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PISHTAZ TEB DIAGNOSTICS. Quality Assurance in ELISA. DR. MEHDI BOUTORABI DR. ALI MIRJALILI. CONTENTS. Kit Selection Preanalytical Considerations Test provision : Equipment calibration and maintenance Kit Verification Performance Characteristics and IFU Troubleshooting Questions.
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PISHTAZ TEB DIAGNOSTICS Quality Assurance in ELISA DR. MEHDI BOUTORABI DR. ALI MIRJALILI
CONTENTS • Kit Selection • Preanalytical Considerations • Test provision : Equipment calibration and maintenance • Kit Verification • Performance Characteristics and IFU • Troubleshooting • Questions
PART 1. KIT SELECTION • TERMS AND DEFINITIONS • CONSIDERING AND LISTING THE FEATURES REQUIRED • GATHER INFORMATION ABOUT THE AVAILABLE PRODUCTS • PUBLISHED, INDEPENDENT TECHNICAL EVALUATIONS
Scopes of Method Evaluation Studies • Evaluationis the determination of the analytical performance characteristics of a new method. • Validationis confirmation by examination and provision of objective evidence that the particular requirements for a specific intended use can be consistently fulfilled. • Verificationis confirmation by examination of objective evidence that specified requirements have been fulfilled. • Demonstrationis a minimum evaluation for a laboratory to use to show that it is able to obtain expected results by following the manufacturer’s instructions. This is appropriate for test systems whose performance characteristics have been well studied and documented.
ISO 15189 Examination processes • . 5.5.1 Selection, verification and validation of examination procedures • 5.5.1.1 General The laboratory shall select examination procedures which have been validated for their intended use.
Reasons for Selecting a New Method • improve accuracy and / or precision over existing methods • to reduce reagent cost • to reduce labour cost • new analyzer or instrument • to measure a new analyte
INITIAL SCREENINGCONSIDERING AND LISTING THE FEATURES REQUIRED . List assay related factors: Less cross-reactivity Instrument features General concerns, better service support. • Cost • Internal costs, • technician labor • External costs • Price of the system • Consumables. • Maintenance contracts Adverse consequences (False negative and false positive results)
INITIAL SCREENINGGATHER INFORMATION ABOUT THE AVAILABLE PRODUCTS .Which methods are being used by your colleagues and ask them for advice. • External quality assessment schemes • How many participants are using each method, • Estimates of imprecision (%CV) • Number of outlying results can reflect unreliability (although sometimes outliers may be due to the use of inconsistent units of concentration). • The method means are particularly useful, as they reflect method biases • In some countries, national organizations or government departments e.g., Department of Health in the United Kingdom.
INITIAL SCREENINGCONSIDERING AND LISTING THE FEATURES REQUIRED . Visit the manufacturer’s website and download information. • Estimate the labor requirements and turnaround times • The maintenance and service requirements • Any environmental specifications, e.g., for laboratory temperature and humidity. • Reagent stability during transport • Check if refrigerated transport is required for the typical shipment time.
INITIAL SCREENINGPUBLISHED, INDEPENDENT TECHNICAL EVALUATIONS • Check for performance • Data and warnings of unexpected results or interferences. • For new tests, check for clinical issues. • Competitive method instructions, regulatory agency websites (such as the FDA), • Published warning and recall notices published by, e.g., the FDA in the USA and MHRA in the UK. • Clinical guidelines ,NICE or Royal Colleges in the UK, or societies in the USA • Clinical and Laboratory Standards Institute (CLSI) method evaluations.
ELISA Instrument Quality assurance Dr. Ali Mirjalili
Five M of Quality FACILITY (SIZE, CONSTRUCTION, LOCATION) Qualifications, Organization, Job description, Training, etc. Qualification, Calibration Machine Post-analytical Preanalytical Material/ Sample Man Storage Label Manual Methodology Motivation SOP, Mfr Bruchure WWW.PISHTAZTEB.COM Analytical (Examination Phase(
Equipment required for ELISA testing 1. Microplate reader. 2. Microplate washer. 3. Liquid dispensing system (multi-channel pipettes may be used). 4. Incubator to incubate the plates.
Microplate Reader wavelength range to that used in ELISA, generally between 400 to 750 nm (nanometres) Some readers (ultraviolet range between 340 to 700 nm. INSTALLATION REQUIREMENTS 1. A clean, dust free environment. 2. A stable work table away from equipment that vibrates (centrifuges, agitators). 3. An electrical supply source, which complies with the country’s norms and standards.
Calibration of the microplate reader • Executed by a technician or trained engineer • Following the instructions provided by each manufacturer. • It is necessary to have a set of grey filters mounted on a plate of equal geometric size • Calibration plates are equipped with at least three pre-established Optic density values within the measurement ranges; low, medium, and high value.
Calibration of the microplate reader 1. Place the calibration plate on the equipment. 2. Carry out a complete reading with the calibration plate. Verify if there are differences in the readings obtained from well to well. If this is the case, invert the plate (180°) and repeat the reading to rule out that differences are attributed to the plate itself. In general, it is accepted that the instrument does not need further calibration if the plate results are as expected at two wavelengths. 3. Verify if the reader requires calibration. If so, proceed with the calibration following the routine outlined by the manufacturer, verifying that the reading’s linearity is maintained as rigorously as possible. 4. If the instrument does not have a calibration plate, verify it by placing a coloured solution in the wells of a plate and immediately carry out a complete reading. Then invert the plate 180° and read the plate again. If both readings display identical, average values in each row, the reader is calibrated. 5. Verify that the reader is calibrated, column by column. Place a clean, empty plate and carry out a reading. If there is no difference between each of the average reading of the first to the last column, it can be assumed that the reader is calibrated. ELISA Check
Basic maintenance Frequency: Daily 1. Review that optical sensors of each channel are clean. If dirt is detected, clean the surface of the windows of the light emitters and the sensors with a small brush. 2. Confirm that the lighting system is clean. 3. Verify that the reader’s calibration is adequate. When the daily operations begin, let the reader warm up for 30 minutes. Next, do a blank reading and then read a full plate of substrate. The readings must be identical. If not, invert the plate and repeat the reading in order to determine if the deviation originated in the plate or the reader. 4. Examine the automatic drawer sliding system. It must be smooth and constant.
Preventive maintenance Frequency: Quarterly 1. Verify the stability of the lamp. Use the calibration plate, conducting readings with intervals of 30 minutes with the same plate. Compare readings. There must be no differences. 2. Clean the detectors’ optical systems and the lighting systems. 3. Clean the plate drawer. 4. Verify the alignment of each well with the light emission and detection systems.
Microplate Washer • The microplate washer has been designed to performwashing operations in the ELISA technique Comprise: • Control subsystem. • Supply subsystem • Extraction or suction system • Advance sub-system
Microplate Washer • INSTALLATION REQUIREMENTS 1. A clean, dust-free environment. 2. A stable work table located away from equipment that generates vibrations, (centrifuges, and agitators). 3. An electric outlet in good condition with a ground pole.
Microplate Washer Calibration • Washer calibration The microplate washer is critical for guaranteeing that the ELISA technique performs as expected. • Position of the needles (supply and aspiration head). Situated very close to the well’s wall. The suction needle should be located in the centre of the well: A needle-base distance is maintained in the well, usually between 0.3 to 0.5 mm. The needles must never be allowed to touch the bottom of the wells to avoid mechanical interferences • Aspiration time. • Distributed Volume. Check that the volume distributed is as close as possible to the maximum capacity of the well; confirm that all the wells are filled uniformly (at the same level). Verify that the distributing needles are clean (free of obstructions). •• Vacuum. The suctioning system must be calibrated efficiently. If the vacuum is too strong, the test can be altered. In fact, it could dry out the wells and considerably weaken the enzyme activity in the wells and completely alter the test result. The majority of washers function with a vacuum ranging between 60 and 70% of atmospheric pressure.
MicroplateWasher Performance Verification • Washing process verification To verify that the washing process is done according to the specifications of ELISA techniques • One of the controls is based on using the peroxidase reagent, which is dispensed using a pipette in the plate wells to be read at 405, 450 and 492 nm. At once the wells are washed and a colourless substrate is added (TMB/H2O2) Tetramethylbenzidine/Hydrogen Peroxide). Whatever conjugate remains will hydrolyze the enzyme and the chromogen will change to blue. After stopping the reaction with acid, the TMB will turn yellow again. The resulting colour intensity is directly related to the washing process efficiency. ELISA Check
Microplate Washer Maintenance Basic maintenance Frequency: Daily 1. Verify the volume distributed. 2. Test the filling uniformity. 3. Verify the aspiration sub-system’s efficiency. 4. Confirm the cleaning of the supply and extraction needles. 5. Clean the washer with distilled water after use, to remove every vestige of salt in the supply and extraction subsystems’ channels. The needles may be kept submerged in distilled water. 6. Verify that the body of the washer has been cleaned. If necessary, clean the exterior surfaces with a piece of cloth, moistened with a mild detergent.
Microplate Washer Preventive Maintenance • Preventive maintenance Frequency: Quarterly 1. Disassemble and clean the channels and connectors. Verify their integrity. If leaks or any vestiges of corrosion are detected, adjust and/or replace. 2. Verify the integrity of the mechanical components. Lubricate according to the manufacturer’s instructions. 3. Test the adjustment of each one of the subsystems. Calibrate according to the manufacturer’s recommendations. 4. Confirm the integrity of the electrical connector and the inter-connection cable. 5. Clean the washer with distilled water after using it in order to remove every vestige of salt in the supply and extraction subsystems’ channels. 6. Verify the integrity of the fuse, and that its contact points are clean.
Pipettes and Best pipetting practice Electronic multi- channel Manual single channel Manual multi- channel Electronic single channel
Ways to optimize pipette performance • Choose the right pipette for the job. • Check for leaks or any other pipette malfunctions • Choose the correct pipette tip • Correct size • Correct style • Have pipettes calibrated and serviced regularly. • Allow all liquids and equipment to equilibrate to ambient temperature before beginning work. • Pre-rinse the pipette tip by aspirating and dispensing the sample liquid at least 3 times before aspirating a sample for delivery. • Immerse the tip vertically into the sample liquid well clear of the container walls and bottom and at a depth of approximately 2 – 5mm below the meniscus.
Tips to minimize pipetting errors Aspirate using a consistent speed, rhythm, and plunger pressure. Hold the tip in the sample for 1 second after aspiration and withdraw the tip slowly and smoothly To dispense touch the pipette tip to the sidewall of the container where sample is to be delivered just above the liquid the sample is being dispensed into. Use consistent speed, rhythm, and plunger pressure to dispense. Put the pipette in it’s stand between pipetting cycles to avoid warming the pipette in your hand – this can affect the volume of liquid dispensed.
PIPETTING GUIDE TO PIPETTING • ONLY USE FIRST STOP ! • DO NOT DRIP • DO NOT PRESS HARD INTO WELL • DO NOT USE TOO ACUTE AN ANGLE • MAKE SURE TIP TOUCHES SIDE OF WELL AND LIQUID
Pipetting tips • Forward Pipetting technique • Reverse Pipetting technique • Highly viscous fluid • Avoid foaming
Pipette preventive maintenance • As part of Preventive Maintenance, each Pipette disassembled according to manufacturer’s diagrams and cleaned thoroughly with the appropriate chemical solutions. • All parts are carefully inspected, piston is polished in most cases, piston seal and o-ring are replaced as well as all defective and malfunctioning parts.
Pipette Calibration Considerations • All pipettes must be tested when first purchased, following any major service and once annually by analyzing ten replicates. • All pipettes must be tested quarterly by analyzing four replicates. Replicate analyses must meet acceptance criteria or use of the pipette should be discontinued until the problem has been corrected. • Inaccuracy [(Corr. Mean - true value) ÷ true value × 100] must be less than 2% • No single replicate may be greater than 2% from the true value. • %CV (Standard Deviation ÷ Corr. Mean × 100) must be less than 1.00 • Three different volumes are tested; 10% of maximum volume, mid volume and maximum volume.
Pipette calibration • Gravimetric • Colourimetric
ISO 13485 Examination processes • . 5.5.1 Selection, verification and validation of examination procedures • 5.5.1.1 General The laboratory shall select examination procedures which have been validated for their intended use.
5.5.1.2 Verification of examination procedures • The laboratory shall document the procedure used for the verification and record the results obtained. • Staff with the appropriate authority shall review the verification results and record the review.
Diagnostic kit development, validation and verification pathway
Aim of this standard • Provide minimum implementation protocol to verify that a particular example of measurement procedure is operating in accordance with the manufacturer claims For precision and trueness
Overview of the protocol • Precision evaluation experiment • Repeatability, Reproducibility • Trueness evaluation experiment • Comparability (20 serum samples) • Recovery of expected values from certified reference materials
Trueness Evaluation ExperimentExample
Practical Example ELISA Kit Verification Reference : The Immunoassay Handbook Theory and applications of ligand binding, ELISA and related techniques Edited by David Wild
Method Evaluation Company Logo
Method Evaluation • Analysis of results from initial kit evaluation • The calibration curve should be fitted as recommended by the manufacturer • Within assay precision • %CV for the controls, samples and calibrators • (Value not OD) • Between-assay differences and stored calibration curve stability • % CV for control and sample • Compare the values generated by the stored calibration curve with those derived from a manual plot of all the calibrators Company Logo
Method Evaluation • Analysis of results from initial kit evaluation (continue) • Drift • Plot the values of controls obtained at the beginning, middle and end of the assay to detect assay drift • Sensitivity • 10 replicates of zero calibrator, Analytical sensitivity is two SD above or below the zero calibrator mean • Accuracy • Compare the results for the external QC scheme samples with those obtained from other methods and all-laboratory trimmed means • Compare the patient samples with current method Company Logo
Method Evaluation • Analysis of results from initial kit evaluation (continue 2) • Dilution • Samples diluted by zero calibrator, Plot the dilution curve, straight line • Verification of Reference Interval • Other information • Check the appearance of the reagents, • Check the ease of using of packaging • Quality of the instructions • Estimate the total assay time • Telephone to customer service and ask one/two questions to check the quality and the speed of their responses WWW.PISHTAZTEB.COM Company Logo
