Current practices in antinuclear antibody testing: results from the Belgian External Quality Assessment Scheme

Blerk, Marjan Van; Campenhout, C M Van; Bossuyt, Xavier; Duchateau, Jean; Humbel, R; Servais, G.; Tomasi, Jean-Paul; Albert, Adelin; Coucke, Wim; Libeer, Jean-Claude Jc

doi:10.1515/cclm.2009.021

Cited by 26 publications

(16 citation statements)

References 35 publications

(42 reference statements)

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“…Recently, ANA detection has been confirmed as the first level test for the serology of SARD by an international expert panel [1]. As a matter of fact, classical ANA testing requires the characterization of positive ANA findings by immunoassays for the detection of specific autoAbs to nuclear and cytoplasmic autoantigenic targets in the framework of the recommended two-tier approach [30][31][32]. However, this diagnostic two-step process has proved to be laborious and created constraints in particular for larger laboratories with high throughput [16,30].…”

Section: Discussionmentioning

confidence: 99%

Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing

Scholz

Großmann

Knütter

et al. 2015

Clinical Chemistry and Laboratory Medicine (CCLM)

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Background: For the serological diagnosis of systemic autoimmune rheumatic diseases, a two-tier approach starting with sensitive antinuclear antibody (ANA) detection by indirect immunofluorescence (IIF) on HEp-2 cells followed by characterization of positive findings with different immunoassays is recommended. To overcome drawbacks of this approach, we developed a novel technique allowing the combination of screening and simultaneous confirmatory testing. For the first time, this creates the basis for second generation ANA testing. Methods: ANA and autoantibodies (autoAbs) to doublestranded DNA (dsDNA), CENP-B, SS-A/Ro52, SS-A/Ro60, SS-B/La, RNP-Sm, Sm, and Scl-70 were determined by IIF and enzyme-linked immunosorbent assay (ELISA), respectively, and compared to simultaneous analysis

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Section: Discussionmentioning

confidence: 99%

Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing

Scholz

Großmann

Knütter

et al. 2015

Clinical Chemistry and Laboratory Medicine (CCLM)

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“…Indirect IIF on human epidermoid laryngeal carcinoma (HEp-2) cells is the preferred method for the screening of ANA within the reco mmended two-stage diagnostic strategy for systemic autoimmune rheumatic diseases (SARD) [9]. Due to the unsurpassed sensitivity of ANA IIF, this method is the ideal diagnostic tool for the screening stage followed by confirmatory testing with different immunological assay technologies [10].Despite the controversial discussion on the need of quantitative ANA results in clinical practice, the determination of endpoint titers is offered by the majority of routine autoimmune laboratories for the serological diagnosis of SARD. In general, endpoint titers higher than 160 are scored positive and respective sera are recommended to be further diluted for their endpoint titer quantification.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Digital immunofluorescence enables automated detection of antinuclear antibody endpoint titers avoiding serial dilution

Roggenbuck¹,

Hiemann

Schierack

et al. 2014

Clinical Chemistry and Laboratory Medicine (CCLM)

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We noticed with great interest the study by Bonroy et al. reporting the evaluation of antinuclear antibody (ANA) detection by an automated interpretation system for indirect immunofluorescence (IIF) images [1]. We would like to comment on their intriguing data regarding the quantification of ANA titer levels based on our experience with the first commercially available automated IIF system AKLIDES ® . We have a long lasting interest in the development of quantification methods for autoantibody assessment by cell-based IIF tests and we generated a software module for the determination of ANA endpoint titers [2,3].With the development of digital IIF reading and novel pattern recognition algorithms, a new era in autoantibody diagnostics by IIF has been ushered in, overcoming such shortcomings like human bias in IIF reading and laborious data management [2,4]. Quantification of fluorescence signals equals IIF testing with other quantitative methods used in autoimmune diagnostics such as enzyme immunoassays. Thus, automated interpretation of cellbased fluorescence tests has become available addressing the rising demand for the standardized assessment of autoantibodies by IIF in routine laboratories [5][6][7][8]. Indirect IIF on human epidermoid laryngeal carcinoma (HEp-2) cells is the preferred method for the screening of ANA within the reco mmended two-stage diagnostic strategy for systemic autoimmune rheumatic diseases (SARD) [9]. Due to the unsurpassed sensitivity of ANA IIF, this method is the ideal diagnostic tool for the screening stage followed by confirmatory testing with different immunological assay technologies [10].Despite the controversial discussion on the need of quantitative ANA results in clinical practice, the determination of endpoint titers is offered by the majority of routine autoimmune laboratories for the serological diagnosis of SARD. In general, endpoint titers higher than 160 are scored positive and respective sera are recommended to be further diluted for their endpoint titer quantification. Endpoint titers should not to be reported higher than 5120 [9].For the first time in ANA IIF reading, digital IIF offers the opportunity to quantify ANA titers by using just one sample dilution. This decreases the hands-on time for an autoantibody-positive sample dramatically avoiding further dilution steps. Bonroy et al. demonstrated the usefulness of this new approach with a novel commercially available interpretation system for the detection of a probability index (PI) [1]. The authors report a significant correlation of the PI with the classical endpoint titer. However, there appears to be no statistical difference between the PI of positive samples with a classical titer of 320 and those with 640. The same phenomenon was reported for samples with classical titers of 1280 compared to samples with titers higher than 1280 hinting to a non-linear dependency.We fully concur with the author's view that quantification of ANA testing can be achieved with automated IIF reading. According to our experience, ...

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“…Sharp-Syndrom). Diese Systemerkrankungen sind charakterisiert durch die Produktion zahlreicher nicht-organspezifischer, vorwiegend antinukleärer (ANA) aber auch antizytoplasmatischer Antikör-per, welche mit Ausnahme einiger Myositis-spezifischer AAK mittels Immunfluoreszenzscreening an HEp-2-Zellen erfasst werden [23][24][25][26][27]. In Abhängigkeit von klinischer Fragestellung und Fluoreszenzmuster an HEp-2-Zellen erfolgt danach die Bestimmung der entsprechend relevanten AAK mittels spezifischer Immunoassays.…”

Section: Das Prinzip: Screening Und Bestätigungunclassified

Der CytoBead-Assay – Eine neue Möglichkeit der multiparametrischen Autoantikörperanalytik bei systemischen Autoimmunerkrankungen

Sowa¹,

Großmann

Scholz

et al. 2014

LaboratoriumsMedizin

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ZusammenfassungBei Verdacht auf Vorliegen einer systemischen Autoimmunerkrankung wird für die serologische Routinediagnostik ein Zwei-Stufen-Verfahren empfohlen. Zuerst werden Autoantikörpern (AAK) mittels sensitiver zellbasierter indirekter Immunfluoreszenz (IIF)-Teste bestimmt. Ein positives Ergebnis muss aufgrund der Möglichkeit von falsch-positiven Ergebnissen mit einem weiteren, spezifischen Test bestätigt werden. Dieses sukzessive Vorgehen ist notwendig, da zurzeit keine Assaytechnik die notwendigen Anforderungen an ein einstufiges Verfahren hinsichtlich Sensitivität und Spezifität erfüllt. Im Sinne einer effektiven AAK-Diagnostik kann heute schon eine simultane Bestimmung von mehreren AAK mittels multiparametrischer Bestätigungstests die Diagnosefindung im Vergleich zu konventionellen, monoparametrischen Tests wesentlich verkürzen. Jedoch erlauben die verfügbaren multiparametrischen AAK-Nachweismethoden nicht die Kombination von Screening- und Bestätigungstesten. Deshalb wurde basierend auf der digitalen Fluoreszenz mit der hier vorgestellten CytoBead Technologie ein neuer Ansatz entwickelt. Ziel war die Kombination der empfohlenen Stufendiagnostik bestehend aus sensitivem Screening und spezifischer Bestätigungsdiagnostik in einer Reaktionsumgebung und darüber hinaus die Möglichkeit der Adaption auf die serologische Diagnostik mehrerer Autoimmunerkrankungen. Durch a) die Nutzung von Standardglasobjektträgern, b) die Kombination von nativen zellulären oder Gewebesubstraten mit antigenbeladenen fluoreszierenden Mikropartikeln (Beads) in einer Reaktionsumgebung, c) die Möglichkeit der manuellen und automatischen Auswertung mittels IIF und d) die Erhebung von quantitativen Fluoreszenzmessergebnissen konnten die Nachteile der bisher bestehenden Testsysteme überwunden werden. Das neue Prinzip ist auf verschiedene multiparametrische AAK-Nachweise wie zum Beispiel die Bestimmung von antinukleären Antikörpern und AAK gegen entsprechende nukleäre und zytoplasmatische autoantigene Zielstrukturen anwendbar. Damit wurde weiterhin die Basis für die simultane AAK-Multiparameterbestimmung für die Serologie der Zöliakie und von ANCA-assoziierten systemischen Vaskulitiden geschaffen.

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Current practices in antinuclear antibody testing: results from the Belgian External Quality Assessment Scheme

Cited by 26 publications

References 35 publications

Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing

Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing

Digital immunofluorescence enables automated detection of antinuclear antibody endpoint titers avoiding serial dilution

Der CytoBead-Assay – Eine neue Möglichkeit der multiparametrischen Autoantikörperanalytik bei systemischen Autoimmunerkrankungen

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