How should I validate my high-sensitivity PNH assay?

The careful screening and selection of specific antibody clones and conjugates has allowed the development of reagent cocktails which are suitable for accurate and reliable high sensitivity flow cytometric detection of PNH RBCs and PNH WBCs. However, a thorough validation of all steps of a PNH assay is necessary in order to accurately and reliably identify and quantify small and large PNH clones.

 Instrument optimization is key and it is important to realize that although FLAER expression is seen in the same PMT as FITC, the compensation is different and typical leukemia/lymphoma settings may not be optimal for a PNH WBC assay. The same applies for the PNH RBC assay which needs assay specific voltage and compensation settings. Once the instrument settings are optimized, a well-designed template should be set up to include time as a parameter, allow for lineage-specific gating, the use of internal controls to check for expected antibody/instrument performance.

 Normal patients may be used to verify that the "PNH gate" does not contain any background events and spiked PNH patient cells may be used to verify the identification of PNH clone and determine sensitivity/lower limit of quantification (LLOQ) of the assay. For labs setting up this assay that do not have access to a PNH sample, one way to check or verify instrument/compensation settings is to perform "fluorescence minus two" staining in which a normal sample is only stained with the gating antibodies (e.g. CD15 and CD45 for granulocytes) but not the GPI-linked markers (e.g. FLAER and CD24 for granulocytes). These cells appear as PNH cells (as they are not stained with the GPI markers and can be used to visualize the location of PNH cells on the dot plot. An optimized PNH assay will allow accurate assessment of large as well as minor PNH clones down to a sensitivity of 0.01%. The laboratory should validate the sensitivity of their assay using dilution studies of patient samples or “fluorescence minus two” controls.

Further Reading:
 1. Sutherland DR, Keeney M, Illingworth A (2012) Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry B Clin Cytom. 82:195-208.