PRINCIPLE OF METHOD: Cultured cells or fragments of chorionic villi are incubated in tissue culture medium containing [35S]-L-cystine. In normal cells the labelled amino acid enters lysosomes and diffuses out again. In cells from cystinosis patients, however, it remains within lysosomes due to lack of a transporter required for its exit. When the cells are harvested, protein is removed by precipitation and the soluble fraction run on a thin-layer chromatogram an excess of label is found in the position of cystine. The harvested cells are in fact suspended in a buffer containing N-ethylmaleimide (NEM) which binds to free -SH compounds. The glutathione-NEM complex thus formed is also separated in the chromatogram and counted. Final results are expressed as a ratio cystine: glutathione-NEM and also as cystine incorporated per ìg protein.
USES AND LIMITATIONS OF THE METHOD: The method is used to diagnose cystinosis both postnatally and prenatally. In fact the use for postnatal diagnosis has been largely superseded by an alternative method employing a cystine binding protein which can be used with freshly prepared white cells and is more rapid and less costly than the isotope incorporation method (available in the Paediatrics Department laboratory, Guy’s Hospital). However, the isotope method is still routinely used for prenatal diagnosis. Heterozygote detection is not offered using this method.
SPECIMEN REQUIREMENTS: Fibroblasts cultured from a skin biopsy are needed. Biopsy material should be collected aseptically into a sterile bottle containing tissue culture medium (available from the laboratory), and sent at room temperature to arrive at the laboratory within 24 hours. Biopsies for tissue culture should not be frozen. Fibroblast cultures established in other laboratories should be sent in plastic 25 cm2 flasks filled with medium.
THE LABORATORY RECOMMENDS USE OF A COURIER SERVICE OR ROYAL MAIL SPECIAL DELIVERY FOR SENDING ALL SPECIMENS TO THE LABORATORY.