Laboratory Techniques

Muscle biopsies are processed using frozen section techniques in order to enable histochemical methods of staining and morphometric evaluation of muscle fibre types, innervation and enzyme biochemistry. Muscle biopsies in formalin fixative and paraffin wax embedding are of limited use, allowing only evaluation of inflammatory conditions and confirming muscle wasting.

Orientation of the small muscle biopsy is of great importance. The classical histological section is a cross section of muscle fibres and fascicles. Longitudinal sections of muscle fibres are of limited use except when looking for neuro-muscular junctions.

It is necessary for the biopsy to be taken to the laboratory immediately following removal from the patient, so as to conserve the potential for enzyme histochemistry. If this is not possible then ammonium sulphate fixative solution may be used but this requires practice and familiarity by technicians and pathologists to ensure that the histochemical techniques work.

The biopsy is orientated to give a transverse section using a dissecting microscope. The biopsy is placed in O.C.T. on a small piece of cork and snap frozen in iso-pentane cooled in liquid nitrogen. The process of snap freezing is important as crystal artefacts will result if the freezing process is too slow. Transverse sections are cut and stored using a cryostat.

The standard histochemical stains, besides H+E, are ATPase and NADH to differentiate between muscle fibres, a modified Gomori to study mitochondria and a battery of other enzyme histochemical stains to study the biochemistry of the muscle including acid phosphatase, succinic dehydrogenase, cytochrome oxidase, phosphorylase. Immunocytochemical staining (e.g., Dystrophins 1-3, Merosin, Adhalin, Utrophin and Spectrin) is also being used to examine for and subtype muscle dystrophies.

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