9.3.1 Critical Elements
Reporting of test results derived from molecular analysis has been described in NCCLS and other documents and is applicable to DNA sequence analysis.42,45,46 Salient features include the name of the individual, date of birth, specimen collection date, and other data. In addition to these, critical to the reporting of results derived from sequence analysis, the following should also be included:
• The gene and/or chromosomal region interrogated should be clearly identified (CFTR gene, etc.).
• That part of the gene and/or chromosomal region interrogated should be specified (exons, splice sites, etc.).
• The sequence and/or variation determined and position assignments should be specified and assigned according to accepted nomenclature. Both the nucleotide and the codon where a sequence change occurs should be indicated and frame shifts should be noted, including the codon position that results in a stop.
• When appropriate, the corresponding change in amino acid sequence should be specified according to accepted nomenclature.
• For human sequence determinations, results should indicate findings relevant to each allele.
• If available, other supportive data should be indicated that had been used in deriving the interpretation (i.e., family history, other clinical findings, homology to related sequences).
• The clinical relevance of the findings (see Section 9.2.2).
• Analytic limitations (see above).
• Interpretive limitations of the analysis (i.e., a negative result does not rule out contributory mutations present elsewhere in the genome).
• All databases used in the analysis should be referenced in the report. If applicable, a website reference for identifying gene-specific databases should be included.
9.3.2 Confidentiality
All patient results should remain confidential and adhere to regulatory requirements. Generally, results should only be made available to the referring healthcare professional. In some cases, results may be communicated directly to the patient. In these situations, specific policies should be established to help ensure that the patient understands the results, the limitations of the test, and recommendations for follow-up.
9.3.3 Preservation of Records
The laboratory should retain records for at least ten years and/or at least as long as mandated by regulatory authority. In some cases, such as tests performed on newborns, it is advisable to maintain records until the age of maturity.
References
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8 Roux KH, Hecker KH. One-step optimization using touchdown and stepdown PCR. Methods Mol Biol. 1997;67:39-45.
9 Roux KH. Optimization and troubleshooting in PCR. PCR Methods Appl. 1995;4(5):S185-194.
10 Burgos JS, Ramirez C, Tenorio R, Sastre I, Bullido MJ. Influence of reagents formulation on real-time PCR parameters. Mol Cell Probes.
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11 Kolmodin LA, Birch DE. Polymerase chain reaction. Basic principles and routine practice. Methods Mol Biol. 2002;192:3-18.
12 Lawrence JG, Hartl DL, Ochman H. Sequencing products of polymerase chain reaction. Methods Enzymol. 1993;218:26-35.
13 Stirling D. Quality control in PCR. Methods Mol Biol. 2003;226:21-24.
14 McPherson MJ, Møller SG, Beynon R, Howe C. PCR Basics: From Background to Bench. 1st ed. Springer Verlag; 2000.
15 Innis MA, Gelfand DH, Sninsky JJ. PCR Application: Protocols for Functional Genomics. Academic Press; 1999.
16 Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols: A Guide to Methods and Applications. Academic Press; 1990.
17 Innis MA, Gelfand DH, Sninsky JJ. PCR Strategies. Academic Press; 1995.
18 Niederhauser C, Hofelein C, Wegmuller B, et al. Reliability of PCR decontamination systems. PCR Methods Appl. 1994;4:117-123.
19 Burkardt HJ. Standardization and quality control of PCR analysis. Clin Chem Lab Med. 2000;38:87-91.
20 Kwok S, Higuchi R. Avoiding false positives with PCR. Nature. 1989;339:237-238.
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22 Persing DH. Polymerase chain reactions: trenches to benches. J Clin Microbiol. 1991;29:1281-1285.
23 Trower MK, Burt D, Purvis IJ, Dykes CW, Christodoulou C. Fluorescent dye-primer cycle sequencing using unpurified PCR products as templates; development of a protocol amenable to high-throughput DNA sequencing. Nucleic Acids Res. 1995;23(12):2348-2349.
24 Heery DM, Gannon F, Powell R. A simple method for subcloning DNA fragments from gel slices. Trends Genet. 1990;6(6):173.
25 Zhen L, Swank RT. A simple and high yield method for recovering DNA from agarose gels. Biotechniques. 1993;14(6):894-898.
26 Werle E, Schneider C, Renner M, Volker M, Fiehn W. Convenient single-step, one tube purification of PCR products for direct sequencing.
Nucleic Acids Res. 1994;22(20):4354-4355.
27 Hanke M, Wink M. Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs.
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28 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
29 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977;74:5463-5467.
30 Sterky F, Lundeberg J. Sequence analysis of genes and genomes. Journal of Biotechnology. 2000;76:1-31.
31 Franca LTC, Carrilho E, Kist TBL. A review of DNA sequencing techniques. Quarterly Reviews of Biophysics. 2002;35:169-200.
32 Tabor S, Richardson CC. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc Natl Acad Sci USA. 1995;92:6339-6343.
33 Parker LT, Zakeri H, Deng Q, Spurgeon S, Kwok PY, Nickerson DA. AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns. Biotechniques. 1996;21;694-699.
34 Vander Horn PB, Davis MC, Cunniff JJ, et al. Thermo sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing. Biotechniques. 1997;22:758-762, 764-765.
35 Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Research. 1998;8:175-185.
36 Naeve CW, Buck GA, Niece RL, Pon RT, Robertson M, Smith AJ. Accuracy of automated DNA sequencing: a multi-laboratory comparison of sequencing results. Biotechniques. 1995;19:448-453.
37 Kijak GH, Rubio AE, Pampuro SE, et al. Discrepant results in the interpretation of HIV-1 drug-resistance genotypic data among widely used algorithms. HIV Medicine. 2003;4(1):72-78.
38 Ad Hoc Committee on Mutation Nomenclature. Update on nomenclature for human gene mutations. Hum Mutat. 1996;8:197-202.
39 Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat.
1998;11:1-3.
40 den Dunnen JT, Antonarakis SE. Nomenclature for the description of human sequence variations. Hum Genet. 2001;109:121-124.
41 den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat. 2000;15:7-12.
42 NCCLS. Molecular Diagnostic Methods for Genetic Diseases; Approved Guideline. NCCLS document MM1-A (ISBN 1-56238-395-7).
NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2000.
43 Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981; 290:457-463.
44 ACMG Laboratory Practices Committee Working Group; ACMG recommendations for standards for interpretation of sequence variations.
Genet Med. 2000;2(5):302-303. (Available at: http://www.acmg.net/resources/policies/pol-027.pdf.)
45 American College of Medical Genetics; Standards and Guidelines for Clinical Genetics Laboratories; 2003. (Available at:
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Summary of Consensus/Delegate Comments and Committee Responses
MM9-P: Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Proposed Guideline Section 5.3, Specimen Quality Assurance
1. Quality Assurance seems incredibly brief and does not address as a guideline how to set up QA related to sequence diagnostics.
• The subcommittee has reviewed Section 5.3 and has found that specimen quality assurance is adequately covered. Quality assurance is addressed throughout the document and in significant detail in Section 8.2, Controls and Reference Standards, and in Section 9.1.1, Quality Assurance and Quality Control.
Section 8.2, Controls and Reference Standards
2. Positive and negative controls are not needed for every sequencing run, since the sequence itself is an internal control.
• The subcommittee agrees with the commenter and it is not stated in the document that positive and negative controls are needed for every sequencing run. However, it has been reported that the FDA has required a company to specify running a control with every run for its FDA-cleared genotyping product.
Section 8.6.1, Sequence Quality Assessment
3. Sequencing of both strands may not be needed for all applications, but can be helpful when looking for heterozygous single nucleotide substitutions.
• The second paragraph of Section 8.6.1 has been revised with the addition of the commenter’s statement as the second sentence.
Section 9.2, Clinical Interpretation
4. The issue of software generated clinical interpretations which interface with wet sequence software are not addressed.
• Recommendations to assessing the validity of such software is beyond the scope of this guideline;
however, for informational purposes the following paragraph has been added in Section 9.2:
Software packages are available that provide clinical treatment recommendations based upon the sequence determined. For instance, several packages are available that provide recommendations for HIV antiviral therapy. While recommendations to assess the validity of such software are beyond the scope of this guideline, discordance in the interpretation of sequence results has been reported among a number of these software packages.37 Therefore, users (laboratory personnel and clinicians) must take steps to understand the usefulness, reliability, and limitations of clinical interpretations generated by such software.
NCCLS consensus procedures include an appeals process that is described in detail in Section 8 of the Administrative Procedures. For further information, contact the Executive Offices or visit our website at www.nccls.org.
NOTES
The Quality System Approach
NCCLS subscribes to a quality system approach in the development of standards and guidelines, which facilitates project management; defines a document structure via a template; and provides a process to identify needed documents through a gap analysis. The approach is based on the model presented in the most current edition of NCCLS document HS1—A Quality Management System Model for Health Care. The quality system approach applies a core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any healthcare service’s path of workflow (i.e., operational aspects that define how a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The quality system essentials (QSEs) are:
Documents & Records Equipment Information Management Process Improvement Organization Purchasing & Inventory Occurrence Management Service & Satisfaction
Personnel Process Control Assessment Facilities & Safety
MM9-A addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other NCCLS documents listed in the grid, please refer to the Related NCCLS Publications section on the following page.
Documents & Records Organization Personnel Equipment Purchasing & Inventory Process Control Information Management Occurrence Management Assessment Process Improvement Service & Satisfaction Facilities & Safety
X MM6 MM5
Adapted from NCCLS document HS1—A Quality Management System Model for Health Care.
Path of Workflow
A path of workflow is the description of the necessary steps to deliver the particular product or service that the organization or entity provides. For example, NCCLS document GP26—Application of a Quality Management System Model for Laboratory Services defines a clinical laboratory path of workflow which consists of three sequential processes: preanalytic, analytic, and postanalytic. All clinical laboratories follow these processes to deliver the laboratory’s services, namely quality laboratory information.
MM9-A addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other NCCLS documents listed in the grid, please refer to the Related NCCLS Publications section on the following page.
Preanalytic Analytic Postanalytic
Patient Assessment Test Request Specimen Collection Specimen Transport Specimen Receipt Testing Review Laboratory Interpretation Results Report Post-test Specimen Management
MM6 x
MM1 MM5 MM6
x
MM1 MM6
x
MM1 MM5 MM6
x
MM1 MM5 MM6
x
MM1 MM5 MM6
x
MM6
x
MM1 MM5 MM6
x
Adapted from NCCLS document HS1—A Quality Management System Model for Health Care.
Related NCCLS Publications
*MM1-A Molecular Diagnostic Methods for Genetic Diseases; Approved Guideline (2000). This document provides guidance for the use of molecular biologic techniques for clinical detection of heritable mutations associated with genetic disease.
MM5-A Nucleic Acid Amplification Assays for Molecular Hematopathology; Approved Guideline (2003). This document addresses guidelines for a variety of amplification-based laboratory tests, including polymerase chain reaction (PCR), transcription-based amplification system (TAS), strand displacement amplification (SDA), ligase chain reaction (LCR), and other methods now widely used in diagnostic hematopathology. This guideline provides a basis for laboratory implementation and quality assurance in this important area of diagnostic molecular medicine.
MM6-A Quantitative Molecular Methods for Infectious Diseases; Approved Guideline (2003). This document provides guidance for the development and use of quantitative molecular methods, such as nucleic acid probes and nucleic acid amplification techniques of the target sequences specific to particular microorganisms. It also presents recommendations for quality assurance, proficiency testing, and interpretation of results.
* Proposed- and tentative-level documents are being advanced through the NCCLS consensus process; therefore, readers should refer to the most recent editions.
(as of 1 October 2004)
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CLMA
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BD
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Optimer Pharmaceuticals, Inc.
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(Rochester, NY) Ortho-McNeil Pharmaceutical (Raritan, NJ) Oxoid Inc.
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SYN X Pharma Inc.
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TheraDoc Theravance Inc.
Thrombodyne, Inc.
THYMED GmbH Transasia Engineers Trek Diagnostic Systems, Inc.
Vetoquinol S.A.
Vicuron Pharmaceuticals Inc.
Vysis, Inc.
Wyeth Research XDX, Inc.
YD Consultant
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