Establishment Of A New Protein C Detection System Based On Chromogenic Substrate Assay And Its Clinical Application

Objective:A protein C(PC)activity detection system with high precision,wide linear range and good stability was developed based on the principle of chromogenic substrate method.And the detection performance evaluation and methodological comparison of the detection system were carried out.Clinical specimens were collected to evaluate the clinical application value of the detection system in deep venous thrombosis(DVT).Methods:1.Chromogenic substrate method was adopted to establish a PC activity detection system.The protein C activator protac~②and the chromogenic substrate Pefachrome~②PCa5297 were selected as the main components of reagent Reagent1(R1)and Reagent2(R2),respectively.Optimal concentration ranges of the two were explored through concentration gradient experiments and the composition and concentration of other components in the kit were also explored.XL-3200c Automated Blood Coagulation Analyzer was used as the detection instrument and the detection parameters of the kit on XL-3200c were optimized to establish a complete detection system.Berichrom PROTEIN C kit from Siemens and CN-6000 Automated Blood Coagulation Analyzer from Sysmex were used as the compared detection system.Standard curves of the newly-built PC detection system and Siemens detection system were established through measuring the change value of optical density of a series of concentrations of PC standard plasma.2.The performance of newly-built PC detection system was evaluated.Normal-level control plasma,pathological-level control plasma,and normal mixed plasma were detected to assess the imprecision of this detection system.The sensitivity,linear range,carryover rate and stability of the newly-built PC detection system were also evaluated,and plasma specimens from 152 apparently healthy individuals were tested using the newly-built PC detection system,to establish the reference interval.3.Specimens of 68 DVT patients and 80 healthy people were used for methodological comparison;patients were recruited from the Affiliated Hospital of Qingdao University between November 2020 and June 2021.The PC activity of the two groups was measured using the newly-built PC detection system and Siemens detection system,respectively.Correlation analysis and Bland-Altman analysis were performed on the results,and the ROC curves were used to compare the diagnostic efficacy of the two detection systems for DVT.4.A total of 148 anonymous plasma samples from 80 apparently healthy subjects and 68 DVT patients were subjected to routine coagulation tests,including activated partial thromboplastin time(APTT),prothrombin time(PT),thrombin time(TT),fibrinogen(FIB),and D-dimer(DD)tests.The test results of PC activity were obtained by the newly-built detection system.ROC curve analysis was used to compare the the clinical diagnostic efficacy of PC activity tests and routine coagulation tests for DVT,as well as the diagnostic efficacy of each test carried out separately and in combination.Results:1.The main components and concentrations of the kit were screened after a series of concentration gradient tests,in which the concentration of protac~②in R1 was0.15 U/m L and the concentration of Pefachrome~②PCa5297 in R2 was 1.5m M.The kit was applied to XL-3200c Automated Blood Coagulation Analyzer with the following reaction parameters:the sample addition ratio was S:R1:R2=60μL(1:1):100μL:100μL,the incubation time was 10min and the detection time was 5min.2.The intra-batch,inter-batch and inter-day imprecision of the newly-built PC detection system met the detection requirements.The linearity range was 150.00%～20.00%(R2=0.9993≥0.99)and the carryover rate was 0.95%.The kit of this detection system is thermally stable for at least 14 days after thermal disruption at 37°C,and the relative deviations are all≤5%.The initial reference interval established in this laboratory was79.00%～148.50%.3.The test results of the newly-built detection system were highly correlated with those of the Siemens detection system(R2=0.9771 in the control group,R2=0.9776in the DVT group),and Bland-Altman plots also showed high consistency between the two detection systems.The area under the curve(AUC)for DVT was 0.888 for the newly-built system,which was higher than that of the Siemens system.4.The differences in APTT,PT,TT,FIB,DD and PC test results between the DVT and control groups were statistically significant(P≤0.01).The results of ROC analysis combined with multiple logistic regression analysis showed that the AUC of coagulation routine combined with PC activity assay was significantly higher than that of each item’s separate detection as well as coagulation routine combined detection.Conclusion:1.In this study,a plasma PC activity detection system was developed,which has the advantages of simplicity,rapidity,wide linear range and high sensitivity.2.This study verified that the measurement results of the newly-built PC detection system and the commercially available detection system had significant correlation and high consistency,which certified the reliability of the results of this detection system.3.This study explored the clinical application value of the newly-built PC detection system.The application of this system in combination with routine coagulation tests in the diagnosis of DVT patients could effectively improve the diagnostic efficacy of DVT.