Click to edit Master subtitle style

Method Validation

Wong Syi Huey 149246 Vickneswaary Sockalingam 149917 Tan Sin Luan 149491 Yeoh Poh York 149780 Vicknesh Ramanaidu 149857 Sim Biow Ing 149894

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INTRODUCTION

Process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. from method validation can be used to judge the quality, reliability and consistency of analytical results. integral part of any good analytical practice.

Results

An

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Principle

A method should be validated when it is necessary to verify that its performance parameters are adequate for use for a particular analytical problem. E.g.:
New method developed for particular problem. Established

method revised to incorporate improvement or extended to a new problem. with time.

When QC indicates an established method is changing Established method used in a different laboratoty or

with different analysts or different instrument.

To demonstrate the equivalence between two methods,

e.g. a new method and new standard.

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Method validation criteria including:

Repeatability Accuracy Linearity Reproducibility Selectivity/ specificity Limit of detection/ quantitation Robustness/ ruggedness Sensitivity Range Intermediate precision
6/5/12 Practicability

and applicability under normal conditions

Strategy for validation of methods

Validity of specific method should be demonstrated in laboratory experiments using samples or standards that are similar to unknown samples analyzed routinely. During method validation, the parameters, acceptance limits and frequency of ongoing system suitability tests or QC checks should be defined.
To indicate when the method and system are

beyond statistical control.

Aim to optimize the experiment so that, with a

minimum number of control analyses, the method and the complete analytical system will provide long-term results to meet the objectives defined in the scope of the method.
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Real-time PCR Validation

In microbiology laboratories, a new set of rules must be applied to determine whether the tests will have acceptable performance. The classical methods of culture and biochemical testing must, like other assays, have appropriate QC and QA practices associated with each assays. After successful completion of the assay development phase, an assay must undergo a thorough validation evaluation prior to any implementation.

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ContinueReal-time PCR Validation

An SOP (standard operating procedure) should be defined and accepted. The SOP must include predefined interpretation and reporting algorithms. Validation studies typically include analyses that confirm the assays accuray, precision, sensitivity and specificity. Validation data summaries are compiled during the process so that if an assay passes and becomes validated, all phases of the development and validation are documented in a single report.

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ContinueReal-time PCR Validation

Validation of a molecular assay use on clinical specimens

Begins with a sensitivity study in the clinical specimen matrices.

Similar to analytical sensitivity evaluation, the evaluation

of clinical or diagnostic sensitivity should use a dilution series consisting of 8 to 20 log concentrations of the whole target organism assayed in triplicate.
Determination of sensitivity in this manner will also

produce the necessary accuracy, reportable range, and linearity data for assessment of the performance of the assay in a matrix.
These studies should be performed for ach potential

matrix for which the developing laboratory will use the assay. 6/5/12

Next, assess inter- and intra assay reproducibility.

Used to determine the accuracy and precision of the assay. Typically expect assay accuracy to exceed 95% (a level that limits the

false-negative rate <5%)

Intra-assay reproducibility: the assay being validated is performed in

triplicate on three specimens.

The coefficient of variance (CV) must be <5% in order for the assay

to be acceptable for use in clinical specimen.

For intra-assay reproducibility: the specimens are test on 3 separate

days.
Alternatively, the positive control used in the assay can also be

examined for longer period of time (e.g. 15 days) for determination inter-assay reproducibility.
The CV must be <5%.

For master mix quality and PCR cycling parameters, the use of a

positive control allows the baseline and threshold settings to be 6/5/12 adjusted so that the analysis is effectively normalized from run to run,

Lastly, assess the robustness of the assay in the specimen matrix that will eventually be used for diagnosis (e.g. blood or serum for bacteria diagnosis).
Include a study performed in a blinded, randomized fashion. Blinded validation study: include a least 30 positive or seeded

specimens and at least 10 negative specimens.

It is important that the specimens include those having a target

organism concentration that is at or near the limit of detection (LOD) of the assay.
The sensitivity assays and initial characterization of the assay

that were performed in the development phase can be used as guidelines during the ensuing validation phase. known to contain the target organism.

Blinded validation study should utilize actual specimens

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The assay to be validated must be tested for each specimen matrix to which it will be applied, because differences in physical properties, inhibitors present and pathogen loads exist among specimen types. of the validated assay against another FDA-approved or validated molecular assay can be performed, the validation study should be ideally be compared to gold standard assay.

Comparison

Since

most real-time PCR assays are performed on extracted nucleic acid, the validation study must take into account the potential loss of the nuclei 6/5/12 acid during the extraction process; thus, the

Validation of a sameday Click to edit Master subtitle style real-time PCR method for screening of meat and carcass swabs for Salmonella

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Meat is one of the major sources of human Salmonella infections. Validation of alternative methods is needed to prove that the performance is equal to established methods. This study describes a validation including comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal) of a same-day, noncommercial real-time PCR method for detection of Salmonella in meat and carcass swabs.

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Results

60 minced veal and pork meat samples, 60 poultry neck-skins, and 120 pig carcass swabs were used to perform comparative trial against a reference method using artificially and naturally contaminated samples. From the finding,
relative accuracy - 99% relative detection level -100% relative sensitivity - 103% relative specificity - 100%

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Collaborative trial included six laboratories testing minced meat, poultry neck-skins, and carcass swabs as un-inoculated samples and samples artificially contaminated with 110 CFU/25 g, and 10100 CFU/25 g. Valid results were obtained from five of the laboratories - one of the non-inoculated samples was found to be false positive with PCR. The PCR method was compared with the BAX Salmonella test on 39 pork samples artificially contaminated with Salmonella and no significant difference were found between the two methods.

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Conclusion

The validation shown that PCR method performed well. The test is currently being implemented for screening of several hundred thousand samples per year at a number of major Danish slaughterhouses to shorten the post-slaughter storage time and facilitate the swift export of fresh meat.

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