3 Linearity and Detection Limit Linearity Detection limit... 4

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1 Design Verification liquicolor 1 Introduction Imprecision, Reproducibility Data from internal study Date from external study (Cholesterol Reference Method Laboratory Network) Linearity and Detection Limit Linearity Detection limit Method comparison Friedewald vs. LDL-CHOLESTEROL Roche-BM vs. LDL-CHOLESTEROL liquicolor LDL CHOLESTEROL vs. Friedewald, performed at University hospital Mainz, Germany Roche Diagnostics vs. LDL CHOLESTEROL, performed at University hospital Mainz, Germany Traceability Interference study Endogeneous substances Anticoagulants Stability Lot real time stability at 8 C: Lot real time stability at 8 C: ot real time stability at 8 C QM-Element: ISO91:2 No Rev. 7 valid of /13

2 1 Introduction The LDL CHOLESTEROL liquicolor test is a direct homogeneous enzymatic assay for the quantitative determination of LDL-cholesterol (LDL). The test combines two steps, the first step eliminating chylomicrons, VLDL and HDL by specific enzymatic degradation. The next step determines the remaining LDL 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 spectrophoto-metrically (555 nm, [ nm] Hg 578 nm) and is directly proportional to the LDL concentration in the sample. For calculation of the LDL concentration a calibrator is employed. 2 Imprecision, Reproducibility The within-run imprecision of the LDL CHOLESTEROL liquicolor test was calculated from 1-fold determinations. The day-to-day imprecision was calculated from the results obtained on 4 different dates. Pooled human sera were employed as sample materials. 2.1 Data from internal study Sample Date 1 Date 2 Date 3 Date 4 Low 1 Medium 1 Low 1 Medium 1 Low 1 Medium 1 Low 1 Medium Mean, 84,7 141,9 84,7 142,4 83,5 141,8 84,7 142,1 SD,,82,99 1,16 1,7 1,8 1,3 1,16 1,2 CV, %,97,7 1,37,75 1,29,73 1,37,84 1 all results expressed in Overall results were as follows: Low Medium Overall mean, Overall SD, Overall CV, % Date from external study (Cholesterol Reference Method Laboratory Network) Three well characterised pool sera were employed as sample material and was tested over a period of 2 consecutive days with the LDL CHOLESTEROL test. The results are summarised in the following table. Day Sample 1 Sample 2 Sample 3 Result, Result, Result, Rev. 7 2/13

3 MEASURED ANALYTE CONCENTRATION (mg/dl ) Mean, SD, CV,% The above results demonstrate the excellent reproducibility of the test. Within-run and day-to-day imprecisions were found to be around or even below 1%. 3 Linearity and Detection Limit The linearity of the LDL 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. 3.1 Linearity High Pool Analytical Data Regressed Data Deviation from Regression Line Content (%) (%) Linear Regression Error Bars = ± 2SD LINEARITY HIGH POOL CONTENT (%) Conclusion: From the above results a linearity range up to 5 LDL has been confirmed. It should be noted, however, that the linearity of the assay depends on the respective analyser application. Rev. 7 3/13

4 3.2 Detection limit A 2-fold determination of a sample (phys. saline) on a HumaStar 1 analyser revealed an absolute mean of -.15 and a SD of.5. From the three-fold standard deviation the detection limit can therefore be calculated on the base of mean +3 SD to Method comparison Independent studies have been performed to verify the comparability of the LDL CHOLESTEROL liquicolor test with the CDC reference method, other LDL tests and the LDL results calculated according to the Friedewald formula. 4.1 Friedewald vs. LDL-CHOLESTEROL The LDL CHOLESTEROL test has been compared against results obtained with by the classical Friedewald formula. 15 serum samples have been tested and evaluated by a non-parametric regression analysis. Y = A * X + B A to 1.35 B, to 1.35 r.938 Low, for Y: 27.1 for X: High, for Y: for X: Median, for Y: 12.2 for X: Method comparison LD L FR IEDEWALD Rev. 7 4/13

5 4.2 Roche-BM vs. LDL-CHOLESTEROL liquicolor The LDL CHOLESTEROL liquicolor test has been compared against results obtained with by a competitor s test (Roche-BM). 15 serum samples have been tested with both methods and evaluated by a non-parametric regression analysis. Y = A * X + B A to 1.51 B, to r.938 Low, for Y: 27.1 for X: 37.9 High, for Y: for X: Median, for Y: 12.2 for X: Method comparison LDL 2 LD L Ch 15 ole stemg rol /dl HU M AN Roche-BM 4.3 LDL CHOLESTEROL vs. Friedewald, performed at University hospital Mainz, Germany The LDL CHOLESTEROL test has been compared against results obtained with by the classical Friedewald formula. 12 serum samples have been tested on an analyzer (CHOLESTEROL, HDL CHOLESTEROL, triglycerides and LDL CHOLESTEROL liquicolor). Results were evaluated by a non-parametric regression analysis. Y = A * X + B A B, r.974 Low, for Y: 32 for X: 29 High, for Y: 263 for X: 239 Median, for Y: 133 for X: 12 Rev. 7 5/13

6 Method comparison University hospital Mainz, Germany FR IEDEWALD 4.4 Roche Diagnostics vs. LDL CHOLESTEROL, performed at University hospital Mainz, Germany The LDL CHOLESTEROL test has been compared against results obtained with by a competitor s test (Roche-BM). 6 serum samples have been tested with both methods and evaluated by a non-parametric regression analysis. Y = A * X + B A B, r.968 Low, for Y: 45 for X: 56 High, for Y: 263 for X: 25 Median, for Y: 135 for X: 134 Rev. 7 6/13

7 mg /d l ROCHE Diagn ostics mg /dl Conclusion: The method comparison studies demonstrated a very well correlation with long-standing classical methods as well as with competitor tests. 5 Traceability The LDL-CHOLESTEROL liquicolor test for the determination of LDL-Cholesterol is calibrated with a calibrator, which is traceable to the CDC reference method. The recovery of the calibrators have been tested as well as the comparison of 5 patient samples. The results are summarised in the tables below. Hitachi 717 Calibration: Reference Human assign. abs assign. abs cal cal Recovery: assign Reference Human Reference Human Reference Human rep rep rep Mean Recovery % 1.3% 12.9% 11.4% Rev. 7 7/13

8 Human LDL [mg/dl] Sample: X: Reference Y: Human Sample: X: Reference Y: Human y =,998x -,593 R 2 =, Reference method LDL [mg/dl] Conclusion: The LDL-CHOLESTEROL liquicolor calibrator is traceable to the international reference method. Rev. 7 8/13

9 6 Interference study 6.1 Endogeneous substances Any potential interfering influence by ascorbic acid, hemoglobin and bilirubin has been studied by adding pure substances to a normal pool serum. The obtained results demonstrate that ascorbic acid up to 5, hemoglobin up to 5 and bilirubin up to 3 do not interfere with the test (Glick value = 1). VLDL triglycerides up to 13, VLDL cholesterol (total) up to 5, chylomicron triglycerides up to 7 and chylomicron cholesterol (total) up to 17 showed only minor or negligible interference (Glick value = 1-2). 6.2 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 LDL Na-Hep. mg/l LDL Na-Fl. mg/l LDL Na-oxal. mg/l LDL EDTA mg/l LDL Conclusion: The results gave clear evidence that none of the anticoagulants has an effect on the test results. 7 Stability The stability of the LDL-CHOLESTEROL liquicolor test has been tested on real-time storage conditions over a period of 21 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. 7.1 Lot real time stability at 8 C: Duration (days) (months) 6M 9M 13M 18M 21M Sensitivity BLK STD mabs./1mg/dl (deviation from T=) (1%) (97%) (1%) (98%) (97%) (93%) Calibrator lot # Assigned value (mg/dl) Specificity Lipid Cont. I mean Lot recovery% 1% 98% 11% 98% 96% 98% Rev. 7 9/13

10 LDL-C(mg/dL) Deviation from T= Lipid Cont. II mean Lot recovery% 1% 98% 98% 98% 99% 99% Linearity % % % % % % % % % % % Lin. material lot # A-12 A-12 A-13 A-14 A-15 A-15 Sensitivity [per 1mg/dL] 14% 12% 1% 8% 6% 4% 2% % Duration (days) Linearity % 1% 2% 3% 4% 5% 6% 7% 8% 9% 1% 6M 9M 13M 18M 21M Rev. 7 1/13

11 L D L - C ( m g / d L ) 7.2 Lot real time stability at 8 C: Duration (days) (months) 6M 9M 13M 18M 21M Sensitivity BLK STD mabs./1mg/dl (deviation from T=) (1%) (98%) (1%) (98%) (97%) (93%) Calibrator lot # Assigned value (mg/dl) Specificity Lipid Cont. I mean Lot recovery% 1% 97% 11% 97% 96% 95% Lipid Cont. II mean Lot recovery% 1% 97% 99% 97% 11% 98% Linearity % % % % % % % % % % % Lin. material lot # A-12 A-12 A-13 A-14 A-15 A-15 De viat ion fro m T= 14% 12% 1% 8% Sensitivity [per 1mg/dL] 6% 4% 2% % Duration (days) L i n e a r i t y % 1 % 2 % 3 % 4 % 5 % 6 % 7 % 8 % 9 % 1 % 6 M 9 M 1 3 M 1 8 M 2 1 M Rev. 7 11/13

12 Deviation from T= 7.3 ot real time stability at 8 C Duration (days) (months) 6M 9M 13M 18M 21M Sensitivity BLK STD mabs./1mg/dl (deviation from T=) (1%) (97%) (99%) (97%) (96%) (92%) Calibrator lot # Assigned value (mg/dl) Specificity Lipid Cont. I mean Lot recovery% 1% 97% 12% 98% 97% 98% Lipid Cont. II mean Lot recovery% 1% 11% 11% 98% 1% 1% Linearity % % % % % % % % % % % Lin. material lot # A-12 A-12 A-13 A-14 A-15 A-15 Sensitivity [per 1mg/dL] 14% 12% 1% 8% 6% 4% 2% % Duration (days) Rev. 7 12/13

13 LDL-C(mg/dL) Linearity % 1% 2% 3% 4% 5% 6% 7% 8% 9% 1% 6M 9M 13M 18M 21M Conclusion: The above results support the claimed stability of the test of 18 months from the date of production. Rev. 7 13/13