Biases introduced by Ficoll-based isolation in acute myeloid leukemia sample analyses support the use of hemolysis

Acute myeloid leukemia (AML) is a heterogeneous malignancy whose characterization relies on immunophenotyping and molecular profiling. While hemolysis is recommended for leukocyte isolation in clinical diagnostics, Ficoll-based density gradient centrifugation is widely used in research and biobanking. Here, we evaluated the impact of Ficoll isolation on commonly performed analyses of AML samples. Ficoll altered flow cytometry-based characterization by systematically enriching lymphocytes and AML blasts while depleting granulocytes. The increased T-cell content impaired AML engraftment in NSG mice, as T cells mediated terminal graft-versus-host disease. Although Ficoll had minimal impact on ex vivo AML blast expansion or chemotherapy response, RNA sequencing identified 1,136 differentially expressed genes compared with hemolysis, with Ficoll-processed samples notably leading to an overestimation of leukemic stem cell gene set expression. Immunogenomic deconvolution highlighted that Ficoll leads to an overestimation of CD8⁺ T-cell and monocyte abundances in sequenced samples. Mutation calling from RNA-seq data revealed substantial discrepancies between methods, including failure to detect a clinically relevant DNMT3A R882 mutation in a Ficoll-processed sample. Together, these findings support the systematic use of hemolysis to preserve cellular diversity and avoid unpredictable biases introduced by Ficoll-based isolation.