The Principles of Clinical Cytogenetics - Extra Materials - Springer

Fluorescence In Situ Hybridization 479
Fig. 16. Examples of normal (A) and abnormal (B) results for the Vysis UroVysion assay used to monitor
for bladder cancer recurrence in urine or bladder wash samples. The normal signal pattern reveals 2 red signals
for the chromosome 3 centromere, two green signals for the chromosome 7 centromere, two gold signals for
9p21, and two aqua signals for the chromosome 17 centromere. These cells were from a male with
microhematuria. The abnormal cell exhibits aneuploidy for chromosomes 3 (red), 7 (green), and 17 (aqua),
consistent with urothelial carcinoma. These results confirmed a recurrence in a 70-year-old male with a history
of bladder cancer.
SPECIAL QUALITY CONSIDERATIONS FOR FISH
Although a few commercially manufactured probe kits have been approved by the FDA for in
vitro diagnostic FISH testing, the majority of materials used for clinical FISH studies are considered
analyte-specific reagents (ASRs) that are exempt from the FDA and must be independently validated
in each laboratory. According to the Standards and Guidelines for Clinical Genetics Laboratories
from the American College of Medical Genetics (ACMG) (5), prior to utilizing a probe for clinical
purposes, probe validation must be performed. The validation should consist of localizing the probe
to the correct chromosomal band on normal metaphase spreads and determining sensitivity and specificity.
For probes that will be used for interphase analysis, normal ranges must also be calculated
from a database of cytogenetically characterized cases to establish the percentage of cells with an
apparent “abnormal” pattern that occur randomly. Thus, depending on the normal cutoff point, probes
might or might not be useful for detecting aberrations for interphase cell analysis. Biannual or continuous
evaluation of performance characteristics of each probe is required.
It is recommended that FISH tests be analyzed by two or more noncolorblind technologists who have
been trained in the scoring of the resulting signal patterns. For metaphase studies, at least 10 intact cells
should be scored, with 1 image saved for documentation. A large number of nuclei (approximately
200) are generally scored for an interphase study, with at least 1 image documenting results. Many
commercially available probe mixes contain internal control probes that identify the chromosome of
interest. In addition, the normal homolog signal can often be used as a control as well. For tests
without internal controls, for example, a Y chromosome probe on a newborn with ambiguous genitalia,
a control sample (for the example given, a sample known to have a Y chromosome present) needs
to be studied along with the test case. Reports should include the names of probes used and proper
ISCN nomenclature (see Chapter 3). When ASRs are employed for FISH studies, the disclaimer
This test was developed and its performance characteristics determined by [laboratory name]. It has
not been cleared or approved by the U.S. Food and Drug Administration
must be included on the final report.