Quantitative immunocytofluorometry--new parameters for the definition of leukemia cells

In our study we used for definition of leukemia/lymphoma cells a new parameter which allows the enumeration of mean fluorescence intensity expressed by the number of antigen molecules per cell. Quantitative immunofluorescence using calibration microbeads was performed in 36 patients with different acute and chronic lymphoid and myeloid leukemia and in 19 healthy volunteers. We showed that quantitative immunophenotyping allowed the definition of aberrant marker densities on neoplastic cells. We demonstrated under- and overexpression of CD8 marker in CD3/CD4/CD8 complex in T acute lymphatic leukemia and T non-Hodgkin's lymphoma and T leukemia of large granular lymphocytes as compared to normal counterparts. We pointed out that certain antigens (e. g. CD10, CD4, CD24) were expressed at different levels on different cell subsets (CD10 in early B-acute lymphatic leukemia and coexpressed in T-acute lymphatic leukemia, CD4 on T cells and monocytes, CD24 on B cells and granulocytes in chronic myeloid leukemia). We showed that quantitative immune fluorescence could provide new data contributing to a more precise definition of cell differentiation. We documented the significant difference between antigen density of early and late markers in B-cell and myeloid malignancies. Further, we demonstrated that quantitative immune phenotyping could help in determination of exact definition of pathologic clone in morphologically immature leukemia population and showed that parameters of this method are also convenient for cytoplasmic marker evaluation. In our study we were able to demonstrate that CD45 quantitative expression appeared to be a more informative parameter than its percentage of antigen-positive cells as a measure of antigen expression only and we pointed out that low and high CD45 densities enabled to differentiate between pathological clone and residual healthy population in examined sample. We showed that quantitative immune phenotyping could be another important parameter for definition of leukemia phenotype suitable for detection of minimal residual disease.