title: Optimization of a histopathological biomarker for sphingomyelin accumulation in acid sphingomyelinase deficiency.
abstract: Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.
authors.affiliation: Department of Pathology, Genzyme, a Sanofi Company, Framingham, MA 01701, USA. tatyana.taksir@genzyme.com
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Taksir
authors.forename: Jennifer
authors.initials: J
authors.lastname: Johnson
authors.forename: Colleen L
authors.initials: CL
authors.lastname: Maloney
authors.forename: Emily
authors.initials: E
authors.lastname: Yandl
authors.forename: Denise
authors.initials: D
authors.lastname: Griffiths
authors.forename: Beth L
authors.initials: BL
authors.lastname: Thurberg
authors.forename: Susan
authors.initials: S
authors.lastname: Ryan
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001881:Borates; D004853:Epoxy Resins; D016591:Histocytological Preparation Techniques / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D008099:Liver / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009542:Niemann-Pick Diseases / Q000378:metabolism* / Q000473:pathology; D009928:Organ Specificity; D010796:Phthalic Anhydrides; D013109:Sphingomyelins / Q000378:metabolism*; D013194:Staining and Labeling; D013634:Tannins; D016610:Tissue Embedding; D014048:Tolonium Chloride; D014118:Toxins, Biological
chemical_list: D015415:Biomarkers; D001881:Borates; D004853:Epoxy Resins; D007202:Indicators and Reagents; D010796:Phthalic Anhydrides; D013109:Sphingomyelins; D013634:Tannins; D014118:Toxins, Biological; C107040:lysenin; C018851:borax; D014048:Tolonium Chloride; C005752:araldite; C004875:EPON