| ObjectiveLymphoma is one of common malignancies.Bone marrow lesions and leukemia often occur in the late stage.Detection of pathological lymphoma,lymphocytes and their subtypes in peripheral blood is helpful for clinical diagnosis and classification of lymphoma.When the lymphocytes undergo a tumor,it difficult to distinguish them from normal cells.Cell morphology and immunological markers are the basis for the detection of lymphoma cells.In this study,a microfluidic cell chip analysis system was established,which combined with a self-designed blood cell image analysis system to detect the morphological and immunological markers of various leukocyte and lymphoma cells and their subtypes in peripheral blood of lymphoma patients.The intelligent analysis of peripheral blood cell types provides assistance for the clinical diagnosis and classification of lymphoma.Methods1.Microfluidic cell chips for sorting lymphoma cells were designed and fabricated.2.B and T lymphoma cell lines were cultured to establish a cell image recognition model.Ten patients with clinically diagnosed lymphoma were collected,and 10healthy volunteers were collected as a control group.3.The hydrodynamic conditions for sorting lymphoma cells are optimized by the distribution of blood leukocytes in the microfluidic chip separation channel.4.The cell image recognition model was established to analyze the cell image signals collected in the microfluidic cell chip,and to realize the two-way detection of lymphoma cell images and immunological markers(CD3,CD19).Results1.Leukocyte and lymphoma cells can be isolated using this microfluidic cell chip.When the flow rate is set to 300 ul/h,there are less than 2 red blood cells in contact with white blood cells,and some cell-free areas larger than red blood cell diameter appear around some white blood cells,and there is no red blood cell aggregation phenomenon.2.Image characteristics of Mino and Jurkat lymphoma cell lines were collected on microfluidic cell chip.Mino cells showed CD19~+,and Jurkat cells showed CD3~+.The results were consistent with immunological markers of B and T lymphoma cells,indicating that microfluidic analysis conditions were suitable for the analysis of immune markers in lymphoma cells.3.Five morphological parameters were analyze of leukocytes and lymphoma cells for energy,entropy,moment of inertia,color and area.Under the parameters of energy,entropy,moment of inertia,color and area,there are significant differences of all kinds white blood cells.Selected morphological parameters can visually reflect the morphology and immune markers of blood cells as an indicator of WBC classification.4.Set the blood cell characteristic parameter analysis process to classify granulocytes,monocytes,B lymphocytes,T lymphocytes,B lymphoma cells,and T lymphoma cells.The detection rate of various types of WBC was more than 80%,and the detection rate of lymphoma cells in clinical patients was 62.5%.Conclusions1.The hydrodynamic conditions of the blood cells in the microfluidic cell chip designed in this study were explored,and the WBC and RBC were separated under the selected conditions,so that the proportion of RBC around the WBC was as small as 1:2.2.The microfluidic analysis conditions are applicable to the intelligent analysis of lymphoma cell morphology in the blood.3.The blood cell image Matlab processing software was designed,and various cell morphological parameters of granulocytes,lymphocytes,monocytes,Mino cells and Jurkat cells under RGB channels were analyzed.4.Based on the difference of CD3 and CD19 markers expressed by various types of leukocytes in blood cells,immunofluorescence labeling was used to identify various leukocyte and lymphoma cell subtypes. |
PDF Full Text Request