This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.
Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people’s hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.
The application of fluorescence in situ hybridization (FISH) to a single embryo cell (blastomere) presents special challenges both in practicalities and in interpretation of the signal pattern. The biopsied cell needs to be spread within a pre-defined area on the slide in order to facilitate its localization following FISH; extreme care needs to be taken in ensuring that the cell is lysed, that the cytoplasm has been dispersed, and that the nucleus is visible and intact; and, as the diagnosis depends on the results from this single cell, stringent scoring and interpretation guidelines should be applied. However, in experienced hands, FISH is a robust technique for pre-implantation genetic diagnosis (PGD) in clinical practice. The principle of PGD by FISH is that target-specific DNA probes labelled with different fluorochromes or haptens can be used to detect the copy number of specific loci, and thereby to detect chromosome imbalance associated with meiotic segregation of chromosome rearrangements (1), including Robertsonian translocations, reciprocal translocations, inversions, and complex rearrangements (2). FISH can also be used to select female embryos in families with X-linked disease, for which there is no mutation-specific test (3, 4). More controversially, FISH has also been used to screen for sporadic chromosome aneuploidy in order to try and improve the efficiency of assisted reproduction (5, 6); however, the predictive value of this test using FSIH is likely to be unacceptably low in most people’s hands and it is not recommended for routine clinical use (7). This article concentrates on the technical aspects and the limitations of FISH applied to clinical single-cell diagnosis.
Methods of spreading and fixing single blastomeres include methanol/acetic acid (5, 8), Tween/HCl (9), and a combination of Tween/HCl and methanol/acetic acid (10). Variations include hypotonic treatment of cells prior to spreading and/or pepsin and paraformaldehyde treatment after fixation. The method should be appropriately validated for the laboratory (14). The Tween/HCl method is described in detail in this article. The Tween/HCl method is technically simple and highly reproducible in different laboratories. This method can be used to prepare single nuclei for the FISH diagnosis of sex determination and chromosome rearrangements with acceptable diagnostic accuracy (7).
Probe mixes can combine directly labelled and indirectly labelled probes, and probes from different manufacturers. Probes for known polymorphic chromosome regions (11, 12), or those known to cross-hybridize significantly with other chromosomes (13) should be avoided, although can be used if shown to be specific and suitable for PGD by prior testing on both reproductive partners. Available fluorochromes/haptens and strategies for discriminating probes include the following: Texas Red (TR), fluorescein isothiocyanate (FITC), SpectrumGreen (Vysis), SpectrumOrange (Vysis), SpectrumAqua, biotinylated probes detected with TR-avidin, FITC-avidin, or Cy-5 streptavidin (visualized using a FarRed filter), a mix of red and green probes to produce a yellow signal, a second round of hybridization and a third color created by sequential capturing of SpectrumOrange using a TexasRed and a SpectrumGold filter).
A probe set containing three probes, specific for the centromere regions of the X and Y chromosomes and one autosome, is recommended for sex determination (14); the autosomal probe is used to establish ploidy and thereby to differentiate between trisomy X (2n, 47,XXX) and triploidy (3n, 69,XXX), and between tetrasomy X (48,XXXX) and tetraploidy (4n, 92,XXXX). A typical probe set applied in this setting is the Abbott AneuVysion mix containing alpha-satellite X, Y, and 18; this probe set has been demonstrated to have a very low polymorphism rate, and therefore pre-treatment work-up of the reproductive partners is not required (14).
The probe mix for any specific rearrangement should: Ideally contain probes at least sufficient to detect all the expected products of the rearrangement with chromosome imbalance. If this is not possible, probe mixes where the undetected unbalanced products have been assessed to be non-viable in a recognizable pregnancy and are likely to have very low prevalence may be acceptable (14). Be tested on cultured lymphocyte metaphases from both reproductive partners. At least ten metaphase spreads should be examined for probe specificity, polymorphisms and cross-hybridization, and, for the chromosome rearrangement carrier, to ensure that the probes hybridize as expected to the different segments of the rearrangement. In addition, at least 100 interphase nuclei from these preparations should be scored to assess signal specificity, brightness, and discreteness (14).
Commercial PGS probe sets are available (e.g. Abbott MultiVysion PB or PGT), targeting the chromosomes most frequently found to be aneuploid in products of conception, and comprising a single probe per chromosome targeted. Typically the nucleus may have a second hybridization with probes for additional chromosomes providing an assay for chromosomes 13, 15, 16, 18, 21, 22, and XY (14). The predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people’s hands and it is not recommended for routine clinical use (7, 15).
Techniques such as comparative genomic hybridization (CGH) applied to single cells are able to test for the copy number of every chromosome (Wilton et al. 2001), and the use of single nucleotide polymorphisms (SNP) arrays and quantitative analysis (Wells et al. 2008) or “karyomapping” genotyping (Handyside et al. 2009) are promising alternative techniques to detect chromosome aneuploidy. However, the resolution limit and accuracy for segment imbalance remain uncertain and it is likely that FISH will continue to be the technique of choice for chromosome rearrangements involving small segments.
The authors have nothing to disclose.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Hydrochloric Acid, 2.5L | VWR | 101254H | ||
Sodium Chloride, 5Kg | VWR | 443827W | ||
Potassium Chloride, 250g | VWR | 26764.232 | ||
di-Sodium Hydrogen Orthophosphate Anhydrous, 500g | VWR | 102494C | ||
Potassium Dihydrogen Orthophosphate, 500g | VWR | 10203 4B | ||
Sodium Hydroxide Pellets, 500g | VWR | 28245.265 | ||
Tween20, 500mL | VWR | 663684B | ||
Ethanol, 2.5L | VWR | 8.18760.2500 | Duty Paid | |
Tri-Sodium Citrate, 5Kg | VWR | 27833.363 | ||
Cow Gum, 250mL | Cow Proofings Ltd. | No longer available | Old stocks can still be found in art shops | |
DAPI/Antifade ES, 0.125μg/mL, 500ul | Cytocell | DES500L | 150μl vial rec’d with each Cytocell TEL Probe | |
Nail Varnish, 12mL | Boots | 10088316001 | ||
Amine-coated Slides, 25 | Genetix | K2615 | ||
DAPI, 0.1mL | Sigma-Aldrich | D-9542 | ||
Vectashield, 10mL | Vector Laboratories | H1000 | ||
Texas-Red-avidin, 0.001mL | Vector Laboratories | A-2016 | ||
FITC-avidin, 0.001mL | Vector Laboratories | A-2011 | ||
Cy-5 Streptavidin, 0.001mL | GE Healthcare | PA45001 | ||
Microcentrifuge Tubes, 1.75mL, box of 500 | Thistle Scientific | AX-MCT-175-C | ||
Microcentrifuge Tubes, 0.6mL, box of 1000 | Thistle Scientific | AX-MCT-060-C-CS | ||
30ml Universal Containers, box of 400 | Sterilin | 128B | Available from NHS Supply Chain, Cat No. KCP053 | |
Hot Block | ||||
Spirit Burner Spirit Burner Socket Spirit Burner Wick |
VWR VWR VWR |
451-0107 451-3112 451-3111 |
||
Plugged Glass Pipettes | Fisher Scientific | PMK-300-052R | ||
Dissecting Microscope | Wild Heerbrugg | |||
Tissues, 100boxes | NHS SUPPLY CHAIN | MJT058 | ||
Metal Culture Trays | ||||
Plastic Melamine Trays | VWR | 682-009 | ||
Oven | ||||
Mouth Pipette Mouthpiece | Scientific Laboratory Supplies | HAE3700 | ||
Tubing | ||||
Syringe Filter, 0.2μm pore size, 25mm diameter | Fisher Scientific | FDM-340-010U | ||
Diamond Pen | VWR | 818-0021 | ||
Hot Block for Slides | Thermo Hybaid | HBOSBB | ||
Hybridization Chamber | ||||
Tissue Roll | NHS SUPPLY CHAIN | MJT004 | ||
Schieferdecker Jar | VWR | 631-9313 | ||
Coplin Jar | VWR | 631-9331 | ||
Forceps, Fine | VWR | 232-2123 | ||
Forceps, Blunt | VWR | 232-2113 | ||
1000mL Beaker | VWR | 213-1642 | ||
Phase Contrast Microscope | Nikon | E200 | ||
Fibre-free Blotting Cards, box of 100 | Fisher Scientific (Raymond Lamb) | SDE1 | ||
Blue-Frosted Microscope Slides, box of 100 | Scientific Laboratory Supplies | MIC3022 | ||
Coverslips, 18x18mm, No. 1, box of 200 | Scientific Laboratory Supplies | MIC3110 | ||
Coverslips, 22x22mm, No. 0, box of 200 | Scientific Laboratory Supplies | MIC3104 | ||
Coverslips, 22x50mm, No. 0, box of 100 | Scientific Laboratory Supplies | MIC3206 | ||
1mL Syringe, box of 100 | NHS Supply Chain | FWC045 | ||
1mL Pipette Tip, 10racks of 1000 | Thistle Scientific | AX-T-1000-C-L-R | ||
Incubator | LEEC | |||
Waterbath | Grant | W22 | ||
Alcohol Thermometer | Various sources available | |||
pH Meter | Jenway | 3305 | ||
pH Electrode | VWR | 662-1759 | BNC Plug to fit Jenway pH meter 3305 | |
Heated Stirrer | Bibby | HC502 | ||
1mL Pasteur Pipettes, box of 500 | Scientific Laboratory Supplies | PIP4202 | ||
Parafilm, 50mm x 75m | VWR | 291-1214 |