A Novel Full-Spectral Flow Cytometric Assay for Detection of Measurable Residual Disease in Patients with B-Lymphoblastic Leukemia
Authors: Li Ke , Chen Man , Gong Meiwei , Lu Peihua , Wang Hui 
Published: December 2024

Introduction: Multicolor flow cytometry (MFC) is one of the most widely utilized methods for measurable residual disease (MRD)detection. As the technology advances, full-spectral flow cytometry (FSFC) which allows the simultaneous analysis of multiple markers is promising to improve the sensitivity of MRD.

Methods: We collected 546 BM from 511 B-ALL patients who detected in our lab from Feb.2024 to Jun. 2024. There were 300 male (58.7%) and 211 female (41.3%), with a median age of 13 years old (range,1-71 years). They were detected at three time points: post-induction (PI; day-35), post-consolidation (PC; day-78), and subsequent follow-up time-points (SFU). We created a novel 13-color single-tube FSFC panel as cTdT-FITC,CD10-PE,CD34-Percpcy5.5,CD19-Pecy7, CD20-Apccy7,cCD79a-APC,CD38-Percpeflour710,CD86-PE dazzle594,CD72-BV421,CD33-BV650,CD123-BV711,CD81-BV786, and CD45-V500. The prepared samples were detected on a three-laser 24 channel flow cytometer(Cytek Biosciences, USA) with a target of 1*106 events, and the data were analyzed with SpectroFlo software. Forward scatter (FSC)/side scatter (SSC) dot plots set the live cell gate, doublets were excluded on FSC-A/FSC-H, and synchronous gating was performed from the single live cells: CD45/SSC for background subsets, B cells were gated by CD19/SSC, cCD79a/SSC and CD72/SSC. By analyzing the developmental pattern of B-lineage cells in dot plots such as CD10/CD20, CD10/CD34, CD10/CD38, CD10/CD81, CD10/CD86, CD34/cTdT, CD38/CD72, CD19/cCD79a, CD33/CD72, CD123/CD34, CD19/CD45, cCD79a/ CD45, CD72/ CD45 and so on, we identified B-ALL MRD cells based on the combined DfN and LAIP strategy. To verify the accuracy of our 13c-FSFC method, 186 BM samples were analyzed in parallel to molecular monitoring. BCR-ABL1, TCF3-PBX1, ETV6-RUNX1, MLL-AF4, MLL-AF10, MLL-ELN, and UBTF-ATXN7L3 fusion genes, PAX5, ZNF384, MEF2D, and IgH rearrangements, MLL-PTD mutation, and IKZF1 deletion were detected by PCR. McNema's test was performed in SPSS Statistics version 27, and P &lt; 0.05 was deemed to be statistically significant. Cohen's Kappa test was also performed in SPSS, Kappa≥0.75 indicates that the diagnostic results of two methods are in good consistency.

Results: It was shown that CD19-directed therapies, such as blinatumomab and anti-CD19-CAR-T, have been receiving increasing attention in the treatment of B-ALL. In order to establish an alternate gating strategy, it is necessary to add cCD79a and CD72 into the panel since CD19 might downregulated. We found that out of 546 bone marrow samples, there were 49 MRD positive samples (8.97%) and 497 MRD negative samples (91.03%). The median tumor burden was 0.15% (range, 0.001%-89.77%). Among the 49 MRD positive samples, abnormalities in CD19 (1 bright , 3 loss) accounted for 8.61%, cCD79a (2 dim) accounted for 4.08%, CD72 (7 dim) accounted for 14.29%, CD34 (4 bright, 18 dim/-) accounted for 44.90%, CD10 (33 bright, 7 dim/-) accounted for 81.63%, CD20 (6 dim) accounted for 12.24%, CD38 (38 dim) accounted for 77.55%, CD81 (36 dim) accounted for 73.47%, cTdT (17 dim/-) accounted for 34.69%, CD123 (2 bright,5 dim) accounted for 20.00%, CD33 (6 positive) accounted for 12.24%, CD86 (9 positive, 8 dim) accounted for 34.69%. Besides CD19, cCD79a, CD72 and CD45, the contribution of these antibodies, in descending order, is CD10, CD38, CD81, CD34, cTdT, CD86, CD123, CD33, CD20. CD10 str/+ and CD38 dim/- and CD81 dim/+ are the most frequent aberrancies, we were able to identify 59.18% abnormal samples in the combination of CD10/CD38 (n=19) or CD10/CD81 (n=10) dot plots. Three MRD positive samples were found to have lost CD19 but were defined by cCD79a or CD72. Out of the186 samples analyzed by molecular method, MRD positive samples account for 8.60% (16/186), while the remaining 91.40% tested negative for MRD. When comparing the results of MRD using FSFC and PCR methods, 179 samples showed consistent analysis results: including 15 MRD positive samples and 164 MRD negative samples. There were seven samples with two inconsistent test results, and no statistical difference between the two methods by McNema's test (P=0.125). According to Cohen's Kappa test, the FSFC method has good consistency with PCR monitoring (Kappa=0.790).

Conclusions: We have established a single-tube 13c-FSFC method for detecting MRD in B-ALL.