Cholesterol Storage

Principle Of Method:
Free cholesterol accumulation is determined qualitatively in cultured cells (skin fibroblasts or chorionic villus cells) which have been pre-incubated in media containing lipoprotein deficient serum and subsequently challenged with media containing pure low density lipoprotein. These cells are fixed and treated with filipin, which forms a fluorescent complex with free cholesterol. Slides are then examined by fluorescence microscopy.

Uses And Limitations Of The Method:
This method is used primarily to screen patients for Niemann-Pick disease type C (NPC). Skin fibroblast cells from NPC patients fit into three recognised biochemical phenotypes: classical, variant and intermediate. All three groups can be detected using this method. The classic and intermediate biochemical phenotypes exhibit an intense, punctate perinuclear fluorescence in > 95% of the cells; the variant phenotype has cells exhibiting a mixture of staining patterns, but a large percentage (> 50%) of cells are characteristic of NPC. The primary defect for this disorder is unknown but is related to a disturbance in intracellular trafficking of cholesterol. Some patients resemble NPC clinically, but exhibit normal filipin staining. In addition this method can detect patients with I-cell disease, but the staining pattern is distinctive from that observed in NPC cells. Positive patients are followed up with measurement of the cholesterol esterification rate in cultured cells.

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.

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