Triple immunofluorescence evaluation of CD15, CD34 and class II expression by flow cytometry in normal and leukemic bone marrows.

BACKGROUND

The CD15/CD34 phenotype has been reported as aberrant and exploited for monitoring minimal residual disease in acute myeloid leukemia (AML) patients. Moreover, CD15/CD34 has been described as a rare phenotype in normal bone marrow (NBM) (< 0.10%). We used a triple immunofluorescence assay to investigate the expression of CD15, CD34 and class II antigens in normal (NMB) and leukemic (LBM) bone marrows.

METHODS

A FACScan for quantitative fluorescence and the PAINT-A-GATE program for multiparametric analysis were utilized. Fifteen normal bone marrow and fifteen leukemic bone marrow samples were studied with a triple immunofluorescence assay. A FACSorter was used for sorting.

RESULTS

Eleven out of 15 normal marrows contained less than 0.67% (range 0.01-0.66) cells which coexpressed CD15, CD34 and class II antigens. Three normal marrows contained more than 0.67% but less than 1.84% (range 1.05-1.83) triple stained cells. The entire group of leukemic marrows coexpressed triple stained cells with a frequency higher than 1% (range 1.1-48.6); furthermore, 10 samples contained the same population at frequencies higher than 10%. The difference between normal and leukemic marrows was statistically significant (p = < 0.001). Triple positive cells (TPc) from NBM were sorted for morphology, which proved to be consistent with myeloid progenitor features (early myeloblasts). This confirms that CD15+ CD34+ Class II+ precursors are commonly expressed in NMB, although at low frequency. Interestingly, 12 (80%) AMLs out of 15 were diagnosed as M1 (5) or M2 (7), while the remaining were M4 (2) and M5a (1). Additionally, all M2 cases were positive at percentages higher than 10%. Apparently CD15, CD34, class II expression correlates mainly with granulocyte differentiation. Two complete remission (CR) LBM, positive at onset for the triple combination, were regularly monitored. In one case the TPc percentage always remained near the normal values found in NBM (0.72%), and this patient is still in CR. In the other, overt relapse was preceded by a progressive increase in the TPc percentage.

CONCLUSIONS

Although the presence in NBM of CD15+ CD34+ and class II+ precursors hinders minimal residual disease detection, we conclude that this unusual combination may distinguish a leukemic population and may allow monitoring of "early relapse".