You are in: eMedicine Specialties > Neurology > Specialized Neurodiagnostic Tests Muscle Biopsy and the Pathology of Skeletal MuscleArticle Last Updated: Nov 2, 2006AUTHOR AND EDITOR INFORMATIONSection 1 of 11
 
Author: Roberta J Seidman, MD, Director of Neuropathology, Clinical Associate Professor, Department of Pathology, Stony Brook University Medical Center Roberta J Seidman is a member of the following medical societies: American Academy of Neurology, American Association for the Advancement of Science, and American Association of Neuropathologists Editors: Milind J Kothari, DO, Professor and Vice-Chair for Education and Training, Department of Neurology, Pennsylvania State University College of Medicine; Consulting Staff, Department of Neurology, Hershey Medical Center; Francisco Talavera, PharmD, PhD, Senior Pharmacy Editor, eMedicine; Glenn Lopate, MD, Associate Professor, Department of Neurology, Division of Neuromuscular Diseases, Washington University School of Medicine; Chief of Neurology, St Louis ConnectCare, Consulting Staff, Barnes Jewish Hospital; Selim R Benbadis, MD, Professor of Neurology, Director of Comprehensive Epilepsy Program, Departments of Neurology and Neurosurgery, University of South Florida College of Medicine, Tampa General Hospital; Nicholas Lorenzo, MD, Chief Editor, eMedicine Neurology; Consulting Staff, Neurology Specialists and Consultants Author and Editor Disclosure Synonyms and related keywords: muscle biopsy, skeletal muscle pathology, muscle pathology, myopathy, encephalomyopathy, neuropathy, muscular dystrophy, dystrophinopathy, myofiber, myositis, dermatomyositis, polymyositis, inclusion body myositis, neuromuscular disease, neuromuscular pathology, neurogenic disorder, muscle fiber, glycogen storage disease, mitochondrial myopathy, congenital myopathy INTRODUCTIONSection 2 of 11
 
Muscle biopsy plays an integral role in evaluation of the patient with neuromuscular disease. With occasional exceptions, it is an essential element in the assessment of a patient with suspected myopathy. In addition to being indispensable for the evaluation of muscle diseases, muscle biopsy is also involved in the evaluation of suspected neuropathic disease, particularly in the distinction of an atypical neurogenic disorder from a primary myopathic one, and for diagnosis of a variety of systemic disorders. The surgical procedure to obtain a muscle biopsy is relatively simple and poses little risk to the patient, but it is a specialized procedure and must be performed properly to optimize the information it can yield for the benefit of the patient. The clinician must first arrive at a rational differential diagnosis by synthesizing information obtained from the clinical history, physical examination, and laboratory and electrodiagnostic studies. This information is used to influence the details of each procedure. The choices of the right time for biopsy, which muscle to select, how many specimens to obtain, and how to handle them immediately following excision are individualized for each patient on the basis of clinical findings. After the biopsy arrives in the pathology laboratory, it undergoes a complex series of studies. The pathologist uses knowledge of the clinical features to assist in interpretation of the constellation of pathologic findings in the biopsy and to help determine whether additional studies are warranted for a given patient. Therefore, muscle biopsy is somewhat complex in that an optimal outcome requires coordination of the clinician, surgical team, pathologist, and technical staff in the pathology laboratory. As muscle biopsy results are often interpreted at specialized centers, a courier service also may need to be involved in the process; this is yet one more link in the chain from procedure to diagnosis. Every muscle pathologist has a series of stories about biopsy procedures that were performed improperly. Many of these situations were salvaged and yielded diagnoses, but on occasion, the specimen was inadequate for the diagnosis under consideration or some aspect of the procedure was performed so improperly that the procedure had to be repeated. Occasional situations exist when the biopsy must be repeated for precise diagnosis and no one is at fault. Some situations in which this may occur include the following:
Unsuitable, suboptimal, or inadequate biopsy specimens usually can be attributed to lack of planning and forethought; no excuse exists for this situation. The single most important point to remember when one contemplates muscle biopsy (also the single most important point of this article) is to call the pathology laboratory in advance for advice on how to proceed. INDICATIONS FOR MUSCLE BIOPSYSection 3 of 11
When a clinical diagnosis of myopathy is considered, muscle biopsy is required (with occasional exceptions). Muscle biopsy is an integral part of the initial evaluation of a patient with possible muscle disease, or myopathy. At present, muscle biopsy is absolutely essential part of the diagnostic investigation of most categories of muscle diseases, including inflammatory and many metabolic and congenital myopathies, as well as most of the muscular dystrophies. Today, the most specific and definitive effective therapies are for inflammatory myopathies. Performing muscle biopsy to diagnose these disorders before the start of therapy is of critical importance for several reasons:
Repeat muscle biopsy is occasionally indicated to evaluate the patient with known inflammatory myopathy who, after improvement with steroid therapy, has increasing weakness. Biopsy findings can help distinguish between exacerbation of the disorder and steroid myopathy. For other disorders with therapeutic options less definitive than those for inflammatory myopathies, several reasons underlie the importance of obtaining a precise diagnosis:
One common indication for muscle biopsy is to distinguish between myopathy and neuropathy. Their classic presentations are clearly distinct; however, in practice, their histories and physical and laboratory findings often overlap. Neuropathy and myopathy may also coexist, making a diagnosis based on clinical findings alone even more difficult than it already is. WHEN A MUSCLE BIOPSY IS NOT INDICATEDSection 4 of 11
The only exceptions to the requirement for muscle biopsy for accurate diagnosis of possible myopathy are suspected dystrophinopathies (also known as Duchenne or Becker muscular dystrophies), some rare congenital and limb-girdle dystrophies (Vogel, 2005), myotonic dystrophy, certain mitochondrial disorders, periodic paralyses, and endocrine myopathies. Dystrophinopathies and certain other muscular dystrophies Recent advances in molecular genetics have eliminated the need for muscle biopsy in some patients with dystrophinopathies. In these patients, mutations, most commonly deletions, can be demonstrated in the gene for dystrophin, a structural protein of skeletal muscle located on the X chromosome. The gene for this protein is extremely large (2 million base pairs); this size usually precludes searching the entire gene for point mutations. Muscle biopsy is still required for definitive diagnosis in approximately 40% of patients with these disorders in whom genetic testing based on current methods is uninformative. Genetic testing is available for fascioscapulohumeral dystrophy and Perlecan deficiency (Schwartz-Jampel syndrome). Therefore, muscle biopsy, for which findings are nonspecific, is generally not indicated to diagnose these disorders. Myotonic dystrophy Myotonic dystrophy now can be diagnosed definitively by means of genetic testing to look for the characteristic increase in triplet repeats in a gene for a protein kinase. This method is far superior to muscle biopsy for diagnosing myotonic dystrophy because the findings on muscle biopsy are not specifically diagnostic; however, they may be generally helpful to support this diagnosis and exclude other disorders. Periodic paralyses Periodic paralyses, uncommon disorders that result from mutations in a variety of genes for muscle membrane ion channels, have unique clinical, biochemical, and electrodiagnostic features. They lack diagnostic findings on muscle biopsy, though dilatation of the T tubule system is found in some patients with hypokalemic periodic paralysis. Muscle biopsy can also demonstrate a nonspecific myopathic picture in these disorders. Typically vacuoles are seen on the biopsy sample. Endocrine myopathies Myopathy can be a feature of disorders of thyroid, parathyroid, and adrenal function. The correct way to diagnose endocrine myopathies is to recognize their clinical presentations and follow this with serologic testing for appropriate components of the hypothalamic-pituitary–endocrine organ axis. Myotonic dystrophy, periodic paralyses, and endocrine myopathies are not considered further in this article. CLINICAL AND LABORATORY FEATURES OF NEUROMUSCULAR DISEASESection 5 of 11
Clinical features Few findings in a muscle biopsy are pathognomonic for a specific diagnosis. Instead, a typical muscle biopsy sample presents a constellation of findings that must be interpreted in light of the clinical history. Therefore, the pathologist must know the clinical features of a given patient to properly assess the clinically significance of the histologic findings in a muscle biopsy sample and to decide whether to pursue additional special studies. The clinical hallmark of neuromuscular disease, whether of neurogenic or myopathic origin, is weakness. Weakness is manifested in age-related variations. For example, in utero weakness may be expressed as decreased fetal movements and may be recognized by a woman who has had previous pregnancies. In the neonatal period, the infant may be floppy. In later infancy and during the toddler years, delay in an acquisition of motor-developmental milestones is likely the major sign of weakness. From childhood through adulthood, diminished muscle power is a characteristic clinical feature of neuromuscular disease. The classical clinical features of myopathy include the following:
Variation of strength with exercise can occur in some patients with muscle disease. This can mean either decremental or incremental change in strength with activity that would not result in this change in a healthy individual.
In contrast to myopathy, the classic clinical features of peripheral neuropathy include the following:
In their conventional clinical presentations, distinguishing muscle disease from peripheral nerve disease is a straightforward matter. In practice, this is not always simple. Several reasons explain why it may be difficult to determine whether a patient has neuropathy or myopathy on clinical evaluation:
Laboratory studies The serum creatine kinase (CK) level is the single most important blood value to obtain when myopathy is being considered. A representative reference range is 24-196 IU/L. The CK level is useful, but not definitive, in determining whether neuropathy or myopathy is present. Extremely elevated levels of CK (>1000 IU/L) almost always indicate muscle disease. Mildly elevated levels (200-600 IU/L) can be observed in either entity, and normal levels are less likely to be found in the patient with myopathy. Patients with myopathy and severely reduced residual muscle mass may have a normal serum CK level. The serum aldolase level may be helpful in further suggesting myopathy. Because of its longer half-life in serum, the serum aldolase level may be elevated in the setting of myopathy when the CK level is normal. Electrodiagnostic studies Electrodiagnostic studies are often extremely useful in determining whether a neuropathic, myopathic, or mixed disorder is present. Changes in nerve conduction velocities and the compound muscle-action potential can be present in neurogenic disorders. Electromyography (EMG) shows different findings in neurogenic and myopathic disorders and can be useful to help distinguish them. Avoiding EMG in a muscle that will undergo biopsy is of critical importance. EMG inflicts damage on the muscle that interferes with proper interpretation of biopsy results for 1-2 months. In patients with suspected myopathy, needle EMG should be performed on only 1 side. TECHNICAL CONSIDERATIONSSection 6 of 11
The technical issues that must be addressed by the physicians involved are the proper selection of a muscle for biopsy, the biopsy procedure and immediate handling of the tissue in the operating room, and studies performed on the biopsy sample. Selection of a muscle for biopsyBiopsy of a clinically involved muscle is important. Some disease processes have a patchy, rather than a diffuse, distribution. To increase the likelihood of sampling the pathologic process, selecting a symptomatic muscle is important. Select a muscle on the basis of the expected distribution of the leading clinical diagnosis. For example, if the leading diagnostic consideration is polymyositis, select a proximal muscle such as the vastus lateralis of the quadriceps, for biopsy. Biopsy a muscle that is not too weak and atrophic (see Media file 1). In this situation, obtaining a sample of end-stage muscle is a risk. In end-stage muscle, loss of myofibers is severe, and they are replaced by fibrovascular and adipose tissue, without residual clues to the process that caused the muscle damage. On occasion, only the presence of a muscle spindle confirms that the specimen is a biopsy sample of skeletal muscle (see Media file 2). Biopsy procedure and immediate handling of tissueThe specimens required and the preferred method of handling may vary among medical centers. Consulting the center that will receive the biopsy sample is essential to learn exactly what is required and the preferred method of handling and shipping the tissue. However, the surgeon must ultimately determine the precise surgical method for each patient. Consider the information below a general guide. These considerations should be tailored to meet the needs of the individual patient and institution. The typical muscle biopsy sample consists of 2 specimens: fresh and fixed. In certain special clinical circumstances, a third sample is required for biochemical or genetic analysis. On occasion, a muscle biopsy sample consists only of a single fresh specimen obtained by means of needle biopsy. This method provides a specimen of limited size. However, this procedure may be the method of choice, as follows:
Fresh specimen A fresh specimen (see Media file 3) is used for histochemical studies in all patients and for immunofluorescence in selected patients, when indicated. It should measure approximately 0.5 X 0.5 cm in cross-section, or 0.5 cm in diameter, and 1 cm in length along the longitudinal axis of the muscle fibers. The sample can be sent to the laboratory on saline-moistened gauze in a sealed container on ice. This technique keeps the specimen cold but does not cause it to freeze. The tissue should not be immersed in sodium chloride solution because this leads to the formation of ice crystals in the myofibers when the sample is frozen. When the specimen arrives in the laboratory, the technologist mounts it in gum tragacanth in the appropriate orientation and snap freezes it in isopentane chilled in liquid nitrogen. Frozen cryostat sections are cut from this sample. In the optimal situation, this fresh specimen is rapidly transported to the laboratory for processing to prevent the tissue from losing any of its enzymatic reactivity or immunogenicity for immunohistochemical studies. However, in most situations, refrigeration of the specimen is probably adequate for most necessary studies after an overnight delay or even a delay of a few days (though a delay longer than overnight is definitely not recommended). Fixed specimen A fixed specimen (see Media file 4) is used for routine microscopy and possible electron microscopy (EM). EM is reserved for special situations in which it may substantially contribute to the diagnosis. The fixed specimen should have dimensions similar to those of the fresh specimen. It must be handled properly to maintain orientation of the fibers, to keep the fibers at rest length, and to prevent contraction. The sample is optimally removed from the patient by using a special clamp designed for this purpose, such as the 10-mm Rayport clamp (see Media file 4). A segment of muscle of the desired dimensions is dissected. The bottom portion of the clamp is inserted below this segment of muscle in the posts-up position so that the length of the fibers runs perpendicular to the jaws of the clamp. After the bottom portion of the clamp is inserted, the top portion of the Rayport clamp can be folded over and the holes fitted onto the bottom posts. The surgeon then excises the fibers 1-2 mm external to the clamp. The specimen is placed in fixative. The preferred fixative is 4% paraformaldehyde. If a special clamp is not available for the procedure, alternative methods of obtaining the fixed specimen are available. It can be obtained in a manner similar to how fresh specimen are obtained and sent to the laboratory fresh, where the technologists perform the procedures needed for immobilization and fixation. Another method involves suturing the specimen to a tongue blade for immobilization prior to fixation. If paraformaldehyde is not available, 10% neutral buffered formalin is an acceptable alternative for most light microscopic purposes. If, however, EM is desired, the specimen initially fixed in paraformaldehyde has ultrastructural preservation better than that of a sample fixed in formalin. Paraformaldehyde is also superior to formalin for immunohistochemical studies for surface markers. If paraformaldehyde is not available and EM is anticipated, a small portion of muscle can be placed directly in 3% glutaraldehyde at the time of biopsy for submission to the EM laboratory. This sample should be maintained at rest length before it is immersed in the fixative to prevent contraction of the muscle. The specimen placed in glutaraldehyde must be small because glutaraldehyde penetrates tissue slowly. After overnight fixation, the technologist separates a small section and submits it in glutaraldehyde for embedment for EM. The remainder is submitted for paraffin processing, with the end of the specimen removed and placed in cross-section and most submitted in longitudinal section. Optional additional fresh specimen An additional fresh specimen is required in selected patients when the presence of a metabolic myopathy or some of the muscular dystrophies is strongly suspected. The sample may be sent to specialized laboratories for assessment of specific enzymatic activities (eg, mitochondrial enzymes) or for measurement of specific protein constituents in muscle (eg, protein dystrophin). This specimen should be of dimensions similar to those of the other specimens and should be snap frozen in liquid nitrogen at the location of the procedure because of the lability of some of these cellular constituents. Store it in a freezer at -70°C. Alert laboratory personnel in advance if the need for this type of specimen is anticipated. Many medical centers are not equipped to perform this service. Studies performed on the biopsy sampleLight microscopy The actual methods for performing the stains can be found in histology textbooks and pathology laboratory manuals. Immunohistochemical stains must be performed by a laboratory set up for this purpose. The manufacturer provides instructions for use of each individual antibody. Frozen sample For every muscle biopsy, a battery of stains is performed on the frozen sample in addition to the routine hematoxylin and eosin (H-E) stain. These assist in the evaluation of neurogenic or other types of atrophy, metabolic diseases, and demonstration of structural changes or inclusions diagnostic of specific disorders. These studies cannot be performed on material that has been fixed and embedded in paraffin. After review of the initial battery of stains, if the clinical and pathologic findings warrant, the pathologist may decide to perform additional special stains. The battery of stains performed on every biopsy includes the following:
Additional special stains that can be performed on the frozen sample when the clinical history and findings in the initial battery of stains warrant include the following:
Paraffin specimen Paraffin sections are usually stained with H-E. This specimen consists of a large surface of fibers oriented in the longitudinal direction and a piece in cross-section. A relatively large amount of tissue usually is exposed in each paraffin section; therefore, this specimen is extremely useful for evaluating for processes with a nonuniform distribution (eg, inflammatory myopathies, vasculitis). The fixed and paraffin-embedded specimen maintains more cellular detail than the frozen specimen, making it the preferred sample for detecting subtle evidence of myofiber necrosis, for determining the type of inflammatory infiltrate present, and for examining the structure of vessels walls. When indicated, special stains can be performed on the paraffin specimen. These include the following:
Electron microscopy While a small sample of every muscle biopsy should be set aside for possible EM, performing EM muscle biopsy samples is not a routine procedure. It is reserved for selected circumstances in which the pathologist determines that EM has the potential of contributing significantly to determining a specific diagnosis. The pathologist uses knowledge of the clinical history and findings of light microscopic studies to decide if EM is indicated. EM is costly, time-consuming, and requires a specialized laboratory and technical expertise. Some technical aspects of EM are described below.
NORMAL SKELETAL MUSCLESection 7 of 11
Basic structure and terminology A layer of dense connective tissue, which is known as epimysium and is continuous with the tendon, surrounds each muscle (see Media file 5). A muscle is composed of numerous bundles of muscle fibers, termed fascicles, which are separated from each other by a connective tissue layer termed perimysium. Endomysium is the connective tissue that separates individual muscle fibers from each other. Mature muscle cells are termed muscle fibers or myofibers. Each myofiber is a multinucleate syncytium formed by fusion of immature muscle cells termed myoblasts. Sarcoplasm, the cytoplasm of each myofiber, is occupied largely by the contractile apparatus of the cell. This is composed of myofibrils arranged in sarcomeres, which are the contractile units of the cell. The sarcomeres contain a number of proteins, including alpha actinin, which form a major portion of the Z band, and actin and myosin, which form the thin and thick filaments, respectively. The remainder of the sarcoplasm, located between the myofibrils, is termed the intermyofibrillar network and contains the mitochondria, lipid, glycogen, T-tubules, and sarcoplasmic reticulum. T tubules and sarcoplasmic reticulum are responsible, respectively, for conduction of electrical signals from the cell surface and intracellular storage and release of calcium required for contraction to occur. Myofiber types The 2 basic myofiber types are type 1 and type 2. The designation of these types is based on their physiologic properties, which are correlated with their cellular structural specializations and are reflected in their histochemical properties (see Media file 6). Type 1 myofibers are the slow fibers. Physiologists refer to them as slow-oxidative, or SO, fibers. They have a slow contraction time following electrical stimulation, and they generate less force than do type 2 myofibers. If the response of a muscle to the application of gradually increasing loads is measured, the slow fibers are recruited first. They are used for sustained, low-level activity. To accomplish this, they are equipped with numerous large mitochondria and abundant intracellular lipid for oxidative metabolism. Type 2 myofibers are the fast fibers. Physiologists call these the fast-glycolytic, or FG, fibers. They have a rapid contraction time following stimulation. If the response of a muscle to the application of gradually increasing loads is measured, the fast fibers are recruited late. They are used for brief-duration activity in carrying heavy loads and are specialized for anaerobic metabolism. These fibers contain smaller, less numerous mitochondria, less lipid, and have higher glycogen stores than type 1 fibers. The subgroups of type 2 fibers are not discussed here. Each muscle has a characteristic ratio of type 1 to type 2 myofibers. For example, in the vastus lateralis, the most commonly biopsied muscle, more than 50% of the fibers, as many as two thirds, are expected to be type 2 myofibers. In the deltoid muscle, another muscle commonly evaluated with biopsy, typically the balance favors type 1 myofibers. In normal muscle, the 2 myofiber types are interspersed in a random interdigitating pattern. The 2 myofiber types are normally similar in size. Information about changes in the myofiber types in a muscle biopsy often provides significant clues in making the diagnosis. Different pathologic processes alter the ratio of the myofiber types and their distributions in the muscle and may selectively affect the size of 1 type or the other or of both equally. Innervation of a particular muscle fiber determines whether it is type 1 or type 2. Therefore, if the type of motor neuron innervating a myofiber is changed, that myofiber acquires a new phenotype from its new innervation. Pathologists take advantage of this fact to evaluate for evidence of neurogenic disease of muscle. In a muscle in which denervation has been followed by reinnervation due to sprouting of residual viable motor neuron terminals, groups of myofibers of a single type are present instead of the random interdigitation normally found. Histology With frozen-section H-E, a cross-section of a frozen sample of normal skeletal muscle stained with H-E (see Media file 7) shows several fascicles surrounded by and separated from each other by a thin layer of perimysium. The muscle fibers are of relatively uniform size and shape, with nuclei located at the periphery of the cell. In normal muscle, less than 3% of fibers should have internal nuclei (located in the center of the fiber). The fibers fit together in a mosaic pattern. At high power (see Media file 8), the endomysium separating the myofibers can be observed as normally so thin and delicate it is almost invisible and the contiguous myofibers appear to have almost no space between them. The sarcoplasm is relatively uniform throughout the cell. On the section stained with NADH (see Media file 9), which stains predominantly mitochondria in the intermyofibrillar network, the type 1 myofibers are darker than type 2 myofibers. In normal muscle, the stain is distributed fairly uniformly throughout the sarcoplasm. High power (see Media file 10) allows observation of the distribution of the stain in a punctate pattern, where it is localized mostly to the mitochondria in the intermyofibrillar network. On the frozen-section fiber-typing stains in Media file 11, which are treated with the stain for myosin ATPase at pH 10.5 (actual pH varies among laboratories), type 2 myofibers are stained brown, and type 1 fibers are stained pink with an eosin counterstain to make them visible. This section demonstrates the normal, random, almost checkerboard distribution of the 2 types of myofibers. The same stain, performed at a pH of 4.3, demonstrates staining of the type 1 myofibers, so the slide would have exactly the reverse pattern of that seen on the image. An alternative to the technically difficult myosin ATPase stain is the immunohistochemical stain for myosin heavy chain. Media file 12 shows the stain for myosin heavy-chain slow, which stains the type 1 myofibers. In Media file 13, a section from the same patient is stained for myosin heavy-chain fast, which stains the type 2 myofibers. With frozen-section PAS staining, PAS is distributed fairly uniformly across a normal myofiber (see Media file 14). It is located mostly in the intermyofibrillar network, which contains much of the intracellular glycogen content. Normally, the type 2 myofibers stain darker with this stain than type 1 fibers, because the type 2 fibers use glycolysis more than type 1 fibers. With the modified frozen-section Gomori trichrome stain (see Media file 15), the myofibers and connective tissue stain slightly different shades of blue-green. Nuclei normally are red. The intermyofibrillar network exhibits punctate red staining, which normally is inconspicuous. With the frozen-section lipid Sudan Black stain (see Media file 16), intracellular lipid appears brown-black and is distributed throughout the intermyofibrillar network. Type 1 myofibers stain darker than the others because of their increased reliance on oxidative metabolism. For this reason, type 1 fibers have a greater lipid content than the type 2 myofibers, which rely more on anaerobic than oxidative metabolism. Paraffin section: The paraffin section is stained with H-E. In a low-power view of the paraffin section (see Media file 17), the fibers are seen in longitudinal section, forming an array of fibers lined up in parallel. At high power (see Media file 18) in normal myofibers, the striations, which are formed by the sarcomeres, are demonstrated readily. One of the earliest changes in myofiber necrosis is loss of the striations. On occasion, this subtle but important finding may be the only pathologic change in a sample. EM: Normal muscle in longitudinal section (see Media file 19) reveals the remarkable ultrastructural architectural order of skeletal muscle. The myofibrils are the contractile machinery of the cell and are arranged in units, the sarcomeres. The boundary of each is a thin dark line, the Z disk or Z band. This is the anchor for the thin filaments, which are actin. The thin filaments are best seen in the pale zones of the sarcomere, known as the I band, adjacent to each Z disk. The broad darker central region of each sarcomere is the A band, formed mostly by the overlap of the thick myosin filaments and the thin filaments. In the center of each sarcomere is a thin dark band termed the M band, flanked by thin pale H zones, where the thick and thin filaments do not overlap. Between the myofibrils, the sarcoplasm contains the intermyofibrillar network. Mitochondria are the moderately dense oval structures located adjacent to the I bands. At high power (see Media file 20), the intermyofibrillar network contains glycogen, which can be seen as dark granular material distributed diffusely through this area. The triads also are visible. Each triad is formed by a segment of the T tubule flanked on either side by the lateral sacs of the sarcoplasmic reticulum. The T tubule is continuous with the sarcolemma, which is the plasma membrane of the myofiber, from which it rapidly transmits the muscle cell action potential throughout the cell. Excitation transmitted from the T tubule to the sarcoplasmic reticulum is responsible for the intracellular release of calcium required for contraction that normally is sequestered from the myofibrils when the muscle cell is at rest. Distinguishing type 1 and type 2 myofibers is possible on the basis of their ultrastructural appearances. Type 1 fibers (see Media file 21) contain abundant, fairly large, prominent mitochondria and abundant fat. The mitochondria are the ovoid structures, and the fat is contained in pale homogeneous round structures. Type 2 fibers (see Media file 22) contain smaller, less abundant, less prominent mitochondria. Glycogen is abundant, and lipid is more difficult to find in these myofibers than elsewhere. These ultrastructural specializations are correlated with the functional roles of the 2 fiber types. Results of improper handling Compare the appearances of improperly handled specimens with those of properly handled specimens. The specimen shown in Media file 23 arrived at the laboratory stuck to dry ice. This improper handling caused uneven freezing of the specimen and freeze artifact, resulting in disruption of the sarcoplasmic features and a loss of information about the state of the myofibers. Media file 24 is from a case in which the muscle specimen was immersed in cold fixative without prior immobilization by a clamp. This allowed the muscle to hypercontract, producing the appearance of contraction bands, a finding that can be associated with myofiber necrosis. However, in this situation, this finding is meaningless. Media file 25 is an EM of a specimen of muscle in which the surgeon was instructed to mince the muscle sample before submitting it in glutaraldehyde. The photograph demonstrates the serious disruption of the normally orderly ultrastructural architecture of the myofiber caused by this procedure. In all 3 of these situations, improper handling of the muscle specimen at the time of biopsy in the operating room could have made it impossible to make a diagnosis. Fortunately, in each of these examples, a diagnosis was possible. INTRODUCTION TO SKELETAL MUSCLE PATHOLOGYSection 8 of 11
Interpretation of a muscle biopsy results can be a challenging task. The opinion of the muscle pathologist is often required in combination with the observation of a variety of histopathologic findings and a consideration of the clinical situation to arrive at a diagnostic formulation that makes sense for a given patient. This process can be difficult because few individual histologic findings are diagnostic of a specific disorder. For example, a biopsy may exhibit myofibers that contain empty vacuoles on H-E. This type of vacuole can be observed in a variety of settings, including glycogen storage disease, colchicine toxic myopathy, critical care myopathy, periodic paralyses, and technical artifact. The pathologist uses a variety of strategies to decide which is the most likely cause of the vacuoles in a given case. Many biopsy samples show numerous findings in varying degrees, each of which is consistent with an assortment of diagnoses. The pathologist must judge the clinical significance of each finding, decide if and how it fits with the other findings in the specimen, and determine what light to cast on the biopsy result to best fit the patient's presentation. Neurogenic changes in muscle biopsy The muscle can show neurogenic changes in disorders that affect motor neurons, including diseases of the anterior horn cell (eg, motor neuron disease), motor neuropathy, peripheral neuropathy, and disorders that affect the intramuscular nerve twigs. One of the common requests accompanying muscle biopsies is to assist in determining whether the patient has neuropathy or myopathy. (See Clinical and Laboratory Features of Neuromuscular Disease for a discussion of this issue.) Neurogenic disorders have the following characteristics on muscle biopsy:
When all of these findings are present and no other abnormalities are found in the specimen, the diagnosis of neurogenic atrophy and reinnervation is straightforward. Often, the biopsy shows a combination of neurogenic and myopathic findings (see Muscle biopsy in myopathy). These may represent myopathy that is secondary to the neuropathic process or a separate primary myopathic process. The pathologist can often surmise the correct interpretation on the basis of clinical findings, but the truth occasionally cannot be determined with certainty. Many biopsy samples with inflammation also demonstrate evidence of neurogenic change. Myogenic denervation, in which the sick muscle fibers lose their innervation, can cause this change. The inflammatory process overruns and entraps the intramuscular nerve twigs in an innocent-bystander mechanism, or the nerves are concurrently inflamed. A broad spectrum of pathologic findings is present in myopathic disorders. Each individual finding is usually nonspecific and can be found in a variety of pathologic processes. A single finding can have many connotations and, in arriving at a diagnostic impression, the pathologist must always interpret the clinical significance of the individual findings. The constellation of pathologic findings in a given clinical setting leads to the diagnosis. In contrast to the pathologic findings in neuropathy, several findings are characteristic of myopathic processes, including the following:
Numerous other ancillary findings can be found in myopathic muscle biopsy samples. Additional histologic abnormalities in the spectrum of myopathic findings include the following:
Some histologic findings mimic abnormalities but actually are normal features of skeletal muscle structure. For example, near the myotendinous junction, the muscle fibers appear fragmented, exhibit increased variability of fiber size, and have an increase in number of internal nuclei (see Media file 48). The pathologist must be vigilant not to misjudge these findings. PATHOLOGY OF MYOPATHIES BY DIAGNOSTIC CATEGORIESSection 9 of 11
Myositis, muscular dystrophies, glycogen storage diseases, mitochondrial myopathies, and congenital myopathies are 5 important groups of disorders that can be diagnosed by muscle biopsy. MyositisThe term myositis refers to inflammatory disease of muscle. In practice, this term most commonly is applied to the idiopathic inflammatory myopathies that are the main focus of this section; however, a comprehensive classification of myositis includes a variety of disorders (see Media file 49). The most common reason for performing a muscle biopsy is to evaluate for the diagnostic consideration of idiopathic inflammatory myopathy. The idiopathic inflammatory myopathies are polymyositis, dermatomyositis, and IBM. The usual clinical presentation of patients with polymyositis and dermatomyositis is a subacute course of progressive weakness affecting proximal muscle groups, occasionally with myalgia, an elevated CK level, and myopathic and irritative findings on EMG. Many patients have serum autoantibodies, some of which are associated with specific clinical syndromes. Patients with dermatomyositis usually have characteristic rashes. Dermatomyositis in adults fairly often is a paraneoplastic syndrome. Polymyositis The following are pathologic features of polymyositis:
The distribution of the pathology in polymyositis can be patchy, so obtaining normal biopsy findings are possible in a patient who has this disorder and do not exclude the diagnosis. A subgroup of patients who are believed to have polymyositis have an abnormal muscle biopsy that does not show inflammation. These patients present with a fairly rapidly evolving myopathy with severe weakness. They tend to have exceedingly high CK levels, often greater than 20,000 IU/L. Some of these patients have autoantibodies in their serologic studies, often anti–signal recognition particle (anti-SRP). The presence of these autoantibodies is the strongest evidence that this disorder is an immune-mediated disease. In this group of patients, the disease is resistant to therapy. Muscle biopsy shows the presence of scattered necrotic fibers, myophagocytosis, and other nonspecific myopathic findings, but inflammatory infiltration is absent. Dermatomyositis Pathologic findings in dermatomyositis occasionally can bear a superficial resemblance to polymyositis, but some important distinguishing features are present. In many patients, the pathology of dermatomyositis is strikingly unique. The following are pathologic features of dermatomyositis:
Inclusion-body myositis IBM is the most common myositis in patients older than 50 years. In contrast to polymyositis and dermatomyositis, which affect more women than men, IBM most often affects men. The clinical course of IBM may be more indolent than the other 2 forms of myositis, and distal muscles are involved most often in IBM. IBM is the inflammatory counterpart of a group of disorders labeled inclusion body myopathy, which includes a variety of inherited myopathies, some with characteristic distinctive clinical presentations (eg, quadriceps-sparing myopathy). These myopathies share many of the pathologic findings of IBM. The following are pathologic features of IBM:
An occasional eosinophil often can be seen in necrotizing and inflammatory myopathies. When many eosinophils are present, begin to search for a specific etiology of the myopathy, such as trichinosis (see Media file 61) or drug reaction (see Media file 62). Muscular dystrophiesMuscular dystrophy is a hereditary disease characterized by progressive degeneration of muscle. Many such diseases exist. The old classification scheme comprised Duchenne, Becker, various other eponymous dystrophies, and a group of dystrophies named for the distribution of affected muscle groups or by their mode of inheritance. As researchers determine the etiology of many of these disorders, a more pathogenetic nomenclature is evolving. Duchenne and Becker dystrophies now are classified as dystrophinopathies because they are caused by mutations in the gene for the protein dystrophin. Similarly, abnormalities of other structural proteins of skeletal muscle are being discovered, so that now, instead of limb-girdle muscular dystrophy, disorders due to abnormalities of membrane proteins, such as sarcoglycans, dystroglycans, dysferlin and others, are recognized. Abnormalities of proteins of the external basal lamina and cytoskeletal proteins are also responsible for some forms of muscular dystrophy. As steady progress is made in determining the genetic basis of many muscular dystrophies, muscle biopsy will become less important as a diagnostic tool for these disorders. Muscle biopsy is still required for most muscular dystrophies, except for approximately two thirds of patients with Duchenne and Becker muscular dystrophies in which the diagnosis can be made by genetic testing of blood samples and a few additional rare forms of muscular dystrophy. Most of the pathologic findings in the routine histologic sections of skeletal muscle in the muscular dystrophies are nonspecific myopathic findings (see Media files 31-39). Occasional features are characteristic of certain dystrophies, such as hypercontracted fibers in Duchenne muscular dystrophy (DMD) (see Duchenne muscular dystrophy) or nuclear clumps in some patients with limb-girdle dystrophy. The skeletal muscles of some patients with oculopharyngeal dystrophy (see Media file 44) contain rimmed vacuoles and eosinophilic inclusions. The specific diagnosis of muscular dystrophies can be confirmed in many patients with special immunohistochemical stains for specific proteins that are abnormal or deficient in these disorders. Many of these disorders are uncommon, so it is necessary to send the muscle biopsy to a laboratory that is prepared to perform these studies if they are indicated. If the immunohistochemistry results point to a certain disorder, the muscle specimen must then be sent to a laboratory that can perform biochemical analysis of the protein for confirmation of the immunohistochemistry and definitive diagnosis. Immunohistochemistry is not useful as a diagnostic tool for some of the uncommon muscular dystrophies, for reasons that are beyond the scope of this article. When the clinical suspicion of the presence of a muscular dystrophy is strong, make arrangements to obtain a specimen of muscle appropriate for biochemical analysis. Please see Optional additional fresh specimen for details on how to proceed. Examples of muscle biopsies from patients with Duchenne or Becker muscular dystrophies, the dystrophinopathies, or congenital muscular dystrophy (CMD) are used to illustrate the pathology of muscular dystrophies. DMD is the most common and most severe of all muscular dystrophies, occurring with a frequency of 1 case in 3500 live male births. It is caused by a mutation on the X chromosome in the gene for the structural protein dystrophin, resulting in an absence of the protein. The gene for dystrophin is large, with 2 million base pairs. Because of the size of this gene, mutations are common, and one third of patients with DMD do not have a family history of the disease. The children are generally healthy until approximately age 3 years, when they develop problems with gait, and from then on experience an inexorably progressive course. Without treatment, all patients are wheelchair bound by 12 years, and most die in the second decade. With steroid therapy, many patients remain ambulatory until the age 15 or 16 years, and survival is prolonged well into the third decade. Muscle biopsy sections from young patients with DMD illustrate the characteristic pathologic findings:
Special immunohistochemical studies for N-terminal, mid-rod, and C-terminal moieties of the dystrophin molecule can be performed. In control skeletal muscle, these studies reveal linear staining of the periphery of the myofibers, consistent with the periodic subsarcolemmal localization of dystrophin (see Media file 67). In a patient with DMD (see Media file 68), all 3 antibodies demonstrate absence of staining in all but an occasional fiber. The rare fibers that stain with antidystrophin antibody actually can produce dystrophin because of a second mutation in the dystrophin gene that restores the reading frame and allows for production of this protein. The observation that occasional fibers in patients with DMD can produce dystrophin serves as the basis for the current efforts to develop novel therapeutic interventions for this disorder. Becker muscular dystrophy Becker muscular dystrophy (BMD), a disease similar to DMD but with a later onset and a course characterized by a slower progression, is also caused by mutations of the dystrophin gene. In BMD, the mutations lead to production of abnormal dystrophin, occasionally in decreased quantities in comparison with normal skeletal muscle and in contrast to the absence of dystrophin of DMD. The course of BMD is more variable than that of DMD, which is fairly stereotypical. In BMD, the severity of the disease is correlated with the portion of the dystrophin molecule affected. The C-terminal end of dystrophin is linked to a beta-dystroglycan of the transmembrane glycoprotein complex that in turn is linked to the external basal lamina of the myofiber. If this region of the dystrophin molecule is absent, the patient experiences a severe course. In general, if the patient has a mutation affecting the mid-rod domain or a mutation affecting the N-terminal end of the dystrophin molecule, which is linked to cytoskeletal actin, the course is more indolent. The muscle biopsy illustrating BMD in this article, below, is from a 22-year-old man with a history of gradually progressive weakness that began in early childhood. At the age of 22 years, he remained ambulatory but could no longer run. Biopsy demonstrated the following:
The findings in this representative biopsy can be observed in most muscular dystrophies. The immunohistochemical findings lend specificity to the histologic diagnosis. In this situation, staining for C-terminal and mid-rod portions of the dystrophin molecule is normal (see Media file 72), but the muscle shows no staining with the antibody for the N-terminal region (see Media file 73). This is highly consistent with the diagnosis of BMD, but confirming this diagnosis by sending a skeletal muscle specimen to a laboratory for Western blot analysis is appropriate. In the situation illustrated here, muscle biopsy was not performed at a facility that could appropriately handle it for such an analysis. However, such strong correlation was present between the patient's clinical course, the findings on routine muscle biopsy, and the immunohistochemical findings that the correct diagnosis was not in doubt. Extensive research has led to a detailed model of the structure of the myofiber membrane and has revealed many of the components of the transmembrane glycoprotein complex. It contains several proteins known as sarcoglycans and others termed dystroglycans. Mutations of each of these proteins, as well as others not mentioned here, now are known to be responsible for many forms of muscular dystrophy. Congenital muscular dystrophy CMD is clinically evident from the neonatal period. Multiple disorders probably fall within this category. In one third of patients, CMD is caused by an abnormality of laminin alpha-2, also known as merosin, which is a component of the basal lamina of skeletal muscle. Muscle biopsy was performed in a 4-month-old floppy boy who was a full-term infant with low Apgar scores. He had mild joint contractures and weakness of upper extremities greater than that of lower extremities. Electrodiagnostic studies showed early myopathic units and borderline nerve conduction velocities. CT scans and MRIs of the brain were normal. Biopsy (see Media file 74-76) showed a range of fiber sizes, instead of the normal uniform size of myofibers. No necrosis was present, but occasional fibers with minor abnormalities on trichrome and NADH stains were slightly suggestive of a mitochondrial disorder. Immunohistochemical findings for dystrophin were normal (see Media file 74), but no staining occurred with an antibody to laminin alpha-2 (see Media file 75). A control stain with a normal muscle sample (see Media file 76) demonstrated the normal pattern of staining for laminin alpha-2. Therefore, the most likely diagnosis was CMD caused by deficiency of laminin alpha-2 (or merosin). A major clinical differential diagnostic consideration in this patient was Werdnig-Hoffmann disease, which is infantile spinal muscular atrophy, a motor neuron disease. At present, the best way to diagnose infantile spinal muscular atrophy is by genetic testing performed with a sample of blood. If the blood test is unrevealing, muscle biopsy can be performed. Glycogen storage diseaseGlycogenoses are inherited inborn errors of glycogen metabolism. Nine of them affect skeletal muscle. The two most commonly encountered by muscle pathologists are type II glycogenosis (acid maltase or alpha glucosidase deficiency) and type V glycogenosis (myophosphorylase deficiency). Type II glycogenosis Type II glycogenosis, which is due to deficiency of acid maltase (acid alpha-glucosidase), has the following 3 basic clinical variants:
The following are muscle biopsy findings in acid maltase deficiency:
Confirming the diagnosis by biochemical assay of the activity of acid maltase from a special sample of skeletal muscle that has been obtained appropriately for this purpose is best; this is the optional additional fresh specimen described in the technical section. The assay can also be performed on fibroblasts or urine. It is also possible to identify the specific mutations responsible for the producing the disease in an individual. Type V glycogenosis In Type V glycogenosis, also known as McArdle disease, due to deficiency of myophosphorylase, the abnormality is restricted to skeletal muscle. The classic presentation is the development of muscle cramps with exercise and episodes of exercise-induced rhabdomyolysis. Venous lactate levels fail to rise during an ischemic exercise test. The following are muscle biopsy findings in patients with myophosphorylase deficiency:
Mitochondrial myopathiesMitochondrial myopathies are disorders with a broad spectrum of clinical presentations. Numerous well-recognized clinical disorders are among this group of diseases (eg, Kearns-Sayre syndrome, myoclonus epilepsy with ragged red fibers (RRFs), mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes, Leber hereditary optic neuropathy). Many of these disorders present with a combination of central nervous system disease and myopathy and are referred to as encephalomyopathies. The common etiology underlying these disorders is the presence of a mutation that affects mitochondrial function. In some of these disorders, the mutations are in the mitochondrial genome; in others, they are in the nuclear genes that encode mitochondrial proteins. Many of these fairly diverse disorders share a common finding on muscle biopsy, the RRF. Genetic elucidation of these disorders has revealed that the RRF is not found in all of these disorders. Nonetheless, it is helpful when present on muscle biopsy. The following are characteristic pathologic findings in skeletal muscle in the mitochondrial myopathies:
Identifying the specific biochemical and genetic abnormalities is possible in many patients with mitochondrial encephalomyopathies if an extra muscle specimen has been properly handled for this purpose. Congenital myopathies and tubular aggregate myopathyCongenital myopathies form a diverse group of disorders with the common feature of distinctive pathologic findings. Each congenital myopathy is named for these findings, as in the following:
Each congenital myopathy may |