Comparisons Of Immunoassay Methods In The Therapeutic Drug Monitoring Of Cyclosporine A

Objective:To compare chemiluminescent microparticle immunoassay（CMIA）,electrochemiluminescence immunoassay（ECLIA）,liquid chromatography-mass spectrometry（HPLC-MS/MS）methods for the determination of cyclosporine A（CsA）in the whole blood.And to evaluate the correlation and consistency between the methods.To analyze the characteristics and advantages of two immunoassay methods in the determination of CsA whole blood concentration of bone marrow tranplantation patients.Meanwhile,to discuss the reference value of the immunological analysis in the therapeutic drug monitoring（TDM）in the adjustment of the dosage regimen for clinicians.Methods:Above all,to validate the three methods by methodology.To draw up the standard curve,determinate the lower limit of quantitation,test the accuracy and precision and specificity of the analysis method,verify the recovery rate and the stability.The whole blood samples of bone marrow transplantation patients which reached the steady state of the valley concentration of oral cyclosporine A were collected from our hospital.The three methods mentioned above were used to measure the 101 whole blood samples,and the steady blood concentration of CsA was obtained.The data were calculated by the software MedCalc 18,the results were expressed in x±s.The results were compared with the paired t test to analyze the differences among the methods.To analyze the correlation between each method,linear correlation ananlysis was used and the linear regression equation were obtained by the least square method.To analyze the consistency between the methods by the Passing-Bablok regression analysis and the Bland-Altman deviation map.Results:The detection range of ECLIA,CMIA,HPLC-MS/MS were 30²000μg·L^-1,30¹500μg·L^-1,and 16¹600μg·L^-1.The intra and inter batch variation of both ECLIA and HPLC-MS/MS were less than 15%,while the intra and inter batch variation in CMIA were larger than 15%.The recovery rates of ECLIA,CMIA and HPLC-MS/MS respectively were 101.17%¹07.17%,93%¹06%and 87.80%¹04.29%.The stability of each method complied with the requirements.The three methods were verified and conformed to the requirements of CsA determination.The steady state plasma concentration of ECLIA was 135.4±69.7μg·L^-1,CMIA 195.7±111.8μg·L^-1,HPLC-MS/MS 144.3±77.7μg·L^-1,the test result of CMIA was significantly higher than ECLIA(t=1.97x10^-4,P<0.01)and HPLC-MS/MS(t=7.49x10^-6,P<0.01).The test results of ECLIA and HPLC-MS/MS were not statistically different（t=0.39,P>0.05）.Compared with HPLC-MS/MS,the linear regression equation of CMIA was Y_HPLC-MS/MS=0.6512X_CMIA+16.774 R²=0.8764（r=0.94）.Compared with HPLC-MS/MS,the linear regression equation of ECLIA was Y_HPLC-MS/MS=1.1061X_ECLIA-5.4731,R²=0.9822（r=0.99）.Compared with ECLIA,the linear regression equation of CMIA was Y_ECLIA=0.5721X_CMIA+23.387,R²=0.8424（r=0.92）.The linear regression analysis demonstrated that the three methods had good correlation.Bland-Altman deviation map showed the bias value of HPLC-MS/MS and ECLIA was 8.9,the 95%points were in the confidence internal,HPLC-MS/MS and ECLIA have perfect consistency.The bias value of HPLC-MS/MS and CMIA was-51.5,HPLC-MS/MS and CMIA have poor consistency.The bias value of ECLIA and CMIA was 60.4,hence,ECLIA and CMIA have poor consistency.By Passing-Bablok regression analysis,the consistency of HPLC-MS/MS and ECLIA have good consistency,and P=0.85,proved that the linearity is good and there is no obvious deviation,and the regression equation is Y_HPLC-MS/MS=-6.074（95%CI:-10.2141 to-3.3422）+1.120X_ECLIA（95%CI:1.0917 to 1.1523）.The consistency of HPLC-MS/MS and CMIA have poor consistency,and P=0.85,proved that the linearity is good and there is no obvious deviation,and the regression equation is Y_HPLC-MS/MS=6.718（95%CI:-0.1926 to 14.2097）+0.695X_CMIA（95%CI:0.6452 to 0.7507）.The consistency of ECLIA and CMIA have poor consistency,and P=0.70,proved that the linearity is good and there is no obvious deviation,and the regression equation is Y_CMIA=-21.246（95%CI:-39.5923 to-10.8099）+1.613X_ECLIA（95%CI:1.4886 to 1.7636）.Conclusion:HPLC-MS/MS,ECLIA and CMIA have good correlation.The consistency between ECLIA and HPLC-MS/MS is good.The results of ECLIA and HPLC-MS/MS could be mutually calculated according to the corresponding regression equations.ECLIA and CMIA could provide a reliable basis for clinical rational usage of CsA.