Combined immunophenotyping and fluorescence in situ hybridization with chromosome-specific DNA probes allows quantification and differentiation of ex vivo generated dendritic cells, leukemia-derived dendritic cells and clonal leukemic cells in patients with acute myeloid leukemia

Abstract

Antileukemic T-cell responses induced by leukemia-derived dendritic cells (DC_leu) are variable, due to varying DC/DC_leu composition/quality. We studied DC/DC_leu composition/quality after blast culture in four DC media by flow cytometry (FC) and combined fluorescence in situ hybridization/immunophenotyping analysis (FISH-IPA). Both methods showed that DC methods produce variable proportions of DC subtypes. FISH-IPA is an elaborate method to study clonal aberrations in blast/DC cells on slides, however without preselection of distinct cell populations for FISH analysis. FISH-IPA data proved previous FC data: not every clonal/blast cell is converted to DC_leu (resulting in various proportions of DC_leu) and not every detectable DC is of clonal/leukemic origin. Preselection of the best of four DC methods for “best” DC/DC_leu generation is necessary. DC_leu proportions correlate with the antileukemic functionality of DC/DC_leu-stimulated T-cells, thereby proving the necessity of studying the quality of DC/DC_leu after culture. FC is the superior method to quantify DC/DC_leu, since a blast phenotype is available in every given patient, even with low/no proportions of clonal aberrations, and can easily be used to study cellular compositions after DC culture.

Keywords::

• Dendritic cells
• acute myeloid leukemia
• FISH
• immunophenotyping
• clonality

Acknowledgements

The authors thank nurses and physicians on the wards for their support, and diagnostic laboratories for the contribution of diagnostic reports of the patients. Part of the results presented in this manuscript were determined in the course of the MD theses of Andreas Kremser and Stefanie Kufner at the University of Munich, Department of Hematopoietic Transplantations of the Ludwig-Maximilian-University of Munich and the Helmholtz-Center Munich.

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