Establishment And Preliminary Clinical Application Of Flow Cytometry In Detecting Renal Injury Markers

Background:Cytometric Bead Array(CBA) is a new molecular biology technology, has been widely applied in medical field for its high sensitivity, strong specificity, advantages of high speed analysis. Cys C, NGAL, IL-18 and KIM-1 were proved to be the model biological markers of acute kidney injury(AKI). A number of studies have shown that they have a close association with chronic kidney diseases(CKD), but these researches only aim at one or two markers. There have no report about combined detection of the four markers in CKD diagnosis. In the past, these markers were usually detected by ELISA, but the complex operation, non-combined detection and need for larger sample limited its use. Here, we establish a CBA method to detect four kidney injury markers through mature antibody labeled microspheres and flow cytometry, simultaneously.Purpose:To establish a method to simultaneous detect multi-markers of renal injury by CBA,proceed the method evaluation, screen CKD plasma samples and preliminary study the role of multi-markers CBA detection in diagnosis and prognosis of CKD.Methods:1. Paired antibodies selection: take four markers as antigens, choose for capture antibody and detection antibody matching each antigen, each paired antibody must be directed against different antigen and formerly used in ELISA kit, luminex or CBA experiment.2. Functional microspheres coating and stability verification: first of all, four markers’ capture antibody were coupled on polystyrene microspheres containing different fluorescence. After the coupling, PE anti-Ig detector was used to detect whether the antibody and microspheres binding or not. Then verification operations were also repeated to evaluate the stability of coated microspheres.3. Multi-marker CBA detection establishment: proceeding pre-experiment by using Cys C and NGAL, under the premise of sufficient PE-Streptavidin, exploring and optimizing their highest detection limit and detection antibody concentration,improving the experimental conditions and experimental procedures at the same time.According to the result of pre experiment, adjusting and optimizing the maximum detection limit and detection antibody concentration continuously. Then the concentration of PE streptavidin was determined by titration. We optimized the four markers standard curves and finally established the method of multi-markers detection of kidney injury by CBA.4. Methodology performance evaluation: performing evaluation referencing to the NCCLS and according to the characteristics of CBA, including sensitivity, detection range test, repeatability test, interference test and recovery test. Sensitivity is the lowest limit of detection, the lowest concentration(0 except) tube of the four markers were double diluted for CBA examination, after completing the detection, recorded MFI values, the corresponding concentration of the minimum MFI value that can be distinguished is the sensitivity. According to the method established for detecting four markers, the highest detection limit were known, sensitivity is the lowest detection limit, ultimately determine the detection range of four biomarkers. Three sets of parallel standard solution were operated at the same time for within-run repeatability test, repeated the content detection of the four markers after 24 h and 48 h for between-run reproducibility test. Evaluated the within-run repeatability test by coefficient of variation(CV) and evaluated the between-run reproducibility test by relative deviation and correlation coefficient. The coefficient of variation within 5%,the relative deviation within ± 10% and the correlation coefficient above 95% is considered acceptable. According to the sample types, this study selected albumin as interfering substance, detecting four markers’ content in single standard solution and mixed standard solution by CBA, evaluated two types of the interference test by interference rate. The interference rate within ±5% is considered acceptable. In this study, a trace amount of known concentration was added to the mixed standard solution, and the recovery rate was calculated according to the concentration of the base sample tubes and analysis sample tubes. The recovery rate betweens 95% to105% is considered acceptable. According to the four kinds of markers’ ELISA kit,the research performed performance index evaluation in CBA and ELISA method.5. Preliminary clinical application of multi-markers CBA detection: CBA and ELISAmethod were used to determine the content of four markers in plasma of CKD patients of different disease degree(Scr is less than or equal to 178umol/L placed in group A,Scr is more than 178umol/L placed in group B) and normal controls. SPSS16.0software was used for statistical analysis, the measurement data was analyzed by normal distribution test and the test of homogeneity of variance. Normal distribution data was expressed in the form of x ±SD, and the analysis of the difference among groups was evaluated by t test. Non normal distribution data was expressed in the form of the median(four percentile interval), the analysis of the difference between groups was evaluated by Kruskal-Wallis rank sum test. Correlation analysis between four markers and GFR/Scr was conducted by Spearman correlation analysis. To evaluate the accuracy of combined detection of four markers in the diagnosis of CKD,ROC curve and a Score-based classification method was used. X2 test was used to compare the clinical data between groups. And P<0.05 was believed that the difference was statistically significant. Finally we evaluate the coincidence degree of four markers between CBA and ELISA, and discuss the role of four markers in the diagnosis and prognosis of CKD.Results:1. The coated effect and stability was respectively test on 0day, 7day, 1month,2month, 3month and 4month after the microspheres being coated. The results showed that the capture antibodies of the four markers were coated on microsphere successfully, and coated microspheres have good stability.2. We successfully established the method to simultaneously detect multi-markes of kidney injury by CBA, standard curve correlation coefficient R2 of four markers can reach up 99.7%. The optimal dilution ratio of standards is 3 times, and the highest detection limit of Cys C, NGAL, IL-18 and KIM-1 were 100000pg/ml, 50000pg/ml,25000 pg/ml and 80000pg/ml respectively. The optimal concentrations of detection antibody of Cys C, NGAL, IL-18 and KIM-1 were 0.05 g/ml, 0.1 g/ml, 0.05 g/ml, 0.1g/ml respectively. The optimal concentration of PE-streptavidin was 0.125 g/ml.3. The sensitivity of Cys C, NGAL, IL-18 and KIM-1 were Cys C 3.81pg/ml,7.62pg/ml, 0.24pg/ ml, 1.52pg/ml respectively. Their detection range was3.81-100000 pg/ml, 7.62-50000 pg/ml, 0.24-25000 pg/ml, 1.52-80000pg/ml respectively. The coefficient of variation of the within-run repeatability test was to be within acceptable limits(CV<5%). between-run repeatability test results showed the relative deviation was in the acceptable range(relative deviation <10%) at 24 h, while the relative standard deviations are not acceptable and the standard curve correlation coefficient is low(R2<85%) in 48 h. In addition, the interference test results showed that four markers had no obvious interference(interference rate within +5%), albumin within 100g/L has no obvious effect on the combined detection of four markers. The recovery test results showed that percent recovery of the four markers were all within100%+5%.4. The content of four markers detected by CBA and ELISA method was different and the difference was statistically significant(P<0.05); but with the same method of statistical analysis, the test results of two methods can draw the same conclusion. The levels of Cys C and NGAL were increased gradually among normal control group、group A and group B, and the difference was statistically significant(P < 0.001); The difference of IL-18 levels was not statistically significant between groups; KIM-1levels in group A significantly increased, and the difference was statistically significant compared with the normal control group and group B(P < 0.001).Correlation analysis showed that Cys C, NGAL was positive correlation with Scr(P <0.001), and was negative correlation with GFR(P < 0.001); KIM-1 showed negative correlation with Scr(P < 0.05), and positive correlation with GFR(P < 0.05); IL-18 was no significant correlation with serum creatinine and GFR. ROC curve analysis showed that in CBA examination, the AUC of Cys C, NGAL, IL-18 and KIM-1 were0.917, 0.951, 0.607 and 0.948 respectively, The AUC of combined detection of Cys C+NGAL+KIM-1 was 0.997; While in ELISA, the AUC of Cys C, NGAL, IL-18 and KIM-1 were 1.000, 0.956, 0.640 and 0.861, and the AUC of Combined detection of Cys C+NGAL+KIM-1 was 0.999. When detected by two methods, the AUC of combined three indicators(Cys C、NGAL and KIM-1) was higher than combined the four.Conclusion:1. The method to multi-marker CBA detection of kidney injury was establishedsuccessfully, methodological evaluation was good and in line with international requirements and relevant professional requirements of CBA.2. Cys C、NGAL and KIM-1 could be used as indicators of sensitivity and specificity in diagnosis of CKD, the changes of the former two levels could be used to evaluate the severity of CKD, while KIM-1 had high accuracy in diagnosis of mild CKD.3. The diagnostic value of cooperative detection of Cys C, NGAL and KIM-1 was greater than cooperative detection of Cys C, NGAL, KIM-1 and IL-18.