多参数流式细胞术（MFC）检测残留微小疾病（MRD）已成为B型急性淋巴细胞白血病（B-ALL）中一种成熟的风险分层因素和治疗修改策略。然而，目前的8色（8c）MFC MRD检测的灵敏度为0.01%，存在假阴性和假阳性。因此，迫切需要一种更敏感、适用的MFC-MRD方法。本研究旨在建立一种单管21c-MFC方法来检测B-ALL MRD，评估其性能，并研究其初步临床应用。我们选择了21个标记物来建立一种单管21c-MFC方法。通过向正常骨髓中添加Nalm-6细胞，验证了该方法的重复性和敏感性。分别使用21c-MFC和8c-MFC检测了对照组（n=6）、B-ALL组（n=7）和完全缓解（CR）组（n=26）的样本。在对照组和B-ALL组中分析了这些标记物的表达特征，并比较了21c-MFC和8c-MFC在检测MRD方面的一致性。该方法的重复性系数为1.91%，灵敏度高达0.005%。在对照组中，CD81、CD97和CD200的表达逐渐下降，而CD44、HLA-DR、CD73和CD72的表达随着正常B细胞的成熟逐渐增加。在B-ALL组中，CD73stro、CD81low、CD44stro、CD123stro和CD58stro显示高频表达。21c-MFC和8c-MFC在检测MRD方面的一致性率达到96%。建立了一种用于B-ALL中MRD检测的单管21c-MFC方法，其灵敏度高于8c-MFC方法。

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.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.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%.A single-tube 21c-MFC method was established for MRD detection in B-ALL and had higher sensitivity than the 8c-MFC method.