Experimental Design
A qualitative study design was adopted with samples chosen from a five-year period. The final patients selected were Black and Coloured South Africans, seen at state run facilities.
Cases
In this study 11 cases with copy number variants (CNVs) detected by array comparative genomic hybridisation (aCGH) were chosen for FISH follow-up testing. The cases selected were also based on the availability of FISH probes. These cases were received in the Division of Human Genetics, National Health Laboratory Service, Johannesburg for diagnostic testing using aCGH between 2018–2022. The cases selected for FISH testing met the following inclusion criteria: (i) Patient seen at any of the Human Genetics clinics described above, (ii) any race or gender as it does not affect the outcome of the FISH testing and (iii) positive or inconclusive aCGH result on post-natal sample.
Array Comparative Genomic Hybridisation (aCGH)
Array Comparative Genomic Hybridisation (aCGH) is a molecular-cytogenetics technique that allows for genome-wide investigation of CNVs. The aCGH uses a two-colour process to measure DNA copy number in a patient sample relative to a reference control sample. The Division utilises the Agilent SurePrint G3 ISCA v2 CGH 8x60K dual-colour chromosomal microarray (Agilent Technologies, Santa Clara, CA) for diagnostic testing of postnatal samples. The workflow was performed according to the manufacturer’s instructions and data analysis by following recommended guidelines [3, 8]. The aCGH results for all cases were visualised using CytoGenomics software.
Fluorescence in situ Hybridisation (FISH)
Peripheral blood samples were processed by the standard conventional cytogenetics method for planting and harvesting, and the cell pellets from this procedure were used to make slides. These slides along with buccal smear slides were processed for FISH testing. The Abbott Hybridisation conditions for cytological Specimens was followed for pre-treatment up to the post-hybridisation steps, for processing the slides with the Vysis IntelliFISH Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL), for the cases selected based on the microarray results and FISH probes available. The Vysis FISH probes (Abbott Molecular Inc., Des Plaines, IL) used for the cases selected are listed in Table 1.
Table 1
Cases Selected for FISH Testing
CASE NUMBER | FISH PROBE | PROBE DESIGN AND COVERAGE |
1–6 | DiGeorge Region LSI TUPLE 1 (HIRA) SpectrumOrange/LSI ARSA SpectrumGreen Probe Set | Dual colour (22q11.2q13.3) |
7–8 | Wolf-Hirschhorn Region LSI WHS SpectrumOrange/CEP 4 SpectrumGreen Probes | Dual colour (4p16.3q10) |
9 | PWS/AS SNRPN SpectrumOrange/CEP15 SA/PML SpectrumGreen | Dual colour (15q11.2q13) |
10–11 | CEP 12 SpectrumOrange Direct Labeled Fluorescent DNA Probe Kit | Centromeric, single colour (12p10) |
Standard Vysis LSI/WCP Hybridization Buffer versus Vysis IntelliFISH Solution Hybridization Buffer
The six cases (1–6) chosen for this purpose were those that came in with a suspected diagnosis of DiGeorge Syndrome. Slides were used in pairs and anonymised and labelled in numerical order and indicating if the standard, Vysis LSI/WCP Hybridisation Buffer (Abbott Molecular Inc., Des Plaines, IL), or the second buffer, Vysis IntelliFISH Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL), was used. Each sample chosen was assessed for two elements of performance i.e., signal intensity and hybridisation specificity. Since the laboratory FISH procedure was already validated using the standard Vysis LSI/WCP Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL), these slides served as the control for the hybridisation time. The slides were processed according to the laboratory developed procedure for the standard Vysis LSI/WCP Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL). Abbott Hybridisation conditions for cytological Specimens were followed for pre-treatment up to the post-hybridisation steps for processing the test slides with the Vysis IntelliFISH Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL). The samples were probed with Vysis DiGeorge Region LSI TUPLE 1 (HIRA) SpectrumOrange/LSI ARSA SpectrumGreen Probe Set (Abbott Molecular Inc., Des Plaines, IL) in duplicate. The preparation of the probe mixture using the two buffers is presented in Table 2.
Table 2
Composition of probe mixture for Vysis LSI/WCP Hybridization Buffer and Vysis IntelliFISH Hybridization Buffer
BUFFER | Vysis IntelliFISH Hybridization Buffer Probe Mixture | Standard Vysis LSI/WCP Hybridization Buffer Probe Mixture |
COMPONENTS | VOLUME ADDED |
Hybridization Buffer | 12 µL | 7 µL |
Water | 2 µL | 2 µL |
Probe | 1 µL | 1 µL |
Total Volume | 15 µL | 10 µL |
The standard ThermoBrite (Abbott Molecular Inc., Des Plaines, IL) program was used for the slides hybridised with the Vysis LSI/WCP Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL). A modified program was used for slides hybridised with the Vysis IntelliFISH Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL) where two hours hybridisation time was established to be ideal. Once it was established that the Vysis IntelliFISH Hybridization Buffer (Abbott Molecular Inc., Des Plaines, IL) worked optimally, the remaining five sample were processed with this buffer and appropriate FISH probe.
Post hybridisation steps included a stringent wash to remove any excess and unbound probe. Slides were then treated with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI II) (Abbott Molecular Inc., Des Plaines, IL) counterstain to remove any background interference.
FISH Analysis
All slides were viewed and analysed under a fluorescent microscope (Olympus BX61, Olympus, Tokyo, Japan). Images for each case were captured using the GSL120 Cytovision automated scanner and capture system (GSL120, CytovVision, AB Imaging, Germany). A total of 100 interphase nuclei were screened from peripheral blood cultures. For buccal smears, 300 interphase cells were screened per slide. All findings were recorded with FISH nomenclature according to the International System for Human Cytogenomic Nomenclature (ISCN) 2020 [9].