Detecting balanced translocations using tissue sections plays an important diagnostic role in cases of hematological malignancies. Manual scoring is often problematic due to truncation and overlapping of nuclei. Reports have described automated analysis using primarily tile sampling. The aim of this study was to investigate an automated fluorescent in situ hybridization analysis method using grid sampling on tissue sections, and compare the performance of dual-fusion (DF) and break-apart (BA) probes in this setting. Ten follicular, 10 mantle cell lymphoma, and 10 translocation-negative samples were used to set the threshold of false positivity using IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes. The cut-off distances of red and green signals to define fusion signals were 0.5, 1.0, and 1.2 lm for the IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes, respectively. The mean false positivity of grid units was 5.3, 11.4, and 28.1%, respectively. Ten to 14 additional samples analyzed blindly and were correctly classified using each probe. Discriminating positive and negative samples using automated analysis and grid sampling was possible with each probe, although different definitions of fusion signals were required due to the different physical distances between the DNA probes. Using the DF probes resulted in lower false positivity, which was less affected by signal numbers per grid units. '
International Society for Advancement of Cytometry
Key termsFISH; automated analysis; cytogenetics; paraffin; lymphoma; FFPE; tissue sections; dual-fusion; break-apart; grid sampling RECURRENT, structural cytogenetic abnormalities, most frequently balanced translocations play an important diagnostic and prognostic role in cases of hematological malignancies (1). Frequently, only formalin-fixed, paraffin-embedded (FFPE) material is available for demonstrating such abnormalities. Several recent reports suggest that out of the available molecular techniques, fluorescent in situ hybridization (FISH) is the most reliable and appropriate for detecting translocations in FFPE samples (2-6). Several groups reported on FISH analysis of FFPE material, which may be performed on isolated nuclei (7-10) or on tissue sections (11-15). The major advantage of the former is that whole nuclei can be analyzed free from truncation artefacts. However, isolation of nuclei is more time-and resource consuming, than analyzing tissue sections and valuable information regarding histological topography is lost. If the sample contains only a small focus of neoplastic cells, their frequency in suspensions of isolated nuclei may drop below the false positivity rate of the FISH analysis.Because of recent advancement of image analysis and computer science, automated FISH analysis is becoming a desired possibility (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). A recent report
