Flow cytometry of peripheral blood cells from patients with either subclinical PNH, PNH/bone marrow failure (BMF), or classic PNH. (A) In these examples, 2-color flow cytometry is used to analyze RBCs (top) and neutrophils (PMNs) (bottom). RBCs are gated on based on staining with phycoerythrin (PE)-conjugated anti-glycophorin, and GPI-APs are gated on using a combination of fluorescein isothiocyanate–conjugated anti-CD55 and anti-CD59. Neutrophils (PMNs) (bottom) are gated on using PE-conjugated anti-CD11b. (Left, top and bottom) Analysis of RBCs and PMNs from a normal volunteer (control). All the normal RBCs and PMNs express CD55 and CD59. Patients with subclinical PNH (middle) have very small clones, typically less than 1% (middle), and they have no biochemical evidence of hemolysis. Patients with PNH/BMF (right) have at least biochemical evidence of hemolysis, but clone size is generally relatively small. In the case illustrated, the clone size of 21% is at the lower limit for biochemical detection of hemolysis. (B) (Right, top and bottom) Analysis of RBCs and PMNs from a patient with classic PNH. The percentage of cells that are deficient in expression of GPI-APs is shown the inner-upper quadrant of the histograms. The percentage of deficient RBCs is invariably greater than the percentage of deficient PMNs because GPI-AP–deficient RBCs are selectively destroyed by complement-mediated lysis, whereas GPI-AP–deficient PMNs have a normal life span. For this reason, the size of the PNH clone is determined by the percentage of GPI-AP–deficient PMNs rather than by the percentage of GPI-AP–deficient RBCs.