Executive Summary
The automation of FISH slide scoring in a high throughput fashion has long been a goal for cytogenetic analysis. The development of CytoVision automated scanning and image analysis has enabled realization of this aspiration. In the current study, automation was utilized across seven assays, in combination with a CLL menu of Kreatech FISH probes. The results demonstrate that the combination of Kreatech FISH probes with a fully automated, unsupervised analysis can successfully be used to classify patient samples according to known CLL-associated genomic abnormalities. The overall comparative analysis per CLL probe set, between manually scored slides and slides analyzed using automation and unsupervised analysis, showed at least 94% concordance, which increased to 100% following subsequent review. Overall, the hands-on time was drastically reduced by 88% (48 hr 45 min) and a permanent record for each sample automatically created. The full automation of the image analysis process using the CytoVision GSL-120 scanning system demonstrates the versatility and effectiveness of the system to be easily integrated into high throughput, routine FISH analysis.
Summary
B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, with a median survival of ~9 years (1). The disease is characterized by a highly variable clinical course. Fluorescence in situ hybridization (FISH) has been shown to detect genomic abnormalities in over 80% of CLL cases (2, 3). A menu of seven specific molecular biomarkers detectable using FISH assays has been established within the Leica Biosystems’ repeat-free Kreatech FISH portfolio to predict clinical outcomes per patient. These seven DNA FISH probes have been selected to detect prevalent genetic aberrations found in B-cell CLL with known diagnostic value. The most common recurrent chromosomal abnormalities in CLL include deletion of 13q14 in 40 - 55% of cases, trisomy 12 in 16 - 20% of cases and deletion of 11q22 and deletion of 17p13 in 14 - 20% and 7 - 11% of cases respectively (4, 5, and 6). These genomic aberrations are used to group patients into the Döhner categories, providing prognostic information on each patient subgroup (7, 8, 9, 10). Five prognostic categories have been defined in a hierarchical model. Those with 17p13 deletions show the worst prognosis, followed by those with 11q22 deletions. Patients with trisomy 12 and those with normal karyotypes show intermediate survival. Patients with 13q14 deletions as the sole abnormality had the longest estimated survival times (7, 8). The automated analysis of common FISH tests in routine cytogenetic laboratories has many potential benefits. Obvious benefits include a more efficient workflow, freeing up valuable laboratory staff time and increased consistency of scoring. In addition, the ability to work more ergonomically and without the need for a darkroom would be attractive for many laboratories and personnel. Moving the analysis away from manual microscopes also has the potential to lower costs, and utilizing on the screen analysis could simplify staff training. Automated routine FISH analysis requires slide preparation to be of a consistent high quality. At the Leica Biosystems’ Amsterdam site a range of high-quality FISH probes are manufactured for commercial sale. During manufacture, the CytoVision GSL-120 platform is used as part of the quality control process. In this study we investigated the use of CytoVision GSL-120 for a set of common genomic aberrations associated with CLL, compared with typical manual dual scoring in order to investigate the potential for use of such analysis in a routine laboratory workflow. This study was designed and conducted to compare fully automated scanning and unsupervised image analysis of patient samples versus manual signal enumeration per probe set within the CLL menu of Kreatech FISH probes.>> Download the full Application Note as PDF
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