1. College of American Pathologists Copyright © 2002 College of American Pathologists (CAP) These materials by the CAP are copyrighted works. All rights are reserved. Participants are permitted to duplicate the materials for educational use only within their own institution. These materials may not be used for commercial purposes or altered in any way. Inspecting a Molecular Pathology Lab Wayne W. Grody, MD, PhD, FCAP March 13, 2003

2. When to Use Molecular Pathology Checklist  DNA/RNA amplification methods, regardless of discipline  E.G Abbott LCX, Genprobe  Commonly missed, requires reinspection  Lab neglects to indicate to CAP via activity menu  Inspector might erroneously decide checklist does not apply  For all home brew tests  Even if performing only 1 test

3. When to Use Molecular Pathology Checklist 2  Clinical molecular diagnostics testing occurs in many disciplines OncologyHLA typing HematologyForensics Infectious diseaseParentage Inherited disease

4. Inspector Requirements  Actively practicing molecular scientists preferred  Familiar with Checklist  Technical and interpretive skills  CAP provides list of potential regional specialty inspectors in Inspector’s packet

5. Follow the Specimen AccessioningBlotting DNA/RNA extractionMicroscopy Probe labelingReporting Amplification/hybridizationComputer entry Electrophoresis orFiling other signal detection

6. Southern Blot  DNA quantitation and purity are monitored  Completeness of restriction endonuclease digestion is monitored  Fragment size evaluated by comparing with molecular weight standards  ELP performed at standard voltages/times for reproducibility  Probes adequately labeled  Positive, negative & sensitivity controls assayed with each run  Interpretive guidelines used

7. Amplification Assays  Negative controls help detect:  contamination - water, contains all but DNA  cross-hybridization - contains wild type template  Positive controls ensure assay performance  Internal probes used to verify specificity  Physical separation of PCR set-up and post- amplification areas

8. In situ Hybridization (ISH)  Results are interpreted in context of tissue morphology  FISH results interpreted in context of karyotype  Tissue and probe controls verify assay sensitivity and specificity  Check microscopes for sufficient resolution and interpretable stain results  Review sampling of cases and controls to evaluate signal, background and morphology

9. Reports  Must contain sufficient information for proper clinical decision making  See Appendix A  For tests that complement other data to establish diagnosis, a narrative must be included that interprets molecular findings In a clinical context

10. What to Look For  Turn-around time  Competency (technical skills, clinical judgement, communication skills) assessed periodically  Direct observation  PT results  Written exam  Look for documentation; sign-off sheets

11. FDA Recommendations Analyte Specific Reagents (ASR)  Molecular probes are an ASR  FDA rulings focus on commercially prepared ASRs  Home brew ASRs acceptable  If test uses a non-FDA approved ASR a disclaimer must be added to test report:  This test was developed and its performance characteristics determined by (laboratory name). It has not been cleared or approved by the US FDA.

12. Common Phase II Deficiency MOL.38100 (Changed) Phase II Are reagents and solutions properly labeled, as applicable and appropriate, with the following elements: 1. Content and quantity, concentration or titer, 2. Storage requirements, 3. Date prepared or reconstituted by lab, 4. expiration date?

13. Checklist Addition MOL.36025 Phase II If reports are based on the use of reagents labeled by the manufacturer as "analyte-specific reagents", is the federally-required clarifying statement part of the report? NOTE: Laboratories must include an FDA-required disclaimer on reports for tests using analyte-specific reagents, whether obtained commercially or produced in-house.

14. Checklist Addition, cont. The mandatory language is "This test was developed and its performance characteristics determined by (laboratory name). It has not been cleared or approved by the U.S. Food and Drug Administration." The CAP recommends additional language, such as "The FDA has determined that such clearance or approval is not necessary. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88) as qualified to perform high complexity clinical laboratory testing."

15. Checklist Addition Quality ControlPhase II  38400 Acceptance limits all QC materials/standards  38550 QC verified before reporting results  38700 Corrective Action when QC exceeds limits  38850 QC tested in same manner as patient

16. College of American Pathologists Laboratory Accreditation Program Common Molecular Pathology Deficiencies in 2002

17. CAP Laboratory Accreditation Program Common Phase II Deficiencies 522 Labs Inspected in 2002 using the Molecular Pathology Checklist

18. MOL.334002.3% MOL.334002.3% MOL.33400 Is there a written policy that no aliquot is ever returned to the original container? COMMENTARY: The laboratory must have a policy that no aliquot is ever returned to the original container.

19. MOL.380002.1% MOL.380002.1% Are new reagent lots checked against old reagent lots or with suitable reference material before or concurrently with being placed in service? NOTE: For qualitative tests, minimum cross ‑ checking includes retesting at least one known positive and one known negative patient sample from the old reagent lot against the new reagent lot, ensuring that the same results are obtained with the new lot. Good clinical laboratory science includes patient ‑ based comparisons in many situations, since it is patient results that are "controlled". A weakly positive control should also be used in systems where patient results are reported in that fashion.

20. MOL.200201.7% MOL.200201.7% Is there evidence of ongoing evaluation of results of instrument maintenance and function, temperature, etc. for all procedures on all shifts?

21. MOL.312001.7% MOL.312001.7% Are enzymatic amplification procedures (e.g., PCR) designed to minimize carry over (false positive results) using appropriate physical containment and procedural controls? NOTE: This item is primarily directed at ensuring adequate physical separation of pre- and post- amplification samples to avoid amplicon contamination. The extreme sensitivity of amplification systems requires that the laboratory take special precautions.

22. MOL.312001.7% MOL.312001.7% NOTE, continued: For example, pre ‑ and post ‑ amplification samples should be manipulated in physically separate areas; gloves must be worn and frequently changed during processing; dedicated pipets (positive displacement type or with aerosol barrier tips) must be used; manipulations must minimize aerosolization; following complete reagent addition to the reaction tubes, the patient samples should be added one at a time. The best way to avoid cross- contamination is to use the following order of preparation within an amplification run: actual samples, followed by positive controls, followed by negative controls.

23. MOL.103501.5% MOL.103501.5% For analytes where graded proficiency testing is not available, are other procedures used to validate performance at least semi ‑ annually? NOTE: Other appropriate performance assessment procedures include: participation in ungraded proficiency survey programs, split sample analysis with reference or other laboratories, split samples with an established in ‑ house method, assayed materials, regional pools, clinical validation by chart review, or other suitable and documented means. It is the responsibility of the Laboratory Director to define such procedures, as applicable, in accordance with good clinical and scientific laboratory practice.

24. MOL.360251.4% MOL.360251.4% If reports are based on the use of reagents labeled by the manufacturer as "analyte-specific reagents", is the federally-required clarifying statement part of the report? NOTE: Laboratories must include an FDA-required disclaimer on reports for tests using analyte-specific reagents, whether obtained commercially or produced in-house. The mandatory language is "This test was developed and its performance characteristics determined by (laboratory name). It has not been cleared or approved by the U.S. Food and Drug Administration."

25. MOL.360251.4% MOL.360251.4% NOTE, continued: The CAP recommends additional language, such as "The FDA has determined that such clearance or approval is not necessary. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88) as qualified to perform high complexity clinical laboratory testing."

26. MOL.313501.3% MOL.313501.3% Are appropriate internal or external validation studies referenced? REFERENCE: Association for molecular pathology statement. Recommendations for in-house development and operation of molecular diagnostic tests. Am J Clin Pathol. 1999;111:449-463.

27. MOL.360001.3% MOL.360001.3% Does the final report include an appropriate summary of the methods, probes and endonucleases used, the loci or mutations tested, the objective findings and a clinical interpretation in an easy ‑ to ‑ interpret format?

28. MOL.101501.1% MOL.101501.1% Does the laboratory participate in an appropriate available CAP or CAP- approved alternative interlaboratory comparison program for the patient testing performed?

29. CAP Laboratory Accreditation Program Common Phase I Deficiencies 522 Labs Inspected in 2002 using the Molecular Pathology Checklist

30. MOL.501502.1% MOL.501502.1% Is there adequate space for technical work (bench space)?

31. MOL.205501.9% MOL.205501.9% Are statistics on all molecular pathology laboratory results (e.g., percentages of normal and abnormal findings, allele frequencies) maintained, and appropriate comparative studies performed?

32. MOL.206001.7% MOL.206001.7% Are these statistics (e.g., percentages of normal and abnormal findings, allele frequencies) and comparative studies reviewed at regular intervals by the Laboratory Director or designee and appropriate corrective action taken, if indicated? Commentary: This process may detect trends, systematic errors, or local population variations that may affect test results or interpretation.

33. MOL.398901.7% MOL.398901.7% When possible, are individual wells (or a representative sample thereof) of thermocyclers checked for temperature accuracy before being placed in service and periodically thereafter? NOTE: If it is not physically possible to check individual wells, a downstream measure of well-temperature accuracy (such as productivity of amplification) may be substituted to functionally meet this requirement.

34. Space Deficiencies1.3 - 1.0% Space Deficiencies1.3 - 1.0% Is there adequate space for:  Instruments1.3%  Administrative functions1.1%  Shelf storage1.1%  Clerical work1.0%

35. MOL.333601.0% MOL.333601.0% Are there written criteria for questioning or rejection of clinically inappropriate test requests? NOTE: For every test offered, there should be documentation describing the appropriate (and inappropriate) clinical indications.

36. MOL.333601.0% MOL.333601.0% COMMENTARY: Many of the disease genes subject to molecular genetic testing are extremely complex as to their size, mutational heterogeneity, penetrance, and expressivity. Especially with regard to presymptomatic testing, application of these tests to patients not carefully screened and counseled can be meaningless or damaging. For certain tests, only those patients with strict family history criteria are eligible. There are also many ethical considerations, such as the policy of not offering predictive genetic tests to children unless there is a viable clinical intervention to be initiated prior to adulthood.

37. MOL.333601.0% MOL.333601.0% COMMENTARY, continued: Because primary care physicians may not be conversant with these matters, it is sometimes left to the molecular diagnostic laboratory to provide consultation. The laboratory therefore should have established guidelines for the rejection or questioning of test requests felt to be inappropriate on clinical or ethical grounds. Reference to the policies promulgated by professional organizations and government agencies, and/or consultation with a medical ethicist, may be of help on a case ‑ by ‑ case basis.

38. MOL.402001.0% MOL.402001.0% Is there an adequate training program for new technologists, and is there a continuing medical laboratory education program?