Muscle Biopsy

The decision to proceed with a muscle biopsy is made after a thorough medical and neurological history, examination, laboratory evaluation, and electromyogram study, thereby permitting the formulation of a preliminary diagnosis and, hence, dictating the studies required of the muscle biopsy specimen. The history characterizes the weakness (e.g., its rate of onset, distribution, time course, precipitants [e.g., exercise-induced], associated symptoms, and family pedigree). The clinical examination quantifies the weakness and identifies any associated features (e.g., myotonia). The laboratory assessment (e.g., serum creatine kinase, urine myoglobin) screens for abnormalities of the blood and urine, and electromyography provide information typically not obtainable in other ways, including (1) the identification of a disorder other than a myopathy (e.g., motor neuron disease, neuromuscular junction defect), thereby avoiding an unnecessary muscle biopsy; (2) further characterization of the myopathy (e.g., its distribution, its associated electrical features [e.g., fibrillation potentials]), thereby further defining the diagnostic possibilities; and (3) the identification of the best muscle for biopsy. As expected, the information obtained from the muscle biopsy specimen is related to the underlying disorder, as well as its severity and rate of progression. When the patient is free of muscle weakness, the muscle biopsy is unlikely to show any significant changes. Other important variables influencing the yield of the muscle biopsy include (1) the particular muscle biopsied; (2) the way in which the muscle biopsy specimen is processed; and (3) the particular studies performed on the specimen. Consequently, this subsection includes a discussion of these important variables. Most important, however, it is necessary for the clinician, the person performing the biopsy, the pathologist, and, when indicated, the referring pathology laboratory to carefully plan their approach to the evaluation of the muscle specimen.

MUSCLE SELECTION AND TECHNIQUE

Myopathic processes do not affect all skeletal muscles equally. Therefore, a risk of sampling error among muscle biopsy specimens is always present. For this reason, it is paramount to select the muscle that will most likely yield the desired information. Important clinical and electrodiagnostic data to consider in making this determination include (1) the degree of involvement of the muscle; (2) the rapidity of onset of the disease process; (3) the muscle's history; (4) the pathological familiarity of the muscle; (5) the accessibility of the muscle; and (6) the electromyographic examination findings. A clinically unaffected muscle should be avoided, because it may not be involved pathologically. A severely affected muscle is also avoided, since it may only show endstage features (e.g., atrophy, fat, and fibrosis) rather than the distinguishing features of a specific disorder. Although a moderately affected muscle should be sought in patients with a slowly progressive disorder, a more severely affected muscle may provide the greatest diagnostic yield in individuals with acute disorder. The chosen muscle additionally should neither be involved

with another disease process (e.g., radiculopathy) nor have suffered a recent (i.e., within 1 month) injection or needle electrode examination (i.e., electromyography). The needle electrode examination should generally be performed on only one side of the body (and this should be clearly labeled in the chart) and is used to help in the identification of a muscle meeting sampling criteria so that the homologous muscle in the contralateral extremity may be sampled. In general, the most frequently biopsied upper extremity muscles are the deltoid and biceps brachii, and the most frequently biopsied lower extremity muscles are one of the quadriceps (e.g., vastus lateralis) because the range of normal for these muscles is well-defined. Although the gastrocnemius is frequently cited as a useful muscle, it should be avoided because of its type 1 muscle fiber predominance, its greater susceptibility to random pathological changes, and its pennate nature. '7! The problem with pennate muscles (i.e., muscles in which the tendon insertion extends throughout the muscle) is that inadvertent sampling near the myotendinous junction can occur. This region tends to have more central nucleation, muscle fiber size variability, and split muscle fibers. As expected, the muscle chosen must be easily accessible.

The technique of muscle biopsy is usually not difficult in the hands of an experienced individual. The skin and subcutaneous tissues are anesthetized (e.g., 2 percent Xylocaine 'Cocaine]), while carefully avoiding muscle infiltration. A small incision is then placed in the belly region (i.e., away from the myotendinous junction) along the long axis of the muscle and is extended only to the fascia. In most cases, three specimens are collected: (1) an unclamped specimen for histochemistry (the most important piece); (2) a clamped specimen for electron microscopy; and (3) a clamped specimen for histopathology. A fourth specimen can be obtained and frozen in the event further studies are deemed necessary.

The specimen for histochemistry should be 2 to 3 cm in length and about as round as a pencil. It should be handled gently by its ends using tweezers and placed in a cool, normal saline-moistened piece of gauze to prevent drying out (soaked gauze may interfere with freezing and produce artifacts). The gauze-wrapped muscle specimen is then placed in a screw-cap vial for later freezing. The specimen should be rapidly transported to the pathology laboratory, otherwise it may lose its enzymatic activity. Many techniques exist for the preparation of the frozen specimen. Typically, it is gently immersed in a Pyrex beaker containing 2 inches of liquid nitrogen-cooled isopentane (minus 140° to 155° C), frozen for roughly 30 seconds, and then removed and immediately placed in a previously cooled specimen container (e.g., another screw-cap vial). When the specimen is sent to an outside reference laboratory, the specimen container should be placed in an insulated shipping container filled with dry ice.

The specimen for electron microscopy can be slightly smaller and should be gently raised (e.g., with Metzenbaum scissors) just high enough to permit the placement of a muscle clamp. The clamp is then locked and the muscle specimen cut just outside the clamped sites. Clamping helps avoid contraction artifact. A less cumbersome technique involves the suturing of the muscle tissue specimen (e.g., with 3-0 silk) to a piece of tongue depressor before excising it (i.e., it is sutured in situ). Once removed, the muscle specimen is then placed in a 4 percent buffered glutaraldehyde fixative (or Karnovsky's fixative).

The specimen for histopathology is obtained similar to that for electron microscopy, with the exception that it is fixed in formalin. The fixed specimens should be shipped separately from the frozen specimen. It is important to include the patient's name, the sampled muscle, the procurement time, and a brief note detailing the clinical presentation and workup findings to date, as well as a list of pending studies. Importantly, when specialized studies are planned (e.g., mitochondrial DNA studies), larger tissue specimens may be necessary. For this reason, the reference laboratory should be contacted before performing the muscle biopsy so that the required amount of tissue is procured.

Individual subsection authors: Genetic studies, James Garbern; Muscle enzymes and biopsy, Mark A. Ferrante; Nerve tissue, William Kupsky; Brain and meningeal tissue, William Kupsky.

The indications for an open biopsy, as opposed to a needle biopsy, are unsettled. There are two major advantages of open biopsies--a larger specimen can be obtained and the specimen can be fixed at its in situ length, thereby preventing contraction artifact. The two major advantages of a needle biopsy are the limited scarring and the ability to sample multiple sites (in either the same or different muscles) in a single session. Disadvantages of this technique include the smaller specimen size and greater orientation difficulty. In general, the experience of the person performing the biopsy and the expertise of the laboratory personnel dictate which technique is utilized.

DIAGNOSTIC STAINING METHODS

Overview. A brief overview of pertinent neuromuscular anatomy and physiology, including muscle fiber types, is a necessary preface to a discussion of the various stains and reactions. Skeletal muscles are composed of muscle fibers and connective tissue elements termed endomysium, perimysium, and epimysium. Epimysium surrounds the muscle belly and also lies between fascicles. Fascicles are surrounded by perimysium and are composed of groups of muscle fibers that are individually surrounded by endomysium. Small arteries, arterioles, veins, and nerve twigs are contained within the perimysium. Unlike the epimysium, which may contain adipose tissue, the perimysium of adults is usually devoid of adipocytes. Muscle fibers are cylindrical, polygonal (in adult cross sections), multinucleated syncytia derived from myoblast fusion. The muscle cytoplasm is termed sarcoplasm, and the muscle membrane is termed sarcolemma. Muscle nuclei (roughly five per fiber in cross section) are located just below the sarcolemma (up to 3 percent may be more centrally placed). Satellite cells are located subjacent to the sarcolemma and are covered by the basement membrane, which surrounds the entire muscle fiber. These function as a reserve cell population (discussed later). Each muscle fiber contains hundreds of myofibrils that are composed of repeating subunits, termed sarcomeres, containing the contractile elements (e.g., actin, myosin). Tubular extensions of the sarcolemma, termed T-tubules, extend transversely into the muscle fiber between the myofibrils. These structures permit the passage of electrical activity into the depths of the muscle fiber, thereby ensuring a maximal contraction. A second intermyofibrillar system of tubules is the sarcoplasmic reticulum, which is

oriented parallel to the myofibrils (i.e., perpendicular to the T-tubules). The ends of these tubules are dilated into cisternae. The T-tubules are surrounded on both sides by the terminal cisternae of the sarcoplasmic reticulum. The nearness of these structures permits the electrical activity traversing the T-tubules to induce calcium release from the sarcoplasmic reticulum, thereby initiating muscle contraction. These tubular structures, as well as the aqueous sarcoplasm and mitochondria, are collectively termed the intermyofibrillar network.

There are two major types of muscle fibers, type 1 and type 2, each with different histochemical and physiological properties that give them specificity for certain activities. Type 1 fibers contain more oxidative enzymes, mitochondria, capillaries, and myoglobin and are hence best suited for aerobic metabolism and nonfatiguing activities (e.g., standing still). Type 2 fibers contain fewer mitochondria, more abundant glycogen and glycolytic enzymes, and are best suited for anaerobic metabolism and, consequently, those activities requiring maximal energy outputs (e.g., bench-pressing). Type 2 fibers are further subdivided into types 2A, 2B, and 2C. The type 2A fibers are more oxidative in nature than the type 2B fibers; type 2C fibers are present in fetal muscle, as well as regenerating muscle. Individual human skeletal muscles are composed of both muscle fiber types. The functional unit of movement is the motor unit, which consists of all the muscle fibers innervated by the same lower motor neuron. Its size, like that of the muscle fiber (discussed later), is dependent on the particular muscle. Although different types of muscle fibers compose an individual human skeletal muscle, the muscle fibers of a given motor unit are all identical in type (since this determination is made by the innervating lower motor neuron). The muscle fibers of a given motor unit normally are randomly distributed among the muscle fibers of other motor units. Although the relative abundance of fiber types among different muscles varies, the proportion of muscle fiber types observed in adults is as follows: type 1 (30 to 40 percent), type 2A (20 to 30 percent), and type 2B (40 to 50 percent); thus, there are roughly twice as many type 2 fibers as type 1 fibers, whereas in children, the ratio of type 1 fibers to type 2 fibers is nearly equal. y Type 1 fiber predominance is present when more than 55 percent of the fibers are type 1, whereas type 2 fiber predominance occurs when more than 80 percent of the muscle fibers are type 2.y The mean diameter of powerful muscles is 85 to 90 microns, as opposed to weaker, more distal muscles that have a mean diameter of 20 microns.y The muscle fiber type also influences diameter. In general, the diameter of type 1 fibers is less than or equal to that of type 2 fibers. Regarding gender, male type 1 fibers are equal to those of females, whereas male type 2 fibers are larger than those of females, a reflection of hormonal influences.y

Frozen Specimen. This, the most important specimen, is utilized for a variety of histochemical stains and reactions, some of which are listed in Table 25-4 . The hematoxylin and eosin stain is useful for characterizing the general morphology (e.g., myocytes, nuclei, connective tissue, blood vessels, nerves, etc.) of the skeletal muscle specimen. The hematoxylin component identifies cell nuclei, and hence nuclear position, and muscle cross-striations; the eosin portion counterstains the cytoplasm reddish pink.

TABLE 25-5 -- SELECTED STAINS AND REACTIONS AND THEIR USEFULNESS IN EVALUATING SKELETAL MUSCLE

STAINS

Hematoxylin and eosin

Hematoxylin: nuclei, cross-striations (purple)

Eosin: cytoplasm (red), connective tissue (darker red)

Modified Gomori trichrome

Nuclei, mitochondria, T-tubules sarcoplasmic reticulum (red); myocytes (blue-green)

Periodic acid-Schiff

Glycogen (purple; type 1 >2; glycogen storage disorders)

Oil red O

Lipid (orange; type 1 >2; lipid storage disorders)

REACTIONS

NADH-TR

T-tubules, sarcoplasmic reticulum, mitochondria

Type 1 (dark); type 2A (intermediate); type 2B (light)

Succinate dehydrogenase

Krebs' cycle enzyme; selectively stains for mitochondria

Cytochrome-c oxidase

Respiratory chain enzyme (orange-brown; type 1 >2)

ATPase (pH 4.3)

Type 1 (dark); type 2 (light)

ATPase (pH 4.6)

Type 1 (dark); type 2A (light); type 2B (intermediate)

ATPase (pH 9.4)

Type 1 (light); type 2 (dark)

Acid phosphatase

Degeneration (stains red; background fir green)

Alkaline phosphatase

Regeneration (stains black; background yellow)

Nonspecific esterase

Acetylcholinesterase (yellow-red; type 1 >2)

Connective tissue appears darker with eosin staining. The modified Gomori trichrome stain stains nuclei and mitochondria red and myocytes blue-green. This stain is useful in identifying ragged-red fibers. The periodic acid-Schiff (PAS) stain causes glycogen to appear purple. This stain readily identifies glycogen storage disorders, as well as capillaries. Oil red O stains lipid orange. Its utility, like that of the Sudan black stain, is in the identification of lipid storage disorders. Nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase (NADH-TR) is an oxidative enzyme reaction that, like other reactions of this type, reflects the concentration of mitochondria within myocytes. This reaction colors myocytes purple-gray. By highlighting the sarcoplasmic reticulum, T-tubules, and mitochondria (i.e., the intermyofibrillar network), this reaction causes the sarcoplasm to take on a granular appearance. Although oxidative enzyme reactions differentiate the different muscle fiber types (i.e., type 1 > 2A > 2B), these reactions should not be used for fiber typing since atrophied type 2 fibers appear darker (i.e., appear like type 1 fibers). Succinate dehydrogenase, a Krebs' cycle enzyme, selectively stains mitochondria, as does cytochrome-c oxidase, a respiratory chain enzyme. Thus, unlike NADH-TR, the tubular elements are not highlighted. These two reactions can be used to determine whether intermyofibrillar aggregates are mitochondrial or tubular elements. The myofibrillar ATPase reaction is the most accurate method of muscle fiber typing. [9 The color characteristics are determined by the preincubation pH. In short, acidic

pHs (e.g., 4.3, 4.6) cause type 1 fibers to appear darker than type 2 fibers, whereas alkaline pHs (e.g., 9.4) cause the type 2 fibers to appear darker. When preincubated at pH 4.3, the type 2A and 2B fibers are lighter than the type 2C fibers, which are intermediate in intensity; at pH 4.6, the type 2A fibers are lighter than the type 2B and 2C fibers, which are intermediate in intensity. Thus, preincubation at pH 4.3 permits the 2C fibers to manifest. Acid phosphatase, a lysosomal enzyme, facilitates the identification of degeneration (since necrotic fibers have increased lysosome content), inflammatory cells, and lysosomal storage disorders (e.g., acid maltase deficiency). Acid phosphatase is stained red, whereas the background is green. The alkaline phosphatase reaction stains regenerating fibers black, as well as normal capillary basement membranes; the background is yellow. Nonspecific esterase highlights endplates, lysosomes, and macrophages. It also identifies recently (i.e., within 6 months) denervated muscle fibers. The denervation-induced atrophy causes these fibers to appear smaller and darker. Sulfonated alcian blue stains amyloid "sea foam" green; it stains mast cells red. The alkaline Congo red stain causes amyloid to appear red, and, when viewed under polarized light, an apple green birefringence is observed.

These stains and reactions, on transverse section, permit the shape, diameter, and intermyofibrillar pattern of the myocyte, as well as the arrangement and proportion of muscle fiber types, to be determined. Adult muscle fibers appear polygonal and their cross sectional diameters vary, depending on the specific skeletal muscle (discussed earlier). Within a given section, they are somewhat uniform. The intermyofibrillar pattern is best demonstrated with the histochemical reactions for oxidative enzymes and should appear uniform. Two examples of the histopathological features that disrupt the intermyofibrillar network include target fibers and central cores. Since the muscle fiber types of different motor units are interspersed, normal muscle shows a checkerboard pattern of light and dark fibers. This same reaction permits the recognition of fiber type disproportion and those disorders confined predominantly to one muscle fiber type (e.g., atrophy, cytoarchitectural abnormalities). When atrophy is identified, it should be further characterized as to muscle fiber type involved. When atrophy is due to a chronic denervating disease, the checkerboard pattern of type 1 and type 2 fibers is altered by collateral sprout-related reinnervation. Thus, the adjacent atrophied myocytes are of the same histochemical fiber type. Conversely, when atrophy is due to disuse, the checkerboard arrangement of muscle fiber types is maintained because collateral sprouting does not occur. Certain cytoarchitectural abnormalities are more pronounced in one muscle fiber type (..TaMe.25-5 ).

Target fibers are a cardinal feature of neurogenic disorders and are composed of three "rings": (1) a central light-staining ring; (2) an intermediate dark-staining ring; and (3) a peripheral normal-staining ring. They are most predominant among type 1 muscle fibers. With central core disease, the NADH-TR reaction produces central unreactive zones, termed central cores, among affected myocytes. Thus, these structures are composed of two rings: a central ring of absent staining surrounded by a normally staining periphery. Again, type 1 fibers are mainly affected. Rod

_TABLE 25-5 -- PREFERENTIAL FIBER TYPE INVOLVEMENT_

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