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The article by Sheriff et al on the use of paraffin wax embedded muscle for the diagnosis of muscular dystrophy1 illustrates some valid points, but some are questionable. Excellent results are illustrated and some retrospective studies of archival material will clearly be possible.
However, many of us in the field of muscle pathology will be alarmed at the statement in the discussion that “ . . .frozen muscle tissue is no longer necessary for the diagnosis of muscular dystrophy, with the exception of LGMD2F”. This statement is premature, inaccurate, and only deals with a limited number of muscular dystrophies. It also takes no account of the fact that the type of neuromuscular disorder is not known before a biopsy is taken, so tissue must be prepared for all possible studies.
Enzyme histochemistry still has an important role, and requires frozen tissue.2 The authors take no account of the importance of immunoblotting, which requires frozen tissue, and that some defective proteins can only be studied on immunoblots (for example, calpain 3, responsible for limb girdle muscular dystrophy 2A).
No evidence of the diagnostic use of the technique is shown; only the known localisation of antibodies in control muscle. No assessment of reduced or partial protein expression is shown although this, in contrast to absent protein, occurs in many muscular dystrophies. It is essential that reduced expression is fully assessed in fixed material before frozen material is dispensed with.
Secondary abnormalities are also useful3,4 and the value of paraffin wax sections for the assessment of these is not known, or not possible. For example, the commercial antibodies to fetal myosin (Novocastra MHCn) and to laminin β1 (Chemicon) produce negative results with antigen retrieval, but both are important in muscular dystrophies.3,5
The figures illustrate excellent morphology of paraffin wax embedded material. However, fig 1G,H shows rounded fibres that may be pathological or artefact; in frozen muscle of dystrophic muscle this is an important pathological feature. In addition, it is well known that wax embedding can cause unacceptable artefacts, and results differ from sample to sample and from laboratory to laboratory.
The number of gene defects responsible for a muscular dystrophy is increasing rapidly. It is not possible to know the type of material that will be required in the future, but a bank of frozen muscle will probably be the most versatile. Contrary to the comments on page 520, adequate freezing, storage, and orientation of frozen material are all possible with care, and fixed frozen sections give equivalent morphology of inflammatory cells.
It has taken decades to ensure that muscle samples are kept out of formalin so that a wide range of techniques can be applied. The role of antigen retrieval will probably increase, but the stage when everything, both now and in the future, can be performed on paraffin wax embedded material has not yet been reached. I hope all clinicians and muscle pathologists will take note of this and not set the clock back.
The purpose of our article was not to belittle the value of frozen material in the advancement of muscle pathology diagnosis (that is, for western blotting), but to stress that we should not ignore humble paraffin wax embedded sections. It is vital to emphasise that with the help of immunohistochemistry they facilitate the accurate diagnosis of many muscular dystrophies and other muscle pathologies, such as nemalin myopathy.
Dr Sewry's comment about negative results for laminin β1 and fetal myosin on paraffin wax embedded sections is currently valid, but antigen retrieval techniques are evolving and new antibodies are being developed, allowing larger antibody panels to be used on paraffin wax embedded tissue.
The question of the ease of interpretation of paraffin wax embedded versus frozen tissue is partly a matter of re-education. Adequate freezing, storage, and orientation of frozen material is no problem in specialist centres; however, referred frozen tissue from centres unaccustomed to dealing with them is often inadequate, and in those situations paraffin wax embedded tissue can provide the diagnosis. Moreover, there is an opportunity to study archival tissue or tissue taken at necropsy when there has been no clinical suspicion of muscle disease.
We are not turning back the clock, merely suggesting that much can be achieved using routinely processed tissue. In an ideal world, any tissue should be preserved for many different techniques (morphological, enzyme, protein, DNA, and RNA analyses). However, we live in a world with constraints. Given a choice of badly frozen/partially thawed muscle or paraffin wax embedded sections for morphological interpretation (with the help of immunohistochemistry) the latter is more likely to be helpful.