| BackgroundLymphocyte transformation test(LTT)is a method to measure the function and status of immune cells of a certain organism by stimulating the proliferation of lymphocytes.The principle is that T cells contain mitogen receptors on their surface and can be stimulated by antigens or mitogens such as phytohaemagglutinin(PHA),cuttlefish protein A(ConA)and American merchant(PWM),etc.After stimulation,mainly cell metabolism and morphology change manifested by increased intracellular protein and nucleic acid synthesis and a series of proliferative reactions,such as cell enlargement,which is 3～4times larger than mature lymphocytes,increased cytoplasm,and vacuolation of cytoplasm,loose nuclear chromatin,obvious nucleoli,and transformation into lymphoblastoid cells.LTT is a widely used test,and it is believed that PHA-induced lymphocyte transformation is mainly a characteristic of thymus-derived T lymphocytes,and the rate of T lymphocyte transformation can reflect the cellular immune status of the body.Therefore,in vivo T-lymphocyte tests are often used in clinical practice to diagnose infections of certain pathogenic microorganisms and cellular immune deficiencies,as well as to observe changes in the course of treatment and to determine prognosis.The attempt of this paper is to improve the method of detecting lymphocyte transformation by observing the flow cytometry and molecular characteristics of lymphocyte transformation based on the morphological counting method.And we try to use the method to detect lymphocyte transformation ability in healthy physical examiners,lymphoma and leukemia,analyze the significance of its clinical application and explore the potential of its clinical application.Methods1、 Whole blood method was used to culture whole blood cells with 1640 culture solution of 10% calf serum,and the experimental samples were stimulated with pha at37℃ 5% CO2 for 72 h,while a blank control was set up,and the erythrocytes in the culture solution were lysed with lysis solution after culture,and the obtained leukocytes were resuspended and observed by flow cytometry,and the lymphocyte transformation culture was observed by setting up a quadrant gate The percentage change of each quadrant gate of lymphocytes in the two groups was observed by setting up quadrant gates,and the differences between the experimental group and the blank group were analyzed using the independent sample t-test of SPSS software,and the most comparable quadrant gate data in the quadrant gates were identified using mean,standard deviation,and comparative value of change magnitude analysis and comparison.2、 DNA extraction was performed using the cytosol after culturing by the above culture method,and the DNA extracted from the blank group and the experimental group were subjected to telomere length determination using qPCR,and the telomere length measured in each blank group and the experimental group were subjected to paired sample t-test to observe the molecular biological characteristics of the lymphocyte transformation experiment.3、 Samples were cultured by using the above culture methods and compared with the classical morphological counting method.The lymphocyte percentages of the two groups after culture were observed using a blood cell analyzer in the samples from healthy physical examiners,and the differences in lymphocyte percentages between the experimental and control groups were analyzed using the independent sample t-test of SPSS to calculate the lymphocyte transformation rate,which was calculated as(lymphocyte percentage of experimental group-lymphocyte percentage of blank group)/lymphocyte percentage of experimental group,and the obtained lymphocyte conversion rate was compared with the normal value of morphological counting method to prove the feasibility of the test.The stability of the experiment was then demonstrated by comparison between different batches.4、 A newly established method was used to observe the differences in lymphocyte conversion rates between these two groups of peripheral blood from hematologic patients and healthy individuals.Then the T-test and normal distribution test were used to compare the percentage of lymphocytes and lymphocyte conversion rate between the case and control groups,which proved the clinical value of the method.We compared the lymphocyte conversion rate of all hematological patients with the peripheral blood culture of healthy people and observed whether the difference was statistically significant;and compared the peripheral blood culture of patients with different hematological diseases,i.e.leukemia and lymphoma,with that of healthy people and observed whether the difference was statistically significant.Results1.The experimental data after gating was observed by flow cytometry,and it was found that the experimental data of the blank group and the experimental group had significant differences,and the lymphocyte transformation could be observed by flow cytometry,and the “%ALL” in quadrant IV was the most comparable with the greatest difference.2.The analysis of the determination of telomere length between the blank group and the experimental group was examined and it was found that the changes between the two groups in their paired analysis showed an increasing trend and the changes were significantly different with a p-value of 0.023,which is less than 0.05 and the difference was statistically significant.3.The percentage of lymphocytes in the experimental group was 37.046±12.654%compared with 15.653±9.469% in the blank group,which was significantly higher,as can be observed with the blood cell analyzer.And the lymphocyte conversion rate of 55 healthy physical examiners analyzed by SPSS was 0.543 ± 0.269,while the normal value of the lymphocyte conversion rate by morphological counting was 0.6-0.8,and their results were also largely consistent.Since the blood of this experimental sample could not be stored for a long time,the experiment was divided into five batches of healthy normal human blood culture specimens with a hematocrit analyzer,and the experiment was considered stable if there were no significant differences among the five batches of healthy normal human blood specimens.The experimental results were analyzed by ANOVA,and the P value was 0.892,which was greater than 0.05,and there was no significant difference between the five batches.4.The 44 copies of the diseased group(including 28 cases in the leukemia group and 16 cases in the malignant lymphoma group)and 30 copies in the healthy group were cultured at the same time,and the results of the data were made in Q-Q plots of the normal distribution test,and the P values of the normal test K-S test for the leukemia group,lymphoma group and healthy group were 0.305,0.272 and 0.641,respectively,and the P values were greater than 0.05,indicating that all three groups were normally distributed.The mean value of lymphocyte conversion rate of samples from patients suffering from hematological diseases was compared with that of samples from healthy individuals,and the results were significantly different(P<0.05.)The leukemia and lymphoma groups were compared with the healthy group,respectively,and the mean value of lymphocyte conversion rate of samples from the leukemia group was compared with that of the healthy group P<0.05,and the mean value of lymphocyte conversion rate of samples from patients suffering from lymphoma was compared with that of samples from healthy individuals P<0.01,all with significant differences.Conclusions1.It was confirmed that lymphocyte transformation can be observed by flow cytometry.2.The blood cell analyzer observation lymphocyte transformation test was successfully established.3.The blood cell analyzer observation lymphocyte transformation test can be applied in the process of diagnose and determine diseases,and has great clinical application value. |
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