Muscle Biopsy and Clinical and Laboratory Features of Neuromuscular Disease 

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.^[1] In addition, muscle biopsy is also sometimes indicated for the diagnosis of various systemic disorders and the evaluation of suspected neuropathic disease, particularly for the purpose of distinguishing between an atypical neurogenic disorder and a primary myopathic disorder.

The surgical procedure to obtain a muscle biopsy is relatively simple and poses little risk to the patient; however, it is a specialized procedure and must be properly performed 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 specimen to obtain, and how to handle the specimen 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 may also need to be involved in the process.

This article serves as a primer on the technical aspects of muscle biopsy, which are critical for the success of this procedure. The article assists in the understanding of how biopsies should be performed, what happens to the tissue that is obtained, and how to evaluate the pathology reports.

Clinical features of neuromuscular disease are highlighted because knowledge of the clinical history is crucial to correctly interpret the histologic findings in a skeletal muscle sample. A discussion of peripheral neuropathy, including clinical features and laboratory findings, is included. Muscle biopsy can sometimes help determine whether a patient has a neurogenic or myogenic disorder, which is a relatively common issue prompting performance of a biopsy.

The general introduction of selected categories of skeletal muscle disease provides information about the classical clinical presentations of different specific groups of myopathies and how these disorders are diagnosed.

A presentation of the structure and histology of normal muscle serves as a basis for comparison with the pathologic alterations observed in muscle. This presentation can be found in the eMedicine article Skeletal Muscle - Structure and Histology.

A discussion of the pathology of the diseases of skeletal muscle is found in the eMedicine article Pathology of Skeletal Muscle. It serves as a basis for understanding the pathophysiology of some of these disorders, assisting the reader in visualizing the effect of disease at the tissue level and demonstrating the formulation of histopathologic diagnoses.

When a clinical diagnosis of myopathy is considered, muscle biopsy is often required (for exceptions to this requirement, please see the Contraindications section below).

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 myopathies and many metabolic and congenital myopathies and 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 prior to the initiation of therapy for the following 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.

Few true contraindications to muscle biopsy are noted. This procedure might be contraindicated in someone with a high risk of intractable bleeding from the procedure or with an obvious infection at the planned biopsy site. Several categories of myopathies do not need muscle biopsy for a diagnosis.

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 dystrophies,^[2] myotonic dystrophy, certain mitochondrial disorders, periodic paralyses, and endocrine myopathies.

Dystrophinopathies and certain other muscular dystrophies

Advances in molecular genetics have eliminated the need for muscle biopsy in most patients with dystrophinopathies by permitting specific diagnoses 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 located on the internal surface of the sarcolemma (muscle plasma membrane). The gene for this protein is extremely large (2 million base pairs). Until recently the size precluded searching the entire gene for point mutations, but now dystrophin gene sequencing is commercially available.

Muscle biopsy is 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 and to exclude an inflammatory myopathy, 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 [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 various 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 specific enough to be diagnostic. Muscle biopsy can also demonstrate a nonspecific myopathic picture in these disorders. For a discussion and examples of nonspecific myopathic findings, see the article Pathology of Skeletal Muscle.

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 Myotonic Dystrophy Organization.

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

Disease of muscle tissue can be expressed 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 a degree of 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 disease.^[3] It was written prior to the explosion of information regarding the molecular genetics of neuromuscular disease, but is nonetheless 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 issues

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 the first image below). 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 the second image below).

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 the requirements 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, usually in the form of multiple minute fragments, obtained by means of needle biopsy. In one center, with a strong research program in muscle disease and a strong motivation to perfect the technique, needle biopsy is the preferred method of muscle biopsy, with a diagnostic yield comparable to open biopsy.^[4] This method generally provides a specimen of limited size that typically is not well-oriented; 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 a patient will not consent to an open biopsy, but will allow a needle biopsy
• When open biopsy is contraindicated

A fresh specimen (see image below) 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).

A fixed specimen (see image below) 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 (V. Mueller, McGaw Park, Ill) (see image above). 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, 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.

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

For examples of these studies in normal skeletal muscle, please refer to the eMedicine article, Skeletal Muscle - Structure and Histology. Examples of these studies in various neuromuscular disorders are found in the eMedicine article Pathology of Skeletal Muscle.

The actual methods for performing the stains can be found in standard 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.

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 include those listed below (some variation on which stains are routinely performed at different institutions is observed).

Hematoxylin and eosin (H&E) is the routine histologic stain used for evaluation of basic tissue organization and cellular structure.

With nicotinamide adenine dinucleotide tetrazolium reductase (NADH) staining, 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 for detecting subtle alterations of intracellular structure in a myofiber that suggest it is not well.

Fiber-typing stains are also used. Muscle is composed of 2 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 2 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.

The Modified Gomori trichrome 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) 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 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.

Human leukocyte antigen class I by immunohistochemistry is used to identify and support the diagnosis of autoimmune or inflammatory myopathies.

Some additional special stains that can be performed on the frozen sample when warranted by the clinical history and findings in the initial battery of stains 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 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 is usually 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 to detect subtle evidence of myofiber necrosis, determine the type of inflammatory infiltrate present, and examine 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 microorganisms, 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

While a small sample of every muscle biopsy should be set aside for possible electron microscopy (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.

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 for EM is possible, 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 and stained with either toluidine blue or methylene blue-azure II. The pathologist reviews these sections 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 electron microscope 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 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 relatively 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.

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. The section Results of improper biopsy procedure in the article Skeletal Muscle - Structure and Histology provides a few examples of suboptimal biopsies to reinforce the point that it is really important to use proper technique and follow established protocols.

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

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.

Unsuitable, suboptimal, or inadequate biopsy specimens can usually 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. 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 exact requirements and the preferred method of handling and shipping the tissue.

The surgical procedure to obtain a muscle biopsy is relatively simple and poses little risk to the patient in the absence of underlying bleeding or clotting disorder.