FISH Lab Quality Control

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.

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.

1. Scan the target area using a low power objective to examine cell distribution.
2. 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.
3. 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.
   

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).

1. Do not evaluate cells that are touching or overlapping.
2. Count diffuse signals if they are separate from other signals.
3. Count split signals - two smaller signals in very close proximity - as one signal. The two signals represent a single chromosome complement.
4. Count two signals connected by a strand of fluorescence as one signal.
5. Do not count cells with zero signals unless they are surrounded by nuclei that contain signals.
6. Do not evaluate nuclei that are not intact.
7. Do not evaluate nuclei with overlapping signals of different color.
8. Do not count non-specific hybridization signals. These signals can be recognized by their lower intensity and different shape.
9. Do not evaluate nuclei with signals located on the periphery of the nucleus.
10. Count only nuclei in which a definite enumeration can be made; skip all others.
11. Record accurately. Recount in order to validate questionable counts.

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.

1. Do not evaluate cells that are touching or overlapping.
2. Count diffuse signals if they are separate from other signals.
3. Count split signals - two smaller signals in very close proximity - as one signal. The two signals represent a single chromosome complement.
4. Count two signals connected by a strand of fluorescence as one signal.
5. Do not count cells with zero signals unless they are surrounded by nuclei that contain signals.
6. Do not evaluate nuclei that are not intact.
7. Do not evaluate nuclei with overlapping signals of different color.
8. Do not count non-specific hybridization signals. These signals can be recognized by their lower intensity and different shape.
9. Do not evaluate nuclei with signals located on the periphery of the nucleus.
10. Count only nuclei in which a definite enumeration can be made; skip all others.
11. Record accurately. Recount in order to validate questionable counts.