Preparation,Evaluation And Application Of The Erythrocyte Sedimentation Rate And D-Dimer Reference Material

Objectives:Erythrocyte sedimentation rate(ESR)is one of the most common hematology routine testing in the laboratory.D-dimer testing is usually used for the detecting of thrombosis.Currently,the comparability of ESR and D-dimer testing results between laboratory is poor.So it is necessary to develop reference materials for the laboratory quality improvement.Methods:1.The development and evaluation of ESR reference materials:?The development of reference materials:the key steps of the experimental conditions based on previous preparation experience for the preparation of reference materials were verified.Those conditions including the concentration of the fixed solution,the time,the synthesis of plasma from dextran and the ratio of red blood cells to simulated blood plasma.Suitable preparation conditions and procedures were selected to prepare 6 sets of ESR reference materials(ESR01-ESR06)and 2 sets of batch numbers(201816 and 201817)with different sedimentation rates according to the validation results.?The evaluation of reference materials:According to ISO Guide 35 "General Principles and Statistical Methods for the Standardization of Standard Samples",CNAS-GL03 "Guidelines for the Evaluation of Samples for Uniformity and Stability of Proficiency Testing Samples" and CLSI EP-30 "Reference Substance Setting and Commutability".The homogeneity?stability,and commutability of those reference materials were evaluated according to the requirements of ISO Guide 35 "General Principles and Statistical Methods for the Standardization of Standard Samples" and JJG 1006-94 "Technical Specification for Primary Standards Materials",5 laboratories using imported instrument systems(Monitor and SRS)were selected to assign value for the reference materials.2.The Application of the ESR Reference Material:?Applying it in internal quality control(IQC):We selected the appropriate home-made reference materials Lots(ESR01,ESR02,and ESR04)and the commercial QC materials(QCOland QC02).Those materials were measured as QC materials on 5 instrument systems(Monitor,SRS,Saikexide,.Yuehua,and Zhongchi)in 9 laboratories(analysis of 3,2,2,1 and 1 laboratories for each detection system).We run the test once every day for a total of 30 days,using imported commercial QC materials as control,and then analyzed the application effects and CV of the home-made reference materials for IQC;The second batch of home-made reference materials(201816 and 201817)and two imported commercial QC materials with similar concentration(201812 and 201815)were tested and applied to the IQC on two systems(Monitor and SRS).The reference materials were tested 4 times for 15 days.We compared and analyzed the results of the home-made reference materials and imported commercial QC materials.?Applying it in external quality assessment(EQA):The home-made reference materials(Lots 201816 and 201817)and commercial quality control materials were simultaneously sent out to 1,099 laboratories in the 2018 national external quality assessment(EQA)program.The commercial quality control materials(201812 and 201815)with similar sedimentation rates were used as control.We analyzed the CV of the home-made reference materials.?Applying it in the Calibration of different detection systems:The comparability between Monitor and SRS systems was analyzed according to EP9-A3.The calibration curve was plotted with the mean value of the reference materials in each system as the x-axis and the assigned value of the reference materials as the y-axis.The results of the clinical samples of each system were converted and the comparability of the systems after conversion was calculated.We compared the comparability of the systems before and after conversion and then evaluated the calibration effects.3.Evaluation of D-dimer Reference Material:We evaluate the homogeneity,stability,and commutability according to ISO Guide 35 "General Principles and Statistical Methods for the Standardization of Standard Samples",CNAS-GL03 "Guidelines for the Evaluation of Proficiency Testing Sample Uniformity and Stability," and CLSI EP-30 Reference Guidelines for Evaluation of Substance Valuation and commutability.4.The Application of the D-dimer Reference Material:?Applying it in internal quality control(IQC):We selected the appropriate home-made reference materials Lots(DD01,DD03 and DD04)and the commercial QC materials of each system(Stago and Sysmex).Those materials were measured as QC materials on 2 systems(Stago and Sysmex).We run the test 4 times every day for a total of 15 days.The accumulated CV and IQC chart were then analyzed.We used the results of QC materials of each system(Stago and Sysmex)as standards to compare the effect of our home-made materials;?Applying it in the calibration of different detection systems:the comparability of the Stago and Sysmex systems using the home-made reference materials at the appropriate concentration level and the commercial materials were analyzed according to CLSI EP9-A3.Comparability of the system under different calibration condition was compared.Results:1.Confirmation of the key conditions for the preparation of ESR reference material:?Confirmation of erythrocyte fixation conditions:The sedimentation rate of the mixed solution changed with the different concentration of the fixative solution.When the concentration of the fixative solution is A,there is adhesion and phenomenon of incomplete sedimentation;When the fixed solution concentration is B,the supernatant becomed red after 15 days;Only when the concentration of the fixed solution is C or D,the appearance and sedimentation rate of the mixed solution met the requirements.here was no significant difference in sedimentation rate of the preparation when red blood cells were fixed for 2h,4h and 18h(overnight).? The concentration of dextran simulated plasma:The reference material was prepared with simulated plasma with different concentrations of dextran.If the fixed mixture Hct is about 10%,it is found that when the concentration is A,the boundary between the cell and plasma layer is unclear.When the concentration was C,there was a serious phenomenon of cell adhesion and polycondensation;when the concentration was B,the supernatant after settlement and the boundary between red blood cells and plasma was clear.There was no erythrocyte adhesion.?Ratio of red blood cells to simulated blood plasma:The sedimentation rate of the reference substance is influenced by the Hct value of the mixed liquid.When the Hct value is lower,the ESR value is higher.When conditions such as conditions and dextran simulated plasma concentration are fixed,the reference substance with a sedimentation rate ranging from 0 to 140 mm/h can be prepared by adjusting the Hct value of the mixed solution to 0 to 20%.3.Evaluation of the ESR reference material and assign value to it:Six batches of reference materials with different sedimentation rate levels(ESR01-ESR06)and import control materials(QC01-QC02)all showed that the homogeneity was P>0.05,and all the uncertainty of homogeneity ubb<method repeatability uncertainty ur;Short-term stability evaluation results showed that there were no trend changes in the home-made and imported quality control materials within 28 days(P>0.05).However,if the home-made material was placed at 2-8? after testing,there will be large fluctuations in sedimentation rate.The detection results at some time points were beyond the 1/2 EQA criteria;Long-term stability results showed that there was no trend change(P>0.05)in both home-made and imported quality control materials within 29 weeks;The commutability evaluation results showed that the correlation between the systems of Monitor and SRS was better(R2>95%).The analysis of deviations showed that the number of samples exceeded the total allowable error was 17/66(25.8%);The self-made and imported quality control materials in the evaluation of commutability all fall within the 95%confidence interval of the regression line.The self-made and imported quality control materials have commutability among the systems,but they need to be combined with clinical analysis.4.Application results of ESR reference materials:?Application results of IQC:The results of the first and second test applications showed that CV and similar levels of imported control materials was comparable,and it can be applied to the IQC of different instruments systems;?Application results of EQA:Compared with imported control materials of similar concentration levels,the CV of the two reference materials groups was much larger than that of the imported quality control group in comparison with most of the groups.However,the CV value of the self-made reference material in the Yakun instrument group was lower than that of the imported control material.In the high-level group,the CV values in the Pleasant,Tianhai,and VACUETTE groups were comparable,and the CV slightly decreased.?Appling it to calibration of different instruments:The results showed that there was no significant change in inter-system correlation R2 before and after calibration.The average absolute deviation appeared from 1.7mm/h to 1.6mm/h,and the average relative deviation decreased from 24.1%to 18.4%,but there was no significant change in deviation distribution.5.Evaluation of D-dimer reference materials:The homogeneity evaluation results of 5 batches of self-made reference materials(DD01-DD05)and imported quality control materials(201711 and 201712)showed that the P values were all greater than 0.05.The uncertainty of homogeneity ubb<method repeatability uncertainty ur,indicating that the self-made and imported blood control materials are of good homogeneity.The results of the stability evaluation showed that there was no trend change in self-made and commercial quality control materials within 27 weeks(P>0.05).Commutability evaluation results:Comparability analysis between systems showed that when the clinical sample D-dimer concentration was 0-10 mg/L,the inter-system correlation was good,R2>95%.When the deviation between systems is based on the lowest RCPA and TEa evaluation criteria,1/35(2.9%)and 2/35(5.7%)samples are beyond the acceptable range,and the comparability between systems is acceptable;the evaluation of commutability shows all batches of the self-made substances fell within the 95%confidence interval of the regression line between systems,indicating that the home-made D-dimer reference material has commutability between the Stago and SRS systems.6.Application results of D-dimer reference material:? Application results of IQC:similar concentrations of self-made reference materials and supporting quality control products IQC test results showed that s and CV was equal in size,from the IQC test application effect.The IQC figure showed that most of the results of self-made and matched quality control materials with similar concentration levels were within 2s and only a few exceeded 3 s.The self-made IQC reference material is similar to the matched quality control materials and can be used for the IQC of Stago and Sysmex systems.?Calibration results of self-made reference materials in different systems:In the concentration range of 0 to 6 mg/L,correlations between systems before and after calibration using self-made substances were 85.1%and 81.9%,respectively,and the correlation decreased slightly.Before the calibration,the evaluation criteria were TEa appropriate,TEa minimum,and RCPA deviation.After calibration 18/42(42.9%),4/42(9.5%),and 3/42(7.1%)samples exceeded the acceptable range respectively.With the TEa appropriate,TEa minimum,and RCPA deviation criteria,15/42(35.7%),9/42(21.4%),and 3/42(7.1%)samples were beyond the acceptable range,respectively.There was no significant change in the comparability of clinical samples before and after the calibration in each system.Conclusions:1.Confirmation of the key conditions for preparation of ESR reference materials,preparation of 6 batches of ESR reference materials,and uniformity,stability and good interoperability between the two import detection systems Monitor and SRS,reference materials can be used During the experiment,the internal quality control,but the quality of the inter-chamber quality assessment compared with the imported quality control material significantly increased,still need to continue to explore the key links affecting its application,the self-made reference material for the two systems Monitor and SRS The external calibration,the consistency between the system after calibration significantly improved.2.The concentration conditions of the D-dimer reference substance were re-determined and 5 reference materials with different concentrations were prepared.The homogeneity,stability,and interoperability between the Sysmex and Stago systems were good.The application results show that the application of quality control products and the corresponding degree of application effect,can be used for internal laboratory quality control,as a calibrator used in Sysmex and Stago two system calibration results show that after calibration,the comparability between the system is not obvious.