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These guidelines were originally written for the ProbeChek™ control slide product, but they are an excellent starting point for establishing quality laboratory procedures using CEP® or LSI® probes for enumeration.
CEP or LSI probe hybridization procedures should be followed as indicated in the package insert provided with the probe product. Use of optimally performing fluorescence microscope equipment, filters, and procedures as indicated in the probe product package inserts are strongly recommended for optimal hybridization and accurate evaluation and interpretation of the CEP and LSI probe signals using the ProbeChek™ Controls. Test specimens should be evaluated using phase-contrast microscopy prior to hybridization to determine if optimum for FISH. Suggested guidelines for slides for FISH are indicated below:
Each hybridized slide should be evaluated against quality parameters determined by the laboratory. Using an appropriate microscope, illumination and filter set, the specificity of the hybridization, the probe signal intensity and the signal to background noise should be evaluated to determine if the hybridization was optimum for analysis. At least 85% of all nuclei in the target area should be easily enumerable. There should be minimal background or nuclear fluorescent "noise". The probe signals should be easily visible using a minimum 400X magnification. The majority of the nuclei on the slide preparation should be disassociated from surrounding nuclei and should not be covered by cytoplasm. The presence of numerous areas of clumped nuclei (greater than 3 nuclei together) may preclude adequate and homogenous nuclei distribution for accurate enumeration. Preparations not meeting these criteria should not be used for signal enumeration. Signal enumeration should be performed separately for each probe under evaluation, however it is reasonable to enumerate CEP X and CEP Y probes simultaneously.
Download Slide Prep Document in PDF Format
Suggested documentation format for slide preparation and in situ hybridization quality control.
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.
Recommended Enumeration Guidelines for Single Color Probe Enumeration
Single color probe enumeration can be performed using a single bandpass filter, or muti-bandpass filter set in order to visualize the counterstain fluorescence (if present).
Two or more probes labeled with different color fluorophores that are hybridized to the same cells may be enumerated singly using single bandpass filter sets or simultaneously using multi-bandpass filter sets depending upon the signal intensity of the probes and the size of the nucleus. Cells with small or constricted nuclei may not be easily enumerated using multi-bandpass filter sets due to the spacial arrangement of the probe signal in the nucleus or due to perceived dim probe signals.
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