Inter-laboratory validation of a harmonized PNH flow cytometry assay

Area of Science:

• Clinical diagnostics
• Hematology
• Flow cytometry

Background:

• A network of 11 laboratories aimed to standardize paroxysmal nocturnal hemoglobinuria (PNH) testing.
• Previous guidelines and studies informed the development and validation of a novel PNH assay.

Purpose of the Study:

• To develop and validate a robust, network-endorsed assay for PNH detection.
• To improve the precision and reliability of PNH testing across multiple laboratories.

Main Methods:

• A network analysis of over 20 samples was conducted to refine the assay protocol.
• Twenty-seven native samples underwent blind testing across participating laboratories, with central re-analysis.
• Assay validation included linearity, precision testing, and determination of assay limits (blank, detection, quantification).

Main Results:

• The final PNH assay protocol demonstrated comparable cell enumeration across laboratories, with no significant discrepancies.
• Biological sample characteristics influenced precision, necessitating adjustments like a red cell gating strategy to eliminate false positives.
• The validated assay achieved a Limit of Quantification of 0.01% for neutrophils and red cells, and 0.1% for monocytes.

Conclusions:

• A robust, network-validated PNH assay has been established, suitable for adoption by other laboratories.
• This assay facilitates high-resolution PNH analysis, enhancing diagnostic capabilities.