80 Quality Control ELISA

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Quality in Transfusion Transmitted Infections (TTI) Testing Kshitija Mittal :

Quality in Transfusion Transmitted Infections (TTI) Testing Kshitija Mittal




Troubleshooting & Resolution

High Background Color:

High Background Color

High background COLOR :

High background COLOR Contaminated wash buffer, washer tubing Incubation too long or at high temp. Reuse of disposable tips Freshly prepared wash buffer, clean tubings with DW Check protocol Discard after use ( never reuse tips that have delivered conjugate ) Cause Resolution

High background COLOR contd… :

High background COLOR contd … Improper washing leading to residual conjugate Colored substrate (contamination with metal ions or conjugate) Improper conc. of stop solution Check performance & vacuum of the washer Fresh substrate to be used Check concentration of stop solution & quality of distilled water Cause Resolution

High background COLOR contd… :

High background COLOR contd … Conjugate too strong or left on too long Substrate solution or stop solution not fresh Reaction not stopped . Check dilution of conjugate Use fresh substrate solution. Stop solution should be clear (if it has gone yellow, this is a sign of contamination and should be replaced). Colour will keep developing if the substrate reaction not stopped

High background COLOR contd… :

High background COLOR contd … Substrate incubation carried out in the light Plate left too long before reading on the plate reader Contaminants from laboratory glassware Substrate incubation should be carried out in dark. Color will keep developing (though at a slower rate if stop solution has been added). Read within 30 min of adding stop solution Ensure reagents are fresh and prepared in clean glassware.

No Color Development:

No Color Development

No Color Development:

No Color Development Forgot to add sample, conjugate or substrate Added in wrong sequence Short substrate incubation Follow test procedure correctly Follow test procedure correctly Follow test procedure correctly Cause Resolution

No Color Development contd…:

No Color Development contd … No color in positive control Inactive microplate Substrate contaminated with stop solution Defective controls or wells, expired microplate Always cover unused strips with desiccant bag inside Prepare fresh substrate ( sensitive to contamination from variety of oxidizing agents) Cause Resolution

False Positive Results:

False Positive Results

False Positive Results:

False Positive Results Lipemic or hemolysed samples Incubation at higher temperature Improper washing, less wash vol. & less soak time Avoid use of hemolysed samples Check temperature before incubation Wash wells with adequate volume Cause Resolution

False Positive Results contd…:

False Positive Results contd … Contamination of reagents Air bubbles during reading Finger tips on plates. STRICTLY FOLLOW the test procedure as per package insert, CHECK pipettes, equipments & tips Remove them by gentle tapping Clean bottom surface of plate with wash buffer, blot to dry. Cause Resolution

False Positive Results contd…:

False Positive Results contd … Inadequate washing. Clogged cannulas in washer Contamination of wells by conjugate Contamination of substrate solution by conjugate Check washer before use to determine they are working properly. Perform routine maintenance Splashing of conjugate on rims of wells during conjugate addition. Always use fresh substrate solution and should be colorless before addition (Do not return excess TMB to primary storage container)

High Color in One Row/Column:

High Color in One Row/Column

High Color in One Row/Column:

High Color in One Row/Column Wash channel of the washer clogged Overflowing of wells in one row/column Contamination from well to well Unclog with appropriate needle Check vacuum pressure of the equipment Check wash performance of wash head Cause Resolution

Controls OUT of Validation Criteria:

Controls OUT of Validation Criteria

Controls OUT of Validation Criteria:

Controls OUT of Validation Criteria Cross-contamination of controls Incorrect filter used Incorrect temp., timing or pipetting Pipette carefully, DO NOT interchange caps Check filter wavelength ( use of reference filter minimises interference due to microwells that are opaque, scratched or irregular) Follow test procedure correctly Cause Resolution

Controls OUT of Validation Criteria:

Controls OUT of Validation Criteria Expired reagents Improper reagents preparation, error in dilution, reagents NOT mixed ALWAYS CHECK kit / reagents expiry before use Check procedure Cause Resolution


Contd …

Low Response :

Low Response

Low Response contd…:

Low Response contd … Use of expired kits Reagent not at room temperature Sample volume too low Moisture in pouches . Check expiry date of kits Make certain all kit components are at RT (22-28 o C). Ensure pipette tips are fitted correctly/ tightly. Check pipette barrels for obstructions. Check calibration of pipettes. Check whether desiccant in pouch is in working condition. Seal unused wells in pouches

Low Response contd…:

Low Response contd … Room temperature too low for substrate incubation Washing step too vigorous Insufficient conjugate concentrate added in preparing working stock Check temperature of the working area. Reduce pressure in wash system Prepare conjugate accurately. Follow working reagent preparation as described by the manufacture

Low Response contd…:

Low Response contd … Failure to add stop solution Reagents not fresh or not at the correct pH Incubation time too short Addition of stop solution increases intensity of colour reaction and stabilizes final colour reaction Ensure reagents have been prepared correctly and are in date Ensure antibody is incubated for the recommended amount of time

Low Response contd…:

Low Response contd … Substrate solutions not fresh or combined incorrectly Less wash vol. per well, NO soak time, wrong buffer used Prepare the substrate solutions immediately before use.


Definitions Quality Control - refers to the measures that must be included during each assay run to verify that the test is working properly . The aim of quality control is simply to ensure that the results generated by the test are correct Quality Assurance - defined as overall program that ensures that the final results reported by the laboratory are correct .

Definitions contd…:

Definitions contd … Quality Assessment - also known as proficiency testing is a mean to determine the quality of the results generated by the laboratory. Quality assessment is a challenge to the effectiveness of the QA and QC programs. Quality Assessment may be external or internal.

Accuracy and precision:

Accuracy and precision Accuracy: is the degree of closeness of measurements of quantity to actual (true) value. Precision: also called reproducibility or repeatability, is the degree to which repeated measurements under unchanged conditions show the same results. Two types of precision: intra-assay precision and inter-assay precision

Accuracy and precision contd…:

Accuracy and precision contd … Accuracy indicates proximity of measurement results to the true value. Precision to the repeatability or reproducibility of the measurement. A measurement system is designated valid if it is both accurate and precise.

PowerPoint Presentation:

High accuracy , but low precision High precision , but low accuracy Accuracy and precision contd …

Accuracy and precision contd…:

Accuracy and precision contd … Accuracy can be affected by all components of an assay. Generally, accuracy determined by comparing results to a reference method. Precision is always examined first in any assay development because assay with great imprecision in early stages is not likely to be of any routine use. Precision is measured as a coefficient of variation (CV)

Coefficient of variation (CV) :

Coefficient of variation (CV) Standard Deviation (SD) of assays generally increase or decrease proportionally as mean increases or decreases CV is therefore a standardization of SD as division by mean removes it as a factor in the variability that allows comparison of variability estimates regardless of magnitude of analyte concentration.

Errors in measurement: Systematic Error:

Errors in measurement: Systematic Error Systematic: errors that consistently affect repeated measurements of the same sample. Manifested in form of bias. May be induced by factors such as variations in incubation temperature, blockage of plate washer, change in the reagent batch or modifications in testing method. The accuracy of measurements is often reduced by systematic errors.

Systematic Errors:

Systematic Errors

Random Error:

Random Error Observance of one result significantly different from other results without any pattern. Reflected in form of poor reproducibility or imprecision. Examples include errors in pipetting , changes in incubation period. Can be minimized by training, supervision and adherence to standard operating procedures. The precision is limited by the random errors

Random Errors:

Random Errors

Validation and calibration:

Validation and calibration Validation is a way of assuring that a system, process or equipment is performing the way it is supposed to do so. Calibration is a process that compares a known (standard) against an unknown (testing device). A validation is a detailed process of confirming that the instrument is installed correctly, that it is operating effectively, and that it is performing without error. Qualification is the process of the actual testing that is perform during validation ( installation qualification (IQ), the operational qualification (OQ), and the performance qualification (PQ ).

Validation Tools:

Validation Tools include positive and negative controls in every test run include additional validation measures where possible eg rapid tests with internal control spot or line independent readings of rapid tests by 2 people use of mechanical readers if available - to reduce subjectivity

Essential elements governing quality in TTI:

Essential elements governing quality in TTI Quality of the specimen used for testing Quality of kits used for testing Calibration and validation of equipment used Use of SOPs for testing Type of Controls used while testing Interpretation of results Validation of results Record keeping Training of staff

quality of specimen used for testing:

quality of specimen used for testing Specimen should be properly labeled Specimen should be clear; Lipemic and hemolysed specimens( cellular constituents may interfere with results ) do not yield reliable results Avoid adding preservatives - some interfere with test results (sodium azide interferes with HRP. ProClin is a broad-spectrum biocide, having good compatibility and stability and low toxicity at in use levels. It eradicates bacteria, fungi, and yeast cells at very low concentration, does not interfere with enzyme reactions, and can be disposed of without rest and is used as preservative in wash buffer and Conjugate solution)

Quality of kits used for testing:

Quality of kits used for testing In general kits with highest sensitivity and specificity should be used in a BTS for TTI testing All kits and reagents should be used within the expiration date Kits which are used should have approval of certifying authority(DCGI,NACO) Never interchange reagents from one kit to another or one lot to another (as they are optimized for individual batch performance)

Sensitivity and specificity:

Sensitivity and specificity For blood screening tests, the key performance parameters are sensitivity and specificity. Sensitivity is assay's capacity to measure smallest amount of target analyte under standard conditions defined. A more sensitive test will identify more infected donors. The most important aspect is ability to detect infection at the earliest time , because this reduces infectious window period and hence risk of transmitting infection to recipients.

Sensitivity and specificity contd…:

Sensitivity and specificity contd … Sensitivity for early infection is best evaluated against panels of samples taken during seroconversion . Another aspect is ability of a given test to detect different serotypes, genotypes, or mutants of the agents of concern . Sensitivity evaluations include use of wide variety of positive samples representing different genotypes

Sensitivity and specificity contd…:

Sensitivity and specificity contd … Test specificity is another critical parameter. Tests with poor specificity result in unnecessary loss of products and their donors, and require additional confirmatory tests. A test’s specificity is best evaluated by testing many nonreactive samples, preferably from donor populations.

Sensitivity and specificity contd…:

Sensitivity and specificity contd … Specificity is assessed by testing 10,000 or more routine donor samples. Samples giving positive results must be further investigated and donors evaluated by follow-up. Thus, the overriding goal should be to maximize safety (sensitivity) and minimize waste (specificity)

Calibration and Validation of equipments used:

Calibration and Validation of equipments used Always use standard equipment in a BTS for TTI testing - ELISA reader & washer, Micro-pipettes, incubators, shakers. Periodic calibration of equipment is vital to maintain quality of test. Periodic servicing of equipment is crucial for optimal use of equipment- ELISA washers. Proper documentation on equipment check and its performance is essential- maintain records (logbook / file).

Controls used in the assay for testing:

Controls used in the assay for testing Internal kit controls : Include the positive control, Negative control. At times may include a calibrator provided by the manufacturer External controls : Include positive samples from the laboratory either pooled or single, diluted or undiluted. Essential to incorporate this to monitor quality in testing procedures Intra-run and Inter-run reproducibility : three slots/run and on three consecutive days

Interpretation of test results:

Interpretation of test results As rule all readings (both quantitative and qualitative) and calculations should be checked by two individuals Validation of every run is essential for proper interpretation of results Proper records - print outs of results, calculations of cut off values, graphs should be maintained Any errors detected should be brought to the notice of the concerned staff and corrective measures instituted promptly


Controls Internal controls Set of controls (Positive & Negative) provided along with the kit To be used only in those batches of kit from which they originate Do not detect minor deterioration of kits

Controls (contd..):

Controls (contd..) External controls Set of controls included from outside Positive (Borderline Reactive) & Negative Detect minor error in the assay performance Sources of External Controls National reference laboratories Commercial control panels In-house prepared external controls Pooled test kit controls Samples collected from other laboratories

Preparation of In-house External Controls:

Preparation of In-house External Controls Select a high titre sero -positive serum/plasma Retest the sample with another kit Heat inactivate the sample @ 56 0 C X 30 min If plasma taken, re-calcify it to obtain serum Make serial doubling dilution of the sample with a sero -negative serum (to keep antibodies in natural serum protein environment) NACO Guidelines

PowerPoint Presentation:

Making Suitable Dilutions

Preparation of In-house External Controls….contd.:

Preparation of In-house External Controls….contd. Select the dilution with ER b/w 1.5 to 2 Prepare external control aliquots of dilution selected above Store at – 20 0 C or below @ 1 year After test run, calculate E Ratio for each dilution ELISA ratio = sample OD / cut off OD Once thawed, control can be kept @ 2-8 0 C, 1 wk NACO Guidelines




CONTD… DILUTION OD VALUE (CUT OFF 0.392) E-RATIO 1:1 1.044 2.66 1:2 0.967 2.47 1:4 0.949 2.42 1:8 0.925 2.36 1:16 0.761 1.94 1:32 0.695 1.77 1:64 0.443 1.13 1:128 0.306 Neg.

PowerPoint Presentation:

E.P.C. Selection of Dilution for External Control

Need for E ratio:

Need for E ratio E Ratio = Cut off values differ depending on the principle of the test, manufacturer guidelines and recommended protocol for the calculation. Some degree of variation in internal controls results in the variation in the cut off values due to Variation in incubation condition Preparation of reagents Plate to plate and well to well variation (antigen coating) Sample OD Cut off OD

PowerPoint Presentation:

OD of the controls would expectedly influence the OD value of the test samples in similar directions. However, relative reactivity of the given sample and the cut off would not vary. This relative reactivity of a test sample in relation to cut off value in a particular direction is expressed as E Ratio. Need for E ratio contd ….

Preparation of QC charts: Levy Jennings charts:

Preparation of QC charts: Levy Jennings charts Include at least 20 runs on the same graph Mean ± 2SD is calculated and plotted on the graph. E ratios are plotted on the Y axis in chart and consecutive dates of runs are plotted on X axis. Change of operator and batch of assay should be recorded

Batch Validation: Determination of Inter-aliquot Variation :

Batch Validation: Determination of Inter-aliquot Variation This process is to check that the batch has been sufficiently mixed and is homogenous so as to minimize the inter aliquot and inter run variation. Higher the dilution, more is the importance of such validation. The mean, Standard Deviation (SD) and Coefficient of variation (CV) of the E-Ratios is calculated CV< 20% (minimal batch variation i.e inter-aliquot variation) tested in at least 20 consecutive runs , the external control is considered suitable for inclusion as external control in subsequent daily runs of EIA. The batch is aliquoted into propylene storage containers (polyethylene containers not recommended as these absorb antibodies and may affect titre of sample).

L J chart –Scope and application:

L J chart –Scope and application Detection of the following Systematic variation Random variation Lot to lot variation Day to day variation

Applications of control charts:

Applications of control charts Highlight the outliers (values outside +/- 2 SD) Reveal batch to batch variation Reveal operator to operator variation Changes in assay performance even when test runs are valid

Systematic Variation:

Systematic Variation Trend- Results change gradually in either direction indicating slowly changing parameters-deteriorating reagents, equipment failing Shift- Results fall sharply on one side of the mean indicating a major change has occurred

Chart showing shift and trend:

Chart showing shift and trend

Interpretation of aberrant results:

Interpretation of aberrant results Control values of six consecutive runs fall on one side of mean (SHIFT) Switching to new lot of kits New reagents Changes in incubation temperature New technical hand Six consecutive points distributed in on general direction (TREND) Deterioration of reagents Slowly faltering equipment.

Random Variation:

Random Variation Causes Transcription errors Sample mix-up Poor pipette precision Poor mixing of samples Reader not calibrated Washing inconsistent

Shewhart Control Charts:

Shewhart Control Charts A Shewhart Control Chart depend on the use of IQC specimens and is developed in the following manner:- Put up at least 20 or more assay runs and record down the O.D./cut-off value Calculate the mean and standard deviations ( s.d .) Make a plot with the assay run on the x-axis, and O.D./ cut-off on the y axis. Draw the following lines across the y-axis: mean, -3, -2, -2, 1, 2, and 3 s.d . Plot the O.D./cut-off obtained for subsequent assay runs Major events such as changes in the batch no. of the kit and instruments used should be recorded on the chart.

Shewhart Chart:

Shewhart Chart +3 sd -3 sd +2 sd -2 sd -1 sd +1 sd VZV IgG ELISA: Target Value = 49 U/ml Antibody Units Target value Assay Run

Westgard rules:

Westgard rules The formulation of Westgard rules were based on statistical methods. Westgard rules are commonly used to analyse data in Shewhart control charts. Westgard rules are used to define specific performance limits for a particular assay and can be use to detect both random and systematic errors. There are six commonly used Westgard rules of which three are warning rules and other three mandatory rules. The violation of warning rules should trigger a review of test procedures, reagent performance and equipment calibration . The violation of mandatory rules should result in rejection of the results obtained with patients’ serum samples in that assay .

Warning rules:

Warning rules Warning 1 2SD : It is violated if the IQC value exceeds the mean by  2SD. It is an event likely to occur normally in less than 5% of cases. Warning 2 2SD : It detects systematic errors and is violated when two consecutive IQC values exceed the mean on the same side of the mean by  2SD. Warning 4 1SD : It is violated if four consecutive IQC values exceed the same limit (mean  1SD) and this may indicate the need to perform instrument maintenance or reagent calibration.

Mandatory rules:

Mandatory rules Mandatory 1 3SD : It is violated when the IQC value exceeds the mean by  3SD. The assay run is regarded as out of control. Mandatory R 4SD : It is only applied when the IQC is tested in duplicate. This rule is violated when the difference in SD between the duplicates exceeds 4SD. Mandatory 10x : This rule is violated when the last 10 consecutive IQC values are on the same side of the mean or target value.

Westgard Rules: 1 3SD:

Westgard Rules: 1 3SD +3 sd -3 sd +2 sd -2 sd -1 sd +1 sd VZV IgG ELISA: Target Value = 49 U/ml Antibody Units Target value Assay Run

Westgard Rules: 10X:

Westgard Rules: 10X +3 sd -3 sd +2 sd -2 sd -1 sd +1 sd VZV IgG ELISA: Target Value = 49 U/ml Antibody Units Target value Assay Run

ELISA Reader:

ELISA Reader Quality Control of Equipment

ELISA Reader….contd.:

ELISA Reader….contd. Photometric instrument filter should be protected from moisture and fungal growth keep silica gel packs in the filter box Calibration is done every six months (supplier) OD of special plates & standard color solution are recorded Results of OD should be within 10% of expected Daily check – negative & positive controls added to each run

ELISA Washer:

ELISA Washer After Use- Fill the rinse bottle with about 500 ml of distilled water. Dispose off the unused wash buffer. Rinse with distilled water, a couple of times and leave about 500 ml in wash bottle. Fix cap tightly.

Water baths & Incubators:

Water baths & Incubators Daily recording of temp. using a calibrated thermometer Acceptable results are the expected temp. ± a narrow range (± 0.5°C) predetermined by the laboratory Distilled water changed regularly (every 8 days) Bacterial cultures can be done periodically



Maintenance of Pipette :

Maintenance of Pipette Do not dispense volatile / corrosive materials as it will disturb the vacuum Volume should be increased or decreased gradually Pipette must always be return to zero position after use Always store pipettes in vertical or erect position in a holder Pipette snout must be cleaned regularly with moist filter paper after use


Contd … Because micropippets are instruments that deliver liquid and multichannel spectrophotometer reads through thickness of liquid, any change in volume in well will result in an alteration of OD reading for the same colored solution ( also true of blotting of plates to eliminate residual washing solution and covering with adhesive strips to avoid evaporation, all of which affect the final volume per well ), they are fundamentally important to the accuracy of the ELISA. They should be checked regularly for leakage, precision and accuracy of delivery volumes

Calibration of Pipette:

Calibration of Pipette All items at ambient room temp. Record the weight of empty beaker Record the temp. of tube with distilled water Pipette a known volume of water (expected volume) Record: [wt of beaker + water] – wt of empty beaker = weight of water

Calibration ….contd.:

Calibration ….contd. weight of water Delivered vol. = temp. factor x sp. gravity of water Repeat this 10 times, changing the tip Calculate mean, SD and CV expected vol. – delivered vol. x 100 % Deviation = expected vol. % deviation < 1.5% CV < 1%


REFERENCES ELISA Guide book by J.R.Crowther (2001) Wikipedia Manual on Quality Standards for HIV Testing Laboratories, March 2007 (NACO) Blood Banking and Transfusion Medicine. Basic Principles and Practice. Anderson, Second Edition Various Package inserts

PowerPoint Presentation:

Thank you

Definitions contd…:

Definitions contd … Test efficacy : performance under ideal conditions Test effectiveness : performance under average conditions

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