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Multicenter validation of a simplified method for paroxysmal nocturnal hemoglobinuria screening
Arianna Gatti1, Luigi Del Vecchio2, Massimo Geuna3
1Haematology Laboratory and Transfusion Center, Western Milan Area Hospital Consortium, Legnano General Hospital, Legnano(Milano), Italy.
European Journal of Haematology
|March 24, 2017
Summary
A new, simplified two-color assay effectively screens for paroxysmal nocturnal hemoglobinuria (PNH) clones. This cost-effective method demonstrates comparable precision, sensitivity, and specificity to complex six-color assays for PNH diagnosis.
Area of Science:
- Hematology
- Clinical Diagnostics
- Flow Cytometry
Background:
- Current Paroxysmal Nocturnal Hemoglobinuria (PNH) diagnostic guidelines involve complex, multi-color flow cytometry assays.
- Screening for PNH can be challenging and costly due to low clone detection rates in at-risk patients.
Purpose of the Study:
- To validate a simplified, one-tube, two-color FLAER-based assay for PNH screening.
- To assess the feasibility and effectiveness of this simplified assay in a multicenter setting.
Main Methods:
- A two-color (FLAER/CD15) assay was compared against a six-color assay in six laboratories.
- Standardized protocols, calibration, and analysis were used across all sites.
- Sensitivity, specificity, and precision were tested using patient samples and normal donors.
Main Results:
- The two-color assay showed excellent agreement with the six-color assay for granulocyte PNH clones.
- Sensitivity reached 0.01% for granulocytes and 1% for monocytes.
- Specificity testing identified potential artifacts from basophils and platelets in monocyte gates.
Conclusions:
- A simplified two-color FLAER/CD15 assay is a validated, feasible, and effective tool for PNH screening.
- The simplified assay offers comparable performance to more complex methods for granulocytes, suitable for peripheral laboratories.
- Reference centers should still perform definitive PNH diagnosis using established techniques across all cell lineages.
