Chapter 18 - Laboratory diagnosis of Niemann–Pick disease type C: The filipin staining test
Section snippets
Introduction and Rationale
Niemann–Pick disease type C (NPC) is an atypical lysosomal storage disorder with wide clinical heterogeneity, resulting from mutations in one of two genes, either NPC1 or NPC2 (Patterson et al., 2012, Vanier, 2010). Not an enzyme deficiency, it is currently conceived as a lipid trafficking disorder. Initial studies by Peter Pentchev and associates and further work from several laboratories (Patterson et al., 2001, Pentchev et al., 1994) demonstrated, in cultured skin fibroblasts of patients, a
Materials
- 1.
A cell culture unit with a CO2 cell culture incubator and an inverted microscope (if possible with phase contrast equipment).
- 2.
An epifluorescence-equipped microscope with an x20 or x25 (and if possible x40) fluo-objectives, and a UV-filter suitable to observe the fluorescence of filipin–cholesterol complexes (excitation 364 nm; emission 475 nm).
The authors have successively used a Leitz Ortholux with a B2 combined filter (excitation filter BP 350-410; Barrier filter LP 470); a Zeiss Axioscop 2 with
General Considerations
Filipin staining itself is a very straightforward procedure, but when the aim is to optimally diagnose NPC, all steps preceding the final staining are critical. Furthermore, fluorescence microscopic evaluation of the results must follow strict rules, since in a small subset of patients, results can overlap with those seen in NPC heterozygotes or patients with other conditions.
The protocol described below was established in the Lyon laboratory in 1988 to adapt the research method to a clinical
Less than optimal or inappropriate conditions for fluorescence microscopic examination
Selecting a proper and sufficiently specific filter is of course essential. This should be checked by examination of a filipin-stained slide from a known “classic” positive control. Other practical considerations related to the use of filipin have been discussed in a previous issue of Meth. Cell Biol. (Maxfield & Wüstner, 2012): an important fact is that filipin is rapidly photobleached with the UV light intensity of most fluorescence microscopes. We fully agree with these authors that
Concluding Remarks
Interpretation can be difficult in a subset (about 15%) of all NPC patients showing a “variant” filipin pattern. This profile is more common in adult onset patients, which are often clinically less typical. Mild alterations of intracellular cholesterol trafficking can also be seen in a variety of other pathological conditions, which further complicates the situation. These inconclusive results occur in about 15% of all referrals, and require complementary sequencing of the NPC1 and NPC2 genes.
Acknowledgments
The authors wish to acknowledge the unfailing expert technical assistance of Ms Marie-Christine Juge, Ms Heliane Cornot, and Ms Guenaelle Piguet-Lacroix throughout the years.
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2023, Blood Cells, Molecules, and DiseasesCitation Excerpt :Interassay variability was 8 %. Free cholesterol accumulation in fibroblast cultures were detected by UV fluorescence microscopy, by specific binding of the Filipin antibiotic to free cholesterol [31]. All biochemical tests were performed in naïve treated patients, except two cases for Lyso-Gb3 (#6 and #11).
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