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Validation of screening methods (2002/657/EC) N. Van Wouwe IPH AFSCA-FAVV.

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Presentation on theme: "Validation of screening methods (2002/657/EC) N. Van Wouwe IPH AFSCA-FAVV."— Presentation transcript:

1 Validation of screening methods (2002/657/EC) N. Van Wouwe IPH AFSCA-FAVV

2 Definition (2002/657/EC) Screening method :  used to detect the presence of a substance or class of substances at the level of interest.  have the capability for a high sample throughput => are used to sift large numbers of samples for potential non-compliant results. Exemple: ELISA, plate test, biosensor, receptor test,…

3 Definition (2002/657/EC) Minimum criteria to use an analytical method as screening method:  must be validated (traceability)  must have a false compliant rate of <5% (β- error) at the level of interest

4 Performance characteristics for method validation (screening) + = determination is mandatory Qualitative method: identifies a substance on basis of its chemical, biological or physical propriety (binary response: +/-, absence/presence) Quantitative method: determines the amount or mass fraction of a substance (response: numerical value of appropriate unit)

5 Validation of screening test Definition of the scope of the method Analyte of group of analytes Range of concentration List of matrices Initial validation with the most often used matrice in national monitoring program Detection capacity (CCβ) Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative method) If possible: different sources of blank material, different technicians, different days on the same spiked sample

6 Validation of screening test Targeted test: for 1 compound  validation for this compound Targeted test: for a family of compounds  validation for 1 representative molecule of the family (antibody) Wide range test: for more than 50 different molecules  Validation for at least a list of representative compounds Common pattern of activity on a specific bacteria? Common way of action (acting target)? Published reference data on validation available?

7 Proposition of the CRL for antimicrobials (in milk) Representative CompoundAntimicrobial class Cefalonium/Cephapirin/CefquinomeCEPHALOSPORINS Penicillin G/CloxacillinPENICILLINS Tetracycline/DoxcyclineTETRACYCLINS Gentamicin/Streptomycin/SpectinomycinAMINOGLYCOSIDES Enrofloxacin/FlumequineQUINOLONES Sulfathiazole/sulfaguanidine/SulfamerazineSULFONAMIDES Erythromycin/TylosinMACROLIDES LincomycinLINCOSAMIDES ThiamphenicolPHENICOLATED Trimethoprim/colistineMISCELLANEOUS

8 Performance characteristics Detection capacity Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative method)

9 Detection capability (CCβ) The smallest content of the substance that may be detected, identified and/or quantified in a sample with an error probability of β  In case of MRPL, CCβ= lowest concentration at which the method is able to detect truly contaminated sample with a statistical certainty of 1-β  In case of MRL, CCβ= concentration at which the method is able to detect the MRL concentrations with a statistical certainty of 1-β

10 Detection capability (CCβ) No permitted limit  Analyse 20 blank materials => CCα = 3x signal/noise Analyse 20 blank materials fortified at CCα => CCβ = CCα x SD RW  Calibration curve procedure (ISO 11843)  Analyse of blank material fortified at 0 MRLP, 0.5 MRLP, 1 MRLP, 1.5 MRLP and 2 MRLP  Plot analytical results (y-axis) vs concentration(x-axis)  CCα = y-intercept (blank) x SD RW  CCβ = CCα x SD RW

11 Detection capability (CCβ) No permitted limit  If no quantitative results Analyse fortified blank samples at and above CCα (n ≥ 20 / concentration level) CCβ = concentration level where only ≤5% false compliant results remain

12 Detection capability (CCβ) CC  Signal or Concentration CC  x SD blank +1.64xSD RW Blank α=1%β=5%

13 Detection capability (CCβ) Permitted limit (MRL)  Analyse 20 blank materials fortified at MRL => CCα = MRL x SD RW Analyse 20 blank materials fortified at CCα => CCβ = CCα x SD RW  Calibration curve procedure (ISO 11843)  Analyse of blank materials fortified at 0.5 MRL, 1 MRL, 1.5 MRL and 2 MRL  Plot analytical results (y-axis) vs concentration(x-axis)  CCα = MRL x SD RW  CCβ = CCα x SD RW

14 Detection capability (CCβ) CC  Signal or Concentration CC  MRL x SD MRL x SD RW β=5%α=5%

15 Performance characteristics Detection capacity Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative method)

16 Selectivity/specificity Specificity: ability of a method to distinguish between analyte being measured and other substances problem of interference?  F(measuring technique, class of compounds, matrices,…)

17 Selectivity/specificity How to test specificity for qualitative screening method?  Analyse 20 different blank samples and 20 positive samples (blind study, same or different days/technicians) True positive (N+) True negative (N-) test result positive Positive agreement (PA) False positive (FP) test result negative False negative (FN) Negative agreement (NA) Specificity= 100* NA/N - Other parameters: Accuracy= 100* (PA+NA)/(N - + N + ) Sensitivity= 100* PA/N+ False positive= 100* FP/(N- + N+) False negative= 100* FN/(N- + N+)

18 Selectivity/specificity How to test specificity for semi-quantitative screening methods?  Select potentially interfering substances (metabolites, derivatives,…)  Analyse relevant blank samples (n ≥ 20)  Analyse fortified blank samples with interfering substances at a relevant concentration  Estimate the effect of the interferences  False identification?  Influence in quantification?  Identification of the target analyte is hindered?

19 Performance characteristics Detection capacity Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative method)

20 Applicability Scope of the method must be define in term of :  Matrix (solid/liquid matrix, type of tissue)  Animal species To introduce a new matrix  Analyse at least 10 different blank material fortified at level of interest for the new matrix (CCβ) + test of interferences If 10 positive results => method applicable for the new matrix If 1 negative result => 10 additional analyses  If 1 negative result=> CCβ must be recalculated for the new matrix

21 Ruggedness Ruggedness: the susceptibility of an analytical method to changes in experimental conditions sample material analytes storage condition environmental condition sample preparation condition

22 Ruggedness How to test ruggedness? (during development)  Identify possible factor that could influence the results (the analyst, solvents, pH, T°, rate of heating,…)  Vary each factor slightly  If one factor is found to influence results of the representative molecule, conduct further experiments => acceptability limits for this factor (in the method protocol) Recommendation of CRL: analyses of 10 blank and 10 spiked samples at the same concentration and with minor change of factor to detect influence on results

23 Stability Test are not necessary if stability data already exist (from other lab or from publication)  To include in the validation report Stability test:  the analyte in solution  the analyte in matrix Aliquots of a fresh solution or sample stored under different conditions (T° and/or storing time)

24 Performance characteristics Detection capacity Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative method)

25 Precision (for quantitative screening) Precision: the closseness of agreement between independent test results obtained under predetermined conditions Expressed in terms of imprecision / standard deviation of test results

26 How to test precision?  Repeatability test  within-laboratory reproducibility test(or intermediate precision)  Reproducibility test (between laboratories: interlaboratory studies) determination of RSD (%) < Precision criteria Precision (for quantitative screening)

27 Repeatability  3 concentrations: 1x; 1,5x; 2x MRPL 0,5; 1x; 1,5x MRL  6 replicates/level  3 times  same conditions Within-laboratory Reproducibility  3 concentrations: 1x; 1,5x; 2x MRPL 0,5; 1x; 1,5x MRL  6 replicates/level  3 times  different conditions (analyst, env. condition,…)

28 Precision (for quantitative screening) ANOVA treatment of data => RSD r & RSD RW Comparison with precision criteria:  Horwitz equation: RSD R (%) = 2 (1-0.5logC)  Criteria for repeatability: RSD r = 1/2 to 2/3 RSD R  Criteria for within-lab reproducibility: RSD RW = 2/3 to 1 RSD R ! For concentration < 100 µg/kg, RSD R becomes too high!

29 Other recommendations False negative rate <5%: Analyses of 20 negative and 20 positive samples in order to test the screening method (see selectivity). One QC sample must be added in routine and results must be added to the validation file Method transfer/Commercial test  Bibliographical survey to compil the evaluation of performance of the test  Collection of data from supplier on validation study  Experimental plan to test skillness of technician to perform the test  Use of QC sample  Participation to proficiency test

30 Exemple: analyse of PCDD/F by CALUX bioassay PCDD/F: 17 toxic congeners to analyse in various matrices (TCDD=most toxic dioxin) Results expressed in TEQ (=Sum (CCixTEFi) i=1-17 ) MRL for each matrix (milk, meat, egg, fish oil,…) MRL expressed in pg TEQ/g fat or ng TEQ/ kg Reference method: GC-HRMS Screening method: immunoassay, bioassay,…

31 Exemple: analyse of PCDD/F by CALUX bioassay Gene expression LIGHT All substances fixing the Ah receptor CALUX bioassay= genetically modified cell-based bioassay (luciferase) Amount of light produced is proportional to the toxicity (TEQ) of extracts

32 Analyse of PCDD/F by CALUX bioassay Advantage:  Rapid  Cheaper than GC-HRMS  Time for analyses Disadvantage:  Various compounds can fix the Ah receptor (PAH, PCB, PHDD/F,…) specificity!!!!

33 Analyse of PCDD/F by CALUX bioassay : protocol Extraction of fat Clean-up on silica acid + carbon columns Fraction with PCBs Fraction with PCDD/F Evaporation Dosing plate Reading plate Fraction with interfering compounds

34 Analyse of PCDD/F by CALUX bioassay : validation Selectivity/specificity Ruggedness/Stability Precision Detection capability

35 Analyse of PCDD/F by CALUX bioassay : selectivity Possible interfering compounds?  PAH : mostly in environmental sample  PCB: fractionation during clean-up  Other compounds? (PHDD/F): dependant of the matrix? (matrix effect?) Results of the selectivity test:  No interferences for feedstuff, milk, egg, fat  Interferences for fish oil CALUX results = 2 x GC-HRMS results

36 Analyse of PCDD/F by CALUX bioassay : selectivity Matrix effect for fish oil

37 Analyse of PCDD/F by CALUX bioassay : ruggedness What are the critical point in the protocol?  Carbon column (interferences)  Solvent (interferences)  Curve (results)  Evaporation time (recovery)  Age of CALUX cell line (RSD)

38 Analyse of PCDD/F by CALUX bioassay : ruggedness Carbon column: amount of carbon used DX fraction PCB fraction Not collected fraction (Rdt PCDD/F= 60%)(Rdt PCDD/F= 80%)

39 Analyse of PCDD/F by CALUX bioassay : ruggedness Evaporation time

40 Analyse of PCDD/F by CALUX bioassay : ruggedness Solvent: tested before use on a TCDD solution (antagonist/agonist effect) Curve: tested with an independant TCDD solution Age of CALUX cells: new cell every 2 months

41 Analyse of PCDD/F by CALUX bioassay : precision Validation protocol Day 1Day 2Day 3Day 4Day 5Day 6 Blank solvent Blank sample1111 Sample at MRL/2332 Sample at MRL6233 Sample at 2MRL323 Quality sample111110

42 Analyse of PCDD/F by CALUX bioassay : precision ANOVA results for the TEQ determination of PCDD/F in feedstuff by CALUX bioassay  At MRL (0.75ng TEQ/kg) : X MRL = ng TEQ/kg S r = => RSD r = 8.4% S RW =0.073 =>RSD RW = 9.7%  At MRL/2 (0.376ng TEQ/kg) : X MRL/2 = ng TEQ/kg S r = => RSD r = 11% S RW =0.051 =>RSD RW = 11%  At 2MRL (1.5ng TEQ/kg): X 2MRL = ng TEQ/kg S r = => RSD r = 6.8% S RW =0.115 =>RSD RW = 7.3% RSD < 30% (2002/70/EC)

43 Analyse of PCDD/F by CALUX bioassay : detection capacity CCβ for the TEQ determination of PCDD/F in feedstuff by CALUX bioassay  CCα = MRL x S RW CCα = x = 0.87 ng TEQ/kg  CCβ = CCα x S RW CCβ = x = 1.04 ng TEQ/kg 2002/70/EC: false negative rate < 1% ! => At a concentration of 1.04ng TEQ/kg, we are sure that the sample is a positive sample with 99% certainty

44 Analyse of PCDD/F by CALUX bioassay :confirmatory range MRL CC  Signal or Concentration CC  NON COMPLIANTC OMPLIANT SUSPICIOUS CC *  MRL  sample  MRL ? α=5%β=5% * =1%

45 Analyse of PCDD/F by CALUX bioassay :confirmatory range Lower limit of the confirmatory range for the TEQ determination of PCDD/F in feedstuff by CALUX bioassay  CC*= MRL-2.33 x SD RW CC*= x = 0.58 ng TEQ/kg Conclusion  Sample lower than 0.58 ng TEQ/kg are negative with 99% certainty (false negative rate < 1%)  Sample above 0.58 ng TEQ/kg must be confirmed by GC-HRMS


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