Muscle biopsy is an established and widely used test that is routinely requested by Neurologists, Rheumatologists, Pediatricians and other physicians during the evaluation of patients with a neuromuscular disorder. In particular, a muscle biopsy is indicated in patients with suspected skeletal muscle disease who present with unexplained weakness, muscle cramps or pain, elevated CPK levels, or myoglobinuria. The muscle biopsy is used to confirm that there is a morphological or biochemical abnormality that correlates with or could account for the clinical symptoms. The pathological classification of these abnormalities aid in establishing specific diagnoses which in turn provide guidance for treatment and counseling.

Selection of which muscle to biopsy is based on the clinical picture. In general, it is best to sample a moderately affected muscle and to avoid extremely involved muscle that may only show minimally informative, end-stage changes such as atrophy, fatty change and fibrosis on histopathological examination. Commonly biopsied sites include the vastus lateralis, gastrocnemius, anterior tibialis, deltoid and biceps muscles. The biceps brachii in the upper extremity and the quadriceps in the lower extremity are preferred as they are less likely to be injected or otherwise injured, and their orientation of muscle is more suitable for examination. Muscles tested by EMG in the preceding month should be avoided, as they may show artifactual changes. During the procedure, the surgeon should be careful not to inject a local anesthetic inside the area of the biopsy specimen, or to otherwise traumatize or manipulate the biopsied region. The procedure is commonly performed either as an open biopsy or as a needle biopsy under local or general anesthesia. Significant complications of the biopsy procedure are uncommon and occur during the immediate post-procedure period. Pain and swelling of the biopsy site are common while development of a biopsy site hematoma and infection are possible but less frequent.

After the muscle biopsy sample is obtained, it is immediately divided. One portion remains unfixed while smaller portions are placed into formalin and glutaraldehyde fixatives. The specimens are then sent to the laboratory for specialized processing and interpretation. Routine evaluation of the muscle biopsy sample involves the examination of formalin-fixed, paraffin processed sections and unfixed frozen sections with standard histological and enzyme histochemical stains at the light microscopic level. Immunohistochemical stains are utilized for the diagnosis of various muscular dystrophies. Electron microscopic examination of the glutaraldehyde-fixed portion of the biopsy is performed when the light microscopic studies are inconclusive.

There are many skeletal muscle abnormalities that can be evaluated with muscle biopsy. One of the first questions asked after a muscle biopsy specimen is deemed to be pathologic is whether the abnormality is myopathic or neurogenic. Myopathic disorders are those that affect primarily the muscle while neurogenic disorders are caused by damage or disease of the motor neurons or peripheral nerves that innervate skeletal muscle and therefore secondarily affect the muscle. The neurogenic disorders include motor neuron disease, spinal muscular atrophies, and acquired and hereditary motor and sensory neuropathies. The myopathic disorders are numerous and include acquired forms, such as the inflammatory myopathies and toxic (drug-induced) myopathies, and hereditary forms, such as some metabolic myopathies, mitochondrial myopathies, muscular dystrophies, congenital myopathies, and myofibrillar myopathies. It is important to confirm the histopathological diagnosis of hereditary myopathies with biochemical and molecular genetic analyses.

Denervation of skeletal muscle, such that occurs in motor neuron disease, spinal muscular atrophies, and acquired and hereditary neuropathies, results in stereotypical histopathological changes of the muscle that allow for the general diagnosis of a neurogenic disorder. The presence of atrophic angulated myofibers, fiber type grouping, groups of atrophic myofibers, and target and targetoid fibers are characteristic (Figure 1). However, the specific nature of the neurogenic disorder usually cannot be determined from the muscle biopsy specimen by itself.

The inflammatory myopathies represent the largest group of acquired and potentially treatable myopathies of adults and children. The major disorders include polymyositis, dermatomyositis, sporadic inclusion body myositis, and necrotizing autoimmune myositis. Polymyositis presents mainly after the second decade while dermatomyositis affects both children and adults. Inclusion body myositis occurs more frequently in men and in individuals over the age of 50 years. Chronic proximal and often symmetrical muscle weakness is common to all of these disorders although distal weakness may predominate in inclusion body myositis.

Figure 2. Skeletal muscle with endomysial infiltrates of lymphocytes and reactive changes, consistent with polymyositis. H&E stain.

Microscopic examination of a muscle biopsy is essential for establishing a definite diagnosis of an inflammatory myopathy. In polymyositis, CD8+ cytotoxic T cells invade MHC-I expressing muscle fibers, leading to fiber necrosis (Figure 2). Dermatomyositis is characterized by complement mediated microangiopathy, with destruction of capillaries, and presence of inflammatory cells in the perifascicular region. Inclusion body myositis is identified by the presence of intracellular vacuoles that contain amyloid in muscle cells expressing MHC-I, with invasion by cytotoxic T cells (Figure 3). Necrotizing autoimmune myositis is characterized by a macrophage-mediated muscle fiber injury in the absence of T cells.

A variety of drugs can cause myopathies with weakness and muscle pain even when used in prescribed therapeutic doses (Figure 4). Some of these offending medications appear to have a direct myotoxic effect, sometimes resulting in widespread muscle necrosis. Statins, fibrates, epsilon-aminocaproic acid, emetine, heroin, amiodarone, iopamidol, zidovudine, and proton pump inhibitors have been observed to exhibit myotoxic side effects.

Figure 5. Type 2 myofiber atrophy. ATPase pH9.4 stain.

Other medications appear to indirectly affect muscle by inducing a metabolic or inflammatory disturbance. Diuretics, purgatives, liquorice, fluoroprednisolone (Figure 5), carbenoolone, amphotericin, D-penicillamine, L-tryptophan, L-DOPA, phenytoin, cimetidine, statins, and aluminum containing vaccinations are examples of this group. Interference with the function of the neuromuscular junction producing drug-induced myasthenic syndromes has been described for a large number of drugs including a variety of antibiotics, cardiovascular drugs, central nervous system acting drugs and anti-rheumatic agents. Muscle biopsy is rarely specific in these cases but may be useful when reviewed in full clinical context with detailed medication history. The most common drug-induced myopathy today is the one induced by statin. Statins can trigger a toxic myopathy or an inflammatory myopathy that persists even after the drug is withdrawn and requires immunotherapy. A muscle biopsy is fundamental to distinguish between the two and direct proper treatment.

Disorders of glycogen, lipid (Figure 6), and purine metabolism are included in the hereditary metabolic myopathies. Patients with these disorders present with exercise associated muscle weakness, pain, cramps, and myogloblinuria. Acid maltase (Pompe’s disease), myophosphorylase (McArdle’s disease), and phosphofructokinase (Tarui’s disease) deficiencies are common glycogen storage disorders of skeletal muscle that can be diagnosed with muscle biopsy by demonstration of abnormal accumulations of glycogen or the corresponding absence of enzymatic activity on histological sections. Carnitine deficiency results in excessive lipid droplet accumulations within muscle cells. Patients with myoadenylate deaminase deficiency have mild and non-specific symptoms.

Mitochondrial disorders are among the most common inherited diseases affecting approximately 1 in 5000 of the population. Neuromuscular features often dominate the clinical picture but multisystem involvement is not uncommon. Myopathy in mitochondrial disease commonly presents as a slowly progressive proximal limb-girdle weakness. Multisystemic clinical syndromes include chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS), mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), and POLG (DNA polymerase gamma) disease. Histological, biochemical, and molecular genetic analyses of muscle biopsies are important to determine the diagnoses of these disorders.

The muscular dystrophies are a group of hereditary disorders in children and adults that present with progressive weakness and wasting of skeletal muscle. Duchenne (Figure 7), Becker, limb girdle, congenital, Emery-Dreifuss, myotonic, fascioscapulohumeral, and oculopharyngeal muscular dystrophies are the most common examples among this group of disorders. The causative gene defects and their corresponding protein alterations have been characterized for many of these disorders. Immunohistochemical staining on muscle biopsies and immunoblotting methods to detect altered protein expression patterns and molecular genetic studies to detect the presence of mutations in specific genes play crucial roles in the diagnosis of these disorders.

Figure 8. Central core myopathy showing characteristic alteration of sarcoplasmic staining. NADH-tetrazolium reductase stain.

The congenital myopathies are rare and usually present at birth or in early childhood with hypotonia and muscle weakness. These clinically, genetically and pathologically heterogeneous disorders were originally defined by characteristic histopathological features present in muscle biopsies. Nemaline myopathy, central core disease (Figure 8), multi-minicore disease, myotublar myopathy, centronuclear myopathy, and hyaline body myopathy are the most common examples of the congenital myopathies. Gene defects have been identified for many of the congenital myopathies. Because of the distinctive histopathological features, muscle biopsy plays an important role in the diagnosis of this diverse group of childhood muscle disorders.

The myofibrillar myopathies are another group of muscle disorders that were defined by distinctive histopathological features, in this case by the presence of unusual cytoplasmic inclusions described as spheroid bodies, sarcoplasmic bodies, cytoplasmic bodies and granulofilamentous material. Accumulation of the protein desmin is common. Electron microscopic examination shows myofibrillar dissolution associated with disintegration of the Z-disk and accumulation of myofibrillar degradation products. These disorders have a variable age of onset, with most occurring in adulthood. The presenting symptom is slowly progressive weakness. Familial cases have been described and show a predominantly autosomal dominant pattern of inheritance. Early diagnosis through a muscle biopsy is important because many of these patients have a cardiomyopathy and may suffer sudden death or develop respiratory insufficiency. The disease is suspected based on the accumulation seen in the biopsy and confirmed with molecular studies which identify mutation of several genes that encode Z-disk-associated proteins.