Establishment, Evaluation And Application Of Ameliorative ALT And AST Reference Method

1 ObjectiveNowadays,"mutual Recognition"of clinical results is the hot spot of quality management in Clinical Laboratory. The precondition of mutual recognition is comparability of clinical results. Only when the reference method is directly or indirectly traceable, can the accuracy and comparability among different laboratories be assured. ALT and AST are very important routine tests in serum enzymes determination. Diagnosis, treatment and prognosis of liver diseases are subject to the accuracy of the test. At present, domestic clinical laboratory usually adopt non-matching analytical system, i.e., using import instrument and calibrator but domestic reagent, or changing the test procedure test system. These practices destroy the traceability of calibrator. The results of different test system or method are significantly different and incomparable. In addition, domestic reagent of ALT and AST are almost phosphopyridoxal free, while IFCC reference method for ALT and AST at 37℃contains phosphopyridoxal. In consideration of it, this research shall base on the reference method recommended by IFCC, to find out the ameliorative reference method for phosphopyridoxal free ALT and AST, and carry out methodological evaluation on this preliminary ameliorative reference method to study its performance. Meanwhile, a enzyme calibrator was prepared and its value for ALT and AST was assigned with this preliminary ameliorative ALT and AST reference method, and find out the bias of test results of open and limited system among different laboratories as well as different reference methods, so as to measure the comparability and matrix effect of ALT and AST results and among different laboratories. 2. MethodsThis ameliorative reference method for phosphopyridoxal free ALT and AST is based on the IFCC primary reference measurement procedure of ALT and AST at 37℃. The precision, accuracy, linearity of the method is measured as per CLSI protocols (EP5-A2, EP15-A, EP6-A). The ameliorative reference method is used to determine fresh serum, which will be the calibrator for the routine analytical systems in the 10 laboratories in Guangzhou. The activity of external quality assessment materials,products produced by factories,fresh sera were measured by the routine analytical systems and the ameliorative reference procedure. The comparison is to find out bias before and after calibration between routine method and ameliorative reference methods, bias between routine method and ameliorative reference method for different medium (human serum and quality control serum). Comparability and matrix effect evaluated this way.3. ResultUltraviolet spectrophotometer, analytic balance, pH meter, electronic thermometer, pipettes and volumetirc flask have been verified and its uncertainty fit to method's requirements. When the temperature controller showed 37.2℃, the temperature in cuvette was equal to 37.0℃. The temperature in cuvette reaches the set temperature after 180 seconds. In transferpettor sample input experiment show switch to dimension 2 when the absorbance measurement indicate 1 and inputting sample by side will generate the best precision. The imprecision within run of the ameliorative reference method is less than 1%, total imprecision less than 2%. Both satisfy precision requirement.The results of JSCC ERM which determine by the ameliorative reference method were within uncertainty tolerance. The results of cfas conformed to target. Bias between involved laboratories'network evaluation result and average is within equivalent area. The upper limit of the measurement range of ALT and AST was 335.8U/L and 363.6U/L respectively. Among the 11 tests, of which the bias of ALT and AST fresh serum before calibration and reference method is over 20%, 10 were enzyme activity computed with theoretical K value. The maximum bias of ALT and AST of fresh serum before calibration was -29.2% and -25.0% respectively, average at 10.7% and 9.3%. Calibrated with enzyme calibrator of the ameliorative reference method, the maximum bias decreases to 15.2% and 12.3%, average at 3.4% and 2.1%. In ALT test result, One external quality assessment materials showed matrix effect on all the routine analytical systems. In AST test result, two external quality assessment materials showed no matrix effect on all the routine analytical systems. The routine analytical systems dependent matrix effects of other materials were observed on other materials.4. ConclusionAmeliorative reference method has been established basically. Precision, accuracy and linear range and other performance all satisfy the requirements. It's an effective way to achieve traceability in different testing systems by using the determined fresh serum as the calibrator. Matrix effect will affect the accuracy and comparability of ALT and AST test. It's important to attach importance to the influence of matrix effect on clinical result traceability.