Clinical evaluation of hsPR3-ANCA ELISA for detection of antineutrophil cytoplasmatic antibodies directed against proteinase 3

Holle, Julia; Csernok, Elena; Fredenhagen, G.; Backes, Monika; Bremer, Jan Phillip; Gross, Wolfgang L.

doi:10.1136/ard.2009.109868

Cited by 24 publications

(13 citation statements)

References 4 publications

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“…Their over the past years continuously improved performance, particularly regarding the analysis of PR3-ANCA, have questioned the usefulness of IIF for ANCA testing [24], [25], [36]. Indeed, the need to run two different assay techniques in the recommended two-tier algorithm increases the workload in an already limited in capacity autoimmune laboratory.…”

Section: Discussionmentioning

confidence: 99%

“…PR3- and MPO-ANCA were detected using commercially available antigen-specific ELISAs according to instructions of the manufacturers (GA Generic Assays GmbH, Dahlewitz, Germany; Orgentec GmbH, Wiesbaden, Germany) as described elsewhere [24], [25]. The PR3- and MPO-ANCA ELISAs of GA Generic Assays GmbH revealed intra-assay variabilities of 5.2% each and inter-assay variabilities of 6.2% each for a serum with 20.0 U/mL PR3-ANCA and 20.0 U/mL MPO-ANCA, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The concept of the fully automated interpretation system Aklides for evaluation of ANCA IIF patterns is based on novel mathematical software algorithms for pattern recognition [25], [26]. To obtain a reproducible IIF read out signal, the excitation light intensity was calibrated employing a recently developed calibration tool [20].…”

Section: Methodsmentioning

confidence: 99%

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Simultaneous Automated Screening and Confirmatory Testing for Vasculitis-Specific ANCA

et al. 2014

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Anti-neutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of small vessel vasculitis, so called ANCA-associated vasculitis. The international consensus requires testing by indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils (ethN) as screening followed by confirmation with enzyme-linked immunosorbent assays (ELISAs). This study evaluates the combination of cell- and microbead-based digital IIF analysis of ANCA in one reaction environment by the novel multiplexing CytoBead technology for simultaneous screening and confirmatory ANCA testing. Sera of 592 individuals including 118 patients with ANCA-associated vasculitis, 133 with rheumatoid arthritis, 49 with infectious diseases, 77 with inflammatory bowel syndrome, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis and 125 blood donors were tested for cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA) by classical IIF and ANCA to proteinase 3 (PR3) and myeloperoxidase (MPO) by ELISA. These findings were compared to respective ANCA results determined by automated multiplex CytoBead technology using ethN and antigen-coated microbeads for microbead immunoassays. There was a good agreement for PR3- and MPO-ANCA and a very good one for P-ANCA and C-ANCA by classical and multiplex analysis (Cohen's kappa [κ] = 0.775, 0.720, 0.876, 0.820, respectively). The differences between classical testing and CytoBead analysis were not significant for PR3-ANCA, P-ANCA, and C-ANCA (p<0.05, respectively). The prevalence of confirmed positive ANCA findings by classical testing (IIF and ELISA) compared with multiplex CytoBead analysis (IIF and microbead immunoassay positive) resulted in a very good agreement (κ = 0.831) with no significant difference of both methods (p = 0.735). Automated endpoint-ANCA titer detection in one dilution demonstrated a very good agreement with classical analysis requiring dilution of samples (κ = 0.985). Multiplexing by CytoBead technology can be employed for simultaneous screening and quantitative confirmation of ANCA. This novel technique provides fast and cost-effective ANCA analysis by automated digital IIF for the first time.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Simultaneous Automated Screening and Confirmatory Testing for Vasculitis-Specific ANCA

et al. 2014

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“…Therefore, a positive test result of a screening assay-has to be confirmed with a specific immunoassay. The confirmation assay has a significantly higher diagnostic specificity and a higher positive predictive value than the screening assay, but does not rule out falsepositive findings completely [35,36] . In the case of emergency situations like ANCA-associated vasculitis involving the kidneys, a maximum diagnostic confidence is needed which requires screening for C/P-ANCA via IIF on neutrophil granulocytes along with the specific determination of myeloperoxidase (MPO)-and proteinase 3 (PR3)-ANCA [1,4,33] .…”

Section: The Principle: Screening and Confirmationmentioning

confidence: 93%

The CytoBead assay – a novel approach of multiparametric autoantibody analysis in the diagnostics of systemic autoimmune diseases

Sowa¹,

Großmann

Scholz³

et al. 2015

LaboratoriumsMedizin

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If there is a suspicion of a systemic autoimmune disease, a two-step assessment of autoantibodies (AAb) is recommended for the serological diagnosis thereof. First, AAb will be determined using sensitive, cell-based indirect immunofluorescence. Then, a positive result must be confirmed with a more specific test due to the possibility of false-positive results. This gradual approach is necessary because there is currently no assay technique that fulfills the requirements for a one-stage procedure for sensitivity and specificity. For effective AAb analysis, simultaneous determination of several AAb with multiparametric confirmatory assays significantly shortens serological diagnosis, compared with conventional monoparametric testing. Yet, currently available multiparametric AAb detection techniques do not offer the combination of screening and confirmatory testing. Thus, a new approach based on digital fluorescence was developed by applying a novel CytoBead technology that is presented here. The aim was to combine the recommended stepwise approach consisting of sensitive screening and confirmation of specific diagnosis in a reaction environment and thereafter the possibility of adaptation to the serological diagnosis of several autoimmune diseases. Using standard microscopic glass slides and the combination of native cellular or tissue substrates with autoantigen-loaded fluorescent microparticles (beads) in a reaction environment, along with the possibility of manual and automatic evaluation by IIF and the quantitative measurement of fluorescent signals, the disadvantages of currently existing test systems could be overcome. This novel concept is applicable for the determination of various multiparametric AAb, e.g., the determination of antinuclear antibodies and the corresponding AAb in molecular cytoplasmic and nuclear autoantigenic structures. Further, this becomes the basis for the simultaneous multiparametric AAb determination for the serology of celiac disease or ANCA-associated vasculitides.

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“…Bei einem positiven Testergebnis mittels Screeningassay erfolgt die Bestätigung der Verdachtsdiagnose mit einem spezifischem Immunoassay. Der Bestätigungstest besitzt eine wesentlich höhere diagnostische Spezifität und einen höheren positiven prädiktiven Wert als der Suchtest, schließt jedoch falsch positive Befunde nicht aus [35,36]. In der Notfalldiagnostik bei Nierenmanifestation ANCA-assoziierter Vaskulitiden ist daher im Interesse einer höchstmöglichen diagnostischen Sicherheit das Screening auf C-/P-ANCA mittels IIF an neutrophilen Granulozyten parallel mit der spezifischen Bestimmung von Myeloperoxidase (MPO)-und Proteinase 3 (PR3)-ANCA gefordert [1,4,33].…”

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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Clinical evaluation of hsPR3-ANCA ELISA for detection of antineutrophil cytoplasmatic antibodies directed against proteinase 3

Cited by 24 publications

References 4 publications

Simultaneous Automated Screening and Confirmatory Testing for Vasculitis-Specific ANCA

Simultaneous Automated Screening and Confirmatory Testing for Vasculitis-Specific ANCA

The CytoBead assay – a novel approach of multiparametric autoantibody analysis in the diagnostics of systemic autoimmune diseases

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

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