The specificity of interphase FISH translocation probes in formalin fixed paraffin embedded tissue sections is readily assessed using automated staining and scoring of tissue microarrays constructed from murine xenografts.
Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.
Tubbs
Raymond R
RR
Dept of Clinical Pathology, The Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44195, USA. TubbsR@ccf.org
Pettay
James
J
Barry
Todd S
TS
Swain
Eric
E
Loftus
Margaret
M
Cook
James R
JR
Skacel
Marek
M
Paine
Gillian
G
Roche
Patrick
P
Grogan
Thomas
T
D000818:Animals; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000235:genetics*; D005557:Formaldehyde; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D009369:Neoplasms / Q000235:genetics; D016612:Paraffin Embedding / Q000379:methods*; D046888:Tissue Array Analysis / Q000379:methods*; D023041:Xenograft Model Antitumor Assays / Q000379:methods*
D015342:DNA Probes; D005557:Formaldehyde