Establishment Of Improved Method Of Assaying Ischemia Modified Albumin And Evaluation

Objective:Acute coronary syndrome (ACS) is characterized of coronary artery atheromatous plaque rupture and erosion and full or half thrombosis. It consists of unstable angina, ST-elevated myocardial infarction and non-ST elevated myocardial infarction. Acute myocardial infarction (AMI) is a severe one that can lead to death easily. Therefore, the early diagnosis of ACS which becomes a focus nowdays plays an important role in clinical therapy and decreasing death rate.Ischemia-modified albumin (IMA) is a new biochemical marker discovered in recent years, which is the first biochemical marker to check myocardial ischemia approved by Food and Drug Administration (FDA) for their highly sensibility and high negative predictive value. And it can be discovered at early myocardial ischemia even before the myocardial cell has necrotized. So it is of great significance at early AMI diagnosis. However, the way of checking IMA is quite simple and there is no a unitive standard, which restricts the application of IMA in clinical. The study focuses on how to improve ACB in the early diagnosis of AMI, which uses 1-Nitroso-2-naphthol (NN) instead of using Dithiothreitol (DTT) as a color reagent, and the establishment of the improved method of assaying IMA and its evaluation of methodology. The improved ACB method provides doctors with accurate diagnosable evidence for early diagnosis, risk stratification and prognosis. So that doctors can treat the patients at early AMI, and decrease the death rate. Methods:1. Establishment of Improved Method of Assaying Ischemia Modified AlbuminThe albumin cobalt binding assay colorimetric method in IMA detection was adopted, but NN was used to replace DTT as color reagent in the improved test. According to the test, the basic reaction situations have been confirmed. They are as follows:①The best absorption wavelength:choose wavelength between 340nm and 800nm; Mensurate its absorbency with 722 spectrophotometer every 10nm, then select the absorption wave crest as the best absorption wave.②The best reacting time: mensurate the absorption every 1 minute in30 minutes and make a stability curve.③The mensuration of series of different concentration of Co2+:R1 was different concentration of 2.0g/LCoCl2, and R2 was NN. Choose 405nm to mensurate absorbency of their mixture with 722 spectrophotometer and make an absorption curve.④The mensuration of series of different concentration of NN:R1 was 0.05g/L CoCl2 3ml, and R2 was different concentration of NN. Mensurate absorbency of their mixture and make an absorption curve.⑤The mensuration of series of different concentration of samples:add different quantity of serum into 0.05g/L CoCl2, then add 0.05g/L NN into their mixture, mensurate absorbency of the mixture and make an absorption curve.⑥The test on the stability of reagent: mensurate a mixed samples for 30 times in the shortest time, and calculate the mean and standard error; Store the reagent at 4℃respectively, then mensurate the same sample everyday. If the mensuration was over (?)±3s, the reagent wasn't valid.⑦The test on the effect of anticoagulant: add the sample into three anticoagulants (Heparinize,3.2% of Sodium citrate and EDTA-K2) and coagulant respectively; Analyze its result and find how the different anticoagulants affected the result of the experiment.2. Methodology evaluation of the Improved Method of Assaying Ischemia Modified AlbuminEvaluate the Methodology of the Improved Method of Assaying Ischemia Modified Albumin according to the evaluation protocol of CLSI.①The evaluation on precision:within-run precision, between-run precision, within-day precision and total imprecision, referring to CLSI evaluation protocols—Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline—Second Edition. Choose a mixed serum, and divide it into 20 parts, then store them in -20℃. And measure two separate runs with two test samples during each of the 20 operating days.②Evaluation of trueness performance:Referring to CLSI document EP9-A2—Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline—Second Edition. Analyze 40 patient samples at 5 operating days, analyze each patient sample in duplicate using both the test method and the comparative method, then analyze the datas from the two methods statistically. When the experiment is completed, record the data in a logical manner. Plot the data and assess the diagram visually and statistically for relative linearity, adequate range, and uniform scatter. Based on the results of the data examination, use either simple linear regression or alternative procedures to estimate expected (average) bias and the confidence interval for expected bias at any desired medical decision level. Then, these estimates can be compared with claims or internal criteria to judge the acceptability of the method. The evaluation on accuracy:③interference test: difference partnership was adopted referring to CLSI evaluation protocol EP7-A2—Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition. Choose a mixed serum, and divide it into two samples averagely. Group 1:Add triglyceride, total bilirubin and hemoglobin into one sample as interference. Group 2: the sample without interference. (Each sample was measured 10 times repeatedly.) Interference value was the mean of group 1 subtracts the mean of group 2. The evaluation standard is whether it is over Groupl's (?)±1.96s;④Linear evaluation: Referring to CLSI evaluation protocol EP6-A—Evaluation of the Linearity of Quantitative Measurement Procedures:A Statistical Approach; Approved Guideline. Choose five samples in different concentration. Select a sample (No.1) whose concentration was higher, and then select a sample (No.5) whose concentration was lower. No.1 and No.5 were mixed in 3:1, whose mixture was called No.2; No.1 and No.5 were mixed averagely, whose mixture was called No.3; No.1 and No.5 were mixed in 1:3, whose mixture was called No.4. After that, measure their absorbency and make curve estimation.⑤Sensitivity test:limit of bland, limit of detection, and limit of quantitation, referring to CLSI document EP17-A—Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline⑥The contrast test on sensitivity of Co2+:tiraes ratio dilute 2.0g/LCoCl2 for15 times, and add two color reagents into it respectively and measure their absorbency, then contrast the sensitivity of the two color reagents by making absorbency curve.3. Application of Assaying Ischemia Modified Albumin in the early diagnosing AMIPatients of acute chest pain onset were ruled in randomly. Define the IMA patients by the standard of WHO, then ROC curves for AMI in diagnosing was built. Calculate the area under the curve. Determine the cut-off value of IMA by the ROC curve when the positive likelihood ratio reached to the top.Results:1. Establishment of Improved Method of Assaying Ischemia Modified Albumin①To select 0.982ABSU as the best absorption wavelength, which was about 410nm. As there was no 410nm in Hitachi auto analyzer,405nm was chosen as the best wavelength.②The reaction can continue for 30mins with good stability.③Absorption of series of different concentration of Co2+ was analyzed, the albumin-Co2+ of linear regression formula was Y=0.148+0.003X, R2=0.984, P=0.001.④Absorption of series of different concentration of NN was analyzed, the linear regression formula was Y=0.0004-0.0415X, R2=0.985, P=0.000.⑤Absorption of series of different samples was analyzed, the linear regression formula was Y=1.120-0.001X, R2= 0.990, P=0.000.⑥The reagent was stable at room temperature and 4℃in 30 days.⑦Heparinize,3.2% of Sodium citrate and EDTA-K2 were adopted in the experiment. Then compare the result with that of using coagulant. The purpose was to evaluate how the different anticoagulants affected the test. Heparinize was the same with Coagulant (P=0.383), however,3.2% of Sodium citrate and EDTA-K2 were different from Coagulant (P=0.022, P= 0.001 respectively).2. Methodology Evaluation of the Improved Method of Assaying Ischemia Modified Albumin①Evaluation on precision of the improved ACB assay:the standard for within-run assays was 0.022, coefficient of variation (CV) was 1.36%; the standard for between-run precision was 0.026, CV was 2.86%; the standard for within-day precision was 0.012, CV was 1.36%, the standard for total imprecision was 0.027, CV was 2.96%;②The evaluation on trueness:r=0.989> 0.975, the range of data was considered adequate; Linear Regression:Y=1.017X-0.010 R2=0.987, P=0.000, the standard error of estimate (Sy.x) is 0.035; The confidence interval for predicted bias includes the defined acceptable bias, the data do not show that the bias of the candidate method is different from the acceptable bias.③interferential test:there was no difference when adding TG(<2.08 mmol/L), Hb(<5.01 g/L), TBIL(<68.12μmol/L);④The formula was Y=1.71-2.16X when carrying on the curve estimation, so the linear was good;⑤Sensitivity test:LoB=0.3205; LoD=LoQ=0.360;⑥The test on sensitivity contrast of Co2+:Through observation, the sensitivity of NN was higher than that of DTT as color reagent. The detection limit of NN and DTT were 0.4883mg/L and 7.8125mg/L, P=0.004.3. Application of Assaying Ischemia Modified Albumin in the early diagnosing AMIThe value in AMI group was obviously higher than that in chest pain group (1.195±0.320 vs 0.855±0.068, P=0.000). Area under the ROC curve was 0.947; standard error under the area was 0.021; 95% of confidence interval was (0.905 0.988. Therefore, the value of IMA is significant in diagnosing early AMI (P<0.001). The higher the value of IMA is, the more possibility the patient has AMI. When the cut-off value of IMA is 0.906, the sensitivity is 93.0%, the peculiarity is 82.9%.Conclusions:1. Improved method of assaying albumin cobalt binding assay colorimetric in IMA detection was established. The improved ACB can be applied in auto analyzer and in manual method, so it is suitable for routine use in any level clinic laboratories.2. Serum and anticoagulant serum with heparinize are used as sample.3. Improved ACB method is good in the accuracy and precision, as well as the better sensitivity than the traditional ACB method.4. This method has better diagnostic value for early diagnosing AMI in the clinical patents of acute chest pain onset.