Quality Assessment of a Large Multi-Center Flow Cytometric Dataset of Acute Myeloid Leukemia Patients—A EuroFlow Study

Abstract

Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a dataset and developed a QA methodology of the EuroFlow Acute Myeloid Leukemia (AML) database, based on the eight-color EuroFlow AML panel consisting of six different antibody combinations, including four backbone markers. In total, 1142 AML cases and 42 normal bone marrow samples were included in this analysis. QA was performed on 803 AML cases using multidimensional analysis of backbone markers, as well as tube-specific markers, and data were compared using classical analysis employing median and peak expression values. Validation of the QA procedure was performed by re-analysis of >300 cases and by running an independent cohort of 339 AML cases. Initial evaluation of the final cohort confirmed specific immunophenotypic patterns in AML subgroups; the dataset therefore can reliably be used for more detailed exploration of the immunophenotypic variability of AML. Our data show the potential pitfalls and provide possible solutions for constructing large flowcytometric databases. In addition, the provided approach may facilitate the building of other databases and thereby support the development of novel tools for (semi)automated QA and subsequent data analysis.

Three‐dimensional spatial mapping of the human hematopoietic microenvironment in healthy and diseased bone marrow

“…Routine diagnostics and research rely heavily on single or double immunofluorescence staining of human BM biopsy specimens, as multiplexing beyond three colors becomes increasingly challenging with human material. In contrast, flow cytometry-based analysis routinely uses panels of at least eight different antibodies for example, for leukemia diagnosis and follow-up [25].…”

High-Dimensional Mass Cytometry Analysis of Embryonic Antigens and Their Signaling Pathways in Myeloid Cells from Bone Marrow Aspirates in AML Patients at Diagnosis

“…Although a small number of cases were included, the present study is original and builds upon the few prior studies dedicated to exploring myeloid cell EAs and SPs in AML by mass cytometry. Herein, we designed a 36-antibody panel according to the recommendations of Euroflow [11], allowing us to distinguish among BM cell populations-particularly the myeloid cell populations-and evaluate expression of EAs and EA-regulated SPs. Combinatorial expression of multiple phenotypic markers facilitated the exhaustive exploration and precise identification of 14 BM cell populations in the absence of morphological parameters, among which included cell populations relevant to the diagnosis of AML: HSCs, CD34 + myeloblasts, more mature CD117 + myeloblasts, monoblasts, and immature monocytic cells.…”