BIOT6002 Lecture 10 Immunoassay Standardisation PDF

Standardisation & Quality Assurance – Part 1 BIOT6002: Lecture 10 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Understand why standardisation and quality assurance (QA) are required for immunoassays. List quality assurance activities. Define the following terms Internal qua...

Standardisation & Quality Assurance – Part 1 BIOT6002: Lecture 10 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Understand why standardisation and quality assurance (QA) are required for immunoassays. List quality assurance activities. Define the following terms Internal quality control External quality control International Standard Calibration Standard Discuss how QA determines assay practicability and reliability Why is standardisation necessary? Widespread use of immunoassay techniques Problems encountered with biological materials used as standards & Quality Control (QC) of materials for quantitation of structurally complex analytes. Why Quality Assurance necessary? Quality assurance encompasses a wide spectrum of quality improving activities designed to ensure reliability of laboratory investigations. Activities include: Performance of appropriate tests Correct preservation of sample Validation & standardisation of method Internal quality control (IQC) & external quality assessment (EQA) Correct interpretation of results Definitions Internal Quality Control (IQC) A set of procedures used to assess laboratory assays. Used to determine reliability of routine assays. External Quality Assessment (EQA) A set of procedures for objectively checking laboratory results by means of an external agency. Standardisation Principles 1. Heterogeneity of analyte in the sample and in the standard can cause discrepancies in sample results Proteins & peptides can exist in a variety of molecular forms (post- translational modifications, sub-units etc). 2. Requirements of the Standard. i. Suitable for use in the widest possible range of assay system with the same type of concentration units. ii. Available in sufficiently large quantities, with consistent stability, to permit continuity of use through time and between laboratories. iii. The standard matrix should behave identically to the sample matrix. Standardisation in Immunoassay International Standard (IS) Established by the Expert Committee on Biological Standardisation (ECBS) of the World Health Organisation Most IS for immunoassay analytes are prepared by the National Institute for Biological Standards & Control https://nibsc.org/ Defined following an international collaborative study. Contains a specific amount of analyte (standardisation of assay kits & laboratory standards). Calibration standard Material of known composition derived from IS Used as a comparator to determine the concentration of the material in a test sample. Preparation of International Standard (IS) Characterised by physio-chemical means. Batch (500-5000 ampoules) prepared (inter-ampoule CV of 0.25%). Lyophilised. Stability assessed in accelerated thermal degradation studies. Concentration (molar, IU) measured by direct comparison with existing IS in an international collaborative study. Statistical analysis. Unit assignment. Acceptance by ECBS and designated an IS. Standardisation: SARS-CoV-2 serology Current estimates of SARS-CoV- 2 cases/deaths: >776 million confirmed cases >7million deaths 220 commercial SARS-CoV-2 serological assays have been developed for laboratory use. Standardisation: SARS-CoV-2 serology WHO released the first International Standard for the anti-SARS- CoV-2 immunoglobulin on Dec 18th, 2020 The International Standard was assigned a potency of 250 IU/lyophilised ampoule for neutralizing antibody activity. This IS could be used to compare binding antibody assays using the reference’s designated 250 binding antibody units (BAU)/lyophilised ampoule. Standardisation: SARS-CoV-2 serology Derived from 11 convalescent patients following confirmed SARS-CoV-2 infections during the first COVID-19 wave in 2020. Ampoules rapidly depleted. A second WHO International Standard for anti-SARS-CoV-2 immunoglobulin (NIBSC 21/340) had to be developed in 2022 (and proposed to the ECBS between Oct 24 and Oct 28, 2022) US SARS-CoV-2 serological standard (pooled samples from four convalescent donors, evaluated in a harmonisation study across eight independent laboratories in the USA, and calibrated against the first WHO IS for anti-SARS-CoV-2 immunoglobulin) considered a crucial resource worldwide. Standardisation in Immunoassay Laboratory standard Used in an individual laboratory Calibrated against an appropriate IS (stock & working standard) Control Material A preparation used to assess reliability of an analytical process (precision and accuracy) Reference material A term which is often applied to both standards and control material. Note: It is important to distinguish roles of standard & control material Immunoassay Calibration Quality Assurance (QA) QA is the study of errors & the procedures used to recognise & minimise them The control of quality in the laboratory depends on selection of: The analytical method Performance characteristics Performance characteristics can be grouped into two types: Those concerned with practicability Those concerned with reliability Assay Practicability Speed Cost Safety Analytical skill required Example A method may be accurate and precise but is too slow, requires expensive equipment and highly skilled analysts. Assay Reliability Precision The agreement between replicate measurements (the measure of variability between results) due to Random Error (error occurring by chance) Accuracy The agreement between the best estimate of a quantity and its true value Inaccuracy due to systematic error is assessed from a mean value Accuracy becomes more difficult to assess when precision is poor Method with systematic error will shows bias. Positive bias (increase in mean) Negative bias (decrease in mean) Assay Reliability Specificity The ability of a method to determine solely the component it is designed to measure. No numerical value but assessed on evidence about the components which contribute to the result. Specificity has a large effect on accuracy. If specificity varies with samples, it will contribute to imprecision Sensitivity - Limit of Quantitation (LOQ) The ability to detect small amounts of the test substances i.e. the smallest reading after zero that can consistently be detected & measured. Calculated as the concentration of the analyte which responds to the mean +/- 3 standard deviations of the response in the absence of analyte Magnetic bead base immunoassay for Diclofenac - Calibration curve of the optimized assay (orange) and precision profile (purple); n = 6; error bars represent single standard deviation. The arrows indicate lower and upper limits of detection, and thus the measurement range of the assay (400 ng/L–300 µg/L) Learning Objectives Understand why standardisation and quality assurance (QA) are required for immunoassays. List quality assurance activities. Define the following terms Internal quality control External quality control International Standard Calibration Standard Discuss how QA determines assay practicability and reliability