Interpretation of Results
Enumeration guidelines for CEP and LSI on interphase nuclei are indicated below. The user is encouraged to apply these guidelines and provide additional guidelines as necessary to improve enumeration precision, accuracy and reproducibility within the laboratory. A slide scanning technique for CEP® and LSI® enumeration is described below. This technique or another, provided it is standardized in the lab, may increase enumeration accuracy and reproducibility.
- Scan the target area using a low power objective to examine cell distribution.
- Select an area on the target where the cells are evenly distributed yet at a density that several nuclei can be evaluated in a 400X field. Avoid areas where the distribution of cells is dense, the nuclei are overlapped or the nuclear borders of the individual nuclei are not defined.
- Use a 40X to 100X Plan Apo oil objective and focus first on the upper left quadrant of the selected field of view. Focus on each valid nucleus in the first viewing field and count the number of signals of one probe color within the nuclear boundary. Signals may be either bright and compact oval shapes, split into two smaller but connected dots, or a stringy diffuse shape. Evaluate split or questionable signals by observing at higher magnification. Record the signal count from each cell. Moving from left to right ( or top to bottom) continue to scan the slide for fields with evaluable nuclei. When the boundary of visible or interpretable nuclei is reached skip to the next field and continue the scanning process. Repeat this scanning process until the appropriate number of nuclei are enumerated (200 to 500 or more depending upon the prevalence of the abnormal cells). Repeat this process for each different probe until data is collected for all enumeration probes.