Development of a 27‐color panel for the detection of measurable residual disease in patients diagnosed with acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) measurable residual disease (MRD) evaluated by multiparametric flow cytometry (MFC) is a surrogate for progression‐free and overall survival in clinical trials and patient management. Due to the limited number of detection channels available in conventional flow cytometers, panels used for assessing AML MRD are typically split into multiple tubes. This cripples the simultaneous and correlated assessment of all myeloblast measurements. In response, we prototyped a single‐tube 27‐color MFC assay for the evaluation of AML MRD, incorporating all recommended markers. Marrow aspirates from 22 patients were processed for analysis using full spectrum flow cytometry (FSFC). The signal resolution of each marker was compared between samples stained with single antibody vs. the fully stained panel. The analytical accuracy for quantifying hematopoietic cells between our established 8‐color assay and the new 27‐color method were compared. Variations within an operator and between separate operators were assessed to evaluate the assays reproducibility. The limited of blank (LOB), limit of detection (LOD), and lower limit of quantification (LLOQ) of the 27‐color method were empirically determined using limiting dilution experiments. The stability of antibody cocktails over a period of 120 h was also studied using cryopreserved marrow cells. The stain indices for all antibodies were lower in the fully stained panel compared to cells stained with one antibody but clear separations between negative and positive signals were achieved for all antibodies. Our results demonstrated a high concordance between the established 8‐color method and the new 27‐color assay for enumerating myeloblasts and MRD interpretation within and between operators. The data further showed that the single‐tube 27‐color assay easily achieved the minimum required detection sensitivity of 0.1%. When antibodies were combined, however, expression intensity of some antigens deteriorated significantly when stored. Our single‐tube 27‐color panel is a suitable, high sensitivity flow cytometric approach that can be used for AML MRD testing, which improves the correlation of aberrant antigens and detection of asynchronous differentiation patterns. Based on the stability study, we recommend the full panel be made prior to staining.

A 19‐color single‐tube full spectrum flow cytometry assay for the detection of measurable residual disease in acute myeloid leukemia

“…The feasibility of using high‐parameter panels in clinical practice raises concerns regarding the acquisition of spectral references and the quality control and stability of antibody cocktails. Recent evidence suggests potential cocktail instability within a short period of only 24 h after preparation [33]. Therefore, it is essential to establish the quality of the antibody cocktail for clinical use, whether conventional or spectral flow cytometry is used.…”

“…panels to detect MRD in AML have recently produced a growing interest. For example, Soh et al proposed a 27-color panel to be run on a 5-laser spectral flow cytometer, and even Cytek Biosciences launched its first commercially available 20-color AML kit[33,34]. Our work shows important differences to these assays, as it exclusively features extensively validated antibody clones and focuses on most important LAIPs.…”

Monitoring Measurable/Minimal Residual Disease in Acute Myeloid Leukemia: Multiparametric Flow Cytometry-Based Approach

“…The availability of new technologies such as CyTOF mass spectrometry and full-spectrum flow cytometry (FSFC) has demonstrated the capacity to utilize many markers (up to 27 colors) for AML MRD. 84 85 Additionally, by increasing the number of events from 1 million to 10 million, the sensitivity of MRD assay can easily be reached to as high as 2 × 10 −6 (0.0002%) MRD detection. 63 Thus, the recent availability of new flow cytometry technologies and newer dyes has improved the assay sensitivity and specificity for MRD assessment by flow cytometry.…”

Full spectrum flow cytometry in the clinical laboratory

“…A particularly useful study reported a 27-color single-tube assay using a 3-laser Cytek Aurora for detection of measurable residual disease in patients diagnosed with acute myeloid leukemia. 16 The lower limit of detection for identifying abnormal myeloblasts was 0.0013% in limiting dilution studies, indicating excellent sensitivity of spectral flow cytometers for detecting rare cell types. Importantly, this study also compared the 27-color panel against an 8-color multi-tube assay run a BD Canto II and found the two assays to be concordant in terms of cell type identification.…”

“…Given the recent deployment of spectral flow cytometry in clinical settings, there are limited use cases to draw upon. A particularly useful study reported a 27‐color single‐tube assay using a 3‐laser Cytek Aurora for detection of measurable residual disease in patients diagnosed with acute myeloid leukemia 16 . The lower limit of detection for identifying abnormal myeloblasts was 0.0013% in limiting dilution studies, indicating excellent sensitivity of spectral flow cytometers for detecting rare cell types.…”