immunohistochemistry (ihc) in muscle pathology

muscle biopsy interpretation has been revolutionized by ihc. immunohistochemistry now has an essential role in the evaluation of the muscle biopsies and in examining proteins localizations. advances in the characterization of sarcolemmal proteins and recognition that defects in the genes encoding such proteins may lie at the heart of the multiple differing forms of muscular dystrophy have been among the most important recent developments in muscle pathology. this technique only reflects localization of proteins neither the related rna synthesis nor the activation of the related gene. ihc is complementary to histology and histochemistry and the result should not be interpreted without other morphological studies and detailed clinical and para clinical evaluations. defects in protein localization may show an abnormality in the gene that encoding that protein, known as a “primary defect” or could be a “secondary defect” due to an abnormality in another gene. ihc has a prominent role in assessing recessively inherited conditions, were both alleles are mutated, by revealing immuno-histochemical abnormalities related to primary defects. in dominant conditions, analysis of the secondary defects is particularly important cause normal allele expression may mask any alteration in the mutant allele as the normal allele produces a normal protein product whose localization is often normal and labeling of the mutant and normal protein could be indistinguishable. thus, analysis of both primary and secondary abnormalities has an important role in the assessment of proteins. with advances in molecular medicine the number of primary defects that can be identified with ihc is growing and new classification of disorders is according to the protein defects (e.g. dystrophinopathy, sarcoglycanopathy, desminopathy). many antibodies are now commercially available for studying muscle proteins, in particular those that detect primary defect in muscular dystrophies. ihc is used to present and localize specific protein components. the principle of the technique is the specific affinity of an antibody for its antigen. as for histochemistry, muscle tissue should be rapidly frozen in isopentane cooled in liquid nitrogen and immunolabelling is done on cryostat sections. 6mm thick sections of muscle are used and thick sections give false-positive reactions. either peroxidase- or fluorescein-labeled antibodies may be used. if only formalin-fixed, wax embedded tissue is available, various antigen retrieval techniques can be tried but this is not recommended for routine diagnostic studies of neuromuscular disorders. mhc class i molecules are located in perifascicular fibers in dm. in pm, mhc expression is more intense and is located mainly in non-necrotic partially invaded cells. interpretation of staining depends on several aspects including: 1) the visualization technique used, 2) controls and 3) maturity of the fibers. necrotic fibers often contain igg, which is detected by an anti-igg secondary antibody and must not be confused with specific labeling. immuno-histochemical assessment of the sarcolem is a fundamental aspect of diagnostic muscle pathology and it is essential to know if the cytoplasmic membrane is preserved. fibers affected by freezing artefact or pathologically damaged fibers may loose their plasma membrane and basal lamina. plasma membrane proteins could be less apparent on regenerating fibers internal labeling of the fibers is seen by some antibodies. studies of dystrophin and all plasma membrane proteins could be accompanied by parallel study of the b-spectrin, indicating overall preservation of the muscle fibers. the majority of cases of duchenne dystrophy show an absence of dystrophin from most fibers, or a very marked reduction. isolated fibers, or small clusters, may show a normal intensity and are known as revertant fibers. these are fibers in which the reading frame has been maintained, skipping the mutation. in becker muscular dystrophy the reading frame is also maintained and labeling is usually reduced on all fibers, or uneven on many of them. in some becker cases dystrophin immunolabelling may be normal and then assessment of secondary changes is important. dystrophin is a large protein and using more than one antibody is important to avoid false results. clinical severity should not be judged from dystrophin immunolabelling. dystrophin-associated proteins (dap) localize to the sarcolemmal in immuno-histochemical analysis. the daps are grouped into sub-complexes: a- and b-dystroglycan, the sarcoglycans (a, b, d and e) and sarcospan, the syntrophins, dystrobervin. immuno-histochemical reduction in one sarcoglycan protein may be associated with secondary reductions of other sarcoglycans or even of dystrophins. complete absence of one sarcoglycan, or the most severe reduction, is an indicator of the defective gene and helps to direct molecular analysis. an absence of the whole sarcoglycan complex may occur with a defect in b-sarcoglycan gene. a secondary reduction of sarcoglycans occurs in duchenne dystrophy. an absence or marked reduction of the plasma membrane protein, dysferlin, can be seen in lgmd2b and miyoshi myopathy. secondary changes in dysferlin expression can also occur in other muscular dystrophies. defects in caveolin-3 gene occur in lgmd1c, in rippling muscle disease and some rare cases with elevated creatine kinase. caveolin-3 is a plasma membrane protein and it’s absence or reduction of the protein can be detected by ihc. defects in lama2 gene encoding the laminin a2 chain of merosin occur in the severe mdc1a form of merosin deficient congenital muscular dystrophy. the clinical severity of cases with a defect in the lama2 gene varies from severe to mild and this is usually reflected in the amount of protein that can be detected. defects in the collagen vi gene are responsible for dominantly inherited bethlem myopathy and ullrich form of congenital muscular dystrophy. as bethlem myopathy is dominantly inherited no alteration in collagen vi immunolabelling is usually seen. in recessive cases of ullrich congenital dystrophy, however, collagen vi may be absent or severely reduced. integrin a7 interacts with laminin a2. defects in the gene cause a mild disorder classified as a congenital muscular dystrophy. emerin is anchored to the inner nuclear membrane and mutations in the gene are responsible for the x-linked form of emery-dreifuss muscular dystrophy. mutations in the sta gene encoding emerin result in absence of the protein from all nuclear membranes. this absence is easily detected by ihc. mutations in the gene for lamin a/c are associated with several phenotypes, including the autosomal dominant form of emery-dreifuss muscular dystrophy, dilated cardiomyopathy, lgmd1b, familial partial lipodystrophy, charcot-marie-tooth type 2b1, mandibuloacral disease, premature aging disorders and restrictive dermopathy. immunolabelling of lamin a/c in dominant emery-dreifuss muscular dystrophy shows no abnormality but secondary changes in laminin b1 may be seen. mutations in the acta 1 gene encoding sarcomeric skeletal muscle actin are known to be associated with a different phenotypes in forms of nemaline myopathy. mutations can cause actin accumulation which may be cytoplasmic and/or nuclear. it is not possible to make a specific histological diagnosis in some dystrophies. thus it is essential that a western blot be carried out.