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Looking for Ferns: Optimization of Digestion Pretreatment in Fluorescence In Situ Hybridization (FISH) Technique on Paraffin-embedded Tissues

Tojo, Marta BSc*; Perez-Becerra, Raquel BSc*; Vazquez-Boquete, Angel BSc*; García-Rivero, Arancha BSc; García-Caballero, Tomas MD, PhD*; Forteza, Jeronimo MD, PhD*; Fraga, Maximo PhD*

Diagnostic Molecular Pathology: March 2008 - Volume 17 - Issue 1 - pp 59-63
doi: 10.1097/PDM.0b013e318134ea1c
Original Articles

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.

*Departamento de Anatomía Patológica y Ciencias Forenses and Servicio de Anatomía Patológica, Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain

Servicio de Genética, Centro Oncológico, A Coruña, Spain

This work was supported by a grant from Instituto de Salud Carlos III (FIS PI 061902).

Reprints: Maximo Fraga, PhD, Departamento de Anatomía Patológica y C. Forenses and Servicio de Anatomía Patológica, Complejo Hospitalario Universitario de Santiago, Spain (e-mail:

© 2008 by Lippincott Williams & Wilkins.