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A Single-Tube 21-Color Multiparameter Flow Cytometry Improve the Sensitivity of B-ALL MRD Analysis by Qingchen Wang, Ran You, Linzi Miao, Yao Lu, Yan Gong, Xu Wu, Huizheng Ge, Shuzhen Zhai, Chenxue Qu

Background: Detection of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) is a well-established risk stratification factor and therapeutic modification strategy in B acute lymphoblastic leukemia (B-ALL). However, current 8 color (8c)-MFC for MRD detection had the sensitivity of 0.01% with false negative or positive. Hence, a more sensitive and applicable MFC-MRD method is urgently needed. The aim of this study is to establish a single-tube 21c-MFC method to detect B-ALL MRD, evaluate its performance, and to investigate its preliminary clinical application.
Methods: We selected 21 markers to establish a single-tube 21c-MFC method. The repeatability and sensitivity of this method was validated by adding Nalm-6 cells to normal bone marrow. Samples from control group (n = 6), B-ALL group (n = 7) and complete remission (CR) group (n = 26) were detected by 21c- and 8c–MFC separately. The expression characteristics of these markers was analyzed in control and B-ALL group, and the consistency of 21c- and 8c-MFC in detecting MRD was compared.
Results: Repeatability of this method was 1.91% of CV and sensitivity was up to 0.005%. In control group, the expression of CD81, CD97, and CD200 gradually decreased and CD44, HLA-DR, CD73, and CD72 gradually increased with the maturation of normal B cells. In B-ALL group, CD73stro, CD81low, CD44stro, CD123stro, and CD58stro showed high-frequency expression. The consistency rate of 21c- and 8c-MFC in detecting MRD was 96%.
Conclusions: A single-tube 21c-MFC method was established for MRD detection in B-ALL and had higher sensitivity than the 8c-MFC method.

DOI: 10.7754/Clin.Lab.2023.230226