28-color single tube for flow cytometric assessment of myeloid maturation, myeloid neoplasia, and acute myeloid leukemia minimal/measurable residual disease

Abstract

Flow cytometry (FC) is an indispensable tool for myeloid neoplasia (MN) diagnosis, and the cell of origin has clinical diagnostic and prognostic significance. However, the complex maturational pathways within the blast compartment complicate the detection of the abnormal population at the minimal/measurable disease (MRD) level using the difference from normal approach. The analysis could be simplified by separating the blast compartment into maturation-defined stages with relatively uniform phenotypes as reference populations. This requires a relatively extensive panel of antibodies to define maturation stages and simultaneously detect the common deviations from the normal pattern. We validated a single tube 28-color clinical assay for MN assessment and acute myeloid leukemia (AML) MRD detection assisted by the precise maturation stage assignment. The new assay uses a previously described 21-antigen backbone, with the additions of CD10, CD36, CD42b, CD45RA, CD90, CD105, and CD371. Validation was performed using 37 normal samples and 151 MN follow-up samples in a split-sample fashion comparing the new assay to the legacy 3-tube, 21-antigen assay. Dilution studies were performed to establish assay sensitivity. A new analysis framework relying on comparison to well-defined maturational stages was established for MRD analysis. The assays showed 100% qualitative concordance with excellent quantitative concordance. Dilution studies yielded a limit of detection of 0.01%. The addition of new antibodies allowed for consistent comparisons of candidate abnormal populations to well-defined normal maturation stages through traditional FC plots and Uniform Matrix Approximation and Projection assessments. This new single-tube, 28-color clinical assay allows for efficient MN assessment in clinical settings. It reliably detects MRD levels of abnormal myeloid cells because the expanded panel allows for precise comparison to defined lineage commitment maturational stages. Lastly, it provides high information density while reducing equipment use, reagent use, and technical labor.