Multicenter validation of a simplified method for paroxysmal nocturnal hemoglobinuria screening.

Background: Paroxysmal nocturnal hemoglobinuria (PNH) diagnostic guidelines recommend single-tube five- to six-color or two-tube four-color assays. PNH clones are detectable in only a fraction of patients at risk, and screening for new PNH cases can be complex and expensive. In this multicenter study, we have validated a simplified, one-tube two-color FLAER-based assay suitable for PNH screening.

Methods: Six laboratories received samples containing spiked PNH leukocyte clones to be analyzed in parallel with a common six-color cocktail (FLAER/CD24/CD45/CD64/CD15/CD14) and a simplified two-color mixture (FLAER/CD15), a shared calibration procedure, and a common analysis protocol. Replicate precision and sensitivity tests were performed on PNH patients, from undiluted to 1:10 000. Specificity tests were performed on normal donors to identify the possible sources of artifacts.

Results: The performance comparison between six-color and two-color assays showed an excellent agreement for granulocyte PNH clones. Dilution experiments showed an accurate detectability down to 0.01% sensitivity level for granulocyte PNH clones and to 1% for monocytes. Specificity experiments disclosed that basophils and platelets can contaminate the monocyte gate and generate false PNH events.

Conclusions: A simplified two-color (FLAER/CD15) PNH screening test has been validated in a highly standardized multicenter study and proved feasible and effective in ongoing regional programs. Precision, sensitivity, and specificity of the simplified test for granulocytes were comparable to the more complex and expensive six-color assay and applicable for screening also in peripheral laboratories. The diagnostic confirmation of PNH should be always performed by a reference center using the established technique on all cell lineages.