Modern flow cytometry techniques using monoclonal antibodies (mAbs) specific to glycosylphosphatidylinositol-anchored membrane proteins have revealed an increase in the proportion of paroxysmal nocturnal hemoglobinuria (PNH)-type cells in a varying (30 to 50) percentage of acquired aplastic anemia (AA) patients who do not manifest any signs of hemolysis. However, the clinical significance of such a small proportion of PNH-type cells remains uncertain because previous studies have used a variety of flow cytometric assays with different mAbs and different definitions of an abnormal increase in the proportion of PNH-type cells, and also, few studies have focused on the correlation of presence of increased PNH-type cells with response to immunosuppressive therapy (IST) based on a large number of newly diagnosed AA patients. We previously demonstrated, using two-color flow cytometry capable of precisely determining the proportion of PNH-type cells less than 0.1%, that such a minor population of PNH-type cells represents a reliable marker for presence of immune pathophysiology in refractory anemia patients (
Blood,100: 3897–3902, 2002
; Blood,102:1211–1216, 2003
). To determine if the PNH-type cells serve as a marker for immune pathogenesis in AA as well, we examined peripheral blood in 99 newly diagnosed AA patients (67 severe AA and 32 moderate AA) for presence of CD55−59−CD11b+ granulocytes and CD55−59−glycophorin A+ RBCs and studied correlation of response to ATG + cyclosporine (CsA) therapy with presence of increased PNH-type cells. When the influence of time from blood collection until treatments of cells with mAbs on the outcome of PNH-type-cell detection were examined, the percentages of PNH-type cells remained unchanged for 2 days for granulocytes and for 6 days for RBCs from the time of blood collection. Minor (>0.003% for CD11b+ granulocytes and >0.005% for glycophorin A+ RBCs) population of PNH-type cells were detected in 66 (66.7%) patients. The proportions of PNH-type cells in patients with increased PNH-type cells (PNH+ patients) were 0.005–0.01% in 10 (15.2%) patients, 0.01–0.1% in 20 (30.3%), 0.1–1.0% in 25 (37.9%), and 1.0–8.67% in 10 (15.2%). When clinical characteristics of PNH+ patients were compared with that of PNH− patients, PNH+ patients (mean, 54 year-old) were significantly older than PNH− patients (mean, 42 year-old, [Italic]P[/Italic]=0.042). 55 of 66 (83.3%) PNH+ patients improved with IST and achieved PR or CR whereas 17 of 33 (52.9%) PNH− patients responded. Kaplan-Meier analysis showed that PNH+ patients had a significantly better chance of response to IST than PNH- patients (Figure). These results indicate that a minor population of PNH-type cells defined by the two-color flow cytometry represents a good marker for response to IST in AA patients. Since CD55−59−glycophorin A+ RBCs less than 0.1% remain constant and detectable in blood stored at 4 ?C for up to 6 days, testing blood cells, particularly RBCs, using this sensitive flow cytometry is useful in identifying high responders to IST in AA patients.
