Study On Standardization Of Anti-Xa Activity Assays And Clinical Application

Part I:Investigation of the status of anti-Xa activity assays in clinical laboratoriesObjective:To investigate the current status and problems of anti-Xa activity assays in domestic clinical laboratories and provide reference for quality improvement.Methods:Considering the regional distribution and instrument types and other factors,the appropriate laboratories were selected to investigate the status quo of anti-Xa activity assays.①Questionnaires were designed and distributed online.The use of instruments and reagents,the number of specimens,the status of performance verification and internal quality control were counted to analyze the current problems that might affect the quality of the results in clinical laboratories.②Investigation substances of different concentrations were prepared and sent out by express,including 202101 to 202102 for monitoring unfractionated heparin(UFH)and 202103 to 202104 for monitoring low molecular weight heparin(LMWH),to analyze the results of anti-Xa activity assays in different measurement systems and the applicability of investigation substances.Results:42 laboratories were selected to investigate the status quo.37 laboratories responded to the questionnaire information and the results of investigation substances.①According to the questionnaire feedback,there were 27 laboratories and 31 laboratories conducting anti-Xa activity assays monitoring of UFH and LMWH,respectively.There were 2 2 laboratories and 26 laboratories using matched instruments and reagents,respectively.There were 21 laboratories and 23 laboratories conducting performance verification,respectively.There were 25 laboratories and 28 laboratories that regularly carried out internal quality control,respectively.②According to the returned results of the investigation substances and grouped by reagent brands,the intergroup coefficients of variation(CVs)of anti-Xa activity assays monitoring UFH were 6.3%to 20.2%and the intergroup CVs of anti-Xa activity assays monitoring LMWH were 7.8%to 14.8%.Conclusions:There are few laboratories carrying out anti-Xa activity assays,and there are various problems such as incompatibility of instruments and reagents,poorly performed quality control and unclear indicators.It is necessary to carry out further research on the standardization of anti-Xa activity assays.Part Ⅱ:Study on the standardization of anti-Xa activity assaysObjective:According to the problems found in the current investigation and latest edition of domestic and foreign guidelines,the performance verification methods and evaluation criteria of anti-Xa activity assays monitoring LMWH were studied,and the influence of different calibration methods on the results of anti-Xa activity assays was discussed,which could provide reference for promoting the standardization process.Methods:Referring to the latest edition of the Clinical and Laboratory Standardization Institute(CLSI)series documents,National Health Standards,and reagent manufacturers’instructions,we conducted the studies with Stago STA-R Evolution instrument and supporting reagents.①The performance verification schemes including precision,linear range,trueness and the limit of detection were designed,and the appropriate evaluation criteria were studied.②Calibration methods studies included UFH calibration and hybid calibration,LMWH calibration and hybid calibration method test results comparison.When monitoring UFH,the plasma of clinical patients(n=43)and 9 reference substances monitoring UFH were used to detected anti-Xa activity assays in UFH calibration and hybid calibration,respectively.When monitoring LMWH,the plasma of clinical patients(n=43)and 9 reference substances containing LMWH were used to detected anti-Xa activity assays in LMWH calibration and hybid calibration,respectively.The allowable bias was set with reference to the current technical level,the absolute bias was ±0.05 IU/mL(anti-Xa activity≤0.40 IU/mL),the relative bias was ± 12.5%(anti-Xa activity>0.40 IU/mL).Good comparability was achieved when more than 90%of the sample biases felled within the allowable range.Results:①The performance verification studies showed that the CVs of intra-and interbatch were 1.6%to 4.3%and 1.6%to 6.1%for precision verification samples(0.15 IU/mL to 1.47 IU/mL),respectively.The slope of linear regression equation was 1.0245,r was 0.9993,the absolute biases of sample results were-0.04 IU/mL to-0.03 IU/mL,and the relative biases were-3.7%to 2.8%.The absolute bias of trueness verification was 0.00 IU/mL,and the relative biases were-4.4%to 2.5%.The biases of the limit of detection results were-0.03 IU/mL to 0.01 IU/mL,all of which felled within the allowable range.②The results of calibration methods studies showed that the absolute biases between UFH calibration and hybrid calibration of clinical specimens were-0.02 IU/mL to 0.02 IU/mL,the relative biases were-5.2%to 2.7%and the comparablity compliance rate was 100%.Compared with LMWH calibration and hybrid calibration,the absolute biases of clinical specimens were-0.02 IU/mL to 0.02 IU/mL,the relative biases were-5.7%to 3.4%,the comparablity compliance rate was 100%.Conclusions:①The performance verification results of STA-R Evolution automatic coagulation analyzer to detect anti-Xa activity assays monitoring LMWH met the requirements of manufacturers or the criteria set in the study.②When STA-R Evolution automatic coagulation analyzer and supporting reagents were used for heparin therapy monitoring,the biases of samples detected by UFH calibration and hybid calibration,LMWH calibration and hybid calibration methods were all within the allowable range.The selection of performance verification items,sample preparation methods,evaluation criteria and the comparison results of calibration methods in this study can provide references for clinical laboratories to carry out standardized performance verification and reasonable selection of calibration methods.Part III:Discussion on the clinical application of anti-Xa activity assaysObjective:The clinical application of anti-Xa activity assays were conducted among the elderly patients with LMWH to prevent thrombosis after joint arthroplasty to explore the correlation between the peak and trough values of anti-Xa activity assays and thrombosis and bleeding.M ethods:According to the clinical treatment measures,enoxaparin was used to prevent thrombosis in elderly patients on the first day after joint arthroplasty.Appropriate inclusion and exclusion criteria were set in the study.Blood samples were collected from enrolled patients on postoperative day 3(peak value)and postoperative day 5(trough value).The plasma was separated within 1 hour,and frozen to-80 ℃ refrigerator within 4 hours until the detection of anti-Xa activity assays.Whether peak and trough values of anti-Xa activity assays were within the recommended range(peak:0.30 IU/mL to 0.70 IU/mL,trough:0.10 IU/mL to 0.20 IU/mL),and the correlation between peak and trough values and thrombosis and bleeding were analyzed.Results:A total of 30 patients providing 30 peak plasma and 30 trough plasma were included.There were 19(63.3%)peak plasma levels of anti-Xa activity assays within the recommended range,and 4(13.3%)trough plasma levels of anti-Xa activity assays within the recommended range.There were 3 patients with thrombosis,and the anti-Xa activity assays of trough plasma were lower than the recommended range.Bleeding occurred in 2 patients,and the peak plasma levels of anti-Xa activity assays was within the recommended range in 1 patient and above the recommended range in 1 patient.Conclusions:For the elderly patients with LMWH prevention of thrombosis after joint arthroplasty,the results of anti-Xa activity assays in peak plasma and trough plasma were available to judge the drug efficacy and remind the clinical to monitor the occurrence of bleeding or thrombosis,but more sample size is still needed.