ldquoScreen-ing of antinuclear antibodies comparison between enzymeimmunoassay based on nuclear homogenates purified orrecombinant antigens and immunofluorescence assayrdquo ClinicalChemistry and Laboratory Medicine vol 42 no 10 pp 1155ndash1160 2004

[133] P M Bayer S Bauerfeind J Bienvenu et al ldquoMulticenterevaluation study on a new HEP2 ANA screening enzyme

immune assayrdquo Journal of Autoimmunity vol 13 no 1 pp 89ndash93 1999

[134] P M Bayer B Fabian and W Hubl ldquoImmunofluores-cence assays (IFA) and enzyme-linked immunosorbent assays(ELISA) in autoimmune disease diagnosticsmdashtechnique ben-efits limitations and applicationsrdquo Scandinavian Journal ofClinical and Laboratory Investigation Supplement vol 61 no235 pp 68ndash76 2001

[135] H-P Lehmann D Block C Markert-Hahn and J W ZolgldquoNew concepts in systemic autoimmunity testingrdquo Scandina-vian Journal of Clinical and Laboratory Investigation Supple-ment vol 61 no 235 pp 84ndash90 2001

[136] H-P Lehmann I Fuhling C Ott B Hudepohl and M HaassldquoHEp2 ANA EIA a new fully automated assay for the screeningof antinuclear antibodiesrdquo Israel Medical Association Journalvol 2 no 8 pp 646ndash648 2000

[137] X Bossuyt ldquoEvaluation of two automated enzyme immunoas-says for detection of antinuclear antibodiesrdquo Clinical Chemistryand Laboratory Medicine vol 38 no 10 pp 1033ndash1037 2000

[138] N Hayashi T Kawamoto M Mukai et al ldquoDetection ofantinuclear antibodies by use of an enzyme immunoassaywith nuclear HEp-2 cell extract and recombinant antigenscomparison with immunofluorescence assay in 307 patientsrdquoClinical Chemistry vol 47 no 9 pp 1649ndash1659 2001

[139] G A Maguire A Ginawi J Lee et al ldquoClinical utility ofANA measured by ELISA compared with ANA measured byimmunofluorescencerdquo Rheumatology vol 48 no 8 pp 1013ndash1014 2009

[140] PGhillani AM Rouquette CDesgruelles et al ldquoEvaluation ofthe LIAISONANA screen assay for antinuclear antibody testingin autoimmune diseasesrdquo Annals of the New York Academy ofSciences vol 1109 pp 407ndash413 2007

[141] L K Peterson D Wells L Shaw M-G Velez R Harbeck andL L Dragone ldquoNovel method for quantitative ANA measure-ment using near-infrared imagingrdquo Journal of ImmunologicalMethods vol 349 no 1-2 pp 1ndash8 2009

[142] K Grossmann D Roggenbuck C Schroder K ConradP Schierack and U Sack ldquoMultiplex assessment of non-organ-specific autoantibodies with a novel microbead-basedimmunoassayrdquo Cytometry A vol 79 no 2 pp 118ndash125 2011

[143] J Smith D Onley C Garey et al ldquoDetermination of ANAspecificity using the UltraPlexŮ platformrdquo Annals of the NewYork Academy of Sciences vol 1050 pp 286ndash294 2005

[144] W H Robinson C DiGennaro W Hueber et al ldquoAutoantigenmicroarrays for multiplex characterization of autoantibodyresponsesrdquo Nature Medicine vol 8 no 3 pp 295ndash301 2002

[145] P J Utz ldquoMultiplexed assays for identification of biomarkersand surrogatemarkers in systemic lupus erythematosusrdquoLupusvol 13 no 5 pp 304ndash311 2004

[146] I Balboni C Limb J D Tenenbaum and P J Utz ldquoEvaluationof microarray surfaces and arraying parameters for autoanti-body profilingrdquo Proteomics vol 8 no 17 pp 3443ndash3449 2008

[147] C Gonzalez B Garcıa-Berrocal M Perez J A Navajo OHerraez and JM Gonzalez-Buitrago ldquoLaboratory screening ofconnective tissue diseases by a new automated ENA screeningassay (EliA Symphony) in clinically defined patientsrdquo ClinicaChimica Acta vol 359 no 1-2 pp 109ndash114 2005

[148] J C Parker and C C Bunn ldquoSensitivity of the Phadia EliAconnective tissue disease screen for less common disease-specific autoantibodiesrdquo Journal of Clinical Pathology vol 64no 7 pp 631ndash633 2011


[149] C Bentow A Seaman A Swart A Shikhman N Goodmanand M and Mahler ldquoEvaluation of a novel chemiluminescentassay (QUANTAFlashCTDScreen Plus) for the rapid detectionof autoantibodies in systemic autoimmune rheumatic diseasesrdquoin Proceedings of the 11th Dresden Symposium onAutoantibodiesDresden Germany 2013

[150] E K L Chan M J Fritzler A Wiik et al ldquoAutoAbSCOrgmdashautoantibody standardization committee in 2006rdquo Autoimmu-nity Reviews vol 6 no 8 pp 577ndash580 2007

[151] X Bossuyt C Louche and A Wiik ldquoStandardisation inclinical laboratorymedicine an ethical reflectionrdquoAnnals of theRheumatic Diseases vol 67 no 8 pp 1061ndash1063 2008

[152] M J Fritzler A Wiik E M Tan et al ldquoA critical evaluation ofenzyme immunoassay kits for detection of antinuclear autoan-tibodies of defined specificities III Comparative performancecharacteristics of academic and manufacturersrsquo laboratoriesrdquoJournal of Rheumatology vol 30 no 11 pp 2374ndash2381 2003

[153] J S Smolen B Butcher M J Fritzler et al ldquoReference serafor antinuclear antibodies II Further definition of antibodyspecificities in international antinuclear antibody referencesera by immunofluorescence and western blottingrdquo Arthritis ampRheumatism vol 40 no 3 pp 413ndash418 1997

[154] E M Tan J S Smolen J S McDougal et al ldquoA critical eval-uation of enzyme immunoassays for detection of antinuclearautoantibodies of defined specificities I Precision sensitivityand specificityrdquoArthritis amp Rheumatology vol 42 pp 455ndash4641999

[155] E M Tan J S Smolen J S McDougal et al ldquoA criticalevaluation of enzyme immunoassay kits for detection of antin-uclear autoantibodies of defined specificities II Potential forquantitation of antibody contentrdquo Journal of Rheumatology vol29 no 1 pp 68ndash74 2002

[156] K James A B Carpenter L Cook R Marchand and R MNakamura ldquoDevelopment of the antinuclear and anticytoplas-mic antibody consensus panel by the Association of MedicalLaboratory Immunologistsrdquo Clinical and Diagnostic LaboratoryImmunology vol 7 no 3 pp 436ndash443 2000

[157] M Mahler M J Fritzler and M Bluthner ldquoIdentification of aSmD3 epitope with a single symmetrical dimethylation of anarginine residue as a specific target of a subpopulation of anti-Sm antibodiesrdquo Arthritis Research amp Therapy vol 7 no 1 ppR19ndashR29 2005

[158] T E W Feltkamp ldquoStandards for ANA and anti-DNArdquo ClinicalRheumatology vol 9 no 1 pp 74ndash81 1990

[159] J Damoiseaux J W C Tervaert R Derksen et al ldquoAutoanti-body standardization in the Netherlands the past the presentand the futurerdquoAnnals of the New York Academy of Sciences vol1173 pp 10ndash14 2009

[160] U Sack K Conrad E Csernok et al ldquoStandardization ofautoimmune diagnostics in Germany activities of the Germangroup in the European autoimmune standardization initiativerdquoAnnals of the New York Academy of Sciences vol 1109 pp 31ndash362007

[161] P L Meroni M Biggioggero S S Pierangeli J Sheldon IZegers and M O Borghi ldquoStandardization of autoantibodytesting a paradigm for serology in rheumatic diseasesrdquo NatureReviews Rheumatology vol 10 pp 35ndash43 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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of


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of



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OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Statistical terms relevant for ANA testing

Statistical measure General explanation Implication for ANA

Sensitivity Statistical measure of how accurately a testcorrectly identifies diseased individuals

ANA is used as screening test High sensitivity isimportant The sensitivity for different AARDvaries (ie higher in systemic lupuserythematosus versus myositis)

Specificity Statistical measure of how well a test correctlyidentifies absence of the disease in question

Importance of specificity depends on pretestprobability In settings with low pretestprobability high specificity is required

Diagnostic efficiency Combination of sensitivity and specificity Not commonly used

False negative (clinically) Negative test result of a diseased individual

ANA is used as screening test False negativeresults are undesirable However in all AARDpatients without a positive ANA test existTherefore a negative result should never be usedto rule out AARD

False positive (clinically) Positive test result of an individual without thedisease in question

In case of low pretest probability false positiveresults significantly impact the posttest probability

False negative (analytically) Negative test result in the presence of therespective analyte See negative positive (clinically)

False positive (analytically) Positive test result in the absence of therespective analyte See false positive (clinically)

Positive predictive value Ratio of true positive to combined true andfalse positives Depends on the prevalence (pretest probability)

Negative predictive value Ratio of true negatives to combined true andfalse negatives Depends on the prevalence (pretest probability)

Positive likelihood ratio

The probability of a positive test results inpatients with the disease divided by theprobability of a positive test result inindividuals without the disease Independentfrom prevalence


Negative likelihood ratio

The probability of a negative test result inpatients with the disease divided by theprobability of a negative test result inindividuals without the disease Independentfrom prevalence


The importance of the likelihood ratio in the laboratory report is controversially discussed but might improve use of ANA test results in the future

a task force who recommended the use of the conventionalIIF HEp-2 platform for ANA detection [12] This recom-mendation was in part based on evidence that the HEp-2 cell substrates are essentially an ldquoarrayrdquo presenting gt100autoantibody targets whereas most high throughput screen-ing arrays are much more limited in autoantibody targetcomposition This has prompted a reevaluation of the ANAIIF method which was reflected by entire sessions dedicatedto HEp-2 ANA testing at international clinical and scientificmeetings

In recent years the first digital imaging systems for ANAIIF have been developed which eliminate some major draw-backs of the method namely the subjectivity of observersreading the slides and the lack of an automated proce-dure [15ndash17] Nevertheless several drawbacks of the HEp-2 IIF methods persist and other technologies for ANAdetection continue to emerge and evolve In this reviewnovel insights and updates on ANA detection are pre-sented and the pros and cons of different methods arediscussed

2 Statistical Considerations

21 Sensitivity and Specificity For diagnostic applicationsit is important to differentiate between analytical sensitiv-ityspecificity and clinical (diagnostic) sensitivityspecificityTherefore the terms clinical sensitivity and specificity falsenegative false positive and predictive values are describedin Table 1 In addition it is widely known and extensivelydocumented that certain autoantibodies can precede thediagnosis or full clinical expression of an underlying diseasefor many years and thus false positive results at a given pointin time might over a subsequent time period become a truepositive [18 19] Consequently the term ldquofalse positiverdquo forautoantibodies needs to be used carefully

22 ROC Analysis and Cut-Off Selection The receiver oper-ating characteristic (ROC) analysis has a broad range ofapplications and was first used for military purposes duringWorld War II [20] In medicine ROC analysis has been


0

02

04

06

08

1

0 02 04 06 08 1

Postt

est p

roba

bilit

y

Pretest probability

IIF(+)IIF(minus)



6

080

008 19 020

42 72 3

87 7 96 21

Pretest probability



IIF(+) IIF(minus)


SPA(+) SPA(minus)

SPA(+)SPA(minus)


























Table3Com


oassaysfor

ANAtesting

Antigen

Automated

screeningassays

Multip

lexassays

Line

immun

oassaysd

otblots

EliA

CTD

EliA

Symph

ony

QUA

NTA

FlashEN

A7

QUA

NTA

Flash

CTDScreen

Plus

Alegria

ANA

Detect

Bio-Plex

2200

ANAALB

IA

AtheNAMulti-Lyte

Anti-N

uclear

Antibod

ies(ANA)

FIDIS

Con

nective

10ANAProfi

leIm

mcoStrip

eANA

Advanced

ANA12

IgG

BlueDot

ANA-

12Pro

(BLO

T)

recomLine

ANAENA

IgG

ANALia

MAX

Thermo

Scientific

Thermo

Scientific

INOVA

Diagn

ostic

sIN

OVA

Diagn

ostic

sOrgentec

Bio-Ra

dZe

usTh

eradiag

Euroim

mun

IMMCO

Diagn

ostic

sD-Tek

AES

KUDiagn

ostic

sMIK

ROGEN

Diagn

ostic

sHUMAN

Diagn

ostic

sU1-R

NP

XX

XX

XX

XX

XX

U1-R

NPSm

XX

XX

XX

U1-R

NP68

kDa

XX

XU1-R

NPA

XX

XU1-R

NPC

XX

XSS-ARo6

0X

XX

XX

X(i)

XX

XX

XX

XX

Ro52TRIM21

XX

XX

XX(

i)X

XX

XX

SS-BLa

XX

XX

XX

XX

XX

XX

XX

Centro

mere

XX

XX

XX

XX

XX

XX

Scl-7

0topo

IX

XX

XX

XX

XX

XX

XX

Jo-1

XX

XX

XX

XX

XX

XX

XX

FibrillarinU

3RNP

XRN

A-Po

lIII

XX

Ribo

somalP

XX

XX

XX

XX

XX

PMScl

XX

XX

PMScl-

75X

PMScl-

100

XX

PCNA

XX

XX

XX

XMi-2

XX

XX

XSm

XX

XX

XX

XX

XX

XdsDNA

XX

XX

XX

XX

XX

Nucleosom

eX

XX

XX

Histon

eX

XX

XX

XX

KuX

XX

XX

SRP5

4X

AMA-

M2

XX

X

Notesomec

ompanies

offer

severallineimmun

oassaysfor

ANAdetection

Mostcom

prehensiv

eassaysfrom

different

companies

ares

hown

AMAA

nti-m

itochon

drialantibod

iesPC

NAP

roliferatingcellnu

cleara

ntigenSRP

signalrecognitio

nparticle

NOTE

RNPandRN

PSm

containthes

ubun

itsRN

P-AR

NP-CandRN

P-68

kDa

(i)Re

ported

outsidethe

UnitedStates

asRo

60andRo

52andas

SS-A

intheU

nitedStates

Con

tainsC

ENP-AandCE

NP-B















































Nuclear





Cytoplasmic


Negative






ble7Au

tomated

digitalA

NAreadingsyste

ms

Instrument

NOVA

View

AKL

IDES

EURO

Patte

rnIm

ageN

avigator

Helios

ZENIT

GSight

Manufacturer

INOVA

diagno

stics

Medipan

Euroim

mun

Immun

ocon

cepts

Aesku

Menarini

LIMSconn

ectio

n(soft

ware)

Yes

(QUA

NTA

Link

)

Yes(syste

mindepend

ent

stand

ardXM

Linterfa

ce)

Yes

(EURO

LabO

ffice)

Yes(direct)

Optionallab

traffi

ccon

trol

Yes(direct)

Optional

AeskuLab

middlew

are

Yes(Ze

nIT)

Slideidentificatio

nviab

arcode

Yesb

yhand

held

scanner

Yesb

yhand

held

scanner

Yesb

yintegrated

scanner

Yes

Yesb

yintegrated

scanner

Yesb

yintegrated

scanner

Loadingcapacity

5slides

(upto

60wells)

5slides

(upto

60wells)

50slides

(upto

500wells)

4slides

(upto

84wells)

20slides

(upto

240wells)

5slides

(upto

70wells)

Imagea

cquisitionspeed

sim45

swellfor

3im

ages

sim40

swell

lt20

swell

sim25

swellfor

4im

ages

10spicture

Custo

mizable

from

1to10

images

gt60

swell

numbero

fpictures5

(smallscan)50

(medium

scan)or

220(fu

llscan)

100

QCforsub

strateandprocess

integrity

cou

ntersta

ining

YesDAPI

YesDAPI

YesProp

idium

iodide

Non

eNon

eNon

eNon

eNon

eNon

e

Automaticpo

snegdiscrim

inationincl

presortin

gof

images

Yes

Yes

Yes

Yes

Yes

Yes

Batchw

iseverifi

catio

nof

negativ

esam

ples

Yes

Yes

Yes

Yes

Yes

Yes

Automaticpatte

rnrecogn

ition

Yes

Yes

Yes

No

No

Yes

Patte

rnAnalysis

metho

dPatte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Nopatte

rnmatching

capabilities

Nopatte

rnmatching

capabilities

Patte

rnrecogn

ition

bymathematical

algorithm

Num

bero

frecognizableA

NAsta

iningpatte

rnlisto

ut

6Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

10Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

8Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Nuclear

rimMixed

patte

rnmito

tic

Non

eNon

e

5Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Cytoplasm

Negative

Analysis

ofmixed

staining

patte

rnLimited

(hom

ogeneous

nucle

olar)

Yes

Yes

No

No

No

Mergedresults

perp

atient

(differentd

ilutio

ns)

Yes

Yes

Yes

Yes

Yes

Yes


ssiblewhilesyste

mprocessesrem

aining

samples

Yes

No

Yes

Yes

Yes

No


nto

minim

izev

ariability

Yes

Yes

Yes

Yes

Yes

No

Integrationwith

slide

processin

gin

1instrum

ent

No

No

No

No

Yes

No











(a)

(b)

(c)















8 Conclusions











References














































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

0 02 04 06 08 1

Postt

est p

roba

bilit

y

Pretest probability

IIF(+)IIF(minus)



6

080

008 19 020

42 72 3

87 7 96 21

Pretest probability



IIF(+) IIF(minus)


SPA(+) SPA(minus)

SPA(+)SPA(minus)


























Table3Com


oassaysfor

ANAtesting

Antigen

Automated

screeningassays

Multip

lexassays

Line

immun

oassaysd

otblots

EliA

CTD

EliA

Symph

ony

QUA

NTA

FlashEN

A7

QUA

NTA

Flash

CTDScreen

Plus

Alegria

ANA

Detect

Bio-Plex

2200

ANAALB

IA

AtheNAMulti-Lyte

Anti-N

uclear

Antibod

ies(ANA)

FIDIS

Con

nective

10ANAProfi

leIm

mcoStrip

eANA

Advanced

ANA12

IgG

BlueDot

ANA-

12Pro

(BLO

T)

recomLine

ANAENA

IgG

ANALia

MAX

Thermo

Scientific

Thermo

Scientific

INOVA

Diagn

ostic

sIN

OVA

Diagn

ostic

sOrgentec

Bio-Ra

dZe

usTh

eradiag

Euroim

mun

IMMCO

Diagn

ostic

sD-Tek

AES

KUDiagn

ostic

sMIK

ROGEN

Diagn

ostic

sHUMAN

Diagn

ostic

sU1-R

NP

XX

XX

XX

XX

XX

U1-R

NPSm

XX

XX

XX

U1-R

NP68

kDa

XX

XU1-R

NPA

XX

XU1-R

NPC

XX

XSS-ARo6

0X

XX

XX

X(i)

XX

XX

XX

XX

Ro52TRIM21

XX

XX

XX(

i)X

XX

XX

SS-BLa

XX

XX

XX

XX

XX

XX

XX

Centro

mere

XX

XX

XX

XX

XX

XX

Scl-7

0topo

IX

XX

XX

XX

XX

XX

XX

Jo-1

XX

XX

XX

XX

XX

XX

XX

FibrillarinU

3RNP

XRN

A-Po

lIII

XX

Ribo

somalP

XX

XX

XX

XX

XX

PMScl

XX

XX

PMScl-

75X

PMScl-

100

XX

PCNA

XX

XX

XX

XMi-2

XX

XX

XSm

XX

XX

XX

XX

XX

XdsDNA

XX

XX

XX

XX

XX

Nucleosom

eX

XX

XX

Histon

eX

XX

XX

XX

KuX

XX

XX

SRP5

4X

AMA-

M2

XX

X

Notesomec

ompanies

offer

severallineimmun

oassaysfor

ANAdetection

Mostcom

prehensiv

eassaysfrom

different

companies

ares

hown

AMAA

nti-m

itochon

drialantibod

iesPC

NAP

roliferatingcellnu

cleara

ntigenSRP

signalrecognitio

nparticle

NOTE

RNPandRN

PSm

containthes

ubun

itsRN

P-AR

NP-CandRN

P-68

kDa

(i)Re

ported

outsidethe

UnitedStates

asRo

60andRo

52andas

SS-A

intheU

nitedStates

Con

tainsC

ENP-AandCE

NP-B















































Nuclear





Cytoplasmic


Negative






ble7Au

tomated

digitalA

NAreadingsyste

ms

Instrument

NOVA

View

AKL

IDES

EURO

Patte

rnIm

ageN

avigator

Helios

ZENIT

GSight

Manufacturer

INOVA

diagno

stics

Medipan

Euroim

mun

Immun

ocon

cepts

Aesku

Menarini

LIMSconn

ectio

n(soft

ware)

Yes

(QUA

NTA

Link

)

Yes(syste

mindepend

ent

stand

ardXM

Linterfa

ce)

Yes

(EURO

LabO

ffice)

Yes(direct)

Optionallab

traffi

ccon

trol

Yes(direct)

Optional

AeskuLab

middlew

are

Yes(Ze

nIT)

Slideidentificatio

nviab

arcode

Yesb

yhand

held

scanner

Yesb

yhand

held

scanner

Yesb

yintegrated

scanner

Yes

Yesb

yintegrated

scanner

Yesb

yintegrated

scanner

Loadingcapacity

5slides

(upto

60wells)

5slides

(upto

60wells)

50slides

(upto

500wells)

4slides

(upto

84wells)

20slides

(upto

240wells)

5slides

(upto

70wells)

Imagea

cquisitionspeed

sim45

swellfor

3im

ages

sim40

swell

lt20

swell

sim25

swellfor

4im

ages

10spicture

Custo

mizable

from

1to10

images

gt60

swell

numbero

fpictures5

(smallscan)50

(medium

scan)or

220(fu

llscan)

100

QCforsub

strateandprocess

integrity

cou

ntersta

ining

YesDAPI

YesDAPI

YesProp

idium

iodide

Non

eNon

eNon

eNon

eNon

eNon

e

Automaticpo

snegdiscrim

inationincl

presortin

gof

images

Yes

Yes

Yes

Yes

Yes

Yes

Batchw

iseverifi

catio

nof

negativ

esam

ples

Yes

Yes

Yes

Yes

Yes

Yes

Automaticpatte

rnrecogn

ition

Yes

Yes

Yes

No

No

Yes

Patte

rnAnalysis

metho

dPatte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Nopatte

rnmatching

capabilities

Nopatte

rnmatching

capabilities

Patte

rnrecogn

ition

bymathematical

algorithm

Num

bero

frecognizableA

NAsta

iningpatte

rnlisto

ut

6Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

10Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

8Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Nuclear

rimMixed

patte

rnmito

tic

Non

eNon

e

5Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Cytoplasm

Negative

Analysis

ofmixed

staining

patte

rnLimited

(hom

ogeneous

nucle

olar)

Yes

Yes

No

No

No

Mergedresults

perp

atient

(differentd

ilutio

ns)

Yes

Yes

Yes

Yes

Yes

Yes


ssiblewhilesyste

mprocessesrem

aining

samples

Yes

No

Yes

Yes

Yes

No


nto

minim

izev

ariability

Yes

Yes

Yes

Yes

Yes

No

Integrationwith

slide

processin

gin

1instrum

ent

No

No

No

No

Yes

No











(a)

(b)

(c)















8 Conclusions











References














































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































Table3Com


oassaysfor

ANAtesting

Antigen

Automated

screeningassays

Multip

lexassays

Line

immun

oassaysd

otblots

EliA

CTD

EliA

Symph

ony

QUA

NTA

FlashEN

A7

QUA

NTA

Flash

CTDScreen

Plus

Alegria

ANA

Detect

Bio-Plex

2200

ANAALB

IA

AtheNAMulti-Lyte

Anti-N

uclear

Antibod

ies(ANA)

FIDIS

Con

nective

10ANAProfi

leIm

mcoStrip

eANA

Advanced

ANA12

IgG

BlueDot

ANA-

12Pro

(BLO

T)

recomLine

ANAENA

IgG

ANALia

MAX

Thermo

Scientific

Thermo

Scientific

INOVA

Diagn

ostic

sIN

OVA

Diagn

ostic

sOrgentec

Bio-Ra

dZe

usTh

eradiag

Euroim

mun

IMMCO

Diagn

ostic

sD-Tek

AES

KUDiagn

ostic

sMIK

ROGEN

Diagn

ostic

sHUMAN

Diagn

ostic

sU1-R

NP

XX

XX

XX

XX

XX

U1-R

NPSm

XX

XX

XX

U1-R

NP68

kDa

XX

XU1-R

NPA

XX

XU1-R

NPC

XX

XSS-ARo6

0X

XX

XX

X(i)

XX

XX

XX

XX

Ro52TRIM21

XX

XX

XX(

i)X

XX

XX

SS-BLa

XX

XX

XX

XX

XX

XX

XX

Centro

mere

XX

XX

XX

XX

XX

XX

Scl-7

0topo

IX

XX

XX

XX

XX

XX

XX

Jo-1

XX

XX

XX

XX

XX

XX

XX

FibrillarinU

3RNP

XRN

A-Po

lIII

XX

Ribo

somalP

XX

XX

XX

XX

XX

PMScl

XX

XX

PMScl-

75X

PMScl-

100

XX

PCNA

XX

XX

XX

XMi-2

XX

XX

XSm

XX

XX

XX

XX

XX

XdsDNA

XX

XX

XX

XX

XX

Nucleosom

eX

XX

XX

Histon

eX

XX

XX

XX

KuX

XX

XX

SRP5

4X

AMA-

M2

XX

X

Notesomec

ompanies

offer

severallineimmun

oassaysfor

ANAdetection

Mostcom

prehensiv

eassaysfrom

different

companies

ares

hown

AMAA

nti-m

itochon

drialantibod

iesPC

NAP

roliferatingcellnu

cleara

ntigenSRP

signalrecognitio

nparticle

NOTE

RNPandRN

PSm

containthes

ubun

itsRN

P-AR

NP-CandRN

P-68

kDa

(i)Re

ported

outsidethe

UnitedStates

asRo

60andRo

52andas

SS-A

intheU

nitedStates

Con

tainsC

ENP-AandCE

NP-B















































Nuclear





Cytoplasmic


Negative






ble7Au

tomated

digitalA

NAreadingsyste

ms

Instrument

NOVA

View

AKL

IDES

EURO

Patte

rnIm

ageN

avigator

Helios

ZENIT

GSight

Manufacturer

INOVA

diagno

stics

Medipan

Euroim

mun

Immun

ocon

cepts

Aesku

Menarini

LIMSconn

ectio

n(soft

ware)

Yes

(QUA

NTA

Link

)

Yes(syste

mindepend

ent

stand

ardXM

Linterfa

ce)

Yes

(EURO

LabO

ffice)

Yes(direct)

Optionallab

traffi

ccon

trol

Yes(direct)

Optional

AeskuLab

middlew

are

Yes(Ze

nIT)

Slideidentificatio

nviab

arcode

Yesb

yhand

held

scanner

Yesb

yhand

held

scanner

Yesb

yintegrated

scanner

Yes

Yesb

yintegrated

scanner

Yesb

yintegrated

scanner

Loadingcapacity

5slides

(upto

60wells)

5slides

(upto

60wells)

50slides

(upto

500wells)

4slides

(upto

84wells)

20slides

(upto

240wells)

5slides

(upto

70wells)

Imagea

cquisitionspeed

sim45

swellfor

3im

ages

sim40

swell

lt20

swell

sim25

swellfor

4im

ages

10spicture

Custo

mizable

from

1to10

images

gt60

swell

numbero

fpictures5

(smallscan)50

(medium

scan)or

220(fu

llscan)

100

QCforsub

strateandprocess

integrity

cou

ntersta

ining

YesDAPI

YesDAPI

YesProp

idium

iodide

Non

eNon

eNon

eNon

eNon

eNon

e

Automaticpo

snegdiscrim

inationincl

presortin

gof

images

Yes

Yes

Yes

Yes

Yes

Yes

Batchw

iseverifi

catio

nof

negativ

esam

ples

Yes

Yes

Yes

Yes

Yes

Yes

Automaticpatte

rnrecogn

ition

Yes

Yes

Yes

No

No

Yes

Patte

rnAnalysis

metho

dPatte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Nopatte

rnmatching

capabilities

Nopatte

rnmatching

capabilities

Patte

rnrecogn

ition

bymathematical

algorithm

Num

bero

frecognizableA

NAsta

iningpatte

rnlisto

ut

6Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

10Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

8Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Nuclear

rimMixed

patte

rnmito

tic

Non

eNon

e

5Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Cytoplasm

Negative

Analysis

ofmixed

staining

patte

rnLimited

(hom

ogeneous

nucle

olar)

Yes

Yes

No

No

No

Mergedresults

perp

atient

(differentd

ilutio

ns)

Yes

Yes

Yes

Yes

Yes

Yes


ssiblewhilesyste

mprocessesrem

aining

samples

Yes

No

Yes

Yes

Yes

No


nto

minim

izev

ariability

Yes

Yes

Yes

Yes

Yes

No

Integrationwith

slide

processin

gin

1instrum

ent

No

No

No

No

Yes

No











(a)

(b)

(c)















8 Conclusions











References














































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































Nuclear





Cytoplasmic


Negative






ble7Au

tomated

digitalA

NAreadingsyste

ms

Instrument

NOVA

View

AKL

IDES

EURO

Patte

rnIm

ageN

avigator

Helios

ZENIT

GSight

Manufacturer

INOVA

diagno

stics

Medipan

Euroim

mun

Immun

ocon

cepts

Aesku

Menarini

LIMSconn

ectio

n(soft

ware)

Yes

(QUA

NTA

Link

)

Yes(syste

mindepend

ent

stand

ardXM

Linterfa

ce)

Yes

(EURO

LabO

ffice)

Yes(direct)

Optionallab

traffi

ccon

trol

Yes(direct)

Optional

AeskuLab

middlew

are

Yes(Ze

nIT)

Slideidentificatio

nviab

arcode

Yesb

yhand

held

scanner

Yesb

yhand

held

scanner

Yesb

yintegrated

scanner

Yes

Yesb

yintegrated

scanner

Yesb

yintegrated

scanner

Loadingcapacity

5slides

(upto

60wells)

5slides

(upto

60wells)

50slides

(upto

500wells)

4slides

(upto

84wells)

20slides

(upto

240wells)

5slides

(upto

70wells)

Imagea

cquisitionspeed

sim45

swellfor

3im

ages

sim40

swell

lt20

swell

sim25

swellfor

4im

ages

10spicture

Custo

mizable

from

1to10

images

gt60

swell

numbero

fpictures5

(smallscan)50

(medium

scan)or

220(fu

llscan)

100

QCforsub

strateandprocess

integrity

cou

ntersta

ining

YesDAPI

YesDAPI

YesProp

idium

iodide

Non

eNon

eNon

eNon

eNon

eNon

e

Automaticpo

snegdiscrim

inationincl

presortin

gof

images

Yes

Yes

Yes

Yes

Yes

Yes

Batchw

iseverifi

catio

nof

negativ

esam

ples

Yes

Yes

Yes

Yes

Yes

Yes

Automaticpatte

rnrecogn

ition

Yes

Yes

Yes

No

No

Yes

Patte

rnAnalysis

metho

dPatte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Nopatte

rnmatching

capabilities

Nopatte

rnmatching

capabilities

Patte

rnrecogn

ition

bymathematical

algorithm

Num

bero

frecognizableA

NAsta

iningpatte

rnlisto

ut

6Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

10Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

8Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Nuclear

rimMixed

patte

rnmito

tic

Non

eNon

e

5Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Cytoplasm

Negative

Analysis

ofmixed

staining

patte

rnLimited

(hom

ogeneous

nucle

olar)

Yes

Yes

No

No

No

Mergedresults

perp

atient

(differentd

ilutio

ns)

Yes

Yes

Yes

Yes

Yes

Yes


ssiblewhilesyste

mprocessesrem

aining

samples

Yes

No

Yes

Yes

Yes

No


nto

minim

izev

ariability

Yes

Yes

Yes

Yes

Yes

No

Integrationwith

slide

processin

gin

1instrum

ent

No

No

No

No

Yes

No











(a)

(b)

(c)















8 Conclusions











References














































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ble7Au

tomated

digitalA

NAreadingsyste

ms

Instrument

NOVA

View

AKL

IDES

EURO

Patte

rnIm

ageN

avigator

Helios

ZENIT

GSight

Manufacturer

INOVA

diagno

stics

Medipan

Euroim

mun

Immun

ocon

cepts

Aesku

Menarini

LIMSconn

ectio

n(soft

ware)

Yes

(QUA

NTA

Link

)

Yes(syste

mindepend

ent

stand

ardXM

Linterfa

ce)

Yes

(EURO

LabO

ffice)

Yes(direct)

Optionallab

traffi

ccon

trol

Yes(direct)

Optional

AeskuLab

middlew

are

Yes(Ze

nIT)

Slideidentificatio

nviab

arcode

Yesb

yhand

held

scanner

Yesb

yhand

held

scanner

Yesb

yintegrated

scanner

Yes

Yesb

yintegrated

scanner

Yesb

yintegrated

scanner

Loadingcapacity

5slides

(upto

60wells)

5slides

(upto

60wells)

50slides

(upto

500wells)

4slides

(upto

84wells)

20slides

(upto

240wells)

5slides

(upto

70wells)

Imagea

cquisitionspeed

sim45

swellfor

3im

ages

sim40

swell

lt20

swell

sim25

swellfor

4im

ages

10spicture

Custo

mizable

from

1to10

images

gt60

swell

numbero

fpictures5

(smallscan)50

(medium

scan)or

220(fu

llscan)

100

QCforsub

strateandprocess

integrity

cou

ntersta

ining

YesDAPI

YesDAPI

YesProp

idium

iodide

Non

eNon

eNon

eNon

eNon

eNon

e

Automaticpo

snegdiscrim

inationincl

presortin

gof

images

Yes

Yes

Yes

Yes

Yes

Yes

Batchw

iseverifi

catio

nof

negativ

esam

ples

Yes

Yes

Yes

Yes

Yes

Yes

Automaticpatte

rnrecogn

ition

Yes

Yes

Yes

No

No

Yes

Patte

rnAnalysis

metho

dPatte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Patte

rnrecogn

ition

bymathematical

algorithm

Nopatte

rnmatching

capabilities

Nopatte

rnmatching

capabilities

Patte

rnrecogn

ition

bymathematical

algorithm

Num

bero

frecognizableA

NAsta

iningpatte

rnlisto

ut

6Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

10Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Positive

unrecogn

ized

8Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Nuclear

dot

Cytoplasm

Negative

Nuclear

rimMixed

patte

rnmito

tic

Non

eNon

e

5Hom

ogeneous

Speckled

Centro

mere

Nucleolar

Cytoplasm

Negative

Analysis

ofmixed

staining

patte

rnLimited

(hom

ogeneous

nucle

olar)

Yes

Yes

No

No

No

Mergedresults

perp

atient

(differentd

ilutio

ns)

Yes

Yes

Yes

Yes

Yes

Yes


ssiblewhilesyste

mprocessesrem

aining

samples

Yes

No

Yes

Yes

Yes

No


nto

minim

izev

ariability

Yes

Yes

Yes

Yes

Yes

No

Integrationwith

slide

processin

gin

1instrum

ent

No

No

No

No

Yes

No











(a)

(b)

(c)















8 Conclusions











References














































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























(a)

(b)

(c)















8 Conclusions











References














































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























8 Conclusions











References














































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















References














































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Review Article Current Concepts and Future Directions for the...

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