Assessment of lysosomal function by quantitative histochemical and cytochemical methods.
Quantitative histochemistry and cytochemistry enables a direct link to be made between metabolic functions such as the activity of lysosomal enzymes and the morphology of a tissue or a type of cell. Several approaches exist such as microchemistry based on (bio)chemical analysis of a single cell or a small piece of tissue dissected from a freeze-dried section. This technique has been routinely used for prenatal diagnosis of inherited enzyme defects and especially of lysosomal storage diseases. Other approaches are cytofluorometry or cytophotometry, which are based on the principle that a fluorescent or coloured final reaction product is precipitated at the site of the enzyme. The amount of final reaction product is analysed per cell or per unit volume of tissue using either a microscope cytofluorometer or flow cytometer for fluorescence measurements or an image analysing system or scanning and integrating cytophotometer for absorbance measurements. In principle, fluorescence methods are to be preferred over chromogenic methods because they are more sensitive and enable multiparameter analysis. However, only a limited number of fluorogenic methods are at hand that give a final reaction product which is sufficiently water-insoluble to guarantee good localisation. The best results have been obtained with methods based on naphthol AS-TR derivatives and with methods for the demonstration of protease activity using methoxynaphthylamine derivatives as substrates and 5'-nitrosalicylaldehyde as coupling reagent. Chromogenic methods are far better with respect to localisation properties and, therefore, most commonly used for quantitative histochemical analysis of lysosomal enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)
van Noorden
C J
CJ
Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands.
D003592:Cytophotometry; D005434:Flow Cytometry; D006801:Humans; D016464:Lysosomal Storage Diseases / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry / Q000201:enzymology*