PEPFAR Quality Control and Method Validation Activity 14 Handout: Linearity

172 Linearity Handout 14A02 Chemistry Linearity Guidelines Linearity studies are performed to determine the linear reportable range for an analyte. The linearity for each analyte is assessed by checking the performance of recovery throughout the manufacturer's stated range of the testing system. This is done using a set of standards containing varying levels of an analyte in high enough and low enough concentrations so as to span the entire range of the test system. Therefore, the demonstration of the linear range requires a series of known concentrations or know relationships established by dilution. Linearities are performed whenever a new analyzer, analyte, or method is introduced into the laboratory, or when an analyzer is replaced. Linearities may also be performed for troubleshooting purposes when quality control is unacceptable and deviations from acceptable data cannot be explained, when major analyzer repair or replacement of components has taken place, or at intervals prescribed by the manufacturer in the instrument's user manual. I. Linearity A. Sample Criteria 1. A minimum of 5 samples that cover the reportable range of the method. 2. When plotted, the values should ideally be equidistant from each other. 3. Quality control, commercial linearity standards, and calibrators (if a different lot number is used to calibrate the instrument) may be used. 4. Patient specimens may be used if a high value near the expected upper range can be found. 5. Sufficient volume of each sample must be available to analyze in triplicate and for possible troubleshooting. B. Preparation 1. If using purchased materials, refer to manufacturer's instructions. 2. If using patient specimens, then perform the dilutions using the manufacturer's recommendation of the diluent to use with out -of- range specimens. 3. Select a patient specimen near the detection limit and another patient specimen near or slightly above the expected upper limit of the working range. Ensure that both specimens meet storage and stability requirements as stated by the manufacturer. 4. Prepare 5 pools for testing as follows: a. Label the low specimen Pool 1 and the high specimen Pool 5. b. Prepare Pool 2 (75/25) with 3 parts Pool 1 + 1 part Pool 5. c. Prepare Pool 3 (50/50) with 2 parts Pool 1 + 2 parts Pool 5. d. Prepare Pool 4 (25/75) with 1 part Pool 1 + 3 parts Pool 5. 5. Care must be taken to mix each pool thoroughly, and to protect the pools from evaporation or other deterioration. Page 1 of 3 Modified from pSMILE.org website: EQU35-A-05_Linearity Rqmt_Chemv.1.1.doc. Last accessed May 3, 2013 HILS1746 Effective Date: 12/6/2016 173 Linearity Handout 14A02 C. Testing 1. A set of linearity standards will be tested in the same manner as patient samples. 2. Testing should be performed in triplicate and performed within a single run. If one value deviates greatly from the others due to random error, it may be removed from the data analysis and repeated. 3. Data should be plotted immediately to identify and correct any outliers. 4. Save the instrument print-outs to be filed with the summary statistics D. Evaluation of data: 1. Record the raw values on the Linearity Experiment Worksheet 2. Calculate Mean (Y) for each data point 3. Determine the Assigned Value (X) for each data point: a. If standards have known values, then insert them into the Assigned Value (X) column. b. If using the known relationship between dilutions, then follow the manufacturer's instructions. c. If using patient dilutions, i. Pool 3 will be used as a true value; therefore, the mean value (Y) obtained will be the assigned value (X). ii. The remaining pools will be calculated using the known relationship between dilutions as follows: i. Pool 1 = mean of Pool 3 x 0 = 0 (Pool 1 must be zero or near zero, or else the actual value must be taken into account) ii. Pool 2 = mean of Pool 3 x 0.5 iii. Pool 4 = mean of Pool 3 x 1.5 iv. Pool 5 = mean of Pool 3 x 2.0 4. Visual Assessment a. Plot the assigned values on the X-axis on graph paper b. Plot the mean of the measured values on the Y-axis. c. Manually draw a straight line through as many points as possible, making sure that the line adheres to the lower points. d. An alternative to creating a graph is to use the Linear-data Plotter located on the westgard website. e. Visually inspect the plot for a linear relationship. f. Note on the graph the observed linearity range. g. Date and sign your initials. h. File the graph in the linearity portion of your method validation binder. 5. Quantifying Errors a. Plot the X and Y data in a regression analysis program. Calculate the slope and the y-intercept using linear regression from the westgard website Page 2 of 3 Modified from pSMILE.org website: EQU35-A-05_Linearity Rqmt_Chemv.1.1.doc. Last accessed May 3, 2013 HILS1746 Effective Date: 12/6/2016 174 Linearity Handout 14A02 b. Record the slope (m) and intercept (b) on the Linearity Experiment Worksheet. c. Using slope and intercept, calculate a predicted Y (Y) value for each X value using the equation Y' = m X + b. d. Subtract each measured Y value from the associated predicted Y value (SE= Y - Y') This difference represents . the systematic error due to non-linearity. Record these values on the Linearity Experiment Worksheet under the ± Diff e. Calculate % Diff (%Diff = : [+ Diff/Y']* 100% E. Determining Acceptability 1. Calculate % Limit by dividing the CLIA %TEA by a factor of 2 2. Calculate ± Limit by either inserting 50% of the CLIA absolute value or by multiplying the %Limit by Y', whichever is greater. 3. Compare that systematic error to 50% of the total allowable error. The systematic error must be less than 50% of the total allowable error. 4. Complete the Acceptability column on the Linear Experiment Worksheet 5. Determine the linear range from the experiment 6. Determine the analytical measuring range (AMR). For each laboratory, it may not exceed the manufacturer's stated AMR. 7. Place all documentation in the appropriate method validation binder. II. References A. GCLP Workshop and Workbook18-20 May 2008, Verification of Performance Specifications, pages 1-33. B. Clinical and Laboratory Standards Institute (CLSI). Evaluation of the Linearity of Quantitative Measurement Approved Guideline-Second Edition, CLSI document EP6-A (ISBN 1-56238-498-8) Clinical and Laboratory Standards Institutes, 940 West Valley Road, Suite 100, Wayne, Pennsylvania 19098-1898 USA, 2005. C. Westgard, James O. (2003). Basic Method Validation (2nd ed.). Madison: Westgard QC, Inc. D. Westgard, James O. (2008). Basic Method Validation (3rd ed.). Madison: Westgard QC, Inc. Page 3 of 3 Modified from pSMILE.org website: EQU35-A-05_Linearity Rqmt_Chemv.1.1.doc. Last accessed May 3, 2013 HILS1746 Effective Date: 12/6/2016