Design Verification Total Protein liquicolor Contents 1 Imprecision .................................................................................................................................................................... 2 2 Linearity and Sensitivity............................................................................................................................................... 2 2.1 Linearity ............................................................................................................................................................................................... 2 2.2 Sensitivity ........................................................................................................................................................................................... 3 3 Recovery in Control Sera .............................................................................................................................................. 3 4 Comparison of Methods ............................................................................................................................................... 3 5 Stability.......................................................................................................................................................................... 4 5.1 Real-time Results ............................................................................................................................................................................. 4 5.2 Temperature Stress Results ......................................................................................................................................................... 5 6 Interference ................................................................................................................................................................... 6 7 Traceability .................................................................................................................................................................... 6 Form 4.2-01.06-04 TOTAL PROTEIN liquicolor | Rev. 006 | valid of 25.11.2020 1/6 1 Imprecision The imprecision (within-run and day-to-day) of the TOTAL PROTEIN liquicolor method was calculated from six determinations on ten consecutive days. Precision level low, medium and high control sera were employed as sample material. Within-run imprecision Hitachi 717 Analyte concentration (g/dl) 4.33 7.89 11.38 Intra-assay Standard Deviation 0.048 0.066 0.11 Inter-assay Standard Deviation 0.117 0.196 0.289 %CV 1.10 0.84 0.92 %CV 2.70 2.48 2.54 2 Linearity and Sensitivity Linearity The linearity of the TOTAL PROTEIN liquicolor method was controlled by employing a high concentration pool 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. Hitachi 717 High Pool Content (%) 0 10 20 30 40 50 60 70 80 90 100 Analytical Data g/dl 0.00 0.73 1.97 3.33 4.54 5.76 7.05 8.50 9.75 11.34 12.53 Regressed Data g/dl -0.47 0.82 2.10 3.38 4.67 5.95 7.24 8.52 9.81 11.09 12.37 Deviation from Regression Line g/dl (%) 0.47 -100.00 -0.09 -11.09 -0.13 -6.18 -0.05 -1.60 -0.13 -2.86 -0.19 -3.24 -0.19 -2.65 -0.02 -0.25 -0.06 -0.57 0.25 2.25 0.15 1.22 LINEARITY 14 Linear Regression 12 Error Bars = ± 2SD 10 8 6 4 2 0 0 20 40 60 80 100 120 Conclusion: The TOTAL PROTEIN liquicolor test is linear up to a protein concentration of 12 g/dl or 120 g/l. Design Verification and Product Data for TOTAL PROTEIN liquicolor Rev. 006 2/6 Sensitivity A 20-fold determination of a 0 g/dl calibrator (phys. saline) on a Hitachi 717 analyser revealed an absolute mean of 0.05 g/dl and a SD of 0.02 g/dl. From the three-fold standard deviation the analytical sensitivity can therefore be calculated to < 0.12 g/dl total protein. 3 Recovery in 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 TOTAL PROTEIN liquicolor method. The means of the fivefold determinations have been calculated and compared with the target values. CONTROL SERUM RECOVERY Control LOT HUMAN fresh Reference HUMAN 12 d 45 °C Target Range Result Deviation Result Deviation Result g/dl g/dl g/dl (%) g/dl (%) g/dl (%) # 018 5.83 4.80 – 6.87 5.41 -7.14 5.42 -6.96 5.38 -7.68 HUMATROL P # 016 8.07 6.75 – 9.40 7.45 -7.66 7.44 -7.86 7.42 -8.03 SERODOS # 6868 6.81 6.2 – 7.4 7.13 4.76 7.15 4.96 7.06 3.70 SERO.Plus # 6795 5.43 4.94 – 5.92 5.33 -1.92 5.33 -1.84 5.27 -2.91 Precinorm # 154916 5.59 5.08 – 6.10 5.54 -0.82 5.57 0.36 5.46 -2.29 Precioath # 199459 5.58 5.07 – 6.09 5.59 0.11 5.59 0.11 5.51 -1.25 36.45 -2.12 36.5 -1.99 36.1 -3.08 serum HUMATROL N Summary 37.31 Deviation 4 Comparison of Methods The TOTAL PROTEIN liquicolor has been compared against a commercially available Total protein method. Control sera as well as patient samples have been employed in the comparison (N=55). The results have been evaluated by a main component analysis. The linear regression obtained could be described as follows: TOTAL PROTEIN liquicolor (Y) = 0.980 * Reference TOTAL PROTEIN (X) – 0.069 Coefficient of correlation: r = 0.996 Method Comparison Total Protein 8,5 TOTAL_PROTEIN_LIQUICOLOR, g/dl 8,0 7,5 7,0 6,5 6,0 5,5 5,0 4,5 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 REFERENCE, g/dl Design Verification and Product Data for TOTAL PROTEIN liquicolor Rev. 006 3/6 Both methods showed a good agreement and no significant deviation could be observed with any specific sample. An evaluation of the method comparison acc. to the model of Passing&Bablok yielded the following results: Variable X : REFERENCE Variable Y : TOTAL_PROTEIN_LIQUICOLOR Sample size = 55 REFERENCE Lowest value Highest value Arithmetic mean Median Standard deviation Standard error of the mean = = = = = = 4,9500 7,9850 6,6926 6,7950 0,6977 0,0941 TOTAL_PROTEIN_LIQUICOLOR Lowest value = Highest value = Arithmetic mean = Median = Standard deviation = Standard error of the mean = 5,0050 8,1850 6,7568 6,8650 0,7091 0,0956 Y = -0,0608 + 1,0191 X Intercept A :-0,0608; 95% CI : -0,2283 to 0,0877 Slope B : 1,0191; 95% CI : 0,9974 to 1,0438 Cusum test for linearity: No significant deviation from linearity (P>0.10) 5 Stability Real time studies have been conducted for three independent production lots of this product on a Hitachi 717. Real-time Results CONTROL HUMAN fresh (Reference) HUMAN 24 month # H 038 Control LOT serum # H 016 Target Range Result Deviation Result g/dl g/dl g/dl (%) g/dl Deviation (%) -10.29 5.24 -10.12 Humatrol N # 018 5.83 4.80 – 6.87 5.23 Humatrol N # 019 5.17 4.7 – 5.64 5.1 -1.35 5.1 -1.35 Humatrol P # 016 8.07 6.75 – 9.4 7.1 -12.02 7.01 -13.14 Humatrol P # 017 6.9 6.28 – 7.52 7.1 2.90 6.99 1.30 Serodos # 6868 6.62 6.02 – 7.22 6.89 4.08 6.85 3.47 Sero.Plus # 6795 5.6 5.1 – 6.1 5.16 -7.86 5.12 -8.57 Sero.Plus # 6796 5.3 4.72 – 5.88 5.55 4.72 5.5 3.77 Precinorm # 159296 5.42 4.94 – 5.9 5.33 -1.66 5.31 -2.03 Precipath # 165215 5.19 4.71 – 5.67 5.32 2.50 5.3 2.12 Summary 54.1 Design Verification and Product Data for TOTAL PROTEIN liquicolor Rev. 006 52.78 52.42 4/6 CONTROL HUMAN fresh (Reference) # HUMAN 26 month # H 025 HUMAN 27 month # H H 040 Control LOT serum 024 Target Range Result Deviation Result Deviation Result Deviation g/dl g/dl g/dl (%) g/dl (%) g/dl (%) Humatrol N # 019 5.17 4.7 – 5.64 5.32 2.90 5.25 1.55 5.23 1.16 Humatrol N # 020 5.34 4.75 – 5.93 5.39 0.94 5.26 -1.50 5.24 -1.87 Humatrol P # 017 6.9 6.28 – 7.52 7.1 2.90 6.98 1.16 6.9 0.00 Serodos # 6869 6.78 6.03 – 7.53 6.85 1.03 6.61 -2.51 6.66 -1.77 Sero.Plus # 6796 5.3 4.72 – 5.88 5.45 2.83 5.37 1.32 5.28 -0.38 Precinorm # 164919 6.64 5.83 – 7.45 6.75 1.66 6.64 0.00 6.56 -1.20 Precipath # 163661 5.04 4.44 – 5.64 5.13 1.79 5.01 -0.60 5.03 -0.20 Summary 41.17 41.99 41.12 40.9 Conclusion: All results support the claimed stability of 24 months from production for the test kit. Temperature Stress Results In addition to the real-time storage, temperature stress tests have been applied on the TOTAL PROTEIN liquicolor. Stressed reagents have been compared with fresh ones, as sample dilutions of a high stock solution have been employed. The manual pipetting regime has been applied. % Dilution of high stock solution 10 10 50 50 75 75 100 100 Fresh reagent. g/dl 1.26 1.25 7.27 7.26 9.73 9.73 12.27 12.7 Stressed for 10 d @ 45°C. g/dl 1.29 1.29 7.06 7.34 10.1 10.04 12.65 13.59 Stressed for 12 d @ 45°C. g/dl 1.27 1.34 6.94 6.82 9.34 10.12 12.24 12.28 Total-Protein, Linearity 15 fresh 10 10 d 45 °C g/dl 5 12 d 45 °C 0 0 20 40 60 80 100 120 % Dilution Conclusion: Stressed reagents did not significantly differ from fresh ones, confirming the stability claim of 24 months. Design Verification and Product Data for TOTAL PROTEIN liquicolor Rev. 006 5/6 6 Interference Interference by triglycerides, hemoglobin, bilirubin and ascorbic acid have been studied by adding known amounts of the potentially interfering substance to a known sample. Recoveries have been analyzed according to the method of Glick et al. (Clin.Chem. 1986, 32 470-5). Trig. Result Recov. Hemo Result Recov. Bili Result Recov. Ascorb Result Recov. mg/dl mg/dl % mg/dl mg/dl % mg/dl mg/dl % acid mg/dl % mg/dl 0 5.74 100 0 5.63 100 0 5.70 100 0 5.81 100 250 5.86 102.1 50 5.69 101.1 4 5.62 98.6 2 5.81 100.0 500 5.98 104.2 100 5.82 103.4 8 5.58 97.9 4 5.83 100.3 750 6.27 109.2 150 5.80 103.1 12 5.55 97.3 6 5.89 101.4 1000 6.31 109.9 200 6.02 106.9 16 5.51 96.7 8 5.84 100.4 1250 6.47 112.6 250 6.02 106.9 20 5.45 95.5 10 5.81 100.0 1500 6.56 114.2 300 6.23 110.8 24 5.42 95.0 12 5.89 101.4 1750 6.61 115.1 350 6.29 111.7 28 5.41 94.8 14 5.86 100.9 2000 6.74 117.4 400 6.40 113.8 32 5.36 93.9 16 5.88 101.1 2250 6.90 120.1 450 6.54 116.2 36 5.31 93.1 18 5.83 100.3 2500 7.11 123.8 500 6.64 118.0 40 5.29 92.8 20 5.85 100.7 Glick 3 2 2 1 Conclusion: Triglyceride yielded a low interference up to 1000 mg/dl. Hemoglobin showed only a low effect up to 250 mg/dl and Bilirubin up to 28 mg/dl, and ascorbic acid did not show any effect up to 20 mg/dl. A pronounced interference was observed with triglycerides, hemoglobin and bilirubin, exceeding the a.m. concentrations. Therefore visibly lipemic, hemolytic and icteric samples require a separate sample blank to correct for potential interferences. 7 Traceability The internal master standard, against which the kit working standards are calibrated, is traceable to the international reference preparation NIST SRM 927e. Design Verification and Product Data for TOTAL PROTEIN liquicolor Rev. 006 6/6
