1. Christopher N. Greene, PhD Newborn Screening and Molecular Biology Branch, Division of Laboratory Sciences NCEH, CDC Tuesday, 9th July 2013 Assay Quality Considerations National Center for Environmental Health U.S. Centers for Disease Control and Prevention

2. Molecular Testing Testing ParametersExamples Target AnalyteGenetic Mutations, Gene Dosage PlatformPCR, qPCR, bead arrays, Fluorescent Probe, DNA Sequencers ComplexitySingle- or multi-plex of single nucleotides, Laboratory Developed Test, ASRs, FDA-approved, Gene Sequencing, Next-Gen Sequencing Multiple molecular assays have been developed for screening procedures. These assays vary on their complexity, the target analyte, as well as the platform used for analysis.

3. Molecular Testing When Adopting a New TestRequired Evidence Test Development - LDTAccuracy, precision, reproducibility, sensitivity, specificity, robustness Test Validation - ASRClinical and analytic performance and test limitations Test Verification – FDA approvedIdentify samples or reference materials of known quantitative or qualitative values to verify test performance Molecular tests require verification or validation of assay results. Level of evidence required is related to the type of assay, i.e. laboratory developed test, analyte specific reagents or FDA approved assays.

4. Laboratory Regulatory and Accreditation Guidelines  US Food and Drug Administration (FDA):  approves kits and reagents for use in clinical testing  Clinical Laboratory Improvement Amendments (CLIA):  Regulations passed by Congress1988 to establish quality standards for all laboratory testing to ensure the accuracy, reliability and timeliness of patient test results regardless of where the test was performed  College of American Pathologists (CAP):  Molecular Pathology checklist  State Specific Regulations

5. Testing Parameters  Accuracy  Sensitivity  Specificity  Precision  Limit of Detection  Range  Reproducibility  Robustness

6. Definitions Accuracy: Agreement between test result and the “true” result –Comparison of results between new method and a reference method –Results of new method on certified reference materials Sensitivity: Ability to obtain positive results in concordance with a positive result of a reference method – can the test detect a positive result

7. Definitions Specificity: Ability to obtain a negative result for a qualitative test in concordance with reference method Precision: Measure of random error in quantitative tests, the closeness of agreement usually expressed as standard deviation of CV

8. Definitions Limit of Detection: Lowest amount of analyte that is distinguishable from background or a negative control Range: Range of test values expected for the test population. For quantitative, high and low range of target, for qualitative, possible detectable alleles of the assay

9. Definitions Reproducibility: Equivalent to precision for qualitative and semi-quantitative tests. Can be used to indicate with-in or between run agreement of test results Robustness: Test precision, given small, deliberate changes in test conditions (e.g. incubation times, temperature variation)

10. Pre-Validation Choose and evaluate assay methodology Determining analytic performance of an assay involves:  Reviewing professional guidelines and relevant literature  Stringent design/analysis of primers and probes  Quality and quantity of extracted nucleic acid  Appropriate platform for the test  Availability of controls or calibrators  Optimization of amplification and detection

11. Verifying Assay Specifications Demonstrate that laboratory can obtain specifications comparable to manufacturer –Accuracy –Precision –Reportable Results Applies to unmodified FDA-approved tests Verify appropriate test results for population May require matrix- appropriate materials during verification

12. Assay Validation Performance characteristics of laboratory developed tests Reagents that are the components of a test that is created without regulatory approval – ASR Use of FDA-approved tests that have been modified for the testing procedure Establish that the assay fulfills its intended use with the appropriate sample type and population

13. Modified FDA Test Alteration of any assay step or analyte for the test that may affect performance Changes could include: –Type or concentration of specimen –Storage specifications –Extraction method –Amplification parameters or platforms –Use of test for other purposes

14. Assay Validation  Required for:  New testing methodology  Assay modification – includes cross-checks for different makes/models of instrumentation  Applies to:  Modified FDA assays  Laboratory Developed methods

15. Controls for Each Run Appropriate positive and negative controls should be included for each run of specimens being tested

16. Molecular Assay Controls  Positive controls:  Inhibitors  Component failure  Interpretation of results  Sources:  Residual positive DBS  PT samples  QC materials through purchase or exchange  Negative controls:  Nucleic acid contamination

17. Common Molecular Assay Problems and Trouble Shooting  Temperature errors  Template/Sequence  PCR inhibitors  Buffers  Bad dNTPs  Bad primers  Bad enzyme

18. Critical Molecular Assay Components  Nucleic Acids: Prepare aliquots appropriate to workflow to limit freeze-thaw cycles  Primers and probes  dNTPs  Genomic DNA  4-8°C: Up to one year  -20°C: Up to seven years  Enzymes  Benchtop coolers recommended  Fluorescent reporters  Limit exposure to light  Amber storage tubes or wrap in shielding (foil)

19. Positive Controls Ideally should represent each target allele used in each run May not be feasible when:  Highly multiplex genotypes possible  Systematic rotation of different alleles as positives  Rare alleles  Heterozygous or compound heterozygous specimens

20. Positive Controls  Assays based on presence or absence of product  Internal positive amplification controls to distinguish true negative from false due to failure of DNA extraction or PCR amplification  PCR amplification product of varying length  Specimens representing short and long amplification products to control for differential amplification  Quantitative PCR  Controls should represent more than one concentration  Control copy levels should be set to analytic cut-offs

21. Quantitative PCR Controls Contamination controls Calibration control with reliable low limit of quantification Measures for consistency, especially for DBS Measure of DNA quality – internal control

22. False Negative: ADO Allele drop-out (ADO): the failure of a molecular test to amplify or detect one or more alleles  Potential causes:  DNA template concentration Incomplete cell lysis DNA degradation  Non-optimized assay conditions  Unknown polymorphisms in target sites  Reagent component failure  Major concern for screening laboratories  Confirmation of mutation inheritance in families is not an option

23. DNA Degradation  Lane 1 + 7: 1kb size standard ladder  Lane 2: 100ng control genomic DNA  Lanes 3-5: Crude cell lysates

24. Question? How can you control for presence of sufficient amount/quality of DNA for a PCR based test in a NBS lab?

25. PCR Amplification Controls Allele-specific amplification Are there problems with this assay? What additional controls would be useful? Allele 1 + 2 Allele 2 Allele 1 Reference Negative

26. PCR with Internal Controls Tetra-primer ARMS-PCR Simultaneous amplification of:  Positive amplification control  Mutation allele  Reference allele Alternative to tetra-primer ARMS is to include an additional primer set to amplify a different control sequence

27. Allele Drop-out in PCR Testing ggacatgatGcactacatgctccaaggtagtggag Cgtgatgtacgaggttccat 5’ ggacatgatGcactacatgctccaaggtagtggag cctgtactaCgtgatgtacgaggttccat 5’

28. Allele Drop-out in PCR Testing SNP in primer site ggacatgatGcGctacatgctccaaggtagtggag gatgtacgaggttccat 5’ ggacatgatGcGctacatgctccaaggtagtggag Cgtgatgtacgaggttccat 5’ Cgt

29. False Negatives: Deletions A G Reverse Primer Forward Primer Reverse Primer Forward Primer

30. False Negatives: Deletions A G Reverse Primer Forward Primer Reverse Primer Forward Primer Deletion

31. False Positives  Potential causes:  Non-optimized assay conditions  Unknown polymorphisms in target sites  Gene duplications  Oligonucleotide mis-priming at related sequences  Psuedogenes or gene families  Oligonucleotide concentrations too high  Nucleic acid cross-contamination

32. How Many Samples? New test or to replace an old test? How is the test to be used? What performance criteria are most important? How available are appropriate samples? For screening: daily load vs. weekly load vs. monthly load – it depends It depends…

33. CLSI Evaluation Protocols recognized by FDA as Consensus Standards EP5-A2: Evaluation of Precision Performance of Quantitative Measurement Methods EP9-A2: Method Comparison and Bias Estimation Using Patient Samples EP10-A2: Preliminary Evaluation of Quantitative Clinical Methods EP12-A User Protocol for Evaluation of Qualitative Test Performance EP15-A2: User Demonstration of Performance for Precision and Accuracy

34. Molecular Assay Proficiency Testing Material Sources  CDC NSQAP  UKNEQS  EuroGentest  CAP  Maine Molecular  SeraCare  Corielle  ECACC  In-house samples  Round-robin with other NBS laboratories

35. Professional Guidelines  American College of Medical Genetics (ACMG)  Standards and Guidelines for Clinical Genetics Laboratories  Clinical and Laboratory Standards Institute (CLSI)  MM01-A2: Molecular Diagnostic Methods for Genetic Diseases  MM13-A: Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods  MM14-A: Proficiency Testing (External Quality Assessment) for Molecular Methods  MM17-A: Verification and Validation of Multiplex Nucleic Acid Assays  MM19-P: Establishing Molecular Testing in Clinical Laboratory Environments

36. Additional Sources