| Adenosine deaminase(ADA)is a kind of catabolic enzyme of nucleic acid,which plays an important role in the diagnosis and differential diagnosis of tuberculosis,malignant tumor,central nervous system disease,liver disease and typhoid.Kinds of analytical methods have been used in determining the catalytic concentration of ADA.But these methods often yield widely different results and need to be standardized.Our objective was to establish candidate reference method of ADA by using enzymatic coupling method and prepared ADA reference material by biosynthetic method.The reference interval for ADA has also been established based on candidate reference method.At last,the established reference method was applied in clinical.Chapter 1 Research of the candidate reference method of adenosine deaminaseObjectiveTo establish the candidate reference method of ADA.Methods1.Review of the related literature about measuring method of ADA in domestic and abroad,to know the development and clinical application of ADA.2.The selection of candidate reference method of ADA:at first,kinds of measuring methods of ADA were studied,then the performance of selected methods was evaluated.3.Requirement of preparing reagents:including the condition of environment,weighing and quality of water.All of these were according to the requirements of reference method.4.Calibration of instruments:before the measurement was carried out,all instruments should be calibrated according to the reference laboratory SOP,such as ultraviolet spectrophotometer,dilution compounding device,pH meter,electronic balance,thermometer,pipettes,glassware and other used equipment.5.Selection and optimization of measuring conditions:including environmental temperature and humidity,measuring temperature,detection wavelength,wave width,slot,incubation time,delay time,detection time and detection points etc.6.Optimization of reaction system:including the pH and the concentration of reagents.Appropriate scope of each influencing factor was determined by single factor analysis.Then the significant factors were selected by Plackett-Burman(PB)method.At last,the relationship of multiple factors and indicators in the experiment was fitted by the polynomial approximation and analyzed by responses surface function of response surface method(RSM).Then the optimal value was calculated.7.pH of the system was validated and the measuring methods of PNP,XOD,POD were optimized.8.Performance evaluation for ADA candidate reference method:precision,linearity,accuracy,ability of anti-interference were evaluated according to CLSI documents EP5-A2,EP6-A and EP7-A.9.Uncertainty for the method was evaluated.Results1.Coupled-enzyme of GLDH and PNP,XOD,POD for measuring ADA were selected for performance evaluation through the literature research.2.The coupled enzyme of GLDH for measuring ADA:reagent blank:average(X)= 1.4 U/L,SD was 0.4 U/L.Sample blank of 5 levels were(unit:U/L):7.8,9.0,11.5,9.3,10.2,respectively.Precision:X = 20.1 U/L and 35.6 U/L,SD = 1.2 U/L and 0.8 U/L,CV = 6.0%and 2.3%.Linear equation was Y=32.615X-19.2214((R2=0.9973),the upper limit was 207.7 U/L.The other method:reagent blank:X=0.6 U/L,SD was 0.6 U/L.Sample blank of 5 levels were(unit:U/L):0.1,-0.3,0.5,0.9,-0.4,respectively.Precision:X = 31.1 U/L and 49.2 U/L,both SD =0.4 U/L,CV = 1.1%and 0.8%.Linear equation was Y=38.8179X-30.9571(R2=0.9993),the upper limit was 240.2 U/L.3.The measurement conditions for established reference method:testing temperature 37.0 ± 0.1℃,wavelength 550 ± 1 nm,wave width 10±0.01mm,slot less than 2 nm,detection point more than 6,incubation time 5 min,delay time120s,monitoring time 180s.4.The significantly influence factors were:adenosine,XOD,4-AA,Na2HP04.5.Concentration of the 4 significant factors:adenosine 4 mmol/L,XOD 3224.2 U/L,Na2HPO4 0.96 g/L,4-AA 1.2mmol/L.6.Performance of the method:the within-run precision:the CVs of two samples(31.2 U/L and 80.9 U/L)were both 0.44%,total precision:the CV was 1.81%for sample 1,0.76%for sample 2.Linear range:0.33-286.51 U/L.Trueness verification:average of testing BCR-647(IRMM)was 194.1 U/L,SD was 0.5 U/L,CV was 0.2%,the target value of BCR-647 was 153.0±5.4 U/L.The ability of anti-interference:triglyceride ≤3.39 mmol/L,Hb≤ 3.75 g/L,total bilirubin≤85.5 umol/L,VitC ≤40 mg/1,both glycyrrhetinic acid and deoxycholic acid≤20 mg/L,baicalein ≤37.5 ug/ml have no influence on the method.7.The uncertainty of ADA candidate reference method was 0.6 U/L at 31 U/L.Conclusion1.The coupled-enzyme of GLDH for measuring ADA was not selected,because the internal and external source of NH3pollution can’ t be eliminated of this method.The coupled-enzyme of PNP,XOD,POD for measuring ADA was selected due to its better stabilization as the optimization basic of candidate reference method for ADA.2.The performance of established candidate reference method for ADA could meet the requirement.3.Response surface method(RSM)is a good method,it can be used in the optimization of clinical measurement methods.Chapter Ⅱ Research on reference material of adenosine deaminaseObjectiveTo prepare the reference material of ADA.Methods1.ADA gene sequence was obtained from Pubmed.2.Three prokaryotic expression vectors(BL21+pET-28b+ADA,BL21+pET-32a+ADA,BL21+pHSIE+ADA)and one eukaryotic vector(GS115+pPICZ a A+ADA)were constructed.3.Performance evaluation of the expressed ADA protein.The optimal system was selected for largely expressing ADA protein,then it was purified by His-Ni column.4.Physical fresh serum was collected as the matrix of the reference material.then purified protein was added to the collected serum,subpackaged and stored at-80 ℃.5.Homogeneity and stability were evaluated according to CNAS-GL03.6.The reference materials were valued by established candidate reference method of ADA,the results were expressed as "target value ± Uncertainty".Results1.The highest activity of ADA expressed by three prokaryotic vectors was 89 U/L and one eukaryotic vector for ADA was 4200 U/L.2.Performance of the prepared reference materials:homogeneity:level 1:26.3 U/L,P =0.231(>0.05),level 2:225.7 U/L,P =0.313(>0.05).Stability:5h at room temperature,12h at 4-8℃,15 days at-20℃,3 months at-80℃.3.Valued by ADA candidate reference method:level 1 26.0±0.9 U/L,level 2:219.1 ± 3.4 U/L.ConclusionActive ADA protein was successfully expressed and used for the preparation of reference materials.They could be traceable to candidate reference method.Homogeneity and stability of the reference material could meet the requirements.It will be our own reference material and will provide the experience for other reference materials.Chapter Ⅲ Establishment of reference interval for ADAObjectiveTo establish the reference interval for ADA on the basis of the candidate reference method.Methods1.Developed the inclusion and exclusion criteria in accordance with CLSI C28-A3 and related references.2.Proper individuals were selected and made the informed consent.Then fasting venous blood was collected at 7:00-10:00 a.m.,serum was separated as samples.3.ADA was tested by Mindary system with self-prepared reagent.Other items were tested by Roche Modular PPI system according to its operation.Then abnormality individuals were excluded.4.Review and statistical analysis of testing results.95%reference interval and 90%confidence interval for higher limit was computed.Results1.650 individuals were selected to this project from 1662 volunteers.2.One outlier was removed.3.Groups:Z=3.0,Z*=4.9 by gender.Z=7.8,Z*=4.9 by age.4.Reference interval of ADA for adults(female 354,male 296)at 18-79 years old in Guangdong province is 0-31 U/L,the confidence interval for higher limit was 30.5-31.1 U/L.ConclusionReference interval of ADA has been established for adults in Guangdong province based on candidate reference method for ADA,which can provide reference for clinical laboratories.Chapter Ⅳ Clinical application of the established candidate reference method of ADAObjectiveTo analysis the comparability of clinical systems with the candidate reference method of ADA.MethodsOne clinical routine system(Roche Modular PPI system,system 1)and other established clinical system(Mindary system,system 2)were selected for comparison experiment with the established candidate reference method according to CLSI EP9-A2 document.40 samples were collected according to CLSI EP9-A2 document and established reference intervalof ADA.The accepted bias of clinical system was half of the total error(±15%).ResultsThe equation of clinical system 1 and reference method was Y1=0.6326X-2.2629(R2 = 0.9571),P =0.054(>0.05),the average bias was-45.7%.Bias at clinical important points(30 U/L,200 U/L)were-44.3%and-37.9%.The equation of established clinical system 2 and reference method was Y2 =1.0280X-1.6211(R2 = 0.9977),P =0.565(>0.05),the average bias was-3.7%.Bias at clinical important points(30 U/L,200 U/L)were-2.6%and 2.0%.ConclusionThe correlation of the two clinical systems and established ADA candidate reference method was good.But it was not comparable for clinical system Y1 with candidate reference method.While clinical system Y2 was comparable with ADA candidate reference method.Reference method could evaluate the routine method effectively. |
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