Ham's acid hemolysin and sucrose lysis tests have been replaced 
by flow cytometric testing for glycosylphosphatidyl inositol (GPI)-
anchored proteins CD14 and CD24, and aerolysin binding. These proteins 
are not expressed on PNH white blood cells and their lack of 
expression is determined by flow cytometric assay. 

The channel-forming toxin, aerolysin, and its inactive precursors, 
proaerolysin, bind selectively with a high affinity to the GPI anchor 
itself. The lack of GPI anchor on blood cell surface will decrease the 
ability of fluorescently labeled protein aerolysin (FLAER) to bind to 
nucleated blood cells in patients with PNH. 

Determination of CD14 and CD24 must be performed on fresh whole blood. 
Both monocytes and granulocytes are analyzed for CD14/CD24 expression 
and aerolysin bindings. Granulocytes are the most sensitive population 
in which to detect GPI-anchored protein deficiency. Two additional 
markers are performed for gating purposes, CD45 (leukocyte common 
antigen) and CD15 (myeloid antigen). 

REFS: 
1)Richards, S et al. Application of Flow Cytometry to the Diagnosis of 
Paroxysmal Nocturnal Hemoglobinuria. Cytometry 2000; 42:223-233. 
2)Dunn, D, et al. Paroxysmal Nocturnal Hemoglobinuria in Patients with 
Bone Marrow Failure Syndromes. Ann Int Med 1999; 131:401-408. 
3) Brodsky RA, et al. Improved detection and characterization of 
paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J 
Clin Pathol 2000; 114:459-66.