Intracellular markers in acute myeloid leukemia diagnosis

In our study we used a new proposed system of CD45 monoclonal antibody in c ombination with the side scatter (SSC) parameter as a very useful gating me thod allowing myeloblast detection especially in cases with low blasts perc entage in examined samples. Immunological demonstration of myeloperoxidase (MPO) in the cytoplasm of AM L blasts is considered to be a reliable and highly sensitive marker. Using a direct single and double immunofluorescence staining method and flow cyto metry we evaluated the intracellular expression of two granular constituent s of myeloid cells - MPO and lactoferrin (LF) in leukemia cells from 18 pat ients at AML diagnosis, two patients in remission after allogenic bone marr ow transplantation and in six controls. Two different fixation/permeabiliza tion techniques were used: Fix&Perm, paraformaldehyde and saponin prior to monoclonal antibody staining in order to verify the sensitivity of two labe ling methods for MPO. Although both reagents used in this study proved to b e efficient tools for the fixation and permeabilization of leukemia cells, the second one was characterized by higher sensitivity in detection of MPO. By double staining of MPO and LF we were able to distinguish undifferentia ted cells from the granulomonocytic maturation compartments in bone marrow, since LF is proposed to be selectively expressed from the myelocyte stage of differentiation onward. Cytoplasmic CD13 expression was detectable in AM L blasts after their buffered-formaldehyde-acetone fixation/permeabilizatio n. According to our results the detection of MPO and CD13 markers in the cy toplasm of leukemia cells is of great importance in the definition of FAB M 0-M1 subtype of AML. Furthermore we described overexpression of CD34 antigen in AML and revealed the characteristic marker combination when CD34 was studied simultaneously with MPO. This finding also coincided with some atypical phenotypic featur es (CD15/MPO, CD7/cCD13, CD2/cCD13, CD33/cCD13, MPO/cCD13) contributing to the differential diagnosis and allowing the immunologic monitoring of patie nts for the presence of residual disease.