eMedicine Specialties > Neurology > Specialized Neurodiagnostic Tests

Muscle Biopsy and the Pathology of Skeletal Muscle

Author: Roberta J Seidman, MD, Associate Professor of Clinical Pathology, Stony Brook University; Director of Neuropathology, Department of Pathology, Stony Brook University Medical Center
Contributor Information and Disclosures

Updated: Jun 30, 2009

Introduction

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 sometimes indicated for 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 biopsies 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. In many of these situations, the samples 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:

• A normal biopsy result without pathologic findings in the setting of a high level of clinical suspicion of a disorder with a patchy distribution, such as polymyositis
• An atypical presentation of a rare metabolic disorder, which would not ordinarily be suspected before biopsy

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 contemplating muscle biopsy is to call the pathology laboratory in advance for advice on how to proceed.

This article is intended to provide an introduction to diseases of skeletal muscle, with a focus on the pathology of these disorders as seen in muscle biopsies. It is also a technical manual for muscle biopsies to assist medical professionals involved in this procedure from both the clinical and surgical sides to understand how biopsies should be performed, what happens to the tissue that is obtained, and how to evaluate the pathology reports that they receive. 

This article serves as a primer on the technical aspects of muscle biopsy, which are critical for the success of this procedure. Clinical features of neuromuscular disease are highlighted because knowledge of the clinical history is crucial for the correct interpretation of the histologic findings in a sample of skeletal muscle. A discussion of peripheral neuropathy, including some clinical features and the histological findings on muscle biopsy in the setting of peripheral neuropathy, is included. The question of whether a patient has a neurogenic or myogenic disorder is sometimes resolved by muscle biopsy and this determination is a relatively common issue prompting performance of a biopsy.

The presentation of the structure and histology of normal muscle serves as a basis for comparison with the pathologic alterations observed in muscle. The general introduction to the clinical features and pathology of selected categories of disease of skeletal muscle is intended to provide information about the classical clinical presentations of different specific groups of myopathies. It provides information about how these disorders are diagnosed. It also serves as a basis for understanding the pathophysiology of some of these disorders and to assist the reader in visualizing the effect of disease at the tissue level and to demonstrate the formulation of histopathologic diagnoses.

Indications for Muscle Biopsy

When a clinical diagnosis of myopathy is considered, muscle biopsy is often required (for exceptions to this requirement, please see When a Muscle Biopsy is Not Indicated).

Muscle biopsy is a fundamental part of the initial evaluation of a patient with possible muscle disease, or myopathy. At present, muscle biopsy is an essential part of the diagnostic investigation of most categories of muscle diseases, including inflammatory and many metabolic and congenital myopathies, as well as many of the muscular dystrophies.

Today, the most specific and definitive effective therapies are for inflammatory myopathies. Sometimes a patient is too seriously ill to delay therapy even a few days; however, whenever possible, perform muscle biopsy to diagnose these disorders prior to the initiation of therapy for the following several reasons:

• The risk of treatment, including steroids, immunosuppressive agents, and, in some cases, intravenous immunoglobulin, is high enough that the diagnosis should be confirmed before therapy is started.
• A delay often occurs between the start of therapy and a clinical response. To persevere in the absence of a prompt clinical response, confidence in the diagnosis before therapy is beneficial.
• Treatment can alter the histopathologic findings. If treatment is started and the biopsy is subsequently performed because of a lack of clinical response to the therapy, the pathologic findings can be difficult or impossible to interpret because the intervention may have altered them.

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 that are less definitive than those for inflammatory myopathies, a precise diagnosis is important for the following reasons:

• Palliative therapies are indicated for some patients.
• Patients with certain disorders are eligible for therapeutic clinical trials.
• Many conditions are hereditary diseases, and diagnosis is required for proper genetic counseling.
• Patients often benefit from prognostic information.
• Biopsy can exclude the presence of a treatable disorder.

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 particularly difficult or even impossible.

When a Muscle Biopsy is Not Indicated

The exceptions to the requirement for muscle biopsy for accurate diagnosis of possible myopathy are suspected dystrophinopathies (particularly when the clinical presentation has features characteristic of Duchenne or Becker muscular dystrophies), some congenital and limb-girdle dystrophies¹ , 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 the majority of patients with dystrophinopathies by permitting specific diagnosis on a sample of blood. In these patients, mutations, most commonly deletions, can be demonstrated in the gene for dystrophin, located on the X-chromosome (Xp21), that codes for a structural protein of skeletal muscle that is located on the internal surface of the sarcolemma (muscle plasma membrane). The gene for this protein is extremely large (2 million base pairs) and until recently this size precluded searching the entire gene for point mutations, but now dystrophin gene sequencing is commercially available.

Muscle biopsy is now usually only performed in patients with clinical syndromes that differ from typical dystrophinopathies, such as adults with limb girdle syndromes, some of whom are found to have abnormalities of dystrophin. This category includes some women who carry a single X chromosome with a dystrophin mutation, who, due to an unfortunate pattern of random inactivation of X chromosomes, have clinical myopathy because the number of muscle fibers expressing the mutant dystrophin gene is great enough to produce symptoms.

Genetic testing of blood samples is also available for abnormalities of genes for other muscle membrane, structural and myofibrillar-associated proteins that can present as limb girdle syndromes, such as any of the 4 sarcoglycans, dysferlin, caveolin-3, calpain, lamin A/C, and fukutin-related protein, among others. Sometimes, muscle biopsy is still performed first to narrow the diagnostic possibilities, followed by directed genetic testing. 

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 dystrophies

Myotonic dystrophy type 1 is definitively diagnosed by means of genetic testing on a sample of blood, which reveals a characteristic increase in the number of CTG triplet repeats in the gene for muscle protein kinase on chromosome 19. Myotonic dystrophy type 2 (proximal myotonic myopathy or PROMM) is due to increased CCTG quadruplet repeats in a zinc finger protein gene on chromosome 3, also detectable by testing on a sample of blood. The findings on muscle biopsy in these disorders are not specifically diagnostic, so if these disorders are suspected based on clinical presentation, genetic testing of blood, rather than muscle biopsy, is indicated.

Periodic paralyses

Periodic paralyses are uncommon disorders that result from mutations in a variety of genes for muscle membrane ion channels that have unique clinical, biochemical, and electrodiagnostic features. They lack specifically diagnostic findings on muscle biopsy, so genetic testing is the way to confirm the suspected diagnosis of one of these disorders. Dilatation of the T-tubule system is found in some patients with hypokalemic periodic paralysis, which produces vacuoles in histological sections, but this finding is not sufficiently specific to be diagnostic. Muscle biopsy can also demonstrate a nonspecific myopathic picture in these disorders.

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 perform 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. For more information about myotonic dystrophy, please visit the International Myotonic Dystrophy Organization.

Clinical and Laboratory Features of Neuromuscular Disease

Why providing detailed clinical information to the pathologist is important

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 analyzed with knowledge of the clinical history. The pathologist must have information about the clinical presentation of a given patient to properly assess the significance of histologic findings in a particular muscle biopsy sample. 

Here is an example to illustrate this point. Clear cytoplasmic vacuoles are often present in muscle biopsies. The most common reason for their presence is technical artifact due to ice crystal formation when a sample is frozen, in which case they have no diagnostic significance. Many myopathic disorders are also characterized by the presence of clear vacuoles. These disorders are as varied as certain glycogen storage diseases, lipid myopathies, periodic paralyses and toxic myopathies that can result from treatment with colchicine, chloroquine, or amiodarone, among other possible diagnoses. 

The knowledge of the clinical history allows the pathologist to decide which diagnostic considerations are reasonable in a given case and assists in the determination of whether additional special studies are indicated. In this example, if a biopsy shows only a few myofibers with vacuoles, the pathologist must decide whether the vacuoles are insignificant technical effects or whether they are the key diagnostic finding. The clinical history provides guidance for the pathologist in interpreting the significance of the finding.

Clinical features

Muscle can clinically express disease in very few ways: (1) weakness or decreased movement, (2) muscle ache, or (3) abnormal variations in power as a result of physical activity.
 
The main 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 can 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 typically the major sign of myopathy. From childhood through adulthood, diminished muscle power is a characteristic clinical feature of neuromuscular disease.

The classical clinical features of myopathy include the following:

• Weakness, which predominantly affects the proximal muscle groups (eg, shoulder and limb girdles)
• Myalgia, or muscle aching, which is present in some patients with inflammatory myopathy (Muscle pain is also found in some patients with metabolic diseases affecting muscle and occurs when the energy supply of the muscle is depleted and lactic acid builds up.)
• Relative preservation of muscle-stretch reflexes
• Absence of abnormalities of somatosensation

Variation of strength with activity 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.

• Fluctuation of muscle power can suggest a metabolic myopathy. For example, in McArdle disease, a deficiency of myophosphorylase causes an inability to mobilize glycogen. A patient with this disorder has pain and weakness during the anaerobic phase of exercise. If the patient can exercise at a low level during the anaerobic phase to avoid drawing on glycogen stores, when the aerobic phase of exercise is finally reached and glycogenolysis is no longer needed, the patient's performance improves.
• Fatigability denotes progressive loss of muscle power with exertion that improves with rest. This is a defining clinical feature of myasthenia gravis, a disorder of impaired neuromuscular transmission. Muscle biopsy is typically not performed for myasthenia gravis.

In contrast to myopathy, the classic clinical features of peripheral neuropathy include the following:

• Weakness predominantly affecting distal musculature
• Decrease of muscle-stretch reflexes, particularly in demyelinating neuropathies
• Fasciculations, when abnormal excitability of the motor neuron is present
• Somatosensory abnormalities

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:

• Some myopathies affect distal muscles. Myotonic dystrophy, inclusion body myositis (IBM), and distal myopathy of Welander are examples of myopathies that can affect distal muscle groups.
• Some neurogenic disorders, including diabetic amyotrophy and motor neuron disease, may affect proximal muscles.
• Some patients may have combined neurogenic and myopathic disorders. For example, a patient with neuropathy related to diabetes mellitus can also acquire an inflammatory myopathy. A patient who has peripheral neuropathy caused by chemotherapy for cancer may develop dermatomyositis. A patient can have radiculopathy caused by degenerative joint disease in the vertebral column and a primary myopathy. In these examples, the clinical findings are complicated, which can make it difficult to arrive at a diagnostic category based solely upon the clinical features.

A superb monograph by Michael H. Brooke provides insight into the clinical evaluation of patients with neuromuscular disease²  It was written prior to the explosion of information regarding the molecular genetics of neuromuscular disease, but nonetheless is a unique and valuable tool. It is out of print, but copies can still be found for purchase and in libraries.

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) often indicate muscle disease. Mildly elevated levels (200-800 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. In large patients with substantial muscle mass, CK levels above the normal range in the absence of disease are not uncommon.

The serum aldolase level can be helpful in providing evidence of myopathy. Because of its longer half-life in serum, the serum aldolase level is sometimes 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/or 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; specific details are beyond the scope of this chapter. 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 a biopsy for 1-2 months. In patients with suspected myopathy, needle EMG should be performed on only 1 side. A subsequent biopsy should be performed on the other side.

Technical Considerations

The technical issues that must be addressed by the physicians involved with muscle biopsies 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 biopsy

Biopsy 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 based on 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 tissue

The specimens required and the preferred method of handling 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, it may be the method of choice under the following circumstances:

• When serial biopsy procedures are required to follow the course of the disease or to monitor the response to therapy in a patient
• When a disease with diffuse distribution is the leading diagnostic consideration so that any sample of tissue is likely to be pathologic
• When a sample of muscle is needed for only biochemical study
• When open biopsy is contraindicated

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 will lead 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, or other mounting medium, 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 immediately 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, the sample remains in satisfactory condition for most necessary studies if refrigerated overnight or even if refrigerated for a few days in the event of an unavoidable delay (although 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 sent to the laboratory fresh (see instructions for fresh specimen, above), where the technologists perform the procedures needed for immobilization and fixation. Another method involves suturing or pinning the specimen to a tongue blade or a piece of cork 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 better ultrastructural preservation than that of a sample fixed in formalin.

If paraformaldehyde is not available and it is anticipated that EM will be needed, 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 (1-2 mm in width or depth) because glutaraldehyde penetrates tissue slowly. If the sample placed in glutaraldehyde is too large, portions of the sample will not be adequately fixed for EM before they become degraded. 

After overnight fixation in paraformaldehyde, 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 useful for selected patients when the presence of a metabolic myopathy or a muscular dystrophy 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, the 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 sample

Light 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. Some of these studies cannot be performed on material that has been fixed and embedded in paraffin. Some of the structures are removed by paraffin processing, so they can only be identified in frozen sections. 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 (there will be some variation on which stains are routinely performed at different institutions):

• Hematoxylin and eosin (H&E): This stain is the routine histologic stain used for evaluation of basic tissue organization and cellular structure.
• Nicotinamide adenine dinucleotide tetrazolium reductase (NADH): With this stain, the activity of this group of enzymes is demonstrated by the transfer of hydrogen to a compound that turns gray-blue when it is reduced. These enzymes are found in mitochondria and endoplasmic reticulum. This stain is used to assist in evaluating for neurogenic atrophy, mitochondrial disorders, and central core disease, among others, and is useful in detecting subtle alterations of intracellular structure in a myofiber that suggest it is not well.
• Fiber-typing stains: Muscle is composed of two main myofiber types: 1 and 2. Many disease processes characteristically affect one type or the other, resulting in atrophy of either type 1 or 2 myofibers. Other processes, such as neurogenic disorders, can alter the distribution of both types.
  • Many laboratories use a myosin adenosine triphosphatase (ATPase) stain at multiple pH levels to demonstrate the different fiber types. This is a difficult, labor-intensive stain to perform.
  • An immunohistochemical stain for the different myosin heavy chains found in type 1 and type 2 myofibers is an alternative method for demonstrating the two types of myofiber. These stains are adequate for myofiber typing in most cases. (Novocastra [Newcastle upon Tyne, England] recommends an immunohistochemical stain for research purposes only.) Immunohistochemical stains are now available for different forms of myosin ATPase.
• Modified Gomori trichrome: This stain is particularly helpful in evaluating for the presence of mitochondrial disorders, inclusion body myositis, and nemaline myopathy, among many other uses.
• Periodic acid-Schiff (PAS): This stains glycogen and other polysaccharides. It is most useful for the diagnosis of glycogen storage diseases. PAS also stains the basal lamina of vessel walls, so it can be useful for evaluating the structure of vessels.
• Fat stains, Sudan Black, oil-red-O, or osmium: These stains are used to demonstrate the presence of neutral lipids in muscle, which are normally present but can exist in abnormal amounts or distribution in carnitine deficiency, some mitochondrial disorders, acquired metabolic disorders (such as in starvation) and nonspecific abnormalities of the myofibers.

Some 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:

• For muscular dystrophies, immunohistochemical studies for dystrophin, sarcoglycans, laminin α-2 (merosin), and other structural proteins can be performed. The results of these then can be used to direct special biochemical analysis that will lead to a specific diagnosis.
• For some metabolic disorders, the enzymatic activities of myophosphorylase, phosphofructokinase, myoadenylate deaminase, succinic dehydrogenase (SDH), and cytochrome oxidase (COX) can be performed.
• A stain for acid phosphatase, a lysosomal enzyme, can be useful for the evaluation for certain metabolic disorders, some toxic disorders, and other circumstances.
• For dermatomyositis, immunofluorescence can be performed to look for membrane attack complex of complement in vessel walls.
• For inflammatory myopathies, immunohistology for major histocompatibility class I, also known as human leukocyte antigens-ABC (or HLA class I) can be performed.

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 cytological 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. Some of these are as follows:

• Special stains for organisms, such as bacteria, fungi, and parasites
• Elastic stains to evaluate for disruption of the elastic lamina of arteries in vasculitis
• Immunohistochemical stains to determine the subtypes of inflammatory cells within an infiltrate and a variety of other purposes
• In situ hybridization for identification of viruses
• Congo red or thioflavin S staining for amyloid

Electron microscopy

While a small sample of every muscle biopsy should be set aside for possible EM, performing EM on 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.

• Fixation: If the specimen is fixed in paraformaldehyde, it is transferred to 3% glutaraldehyde after sufficient time has passed for the paraformaldehyde to penetrate the tissue. This depends on the size of the specimen, but overnight fixation is more than satisfactory for this. Glutaraldehyde may provide a bit more cross-linking of the membranes, which is needed for EM.  
  • If paraformaldehyde is not available, the tissue, held at rest length by pinning to cork, can be placed directly in glutaraldehyde. Because glutaraldehyde does not penetrate the tissue as well as paraformaldehyde, a specimen placed in glutaraldehyde must be small, approximately 1-2 mm in width and depth. Glutaraldehyde makes tissue brittle and interferes with immunohistochemical studies, so it is not appropriate for the paraffin specimen.
  • If the tissue is fixed in formalin, it is not as well preserved for EM as it is with paraformaldehyde or glutaraldehyde. Performing EM on tissue fixed only in formalin is possible, but this is suboptimal. Cutting tissue out of a paraffin block or removing it from a slide is possible for EM, but the likelihood of obtaining useful results with these methods is limited.
  • Embedding the tissue: After fixation, the tissue is divided into 1-mm³ samples, postfixed with osmium tetroxide, and embedded in epoxy resin. Samples are oriented in either longitudinal or transverse direction prior to polymerization of the resin. The process of embedment requires 2 days.
  • Survey sections: Survey sections for light microscopy, 1 micron in thickness, termed semithin or thick sections, are cut from the material embedded in plastic. The pathologist reviews these and areas of interest are chosen for EM.
  • Thin sections: An ultramicrotome with a diamond knife is used to cut sections for ultrastructural study. These then are stained with uranyl acetate and lead citrate. They are placed in an EM and examined.
• Selected clinical circumstances in which EM is useful include the following:
  • When seeking evidence to support a diagnosis of dermatomyositis, EM can be used to look for tubuloreticular inclusions (TRIs) in endothelial cells. If light microscopic findings are diagnostic, EM is not necessary.
  • EM can be used to identify inclusions found by light microscopy.
  • EM can help to characterize stored material found on light microscopy and define its intracellular localization.
  • EM can be used to analyze structural abnormalities found by light microscopy.
  • EM can assist in the diagnosis of mitochondrial myopathy.
  • EM is only rarely indicated for a muscle that is normal at the light level. If normal muscle is found with all of the light microscopic studies, then this is exactly what EM will show, only larger. The only common exception to this guideline is in the setting of a strong clinical suspicion for dermatomyositis with normal light microscopic studies. If TRIs are found, they can lend some support to this diagnosis.

Normal Skeletal Muscle

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 is the major constituent of the Z band, and actin and myosin, which are the major components of 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 the intracellular storage and release of calcium required for contraction to occur.

Myofiber types

The two 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-twitch fibers. Physiologists refer to them as slow-oxidative (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-twitch fibers. Physiologists call these the fast-glycolytic (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 intense activity and for 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 commonly biopsied muscle, the balance typically favors type 1 myofibers. In normal muscle, the two myofiber types are interspersed in a random interdigitating pattern. The two myofiber types are normally similar in size.

Information about changes in the myofiber types in a muscle biopsy often provides significant diagnostic clues. Different pathologic processes alter the ratio of the myofiber types and their distributions in the muscle and may selectively affect the size of one 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 and ultrastructure of normal muscle

On a frozen H&E section, 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 frozen PAS section, PAS-positive material 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. 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; however the exact staining in a given case is dependent upon recent carbohydrate ingestion and exercise, so this stain cannot be relied upon for fiber-typing.

With the modified Gomori trichrome stain performed on a frozen section (see Media file 15), the myofibers and connective tissue stain slightly different shades of blue-green. Nuclei are normally red. The intermyofibrillar network exhibits punctate red staining because mitochondria stain red, which is usually inconspicuous.

With the Sudan Black stain for lipid, performed on a frozen section (see Media file 16), intracellular lipid appears blue-black and is distributed throughout the intermyofibrillar network. Type 1 myofibers stain darker than the others because of their high 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.

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 readily demonstrated. 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.

Normal muscle in longitudinal section of an EM (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, whose major constituent is 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.

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 an artifact and has no bearing on the diagnosis.

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 these 3 examples, 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, it was still possible to arrive at a diagnosis.

Introduction to Skeletal Muscle Pathology

Interpretation of a muscle biopsy can be a challenging task. This process can be difficult because few individual histologic findings are specifically diagnostic of a single disorder. Most muscle biopsies exhibit a constellation of pathological findings that must be synthesized to arrive at a diagnosis. The muscle pathologist must analyze and interpret the histopathologic features in the individual clinical context to arrive at a diagnostic formulation that makes sense for a given patient.

For example, a biopsy might exhibit myofibers that contain empty vacuoles on H&E sections. 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, among others. 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:

• Angulated atrophic fibers (see Media file 26)
• Fiber-type grouping (see Media file 27): This finding occurs when denervation and reinnervation have taken place. Innervation of a myofiber determines its type. If a motor unit that was originally innervated by a type 1 nerve loses its innervation, a number of isolated angulated atrophic fibers are initially scattered about a small region of the muscle. If a neighboring intact type 2 motor neuron sprouts and reinnervates these myofibers, all of the muscle fibers in the region become type 2. The muscle loses the normal random checkerboard distribution of myofiber types.
• Group atrophy (see Media file 28)
• Target fibers (see Media file 29)
• Nuclear clumps (see Media file 30)

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 based on clinical findings and the balance of the pathological features, but the truth cannot always be determined with certainty.

Many biopsy samples with inflammation also demonstrate evidence of neurogenic change. A few possible mechanisms account for this neurogenic change: (1) Myogenic denervation, in which the sick muscle fibers lose their innervation; (2) innocent bystander mechanism in which the inflammatory process overruns and entraps the intramuscular nerve twigs resulting in denervation; and (3) the nerves are concurrently inflamed. An unrelated neurogenic disorder is also possible.

Muscle biopsy in myopathy

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:

• Myofiber necrosis (see Media file 31)
• Myophagocytosis (see Media file 32)
• Regeneration (see Media file 33)
• Rounded atrophic fibers (see Media file 34)
• Myofiber hypertrophy and splitting (see Media file 35)
• Increase in internal nuclei: Myofiber nuclei are normally located at the periphery of the cell; greater than 3% internal nuclei is abnormal (see Media file 36).
• Fibrosis (see Media file 37)

Numerous other ancillary findings can be found in myopathic muscle biopsy samples. Additional histologic myopathic findings include the following:

• Nuclear chains (see Media file 38)
• Moth-eaten fibers (see Media file 39)
• Ring fibers (see Media file 40)
• Whorled fibers (see Media file 41)
• Vacuoles (see Media file 42)
• Inclusions (see Media file 43-44)
• Inflammation (see Media file 45-47)

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 misinterpret these normal findings.

Clinical Features and Pathology of Myopathies by Diagnostic Categories

Myositis, muscular dystrophies, glycogen storage diseases, mitochondrial myopathies, and congenital myopathies are 5 important groups of disorders that can be diagnosed by muscle biopsy.

Myositis

The term myositis refers to inflammatory disease of muscle. In practice, this term is usually 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). Good basic reviews of the clinical and pathological features of inflammatory myopathies exist.^3,4,5

The most common reason for performing a muscle biopsy is to evaluate for the diagnostic consideration of idiopathic inflammatory myopathy. The 3 major idiopathic inflammatory myopathies are polymyositis, dermatomyositis, and inclusion body myositis (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,⁶ some of which are associated with specific clinical syndromes. Patients with dermatomyositis usually have characteristic rashes and other cutaneous findings. Dermatomyositis in adults can be a paraneoplastic syndrome,⁷ so some testing for malignancy is necessary when an adult patient has this disorder. Dermatomyositis can develop prior to, concurrently with, or following the diagnosis of a neoplasm.

Polymyositis

The following are the key diagnostic pathologic features of polymyositis:

• Endomysial inflammation (see Media files 45-46): The inflammatory infiltrates in polymyositis are predominantly endomysial, and they are enriched with T-lymphocytes, particularly T-suppressor/cytotoxic (CD8) lymphocytes.
• Invasion of nonnecrotic myofibers by autoaggressive lymphocytes (see Media file 50): This is a key diagnostic finding in which T cells attack intact myofibers. This is believed to be the pathologic correlate of the main factor in the etiopathogenesis of polymyositis.^8,9,10 This represents the fundamental distinction between inflammation that can occur as a secondary phenomenon and inflammation that is the primary pathologic process. In the former case (eg, muscular dystrophy), inflammation is usually found associated with fibers that are already degenerating. In polymyositis, inflammation can be found associated with healthy, intact fibers.
• The expression of major histocompatibility complex (MHC) class I or human leukocyte antigen (HLA) class I on the surface of myofibers is reportedly common to all inflammatory myopathies and is not specific for polymyositis.

The following pathological features are also often found in polymyositis, but they are not diagnostically specific and can be found in a variety of myopathic disorders:

• Myofiber necrosis (see Media file 47)
• Myophagocytosis (see Media file 32): This is the removal of the dead cellular elements by macrophages.
• Internal nuclei (see Media file 36): These are a nonspecific myopathic finding.
• Myofiber atrophy: Myopathic atrophic fibers are generally of both myofiber types and rounded in contour. In some patients with polymyositis, the atrophy affects primarily type 2 myofibers. In patients with inflammatory myopathies, type 2 myofiber atrophy can also be the result of treatment with steroids.
• Regeneration (see Media file 33)
• Fibrosis: This is a feature of chronic polymyositis.

The distribution of the pathology in polymyositis can be patchy, so it is possible for a patient who has this disorder to have a normal muscle biopsy and this does not completely exclude the diagnosis. As in all of medicine, neuromuscular diagnosis is a synthesis of clinical, laboratory, and biopsy features and physicians must use judgment in interpreting the clinical significance of each individual result.

Some patients who have an abnormal muscle biopsy that does not show inflammation are believed to have an autoimmune myopathy that is a variant of polymyositis. 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 to anti–signal recognition particle (anti-SRP). The presence of these autoantibodies is the strongest evidence that this disorder is an immune-mediated disease. This disorder 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 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:

• Chronic inflammation (see Media file 51): The infiltrates most often are concentrated in a perimysial perivascular distribution. More B-lymphocytes and T-helper (CD4) lymphocytes are present than in polymyositis. If the histological features are otherwise characteristic, the diagnosis is made regardless of the exact cell types within an individual infiltrate in a single case, so if typing the lymphocytes does not contribute to the diagnosis, it is not performed.
• Perifascicular atrophy (see Media file 52): This atrophy affects the fibers at the periphery of the fascicle and is believed to be a product of muscle ischemia at the capillary level. It is found somewhat more often in juvenile dermatomyositis, but can be observed in the adult variant of this disorder. Although perifascicular atrophy is not absolutely specific for dermatomyositis, it is found infrequently in other disease processes.
• Myofiber necrosis and/or regeneration: This can occur in a perifascicular distribution.
• Complement deposition in microvessel walls (see Media file 53): The deposition of the membrane attack complex of complement (C5b-9) is found in the walls of the microvessels early in the disease process, even before other pathologic findings are present. This immune attack on vessel walls, with an immunologic cascade involving humoral immunity, may be the pathogenetic mechanism of dermatomyositis, according to the pioneering research of Andrew Engel and his colleagues.¹¹ Treatment with steroids promptly eliminates this finding.
• TRIs in endothelial cells (see Media file 54): This finding is seen only at the ultrastructural level and is no longer present after treatment.
• HLA class I expression on the surfaces of myofibers

Inclusion body myositis

Inclusion body myositis (IBM) is the most common idiopathic inflammatory myopathy in patients older than 50 years. In contrast to polymyositis and dermatomyositis, which affect more women than men, IBM more often affects men, with a male:female ratio of 1.5:1. The clinical course of IBM is typically more indolent than polymyositis or dermatomyositis. It is not unusual for a person to have severe muscle atrophy at the time of presentation for medical care. This indicates that the disorder has been present for some time before the individual seeks medical evaluation. Distal muscle involvement, particularly with weakness of finger flexors, is a common feature of IBM, but unusual in other inflammatory myopathies. IBM is usually resistant to therapy, although some patients do respond to immunomodulatory therapies. 

IBM is the inflammatory counterpart of a group of disorders known as inclusion body myopathies, 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 hereditary forms of IBM (HIBM) are not, however, inflammatory myopathies and they do not typically exhibit evidence of mitochondrial abnormalities. A succinct review of inclusion body myositis summarizes the key clinical, histological, pathogenetic, and treatment issues.¹²

The following are pathologic features of IBM:

• Chronic inflammation: The inflammatory process is similar to that of polymyositis, with an endomysial location of the inflammation and infiltrates that are enriched in CD8 lymphocytes, which are cytotoxic/suppressor T-lymphocytes.
• Invasion of nonnecrotic myofibers by autoaggressive lymphocytes (see Media file 55)
• Myofiber hypertrophy (see Media file 56): Hypertrophy in a myositis biopsy should prompt a consideration of the possibility of IBM.
• Atrophy: Some of the atrophic fibers in IBM share features with those of neurogenic atrophy.
• Rimmed vacuoles (see Media file 57): These appear on H&E sections as ovoid sarcoplasmic vacuoles lined by blue granular material. The granular material is red with the trichrome stain.
• Eosinophilic inclusions (see Media file 58-59): These inclusions are dense and red (eosinophilic) on H&E sections, they can be cytoplasmic or nuclear, and they may be found within rimmed vacuoles. They can stain positive with a Congo red stain for amyloid and with stains for amyloid precursor protein, ubiquitin, and other proteins typically associated with neurodegenerative disease.
• Tubulofilamentous inclusions (see Media file 60): These are the ultrastructural counterparts of the eosinophilic inclusions observed by light microscopy.
• HLA class I expression on the surface of myofibers
• Myofiber degeneration, myophagocytosis, internal nuclei, endomysial fibrosis (see Media files 56-57). Patients with IBM commonly seek medical care at a relatively late stage in the disease process, so the biopsies often demonstrate severe loss of muscle mass with prominent fibrosis and even adipose replacement of the muscle.
• Occasional ragged red fibers (see Media File 87) indicative of mitochondrial alterations are commonly found in 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 dystrophies

A muscular dystrophy is a potentially hereditary disease characterized by progressive degeneration of muscle. Many such diseases exist. The old classification scheme was somewhat random and comprised Duchenne, Becker, and various other eponymous dystrophies and dystrophies named for the distribution of affected muscle groups (such as oculopharyngeal muscular dystrophy or scapuloperoneal muscular dystrophy), all subclassified by their modes 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 the generic term 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. 

Some congential muscular dystrophies are caused by mutations in genes that are responsible for glycosylation of α-dystroglycan, a membrane protein.¹³  These latter disorders belong to the newest category of disorders that has been recognized; they result from abnormalities of genes for proteins involved in the post-translational modification of membrane proteins, rather than from defects that alter the amino acid sequence of membrane or cytoskeletal proteins.

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 many muscular dystrophies, where immunohistochemistry for specific muscular dystrophy proteins can narrow the diagnostic possibilities.¹  Specific and directed biochemical or genetic testing can then be performed for definitive diagnosis.  

For patients with classic presentations of dystrophinopathies with either classic Duchenne or Becker muscular dystrophy, the diagnosis can be made by genetic testing of blood samples, so muscle biopsy is not usually necessary. In certain cases of other types of muscular dystrophies with characteristic clinical presentations, genetic testing can bypass the need for muscle biopsy for definitive diagnosis. For patients with no specific clinical features of myopathy that, in some cases, will finally be diagnosed as a specific dystrophy, muscle biopsy is still an important diagnostic tool.

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 somewhat characteristic of certain dystrophies, such as hypercontracted fibers in Duchenne muscular dystrophy (see Duchenne muscular dystrophy) or nuclear clumps in some patients with limb-girdle muscular dystrophy (see Limb-Girdle Muscular Dystrophy). The skeletal muscles of certain patients with oculopharyngeal dystrophy (see Media file 44) contain rimmed vacuoles and eosinophilic inclusions similar to IBM.

The specific diagnosis of muscular dystrophies can be confirmed in many cases by special immunohistochemical stains for specific proteins that are abnormal or deficient in these disorders. Many of these disorders are uncommon, so this testing is only available in a limited number of specialized centers and 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 findings point to a certain disorder, the muscle specimen must then be sent to a facility that can perform biochemical analysis for the protein or direct genetic testing (or in some cases, a blood sample can be submitted) for confirmation of the immunohistochemistry results and final 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, making arrangements to obtain a specimen of muscle appropriate for biochemical analysis is prudent. Please see Optional additional fresh specimen for details on how to proceed.

Examples of muscle biopsies from patients with Duchenne or Becker muscular dystrophies (dystrophinopathies), and congenital muscular dystrophy (CMD) due to laminin α-2 deficiency are presented below to illustrate the pathology of muscular dystrophies.

Duchenne muscular dystrophy

Duchenne muscular dystrophy (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 protein dystrophin, resulting in an absence of the protein. Dystrophin is a structural protein that is normally located on the inner aspect of the sarcolemma (muscle plasma membrane). 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 (boys) are generally healthy until approximately 3 years of age, when they develop problems with gait. In the early juvenile years, when normal development proceeds with a steep upward curve in increasing strength, many children with DMD appear to stabilize for a short time, but after that, they 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, but nonspecific pathologic findings:

• Endomysial and perimysial fibrosis (see Media file 63)
• Increased variability of myofiber size caused by the presence of both atrophy and hypertrophy (see Media file 63) with fiber splitting (see Media file 64)
• Myofiber necrosis (see Media file 65)
• Increased internal nuclei
• Opaque fibers (see Media file 66): These are characteristic of DMD, although they can be found in other disorders and can be a technical effect. Opaque fibers are enlarged, densely eosinophilic fibers that are hypercontracted. Their presence in DMD caused investigators to postulate that membrane defects might be present in DMD, which were later demonstrated, and this finding eventually led to the discovery of the cause of DMD.^14,15 In DMD, the lack of dystrophin leads to membrane instability, which is responsible for the cascade of cellular events that causes cycles of necrosis, regeneration, and progressive fibrosis of the muscle.

The specific diagnosis of DMD relies on special immunohistochemical studies for N -terminal, mid-rod, and C -terminal regions of dystrophin. In control skeletal muscle, these studies reveal linear labeling of the periphery of the myofibers, consistent with the regular periodic subsarcolemmal localization of dystrophin (see Media file 67). In a patient with DMD (see Media file 68), all 3 antibodies demonstrate absence of labeling of all but an occasional fiber.

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 stereotypical. In BMD, the severity of the disease generally correlates with the portion of the dystrophin molecule affected. The C -terminal end of dystrophin is linked to β-dystroglycan of the transmembrane glycoprotein complex that is linked to α-dystroglycan, which is, in turn, anchored to the external basal lamina of the myofiber. If the C -terminal 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, 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:

• Myofiber necrosis (see Media file 69): Mild, focal, chronic inflammation is associated with some necrotic fibers in this biopsy. Inflammation occasionally leads to a mistaken consideration of an inflammatory myopathy. In the patient above, the clinical history strongly suggests dystrophy instead of inflammatory myopathy, which should prompt a pathologist to avoid hastily forming an erroneous conclusion. With dystrophy, the inflammation is often restricted to an association with necrotic fibers, whereas in myositis, it can be found elsewhere in the muscle; this key finding can sometimes help to distinguish the inflammation in a dystrophy from that of myositis. This assessment can be difficult, and exceptions to this guideline exist. In inflammatory myopathies, there should be myofiber surface expression of HLA class I antigen, which should be absent in a dystrophy.
• Increased variability of myofiber size with myofiber atrophy and hypertrophy (see Media file 70) and fiber splitting (see Media file 71)
• Myofiber regeneration (not shown)
• Increase in internal nuclei (see Media file 70-71): In this patient, the increase in the percentage of fibers with internal nuclei is mild.

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. Sending a sample of blood for genetic testing is even better for confirmation of the diagnosis.

Extensive research has led to a detailed model of the structure of the myofiber membrane and has elucidated the components of the transmembrane glycoprotein complex. It contains several proteins known as sarcoglycans and others known as dystroglycans. Mutations of each of these proteins, as well as others not mentioned here, are now known to be responsible for many forms of muscular dystrophy.¹⁶

Congenital muscular dystrophy

Congenital muscular dystrophy (CMD) is clinically evident from the neonatal period. Multiple disorders fall within this category. In one third of patients, CMD is caused by an abnormality of laminin α-2, also known as merosin, which is a component of the basal lamina of skeletal muscle. Some forms of CMD are now known to be due to mutations that result in defective glycosylation of α-dystroglycan, a membrane protein to which laminin α-2 binds.¹³

Congenital muscular dystrophy is in the differential diagnosis of the floppy infant syndrome. Following is an illustrative case:

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 reportedly normal.

Biopsy (see Media file 74-76) showed a range of fiber sizes, instead of the normal fairly uniform size of myofibers. Necrosis was absent, but occasional fibers exhibited minor nonspecific abnormalities on trichrome and NADH stains. Immunohistochemical studies for dystrophin were normal (see Media file 74), but no labeling occurred with an antibody to laminin α-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 α-2. Therefore, the most likely diagnosis was CMD caused by deficiency of laminin α-2 (or merosin) (see Media file 84).

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.

In Werdnig-Hoffmann disease, as in CMD, muscle biopsy demonstrates a range of myofiber sizes. In Werdnig-Hoffmann disease unlike CMD, the largest fibers (see Media file 77) tend to cluster. In biopsy samples from patients with Werdnig-Hoffmann, the largest and smallest fibers are both type 1 myofibers (see Media file 78); this finding does not occur in CMD. An important caveat is that these changes in myofiber distribution are generally not present until the infant is several months old. Therefore, when possible, defer biopsy as long as possible, or prepare the family for the possibility of repeat biopsy if findings on the first are not specifically diagnostic.

Glycogen storage disease

Glycogenoses are inherited inborn errors of glycogen metabolism. Nine of them affect skeletal muscle. The 2 most commonly encountered by muscle pathologists are type II glycogenosis (acid maltase or alpha glucosidase deficiency) and type V glycogenosis (myophosphorylase deficiency).

There are 2 types of clinical presentations of glycogen storage diseases: 

• The first is characterized by symptoms of weakness and/or cramps during the anaerobic phase of exercise. This syndrome is due to defects in the enzymes required for mobilization of glycogen for energy production during exercise or abnormalities of glycolytic enzymes. 
• The second type is characterized by a progressive proximal myopathy without significant exercise-induced symptoms. This syndrome is caused by defective enzymes involved in the glycogenosynthetic pathway or in the breakdown of glycogen not directly in the pathway for energy production during exercise.

Type II glycogenosis

Type II glycogenosis, which is due to deficiency of acid maltase (acid alpha-glucosidase), is the only glycogen storage disease that is also a lysosomal storage disease. This disorder has the following 3 basic clinical variants:

• A severe, fatal, infantile form, also known as Pompe disease, affects multiple organs, including heart, liver, kidneys, leukocytes, central nervous system, and skeletal muscle. Glycogen storage is demonstrated in most tissues in this disorder.
• A juvenile variant presents with weakness affecting muscles of proximal limbs.
• In adult-onset acid maltase deficiency, weakness, and fatigue occur with progressive respiratory failure. The age of onset and severity of the clinical presentation are generally correlated with the severity of the enzymatic deficiency.

The following are muscle biopsy findings in acid maltase deficiency:

• Clear vacuoles on H&E sections, usually distributed throughout the myofiber (see Media file 79)
• PAS-positive staining of these vacuoles, with disappearance of staining following digestion with diastase
• Acid phosphatase stain demonstrates excessive staining for lysosomes (see Media File 110), which correspond to the vacuoles seen with the H&E stain
• Intralysosomal storage of glycogen on EM (see Media file 80)

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 producing the disease in an individual.

Type V glycogenosis

In type V glycogenosis (McArdle disease), due to deficiency of myophosphorylase, the abnormality is restricted to skeletal muscle. The classic presentation is the development of muscle cramps during the anaerobic phase of 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:

• Clear vacuoles on H&E section, particularly in the subsarcolemmal location (see Media file 81)
• PAS-positive staining of these vacuoles (see Media file 82), with disappearance following digestion with diastase (see Media file 83)
• Storage of excessive amounts of free glycogen within myofibers on EM (see Media file 84)
• Evidence of absence of myophosphorylase activity (see Media file 85) on special histochemical staining, with normal activity in a simultaneous control specimen (see Media file 86).  

Mitochondrial myopathies

Mitochondrial myopathies are disorders with a broad spectrum of clinical presentations due to involvement of a variety of organ systems. They affect organs that are highly dependent upon aerobic metabolism and therefore commonly affect heart, skeletal muscle, eye, and brain. Renal and gastrointestinal involvement occur in some. Many patients with disorders of mitochondrial function have basal elevation of serum lactate because a block in oxidative phosphorylation causes slower turning of the Krebs cycle, which then results in decrease conversion of pyruvate to acetyl-CoA. The excess pyruvate that results is converted to lactate. 

Numerous well-recognized clinical disorders are among this group of diseases, such as Kearns-Sayre syndrome, myoclonus epilepsy with ragged red fibers (MERRF), mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes (MELAS), and Leber hereditary optic neuropathy (LHON). 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 the mitochondrial disorders is the presence of mutations that affect 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.

This is an extremely complex area because of the large variety of potential clinical presentations of these disorders. Many of the disorders can be due to multiple different mutations and an individual mutation can cause more than one type of clinical disorder. Numerous excellent reviews address this complex topic and make it possible for clinicians to develop an approach to diagnosis.^17,18,20,21

Many of these fairly diverse disorders share a common finding on muscle biopsy: the ragged red fiber (RRF). A ragged red fiber is one with intense peripheral red staining with the Gomori trichrome stain. This red staining corresponds to aggregates of abnormal mitochondria. RRFs are not found in all mitochondrial disorders, so their absence does not exclude the presence of a mitochondrial disorder. Nonetheless, it is diagnostically important when they are present in a muscle biopsy from a patient with a clinical presentation that is suggestive of a mitochondrial disorder.

Ragged red fibers can occur as an age-related change in individuals 50 years of age and older, so this finding in an older person must be interpreted with caution.

The following are characteristic pathologic findings in skeletal muscle in the mitochondrial myopathies:

• On trichrome stain, RRFs have a peripheral rim of red material caused by the subsarcolemmal aggregation of mitochondria (see Media file 87).
• Dense peripheral staining for the activity of succinic dehydrogenase (SDH), which is a mitochondrial enzyme involved in the electron transfer chain, can be seen in RRFs (see Media file 88). When visualized with the SDH stain, they are referred to as ragged blue fibers.
• The presence of many fibers with absence of the activity of cytochrome oxidase (COX), which is complex IV of the respiratory chain enzymes, is a characteristic finding (see Media file 88).
• Combined SDH/COX staining demonstrates that many of the COX-negative fibers are the RRFs (see Media file 89).
• EM shows both an increase in mitochondria and morphologically abnormal mitochondria (see Media files 90-92).

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 myopathy

Congenital myopathies are a diverse group of disorders with the common feature that each has its own characteristically distinctive morphological pathological finding. Each congenital myopathy is named for these findings, as in the following:

• In central core disease, the central region of many myofibers has abnormal structure.
• In nemaline myopathy, the fibers contain aggregates of rodlike material seen on trichrome stain.
• In centronuclear (or myotubular) myopathy, the main pathologic finding is myofibers with centrally located nuclei and fibers that appear immature.
• In congenital fiber type size disproportion, type 1 myofibers are small and type 2 myofibers are of normal size.

Each individual congenital myopathy is probably actually a group of disorders with a common morphology on biopsy. Some have multiple characteristic clinical presentations, rates of progression, and modes of inheritance. Currently, the genetic and molecular bases of the defects are being identified, providing further evidence that they are heterogeneous disorders.

Nemaline myopathy

Nemaline myopathy is a disease with both autosomal dominant and recessive modes of inheritance. A severe infantile form exists, and milder forms present later in life. Mutations in 5 different genes (so far) are associated with nemaline myopathy: alpha-actinin (chromosome 1q42), the nebulin gene (chromosome 22q2), alpha tropomyosin 3, beta-tropomyosin, and troponin-1.^22,23,24,25 A form of nemaline myopathy is associated with HIV infection. Small numbers of nemaline rods are found relatively frequently in muscle biopsies, so their presence is not specifically diagnostic. They are a normal finding in myofibers at the myotendinous insertion. 

The biopsy discussed here, from an 8-year-old boy with weakness since infancy, high arched oral palate, myopathic face with mild weakness of proximal muscle groups of the extremities and difficulty keeping up with his peers on the playground, demonstrates the characteristic findings of nemaline myopathy.

• H&E stain (see Media file 93) reveals a biopsy that appears normal except for a slight increase in internal nuclei.
• Trichrome stain (see Media file 94) shows the presence of inclusions in many fibers; on high power (see Media file 95), these have a rodlike structure.
• Myosin ATPase (see Media file 96) shows a predominance of type 1 myofibers.
• EM (see Media file 97) shows that the rods are dense fibrillar structures that extend from the Z bands.

Central core disease

Central core disease is another disorder that is actually a group of disorders. Many patients with central core disease are susceptible to malignant hyperthermia when certain anesthetics are administered. Some patients with central core disease possess mutations in a gene for the ryanodine receptor, which is a calcium channel in the sarcoplasmic reticulum.²⁶

In central core disease, H&E section (see Media file 98) shows many myofibers with faint central abnormalities. Myosin ATPase (see Media file 99) demonstrates that many of the type 1 myofibers have central round areas that do not stain. These are the central cores. They also show absence of staining with the NADH stain, not illustrated here.

Tubular aggregate myopathy

Tubular aggregate myopathy is an unusual disorder that is not always classified as a congenital myopathy, but it has such a distinctive histopathologic picture that it is presented in this section. In a rare familial syndrome, affected patients have fluctuating weakness. Tubular aggregates also are found in association with muscle cramps, diabetes mellitus, and alcoholism.

In tubular aggregate myopathy, inclusions are quite prominent, as demonstrated in the following:

• H&E (see Media file 43): Many fibers have large, pale intracytoplasmic inclusions.
• PAS (see Media file 100): These inclusions are PAS positive.
• Fiber-typing stain (see Media file 101), in this case myosin ATPase: In some subgroups of this myopathy, inclusions are usually found only in type 2 myofibers, as illustrated here. This is highly unusual. In most disorders with inclusions that are fiber-type specific, the inclusions are usually found in type 1 myofibers.
• NADH (see Media file 102): Inclusions are dark with this stain.
• SDH (see Media file 103): Tubular aggregate myopathy is the rare disorder in which inclusions are positive with the NADH stain but are negative for SDH. They are negative for the latter stain because the tubular aggregates are composed of sarcoplasmic reticulum membrane. SDH is found exclusively in mitochondria.
• EM of tubular aggregates in cross section (see Media file 104): Their tubular structure should be appreciated easily in this view.
• EM of tubular aggregates in slightly tangential longitudinal section (see Media file 105): This image demonstrates continuity of the tubules with the lateral sacs of the sarcoplasmic reticulum.

Conclusion

The intent of this chapter was to provide an introduction to the clinical and pathological features of neuromuscular disease, focusing on myopathic disorders, and a detailed primer on muscle biopsy and its role in the evaluation of patients with neuromuscular disease. The importance of clinical context for planning of the muscle biopsy, interpretation of the histological features, and decisions about what special studies are warranted for a given biopsy were highlighted throughout the chapter. A section about normal muscle structure was included to assist the reader with appreciation of the pathological findings.

Muscle biopsy is a diagnostic tool of great potential in the diagnosis of neuromuscular disease. When performed properly, it can yield information of great benefit to the patient and clinician and serve as a basis for providing treatment, genetic counseling, and prognostic information.

This article is a reminder that if clinicians, surgeons, and pathologists share information and work cooperatively, the patients are the ones who benefit.

Multimedia

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Media file 1: Hematoxylin and eosin (H&E) paraffin section of a muscle biopsy sample reveals end-stage muscle. Fibrovascular and adipose tissue have entirely replaced the muscle, which can therefore impart no information about the patient's underlying pathologic process.

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Media file 2: Hematoxylin and eosin (H&E) paraffin section from the same patient as in Image 1. Structure in the center of the image, consisting of a cluster of small muscle fibers surrounded by a capsule, is a muscle spindle; this finding confirms that the specimen is indeed skeletal muscle.

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Media file 3: Fresh specimen is mounted on cork by using gum tragacanth. It is poised above a vial of isopentane, which is chilled with liquid nitrogen coolant. The specimen is frozen by immersing it into the isopentane. In this photograph, the fibers are oriented longitudinally in the vertical plane. Tissue is sectioned by using a cryostat for cross-sections.

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Media file 4: Specimen of skeletal muscle on a 10-mm Rayport clamp is fixed in paraformaldehyde. On this image, the longitudinal axis of the fibers is oriented in the horizontal plane. A small piece of muscle that is not clamped lies obliquely over the clamped portion of the specimen.

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Media file 5: Basic constituents of skeletal muscle.

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Media file 6: Comparison of the histologic features of type 1 and type 2 myofibers. SO represents slow oxidative, and FG represents fast glycolytic, which are physiologic characteristics of type 1 and type 2 fibers, respectively.

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Media file 7: Hematoxylin and eosin (H&E) frozen cross-section of skeletal muscle shows that the sample is composed of several fascicles of muscle. Each fascicle is surrounded by thin, delicate perimysium. The myofibers are of relatively uniform size and shape and fit together in a mosaic pattern. The fibers, which appear to be in almost direct contact with one another, are separated by thin, almost invisible endomysium. When fibrosis is present, the muscle fibers appear separated. The myofiber nuclei are normally located at the periphery of the cells, and the cytoplasm is fairly uniformly distributed.

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Media file 8: Normal muscle. High power hematoxylin and eosin (H&E) cross-section of a frozen section of muscle reveals the thin, delicate endomysial connective tissue, the normal appearance of the sarcoplasm, and the peripherally placed nuclei. Capillaries, normally indistinct, are found at the corners between myofibers.

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Media file 9: Normal muscle. Nicotinamide adenine dinucleotide tetrazolium reductase (NADH) stain demonstrates 2 populations of myofibers. Type 1 myofibers stain more darkly than type 2 myofibers because of the greater use of aerobic metabolism by type 1 fibers. In normal muscle at low power, the sarcoplasm stains fairly uniformly across the cell.

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Media file 10: Normal muscle. High-power view of the nicotinamide adenine dinucleotide tetrazolium reductase (NADH) stain in normal muscle reveals the punctate distribution of the stain throughout the cell because of its predominant colocalization with mitochondria in the intermyofibrillar network. Dark fibers are type 1 myofibers.

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Media file 11: Normal muscle. Myosin adenosine triphosphatase (ATPase) at pH 10.5 stains type 2 myofibers brown. Type 1 fibers are stained with an eosin counterstain so that they are visible. Normal muscle contains a random checkerboard-like interdigitation of the 2 myofiber types. The 2 types of myofiber are similar in size. In the field shown, more type 1 myofibers than type 2 are present. This is a characteristic feature of the deltoid muscle.

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Media file 12: Normal muscle. Immunohistochemical fiber-typing stain for myosin heavy chain, slow type, in which type 1 myofibers are brown. The eosin counterstain makes the type 2 myofibers visible with a pink color. Three arrows indicate 3 type 1 myofibers, which are also seen in image 13. If a laboratory is equipped to perform immunohistochemical studies, this is a technically easier stain to perform than myosin adenosine triphosphatase (ATPase) stains. Another advantage of the immunohistochemical stain is its relative permanence, whereas myosin ATPase stains fade in a few months.

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Media file 13: Normal muscle. Immunohistochemical stain for myosin heavy chain, fast type. In this image, type 2 myofibers are brown and the type 1 myofibers are pink due to the eosin counterstain. Compare this image with image 12, which shows the same field. The identical 3 fibers indicated by arrows in Image 12 are indicated by arrows. Here, the areas indicated by the arrows are pink, confirming their identity as type 1 myofibers.

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Media file 14: Normal muscle. High-power view of the periodic acid-Schiff (PAS) stain shows the normal color and pattern of this stain, which stains sugar moieties so that glycogen, mucopolysaccharides, and glycoproteins are highlighted. This method is most useful for evaluating glycogen storage disease. It also shows the basal lamina of blood vessels so that the PAS stain also provides information about vascular structure. It can highlight fibers that are degenerating or necrotic and demonstrate some inclusions.

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Media file 15: Normal muscle. The modified Gomori trichrome stain is valuable in evaluating mitochondrial myopathies, inclusion body myositis, and some other disorders with intracellular inclusions. The nuclei and mitochondria stain red, the cytoplasm is mostly blue-green, and the connective tissue is green.

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Media file 16: Normal muscle. Sudan Black stain for lipid demonstrates slightly more staining of type 1 myofibers because of their higher lipid content due to their greater dependence on aerobic metabolism compared with type 2 myofibers. Hereditary and acquired disorders of lipid metabolism show excessive staining of fibers. Some of the mitochondrial myopathies are associated with increased intracellular lipid content.

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Media file 17: Normal muscle. Hematoxylin and eosin (H&E) paraffin section shows the fibers aligned linearly in longitudinal section. Most of the nuclei are myofiber nuclei, but some are also capillary endothelial nuclei. Paraffin section provides more cytologic detail than frozen material, so it improves identification of cells involved in inflammatory disorders. Detailed structure of vascular walls can be seen in paraffin sections. This section is usually larger than frozen section and therefore offers more material for examination.

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Media file 18: Normal muscle. High power hematoxylin and eosin (H&E) paraffin section shows myofibers in longitudinal section. Striations produced by the sarcomeres are clearly visible. One of the earliest signs of myofiber necrosis is the loss of these striations.

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Media file 19: Normal muscle. Electron photomicrograph of skeletal muscle in longitudinal section shows the pleasing ultrastructural organization of internal cytoplasmic contents of the cell. Sarcomeres are seen as units bounded by thin, dark lines (Z bands). Broad, light zones are I bands, which are formed by predominantly thin actin filaments. Broad, dark areas are A bands formed by the overlap of thick myosin and thin filaments. Thin, pale lines are in the central region of sarcomeres, where only thick filaments are present. Between myofibrils in the intermyofibrillar network, the cell contains glycogen, lipid, mitochondria, and triads. Mitochondria are dark, ovoid structures found mostly next to the I bands. Triads, located at A-I junctions, are seen better on Image 20 than here.

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Media file 20: Normal muscle. High-power electron photomicrograph shows 2 triads in the middle third of the picture. They are composed of 2 lateral sacs, which are expanded regions of sarcoplasmic reticulum, on either side of a central portion of T-tubule. The T-tubule is continuous with the sarcolemma at the surface of the muscle cell. Triads are seen at the A-I junction. Several ovoid mitochondria are noted. Pinpoint granular material is glycogen.

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Media file 21: Normal muscle. Electron microscopy of a type 1 myofiber shows abundant and fairly large mitochondria. Round, pale homogeneous bodies adjacent to the mitochondria are lipid droplets, which are abundant in type 1 myofibers compared with type 2 myofibers shown in Image 22.

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Media file 22: Normal muscle. Electron photomicrograph of a normal type 2 myofiber shows the small, less conspicuous mitochondria and low lipid content of type 2 fibers compared with the type 1 myofiber shown in Image 21.

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Media file 23: Improper handling of a muscle biopsy sample. Hematoxylin and eosin section is from a muscle sample that arrived in the laboratory stuck to dry ice; this improper handling caused uneven freezing of the sample. Compare this with the normal structure of muscle in hematoxylin and eosin–stained sections of muscle that were properly handled. This error can interfere with diagnosis.

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Media file 24: Improperly handled muscle biopsy sample, hematoxylin and eosin (H&E) paraffin section. This specimen was dropped into cold fixative in the operating room without first immobilizing it at rest length. The result was artifactual hypercontraction of the myofibers, mimicking acute infarction of muscle.

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Media file 25: Improperly handled muscle biopsy sample, electron micrograph. The surgeon was instructed to mince the muscle and place it in glutaraldehyde in the operating room. Compare the internal architectural disarray with the normal internal ultrastructural appearance of muscle in Images 19-22.

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Media file 26: Neurogenic atrophy. Nicotinamide adenine dinucleotide tetrazolium reductase (NADH) frozen section shows an isolated angulated atrophic fiber that is dark with this stain. This is a characteristic image of a denervated fiber.

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Media file 27: Neurogenic process, fiber-type grouping, myosin adenosine triphosphatase (ATPase) pH 10.5. When reinnervation occurs, myofiber types cluster instead of exhibiting the normal random checkerboard distribution of the two myofiber types as seen in Images 11-13. Left side shows a field composed exclusively of type 2 myofibers stained brown. Right side shows a field of type 1 myofibers stained pink. This is a reinnervation pattern.

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Media file 28: Neurogenic atrophy, hematoxylin and eosin (H&E) frozen section. Group atrophy: a small group of angulated atrophic fibers is seen at the center of this field.

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Media file 29: Neurogenic process, target fibers, on nicotinamide adenine dinucleotide tetrazolium reductase (NADH) stain. Several target fibers are distributed throughout the image. These fibers contain central round, clear central areas with a peripheral dark rim. These fibers occasionally have a third zone of intermediate density and are observed in acute denervation and reinnervation.

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Media file 30: Neurogenic process, nuclear clumps, hematoxylin and eosin (H&E) paraffin section. The arrow indicates a dark blue structure that is a cluster of nuclei referred to as a nuclear clump. Several other nuclear clumps are present in this image. They are typically a feature of neurogenic atrophy, although they can also be observed in myopathies. This image also shows a few angulated atrophic myofibers and an increase in internal nuclei.

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Media file 31: Myofiber necrosis, hematoxylin and eosin (H&E) paraffin section. Two necrotic myofibers are present in the center of this image. They are mildly swollen and their cytoplasm is paler than the surrounding viable myofibers and has a homogenized appearance without striations, which are faintly detectable in the intact fibers in the upper left part of the image.

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Media file 32: Myophagocytosis, hematoxylin and eosin (H&E) frozen section. A single myofiber, approximately in the center of the field, has pale cytoplasm and numerous nuclei, some associated with foamy cytoplasm. These cells are macrophages that are removing cellular debris.

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Media file 33: Regeneration, paraffin section, hematoxylin and eosin (H&E) stain. Small cluster of regenerating fibers courses through the center of this image. Regenerating fibers have basophilic, or slightly blue, cytoplasm. Nuclei are prominent and contain conspicuous nucleoli.

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Media file 34: Myopathic rounded atrophic fibers on hematoxylin and eosin (H&E) frozen section. Specimen from a patient with muscular dystrophy has greater-than-normal variability in fiber size due to both hypertrophy and atrophy. In contrast to fibers in neurogenic atrophy, these atrophic fibers are rounded. (See also Image 70.)

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Media file 35: Myofiber hypertrophy and splitting, hematoxylin and eosin (H&E) stain. Fibers in this slightly tangential cross-section are enlarged, and a few exhibit splitting in which they are divided into 2 fibers. Ingrowth of endomysium and blood vessels can be observed.

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Media file 36: Internal nuclei on hematoxylin and eosin (H&E) section. Most of the fibers here are large and contain several nuclei in the middle of the cytoplasmic compartment. In normal skeletal muscle, as many as 3% of the myofibers can have internal nuclei, and most are at the periphery of the cell.

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Media file 37: Fibrosis, hematoxylin and eosin (H&E) frozen section. Sample from a 10-year-old child with probable congenital muscular dystrophy shows severe endomysial fibrosis. One clue to fibrosis is the separation of myofibers. In normal muscle, the endomysium is so thin it is almost invisible, and the myofibers appear to be almost in direct contact with each other. This sample also shows increased variability of fiber size due to atrophy and an increase in internal nuclei.

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Media file 38: Nuclear chain on hematoxylin and eosin (H&E) paraffin section. One fiber near the center contains a row of nuclei lined up, forming a chain. This is a nonspecific, common finding in myopathy.

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Media file 39: Moth-eaten fibers on nicotinamide adenine dinucleotide tetrazolium reductase (NADH)–stained section. Many fibers on this section have irregular distribution of the intermyofibrillar network, with many irregular patchy pale areas, in a pattern reminiscent of that formed in a sweater besieged by moths. This is a nonspecific myopathic pattern and indicates that these fibers are not well.

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Media file 40: Nicotinamide adenine dinucleotide tetrazolium reductase (NADH) frozen section showing a ring fiber in the center of the field. Myofibrils at the periphery of the fiber are oriented circumferentially instead of longitudinally in the muscle cell. This finding is reported in myotonic dystrophy, but it is a nonspecific finding. This specimen is not from a patient with myotonic dystrophy.

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Media file 41: Whorled fibers on nicotinamide adenine dinucleotide tetrazolium reductase (NADH) stain. Two whorled fibers observed on frozen section are characterized by a coiled appearance of the sarcoplasm.

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Media file 42: Inclusions on hematoxylin and eosin (H&E) paraffin section. Many of the myofibers in this section have fairly large inclusions that consist of homogeneous pale material. This slide is from a patient with tubular aggregate myopathy.

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Media file 43: Inclusions on hematoxylin and eosin paraffin section. Many of the myofibers in this section have fairly large inclusions that consist of homogeneous pale material. This slide is from a patient with tubular aggregate myopathy.

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Media file 44: Inclusion in oculopharyngeal dystrophy, hematoxylin and eosin (H&E) frozen section. The small myofiber indicated by the black arrow has an ovoid eosinophilic inclusion with a faint blue rim. This section also shows greater-than-normal variability of fiber size due to the presence of both atrophy and hypertrophy, an increase in internal nuclei, and a single small fiber (dark red) that is probably degenerating. The green arrows indicate a split fiber, which occurs when there is myofiber hypertrophy.

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Media file 45: Polymyositis, hematoxylin and eosin (H&E) frozen section. The numerous small dark blue cells constitute a dense, chronic, endomysial lymphocytic inflammatory infiltrate. This section also shows many rounded atrophic myofibers, an increase in internal nuclei, and moderate endomysial fibrosis.

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Media file 46: Polymyositis on hematoxylin and eosin (H&E) frozen section. Endomysial chronic inflammation is present among intact myofibers that are remarkable only for increased variability of fiber size.

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Media file 47: Polymyositis, immunohistochemistry for CD3 (T-lymphocytes) with hematoxylin counterstain. All of the brown cells percolating throughout the endomysium are T-lymphocytes. The myofibers in this field are not necrotic. The lower region of the largest myofiber in the field appears to be invaginated by lymphocytes. There is myofiber atrophy and mild-to-moderate endomysial fibrosis.

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Media file 48: Myotendinous junction on hematoxylin and eosin (H&E) paraffin section. Segment of dense epimysial connective tissue traverses the field, forming a partial U shape. Myofibers in its vicinity vary in size and shape and have increased numbers of internal nuclei. These findings are expected at a normal myotendinous junction and therefore should not be misinterpreted.

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Media file 49: Forms of myositis. Fasciitis, which can have overlapping clinical features with myositis and can often be diagnosed on muscle biopsy, could also be included on this list. Muscle pathology in the eosinophilia myalgia syndrome (which can also be listed in the eosinophilic myositis category), eosinophilic fasciitis (Schulman syndrome), and relapsing perimyositis belong in this category.

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Media file 50: Polymyositis. Panel A, hematoxylin and eosin (H&E) paraffin section shows an attack on a nonnecrotic myofiber by autoaggressive lymphocytes. Panel B, immunohistochemistry for CD3 (T-lymphocytes) demonstrates segmental infiltration of a non-necrotic myofiber by T-lymphocytes. Labeling for CD8 or cytotoxic/suppressor T-lymphocytes would be more specific.

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Media file 51: Dermatomyositis, hematoxylin and eosin (H&E) paraffin section. In dermatomyositis, the characteristic inflammation is perivascular within the perimysial connective tissue. In this image, a perivascular lymphocytic infiltrate is associated with a blood vessel, indicated by the arrow. A few red blood cells within the lumen assist in identifying this as a blood vessel. Notice that the surrounding myofibers and endomysium are not inflamed.

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Media file 52: Dermatomyositis, hematoxylin and eosin (H&E) paraffin section. The large fascicle in this image shows myofiber atrophy that is located predominantly along one side of the periphery, indicated by the arrows. This is perifascicular atrophy, a finding that is highly characteristic of dermatomyositis, although this can be seen in other disorders. The cause of perifascicular atrophy is thought to be ischemia at the capillary level. This characteristic finding of dermatomyositis is most often associated with the juvenile form but it is also frequently observed in adult dermatomyositis.

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Media file 53: Dermatomyositis, immunofluorescence for the membrane attack complex of complement (MAC). The bright ring of yellow-green fluorescence at the center represents MAC in the wall of a microvessel. This finding is not present after treatment with steroids.

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Media file 54: Dermatomyositis, tuboreticular inclusion in endothelial cell, on electron microscopy. Endothelial cell, identified by the presence of many pinocytotic vesicles, contains an inclusion composed of an aggregate of undulating tubules.

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Media file 55: Inclusion body myositis, attack on nonnecrotic myofiber by autoaggressive lymphocytes, hematoxylin and eosin (H&E) paraffin section. The myofiber in the center of the field is not necrotic. This fiber is infiltrated by lymphocytes. This is a segmental attack on a non-necrotic myofiber by autoaggressive lymphocytes. Immunohistochemistry would identify the lymphocytes as T-cells, most likely CD8 or cytoxic-suppressor T-lymphocytes.

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Media file 56: Inclusion body myositis (IBM), hypertrophy, and other myopathic features, on hematoxylin and eosin (H&E) frozen section. Several fibers are large, and several are split. Split fibers can be identified as groups of 2 or more fibers that fit together in a single nest and share an imaginary outline, as opposed to the normal situation where fibers fit together in a mosaic pattern. Atrophy is also characteristic of IBM. Here, the atrophy shares characteristics with neurogenic atrophy in that the atrophic fibers are angulated and occur in groups. A few dead fibers undergoing myophagocytosis are found. Increase in internal nuclei and mild endomysial fibrosis are present.

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Media file 57: Inclusion body myositis (IBM), rimmed vacuoles, on hematoxylin and eosin (H&E) frozen sections. The arrows indicate 3 rimmed vacuoles, which are cytoplasmic vacuoles lined by blue granular material. The right and left panels are from 2 different cases of IBM.

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Media file 58: Inclusion body myositis, eosinophilic inclusion, on hematoxylin and eosin (H&E) frozen section. Two large myofibers in the central region of the image contain dense eosinophilic inclusions surrounded by basophilic granular material. These inclusions may be cytoplasmic, within rimmed vacuoles, or they may be present within nuclei. These inclusions label for amyloid precursor protein, ubiquitin, tau, and prion protein, which are characteristic of central neurodegenerative diseases.

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Media file 59: Inclusion body myositis, eosinophilic inclusions. Panel A, hematoxylin and eosin (H&E) frozen section shows a large, fairly homogeneous eosinophilic inclusion within a myofiber, indicated by the arrow. In panel B, a Congo red stain from a different case demonstrates an inclusion that is positive for Congo red, indicated by the arrow. (See also Image 58.)

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Media file 60: Inclusion body myositis, ultrastructure of tubulofilamentous inclusion. Characteristic tubulofilaments of the inclusions of inclusion-body myositis are seen in both cross section and longitudinal section in the cytoplasm of a myofiber in the center and lower left quadrant of this electron micrograph. They are 15-18 nm in diameter.

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Media file 61: Trichinosis, hematoxylin and eosin paraffin section. Larva of Trichinella spiralis occupies the center of the image. Below it, a chronic perivascular infiltrate courses horizontally across the field.

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Media file 62: Drug reaction on hematoxylin and eosin (H&E) section. Numerous eosinophils, identified by their bright red granular cytoplasm, are located in the connective tissue between myofibers; bright red extracellular granules are the result of release of granules by eosinophils. Clinicopathologic correlation confirms the histological findings to be most consistent with a drug reaction.

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Media file 63: Dystrophinopathy, Duchenne muscular dystrophy, myopathic features, on hematoxylin and eosin (H&E) frozen section. This image shows marked endomysial and perimysial fibrosis and increased variability of fiber size due to the presence of atrophy and hypertrophy of myofibers.

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Media file 64: Dystrophinopathy, Duchenne muscular dystrophy, on hematoxylin and eosin (H&E) frozen section. The 2 myofibers that occupy the majority of this image originate from a single hypertrophied fiber that split by the ingrowth of connective tissue. A fiber-typing stain would demonstrate that these 2 fibers are of the same myofiber type (not shown).

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Media file 65: Dystrophinopathy, Duchenne muscular dystrophy, hematoxylin and eosin (H&E) frozen section. A necrotic myofiber, with pale cytoplasm, is undergoing myophagocytosis. Image also shows an increase in internal nuclei, increased variability of myofiber size, 2 opaque fibers just below the necrotic fiber, and prominent fibrosis.

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Media file 66: Dystrophinopathy, Duchenne muscular dystrophy, opaque fibers, hematoxylin and eosin (H&E) section. Occasional fibers scattered throughout this biopsy sample are large, dark red, and glassy. Termed opaque fibers, they represent hypercontracted fibers. The image also shows increased variability of myofiber size, focal myofiber necrosis, mild increase in internal nuclei, and extensive fibrosis of both perimysium and endomysium.

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Media file 67: Normal muscle, dystrophin immunohistochemistry, on frozen section. Sample of normal muscle stained with an antibody to dystrophin shows the normal subsarcolemmal localization of this protein demonstrated by the linear peripheral brown staining of every myofiber.

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Media file 68: Dystrophinopathy, Duchenne muscular dystrophy, dystrophin immunohistochemistry. Most of the myofibers of this muscle fail to label with antibodies to the dystrophin molecule. A single fiber does stain for dystrophin. This is a revertant fiber, one in which a second mutation in the large dystrophin gene has restored the ability of this myofiber to produce dystrophin. The identical result is obtained with antibodies specific for the N-terminal, C-terminal, and mid-rod portions of the dystrophin molecule. Image 67 shows the pattern of staining expected when dystrophin is present, for comparison.

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Media file 69: Dystrophinopathy, Becker muscular dystrophy, hematoxylin and eosin (H&E) paraffin section. A necrotic myofiber undergoing myophagocytosis occupies the central zone of this image. Focal, mild, chronic inflammatory response is observed surrounding the fiber. Striations are visible in the adjacent intact myofibers.

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Media file 70: Dystrophinopathy, Becker muscular dystrophy, hematoxylin and eosin (H&E) frozen section. Muscle biopsy demonstrates greater-than-normal variability of myofiber size due to atrophy and hypertrophy. Most of the atrophic fibers have rounded contours. A mild increase in internal nuclei is also present. (See also Image 34.)

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Media file 71: Dystrophinopathy, Becker muscular dystrophy, hematoxylin and eosin (H&E) frozen section. A split fiber is observed in the center. Internal nuclei are increased in number.

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Media file 72: Dystrophinopathy, Becker muscular dystrophy, dystrophin immunohistochemistry. This section, stained with an antibody to the C-terminal region of dystrophin, demonstrates a normal pattern of labeling of all of the myofibers, including the split fiber above the center of the picture.

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Media file 73: Dystrophinopathy, Becker muscular dystrophy, dystrophin immunohistochemistry. This section, stained with an antibody to the N-terminal region of dystrophin, demonstrates absence of subsarcolemmal staining, indicating that this portion of dystrophin is absent. Compare with Image 72, from the same patient, which demonstrates the presence of the C-terminal portion of dystrophin. The diffuse light brown color of the myofibers is a high background and has no diagnostic significance.

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Media file 74: Congenital muscular dystrophy due to laminin α-2 (merosin) deficiency, dystrophin immunohistochemistry. This preparation demonstrates normal labeling with antibody to dystrophin, which is present with all 3 antidystrophin antibodies. This muscle sample is not normal. Moderate variability of fiber size and fibrosis, which separates the myofibers, are present.

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Media file 75: Congenital muscular dystrophy due to deficiency of laminin α-2 (also known as merosin), laminin a-2 immunohistochemistry. The myofibers show no labeling with an antibody to laminin α-2 (merosin), a protein normally found within the basal lamina at the surface of myofibers. Image 76 shows a normal control.

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Media file 76: Normal muscle, laminin α-2 (merosin) immunohistochemistry. A normal pattern of labeling with the antibody to laminin α-2 (merosin) is demonstrated as a red reaction product at the sarcolemma (plasma membrane of the myofibers).

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Media file 77: Werdnig-Hoffmann disease (infantile spinal muscular atrophy [SMA] I), on hematoxylin and eosin (H&E) frozen section. The myofibers range from abnormally small to abnormally large. Clustering of the largest fibers (on the right side) is a characteristic finding of this disorder.

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Media file 78: Werdnig-Hoffmann disease, myosin adenosine triphosphatase (ATPase) at pH 4.3. Type 1 myofibers are brown, and type 2 myofibers are pink. The smallest and largest fibers are type 1, and the large type 1 fibers are clustered. These are the characteristic pathologic findings of this disorder.

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Media file 79: Type II glycogenosis, acid maltase deficiency, hematoxylin and eosin (H&E) paraffin section. Several myofibers throughout this sample have numerous clear cytoplasmic vacuoles.

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Media file 80: Type II glycogenosis, acid maltase deficiency, on electron micrography. Huge aggregates of membrane-bound intralysosomal glycogen are observed in this myofiber.

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Media file 81: Type V glycogenosis, myophosphorylase deficiency, hematoxylin and eosin (H&E) frozen section. The myofiber in the central region of the image contains several subsarcolemmal clear vacuoles.

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Media file 82: Type V glycogenosis, myophosphorylase deficiency, periodic acid-Schiff (PAS) frozen section. Most myofibers in this section contain large vacuoles with PAS-positive material. These vacuoles correspond to the clear vacuoles in image 81.

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Media file 83: Type V glycogenosis, myophosphorylase deficiency, periodic acid-Schiff (PAS) stain with diastase, which digests glycogen. On this photomicrograph, 2 vacuoles in a myofiber originally stained with PAS are empty after treatment with diastase. This finding proves that the material in the vacuole is glycogen.

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Media file 84: Type V glycogenosis, myophosphorylase deficiency, ultrastructure. A huge pool of free glycogen in the center of the image is in the subsarcolemmal zone of the myofiber seen in the top half of the image. Also note a mild increase in intermyofibrillar glycogen.

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Media file 85: Type V glycogenosis, myophosphorylase deficiency on a frozen section stained for myophosphorylase activity. The myofibers show absence of staining for a product of the activity of myophosphorylase, which establishes the diagnosis. Image 86 demonstrates the normal control for this stain.

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Media file 86: Normal muscle on a frozen section stained for myophosphorylase activity. Normal skeletal muscle specimen shows the delicate black reaction product formed by myophosphorylase activity. Compare with Image 85, from a patient with myophosphorylase deficiency.

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Media file 87: Mitochondrial myopathy, Gomori trichrome frozen section. Observe the classic ragged red fiber in the center of the field. The peripheral rim of red staining represents aggregates of mitochondria.

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Media file 88: Mitochondrial myopathy, on a frozen section stain for cytochrome oxidase (COX) activity. In this section, three populations of myofibers can be identified. Type 1 myofibers stain the darkest golden brown, and type 2 fibers are lighter tan-brown. The third population consists of pale fibers; these are COX-negative fibers.

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Media file 89: Mitochondrial myopathy, combined staining for the activities of cytochrome oxidase (COX) and succinic dehydrogenase (SDH), frozen section. Here, the fibers that are positive for both COX and SDH are taupe. This results from combining the brown color of the stain for COX with the blue color of the SDH stain. (See Image 88 for the color of the COX-positive fibers without the simultaneous SDH stain.) Blue fibers are those that stain for SDH activity alone because of absence of COX activity. Several blue fibers have subsarcolemmal dense blue stain. SDH is a mitochondrial enzyme, so the dark blue rim represents aggregates of mitochondria. These fibers correspond to the ragged red fibers on trichrome stain. All of these fibers in this COX/SDH preparation are negative for COX.

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Media file 90: Mitochondrial myopathy, ultrastructure. A large number of enormous mitochondria can be seen in the intermyofibrillar network of this myofiber. These mitochondria are larger than entire sarcomeres. Normal mitochondria are much smaller than sarcomeres. For comparison, see Images 20-22, which show normal mitochondria.

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Media file 91: Mitochondrial myopathy on electron micrography. At high magnification, some of the mitochondria in Image 90 contain paracrystalline material. They lack the normal configuration of cristae formed by the inner mitochondrial membrane of normal mitochondria.

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Media file 92: Mitochondrial myopathy on electron micrography. Many morphologically abnormal mitochondria contain dense crystalline inclusions.

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Media file 93: Nemaline myopathy, hematoxylin and eosin (H&E) frozen section. This biopsy from a patient with nemaline myopathy is remarkable only for a mild increase in internal nuclei on H&E section. Small, faint, clear holes in the myofibers represent slight ice crystal artifact.

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Media file 94: Nemaline myopathy, Gomori trichrome frozen section. Many fibers contain tiny inclusions. They appear dark blue in this image, but when viewed with a microscope, they are red. These inclusions are the nemaline rods of this congenital myopathy.

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Media file 95: Nemaline myopathy on trichrome stain. One myofiber contains 2 clusters of nemaline rods. They appear blue-red and have a slightly elongated, granular appearance.

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Media file 96: Nemaline myopathy on myosin adenosine triphosphatase (ATPase) pH 10.5 frozen section. A marked predominance of type 1 myofibers is present; this is a common occurrence in some congenital myopathies. Type 1 myofibers are pink and type 2 fibers are brown. Clear holes in many of these fibers are from freezing artifact.

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Media file 97: Nemaline myopathy on electron micrography. Many electron-dense fibrillar structures originate in Z bands. This muscle biopsy sample was not clamped before fixation; therefore, the usual orderly ultrastructural architecture of the myofibers was not preserved. Nonetheless, in this patient, the nemaline rods are observed and identified easily.

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Media file 98: Central core disease on hematoxylin and eosin (H&E) frozen section. Look closely, particularly at the large fibers at the top of the image; a faint decrease in staining can be detected in the center of some myofibers. These are the central cores, which are almost invisible on H&E section. In this patient, because of atrophy of occasional fibers, variability of myofiber size is greater than normal.

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Media file 99: Central core disease on myosin adenosine triphosphatase (ATPase) pH 10.5 frozen section. Two pale myofibers contain central clear round areas. The central cores are found in type 1 fibers.

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Media file 100: Tubular aggregate myopathy on periodic acid-Schiff (PAS) frozen section. Many of the myofibers in this image contain fairly large inclusions that are PAS positive.

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Media file 101: Tubular aggregate myopathy on myosin adenosine triphosphatase (ATPase) pH 10.5 frozen section. Type 2 myofibers are brown; these contain inclusions.

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Media file 102: Tubular aggregate myopathy, nicotinamide adenine dinucleotide tetrazolium reductase (NADH) frozen section. Many type 2 myofibers, identified by their light staining compared with type 1 fibers, contain inclusions that are dark with this stain.

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Media file 103: Tubular aggregate myopathy, succinic dehydrogenase (SDH) stain. The inclusions, which are the tubular aggregates, can be seen, but they do not stain for SDH. The finding of a nicotinamide adenine dinucleotide tetrazolium reductase (NADH)–positive, SDH-negative inclusion is exceptional and highly suggestive of this diagnosis.

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Media file 104: Tubular aggregate myopathy, electron micrograph. Cross-section reveals the tubular nature of the inclusions. A tubular aggregate occupies the central zone of the image.

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Media file 105: Tubular aggregate myopathy, electron micrograph. A tubular aggregate, in slightly tangential section, runs from the upper left to lower right. The middle of the image, on the left side of the aggregate, demonstrates continuity of the tubules with the sarcoplasmic reticulum.

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Media file 106: Perifascicular atrophy, in which atrophic fibers are located at the periphery of the muscle fascicle, as observed in the 2 fascicles in this photograph, is one of the characteristic histopathologic findings that support a diagnosis of dermatomyositis.

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Media file 107: This muscle specimen is from debridement of a traumatic rupture of a fibularis longus muscle (formerly known as peroneus longus) due to an accident during a baseball game. Before the injury, the patient had no evidence of any underlying neuromuscular disorder. The biopsy findings are consistent with muscle necrosis and reactive change to the necrosis.

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Media file 108: The accompanying photograph is from an immunohistochemical stain using an antibody to dystrophin. It shows many fibers that are negative for dystrophin and a cluster of fibers in the center of the field that have a brown rim, indicating that they do produce dystrophin. The presence of these revertant fibers is expected in Duchenne dystrophy. It is the result of a small number of fibers that have a second mutation in the large dystrophin gene that restores the reading frame and permits production of dystrophin in these fibers.

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Media file 109: Upregulation of utrophin expression in a dystrophinopathy; immunohistochemistry for utrophin. Panel A, on the left, demonstrates the normal pattern of staining for utrophin in children and adults. The myofibers exhibit no detectable staining, while the blood vessels stain brown. Panel B, on the right, demonstrates upregulation of utrophin expression in a case of a dystrophinopathy. The myofibers exhibit extensive brown staining of the sarcolemmas (plasma membranes), indicative of the presence of utrophin, which may be an attempt to compensate for the lack of dystrophin in these myofibers. The clear vacuoles within the cytoplasm of the myofibers represent mild to moderate ice crystal artifact.

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Media file 110: Acid phosphatase histochemistry. Acid phosphatase is a lysosomal enzyme. Panel A is from a case of adult onset acid maltase deficiency. Two myofibers, indicated by the arrows, exhibit excessive dark red staining that indicates that lysosomes are increased in number and size. The abnormally increased staining here corresponds to vacuoles within the myofibers that contain excessive glycogen. The majority of the myofibers in the biopsy exhibit normal staining. Panel B is from a case of chloroquine myopathy, with markedly increased red staining in many myofibers, corresponding to the widespread increase in number and size of lysosomes in this case.

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

I would like to thank all of the clinicians and surgeons who allow me to share in the care of their patients with neuromuscular disorders, Karin Thompson and the other technologists whose conscientiousness and expertise in handling and processing the biopsies are essential to the successful evaluation of the patients, and Dr. Nancy Peress, who generously devoted years of her life to training me in neuropathology and subsequently working with me as a treasured colleague and advisor.

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