| Objective:There are an increased percentage of immature monocytes in the bone marrow and peripheral blood from Acute Myelomonocytic Leukemia(AMML/M4) patients,and the size and shape of these cells change irregularly. It is difficult to identify the leukemia cells from normal cells in cell morphology. Thus, auxiliary examinations such as myeloperoxidase(MPO) staining and flow cytometry are often applied to distinguish leukemoid monocytes from granulocytes, which provide experimental support for clinical diagnosis of M4. In this study, a microfluidic chip analysis system combined with cell image processing technology was established to detect specific markers in leukemia cells in M4 patients, to achieve intelligent acquisition individual information.Methods:(1) The microfluidic cell chip was designed and prepared for single-cell analysis of the expression of cell surface antigen and the activity of intracellular enzyme.(2) A total of 48 clinically diagnosed M4 patients and 52 patients with normal myelogram were included as the test and control groups, respectively.(3) A method based on microfluidic chip approach was established to detect CD14+ monocytes and determine the positive rate and degree of MPO expression in the cells. Then, the microfluidic chip technique was used to compare the MPO expression in CD14+ monocyte from M4 patients and that from control group.(4) Cell image processing models were set up for image analysis of cell signals acquired by microfluidic single chip real-time detection system to quantitative analyze MPO expression in monocytes of M4 patients and control subjects.Results:(1) The designed microfluidic single cell analysis chip allowed the entry of granulocytes into the corresponding microfluidic channels. Thus, blood cells were separated. Numerous ghost corpuscles surrounded the separated white blood cells(WBCs). WBC morphology did not show obvious changes.(2) We compared the microfluidic chip technique with traditional methods and found that the peripheral blood leukocyte cells separated by microfluidic chip methodwere smaller than the cells stained by traditional benzidine method. Moreover, the morphology of the stained MPO particles is different. But results indicate that there was no significant difference in the positive rate and degree of MPO expression in the cells determined by the two methods(p > 0.05).(3) The positive rate of MPO expression and the activity of CD14+ monocytes in the bone marrow of M4 patients were significantly higher than those in the bone marrow from the control(P<0.05).(4) The image processing model that we established could comprehensively analyze the cell signals collected by microfluidic chip. Different models are suit for different dyeing images and that is B/R/I component model for DAPI /CD14/MPO staining images respectively.(5) With I component as a clinical reference value to analyze the MPO expression in CD14+ monocyte from M4 patients, we found that the output result contains large amount of information and could visually display the MPO expression of each CD14+ monocytes and quantify its expression.Conclusions:(1) We reported a stable and simple method based on microfluidic chip technology to analyze the dual expression of CD14 and MPO in monocytes. The MPO expression in CD14+ monocyte from M4 patients was significantly higher than that from the control group, which could be used as an auxiliary examination marker for clinical diagnosis.(2) A method that with different color processing model for the image which will be detected was reported. HSI color model was used for simulated the color image perceived by human visual system. Then I channel model coupled with R/G/B channels and gray-scale model was established to qualitatively analyze two-dimensional images of the models, which was for selecting the analysis models corresponding to different dyeing methods. Last, three-dimensional images were quantitatively analyzed to intuitively describe the expression of MPO, DAPI, and CD14 in cells. |
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