Non-enzymatic Stains (Frozen Sections)

The hematoxylin and eosin (H&E) stain is arguably the single most useful stain used in the evaluation of frozen sections of skeletal muscle biopsies. H&E stained cryostat sections provide information about the presence and pattern of myofiber atrophy (e.g., group atrophy in neurogenic disorders, selective perifascicular atrophy in classical dermatomyositis), myofiber degeneration and necrosis, regenerative change, vacuoles, inflammatory infiltrates and chronic structural changes of the type seen in many muscular dystrophies. Blood vessels and structural vascular abnormalities (e.g. vasculitis) are also well demonstrated in H&E sections. In normal muscle (Fig. 1.3a), individual myofibers have a polygonal configuration in cross section, with diameters ranging from approximately 30–70 mμ in adult women and 40–80 mμ in adult men [19]. Myofiber nuclei stain blue in H&E sections. Normal myofiber nuclei are small, and typically lie at the periphery of the fiber in transverse sections. Contractile proteins give the sarcoplasm a light pink appearance. Delicate basophilic stippling is visible in well preserved specimens at higher magnification, imparted by the presence of mitochondria and “sarcotubular” components (sarcoplasmic reticulum and T-tubules) in the intermyofibrillar network. Type 1 myofibers stain a bit more darkly than type 2 fibers in H&E stains, although fiber typing is best done with other stains (see below). When muscle fibers regenerate following myofiber injury, the sarcoplasm often stains light blue, owing to the presence increased cytoplasmic ribonucleoprotein associated with protein synthesis. Enlarged nuclei with discernible nucleoli are another characteristic of regenerating myofibers. These changes are illustrated later under the heading “Interpretation of the Biopsy.” Increased numbers of internalized nuclei can be seen in a number of myopathic disorders (discussed below) but are also common near myotendinous insertion sites, and in this location should not be interpreted as evidence of myofiber injury.

The Gomori trichrome stain is familiar to most pathologists as a useful stain for demonstrating collagen, which stains with a light green color in frozen sections. In frozen sections of skeletal muscle, the modified Gomori trichrome stain developed by Engel and Cunningham [9] plays an even more important role in highlighting a variety of normal and abnormal structures within the sarcoplasm of muscle fibers. Normal mitochondria and sarcotubular elements, which are visible as punctate blue structures in H&E sections, stain red in the Gomori trichrome stain owing to the affinity of sarcoplasmic membranous structures for the Chromotrope 2R dye used in the procedure (Fig. 1.3b). Abnormal collections of mitochondria produce characteristic “ragged red” change in type I myofibers in many patients with mitochondrial disorders (discussed below under “Interpretation of the Biopsy”). Tubular aggregates, discussed below in the paragraphs dealing with biopsy interpretation, are also highlighted in the Gomori trichrome stain . Additional structures that are reliably highlighted in the trichrome stain include nemaline rods and cytoplasmic bodies (both derived from sarcomeric Z-bands), and the membranous debris present in sarcoplasmic vacuoles in patients with inclusion body myositis. Figures 1.18–1.20, which will be discussed later, contain examples of some of the abnormal structures that are highlighted in Gomori trichrome-stained cryostat sections. The appearance of collagen in Gomori trichrome-stained cryostat sections is illustrated in Fig. 1.21, which will also be discussed later.

The Oil Red O stain is one of several stains available for demonstrating sarcoplasmic neutral lipid in frozen sections. Neutral lipid is present in normal skeletal muscle, particularly in type I (slow twitch) fibers. Normal sarcoplasmic lipid appears as fine red dots in the Oil Red O stain (Fig. 1.4a), and abnormal lipid stores appear as correspondingly larger red droplets (Fig. 1.4b). “Bleeding” of stained lipid over the section can occur when the muscle biopsy contains a significant amount of adipose tissue, and care must be taken to not misinterpret this phenomenon as abnormal sarcoplasmic lipid accumulation. After staining, sections are covered with glycerol or a comparable aqueous medium and coverslipped.

Periodic Acid-Schiff (PAS) stains have been used for decades to demonstrate carbohydrates in tissues, including glycogen and other molecules with carbohydrate components (e.g., glycoproteins and glycolipids). PAS stains are especially important in the evaluation of biopsies from patients with suspected glycogen storage diseases. Carbohydrate-rich structures, including glycogen, stain magenta in the PAS stain (Fig. 1.4c, d). Incubation of the tissue with diastase before staining will remove particulate glycogen, but will leave other carbohydrate-bearing structures (e.g. basement membranes) intact. The PAS reactivity basement membranes and of abnormal filamentous forms of glycogen of the type that accumulate in type IV glycogenosis and polyglucosan body disease is not affected by diastase digestion. It is worth noting that fixation of tissue in aqueous fixatives prior to staining often removes delicate particulate glycogen from the tissues, preventing adequate demonstration of abnormal glycogen stores.

The Congo Red stain is an essential stain for demonstrating amyloid deposits. The term “amyloid” does not refer to a specific protein, but rather to a heterogeneous group of proteins that have in common a tendency to aggregate into a β-pleated sheet configuration. Amyloid deposits can be encountered in skeletal muscle in patients with sporadic systemic amyloidosis, most commonly associated with plasma cell dyscrasias and abnormal immunoglobulin light chain (particularly lambda light chain) production. Less commonly, amyloid deposits can be seen in hereditary forms of amyloidosis associated with abnormal deposits of transthyretin and, less commonly, a number of other proteins. Interestingly, amyloid deposition is also seen in two forms of autosomal recessive limb girdle muscular dystrophy – LGMD 2B (dysferlinopathy) [20] and LGMD 2 L (anoctamin-5 deficiency) [21]. In all of the preceding conditions, the amyloid is deposited extracellularly – most commonly within vessels walls and around individual myofibers. Intracellular amyloid deposits also occur in some conditions, notably variants of inclusion body myositis (IBM) . In this disorder, the intracellular amyloid deposits are composed of β-amyloid, the type of amyloid encountered in the central nervous system in Alzheimer’s disease and amyloid angiopathy.

Amyloid deposits appear as homogeneous, eosinophilic deposits in in H&E stained sections, and have a salmon-pink color in Congo red stains . Under polarized light, the amyloid deposits contain areas of characteristic “apple green” birefringence (Fig. 1.5a). Fluorescence microscopy with Texas Red filtration is an even more sensitive technique for detection of amyloid deposits [22]. Under fluorescence illumination with Texas Red filtration, amyloid deposits appear bright red (Fig. 1.5b).

Enzyme Histochemical Stains (Frozen Sections)

Staining for myosin adenosine triphosphatase (ATPase) activity has long been the most reliable method for distinguishing slow twitch (type 1) myofibers from fast twitch (type 2) fibers. The staining procedure involves the cleavage of a phosphate group from ATP in a frozen muscle section in a solution of aqueous calcium chloride, producing an insoluble calcium phosphate precipitate at the site of the reaction. The tissue is then incubated in a solution of cobaltous chloride, during which the calcium in the calcium phosphate is replaced by cobalt (Co²⁺). The section is then briefly incubated in a solution of ammonium sulfide, which generates a black cobaltous sulfide reaction product in the muscle fiber [1, 10, 11]. Incubation of the section in an alkaline barbital solution (pH 9.4 in our laboratory) results in the selective activation of myosin ATPase in type 2 fibers, causing them to stain darkly, while type 1 fibers remain pale. Pre-incubation of tissue sections in a barbital buffer at pH 4.3 produces a reciprocal staining pattern – that is, darkly staining type 1 fibers and pale type 2 fibers. In addition, in the ATPase 4.3 preparation, a small subpopulation of intermediate-staining type 2c fibers is often visible. Finally, pre-incubation of the tissue section at pH 4.6 produces a pattern in which type 1 fibers stain darkly, type 2a (fast twitch oxidative) fibers are pale, and type 2b (fast twitch glycolytic) fibers stain with intermediate intensity (Fig. 1.6a–c). The use of standard mounting medium in the preparation of slides stained for ATPase activity will result in fading of the reaction product over time. Staining intensity can be preserved by cover-slipping the ATPase-stained sections with Canada balsam. In normally innervated muscle, the distribution of type 1 and type 2 fibers is random, as illustrated in Fig. 1.6a–c. A loss of randomness in the distribution of myofibers is called “fiber type grouping” and indicates reinnervation of previously denervated muscle (Fig. 1.6d). The ATPase stain is extremely helpful in characterizing conditions associated with various patterns of type-specific myofiber atrophy or hypertrophy, and in the identification of patients with myopathy with thick filament loss (“critical illness”) myopathy. Some of these patterns are illustrated later under “Interpretation of the Biopsy” in Fig. 1.12.

The nonspecific esterase stain relies on the hydrolysis of an exogenous alpha-naphthyl acetate substrate by endogenous esterase to yield naphthol, which in turn forms an insoluble azo dye when incubated with basic fuchsin, which appears brick red under the microscope [12]. The stain highlights normal neuromuscular junctions due to the presence of acetylcholinesterase in those structures, providing a reliable internal control (Fig. 1.7a). Recently denervated myofibers are highlighted in the esterase stain, owing to increased cytoplasmic esterase activity (Fig. 1.7b). The esterase stain also highlights macrophages and, in some cases, sarcoplasmic vacuoles associated with abnormal lysosomal activity, although these are usually demonstrated even more clearly in sections stained for acid phosphatase activity (discussed below). Type 1 fibers generally stain a bit more darkly than type 2 fibers in the esterase stain.

The acid phosphatase stain is based on the hydrolysis of naphthol AS-B1 phosphate by endogenous acid phosphatase to form naphthol, which, like the naphthol generated in the esterase stain, forms an insoluble azo dye in the presence of basic fuchsin [1, 11]. The red reaction product highlights macrophages (Fig. 1.7c) and other structures that have lysosomal activity, such as degenerating myofibers and pathological sarcoplasmic inclusions associated with abnormal lysosomal activity (Fig. 1.7d). The stain is generally a more sensitive marker of lysosomal activity than the nonspecific esterase stain.

The alkaline phosphatase stain is another “hydrolytic” stain that relies on the hydrolysis of an alpha-naphthyl acid phosphate substrate by endogenous alkaline phosphatase to generate naphthol, which reacts, in turn, with a diazo salt (fast blue RR salt) to form a black reaction product at sites of alkaline phosphatase activity. Glycerol or a comparable aqueous mounting medium should be used to coverslip the section, as alcohols or xylene dissolve the reaction product [13]. The alkaline phosphatase reaction generates gas that can produce distracting bubbles if the section is coverslipped prematurely. To minimize this, it is advisable to wait at least 45 minutes before placing the coverslip over the stained section. The stain highlights the sarcoplasm of regenerating myofibers (Fig. 1.7e), as well as occasional myofibers in infantile denervation of the type seen in spinal muscular atrophy type 1 (Werdnig-Hoffmann disease); normal muscle fibers lack alkaline phosphatase activity. Increased alkaline phosphatase reactivity can also be seen in connective tissue in many inflammatory disorders of skeletal muscle. Increased perimysial connective tissue reactivity, in particular, is a feature of some inflammatory myopathies associated with the presence of circulating anti-Jo1 or other anti-tRNA synthetase antibodies (Fig. 1.7f).

The nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stain is an extremely useful member of the “oxidative” family of stains. The NADH-TR enzyme is a flavoprotein involved in electron transfer in the normal respiratory chain. Both mitochondrial and cytosolic forms of NADH-TR are present in human skeletal muscle, the latter associated with sarcoplasmic reticulum. The NADH-TR staining reaction is based upon the enzymatic removal of hydrogen from exogenous NADH substrate, and the transfer of that hydrogen to nitro blue tetrazolium. The transfer of hydrogen to nitro blue tetrazolium, in turn, generates an insoluble dark blue reaction product, which marks the site of enzyme activity. Sections are coverslipped with an aqueous mounting medium [1, 11, 14]. Mitochondria have NADH-TR activity, and for this reason normal type 1 fibers stain more darkly than type 2 fibers. In optimally stained specimens, some differences in the staining intensity of type 2a versus type 2b fibers may be apparent, with type 2a fibers staining a bit more darkly than type 2b fibers, but this distinction is much more reliably made in the ATPase 4.6 stain, The NADH-TR stain is useful for demonstrating architectural abnormalities within muscle fibers, such as target/targetoid change, sarcoplasmic cores, moth-eaten fibers, sarcoplasmic “whorling”, ring fibers and lobulated fibers (discussed later). Denervated myofibers are also variably highlighted in the NADH-TR stain. Abnormal mitochondrial aggregates are sometimes highlighted in the NADH-TR stain, although these are more reliably detected in the succinate dehydrogenase and/or cytochrome c oxidase stains, discussed below. The stain is also useful for the identification of tubular aggregates, structures that are derived from sarcoplasmic reticulum and are sometimes confused with ragged red change in Gomori trichrome-stained sections. An example of a normal NADH-TR stain is illustrated in Fig. 1.8a, and examples of abnormalities highlighted in the NADH-TR stain are illustrated later, under the heading “Interpretation of the Muscle Biopsy.”

The succinate dehydrogenase (SDH) stain identifies a flavoprotein enzyme that is complex II in the mitochondrial respiratory chain. The enzyme is anchored to the inner mitochondrial membrane and is encoded exclusively by nuclear – as opposed to mitochondrial – DNA. SDH normally catalyzes the conversion of succinate to fumarate and the closely linked transfer of electrons to ubiquinone to form ubiquinol in the mitochondrial electron transport chain. In vitro, the SDH stain relies on the ability of SDH to release hydrogen from its sodium succinate substrate and reduce aqueous nitro blue tetrazolium to form a blue tetrazolium precipitate. The stained sections are coverslipped using an aqueous mounting medium [1, 11, 14, 15] The SDH stain is a specific stain for mitochondria, and a much more sensitive marker for mitochondrial aggregation than either the Gomori trichrome stain or the NADH-TR stain. Myofibers harboring abnormal mitochondrial aggregates appear as “ragged blue” fibers in the SDH stain. A normal SDH stain and an SDH stain in a patient with abnormal mitochondrial accumulation are illustrated in Fig. 1.8b, c.

The enzyme cytochrome c oxidase (COX) is complex IV in the mitochondrial respiratory chain. The enzyme is composed of multiple subunits, three of which are encoded by mitochondrial DNA in mammalian tissue. The COX complex is located in the inner mitochondrial membrane, where it transfers electrons from the cytochrome c protein to molecules of dioxygen to form water. The COX stain is based on the ability of the enzyme to transfer electrons from an artificial diaminobenzidine tetra hydrochloride substrate to produce an insoluble brown diaminobenzidine precipitate (Fig. 1.8d). Stained sections are coverslipped using an organic mounting medium [1, 11, 14, 15]. Like SDH, COX is a specific marker for mitochondria. Its usefulness in diagnostic work is based on the fact that many mitochondrial disorders are associated with COX deficiency, characterized by failure of affected myofibers to stain in the COX preparation (Fig. 1.8e). In most mitochondrial disorders associated with COX deficiency, SDH reactivity is preserved, and staining sections sequentially for COX and then SDH activities [15] makes detection of COX-deficient fibers a bit easier, with COX-deficient fibers standing out as blue fibers surrounded by brown-staining fibers with intact COX activity (Fig. 1.8f). COX-deficient fibers are common in a number of conditions including primary mitochondrial disorders, including inclusion body myositis and polymyositis with mitochondrial abnormalities [23]. COX deficient myofibers are also seen in some cases of dermatomyositis, wherein there is a selective loss COX activity in perifascicular myofibers [23, 24]. COX is a fairly unstable enzyme, and care must be taken to distinguish artifactual loss of COX activity associated with improper handling of muscle tissue from true COX deficiency; this staining pattern is illustrated later in Fig. 1.24c, under the discussion of Artifacts in Skeletal Muscle Biopsies.

Two standard enzyme histochemical procedures are available for the detection of enzymes associated with glycogen metabolism – muscle-specific phosphorylase (myophosphorylase) and phosphofructokinase. Myophosphorylase is an enzyme that catalyzes the conversion of glycosyl residues in glycogen to glucose-1-phosphate, which is then used in the generation of ATP. Detection of myophosphorylase is based on the ability of the enzyme to add glucose residues to a glycogen primer in the presence of a large amount of glucose-1-phosphate substrate (the reverse of the reaction sequence that occurs in vivo). The resultant glycogen is then stained with Lugol’s iodine solution, resulting in a gray-brown reaction product. Sections are coverslipped using an aqueous mounting medium [11, 16]. Patients with myophosphorylase deficiency (type V glycogen storage disease, also known as McArdle’s disease) classically present with histories of cramps, exercise intolerance and episodes of rhabdomyolysis following vigorous exercise. In muscle biopsies from such patients, no phosphorylase reaction product is detectable in intact muscle fibers, which appear yellow under the microscope. Myophosphorylase is a labile enzyme that is subject to artifactual degradation, and it is important to distinguish such artifactual losses of enzyme activity from true phosphorylase deficiency. Smooth muscle cells in intramuscular blood vessels express a different isoform of phosphorylase than skeletal muscle, and the identification of residual phosphorylase activity in blood vessel walls serves as an internal control that distinguishes artifactual loss of enzyme activity from selective myophosphorylase deficiency. Of note, the reaction product in the myophosphorylase stain will fade over time, and it is helpful to make a notation of a “positive” result on the slide label. The color of the reaction product can be restored by re-incubating the section in Lugol’s solution.

Phosphofructokinase (PFK) is a glycolytic pathway enzyme that converts fructose-6-phosphate to fructose-1,6-diphosphate. Patients with PFK deficiency (type VII glycogen storage disease, or Tarui’s disease) have a clinical presentation similar to that of patients with myophosphorylase deficiency. The PFK stain is based on the conversion of exogenous fructose-6-phosphate to fructose-1,6-diphosphate by endogenous PFK. A metabolite of fructose-1,6-diphosphate – diphosphoglyceric acid – is formed in vitro, which then reacts with exogenous nicotinamide adenine dinucleotide to generate reduced nitro blue tetrazolium, the latter forming the usual blue precipitate. Sections are coverslipped using an organic mounting medium [1, 11, 17]. PFK is a labile enzyme that deteriorates rapidly in improperly handled biopsies. Patient sections stained for PFK should include a negative control (no added substrate) and a control section using fructose-1,6-diphosphate as a substrate, the latter always generating a blue reaction product, even if the patient is PFK-deficient. Patient sections stained for PFK activity should always be paired with a normal control section.

A final enzyme histochemical stain that is routinely performed in our laboratory is the myoadenylate deaminase (MAD) stain . MAD is an enzyme that removes an ammonia molecule from adenosine monophosphate (AMP) to convert AMP to inosine monophosphate. Deficiency of the enzyme should be suspected when patients fail to generate a normal amount of ammonia during exercise testing. The clinical significance of MAD deficiency appears to vary from patient to patient. While some patients with MAD deficiency are asymptomatic, MAD deficiency has been associated in other patients with a clinical syndrome of exercise intolerance and exercise-induced muscle cramps and pain, and, in severe cases, rhabdomyolysis. A susceptibility to malignant hyperthermia syndrome has been suggested by some authors [25, 26]. The MAD stain is based on the generation of ammonia from an exogenous AMP substrate and subsequent reduction of nitro blue tetrazolium to form a blue reaction product. Staining for MAD is always paired with a negative control, in which aqueous citrate is added to the reaction in place of AMP substrate. Stained sections are coverslipped using an aqueous mounting medium [1, 18]. In patients with MAD deficiency, no blue reaction product is present in the sections stained with the AMP substrate. In addition to detecting MAD deficiency, the MAD stain reliably highlights tubular aggregates (see discussion of abnormal sarcoplasmic inclusions under “Interpretation of the Biopsy”, below).

Non-enzymatic Staining of Fixed Skeletal Muscle (Paraffin Sections)

Although most of the useful diagnostic information in muscle biopsies at the light microscopic level is provided by the evaluation of stained cryostat sections, properly processed paraffin embedded tissue can also be of help in some conditions, particularly those characterized by the presence of inflammatory infiltrates and microorganisms. The morphology of inflammatory cells is generally better preserved in fixed, paraffin embedded tissue than in cryostat sections, and immunohistochemical markers (discussed below) often label inflammatory cells more clearly in paraffin-embedded tissue than in frozen tissue. Paraffin sections of skeletal muscle biopsies are routinely stained with H&E, Masson trichrome and Congo red stains in our laboratory. The H&E and Congo red stains have the same applications in paraffin sections as they do in cryostat sections. The Masson trichrome stain is a sensitive stain for highlighting connective tissue in paraffin sections. An additional stain, the Morin stain , has proven to be helpful in the detection of aluminum deposits in patients with macrophagic myofasciitis (see discussion of cellular infiltrates under “Interpretation of the Biopsy”, below) [27].