Muscle Biopsy and Clinical and Laboratory Features of Neuromuscular Disease
- Author: Roberta J Seidman, MD; Chief Editor: Erik D Schraga, MD more...
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, 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 a neurogenic disorder with an atypical presentation 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 specimens 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 specimen 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.
Muscle biopsy therefore is somewhat complex, in that an optimal outcome requires coordination of the clinician, surgical team, pathologist, and technical staff in the pathology laboratory. Because muscle biopsies are often interpreted at specialized centers, it may also be necessary to involve a courier service in the process.
A portion of this article serves as a primer on the technical aspects of muscle biopsy, which are critical for the success of the procedure. The aim is to help the reader understand how biopsies should be performed and what happens to the tissue that is obtained, as well as to provide some background information to assist in the comprehension of the pathology reports.
Clinical features of neuromuscular disease are highlighted because knowledge of the clinical history is crucial for correct interpretation of 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 several different specific groups of myopathies and how these disorders are diagnosed.
It is helpful to compare the structure and histology of normal skeletal muscle (see Skeletal Muscle - Structure and Histology.) with the pathologic alterations observed in muscle (see Skeletal Muscle Pathology.) This knowledge provides 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 process of formulation of histopathologic diagnoses.
When a clinical diagnosis of myopathy is considered, muscle biopsy is often required (for exceptions to this requirement, see Contraindications below).
Muscle biopsy is a fundamental part of the evaluation of a patient with possible muscle disease, also known as 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, among myopathies, the most specific and effective therapies are for the inflammatory myopathies. Sometimes, a patient is too seriously ill to allow delay therapy even a few days; however, whenever possible, perform muscle biopsy before initiating 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 for evaluation of the patient with known inflammatory myopathy who, after improvement with steroid therapy, develops increasing weakness. Biopsy findings can help distinguish between recurrence of the inflammatory 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. The classic presentations of these two processes are clearly distinct; however, in practice, the 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 is 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 require muscle biopsy for a diagnosis. This section, accordingly, discusses circumstances in which a muscle biopsy is not required for diagnosis of a myopathy, rather than actual contraindications to muscle biopsy.
The exceptions to the requirement for muscle biopsy for accurate diagnosis of possible myopathy include the following:
Suspected dystrophinopathies, particularly when the clinical presentation has features that are highly characteristic of Duchenne or Becker muscular dystrophies
Some congenital and limb-girdle dystrophies 
Certain mitochondrial disorders
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 ultimately found to have abnormalities of dystrophin. This category includes some women who carry a single X chromosome with a dystrophin mutation, in whom an unfortunate pattern of random inactivation of X chromosomes gives rise to 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 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 four sarcoglycans, dysferlin, caveolin-3, calpain, lamin A/C, and fukutin-related protein. 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 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; thus, if these disorders are suspected on the basis of clinical presentation, genetic testing of blood, rather than muscle biopsy, is indicated.
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. Because they lack specifically diagnostic findings on muscle biopsy, genetic testing is the proper 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. (See Pathology of Skeletal Muscle.)
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. More information about myotonic dystrophy is available from the Myotonic Dystrophy Organization.
Open muscle biopsy is performed with local anesthesia for most adult patients. General anesthesia is typically required for infants and young children.
If surgery is performed under local anesthesia, care should be exerted not to inject the anesthetic directly into the biopsy site. This minimizes the risk of introducing a track from the needle and the anesthetic into the muscle, which can interfere with interpretation of the biopsy. The local anesthetic should be injected just under the skin in an oval area, whose length is approximately 1.5 times that of the incision.
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 in the light of the clinical history. To properly assess the significance of histologic findings in a particular muscle biopsy sample, the pathologist must have information about the clinical presentation of a given patient.
As an example, 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. However, clear vacuoles are also present in many myopathic disorders, including (but not limited to) certain glycogen storage diseases, lipid myopathies, periodic paralyses, and toxic myopathies that can result from treatment with colchicine, chloroquine, or amiodarone.
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 the example above, 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.
Against this background, 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.
Signs and symptoms
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 hypotonic (the "floppy infant"). In later infancy and during the toddler years, delay in 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; it 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, myophosphorylase deficiency leads to 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 because free fatty acids can be used for generation of energy, 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. Myasthenia gravis is usually diagnosed on the basis of the clinical presentation, electrodiagnostic studies showing a decremental response in the muscle with repetitive stimulation of the motor nerve, and the presence of serum antibodies to the acetylcholine receptor.
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
Concomitant somatosensory abnormalities
In their conventional clinical presentations, distinguishing muscle disease from peripheral nerve disease is a straightforward matter. In practice, however, this distinction is not always simple. There are several reasons why it may be difficult to determine whether a patient has neuropathy or myopathy on clinical evaluation.
One reason is that some myopathies affect distal muscles. Myotonic dystrophy, inclusion body myositis (IBM), some of the myofibrillar myopathies, and distal myopathy of Welander are examples of myopathies that can affect distal muscle groups.
In addition, 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 diabetic neuropathy can also acquire an inflammatory myopathy. A patient who has peripheral neuropathy caused by chemotherapy for cancer may develop dermatomyositis. A patient can have both 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, A Clinician's View of Neuromuscular Diseases, provides insight into the clinical evaluation of patients with neuromuscular disease. Although it was written before the explosion of information regarding the molecular genetics of neuromuscular disease, it is nonetheless a unique and valuable tool. It is out of print, but copies can still be found for purchase and in libraries.
The serum creatine kinase (CK) level is the single most important blood value to obtain when myopathy is being considered. A representative normal 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 (>1500 IU/L) usually indicate muscle disease. Mildly elevated levels (200-800 IU/L) can be observed in either neurogenic or myopathic disorders, 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. If the CK level is known to be abnormal, there is no reason to obtain testing for the aldolase level.
Electrodiagnostic studies are often extremely useful in determining whether a neuropathic, myopathic, or mixed disorder is present.
Changes in nerve conduction velocities, the compound muscle-action potential, or both 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 can interfere with proper interpretation of a biopsy for 1-2 months. In patients with suspected myopathy, needle EMG should be performed on only one side; a subsequent muscle biopsy should be performed on the other side.
Selection of 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. Selection of a muscle should be based on the expected distribution of the leading clinical diagnosis. For example, if the leading diagnostic consideration is polymyositis, a proximal muscle, such as the vastus lateralis of the quadriceps, is the best choice for biopsy.
When possible, biopsy a muscle that is not too weak and atrophic (see the first image below), because this may result in obtaining a sample of end-stage muscle. 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 tissue handling
It is most important to perform a muscle biopsy with a minimum of trauma to the muscle tissue in order to decrease the risk of causing disruption of the muscle architecture and to minimize the risk of introducing contraction band artifact. Electrocautery should not be used while obtaining a specimen for muscle biopsy.
The specimens required and the details of the preferred method of biopsy handling vary among medical centers. It is essential to consult the center that will receive the biopsy sample so as 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. The information below should be considered 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 two specimens: fresh and fixed. In certain special clinical circumstances, a third sample for biochemical analysis may contribute to the specific diagnosis.
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, a strong motivation to perfect the technique, and physicians experienced with the procedure, needle biopsy is the preferred method of muscle biopsy, with a diagnostic yield comparable to open biopsy.
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 only for 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 the image below) is used for histochemical studies in all patients and for immunofluorescence in selected patients, when indicated. It should measure approximately 0.5 × 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; in this way, it is kept cold with freezing. 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 another mounting medium) in the appropriate orientation and snap-freezes it in isopentane chilled in liquid nitrogen. Frozen cryostat sections are cut from this sample.
Optimally, this fresh specimen is then immediately transported to the laboratory for processing to prevent the tissue from losing any of its enzymatic reactivity or immunogenicity for immunohistochemical studies. In most situations, however, 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 (though a delay longer than overnight is definitely not recommended).
A fixed specimen (see the 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, IL). 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 muscle 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. The sample can be sent as a fresh specimen to the laboratory, 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. To prevent contraction of the muscle, the sample should be maintained at rest length before it is immersed in the fixative. The specimen to be immersed must be small (1-2 mm wide or deep) because glutaraldehyde penetrates tissue slowly. If it 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 is strongly suspected. The sample may be sent to specialized laboratories for assessment of specific enzymatic activities (eg, mitochondrial enzymes). Such a sample can be used for measurement of specific protein constituents in muscle (eg, the protein dystrophin), but in most cases, the availability of genetic testing on a sample of blood has made Western blot analysis of muscle proteins unnecessary.
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 biopsy sample
For examples of these studies in normal skeletal muscle, see Skeletal Muscle - Structure and Histology; for examples of these studies in various neuromuscular disorders, see Skeletal Muscle Pathology.
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, at Stony Brook Medicine, a battery of stains is performed on the frozen sample in addition to the routine hematoxylin and eosin (H&E) stain. These assist in screening for and evaluating neurogenic or other types of atrophy and metabolic diseases, as well as demonstrating 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 and materials are dissolved 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. (There is some variation with regard to which stains are routinely performed at different institutions.)
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 it 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 two main myofiber types: 1 and 2. Some 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.
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, including 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 (eg, in starvation), and nonspecific abnormalities of the myofibers.
Human leukocyte antigen (HLA) class ABC by immunohistochemistry is used to identify and support the diagnosis of autoimmune or inflammatory myopathies. Other terms for HLA class ABC are HLA class I and major histocompatibility complex (MHC) class I.
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 then can be used to direct special biochemical or genetic 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 can be performed
A stain for acid phosphatase, a lysosomal enzyme, can be useful for the evaluation of certain metabolic disorders and some toxic disorders, as well as in some other circumstances; immunohistochemistry for p62 and LC3, which can be performed on cryostat sections or on formalin fixed paraffin embedded tissue, can also perform this function
For dermatomyositis, immunofluorescence or immunohistochemistry can be performed to look for membrane attack complex of complement in vessel walls
For immune-mediated myopathies, which are usually inflammatory myopathies, immunohistology for MHC class I (ie, HLA class ABC or HLA class I) can be performed, if this is not already part of the routine panel of studies
Paraffin sections are usually stained with H&E. This specimen consists of a fairly large surface of myofibers (muscle cells) 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).
Because the fixed and paraffin-embedded specimen maintains more cytologic detail than the frozen specimen does, it is the preferred sample for detecting subtle evidence of myofiber necrosis, determining the type of inflammatory infiltrate present, and examining the structure of vessels walls.
When indicated, special stains can be performed on the paraffin specimen, including (but not limited to) the following:
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
Although 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; rather, 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. The length of time required for this process depends on the size of the specimen, but overnight fixation is more than satisfactory for the purpose. 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 does, 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; thus, 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 would be 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.
After fixation, the tissue is divided into samples of 1 mm3, postfixed with osmium tetroxide, and embedded in epoxy resin. Samples are oriented in either a longitudinal or a transverse direction before polymerization of the resin. The process of embedment requires 2 days.
Survey sections for light microscopy with a thickness of 1 μ m, 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.
An ultramicrotome with a diamond knife is used to cut thin 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 evidence is being sought 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-microscopic level; if normal muscle is found with all of the light-microscopic studies, then this is usually what EM will show, only larger; the only relatively common exception to this guideline occurs when there is a strong clinical suspicion for dermatomyositis but light-microscopic findings are normal; if TRIs are found, they can lend some support to this diagnosis, but if the patient has already been treated with steroids, it is unlikely that TRIs will be present
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. (For examples of suboptimal biopsies that underscore the great importance of using proper technique and following established protocols, see Skeletal Muscle - Structure and Histology.)
Occasional situations exist when the biopsy must be repeated for precise diagnosis and no one is at fault. Such situations 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 (eg, 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, a lack for which there is no excuse. 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 for learning the 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.
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