Draft Genetic Test Review

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Cystic Fibrosis
Clinical Utility

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CLINICAL UTILITY

Question 26: What is the natural history of the disorder?
Question 27: What is the impact of a positive (or negative) test on patient care?
Question 28: If applicable, are diagnostic tests available?
Question 29: Is there an effective remedy or acceptable action, or other measurable benefit?
Question 30: Is there general access to that remedy or action?
Question 31: Is the test being offered to a socially vulnerable population?
Question 32: What quality assurance measures are in place?
Question 33: What are the results of pilot trials?
Question 34: What health risks can be identified for follow-up testing and/or intervention?
Question 35: What are the financial costs associated with testing?
Question 36: What are the economic benefits associated with actions resulting from testing?
Question 37: What facilities/personnel are available or easily put in place?
Question 38: What educational materials have been developed and validated, and which of these are available?
Question 39: Are there informed consent requirements?
Question 40: What methods exist for long term monitoring?
Question 41: What guidelines have been developed for evaluating program performance?

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CLINICAL UTILITY

Question 32: What quality assurance measures are in place?

Summary A quality assurance plan assists laboratories in ensuring reproducible, high quality results in a timely manner that are clinical useful to patients and providers. The components of a generic molecular quality assurance program are well described and are available from national and state regulatory agencies and professional organizations. Specific professional guidelines for prenatal cystic fibrosis quality assurance do not yet exist, but ACMG is in the process of producing such a guideline. Quality assurance oversight is provided by the laboratory certification process administered by Federal or State agencies (CLIA and New York State) or by professional organizations (College of American Pathologists).

Definition
Quality assurance is the program that laboratories develop to ensure reproducible, high quality results in a timely fashion, which are clinically useful to patients and providers. A major goal is to minimize the human error that accounts for the majority of laboratory errors.

Standards and guidelines and checklists
Clinical molecular genetic testing laboratories must follow good laboratory practice guidelines and subscribe to external quality assessment programs. Guidelines, recommendations, and checklists are available from national and state regulatory agencies and professional organizations regarding quality control/quality assurance, inter-laboratory comparison/proficiency testing, and laboratory personnel requirements (Table 4-1). While multiple standards and guidelines exist, only three are enforceable and require laboratory inspection for certification. These include: Clinical Laboratory Improvement Amendments (CLIA), College of American Pathologists (CAP), and New York State. All other guidelines are efforts of genetics and other professional organizations to regulate the genetic testing industry, but are without enforcement.

Table 4-1. Guidelines, Recommendations, and Checklists that Address Quality Assurance

Guidelines, Recommendations and Checklists	Source / Reference
Clinical Laboratory Improvement Amendments of 1988	Federal Register 1992;57:7002-3
Genetic Testing Under the Clinical Laboratory Improvement Amendments	Federal Register 2000;65: 25928-24934
New York State Department of Health Laboratory Standards (9/00)	http://www.wadsworth.org/labcert/clep/Survey/standardsmenu.htm  (last accessed 3/2007)
Molecular Diagnostic Methods for Genetic Diseases: Approved Guidelines	National Committee for Clinical Laboratory Standards MM1-A Vol 20 #7
CAP Checklist	College of American Pathologists Www.cap.org (last accessed 3/2007)
Standards and Guidelines for Clinical Genetics Testing	American College of Medical Genetics http://genetics.faseb.org/cgi-bin/acmgm/rd.pl?pgm=2 (last accessed 3/2007)
European Concerted Action on Cystic Fibrosis	(BMH-4-CT96-0462)

To address concerns raised through the ACMG/CAP proficiency testing program for cystic fibrosis (i.e. variability of from one to 70 mutations in the cystic fibrosis test panel), laboratory standards and guidelines for cystic fibrosis carrier testing have now been developed (Grody et al, 2001). Cystic fibrosis mutation analysis is a complex laboratory procedure which benefits from a uniform testing policy and test interpretation. Because of this, testing should to be restricted to laboratories with the necessary expertise, experience, and resources. Most components of generic quality assurance are well-established and consistent among the published guidelines. However, some are controversial. For example, the qualifications for clinical laboratory directors have not been universally agreed upon by professional and licensing organizations. Overall, the components of quality assurance can be sub-divided into three stages: pre-analytical, analytical, and post-analytical.

Pre-analytic
The pre-analytic components of a quality assurance program include those activities that occur prior to the sample being tested. A general overview of these components is provided below.

• Informed consent: This is one of the most controversial areas of genetic testing. Issues surrounding informed consent vary depending upon the guideline and when it was written, and. The NCCLS describes informed consent as a voluntary process that is non-coercive and easily understood. The information should include risks and benefits of testing and specific information about test performance. Consent requirements are based on applicable state and federal laws. New York requires that informed consent state the purpose of testing, include genetic counseling, the meaning of a positive test result in the context of disease, the positive predictive value of the test, the test disclosure process, and the stipulation that no additional testing be allowed on the specimen without consent. Neither CAP nor ACMG specifically addresses the informed consent requirement in either the checklist or technical standards and guidelines. More recently, the ACMG Laboratory Standards and Guidelines for Cystic Fibrosis Carrier Screening (Grody et al., 2001) included a recommendation that informed consent be obtained. The Task Force on Genetic Testing requires written informed consent, and CLIAC states that an authorized person obtain the informed consent. While there is now general agreement that informed consent should be obtained, the controversial issue has been over who is responsible. According to the Genetic Testing Workgroup of CLIAC, informed consent is now required for all genetic tests. It is the responsibility of the person ordering the test to obtain informed consent from the patient. The level of informed consent depends on whether the test is used for predictive or diagnostic purposes. The laboratory should be available to assist in determining the appropriate level of consent. The requisition must include a space for the person ordering the test to signify that the appropriate level of consent was obtained. The inclusion of a re-use acceptance (opt-out) check-off box should also be on the consent form.

• Confidentiality: All genetic testing is confidential. HIPAA Final Regulation, published December 28, 2000, addresses confidentiality of personal health information (Question 44).

• Specimen types: Specimen types include blood or buccal swabs for carrier testing; direct or cultured amniocytes or chorionic villi samples for prenatal diagnosis. Each laboratory determines the exact sample type and amount required for its testing method, and furnishes that information to referring centers. Prenatal samples are usually tested during the first trimester. For fetal testing, maternal cell contamination studies must be performed on fetal and maternal specimens using highly polymorphic STR markers to detect the presence of contamination.
• Standard information for the requisition slip: This include patient-specific information such as name, date of birth, sex, ethnicity and week of gestation, and sample information such as sample type, date of collection, and indication for testing. Also included is reporting and billing information, such as referring physician/health professional and source of payment.
• Criteria for sample rejection: Each laboratory develops its own written criteria for sample rejection.
• Accessioning. Each specimen is assigned a unique identifier. Specimens from the same patient will have individual identifiers.
• Specimen transport and storage: Each laboratory determines its own criteria based upon experience and furnishes that information to clients.

Analytic

• Test validation and characterization: All guidelines agree that the laboratory is responsible for documenting the validity of its tests. However, the components of test validation have only been addressed by the New York State guidelines and guidelines proposed by the US Food and Drug Administration. Literature review and analytical/clinical studies provide necessary information, including description of the mutations tested, the performance properties of the test, the clinical utility, and limitations. One controversial area in test validation surrounds the number of probands (positive controls) that must be tested in order to validate a test. The Genetic Testing Workgroup for CLIAC recommended that the appropriate number of positive probands required for test validation should be subject to professional guidelines rather than regulations and be disease-specific. These recommendations have been endorsed by ACMG.

New York State requirements for test validation The following information is required by New York State for each new test:

• A description of the disease, the gene, the test, the principle of the test, and indications for testing.
• Assay description, including: all information relevant to the test, DNA extraction protocol, dilution, quantitation; reagent recipes; vendor/catalog information; reagent quality control (in/out dates, storage requirements); required equipment/vendors; step-by-step protocol; primer list with sequences, source of primers; description of positive controls, source, how verified; description of negative controls; technical limitations and troubleshooting guide; equipment, and procedures for quality control.
• Description of expected results from controls and what an indeterminate result looks like.
• Sample requisition form, including physician name, address, phone number, fax, date specimen collected, patient name, and accession number.
• Sample reports for negative, positive, indeterminate or rejected results, including interpretive statement explaining test results for each example, test limitations and relevant disclaimers, specimen information, and signature of laboratory director.
• Consent form.
• Explanation of how validation studies were performed, results, and interpretation. High quality original results showing homozygous normal, carrier, homozygous mutant.
• Reproducibility, sensitivity, specificity, positive predictive value.

Proposed FDA requirements for test validation The following information has been proposed as the components of a validation study by the FDA that would be completed prior to offering testing. Laboratories would submit an application for each new test based on FDA guidelines.

• Intended use of test
• Indications of test
• Method category
• Methodology, specific
• Examples of test results
• Analytical validity (control specimens, number tested, types of specimens, results, sensitivity, specificity, accuracy, reproducibility, confirmation, proficiency testing, statistical analysis)
• Quality control procedures (external controls, checks of results, repeat specimens, frequency of QC assessments)
• Clinical validity (literature citations or study results and summary)
• Clinical interpretation (report templates, information for risk analysis)
• Limitations (technical, biological)
• Clinical utility (interventions available for positive test result; level of efficacy)
• External proficiency testing: The goal of proficiency testing, which is currently the main indicator of quality assurance, is to allow laboratories to identify individual areas of weakness and take steps to improve. The College of American Pathologists requires participation in proficiency testing as part of the laboratory accreditation process. The ACMG/CAP proficiency testing program has provided participating laboratories in the MGL (molecular genetic laboratory) survey with two or three cystic fibrosis challenges once or twice yearly since 1995. Interpretive questions are also included in this survey. CAP estimates that approximately 85% of molecular genetic testing laboratories participate in this program. Proficiency test performance is anonymously reviewed and analyzed. The ACMG/CAP Committee develops a report for each participating laboratory. The performance of clinical molecular laboratories on these proficiency tests will be graded beginning in 2002, and consumer groups will have access to aggregate information. The proficiency testing program will also provide more challenges for laboratories. Laboratories that test patients from New York State must obtain a license from the New York State Department of Health. While the New York State program does not provide proficiency testing for clinical molecular genetic laboratories, it does require these laboratories to participate in an established proficiency testing program, internal or external, at least twice each year. New York State certified laboratories must undergo on-site inspections every other year and submit validation materials for each assay performed.

External proficiency testing programs outside the United States

The European Concerted Action on Cystic Fibrosis has surveyed over 150 clinical molecular genetic laboratories in Europe for cystic fibrosis test performance and has made the following recommendations for quality improvement:

• Develop quality systems leading to their accreditation by national or European agencies
• Participate regularly in external quality assessment schemes
• Test for at least 80% of the mutations found in the laboratory's region
• Use validated testing methods appropriate to detect at least 80% of the mutations based on technical and economic considerations
• Monitor the indications for cystic fibrosis testing
• Provide relevant information to the clinical staff for risk calculation and counseling
• Network with other laboratories in their region, country or at the European level

The European Molecular Genetics Quality Network (EMQN) is focused on improving the standards of European clinical molecular genetics laboratories by providing external quality assessment programs and best practice guidelines. A basic difference between the ACMG/CAP proficiency testing program and that of EMQN is survey administration. The ACMG/CAP program coordinates all disease-specific challenges from a single source, while EMQN identifies a management group to develop disorder-specific proficiency testing and a national partner to disseminate the results to participating laboratories. This program has not yet begun the cystic fibrosis module, which is intended to allow continuation of the trial developed by the European Union funded Concerted Action on Cystic Fibrosis (ECACF).

• Control of PCR contamination: A major concern for any clinical molecular laboratory is false-positive results due
  to contamination by PCR products. This concern can be addressed by following the recommended guidelines for laboratory design, laboratory practice, selection and preparation of controls. This quality assurance standard is generic but applies to cystic fibrosis testing.
• Laboratory design: physically separated into three areas: reagent preparation, specimen preparation, and PCR and product detection
• Laboratory practice: the use of positive displacement pipettors, cotton plug tips, gloves, lab coats, and careful preparation of reagents
• Selection and preparation of controls: Include positive controls for each allele targeted in the test (e.g., a 25 mutation panel should have 25 positive controls). Positive controls should amplify weakly to minimize large quantities of PCR product. No-DNA controls should be included in every run. Assays based on the presence or absence of PCR product must include a known positive control as an amplification control. Include a sizing ladder if the assay is based on fragment size. Include appropriate controls in mobility shift assays. Confirm unexpected results.

Post-analytic
Some issues of post-analytical testing, such as reporting, mutation nomenclature, and retention of records, are held in general agreement by various professional societies and regulatory groups.

• Laboratory reports: Laboratory reports are to the physician or healthcare professional, not the patient. The report should echo any information collected on the requisition slip that is used for identification or as part of the interpretation. In addition, test-specific information should be included such as laboratory identifiers, testing method, test result, interpretation, recommendations (e.g., genetic counseling) and the signature of the laboratory director. The ACMG recommendations for cystic fibrosis testing (Grody et al., 2001) have included model reports for negative results, including residual risk based on ethnicity, positive report templates, and complex interpretations.
• Nomenclature for mutations: The nomenclature developed by the Ad Hoc Committee on Mutation Nomenclature and Antonarakis et al. is recommended. The nomenclature established for cystic fibrosis mutations follows these guidelines and is found in the cystic fibrosis mutation database at http://www.genet.sickkids.on.ca/cfr.
• Retention of records and specimens: The CLIAC Workgroup recommended that a minimum of 10 years was appropriate for records retention of both positive and negative results. However, guidelines for specimen retention time have not been agreed to. There is some controversy over specimen retention, particularly surrounding the opt-out requirement.
• Genetic counseling: All current standards and guidelines address the responsibility of the laboratory to recommend genetic counseling, when appropriate. However, none require the laboratory to actually provide genetic counseling to patients. The laboratory can help guide healthcare professionals to genetic counseling resources.

Is there ongoing review of quality assurance?
The CAP ACMG Molecular Genetics Resource Committee has the main responsibility for the review of the external proficiency testing results. In addition, the ACMG Quality Assurance Subcommittee of the Laboratory Practice Committee reviews these same proficiency testing results at semi-annual meetings. This Committee is composed of clinical laboratory directors (including molecular, biochemical, and cytogenetic laboratories) and representatives from the ACMG/CAP proficiency testing program. Threshold indicators are set for addressing laboratory problems related to specific disease testing, including cystic fibrosis. An additional charge for this committee is to develop of disease-specific technical standards and guidelines. A workgroup composed of laboratory directors with extensive experience in cystic fibrosis testing is in the process of developing these guidelines, anticipated to be released in 2002. Additional goals of the Committee include the development of technology-specific guidelines targeting the methodology used most commonly in laboratories performing cystic fibrosis testing.

Concerns about the quality of genetic testing: The McGovern Report
Although all laboratories performing genetic testing must comply with general regulations under the Clinical Laboratory Improvement Amendments (CLIA), the Task Force on Genetic Testing concluded that the current CLIA requirements are insufficient to ensure quality of molecular genetic testing. The McGovern Report (McGovern et al., 1999) concluded that a number of laboratories had suboptimal quality assurance practices. This survey included 245 molecular diagnostic laboratories across all settings performing testing for over 90 genetic diseases. The main outcome measure was a QA score based upon ACMG Laboratory Practice standards. The report concluded that many molecular genetic laboratories are not performing well. Unfortunately, the study did not focus on clinical laboratories that special in testing for genetic disease. Nevertheless, the report did provide some useful information. It found that 33 percent of the lower scoring laboratories were headed by non-board-certified directors and that 45 percent of lower-scoring laboratories were in a research setting. Indicators of good performance included a larger menu of tests, a larger number of tests performed annually, and a clinical laboratory certified by CLIA '88. While concerns have been raised about the quality of molecular genetic testing, few data exist to substantiate these fears. In a recent report (Hofgartner et al., 1999) which surveyed 42 genetic testing laboratories, significant problems during genetic testing were found to occur infrequently (<0.5% in most laboratories), and problems resulting in patient harm were found to be rare (0.0008%).

CDC recommendations for quality assurance programs
The following are genetic testing quality assurance recommendations developed for the Centers for Disease Control and Prevention. They include:

• conduct pilot research to develop positive controls and test samples for pilot performance evaluation programs >
• develop pilot evaluation programs to supplement what already exists, particularly for diseases and/or methodologies not covered by existing programs
• establish laboratory-oriented, disease-specific consortia to provide quality assurance support as a forum for information networking, and providing methods validation through results comparison;
• To establish and link laboratory oriented and disease-specific databases with other appropriate internet resources
• To improve training and continuing education for clinicians, laboratory scientists, and technicians

References

Bradley L, Johnson D, Chaparro C, Robertson N, Ferrie R. 1997. A multiplex ARMS test for 10 cystic fibrosis (CF) mutations: Evaluation in a prenatal CF screening program. Genet Test 2:337-341.

Dequeker E, Cassiman J-J. 2000. Genetic testing and quality control in diagnostic laboratories. Nat Genet 25:259-260.

Dequeker E, Cuppens H, Dodge J, Estivill X, Goossens M, Pignatti PR, Scheffer H, Schwartz M, Schwarz M, Tummler B, Cassiman JJ. 2000. Recommendations for the quality improvement of genetic testing in cystic fibrosis. European Concerted Action on Cystic Fibrosis. Eur J Hum Genet 8 (suppl. 2):S2-24.

Elles R. 1997. An overview of clinical molecular genetics. Mol Biotechnol 8:95-104.

Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ. 2001. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 3:149-154.

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Hofgartner W, Tait J. 1999. Frequency of problems during clinical molecular-genetic testing. Am J Clin Pathol 112:14-21.

Hofgartner W, Tait J. 1999. Characteristics of clinical molecular-genetic testing laboratories in the United States. Clin Chem 45:1288-1290.

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McGovern M, Benach M, Wallenstein S, Desnick R, Keenlyside R. 1999. Quality assurance in molecular genetic testing laboratories. JAMA 281:835-840.

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