The detection of bone marrow morphology with Wright staining showed active hyperplasia of nucleated cell. Moreover, the proportion of granule cells of was significantly reduced (0.5%) and erythroid cells (1%) had normal morphology. Mature red blood cells were slightly different in size, and the proportion (33%) of monocytes increased with uneven distribution. Primitive naive monocytes accounted for 23%, lymphocytes accounted for 67.5%, and immature lymphocytes accounted for 3.5% of the total proportion. Only 16 megakaryocytes were observed in the smears of blood with platelets distributed in piles. The smears also showed an increase in lymphocytes with primitive monocytes accounting for 3% of the total cell proportion (Fig. 1A).
HE staining showed that the proportion of bone marrow nucleated cells was regular, while the proportion of myeloid blast cells increased (accounting for 50–60%) with diffusive distribution (Fig. 1B). The mature granular and red cells were scattered distributed barely observed in the samples. Few megakaryocytes were observed. A few lymphocytes were distributed in clusters, and reticular fiber staining showed MF2 grade, focal. It was also shown that bone marrow samples were 100% negative for pathological cells, 22% positive for specific esterase ASD (score 22), negative for non-specific esterase α-NAE, extrairon + + for iron staining (Fig. 1C). The results of immunohistochemistry detection showed mature lymphocytes were positive (stained brown) for CD34, CD117, MPO, while negative for CD3, CD20, and CD5 (Fig. 1D and Fig. 1E).
The analysis of karyotype identified nine mitosis phases, all of which were normal karyotype: 46,XX[9].
The flow cytometry showed that T cell occupies 14.22% of nuclear cells (Fig. 2), positive in the detections of CD34, CD117dim, CD123, CD43, CD13, CD38, CD33, and HLA-DR, partially positive in the detections of CD19dim and CD200dim, and negative in the detections of CD5, CD7, CD2, CD10, CD20, CD138, CD81, CD23, CD11c, FMC7, CD103, CD22, CD25, CD36, CD11b, CD16, CD14, CD64, CD15, CXCR4, CD79b, cCD3, cmpo, cCD79a, mKappa, which were identified as abnormal myeloid protocells (Fig. 2). CD19 + cells accounted for 58.1% of the total nuclear cells, positive in the detections of CD19dim, CD5dim, CD81dim, CD200, CD79b, CD43, CD23, CD22dim, HLA-DR, mKappadim, partially positive in the detections of CD20dim, CXCR4, and negative in the detections of CD34, CD117, CD38, CD7, CD2, CD10, CD138, FMC7, CD103, CD25, CD36, CD11b, CD16, CD13, CD33, CD123, CD11c, CD14, CD64, CD15, cCD3, cMPO, cCD79a, and mLambda, which were identified as abnormal monoclonal B lymphocytes (Fig. 2). Based on the flow cytometry detections, two groups of abnormal cells existed in all the nucleated cell, of which CD19 + cells identified as abnormal monoclonal B lymphocytes (CD5 + CD10-) accounted for 58.10% of all the cells. The phenotype of the cells was also in consistence with B-CLL (score 5). In addition, 14.22% abnormal myeloid primitive cells can be observed, which needed further examinations for verification.
As shown in Fig. 3A, FISH detections indicated that no rearrangement was detected in TP53/CEP17 (17P13.1/17p11.1-q11.1), RB1 (13q14), ATM (11q22.3), 12 (12p11.1-q11.1), and MYB (6q23) genes. However, regarding MLL (11q23) gene, the data showed that three signals were detected (one red and two green) and the positive rate was 39.5%. MLL gene was labeled with red signal at 3’ end and green signal at 5’ end. The fusion of genes will result in two yellow signals. The detection of one red and two green signals((5’MLL × 2, 3'MLL × 1) [79/200]) represented the occurrence of gene rearrangement in MLL gene ((KMT2A) (Fig. 3A).
Molecular detection using RT-qPCR showed that of all the 54 fusion genes related to leukemia, only KMT2A-ELL fusion gene t (11:10) (q23:p13.1) was detected (Fig. 3B). Further detections also showed that the sample were negative for B lymphocyte immunoglobulin heavy chain (IGH) gene rearrangement and T lymphocyte receptor (TCRβ/TCRγ) gene rearrangement were.
High-throughput sequencing with the patient’s bone marrow sample detected the expression status of all exon regions of 193 genes closely related to hematological tumors, including single nucleotide variation (SNV) and small insertion/deletion (INDEL). The results showed a missense mutation of p.V157F in the coding sequence of TP53 gene, and a frameshift mutation of p.V220fs and p.A382fs in the coding sequence of WT1.