FISH for Pre-implantation Genetic Diagnosis

1. Lysing and Spreading a Nucleus from a Biopsied Blastomere

1. Cell lysis buffer for spreading cells (0.2% Tween20 in 0.01 M HCl, pH 2.0) should be prepared 24 hours in advance and stored at -20°C. Prepare 100 mL and filter 20 mL into a 30 mL sterile universal container using a sterile 20 mL syringe and syringe filter. Dispense 1 mL aliquots into 10-15 1.7 mL sterile microcentrifuge tubes, close and label the tubes prior to freezing. It is recommended to have two different batches is use, the new batch and the previous batch, which has been tested and can be used if the new batch is unsatisfactory.
2. Defrost the lysis buffer at room temperature 30 minutes before the biopsy procedure. For practical purposes the working temperature will be between room temperature and hand temperature.
3. Score a small circle (approximately 5 mm diameter) on the underside of an amine-coated slide (e.g. Genetix) using a diamond pen and pre-label the slide with the case number, unique slide number, and biopsy date. Use a separate slide for each blastomere in numerical order, and label with the embryo number. Slides should be labeled with a hard pencil such as 4H, and “blotted” with a latex glove to remove any graphite dust.
4. Place a small volume of lysis buffer within the circle.
5. Transfer the biopsied blastomere into the lysis buffer. If necessary add lysis buffer within the circle until the cell begins to lyse; the cell should lyse completely and the cytoplasm disperse before the buffer dries.
6. Observe the nucleus to ensure that it remains within the circle and is not lost; if the cell does not have a nucleus or has multiple nuclei, biopsy another cell.
7. Leave the slide to air-dry at room temperature.

2. In Situ Hybridization of a Single Blastomere Nucleus

1. Prepare an ethanol series (70, 90 and 100%) made up in sterile distilled water.
2. Turn on and set the hot block (e.g. Hybaid Omnislide or Vysis Hybrite) to 75°C.
3. Defrost probes, vortex, and centrifuge. The reagents should be witnessed and verified to be correct before making up the probe mixture. Pipette volumes as specified by the manufacturer to make up the probe mixture in a 0.65 mL sterile microcentrifuge tube and vortex and centrifuge before use. The total volume of probe mixture should be sufficient to allow 2 μL per nucleus to be tested and rounded up to the nearest 10 μL to allow a safety margin.
4. Pre-wash the slides in coplin jars using phosphate-buffered saline (PBS) (pH 7.0: 0.14 M NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) for 5 min at room temperature.
5. Rinse the slides twice in sterile distilled water.
6. Dehydrate the slides with the ethanol series (70, 90, and 100%) for 2 min each at room temperature and air-dry. Ensure slides are fully immersed and if any graphite dust floats to the surface, soak up with a clean tissue.
7. Record the position of the nucleus within the circle by visualizing with a phase contrast microscope.
8. Dehydrate with 100% ethanol for 2 min at room temperature and air-dry.
9. Apply 2 μL of probe mixture, and cover with a 9 x 9 mm coverslip (one quarter of an 18 x 18 mm no.1 coverslip).
10. Seal the edges of the coverslip with rubber cement (e.g. Cow Gum; Cow Proofing).
11. Codenature the slides on a hot block at 75°C for 5 min, and then hybridize the slides overnight (16-20 h) in a humidified chamber at 37°C. Probe mixes that consist entirely of centromere probes (i.e. for sex-linked cases) will give a satisfactory result after 60 min of hybridization.
12. Prepare a water bath with sufficient coplin jars and heat to 71°C.
13. Prepare a 0.4x standard saline citrate (SSC) stringent wash solution (pH 7.0 at 71°C, 0.06 M NaCl, 6 mM C₆H₅Na₃O₇.2H₂O) and heat in the water bath.
14. Dispense 50 mL of stringent wash per coplin jar required and check that the temperature is 71°C immediately prior to use using a clean thermometer.
15. Carefully remove the rubber cement from each slide and rinse off the coverslip using 4x SSC/0.05% Tween20 (pH 7.0) at room temperature.
16. Wash the slides in the 0.4x SSC stringent wash at 71°C for 5 min. Wash no more than 6 slides per coplin jar.
17. Wash the slides in 4x SSC/0.05% Tween20 at room temperature for 2 min.
18. If the probe mix contains indirectly labelled probe(s), drain the slides of excess liquid and apply 20 μL of fluorescently conjugated antibody under a 20 x 20 mm square of Parafilm.
19. Incubate in a humidified chamber at 37°C for 15 min.
20. Remove the Parafilm and wash once in 4x SSC/0.05% Tween20 at room temperature for 2 min.
21. Wash twice for 2 min in PBS at room temperature and drain the slides.
22. Apply 6 μL of DAPI/Vectashield (160 ng of 4′,6-diamidino-2-phenylindole dihydrochloride in 1 mL of Vectashield mounting medium, Vector Laboratories) to a 22 x 22 mm no. 0 coverslip and invert the slide over the coverslip.
23. Blot and seal the edges of the coverslip with clear nail varnish.

3. Analysis using a Fluorescence Microscope Suitably Equipped with Appropriate Filters for the Probes Used.

1. Score signals by direct visualization using a fluorescence microscope and single band-pass filters for each fluorochrome in the assay. Each nucleus should be scored by two analysts. A general guideline should lead to scoring of a single signal where two closely spaced signals are less than one domain (signal-width) apart; however, judgment based on experience needs to be exercised to interpret signals of varying size, intensity, and separation.
2. Use imaging software (e.g. Isis, MetaSystems, Altlussheim, Germany; CytoVision, Genetix) to capture an image of the nucleus for confirmation of the visual diagnosis, and for image archiving as part of a laboratory quality assurance plan.

4. Representative Results:

Metaphase and interphase nuclei from cultured peripheral blood lymphocytes should be examined to confirm that the selected probes are specific for the translocation chromosomes, informative for the breakpoints (the subtelomere probes should hybridize only to the translocated segments and the centromere probe(s) to the centric segment(s)) and the signals in interphase nuclei should be bright and discrete. Scoring the number of signals for each probe in 100 interphase nuclei from each partner is recommended to assess the efficiency of the assay. In this case 2 signals were scored in 95%-99% of nuclei for each probe. Figure 1 shows a metaphase and interphase nucleus from a preparation for a reciprocal translocation between the short arms of chromosomes 5 and 9.

Signals in interphase nuclei from embryo blastomeres should be bright and discrete and scored using separate band pass filters for the colors used. Figure 2 shows a blastomere nucleus with a normal signal pattern (2 copies for all the loci tested) consistent with a normal or balanced chromosome complement for chromosome 5 and 9, and a nucleus with an abnormal signal pattern consistent with an unbalanced product of the translocation that has monosomy (one copy) for the translocated segment of chromosome 5 and trisomy (3 copies) for the translocated segment of chromosome 9.

Figure 1. A metaphase spread and an interphase nucleus prepared from cultured peripheral blood lymphocytes from a carrier of a reciprocal translocation between the short arms of chromosomes 5 and 9 with breakpoints at 5p14.3 and 9p24.1: 46,XY,t(5;9)(p14.3;p24.1).ish t(5;9)(5ptel48-,9ptel30+;9ptel30-,5ptel48+,9cen+). FISH probes were selected for both translocated segments (5p14.3→5pter, red Cytocell subtelomere5ptel48 TexasRed ; 9p24.1→9pter, green Cytocell subtelomere 9ptel30 FITC) and the centric segment of chromosome 9 (9p24.1→9qter, blue Abbott CEP 9 alpha satellite SpectrumAqua).

Figure 2. Signals in interphase blastomere nuclei from day-3 embryos captured using a different filter for each fluorochrome and merged to form a composite image. (A) Two signals for each probe indicating two copies of each locus, which is consistent with a normal or balanced complement for the translocation chromosomes. (B) One red signal, three green signals and two blue signals indicating one copy of the translocated segment of chromosome 5, three copies of the translocated segment of chromosome 9 and two copies of the centric segment of chromosome 9, which is consistent with adjacent-1 segregation of the translocation resulting in an unbalanced product with monosomy for 5p14.3→5pter and trisomy for 9p24.1→9pter.