PEPFAR Quality Control and Method Validation Activity 14 Handout 3: AST Validation Plan

168 Handout 3: AST Validation Plan 1405 Chemistry Validation Plan Validation Plan for Roche Cobas c501 Chemistry Analyzer I. Overview 1. Precision 2. Accuracy 3. Linearity 4. Sensitivity 5. Specificity 6. Reference Range 7. Method Approval II. Plan: The validation will be conducted on the Roche Cobas c501 Chemistry Analyzer (serial number 12345) for the following analytes and methods: AST 1. Precision a. Precision is reproducibility - the agreement of the measurements of replicate runs of the same sample. It is the process of determining the range of random error. The precision is measured in terms of coefficient of variation (CV). b. Random Error will be evaluated by running between day (intermediate precision) and within day (repeatability) precision using normal (QC Multi 1) and abnormal (QC Multi 2) control samples. Between-day precision will be tested by running each sample once per day for 20 days. Within day precision will be tested by running each sample 20 times in one day. The mean, standard deviation (SD), and CV of the replicates will be calculated. c. Acceptability criteria: The % CV for each assay is expected to be equal to or less than the manufacturer' s performance specifications for precision. In the event that an assay does not perform as expected, the %CV will be compared to the allowable random error (33% of CLIA Total Allowable Error Limits for between day and 25% of CLIA Total Allowable Error Limits for within day). . Analyte Mfg Precision 33% of TEA 25% of TEA AST 1.3% 6.67% 5% 2. Accuracy/Correlation a. Accuracy is the true value of a substance being measured. Verification of accuracy is the process of determining that the test system is producing true, valid results. b. Accuracy will be determined by a minimum of 40 samples, tested in duplicate. These will primarily be patient samples, but may include commercial proficiency testing or control samples in order to provide material that covers the reportable range. The samples will be tested on a XYZ Chemistry Analyzer located at Cape Clinic Laboratory chemistry section. The samples will be tested in duplicate on both the test and comparative instruments and duplicates will be averaged. Ideally, testing will occur on both instruments within 2 hours. c. Acceptability criteria: Linear regression analysis will be used to determine if the methods are accurate within the specified TEa when the Correlation Coefficient (r) is >0.975. If the Correlation Coefficient is < 0.975, then more patient data must be collected. If the Correlation Coefficient remains < 0.975, then paired data calculations or another regression analysis technique will be used. Modified from the pSMILE.org website. EQ-35-A01-Chem_Val_Plan_Template. Last accessed May 3, 2013 HILS1746 Effective Date: 12/6/ 2016 169 Handout 3: AST Validation Plan (1405) 3. Linearity a. A quantitative analytical method is said to be LINEAR when measured results from a series of sample solutions are directly proportional to the concentration or activity of an analyte in the test specimens. This means that a straight line can be used to characterize the relationship between measured results and the concentrations or activity levels of an analyte for some stated range of analyte values. b. Linearity verification will be determined using a patient sample previously analyzed on the XYZ analyzer. A known high near or slightly above the upper measurement range and a known low near the limit of detection will be used. i. Samples will be run in triplicate. ii. The mean value for each point will be calculated. iii. The recovered mean values will be plotted versus the corresponding known theoretical X values. A best-fit straight line will be drawn to connect the points on the graph with greater emphasis on the first three points when drawing the best-fit line. iv. The plot will be visually inspected for a linear relationship. v. The predicted Y value will be subtracted from the associated recovered mean value. The absolute difference and % difference will be calculated. This difference is the systematic error due to non-linearity. vi. Systematic error will be compared to 50% of the total error. b. Acceptability criteria: i. Visual assessment of the best-fit line on the linearity plot must demonstrate a linear relationship. ii. The method is linear if the % difference (or absolute difference) between the predicted Y' and the recovered mean is less than the allowable error for each specimen point. iii. The systematic error must be less than 50% of the total error. 4. Sensitivity is the lowest concentration of an analyte that can be measured (Lower Limit of Detection). For an FDA-approved, unmodified method, the manuf acturer's stated sensitivity will be used. 5. Specificity is the determination of the effect of interfering substances. For an FDA-approved, unmodified method the manufacturer's stated specif icity will be used. 6. Reference Ranges a. Reference ranges are measured sets of values determined to occur in a healthy, non-diseased population. The laboratory must verify that their choice of reference ranges is valid for their study population. To verify or transfer a published range, the lab must analyze specimens from 20 healthy, non-diseased individuals for each subgroup. If 2 or fewer results fall outside the published range, it is considered verified. If, however, more than 2 results fall outside the published range, a more extensive study must be conducted. Activity 14: Introduction to Method Evaluation HILS1746 Modified from the pSMILE.org website. EQ-35-A01-Chem_Val_Plan_Template. Last accessed May 3, 2013 Effective Date: 12/6/2016 170 Handout 3: AST Validation Plan (1405) b. The reference range studies have been verified for the following populations: i. Adult reference ranges The population sample used will be from the Cape Clinic Employee's Health Clinic Forty . individuals (twenty adult males and twenty adult females) screened using a patient questionnaire and identified to be in good health will be used for the reference interval study. Any individual answering in the affirmative to unexplained weight loss, fever, jaundice, recent operation, pregnancy, or medications will be excluded from the experiment. A red top tube will be collected and transported to the laboratory in accordance with Cape Clinic's collection and handling procedures for immediate analysis. c. Acceptability criteria: i. Establishment: ranges will be determined using a non- parametric statistical method to determine the 95% reference limits. For most analytes the lower and upper reference limits are defined as the 2.5 and 97.5 percentiles, respectively. ii. Verification: ranges will be considered verified if 90% of values fall within the proposed range iii. Verification of pediatric ranges will be dependent on the ability to collect sufficient pediatric samples in each age category. Additional time may be required, or fewer samples may be acceptable. The Medical Director will give final approval of the acceptability of pediatric reference range verification. 7. Method Approval The final decision regarding methodology validation and acceptance is made after a careful review of all the studies performed as part of the complete method validation process. The Laboratory Director shall make the ultimate decision on method validation. Method acceptance is based on the results from the above studies, plus a n evaluation of the new method's cost effectiveness, turn-around-time, laboratory staff training needs, and any other relevant operational considerations. 8. References a. NCCLS EP9-A2 Vol.22 No.19 (Method Comparison and Bias Estimation Using Patient Samples), Sep 2002. b. NCCLS C28-A Vol. 15 No. 4 (How to Define and Determine Reference Intervals in the Clinical Laboratory), Jun 2000. c. NCCLS C10-A2 Vol. 22 No. 29 (Preliminary Evaluation of Quantitative Clinical Laboratory Methods), Dec 2002. d. NCCLS C6-A Vol. 23 No. 16 (Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach), April 2003. e. NCCLS C5-A2 Vol. 24 No. 25 (Evaluation of Precision Performance of Quantitative Measurement Methods), Aug 2004. f. NCCLS EP21-A Vol. 23 No. 20 (Estimation of Total Analytical Error for Clinical Laboratory Methods), Apr 2003. g. CAP Commission on Laboratory Accreditation, Laboratory General Checklist, 2007. h. Fawzi, W.W, et al. Vitamins and Perinatal Outcomes among HIV- Negative Women in Tanzania. N Engl J Med 2007; 356: 1423-31. i. Roche Cobas package inserts for ALT, AST, ALB, 2006-08 and 2006- 05. Activity 14: Introduction to Method Evaluation HILS1746 Modified from the pSMILE.org website. EQ-35-A01-Chem_Val_Plan_Template. Last accessed May 3, 2013 Effective Date: 12/6/2016