Analytical aspects of the antinuclear antibody test by HEp-2 indirect immunofluorescence: EFLM report on an international survey

Author:

Vercammen Martine12ORCID,Bonroy Carolien34ORCID,Broeders Sylvia5,Chan Edward K.L.6,Bizzaro Nicola7,Bogdanos Dimitrios P.89,Andrade Luis1011ORCID,Coucke Wim5ORCID,de Melo Cruvinel Wilson12,Kozmar Ana13,Kuhi Liisa14,Lutteri Laurence15ORCID,Rego de Sousa Maria Jose16,Schouwers Sofie17ORCID,Van Hoovels Lieve181920ORCID,Bossuyt Xavier1920

Affiliation:

1. Department of Laboratory Medicine , AZ St-Jan Hospital Bruges-Ostend , Bruges , Belgium

2. Reproductive Immunology and Transplantation , Vrije Universiteit Brussel , Brussels , Belgium

3. Department of Laboratory Medicine , University Hospital Ghent , Ghent , Belgium

4. Department of Diagnostic Sciences , Ghent University , Ghent , Belgium

5. Sciensano , Quality of Laboratories , Brussels , Belgium

6. Department of Oral Biology , University of Florida , Gainesville , FL , USA

7. Laboratory of Clinical Pathology , Azienda Sanitaria Universitaria Integrata di Udine , Tolmezzo , Italy

8. Department of Rheumatology and Clinical Immunology, Faculty of Medicine, School of Health Sciences , University of Thessaly , Volos , Greece

9. University General Hospital of Larissa , Larissa , Greece

10. Rheumatology Division, Escola Paulista de Medicina , Universidade Federal de Sao Paulo , Sao Paulo , Brazil

11. Immunology Division , Fleury Medicine and Health Laboratories , Sao Paulo , Brazil

12. Imunologia , Pontificia Universidade Catolica de Goias , Goiânia , Brazil

13. Department of Laboratory Diagnostics , University Hospital Centre Zagreb , Zagreb , Croatia

14. Central Laboratory , East Tallinn Central Hospital , Tallinn , Estonia

15. Department of Clinical Chemistry , CHU Sart-Tilman , Liège , Belgium

16. Laboratory Medicine , Centro de Medicina Laboratorial Germano de Sousa , Lisboa , Portugal

17. Department of Laboratory Medicine , GZA Hospital , Antwerp , Belgium

18. Department of Laboratory Medicine , OLV Hospital Aalst , Aalst , Belgium

19. Clinical and Diagnostic Immunology, Department of Microbiology, Immunology and Transplantation , KU Leuven , Leuven , Belgium

20. Department of Laboratory Medicine , UZ Leuven , Leuven , Belgium

Abstract

Abstract Objectives Detection of antinuclear antibodies (ANA) by indirect immunofluorescence assay using HEp-2 cells (HEp-2 IFA) is used to screen for various autoimmune diseases. HEp-2 IFA suffers from variability, which hampers harmonization. Methods A questionnaire was developed to collect information on HEp-2 IFA methodology, computer-assisted diagnosis (CAD) systems, training, inter-observer variability, quality assessment, reagent lot change control, and method verification. The questionnaire was distributed to laboratories by Sciensano (Belgium), national EASI groups (Italy, Croatia, Portugal, Estonia, Greece) and ICAP (worldwide). Answers were obtained by 414 laboratories. The results were analysed in the framework of the recent EFLM/EASI/ICAP ANA recommendations (companion paper). Results Laboratories used either HEp-2, HEp-2000, or HEp-20-10 cells and most laboratories (80%) applied the same screening dilution for children and adults. The conjugate used varied between laboratories [IgG-specific (in 57% of laboratories) vs. polyvalent]. Sixty-nine percent of CAD users reviewed the automatic nuclear pattern and 53% of CAD users did not fully exploit the fluorescence intensity for quality assurance. Internal quality control was performed by 96% of the laboratories, in 52% of the laboratories only with strongly positive samples. Interobserver variation was controlled by 79% of the laboratories. Limited lot-to-lot evaluation was performed by 68% of the laboratories. Method verification was done by 80% of the respondents. Conclusions Even though many laboratories embrace high-quality HEp-2 IFA, substantial differences in how HEp-2 IFA is performed and controlled remain. Acting according to the EFLM/EASI/ICAP ANA recommendations can improve the global performance and quality of HEp-2 IFA and nurture harmonization.

Publisher

Walter de Gruyter GmbH

Subject

Biochemistry (medical),Clinical Biochemistry,General Medicine

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