Evaluating Alpha-1 Antitrypsin Deficiency testing across Europe to ensure diagnostic accuracy: an EARCO multicentre...

Alpha-1 Antitrypsin Deficiency (AATD) is a rare, underdiagnosed genetic disorder caused by pathogenic variants of the SERPINA1 gene, which can lead to reduced levels and/or dysfunctional forms of the Alpha-1 Antitrypsin (AAT) protein. Accurate AATD diagnosis is critical due to its implications for pulmonary and hepatic health. However, diagnostic practices vary across laboratories, particularly in the detection and reporting of rare variants. This study aimed to evaluate the consistency and reliability of AATD diagnostic testing across European laboratories through a non-commercial external quality assessment (EQA)-like initiative.

Seven European laboratories participated in six assessment rounds between 2019 and 2021. Each round involved blinded analysis of serum and dried blood spot (DBS) samples using local standard operating procedures. Tests included AAT and C-reactive protein quantification, isoelectric focusing for AAT phenotyping, and genotyping/sequencing for common and rare SERPINA1 variants. Interrater agreement was assessed using Fleiss' kappa statistic. Allele error rates were calculated for each variant and Kruskal-Wallis tests were used to compare AAT concentrations across laboratories.

For common variants (M [normal], S [p.E288V], and Z [p.E366K]), interlaboratory concordance was high, with Fleiss' kappa values indicating almost perfect agreement (0.85-1.00). Genotyping and sequencing error rates for these variants were zero. However, detection of rare variants (e.g., M_Procida [p.L65P], M_Heerlen [p.P393L], and M_Wurzburg [p.P393S]) was inconsistent, with error rates ranging from 0.14 to 0.29. Sequencing was more effective than phenotyping or simple genotyping for rare variant identification. AAT concentration measurements were generally consistent across laboratories, though DBS samples showed more variability. Reporting nomenclature also varied widely, highlighting the need for standardisation.

This initiative demonstrated strong interlaboratory agreement in detecting common AATD variants but revealed challenges in identifying and reporting rare variants. The findings support the need for SERPINA1 sequencing in AATD diagnostics and the adoption of standardised nomenclature. Centralised testing in validated reference laboratories is also recommended to ensure diagnostic accuracy and consistency across Europe.