Pikamenetelmät ja niiden validointi Ajankohtaista laboratoriorintamalla 10.10.2012, Helsinki FT, dos Mika Tuomola
Pikamenetelmät Elintarviketurvallisuuden edistäminen hyödyntämällä nopeasti uusia tieteellisiä tuloksia ja teknologisia innovaatioita Pikamenetelmien täytyy antaa luotettavia tuloksia olla viranomaisten ja teollisuuden toimivaksi hyväksymiä olla käyttötarkoitukseensa validoituja
Pikamenetelmien etuja Nopeat tulokset Yksinkertainen workflow Alhainen kustannus per tulos Automaation mahdollisuus On-site analytiikan mahdollisuus Parantuneet analytt. ominaisuudet (määritysherkkyys, multipleksaus,..)
Screening: mostly +/- results Many alternative methods provide quantitative results, but rapid methods are used in particular for qualitative purposes Rapid screening for tentative +/- results Actionable result (present/not present; above/below threshold) clinical sensitivity important (% of true positive results) clinical specificity less important (% of true negative results) Challenges with statistical methods (binary results; noncontinuous variables; IUPAC protocols etc not applicable)
Esimerkki: Salmonella-analyysi Referenssimenetelmä: tulos >4 päivää näyte esirikastus 16-20 h Selektiivinen rikastus 24 h Viljely maljoilla Nopea immunomääritys: tulos 1 päivässä näyte esirikastus 18-19 h selektiiv. rikastus 4-5 h ELISA Nopea PCR tai muu DNA:n monistusmenetelmä: tulos <1 päivässä näyte esirikastus 8-18 h PCR
Esimerkki: Allergeenien analytiikka Direktiivi 2003/89/EC (+ lisäykset 2006/142/EC) Gluteenia sis. viljat Maito (laktoosi) Kananmuna Äyriäiset Kalat Maapähkinät Soija Pähkinät Seesaminsiemenet Selleri Sinappi Nilviäiset Lupiini Sulfiitit Testistripsit Pikamenetelmiä on ollut käytössä jo >10 vuotta! Toimivia instrumentaalimenetelmiä on luotu vasta äskettäin (LC-MS/MS) Validoituja menetelmiä olemassa vain vähän Referenssimateriaaleja olemassa vain vähän Eri kiteissä eri kalibraattorit Raja-arvot? ELISA kitit Allergeeni-PCR
Allergeenien tetauksessa huomioitavaa Erilaiset matriisit huomioitu validaatiossa? DNA:n löytyminen ei välttämättä tarkoita, että varsinaista allergeenia olisi läsnä (epäsuora menetelmä) Onko detektiokohde näytematriisille relevantti? Esim. maitotestin kohde voi olla kaseiini tai beta-laktoglobuliini Herkkyys: Monet vanhemmat menetelmät on kehitetty elintarvikeväärennösten havaitsemiseen herkkyys ei riitä allergeenianalytiikkaan Liian herkkä testi mitä tehdään positiivisen tuloksen kanssa? Raaka vs. kypsennetty havaitseeko testi molemmat? Hydrolysoinnin/fermentoinnin vaikutus?
Validation? Method provider has presented validation data, should it not be enough? Do all the users need to perform full method validation? Solution: Independent validation review by 3rd party, supported by independent external results NordVal, AOAC, MicroVal, AFNOR Only method verification or possibly limited validation required
Method validation - definition Thompson et al. (2002): Investigation whether the analytical purpose of the method is achieved; the acquisition of analytical results with an acceptable uncertainty level NordVal, microbiological methods: The procedure to demonstrate if the alternative method provides equivalent or better results compared to the reference methods NordVal, chemical methods: The procedure to demonstrate if the proprietary method provides adequate results and is in compliance with its claims
Advantages of validation and independent review Proves that the new method performs according to specified requirements Comparison against established reference methods (if possible) Provides useful information to the users for selecting a method that fits their purposes Adoption of new methods requires significantly less work in the end-user laboratories Allows a degree of control over the quality of the method being offered on the market Positive influence on the penetration of rapid methods
Validation regulation Microbiology: EU Regulation EC 2073/2005 Comparison to ISO/CEN reference method Validation according to EN ISO 16140 or other internationally accepted protocols Certification by a third party Chemicals: limited or no regulation Reference methods do not always exist No ISO protocol NordVal protocol No. 2 Certification possible by a third party M. Tuomola 2011
Veterinary drug residues: EU requirements for validation: 2002/657/EC, CRL guideline 20/1/2010 Qualitative Quantitative Screening Confirm. Screening Confirm. Ruggedness + + + + CCβ (detection capability) + + + + CCα (decision limit) - + - + Trueness/recovery - - - + Precision - - + + Selectivity/specificity + + + + Allergens: no regulation but now at least a harmonised guideline: Abbott et al., 2010: Validation Procedures for Quantitative Food Allergen ELISA Methods: Community Guidance and Best Practices J. AOAC Int, 93(2), 442-450
NordVal: Microbiological methods Method comparison against the reference method Ruggedness (e.g., ph, timing of steps, interferences) Batch-to-batch variation (control system check) Method performance characteristics field of application, selectivity (inclusivity/exclusivity), limit of detection, accuracy, sensitivity, false positives/negatives agreement between the methods, κ quantitative methods: repeatability, trueness, recovery Collaborative study Valid results from at least 8 laboratories (include 10-12 labs). Comparing accuracy, sensitivity, agreement M. Tuomola 2011
NordVal: Qualitative chemical methods Method comparison study Ruggedness (e.g., ph, timing of steps, interferences) Batch-to-batch variation (control system check) Method performance characteristics field of application, limit of detection, sensitivity, accuracy, inclusivity, specificity, false positives/negatives agreement between methods or comparison against expected results (reference materials, spiked samples) Intermediate study At least one additional laboratory confirms performance and transferability (multiple matrices, accuracy, sensitivity, agreement) M. Tuomola 2011
NordVal: Quantitative chemical methods Method comparison study Ruggedness (e.g., ph, timing of steps, interferences) Batch-to-batch variation (control system check) Method performance characteristics field of application, range, limit of quantification, specificity, inclusivity, trueness, repeatability, recovery agreement between methods or comparison against expected results (reference materials, spiked samples) Intermediate study At least one additional laboratory confirms performance and transferability (range, precision, trueness) M. Tuomola 2011
Verification Once the proprietary method has been selected, the laboratory needs to verify the correct implementation of the method NMKL: Verification = A test of whether an individual laboratory can carry out the analyses in accordance with the requirements established in the validation Requirement for accredited laboratories according to Clause 5.4.2. of EN ISO 17025
Validation/verification levels NMKL Procedure No 4, 2009: Validation of chemical analytical methods Degree of external validation 1 The method has been externally validated in a collaborative study. 2 The method has been externally validated in a collaborative study, but is used on a new matrix or with a new instrument. 3 The method is well established but has not been externally validated in a collaborative study. 4 The method has been published in scientific literature, and states important performance characteristics. 5 The method has been published in scientific literature, but lacks important performance characteristics. Recommended internal verification Verification of trueness and precision. Verification of trueness and precision, and possibly also limit of quantification. Verification, and possibly a more extensive internal validation. Verification, and possibly a more extensive internal validation. Full internal validation. 6 The method is developed internally. Full internal validation.
Worth keeping in mind (1) End-users often do not read the certificates and assume that the methods are validated for all the possible uses Validation scope lists the food matrices which have been tested (other matrices may work differently or not at all) Limitations are not often well described (possible restrictions with specificity, effects of interfering agents,..) Partipication to proficiency testing schemes is extremely useful for ensuring compliance with regulation or other specifications
Worth keeping in mind (2) Reference methods which have been used in method comparison are not always the same and do not necessarily produce identical results (ISO/CEN vs AOAC/BAM) The alternative method should give results comparable to the reference method, but sometimes the performance may exceed the gold standard Occurrence of true positive results that can not be confirmed
Kiitos mielenkiinnosta! FT Mika Tuomola mika.tuomola@optimica.fi