1 Introduction Imprecision Within-run imprecision, results Day-to-day imprecision, results... 2

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1 Design Verification liquicolor 1 Introduction Imprecision Within-run imprecision, results Day-to-day imprecision, results Linearity and Detection Limit Linearity Detection Limit Method comparison Comparison against -cyclodextrin and polyanion methods Comparison against classical phosphotungstate precipitation method Traceability Recovery of control sera Interferences Endogeneous substances Anticoagulants Stability Lot real time stability at 8 C: Lot real time stability at 8 C: Lot real time stability at 8 C:... 1 Form: Rev. 9 valid of /11

2 1 Introduction The HDL-CHOLESTEROL liquicolor test is a direct homogeneous enzymatic assay for the quantitative determination of HDL cholesterol (HDL). The test combines two steps, the first step eliminating chylomicrons, VLDL and LDL by specific enzymatic degradation. The next step determines the remaining HDL by the well-known enzymatic reaction cascade cholesterol esterase, cholesterol oxidase and peroxidase. In the final reaction hydrogen peroxide oxidises a chromogen (N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline) under the catalytic action of peroxidase. The resulting colour change is monitored spectrophotometrically (593 nm, [57 61 nm], Hg 578 nm) and directly proportional to the HDL concentration in the sample. For calculation of the HDL concentration a calibrator is employed. 2 Imprecision The within-run imprecision of the HDL-CHOLESTEROL liquicolor test was calculated from 3-fold determinations. The day-to-day imprecision was calculated from the results obtained on 2 consecutive days. Pooled human sera as well as commercially available controls were employed as sample materials. 2.1 Within-run imprecision, results Pool serum high Pool serum medium Pool serum low Mean, mg/dl Standard deviation, mg/dl Coefficient of variation, % Day-to-day imprecision, results Pool serum Control I Control II Mean, mg/dl Standard deviation, mg/dl Coefficient of variation, % Linearity and Detection Limit 3.1 Linearity The linearity of the HDL CHOLESTEROL liquicolor method was controlled by employing a high concentration control serum and dilutions with phys. saline. The analysed concentrations were calculated vs. the regression line. Deviation from the regression line are expressed in absolute and relative values. As an example data measured on the Hitachi 717 are reported. High Pool Analytical Data Regressed Data Deviation from Regression Line Content (%) mg/dl mg/dl mg/dl (%) Rev. 9 2/11

3 MEASURED ANALYTE CONCENTRATION (mg/dl ) 25 2 Linear Regression Error Bars = ± 2SD LINEARITY HIGH POOL CONTENT (%) From the above results a linearity range up to 15 mg/dl HDL-CHOLESTEROL has been confirmed. 3.2 Detection Limit A 2-fold determination of a sample (phys. saline) on a HumaStar 1 analyser revealed an absolute mean of.67mg/dl and a SD of.11mg/dl. From the three-fold standard deviation the detection limit can therefore be calculated on the base of mean +3 SD to 1.mg/dl. 4 Method comparison 4.1 Comparison against -cyclodextrin and polyanion methods 27 specimens have been determined by this method and by two other accepted methods (method A: - cyclodextrin/peg enzymatic method; method B: polyanion/surfactant method). Discrepant results have been retested and confirmed by ultracentrifugation method. The HDL-CHOLESTEROL liquicolor test showed an excellent correlation with each other methods. N = 27 r =.996 Y = a*x + b (method A) a =.97, b = 1.25 Y = a*x + b (method B) a =.97, b = 4.2 A total of 6 discrepant results have been observed with method A. When the respective samples have been further investigated by fractionation it could be demonstrated that the HDL-CHOLESTEROL liquicolor results best reflect the true HDL concentration while method A yielded low results. 4.2 Comparison against classical phosphotungstate precipitation method 5 samples have been determined by the "classical" phosphotungstate precipitation method (e.g. HDL Cholesterol HUMAN, cat.-no.: 118 & 117) and by the HDL Cholesterol liquicolor test. The data obtained have been evaluated by means of a non-parametric regression analysis according to Passing&Bablok. The regression analysis yielded the following equation: Rev. 9 3/11

4 Y = a*x + b a =.9641 Confidence interval 95%.923 to 1.1 b = Confidence interval 95% to 3.6 Method comparison HDL CHOLESTEROL HDL Cholesterol liquicolor, mg/dl The above results demonstrate that HDL CHOLESTEROL liquicolor test recovers the independent controls within the assigned target range and that there is no deviation against a comparison method. 5 Traceability The HDL-CHOLESTEROL liquicolor test for the determination of HDL-Cholesterol is calibrated with a calibrator, which is traceable to the CDC reference method. The recovery of the calibrators as well as the comparison of 5 patient samples have been tested. The results are summarised in the tables below. Hitachi 717 Calibration: Reference Human assign. abs assign. abs cal-1. mg/dl 1. mg/dl cal mg/dl mg/dl Recovery: assign.. mg/dl 57.8 mg/dl 71.4 mg/dl Reference Human Reference Human Reference Human rep rep rep Mean Recovery % 1.7% 13.9% 11.% Rev. 9 4/11

5 Human HDL [mg/dl] Sample: X: Reference Y: Human Sample: X: Reference Y: Human , y =,987x -,46 R 2 =, Reference method HDL mg/dl The HDL-CHOLESTEROL liquicolor calibrator is traceable to the international reference method. 6 Recovery of control sera Commercially available control sera have been employed. The control sera have been reconstituted/prepared according to the manufacturer s instructions. Fivefold determinations of each control serum have been performed with the HDL CHOLESTEROL liquicolor test method. The means of the fivefold determinations have been calculated and compared with the target values. Rev. 9 5/11

6 CONTROL SERUM RECOVERY HUMAN fresh Reference HUMAN 8 h 25 C Cal. factor Control LOT Target Range Result Deviation Result Deviation Result Deviation serum g/dl g/dl g/dl (%) g/dl (%) g/dl (%) Serodos # SeroDOS plus # Precinorm L # Lip control 1 # Lip control 2 # Sum No significant differences could be observed between fresh and stressed reagents, confirming the stability of the reagents. Controls were all recoverd well within the specified ranges. 7 Interferences 7.1 Endogeneous substances Any potential interfering influence by ascorbic acid, hemoglobin, bilirubin and turbidity (VLDL-cholesterol and VLDL-triglycerides) has been studied by adding pure substances to a normal pool serum. The data are summarised in the following table. Ascorbic acid, % %.4 %.8 %.12 %.16 %.2 % Found HDL, mg/dl Hemoglobin, mg/dl Found HDL, mg/dl Bilirubin, mg/dl Found HDL, mg/dl VLDL triglycerides, mg/dl Found HDL, mg/dl VLDL cholesterol, mg/dl Found HDL, mg/dl The above results demonstrate that ascorbic acid up to.2 %, hemoglobin up to 5 mg/dl, bilirubin up to 3 mg/dl do not interfere with the test (Glick value = 1) while VLDL-triglycerides up to 13 mg/dl showed only minor interference (Glick value = 2). 7.2 Anticoagulants To pooled plasma different additives in different concentrations were added to show, if they affect the test results. The results are summarised in the following table: Concentr Anticoag. Na-Citr. mg/l HDL mg/dl Na-Hep. mg/l HDL mg/dl Na-Fl. mg/l HDL mg/dl Na-oxal. mg/l HDL mg/dl EDTA mg/l HDL mg/dl The results gave clear evidence that none of the anticoagulants has an effect on the test results. 8 Stability The stability of the HDL-CHOLESTEROL liquicolor test has been tested on real-time storage conditions over a period of 2 months for three independent production lots with different commercial controls. The measurement was made on Hitachi 717. The individual results are summarised in the following tables. Rev. 9 6/11

7 8.1 Lot real time stability at 8 C: Duration (month) Sensitivity Blank STD mabs./1mg/dl (deviation from T=) (1%) (13%) (99%) (12%) (11%) (99%) (98%) (93%) Calibrator lot # Assigned value (mg/dl) Specificity Lipid Cal.-D 6.2 6, Lot , Target: 6.5 mean , recov.% 1% 99% 99% 1% 1% 99% 99% 1% Lipid Cont. I , Lot , Target: 82. Mean , recov.% 97% 98% 11% 1% Lipid Cont. I Lot Target: 85. mean recov.% 13% 14% 14% 15% 18% Lipid Cont. II , Lot , Target: 5.5 mean , recov.% 97% 96% 1% 98% Lipid Cont. II Lot Target: 52.5 mean recov.% 99% 98% 99% 1% 99% Precision n=2 mean Liquid Cont. N SD CV n=2 mean Liquid Cont. SD AN CV Linearity % % % % % % % % % % % Lin. material lot # X-3 X-3 X-4 X-4 X-4 X-4 X-4 X-4 Rev. 9 7/11

8 HDL-C(mg/dL) Deviation from T= 14% Sensitivity [per 1mg/dL] 12% 1% 8% 6% 4% 2% % Duration (month) 2 Linearity % 1% 2% 3% 4% 5% 6% 7% 8% 9% 1% M 3M 6M 9M 12M 15M 18M 2M 8.2 Lot real time stability at 8 C: Duration (month) Sensitivity Blank STD mabs./1mg/dl (deviation from T=) (1%) (12%) (11%) (12%) (11%) (97%) (97%) (92%) Calibrator lot # Assigned value (mg/dl) Specificity Lipid Cal.-D Lot Target: 6.5 mean recov.% 1% 11% 1% 1% 12% 12% 11% 1% Lipid Cont. I Lot Target: 82. mean recov.% 97% 11% 99% 12% Lipid Cont. I Lot Target: 85. mean recov.% 14% 13% 17% 15% 16% Rev. 9 8/11

9 Lipid Cont. II Lot Target: 5.5 mean recov.% 98% 97% 98% 1% Lipid Cont. II Lot Target: 52.5 mean recov.% 99% 98% 13% 11% 99% Precision n=2 mean Liquid Cont. N SD CV n=2 mean Liquid Cont. AN SD CV Linearity % % % % % % % % % % % Lin. material lot # X-3 X-3 X-4 X-4 X-4 X-4 X-4 X-4 14% Sensitivity [per 1mg/dL] De via tio n fro m T= 12% 1% 8% 6% 4% 2% % Duration (month) Rev. 9 9/11

10 HDL-C(mg/dL) 2 Linearity % 1% 2% 3% 4% 5% 6% 7% 8% 9% 1% M 3M 6M 9M 12M 15M 18M 2M 8.3 Lot real time stability at 8 C: Duration (month) Sensitivity Blank STD mabs./1mg/dl (deviation from T=) (1%) (12%) (11%) (11%) (11%) (98%) (93%) (93%) Calibrator lot # Assigned value (mg/dl) Specificity Lipid Cal.-D Lot Target: 6.5 mean recov.% 13% 11% 99% 12% 11% 1% 97% 99% Lipid Cont. I Lot Target: 82. mean recov.% 11% 1% 11% 13% Lipid Cont. I Lot Target: 85. mean recov.% 15% 14% 14% 14% 14% Lipid Cont. II Lot Target: 5.5 mean recov.% 1% 97% 99% 1% Lipid Cont. II Lot Target: 52.5 mean recov.% 11% 99% 11% 99% 97% Precision n=2 mean Liquid Cont. N SD CV n=2 mean Liquid Cont. AN SD CV Rev. 9 1/11

11 HDL-C(mg/dL) Deviation from T= Linearity % % % % % % % % % % % Lin. material lot # X-3 X-3 X-4 X-4 X-4 X-4 X-4 X-4 14% Sensitivity [per 1mg/dL] 12% 1% 8% 6% 4% 2% % Duration (month) 2 Linearity % 1% 2% 3% 4% 5% 6% 7% 8% 9% 1% M 3M 6M 9M 12M 15M 18M 2M The above results clearly demonstrate the stability of the test for more than 18 months after production. In addition to the real-time stability studies, temperature stress tests are routinely performed on each production batch as part of the end control of the product. Rev. 9 11/11