Methods Verification Transfer Of Validated Methods Into .
Methods verificationTransfer of validatedmethods intolaboratories workingroutineDr. Manuela Schulze1
1. Introduction2. Definitions and differencesvalidation – verification3. How to perform verification inGMO detection laboratoriesDr. Manuela Schulze2
Regulation (EC) No 882/2004Recital 17General obligations with regard to theanalysis of official samplesLaboratories involved in the analysis of official samplesshould work in accordance with internationally approvedprocedures or criteria-based performance standards anduse methods of analysis that have, as far as possible, beenvalidated.VALIDATED METHODS are needed !!!Dr. Manuela Schulze3
Regulation (EC) No 882/2004Article 12General obligations with regard to the analysis ofOfficial samplesLaboratories involved in the analysis of official samples shouldoperate and should be accredited in accordance with theEN ISO/IEC 17025 on „General requirements for thecompetence of testing and calibration laboratories‟Accreditation of laboratories is mandatory !!!Dr. Manuela Schulze4
ISO/IEC 17025:2005General requirements for thecompetence of testing and calibrationlaboratories5.4.2 Selection of methods5.4.5 Validation of methodsDr. Manuela Schulze5
Advantages of validated methodsProviding- reliable and accurate test results- comparable results between different laboratoriesrequirement for the accreditation according toISO/IEC 17025 Chapter 5.4.5 „validation of methods‟Dr. Manuela Schulze6
ISO/IEC 17025: 2005Section 5.4.2 Selection of methods1. Methods published in international, regional or national standards shall(i.e. must) preferably be used.Latest valid edition2. Published byreputable technical organisations,in relevant scientific texts or journals oras specified by the manufacturer of the equipment3. Laboratory-developed methods or methods adopted by the laboratoryDr. Manuela Schulze7
Regulation (EC) No 882/2004Article 11Methods of sampling and analysis1. Sampling and analysis methods used in the context of officialcontrols shall comply with relevant Community rules or,(a) if no such rules exist, with internationally recognised rules orprotocols,for example those that the European Committee for Standardisation(CEN) has accepted or those agreed in national legislation;or,(b) in the absence of the above, with other methods fit for the intendedpurpose or developed in accordance with scientific protocols.1. Methods fixed in legislation2. ISO methods / CEN methods3. Other appropriate methods (“fit for purpose”)Dr. Manuela Schulze8
Regulation (EC) No 882/2004Annex IIICHARACTERISATION OF METHODS OF ANALYSIS1. Methods of analysis should be characterised by the following criteria:(a) accuracy;(b) applicability (matrix and concentration range);(c) limit of detection;(d) limit of determination;(e) precision;(f) repeatability;(g) reproducibility;(h) recovery;(i) selectivity;(j) sensitivity;(k) linearity;(l) measurement uncertainty;Picture by Dr. Manuela Schulze(m) other criteria that may be selected as required.Dr. Manuela Schulze
Validation(ISO 17025, 5.4.5.3 note 3)Validation is always a balance betweencosts,risks andtechnical possibilities.There are many cases in which the range anduncertainty of the values can only be given ina simplified way due to lack of information.Dr. Manuela Schulze10
Method validationMethod validation is a processDevelopment ofa new methodOptimisation of themethodEstablishmentof the method
HARMONIZED GUIDELINES FORSINGLE LABORATORY VALIDATIONOF METHODS OF ANALYSIS(IUPAC Technical Report)Prepared for publication byMICHAEL THOMPSON, STEPHEN L. R. ELLISON AND ROGER WOODIn: Pure Appl. Chem., Vol. 74, No. 5, pp. 835–855, 2002. 2002 IUPACGuideline which provide minimum recommendations onprocedures that should be employed to ensure adequate validationof analytical methods.Dr. Manuela Schulze12
Method validation is a processIn-House(single)validation ofthe methodInterlaboratoryValidation of themethod perinterlaboratory(collaborative) uction indifferentlaboratories
Definitions (1)Validation is the confirmation by examination and provision ofobjective evidence that the(ISO 9000:2005 section 3.8.4) .requirements for a specificintended use or application have been fulfilled(ISO 17025:2005 section 5.4.5.1). particular requirementsfor a specific intended use are fulfilledValidationVerificationVerification is the confirmation, through the provision ofobjective evidence that specific requirements have beenfulfilled (ISO 9000:2005 section 3.8.4)Dr. Manuela Schulze14
Definitions (2)ValidationVerification“Full” validation “usually by interlaboratory method performance study(collaborative study)A laboratory using a collaboratively studied method,which has been found to be fit for the intended purpose,needs only to demonstrate that it can achieve theperformance characteristics stated in the method byverification.Dr. Manuela Schulze15
Definitions (3)ValidationVerificationValidation and verification applyto a defined protocolfor the determination of a specified analytefor a specific range of concentrationsin a particular type of test materialfor a specific purposeDr. Manuela Schulze16
Validation / VerificationRealistic operation situations matrix analyte concentration staff equipment reagents environmentDr. Manuela Schulze17
VerificationVerification is applicable only for standard/accepted methodswhich have been validated before.The laboratory has to- demonstrate that the performance parametersspecified in the method have been met with thematrices to which the method is being applied.Most often the critical parameter are the trueness and theprecision (ALACC Guide 2007).Dr. Manuela Schulze18
Definitionsaccuracytrueness and precisionTrueness: Closeness of agreement between the averagevalue obtained from a series from test results and anaccepted valueAccuracy: Closeness of agreement between a test result andthe accepted reference valueDr. Manuela Schulze19
trueness (bias)precisionaverage valuestandard deviation[Source: wikipedia.com]Dr. Manuela Schulze20
high truenesshigh precisionhigh truenesslow precisionlow truenesshigh precision(note: no picture for low trueness and low precision)Dr. Manuela Schulze21
Accuracy is calculated as the percentage of recovery by theassay of the known added amount of analyte in the sample,or as the difference between the mean and the accepted truevalue, together with confidence intervals.Accuracy involves a combination ofrandom components anda common systematic error or bias component.Dr. Manuela Schulze22
The validation/verification parameter which have to be considered aredependent on- the data already available.The extent of validation/verification is dependent on- the parameters which have already been tested.Laboratory own methods need the “whole validation”.Procedures and responsibilities for validation/verification have to bedescribed in the quality management system of the laboratory andrecords about the process of validation/verification have to be kept.Dr. Manuela Schulze23
StrategySelection of method(s) neededCheck of available methods- fit for purpose for the laboratory- validation status of the methodsConsider the modular approach- step by step- extraction method- PCR method- real time PCRDr. Manuela SchulzePicture by Dr. Manuela Schulze24
Available methods Booklet: Compendium of reference methodsfor GMO analysis, developed by EURLGMFF in collaboration with the ENGL
On-line GMO detection method /
The European Union Reference Laboratory for GM Food and Feed (EU-RLGMFF) is responsible for GMO detection methods for food, animal feed andseeds in the EU s authorisation process.The EU-RL GMFF regularly publishes information on the status of validationdossiers on the internet. gmo-crl.jrc.ec.europa.eu/statusofdoss.htmOnce the validation process is complete, the full validation report and theprotocol of detection and quantification are published on the EU-RL GMFFweb site.Dr. Manuela Schulze27
Preparation of the verification (1)Prerequisite: An appropriate validated method is available!1. Do you have the same matrix to analyse?Extraction: Sometimes different materials from the same plant needadditional stepse.g. potato leaf: CTAB extraction methodwithout CTAB precipitationstep workspotato tuber:Picture by Dr. Manuela SchulzeDr. Manuela Schulzea CTAB precipitation stepshould be included28
Preparation of the verification (2)Prerequisite: An appropriate validated method is available!2. Check the possibility to introduce the validated method withoutchanges in your laboratory do you have all the equipment? If not, do you intend to introduce the equipment? If not, how can you achieve the result by a modification oradaptation? Do you have all chemicals needed? If not, which chemicals are mandatory for a first test? How can you achieve the result by a modification oradaptation?Also the price can be a reason for a modification.Dr. Manuela Schulze29
ChemicalsPCR-Mastermix: Compareespecially the MgCl2 concentration of the validatedmethod and your ownmastermix.The mastermix intended tobe used must be compatiblewith your thermal cycler.Note: Try to minimize thedifferent numbers ofmastermixes in yourlaboratory.Probe: If necessary, adapt the detection wavelength if you have tomodify the label of the probeDr. Manuela Schulze30
Preparation of the verification (3)3. Check the availability of positive and negative materialDr. Manuela Schulze31
Reference materialCertified ReferencematerialRM and CRM provide essential traceability inmeasurements and are used for, e.g.demonstrate the accuracy of resultscalibration of equipmentmonitoring of laboratory performance (qualitycontrol)Validation of methodsComparison of methodsDr. Manuela Schulze32
Reference materialCertified ReferencematerialISO Guide 30:1992/Amd.1:2008Certified reference materialCRMReference material characterized by a metrological valid procedure for oneor more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceabilityDr. Manuela Schulze33
Reference materialCertified ReferencematerialISO Guide 30:1992/Amd.1:2008Reference materialRMMaterial, sufficiently homogeneous and stable with respectto one or more specified properties, which has beenestablished to be fit for its intended use in a measurementprocessDr. Manuela Schulze34
Reference materialCertified ReferencematerialISO Guide 31:2000Reference materials – Contents of certificates and labelsISO Guide 34:2009General requirements for the competence of referencematerial producersDr. Manuela Schulze35
Positive MaterialsCheck available certified reference materialsCheck available reference materialsCheck available positive materialFor the verification of an extraction method a matrix matched material isuseful, for the verification of a PCR method a DNA-Standard can bechosen.For the verification of an extraction method there is no need forgenetically modified material.Sometimes you have to consider to use spiked material.Dr. Manuela Schulze36
Controls (ISO/EN 24276:2006,drafted update/actual revision)
Controls (ISO/EN 24276:2006)
Picture by Dr. Manuela SchulzeDr. Manuela Schulze39
Certified Reference material producers e.g. European Commission Institute forReference Materials and Measurements (IRMM)irmm.jrc.europa.eu/reference materials catalogue/catalogue/Pages/index.aspx
Positive MaterialsAmerican Oil Chemists Society (AOCS)https:secure.aocs.org/crm/index.cfmDr. Manuela Schulze41
Positive MaterialsPossible sources:- botanical gardenMillennium Seed Bank Kew Royal botanic garden www.kew.orgor DNA-bank e.g. botanical garden/German Science Foundation BerlinDahlem ww2.bgbm.org/herbarium/dna/defaut.cfm- Commercial companies: e.g. divider of IRMM material- Commercial companies producing genetically modified organisms- Official authorities responsible for the control of deliberate releasestudies- Research institutes- EU-RL GMFFDr. Manuela Schulze42
Positive Materials?Picture by Dr. Manuela SchulzeDr. Manuela Schulze43Picture by Dr. Manuela Schulze
Positive Materials?Picture by Dr. Manuela SchulzePicture by Dr. Manuela SchulzePicture by Dr. Manuela SchulzePicture by Dr. Manuela SchulzeDr. Manuela Schulze44
Preparation of the verification (4)4. Document all the changes you have to consider in yourverification e.g. because you have a different thermal cycler5. Select the parameters which you have to verifyDr. Manuela Schulze45
How to meet ISO 17025 requirements for methodverificationprepared byAnalytical Laboratory AccreditationCriteria Committeeon request of AOACGuideline available ashttp://www.aoac.org/alacc guide 2008.pdfDr. Manuela Schulze46
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DefinitionsSensitivity relates to the ability of the test to identifypositive results.sensitivityspecificitySpecificity relates to the ability of the test to identifynegative results.Dr. Manuela Schulze49
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Example: Verification of validated realtime PCR methodsGuidance document:Verification of analytical methods for GMO testing when implementinginterlaboratory validated s.htmDr. Manuela Schulze52
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Example: Verification of validatedreal time PCR methodsPossible performance characteristics to be considered in the verification:- Specifity- Robustness- Dynamic range- Amplification efficiency- R2 coefficient- Trueness- Precision- LOD- mDr. Manuela Schulze54
Preparation of the verification (5)6. Set up a verification plan (documentation of the result of theconsiderations)- draft a version of the method as it will be performed in thelaboratory- write down the persons foreseen- fix the number of experiments, replicates,- make sure that the word “replicates” or repetitions isunambiguous(analytical sample, test sample, DNA-extractions replicates, PCRreplicates, )Dr. Manuela Schulze55
Determination of 1000 corn weight,documentation of subsamples
DocumentationBase for transparency, tracing back When did ?Who ?What ?By using– Which material, reagents ?– Which equipment ?– Which methods and why ?Not documented means not done!
Collaborative studyTest of the same method,Ring trialProficiency testTest of the laboratoryproficiency, test of themethod used as in theroutine by the laboratory- inter-laboratory studies- analysis of unknown samplesprovided by an external sourceDr. Manuela Schulze58
Collaborative studyProficiency testInterpretation of PT outcomeKey evaluator: z-scoreAcceptable: -2Indicates that results are equal to those of the otherlaboratories participated in the PTWhat if falling out of the score?- Compare with other failing laboratories (similar methods,equipments.)- Handle as “non-conformity”Dr. Manuela Schulze59
Collaborative studyProficiency testBenefits for laboratoriesParticipation in proficiency testing programs allows laboratories to: identify areas where improvement in their testing and measurementmethods is needed identify further training of their staff foster confidence in the performance of their testing and measurements assure laboratory performance in their accredited test and calibrationresults is mandatory for accredited laboratories is a confirmation of the correct verification of methodsDr. Manuela Schulze60
After verificationPicture by Dr. Manuela SchulzeDr. Manuela Schulze61
ISO/IEC 17025 Chapter 5.4.5 „validation of methods‟ . procedures that should be employed to ensure adequate validation of analytical methods. Interlaboratory Validation of the method per . Verification, introduction in different laboratories. Dr. Manuela Schulze 14 Validation is the confirmation by examination and provision of objective .
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The resulting guideline 'Verification of analytical methods for GMO testing when implementing interlaboratory validated methods' was published in July 2011. The 30th ENGL Steering Committee, held on 3rd - 4th of February 2016, established the working group on "Update of Method Verification Document" with the mandate of:
validated awards is a matter of prime importance to the OU. The OU will take any action it considers necessary under its Royal Charter to protect the quality of validated programmes of study and the standard of its validated awards. A1.6 Quality assurance As a UK University, the OU is subject to the requirements and expectations of UK higher education, as represented by the Quality Assurance .
4 Key Takeaways Author, manage requirements in Simulink Early verification to find defects sooner Automate manual verification tasks Workflow that conforms to safety standards Static Source code verification High Level Design Detailed Design Coding Integration Testing Unit Testing Verified & Validated System System Requirements
Basic Heat and Mass Transfer complements Heat Transfer,whichispublished concurrently. Basic Heat and Mass Transfer was developed by omitting some of the more advanced heat transfer material fromHeat Transfer and adding a chapter on mass transfer. As a result, Basic Heat and Mass Transfer contains the following chapters and appendixes: 1.
Page 1 of 44 Chapter 6 Transfer Pricing Methods 6.1. Introduction to Transfer Pricing Methods 6.1.1. This part of the Chapter describes several transfer pricing methods that can be used to determine
S:\FA\2014-2015\Verification 2014-2015\Forms\V6 HH Resources Dep Verification 14-15.doc Track Code: 5G 02/27/14 1 2014-2015 Household Resources Verification Worksheet Dependent Student Your 2014-2015 Free Application for Federal Student Aid (FAFSA) was selected by the Federal processor for review in a process called verification.
Anatomy is the study of the structure of living things. b. Physiology is the science of the functioning of living organisms and their component parts. SELF-ASSESSMENT EXERCISE 2 i. Factors that determine divisions in anatomy are: a. Degree of structural detail under consideration 5. HEM 604 BASIC ANATOMY AND PHYSIOLOGY OF HUMAN BODY b. Specific processes c. Medical application ii. The analysis .
