Blood cell flow cytometry in paroxysmal nocturnal hemoglobinuria: a tool for measuring the extent of the PNH clone.

The membrane expression of nine glycosyl phosphatidyl inositol (GPI)-linked molecules was analyzed by flow cytometry on circulating cells from 18 patients affected by paroxysmal nocturnal hemoglobinuria (PNH). The results allowed us to select CD66b, CD14, CD59, CD24 and CD59 monoclonal antibodies as the most suitable reagents for discriminating between normal and PNH cells in PMN, monocytes, RBC and B or T lymphocytes, respectively. In order to assess whether the analysis of distinct cell populations could provide differential information on the extent of the disease, we compared the proportion of residual normal cells in RBC, monocyte and PMN populations. The mean percentage of unaffected cells was higher in RBC as compared to PMN (50.5 +/- 18.7 vs 17.7 +/- 19.7, P < 0.0001). The proportion of normal PMN was, in turn, significantly greater than that of normal monocytes (17.7 +/- 19.7 vs 8.7 +/- 11.0; P < 0.05). The percentage of CD14+ monocytes was directly related to Hb concentration and platelet (Plt) count, and inversely to percent lysis at the Ham's test. The percentage of CD66b+ PMN was directly related to Plt count and Hb level, while the percentage of CD59+ RBC was associated, in an inverse fashion, only to the Ham's test. No significant correlation was found between cell marker expression and PMN count, reticulocytosis, bilirubin and serum LDH. By dividing the patients into two groups, according to high (> 10 percent) or low (< 10 percent) percentage of CD14+ monocytes, a statistical analysis showed that the main hematological parameters were significantly different.