Establishment Of A Method For Measurement Leukocyte Phagocytosis Based On 16S RDNA And It's Clinical Significance

Objectives:In order to accurately evaluate the phagocytic ability of the body and explore the pathogenesis of the diseases with phagocytic dysfunction susceptibility to infection,we established a new method for accurate and objective and quantitative measurement phagocytosis of leukocyte based on 16S rDNA.The method was used to evaluate the phagocytic function of healthy young people and elderly people,and a new criteria,bacterial DNA change rate(CRD),was established to determine the phagocytosis of leukocytes in whole blood.The reliability of the new method is evaluated from the aspects of specificity,resolution and repeatability.The correlation between the method and microscopic method is compared to ensure the accuracy of our method in order to objectively evaluate the basic immune status of the body.The total phagocytic capacity of smokers and patients with chronic obstructive pulmonary disease(COPD),diabetes mellitus were determined by the method.At the same time,the phagocytosis ability of single white blood cell was evaluated based on white blood cell counts,and the pathogenesis of diseases susceptibility to infection were discussed in combination with relevant clinical laboratory indicators.Moreover,the relationship between diseases and phagocytosis of single white blood cell was analyzed,in order to explore the relationship between the development of clinical diseases and phagocytosis at the cellular and molecular level.Methods:1.Blood culture specimens of 21 positive cases and 12 negative cases were collected in the clinical laboratory.The bacterial strains in the blood culture bottles were detected by real-time PCR based on 16S rDNA gene.To determine the specificity of16S rDNA bacterial universal primers and minimize the waiting time between positive blood culture and pathogen detection,and quantify the sensitivity of real-time PCR for detection of pathogens in blood culture bottles.2.Bacteria were incubated with whole blood leukocytes.Based on 16S rDNA bacterial universal primers,real-time PCR was used to measure the phagocytosis of leukocytes,and the rate of change of bacterial DNA(CRD)was considered as a criterion of phagocyte function.The whole blood of a healthy volunteer was taken to interact with E.coli five times at same time on different days to measure leukocyte phagocytosis and verify repeatability of the method.At the same time,the leucocyte phagocytosis rate of healthy youth group and aged group were measured by traditional microscopy and the new method.The difference of leucocyte phagocytosis between healthy youth group and aged group,smokers and non-smokers,were compared,and the clinical application value of the new method was evaluated.3.Our method was used to measure the phagocytic function of leucocytes in 64patients with non-disease-selective leukocytosis and 59 healthy volunteers,and to evaluate the effect of the counts of leucocytes on the phagocytosis.Blood samples from20 COPD patients and 20 healthy subjects were collected to measure phagocytosis of leukocytes.The phagocytosis of single cell was calculated according to the neutrophil count,and the phagocytosis of white blood cells in disease group and healthy group were compared.Then Logistic regression was used to analyze the association between disease and single neutrophil phagocytosis.4.The white blood cells were incubated with the test strains to simulate the phagocytosis in vivo.The phagocytic rates of total white blood cells,neutrophils and monocytes in diabetic patients were measured respectively,and the phagocytic ability of single monocytes and neutrophils were evaluated.In addition,the factors that might affect the phagocytosis of cells were analyzed and discussed.Results:1.16S rDNA universal primers had a highly specific for bacterial detection.21blood culture positive specimens were collected,including 10 gram positive cocci and11 gram negative bacilli.After real-time PCR detection of bacterial DNA in culture medium,19 cases were positive results,with Ct value ranging from 8.46-30.12,and 2cases were negative results without amplification curve detected.Among 12 blood culture negative specimens,1 case showed obvious amplification curve,while the other11 cases showed no amplification curve,and the results were negative.The sensitivity of real-time PCR to the detection of blood culture specimens was 91%in the experiment.2.The established real-time PCR method has a high sensitivity,objectivity and reproducibility in measuring leucocyte phagocytosis,and the new method has a significant correlation with microscopic method(R=0.818,P<0.01).Phagocytosis of leucocyte was measured from a healthy volunteer five times,the results were 0.9754,0.9222,0.9410,0.9514 and 0.9094,respectively.The mean was 0.9399,the standard deviation was 0.0257,and the CV(Coefficient of Variance)was 2.7%,CV%<10.The results show that this method has a good reproducibility in measuring leukocyte phagocytosis.Analysis by independent sample t-test,the results showed that the rate of leukocyte phagocytosis in the aged group was significantly lower than that of the young healthy group(P<0.05).3.Based on the count of white blood cells,neutrophils and monocytes,we compared the phagocytic rate between smokers and non-smokers.After statistical analysis,the phagocytosis rate of single mononuclear cells was significantly lower than that of non-smoking group(P<0.05).Spearman bivariate correlation analysis found that there was a significant positive correlation between the phagocytic rate and smoking years(R=0.488).4.The phagocytic capacity of the leukocytosis group and COPD group were significantly lower than that of the healthy control groups(P<0.001).We excluded the test sample CRD with abnormal neutrophil counts in blood routine examination(<2×10~9/L or>7×10~9/L).The adjusted samples CRD was also significantly lower in the disease groups than in the healthy control group(P<0.001).Moreover,there was a significant difference in single neutrophils phagocytic capacity between the disease group and healthy control group(P<0.05).Logistic regression analysis showed that phagocytosis of single neutrophils and CRD were closely related to diseases(P?0.05).5.The phagocytosis of total white blood cells,neutrophils,monocytes and single white blood cells in diabetic group was significantly lower than that in healthy control group(P<0.05),while there no significant difference of the phagocytosis ability of single neutrophils and mononuclear cells between diabetic group and healthy control group.Taking the phagocytosis rate of neutrophils and monocytes as the interactive variable,logistic regression analysis showed that the two variables could not produce interaction effect,they played their respective roles independently,and the phagocytosis did not affect each other.Conclusions:1.Based on 16S rDNA bacterial universal primers,real-time PCR was used to measure the phagocytosis of leukocytes.The method is accurate,reliable and objective,and has potential clinical application value.2.The bacterial DNA change rate(CRD),a new criterion for evaluating the body's phagocytosis,was established in this experiment and found to be decreased in patients with diseases.The phagocytic ability of individual cells was evaluated based on the cell count,and the phagocytic capacity of the body could be comprehensively evaluated by combining the phagocytic rate of total white blood cells and individual cells,so as to master the function of the innate immune system.3.This method is suitable for measuring the phagocytic ability of subjects susceptible to infection,such as smoking,COPD and diabetes.It can be used to evaluate the basic immune status of the body.4.Phagocytosis of single leukocytes is likely to decrease due to diseases,although the number of leukocytes increases under pathological conditions.