Vol. 13: Spring, 1996
Molecular Cytogenetics (FISH)
Impact on the Laboratory of Regulatory Issues Regarding FISH
In the previous article, Lynda Fox has described regulatory issues regarding FDA approval (or lack thereof) of FISH technology. I would like to now describe how the American College of Medical Genetics (ACMG) Laboratory Practices Committee has addressed the issue in an effort provide guidance for those of us performing these tests treat them in "real life".
First, all laboratory practice is regulated by the Health Care Finance Adminstration (HCFA) through the Clinical Laboratories Improvement Act of 1988 (CLIA'88). These regulations call for all laboratory procedures to be properly validated, but they do not specify the details of how this should be done.
In order to help clarify what is acceptable as validation of FISH probes and techniques, the ACMG, through its Laboratory Practices Committee, has recently (February 1996) adopted guidelines describing the appropriate steps. These new standards cover both interphase and metaphase FISH analysis at three levels: FDA approved; "home brew"; and "Research Only" reagents.
FDA approval for "in vitro diagnostic use" is granted for probes for each specific use (i.e., diagnosis). As an example, approval might be granted for the the use of a chromosome 18 alpha satellite probe for the detection of aneuploidy in uncultured amniocytes, but the probe would not be approved for detection of trisomy 18 in lymphocyte nuclei from a newborn, even though the target sequence (chromosome 18 specific alpha satellite DNA) and the milieu harboring the sequence (interphase nuclei) are the same.
This use-by-use approval creates a tremendous financial burden to the manufacturer in the form of expensive clinical trials if "in vitro diagnostic use" is to be approved for their products. In practice, manufacturers have only pursued a few, large-market probes with clinical trials. The majority of their products remain "for research use only" (RUO).
This means that most clinical testing is performed with RUO or "home brew" probes, and places the burden of assay development and validation squarely on the shoulders of the laboratory. Each new lot of probe must be validated in each laboratory according to the CLIA'88 regulations. Validation consists of establishing the analytical sensitivity and specificity.
The ACMG guidelines provide a procedural framework for probe validation. However, a major dilemma facing laboratories is that probes are very expensive reagents and are labile, so that, unless a laboratory performs a large number of tests, large quantities of the same lot cannot be stored. For some tests, more reagent may be used in validation than in actual clinical testing!
In addition to the initial validation of probes, CLIA'88 also calls for biannual calibration verification. For some tests, such as aneuploidy detection in uncultured amniocytes, this verification is easily obtained by comparison to the "gold standard" of conventional cytogenetics. However, other FISH testing is performed reflexively, especially in cancer specimens. In these cases, FISH is performed because conventional cytogenetics was normal or ambiguous (e.g. the finding of a single trisomy 8 cell in a patient with leukemia). Here there is no "gold standard" to compare with, since the purpose of FISH is to detect an abnormality which was undetectable by conventional cytogenetics.
The College guidelines suggest that ongoing clinical correlations, i.e., does the clinical outcome match the test result, can serve as the biannual calibration. This may be possible in the case of laboratories affiliated with clinics, but in the case of reference laboratories, it will be extremely difficult because access to clinical follow up data is not possible to obtain for many patients.
In summary, we clinical laboratorians find ourselves to be outlaws. We are performing clinical FISH tests with non-FDA approved reagents, which are prohibitively expensive to properly validate, and, in some cases, are difficult to impossible to be monitored and calibrated. How all of these issues will sort out will hopefully become clear in the months ahead. As of now, though, we seem to be living the Chinese curse, "May you live in interesting times."
Contributed by John Stone, Ph.D.(AZ)
The Genetic Drift Newsletter is not copyrighted. Readers are free to duplicate all or parts of its contents. The Genetic Drift Newsletter is published semiannually by the Mountain States Genetics Network for associates & those interested in Human Genetics. In accordance with accepted publication standards, we request acknowledgement in print of any article reproduced in another publication. The views expressed in the newsletter do not necessarily reflect local, state, or federal policy. For additional information, contact Carol Clericuzio, M.D., Editor, Department of Pediatrics, The University of New Mexico, Albuquerque, NM, 87131
Table Of Contents: Molecular Cytogenetics (FISH)
Introduction & Basic Techniques
Applications of FISH Technology
FISH Applications in Cancer Cytogenetics
FISH in Microdeletion Syndromes
FISHing in Unknown Waters
Regulatory Issues and FISH
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