Cytogenetic techniques

Cytogenetics is the microscopic examination of chromosome shape, size, structure and movement. Originally, plant chromosome methodology was used to study human chromosomes but because the chromosomes did not separate well, the human chromosome number was originally incorrectly reported as being 48 (Hsu, 1952). However Hsu's discovery by accident, that cells treated with hypotonic solution swell, causing thdr chromosomes to separate well enough to be counted, allowed this mistake to be rectified and in 1956, Tijo & Levan reported the correct human diploid number to be 46 fi'om the study of lung tissues of four fetuses. Ford and Hammerton (1956) confirmed these results when they reported the human haploid number to be 23, fi'om studies of spermatocytes.

2.2.1.1 Cell culture

In this study, chromosome preparations were obtained fi'om lymphocyte cultures firom infertile males. Turner patients and patients with premature ovarian failure. In addition, chromosome preparations were obtained from two cultured gonadal tissue samples and three skin biopsies fi'om Turner syndrome patients. All of these preparations were used for karyotyping by G-banded analysis and were stored for any

necessary FISH studies. For G-banded analysis, thirty metaphase spreads were analysed from each patient, 12 fully and 18 counted. This detects 10% mosaicism with 95% confidence (Hook, 1977). When two cell lines were found, a total of 50-100 metaphases were analysed to establish a level of mosaicism. Buccal cells were collected in the form of a mouthwash and were used for FISH analysis.

2.2.1.1.1 Peripheral blood lymphocyte culture

Blood for culturing was collected in sterile lithium heparin tubes. Short term blood cultures depend on the ability of mitogens to induce resting T lymphocytes to divide and under aseptic conditions, 0.25mls of heparinised whole fresh blood was added to a 10ml blood tube, which contained 5mls of warmed Iscoves modified DMEM medium with 1% GPS (Glutamine, Penicillin, Streptomycin), 0.5mls fetal calf serum (FCS) and lOOpl of phytoheamagglutinm (PHA). The contents of the tube were mixed by incubating in an upright position at 37°C for 72 hours.

2J.1.1.2 Synchronised blood culture

This method yields a high mitotic index of long prometaphase chromosomes. Initially, lOOpl of 15mg/ml thymidine was added to the blood culture after 48 hours to block cell division at the Gi/S phase of the cell cycle. After mixing the contents of the tube, the culture was incubated at 37°C for a further 16 hours. The culture was ‘unblocked’ by decanting off the old medium and washing in fresh medium, at lOOOipm in a centrifuge. This medium was decanted off and replaced with 5mls of fresh warmed medium and 0.5mls of FCS. After a further 5 hours» lOOpl o f O.lmg/ml colchicine was added to the culture and this was mixed up and incubated for 10 minutes befi)re harvesting could commence. The addition of colchicine was later replaced by the addition of lOOpl of

lOjig/ml colemid® as colchicine is toxic in powder form. 2.2.1.13 Solid tissue culture

Gonadal tissue or skin was finely dissected in a petri dish using a scalpel. The prepared tissue was resuspended in 1ml of Ham’s FIO medium (100ml FIO, 12.5ml FCS, 1ml Ultroser, 1.25ml PS) and transferred to the flat bottom of four leighton tubes. A long narrow coverslip was carefully placed over the tissue using forceps. Cultures were allowed to settle overnight before being topped up with 1.5ml of medium. Cultures wCTe

media changed after 5 days and then every 2 days, during Wiich growth was carefully monitored.

Usually cultures would need subculturing before they were ready for harvest. The medium was decanted off from the leighton tube and the cells were washed out with O.Smls of trypsin/EDTA. This removed all traces of FCS found in the medium which interferes with the action of the trypsin. Fresh trypsin/EDTA (0.5ml) was added to the tube and was incubated at 37°C for a maximum of 5 minutes. The cells wa*e checked under an inverted microscope to ensure that they had lifted off from the bottom of the tube. Fresh medium (2ml) was added to the tube to stop the action of the trypsin. The resulting cell suspension was subcultured into another tube and when the cells were confluait and therefore ready for harvest, 50pl of colchicine was added for 90 minutes.

2J.AJ2 Harvesting methods

Harvesting methods today are based on those originally developed by Tijo and Leven (1956), Nowell (1960) and Moorhead et al. (1960). fri this study, metaphases were harvested from peripheral lymphocytes and solid tissue cultures.

2.2.1.2.1 Harvesting extended chromosomes from blood cultures

To harvest from blood cultures, the blood tube was initially centrifuged at lOOOrpm for 5 minutes. The supernatant was discarded leaving a pellet in the bottom of the tube and 5mls of 0.075M KCL were added, the pellet was resuspended and the mixture incubated at 37°C for 10 minutes. This was followed by centrifiigation at lOOOrpm for 5 minutes and the supernatant was discarded. Next fresh fixative, which was 3; 1 methanol : glacial acetic add, was added very slowly and with vigorous agitation, until the pellet turned darker and stopped frothing. This was topped up with about 5mls fresh fixative before centrifiiging at lOOOrpm for 5 minutes. At least two ftirther changes of fixative followed by centrifugation were required to obtain a white pellet and clear supernatant. After the final cetiifugation most of the fixative was decanted off leaving a small amount of diluted pellet, vdiich was dropped onto cold (from refiigerator) clean slides. (Slides had been previously soaked in methanol, with a drop of concentrated HCL and wiped dry with gauze).

Slides were checked under a phase contrast microscope using a lOx objective to check the cell density and quality of spreading. If the number of cell divisions were high and poorly

spread they could be improved by adding more fixative to the pellet. To improve spreading, and particularly when cytoplasm was present, a drop of fixative was added to the slide after the drop of cell suspension. In extreme cases, slides were spread over steam so that a layer of water vapour covered the slide, Wach aided spreading. Suspensions were stored at -20°C .