University of Pittsburgh Cancer Institute (UPCI)

Cytogenetics Services and Techniques

Cytogenetic Services

• Classical karyotyping of human and mammalian cell cultures
• Monitoring of established cell lines using classical and/or molecular cytogenetic techniques to rule out interspecies culture contamination.
• Special studies using a variety of banding techniques, including G-, C-, G11-, Q-, R-, DA/DAPI-, and AgNOR-banding.
• Molecular cytogenetic analysis, including:
  
  1. chromosomal fluorescence in situ hybridization (FISH) for mapping cancer-related genes, transgenes, viral integration sites, and oncogene amplifications and to identify marker chromosomes, the origin of which is unidentifiable by classical cytogenetic methods, and
  2. interphase FISH for examining the molecular karyotypes of archived (frozen, paraffin-embedded) or otherwise nondividing cells.
• Primary cell culture of human and mammalian cells and other cell culture as required for the analyses listed above.
• Quality assurance (QA) of cell lines in conjunction with the BGPF. The Cytogenetics Facility carries out two parts of the three-part UPCI Cell Line QA Standard Operating Procedure:
  1. Karyotyping to rule out interspecies cross-contamination and karyotypic evolution;
  2. Mycoplasma testing by two methods, as recommended by the ATCC (PCR-based detection and direct visualization by staining); and
  3. The BGPF carries out microsatellite analysis for identity testing to compare with the original cell line and/or repository standards.

Cytogenetic Techniques

• Chromosome harvesting (directly from research biopsies, blood samples, bone marrow aspirations, or following monolayer or suspension cell culture), slidemaking, and standard trypsin-Giemsa banding of mitotic tumor cells, embryonic stem cells, or cell lines.
• Classical chromosome analysis to identify modal chromosome number, chromosome pattern, and designate karyotype signature.
• Use of special stains and banding techniques, including C-, G11-, Q-, R-, DA/DAPI-, and AgNOR-banding to identify marker chromosomes and/or the species of origin of the specimen, the origin of which may be unidentifiable by routine cytogenetic methods.
• Chromosomal instability assays for aneuploidy, chromosome breakage, etc.
• Chromosomal FISH using gene-, region-, virus- or chromosome-specific DNA probes to map genes, transgene or viral insertion sites, or oncogene amplifications or to identify marker chromosomes that cannot be identified readily by classical cytogenetic methods.
• Interphase FISH for examining the molecular karyotypes of archived or otherwise nondividing cells, after preparation of nuclei from paraffin blocks or fresh or frozen tissues
• Comparative genomic hybridization to identify regions characterized by genomic gain or loss.
• Microscopic analysis, photomicrography, and/or digital imaging requisite for documenting the results of these studies.