Explain and apply the use of calibrators and controls in immunoassay Criteria for assay run acceptance/rejection, including Westgard rules

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1 RCPA Part II Exam Study Group WEEK 8 Objectives Explain and apply the use of calibrators and controls in immunoassay Standardisation Traceability of calibrators Choice of control samples Criteria for assay run acceptance/rejection, including Westgard rules Inter-assay and intra-assay variation Co-efficient of variation Random versus systematic errors Bias The basis of Internal QC is that the lab regularly runs control samples together with test samples. Control values are then plotted in a control chart. Identification of trends/shifts in reported control values If the control value is outside the limits decisions must be made regarding analytical result reporting and remedial action Question 1 a) What is the difference between a calibrator and a control? A calibrator is a material or device with known quantitative/qualitative characteristics e.g. concentration, activity, reactivity. Calibrators are used to calibrate, graduate or adjust a measurement procedure. In Immunoassays calibrators are commonly samples where the concentration of the analyte is known and they are used to establish the relationship between the amount of signal produced in the assay and the analyte concentration. A control is used to monitor the status of an analysis to maintain its performance within desired limits Immunoassay controls are commonly samples that contains a known concentration of the analyte and is used to monitor the performance of your assay by assessing the precision over time, typically using a Levy Jennings chart

2 b) Describe different types of controls Control samples may be standard solutions, real test samples, blank samples, in- house controlled materials and/or certified reference materials. Type I certified reference material (CRM) i. CRM470 although now the international reference material for many proteins (Ig, C3,C4) is not generally available to diagnostic laboratories. Many commercial preparations available are calibrated against this (secondary standards) Type II Reference material- standard solution or in- house material i. This may include samples tested by other labs Type III Blank sample Type IV Test sample Question 2 a) Name 6 common Westgard rules 12s 13s 22s 14s : ie 2 x2sd violations +2SD and - 2SD violations 41s :4 values on the same side of mean (+/- 1SD) 101s :10 values on same side of mean (+/- 1SD) b) For the above rules what are the criteria for acceptance/rejection?

3 12s 13s 22s i. (warning limit) i. Outcome: No analytical results can be reported. All samples 14s : ie 2 x2sd violations +2SD and - 2SD violations 41s :4 values on the same side of mean (+/- 1SD) 101s :10 values on same side of mean (+/- 1SD) analysed since last control value in control was obtained must be

4 Question 3 a) What is the difference between accuracy and precision? i. Accuracy is the ability to measure the analyte correctly on any given occasion. i. Precision is the degree to which repeated measurements taken in unchanged conditions show the same results (also known as reproducibility, repeatability). For any given assay the precision is lower as the analyte concentration approaches the lower level of sensitivity (i.e. limit of measureability). Precision may worsen as a result of reagent deterioration, apparatus instability, operator problems. ii. In the below diagram #2 represents random error and #3 systematic error. b) Define coefficient of variation Statistically precision is expressed as the CoV= SD/mean x100% Typically CV is used to express intra- assay and inter- assay variation. c) Precision may worsen as a result of reagent deterioration, apparatus instability, operator problems. d) What is the intra- assay CV and how is it calculated? What range of intra- assay CV is considered acceptable? Also known as repeatability Measuring the same sample multiple times will produce a spread of results

5 Intra- assay CV attempts to measure the magnitude of imprecision for a single assay on the same platform on the same day To measure aliquot a single sample multiple times e.g. 20 X and run on the same run (e.g. same plate of an ELISA). Calculate the mean of the results, then SD and then CV (SD/mean)x100% Intra- assay CV <10% are generally considered acceptable. e) What is the inter- assay CV and how is it calculated? What range of inter- assay CV is considered acceptable? This is the measure of imprecision over time (on different days). Usually done at 2 different concentrations e.g. high and low. Take 2 samples (One HIGH and one LOW) and aliquot multiple times e.g. each sample aliquoted into 20 tubes. Run one aliquot of each HIGH and LOW sample every day on the same assay/platform for 20 consecutive days. Following this examine the data to calculate (for each sample) mean, SD and CV as above. Inter- assay CV of <15% are generally considered acceptable. f) List some assays with known lower precision: ACL ELISAs often have CV>25%; In such situation qualitative reporting is suggested e.g. LOW, MODERATE, HIGH.

6 Question 4 (Derived from D.Suan) Your RCPA college project is due. You decide to conduct an internal audit into the performance of anti- NMDA Ab ELISA. The Westgard plot shows this: a) What does the graph represent? The graph shows assay drift representing sysematic rather than random error b) Describe sources of random error in the Immunopathology Lab: Sample or reagent pipetting error Air bubbles in reagent or reagent lines Inadequately mixed reagents Air bubbles in water supply Unstable enzyme incubation temperatures Unstable electrical supply Misplacement of control samples within run Incorrect handling of control products Inappropriate storage of control products Operator technique c) Describe sources of systematic error in the Immunopathology Lab: Gradual: Accumulation of debris in sample or tubing Gradual deterioration of reagent Deterioration of control product Deterioration of incubation chamber temperature Deterioration of light filter integrity Deterioration of calibration Deterioration in light source

7 Abrupt: Change in reagent lot Change in reagent formulation Change in calibrator lot Incorrect callibrator values Incorrect calibration/re- calibration Major instrument maintenance Sudden change un incubator temperature Change in room temperature/humidity Failure in sampling system Failure in reagent dispensing system Sudden failure/change in light source d) How would you investigate the above problem? Check other analytes run on this ELISA plate for a systemic issue with the ELISA platform Check the lot validation for the ELISA wells Check the machine for its settings: temp, times Check the spectrophotometer Check all reagents, calibrators, controls for expiry dates Change to a new aliquot of controls Change to new reagents Change to a new calibrator e) Give examples of how random and systematic error may be represented using f) Westgard rules. Westgard Rule Error type 12S 13S Random or Systematic Random or start of Systematic 22S R4S 41S 10X Systematic Random Systematic Systematic