Quality Control and Laboratory Issues

In the clinical laboratory, the availability of two or more unrelated test methods for the diagnosis of each sarcoma type can facilitate the confirmation of unexpected results or the resolution of discrepant results. One key issue regarding the accurate molecular diagnosis of sarcomas is the selection of the appropriate procedure. This is largely determined by the type of sarcoma under consideration, the tissue specimen type, and the specimen quality. For fusion genes with limited structural variations, such as synovial sarcoma, ARMS, clear cell sarcoma of soft parts, extraskeletal myxoid chondrosarcoma, DSRCT, alveolar soft part sarcoma, and inflammatory myofibroblastic tumor, RT-PCR and real-time RT-PCR represent the first choice method. Interphase FISH is the best choice for sarcomas having fusion genes with chromosomal and intramolecular variations, such as myxoid/round cell liposarcoma, DFSP, and ES/PNET. Ideally, all specimens with a working diagnosis of sarcoma should have viable tissue sent for karyotyping. A portion of the diagnostic specimen can be temporarily stored in culture medium at room temperature or 4°C until a provisional histologic diagnosis is made, and then sent for karyotyping if appropriate. Whenever possible, a portion of the tumor specimen should be frozen and stored, to provide the best source of nucleic acid suitable for virtually all types of molecular tests (Southern blot, RT-PCR, real-time RT-PCR, and genomic PCR) used for molecular testing. Unfixed, fresh or frozen tissue also is suitable for protein analysis, although this is rarely used clinically. Touch preparations for FISH should be made and stored for possible use. When only paraffin-embedded archival material is available, interphase FISH and real-time RT-PCR are the best testing choices.

Turnaround time varies significantly among the different testing methods. Cytogenetic karyotyping requires short-term culture (3 to 10 days). The length of time for culturing reflects the quality as well as the quantity of the specimen source, as well as the tumor proliferation rate in vitro. Turnaround time for interphase FISH also depends on specimen type. Touch preparation and frozen sections allow for rapid FISH analysis (1 to 3 days). For archival tissue sources, an additional 2 or 3 days are required to obtain the nuclear suspension or the tissue sections. RT-PCR has a 2- or 3-day turnaround time for common fusion gene detection but requires a longer time for more complex fusion variant detection. Real-time RT-PCR offers a shorter turnaround time (1 or 2 days) because it does not require post-PCR manipulation steps or confirmatory testing. Longer turn-around times may be expected if the laboratory batches specimens and only runs the test once per week, for example.

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