Class II Special Controls Guidance Document: Factor V Leiden DNA Mutation Detection Systems - Guidance for Industry and FDA Staff
Document issued on: March 16, 2004
For questions regarding this guidance, contact Yun-Fu Hu at 301-796-6170, firstname.lastname@example.org
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Table of Contents
Guidance for Industry and FDA Staff
Class II Special Controls Guidance Document:
|Identified risk||Recommended mitigation measures|
|Improper medical management||Sections 6, 7, and 8|
|Misdiagnosis, improper treatment and|
drug selection and dosing
|Sections 6, 7, and 8|
Genetic testing for the FVL mutation has been performed for a number of years, but has chiefly been confined to in-house developed testing. New technological developments and increased demand for genetic tests have led to the demand for marketing of devices capable of detecting the FVL mutation and associated disease-specific polymorphisms. This guidance document makes recommendations for manufacturers who are preparing submissions for tests to be marketed in interstate commerce, without specific regard to the technology used to detect the mutations or polymorphisms (see Section 7).
The following are areas that we believe should be addressed in the preparation of a submission for a device incorporating technology designed to detect the FVL mutation.
The intended use should specify what the test is intended to measure, why it is measured, and should specify populations to which the test is targeted, where appropriate. In this case, the intended use should clearly specify the FVL mutation. References to professional society recommendations are acceptable.
Some tests may have multiple intended uses. FDA recommends a separate application for each intended use that requires unique and separate supporting studies. You should consult the appropriate review divisions in FDA for advice on submitting tests with multiple intended uses.
Consideration of preanalytical factors is essential for high-quality genetic tests. Manufacturers intending to provide reagents for extraction and preparation of DNA for testing should validate each step in the preanalytical process for its effects on reproducibility, robustness, and stability of product. Manufacturers who do not intend to provide these reagents in their kits should provide specifications for assessing the quality of the assay input DNA and other required reagents, so that the user can select appropriate reagents. Justification for specifications given should also be provided in the submission.
Other preanalytical factors to be considered are the source of DNA (blood, PBMC, buccal swab, etc.), validation of its suitability for extraction at an acceptable quality level (e.g., heparin-preserved vs. EDTA-preserved blood, stored vs. fresh sample), and validation of acceptable storage conditions and stability for the sample and the extracted product.
You should perform analytical studies that demonstrate that the device detects the mutations it claims to detect, and does not detect mutations when none are present. The sample type used to perform analytical studies should include patient samples to show that the device will perform as claimed when patients are tested and that the entire process is controlled. FDA recognizes that in some situations, the FVL mutation may not be present in the sampled population at high enough rate to ensure significance of test results. In these cases, you may use archived and/or retrospective samples in addition to prospective patient samples in order to expand the number and proportion of mutations available. In certain cases, you may use “artificial” samples in which DNA containing the mutation has been added at a level simulating that which would be found in a natural sample. You should choose the sample number to achieve a stated statistical confidence that the test performs as expected. For all samples, you should give starting material, extraction method, concentration, and purity. Both heterozygous and homozygous mutant samples are acceptable; homozygous samples may decrease the overall number of samples needed for testing if chromosome count is used as a metric.
The known FVL mutation is G1691A. Additional rare mutations in Factor V are known at A1692C, G1689A and A1696G. We recommend that you assess the possibility of the rare Factor V mutations to give false FVL results and report this as a limitation, if applicable.
FDA recommends that you describe in detail the methodology that will be used to detect mutations. If sample extraction matrices are provided, you should describe this methodology as well. Illustrations or photographs of non-standard equipment or methods can be helpful in understanding novel methodologies.
Controls and Calibrators
FVL molecular mutation testing should include both positive and negative controls. For different technologies, these controls may differ, but the user should be able to determine if critical reactions have proceeded properly and without contamination or cross-hybridization. Controls should approximate sample DNA concentration in order to adequately exercise the system.
We recommend that you implement the calibration of systems where it can aid in generation and interpretation of results. Depending on the technology selected, calibration may or may not be critical for proper use of the test.
You should validate the analytical sensitivity of your test, i.e., what the minimum amount of input nucleic acid is, and approximate the amount of sample required to generate this minimum input. If the assay has an upper, or saturation, limit, you should also validate and state this in labeling.
You should assess interference in FVL mutation detection from any input into the system. Some common known interferences may occur from extraction technique, original sample matrix, excess or inadequate Mg2+ concentration, etc. You should investigate other potential interferences and describe them in labeling, if necessary.
FDA recommends manufacturers fully examine the reproducibility and robustness of their device. NCCLS EP-5A describes an acceptable reproducibility testing plan. FDA also recommends that 3 or more sites with varying molecular experience be used to test reproducibility of panels of mutations and wild-type sequences, using the procedure that you will describe in final device labeling. If extraction reagents are not included in the test kit, each site should use and validate their own extraction procedures and demonstrate that the resulting input material (e.g., DNA) meets manufacturer-supplied specifications. Preferably, you should include multiple operators, multiple product lots, and instruments, if these are part of the device, to adequately test the expected performance of the system. If training will be necessary for users to perform the test once it is marketed, you should provide information on operator training. If such training is not expected to be necessary for users, you should not provide additional training (other than the package insert) at the testing sites.
Instrumentation that is specific and dedicated to the device should be analyzed according to the “Guidance for FDA Reviewers and Industry: Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices” document. You should include a copy of instrumentation manuals for specified instrumentation. If general purpose instrumentation is to be used, you should provide specifications for the required instruments to be used in labeling.
Prospective clinical testing to determine clinical validity and utility will generally not be necessary for the FVL mutation and polymorphisms. You should use clinical samples, however, as often as practicable in analytical testing, in order to demonstrate that correct results can be obtained from clinical material. Because the mutation is well-known, and high quality clinical literature and professional society recommendations supporting clinical utility of mutations and polymorphisms tested exists, these may be substituted for prospective clinical studies, providing that there is some bridge in this literature to an accepted reference method. You should succinctly summarize these materials and provide references to the agency in the submission. If clinical testing is performed, you should select patients prospectively in order to maximize the number of mutations detected, e.g., from patients referred for thromboses, etc. You should describe inclusion and exclusion criteria, and these should conform to the intended use population.
FDA recommends that examples of test reports as would be supplied to the ordering personnel be provided to the agency. Reports should be consistent with current recommendations of genetics professional societies and should contain adequate interpretation guidelines for the use of the ordering physician/counselor. FDA recommends that FVL mutations be reported as “present” or “absent” rather than “normal,” “wild-type,” or “mutant.”
The abundance of technologies that could be used to detect the FVL mutation raises the possibility that assays may vary significantly in terms of methodology, instrumentation, and sample source, and make direct comparability difficult to assess. In order to facilitate product review, you should consider comparing the new assay to a reference method or “gold standard,” for example,, bidirectional DNA sequencing, to define performance. We recommend that you validate and document the accuracy of the reference method used (e.g., percent correct sequence calls or “phred score”). You may then claim the sensitivity and specificity of your device. We recommend that you describe the inclusion and exclusion criteria for all samples tested. Actual comparison of the new device performance to that of a previously cleared device is optional. However, if you wish to include this comparison in the new product labeling, you should also include a description of the study in the submission. You may perform discrepancy resolution for comparison studies, but you should use original unresolved results for all performance calculations unless the resolver is applied to all samples, to avoid bias.
The premarket notification should include labeling in sufficient detail to satisfy the requirements of 21 CFR 807.87(e). The following suggestions are aimed at assisting you in preparing proposed labeling that satisfies the requirements of 21 CFR 807.87(e) and final labeling.5
Directions for use
You should provide clear and concise instructions that delineate the technological features of the specific device and how the device is to be used on patients. Instructions should encourage local/institutional training programs designed to familiarize users with the features of the device and how to use it in a safe and effective manner. Devices incorporating nucleic acid amplification should provide work-flow recommendations in labeling.
We recommend that you provide a description of quality control recommendations in the labeling.
Precautions for interpretations
We recommend that you address the limitations of your assay with statements in the labeling, such as:
- The presence of rare mutations in the Factor V gene may result in false positive reports for FVL.
- This test should not be used alone to diagnose thrombophilias. It is recommended that activated protein C (APC) testing be done alongside the genetic tests.
We recommend that you assess the stability of your reagents and recommended samples and DNA inputs.
You should provide device performance in comparison to an accepted reference method or gold standard in the form of 2 X 2 tables, sensitivity and specificity percentages, or other illustrative examples. You should calculate sensitivity and specificity from all tested samples. Failed assays (e.g., inability to sequence sample) should be considered disagreements for the purposes of reporting performance characteristics.
2Refer to Indications for Use Form (PDF File Size: 1.03MB) for the recommended format.
3If FDA makes a substantial equivalence determination based on acceptance criteria, the subject device should be tested and shown to meet these acceptance criteria before being introduced into interstate commerce. If the finished device does not meet the acceptance criteria and, thus, differs from the device described in the cleared 510(k), FDA recommends that submitters apply the same criteria used to assess modifications to legally marketed devices (21 CFR 807.81(a)(3)) to determine whether the finished device requires clearance of a new 510(k).
4See Required Elements for a Declaration of Conformity to a Recognized Standard (Screening Checklist for All Premarket Notification [510(K)] Submissions).
5Although final labeling is not required for 510(k) clearance, final labeling must comply with the requirements of 21 CFR 809.10 before an in vitro diagnostic device is introduced into interstate commerce.