Development Of A Reference Material Of ?2 Microglobulin And Proficiency Testing Of Homocysteine Detection

80%of clinicians' diagnosis depends on the test results.?2 microglobulin is one of the most important diagnostic markers for kidney disease,and homocysteine(Hcy)is a diagnostic indicator for stroke.Due to the lack of relevant reference materials,the testreagents can't be traced to the source,leading to large deviations in the results of clinical testing laboratories,seriously affecting the diagnosis and treatment of patients.Due to the large molecular weight and complex structure of present,there are only 7 first-class pure protein standard materials in China.In this study,two different principles of ?2 microglobulin standard substance evaluation methods are established.One is the hydrolysis method,which uses Phe,Va1,Pro three amino acids for quantification.The optimal hydrolysis time is 36 h,and the mass spectrometr conditions of amino acids and their labeled amino acids are optimized.The result of the hydrolysis method is 199.35 ?g/g.The other is the enzyme digestion method,IQVYSR(IR),VNHVTLSQPK(VK),SNFLNCYVSGFHPSDIEVDLLK(SK)is selected as three characteristic peptides for quantification,and carries out qualitative analysis and purity analysis on the synthetic peptides used for quantification.In this experiment,the optimized enzyme digestion experiment is carried out,and the optimal ratio of protein to enzyme is 50:1,and the liquid phase conditions and mass spectrometry conditions of the optimized peptide were obtained.The result of enzyme digestion is 193.78 ?g/g.By verifying the consistency of the results of the two quantitative methods and calculating the uncertainty introduced in the experimental process,the final value of the standard substance of ?2 microglobulin is(198.01±6.82)?g/g(k=2).This study laid a good foundation for the research of ?2 microglobulin reference materials.This study organized the proficiency testing of 102 clinical laboratories(including 45 top three hospitals,56 top two hospitals,and one independent laboratory)in the East China region for Hcy,an important indicator of stroke.The reference samples were quantified through 6 reference laboratories and verified for interchangeability.Through the application of self-developed homocysteine reference materials,Hcy provides a standard value with metrological traceability guarantee for the proficiency testing work.In the proficiency testing additional study,25 experiments used Hcy standard materials to calibrate the measurement system to test the test,and the failure rate dropped to 16%.The test results of Hcy2 samples were all passed,and the failure rate was reduced to 0%.Only one laboratory test of Hcy3 samples failed,and the failure rate was reduced to 4%.After calibration,Hcy measurement deviation is significantly reduced,and a complete traceability chain is established from the source SI unit to the end user.