Muscle Biopsy Evaluation

Muscle Biopsy Technique

The two techniques for obtaining a muscle biopsy specimen include the traditional open biopsy and the needle biopsy (3,4). Either technique can be performed under local anesthesia; however, most clinicians in the United States use general anesthesia for open biopsies and local anesthesia for needle biopsies.

Muscle Biopsy Site Selection

Selection of the muscle is based on distribution of muscle weakness found clinically in addition to elec­trodiagnostic findings, if obtained. In a dystrophic myopathy, the muscle biopsy should be clinically affected, but not so severely affected that it is largely replaced by fat and connective tissue with minimal residual muscle fiber present for evaluation. The inser- tional activity on EMG or muscle imaging studies can be helpful in this respect. Sufficient normative infor­mation about proportional fiber type and fiber diam­eter should be available with age-appropriate norms. A diagnosis of congenital myopathy with fiber-type disproportion cannot be made without careful con­sideration of the normal fiber-type predominance in a given muscle. For example, the vastus lateralis is two-thirds type II fibers (with equal proportions of type IIa and type IIb fibers) and one-third type I fibers. The anterior tibialis, on the other hand, contains a predominance of type I fibers, and the anconeus is mostly type I fibers. In addition, some muscles, such as the quadriceps and biceps, have longitudinally running fibers that facilitate orientation of the speci­men for preparation of cross-sectional slices.

For routine diagnostic studies, the vastus lat­eralis muscle in the lower extremity and the triceps, biceps, or posterior deltoid in the upper extremity are often preferred. When proximal muscles are severely affected or only distal muscles are involved, the exten­sor carpi radialis or anterior tibialis muscles are often biopsied.

HistologyZHistochemistry

The histopathologic study is likely to provide informa­tion on whether the basic disease process is primarily a myopathy or a neurogenic process. In some instances, the diagnosis of specific disorders (such as dystrophic myopathy or inflammatory myopathy) is delineated. When analyzing paraffin sections, basic pathologic reactions of muscle may include fiber necrosis, cen­tral nuclei indicative of regeneration, abnormalities of muscle fiber diameter (atrophy, hypertrophy, abnor­mal variation, and fiber size), fiber splitting, vacuo­lar change, inflammatory infiltrates, and proliferation of connective tissue/fibrosis. A dystrophic myopathy frequently is characterized by the presence of normal fibers, hypertrophied fibers, degenerating fibers, atro­phic fibers, regenerating fibers, and connective tissue and fatty infiltration. Neurogenic changes may be char­acterized by small or large groups of atrophic fibers, with or without target fibers, and frequently by hyper­trophy of the nonatrophic fibers. Pyknotic nuclear clumps, target fibers, and darkly staining angulated fibers are consistent with a neurogenic process. Red- rimmed vacuoles suggest inclusion-body myositis. Ragged red fibers are consistent with a mitochondrial myopathy.

Frozen sections can be assessed with standard H & E NADH, ATPase, and trichrome stains. Other stains include include PAS (for glycogen), Oil Red-O (for lipid), congo red (for amyloid), acetylcholinesterase (for motor end plates), myophosphorylase (for McArdle's), acid phosphatase (for type II glycogenosis), cytochrome oxidase C, succinic dehydrogenase (mitochondrial enzymes), and specific immunostains for dystrophin and sarcolemmal membrane associated proteins.

A variety of histochemical stains, including NADH stains and ATPase stains, at different pH values can be used to differentiate fiber types (types I, IIa, IIb, IIc). Based on the histochemical analyses, information is obtained about pattern of fiber types (eg, normal predominance, fiber type predominance, selective fiber type involvement, or reinnervation evidenced by fiber type grouping), analysis of muscle fiber diam­eters (eg, fiber hypertrophy or atrophy, increased var­iability in fiber diameter, or denervation atrophy with narrow range of diameters among atrophic and nona- trophic fibers), or alterations in the muscle fiber (eg, central nuclei, necrosis, splitting or branching, regen­eration or the presence of a variety of other accumu­lations and fiber alterations, both specific to certain conditions and nonspecific).

Congenital myopathies are a group of structural myopathies whose diagnosis is based on classic histo­logic characteristics seen on muscle biopsy (eg, centro- nuclear or myotubular, central core, nemaline rod, and fiber type disproportion myopathies).

Immunoblotting and Immunostaining

Imunoblotting of a muscle sample provides informa­tion about amounts of specific muscle protein, such as dystrophin or other structural proteins important in maintaining structural integrity of the muscle mem­brane. Immunoblotting can be performed with as lit­tle as 10 mg of frozen tissue. Quantitative dystrophin analysis using Western blot technique can differentiate DMD from BMD and thus help determine the progno­sis in a young symptomatic patient—information not precisely determined by standard molecular genetic analysis of the dystrophin gene.

The progressive loss of muscle fibers evident in muscular dystrophy is now thought to be caused by primary muscle sarcolemmal membrane abnormalities due to inherited structural abnormalities (abnormal molecular weight, deficiency, or absence) of dystro­phin or dystrophin-associated transmembrane gly­coproteins. Membrane instability leads to membrane injury from mechanical stresses, transient breaches of the membrane, and membrane leakage. Ultimately, after multiple cycles of degeneration and regeneration, irreversible muscle cell death occurs. The muscle fiber is then replaced by connective tissue and fat, and this fibrotic replacement of the muscle may be exceedingly aggressive. This has given rise to the concept of dis­eases of the dystrophin-glycoprotein complex. Primary genetic abnormalities lead to abnormalities of intra­cellular dystrophin, transmembrane sarcoglycans, or transmembrane dystroglycans. An abnormality in the muscle protein merosin, located in the extracellular matrix, gives rise to one of the forms of congenital muscular dystrophy. Immunoblotting and/or immuno- fluorescent staining of the proteins of the dystrophin- glycoprotein complex allows many LGMD patients to be subtyped.

Electron Microscopy

Electron microscopy (EM) is used to evaluate ultra- structural changes of muscle fiber organelles/internal components, as well as changes in the muscle fiber. At times, this may provide additional complimentary information to the histologic and histochemical assess­ment of muscle fibers that may be diagnostically rel­evant. For example, ultrastructural alterations of the mitochondria may provide important information and direct additional metabolic studies in the workup of mitochondrial myopathy. In a congenital structural myasthenic syndrome, ultrastructural alterations at the neuromuscular junction may be present by EM, either presynaptically or postsynaptically.

Metabolic Studies

Depending on clinical suspicion and histologic and ultrastructural changes on muscle biopsy, additional metabolic studies may be obtained to evaluate for the presence of metabolic myopathies, including glycog­enoses, lipid disorders, or mitochondrial myopathies.