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:
- Cells should be evenly distributed across the slide. Areas of cell clumping indicate poor slide preparation or sample preparation. There should be a minimum of eight cells per 400X (total magnification) field. Fewer cells than this indicates the cell concentration is too low for optimum enumeration.
- Nuclei and metaphase chromosomes should not be phase bright. Phase bright cells indicate slide preparation conditions were not optimum. Always optimize slide preparation conditions using a good quality lymphocyte preparation prior to preparing the slides.
- There should be no visible cytoplasm surrounding nuclei and metaphasess. Cytoplasm can preclude hybridization and lead to inefficient hybridization and inaccurate results.
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.
Suggested documentation format for slide preparation and in situ hybridization quality control.