In order to solve the problem of the micro flow cell clogging, and to improve the reliability of the flow cytometry system, a new method was proposed for hydrodynamic self-cleaning system. By analyzing the flow cell focus principle, we considered that to obtain stable single cell flow, the stable pressure in the flow chamber must be ensured. Therefore, we established a diagnosis method of clogging by the pressure detecting, and designed a self-cleaning system. Then we built up corresponding experimental systems. Experiments and testing showed that the self-cleaning system could improve the flow and resolve the clogging problem.
The process of multi-parametric flow cytometry data analysis is complicate and time-consuming, which requires well-trained professionals to operate on. To overcome this limitation, a method for multi-parameter flow cytometry data processing based on kernel principal component analysis (KPCA) was proposed in this paper. The dimensionality of the data was reduced by nonlinear transform. After the new characteristic variables were obtained, automatical clustering can be achieved using improvedK-means algorithm. Experimental data of peripheral blood lymphocyte were processed using the principal component analysis (PCA)-based method and KPCA-based method and then the influence of different feature parameter selections was explored. The results indicate that the KPCA can be successfully applied in the multi-parameter flow cytometry data analysis for efficient and accurate cell clustering, which can improve the efficiency of flow cytometry in clinical diagnosis analysis.
ObjectiveTo develop a method to quantitatively determine the microparticles (MP) from different sources in plasma by nine-color flow cytometry. MethodsAnnexin-V and 8 antibodies including CD235a, CD41a, CD45, CD34, CD66b, CD20, CD3 and CD14 were used to establish nine-color flow cytometric panel.Platelet poor plasma samples were single-stained and stained with 1 of 8 antibodies lacking respectively, and then we determined the detector voltages and compensations.From December 2014 to January 2015, we detected and analyzed 10 plasma samples from normal adults, and repeatability test and dilution tests were done. ResultsIn staining lacking 1 of 8 antibodies, the percentage of positive MP populations change was all less than 15% based on the population number in single-stained experiment.In dilution tests, there were good linear correlations between MPs from platelets and erythrocytes.In repeatability test, the coefficient of variation of MP from erythrocytes, platelets and granulocytes was all less than 10%.In the platelet poor plasma samples from normal adults, MP from platelets, erythrocytes, endotheliocytes, monocytes, granulocytes, B and T lymphocytes could be detected, and the average concentration of them were respectively 132.6/μL[(60.6-288.9)/μL], 35.4/μL[(22.0-99.7)/μL], 21.6/μL[(3.3-45.5)/μL], 13.9/μL[(7.3-35.1)/μL], 60.0/μL[(22.5-101.2)/μL], 21.9/μL[(6.0-33.4)/μL]and 1.2/μL[(0.7-2.8)/μL]. ConclusionsQuantitatively determining MP from different sources in plasma by nine-color flow cytometry has been successfully developed.This method is simple and fast, and can be applied in clinical detection.
Objective To detect the difference between the peroxidase (POX) by cytochemical staining and cytoplasm myeloperoxidase (cMPO) by flow cytometry in acute leukemia cells, and provide a more accurate basis for the classification of leukemia. Methods The positive rate of POX in acute leukemia cells was detected by cytochemical staining. The positive rate of cMPO in acute leukemia cells was detected by flow cytometry. Then the positive rate of POX and cMPO, and the positive cells score were analyzed. Results The positive rate and the positive cells scores between POX and cMPO in acute lymphoblastic leukemia were significantly different (P<0.05), the positive rate and the positive cells scores of POX were significantly higher than those of cMPO. The positive rate between POX and cMPO in acute non-lymphoblastic leukemia (ANLL) had significant differences (P<0.05), the positive rate of cMPO was higher than that of POX; but no difference was found between POX and cMPO positive cells scores in ANLL (P>0.05). In acute myelocytic leukemia (AML)-M1 subtype, significant difference was found in the positive rate between POX and cMPO (P=0.006); cMPO positive rate was significantly higher than that of POX, but the POX positive cells score was significantly higher than that of cMPO (P=0.001). There were no significances of positive rate and positive cells score in AML-M2, AML-M3, AML-M4, AML-M5 subtypes between POX and cMPO (P>0.05). Conclusions There are not major differences between positive rate of POX and cMPO, as well as the positive cells scores in acute leukemia, especially acute myelocytic leukemia. We can choose the better method according to the actual situation and the sensitivity requirements. The two methods should be replenished by each other and used alternately.
Objective The aim is to sort CD90+ subpopulation cells in human liver cancer cell lines and investigate efficiency of magnetic cell sorting (MACS) on sorting the liver cancer stem cells. Methods ①Expressions of CD90. Immunohistochemical method was used to determine the expressions of CD90 in normal liver tissues in 8 cases, liver cancer and adjacent liver cancer tissues in 58 cases. ②Screened the cell lines. Huh-7, MHCC97-H, Bel-7402, and SMMC-7721 cell lines were divided into blank control group and experimental group (5.5×105 cells per hole, 1 hole), cells of the experimental group were added with 5 μL CD90–PE while cells of the blank control group were treated with 5 μL CD90–PE non fluorescent antibody. Determined the proportion of CD90+ cells in the 2 groups by flow cytometry (FCM). ③MACS. Huh-7 and MHCC97-H cell lines were labeled with magnetic beads respectively and sorted by MACS, 1 mL cell suspensionsorted by magnetic sorting (MS) was collected as CD90– group, and 1 mL PBS after MS wash was collected as CD90+ group, as well as blank control group and experimental group. Determined the proportion of CD90+ cells in 4 groups by FCM. Two times of MACS were performed in Huh-7 cells. ④Serum free culture and serum culture. Huh-7 cells were divided into serum-free culture group and serum culture group (1 hole), and proportions of CD90+ cells were determined by FCM at 1 week after culture. Results ①The positive rate of CD90 was 0 (0/8), 65.5% (38/58), and 20.7% (12/58) in normal liver tissues, liver cancer tissues, and adjacent liver cancer tissues respectively, and the positive rate of CD90 was higher in liver cancer tissues than those of normal liver tissues (χ2=6.78, P<0.05) and adjacent liver cancer tissues (χ2=20.83, P<0.05). ②For Huh-7, MHCC97-H, SMMC-7721, and Bel7402 cell lines, the proportions of CD90+ cells in the experimental group was 0.851%, 1.090%, 2.710%, and 4.050% respectively, the proportions of CD90+ cells in the blank control group was 0.241%, 0.688%, 1.890%, and 2.080% respectively, so we chose Huh-7 and MHCC97-H cell lines to perform MACS. ③Results of MACS for Huh-7 cell line. For the first MACS, the proportions of CD90+ cells in the blank control group, experimental group, CD90– group, and CD90+ group was 0.241%, 0.851%, 0.574%, and 1.100% respectively. For the second MACS, the proportions of CD90+ cells in the blank control group, experimental group, CD90– group, and CD90+ group was 0.032%, 0.961%, 0.426%, and 9.700% respectively. Conclusions The normal liver tissues do not express the CD90, but the liver cancer tissues express CD90 highly. There is a few CD90+ cells in Huh-7 and MHCC97-H liver cancer cell lines. The MACS has a certain effect on improving the proportion of CD90+ cells in the cell lines. The serum-free suspension culture has no effect on enriching CD90+ cells.