**4.1. Accuracy of PNH assays**

Type III RBCs. Type I RBCs are normal red blood cells with bright CD59 expression and a lifespan of approximately 120 days. Type III PNH RBCs have complete CD59 deficiency, which results in no protection from complement-mediated lysis and a shortened lifespan of 10–15 days. Type II PNH RBCs have partial CD59 deficiency resulting in partial protection from complement-mediated lysis. Just as the expression of CD59 on Type II RBCs varies considerably from patient to patient, the lifespan of Type II cells reflects this being intermediate between Type I normal RBCs and Type III PNH RBCs. Since the clinical significance of Type II PNH RBCs and Type III PNH RBCs is well established, it is recom-

mended to report them separately and combined as the total PNH RBC clone.

**Figure 4.** Example of CD157-negative, non-PNH case, 7-color FLAER/CD24/CD14/CD157-based protocol.

10 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

**3.** PNH clone size in both lineages for the WBCs (neutrophils and monocytes). The PNH monocyte clone is often larger than the neutrophil PNH clone and reporting only the PNH neutrophil/granulocyte clone may underestimate the PNH clone size in the WBCs. Neutrophils The accuracy of a measurement is described by its trueness, which refers to the closeness of agreement between the average value of a large number of test results and the true or accepted reference value [25]. For PNH assays, we do not have cellular reference standard, therefore accuracy cannot be determined directly. Alternatively, interlaboratory comparison and/or external quality assessment represent the only available option for assay validation and mandatory step for ISO accreditation [26].
