Flow cytometric testing for paroxysmal nocturnal hemoglobinuria: CD64 is better for gating monocytes than CD33.

BACKGROUND

Paroxysmal nocturnal hemoglobinuria (PNH) is diagnosed by documenting partial or complete absence of glycosyl phosphatidyl inositol (GPI)-associated ligands in neutrophils, monocytes, and red blood cells (RBCs). The monocytes can be separated by their bright expression of either CD33 or CD64. This paper compares the utility of CD33- vs CD64-based monocyte gating in flow cytometric testing for PNH.

METHODS

One hundred and nineteen cases tested for PNH by flow cytometry were included in the study. Both the total number of monocytes and the number of GPI-deficient monocytes gated with CD33 or CD64 were compared. The clustering pattern and any other unusual patterns were noted and investigated.

RESULTS

CD64 staining showed more distinct separation of the monocyte cluster than did CD33 staining. The difference between the number of monocytes gated by CD33 and CD64 staining ranged from -26 to +32% (median 1.60%, average 1.69%). Six patients had GPI-deficient monocytes by both CD33- and CD64-based gating, ranging from 0.02 to 83.23%. There were no patients who showed GPI-deficient monocytes by one but not the other gating. The presence of blasts in patients with acute leukemia resulted in abnormal cluster patterns, both by CD33- and CD64-based gating.

CONCLUSIONS

CD64-based gating showed more distinct clustering of monocytes than CD33-based gating, allowing for objective separation. The number of monocytes in total and GPI-deficient monocytes derived from both gating strategies was comparable.