One Tube 24 Color Full Spectral Flow Cytometry and Multi-Dimensional Software to Study the Maturation Pattern and Antigen Expression of the Myeloid

Introduction Flow cytometry(FC) plays an important role in the diagnosis of hematologic diseases and the study of cell maturation. Spectral multicolor flow cytometry(SMFC) has shown an advantage over traditional FC that more fluorescent markers could be detected simultaneously, more antigen combinations could be made, and the expression of cells could be scrutinized. However, published studies focused on lymphocyte subsets and the differentiation between hematogones and B-acute lymphoblastic leukaemia/lymphoma(ALL/LBL) minimal residual disease(MRD) detection, and there are few studies on myeloid development and expression. Besides, more powerful and cutting-edge software are needed for complicated combinations from SMFC because traditional dot plots are unable to meet the demand of analysis. Here we design a one-tube 24-color panel combining with the multidimensional data analysis software to study the expression and maturation of normal and malignant myeloid cells including minus subgroups. We hope to improve the sensitivity of MRD by FC and explore more information about myeloid diseases, finally promote the development of artificial intelligence(AI) in clinical FC diagnosis. Methods: the one-tube 24-color panel was designed according to our experience and Euroflow recommendation. it is composed of backbones including CD45 and myeloblast markers CD34, CD117 and HLA-DR, adding myeloid markers CD33, CD13, CD371, CD15, CD64, CD11c,CD14, CD36 and CD11b, routine leukaemia associated immunophenotyping(LAIP) or different from normal(DFN) markers CD4, CD19, CD7, CD2, CD56,CD96,CD123, CD38, CD200, CD71 and CD9. The control database consisted of 20 normal bone marrow(BM) specimens, including 8 healthy donors and 12 patients with other diseases that were in complete remission(CR) after treatment. To verify the effectiveness of the panel, 4 BM samples from acute myeloblastic leukaemia(AML) patients with MRD positive or relapsed status were selected, with malignant myeloblasts of 0.23% (sample A1), 4.3%, 30.31% (and 6.47% abnormal mast cells, sample A3), 0.29% (16.49% basophils) (sample A4), respectively. The data was acquired by a 3 laser 38-color Cytek spectral FC, and analyzed by Kaluza and Flowjo software. The results were compared with those of conventional 3 - laser 8 - color Canto FC. Results when analyzing the common antibodies acquired by two kinds of FCs and software, the similarities and good correlations were shown about the percentage of myeloid subsets