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Technical Working Group on DNA Analysis Methods

Guidance for industry in the manufacture and clinical evaluation of in vitro tests to detect in vitro nucleic acid sequences of HIV-1-Draft Guidance Guidance for industry and/or FDA reviewers staff—Premarket approval applications for assays pertaining to Hepatitis C virus (HCV) that are indicated for diagnosis or monitoring of HCV infection or associated disease-Draft Guidance

Recommendations for in-house development and operation of molecular diagnostic tests

Guidelines for a Quality Assurance Program for DNA Analysis

Wayne, PA; www.nccls.org

ABMG/ABGC/ACMG; II Administrative office: 965G Rockville Pike, Bethesda, MD, 2G814-3998

Lenexa, KS 66285-58G4

www.nhgri.nih.gov/

Policyandpublicaffairs/Elsi/tfgentest

www.fda.gov/cber/gdlns/nashiv.pdf;

www.fda.gov/cdrh/ode/1353pdf

performance of different in vitro nucleic acid methodologies, including standard PCR, either uniplex or multiplex. Optimization will not be discussed in this chapter and the reader is recommended to review these citations for further information. When optimization is carried out for each step separately, it is important to realize that when all steps are placed together, reoptimization is required in most of the cases. After optimization of the assay, it is necessary to evaluate and document preanaytical variables that might have an impact on the performance characteristics of the assay. Some of the most common preanalytical variables are specimen type, transport, storage, and handling requirements, as well as interfering substances such us lipids, hemoglobin, billirubin, and so forth.

The use of internal controls to detect the presence of inhibitors or nucleic acid degradation is extremely important. One of the major advantages of using internal controls is that they can mimic the nucleic acid present in a patient specimen and be subject to the entire testing protocol from nucleic acid extraction to detection. A common approach to development of internal controls is the creation of synthetic materials such as in vitro synthesized plasmids that would contain a modified target sequence, which could be spiked into the specimen before

Table 3

Checklist for Verification of Laboratory Developed Tests

Name of test Intended use

Indications for use Method category Testing procedure

Test results Analytical validity

Quality control and quality assurance Assay limitations Clinical data

Clinical validity

Reporting of tests results (clinical interpretation) Clinical utility

State the name of the test, including the trade or proprietary information if exists; be sure that the name identifies the particular disease or condition or gene locus to be analyzed What the test measures and for what purpose; identify the particular parameter(s) that the test measures, point mutation/deletion/insertion, and indicate the use of the test (e.g., diagnosis, prenatal, carrier status, screening, presymptomatic, etc) Provide clinical condition(s) CF. Use reference standard definitions as found in OMIM. Identify test method

Information with regard to specimens, specimen handling, analyte extraction/isolation, analyte storage, description of the test procedure, data reports, expected results, technical interpretation of results Representative examples of results

Analytical sensitivity, analytical specificity, precision, etc.

Delineate the QC and QA program

Briefly delineate and discuss potential limitations

Primary objective of the study, clinical condition evaluated, patient population demographics, sample size estimate Clinical sensitivity, clinical specificity Clinical interpretation

Potential clinical benefit to patient and physician testing. The most common internal controls are synthetic segments of nucleic acid that use the same primer sequence as the target molecule, with an internal portion of the sequence that is unique to the internal control, as to allow separate detection from the target and quantitative standard with the use of specific probes for each species. In addition, this internal control can be used as an internal calibrator. An internal calibrator contains a predetermined amount of the modified target that can be added directly to a clinical specimen and undergo the same manipulation as the target of the patient specimen. One caveat for internal controls is that they must have the same or a very similar efficiency of amplification. Moreover, when the internal control fails to be detected, it is not possible to determine the specific reason (i.e., inhibitors or other amplification problems). In addition, it is important to use a low amount of the internal control to avoid competition with the target of the patient specimen. When detecting and/or quantifying RNA from tissue or cells, amplification of a housekeeping gene can be used as an internal control. Most of the housekeeping genes are highly abundant and might not be the most appropriate internal control. The use of RNA from a gene with approximate abundance as the target sequence is preferred.

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