Introduction

Muscle biopsy plays an important role in evaluation of patients with neuromuscular disease, and it is required to provide definitive diagnosis in many neuromuscular disorders. In addition, muscle biopsy can sometimes help to distinguish between a neurogenic or myogenic disorder, and it may give important information on the course of the disease (acute or chronic) and on disease stage and progression.

At present, muscle biopsy is necessary for diagnosis of several categories of muscle diseases, including hereditary disorders (i.e., muscular dystrophies, myotonic dystrophies) and acquired myopathies, such as inflammatory myopathies, and toxic and drug-induced myopathies (Table 1).

Table 1 Classification of primary muscle disease
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Muscle biopsy techniques

Muscle biopsies can be performed through open or needle technique.

Open muscle biopsy

Open muscle biopsy consists in an incision of 4–6 cm along the long axis of the limb through the skin, subcutaneous tissue, and muscle fascia. After the muscle sample is excised, absorbable sutures close the tensor fascia lata and the subcutaneous tissue. The technique is performed under local anesthesia with previous injection of 1–2 mL lidocaine into the skin and subcutaneous tissue and then into subcutaneous tissue and muscle fascia. It is important not to infiltrate the muscle with lidocaine to avoid alteration of biochemical results. The complications of the procedure include muscle herniation, hematoma formation, wound dehiscence, and infection.

Muscle needle biopsy

Muscle needle biopsy was developed by Bergström in 1962 [1] as an alternative to open biopsy. After local anesthesia, a 4–5-mm stab incision is made in the skin and underlying fascia with a surgical blade (#11) to facilitate passage of the biopsy needle. When it is inside the muscle, the cutting window is opened by sliding the inner cannula that it is pushed back to cut the sample. Some clinicians repeat the procedure to obtain more muscle samples while others prefer to rotate the needle to obtain different samples with one needle insertion [2]. During the procedure the patient can feel deep pressure due to the pump. Sometimes, if the muscle is not relaxed, they can complain of a cramp sensation. Derry et al. [3] assessed patient perception of the procedure and found a mild to moderate degree of discomfort. Patients with inflammatory myopathies complain of greater discomfort than others [3].

At the beginning, the major limitation of the needle biopsy technique was sample size, which was smaller than that obtained with open biopsy. Over the years, several modifications to increase the sample size have been introduced, more specifically the application of suction during the procedure [4–6]. Melendez et al. [7], using a 6-mm Bergström needle with wall suction technique on 55 subjects, obtained mean biopsy sample of 233 mg compared with 25 mg obtained with the original technique. Other limitations of needle biopsy are the impossibility of direct visualization of the sampling site and difficulty in identifying the correct orientation of the fibers of the sample obtained due to its small size. However, a recent study on more than 13,000 patients demonstrated that, using needle biopsy, it is possible to obtain an appropriate sample in >99% of cases for the analyses required [2]. Moreover, needle biopsy is a less invasive procedure than open biopsy and presents low complication rates, and the scar is shorter. Needle biopsy is particularly indicated for diagnosis of neuromuscular disease in infancy and childhood or in inflammatory myopathy to evaluate disease progression and response to treatment.

Muscle selection

The selection of the muscle to biopsy is determined by the distribution of muscle weakness. It should be a muscle affected by the disease but not be too weak or too atrophic, since in end-stage muscle where loss of myofibers is severe, muscle tissue is replaced by fibrovascular and adipose tissue [8]. More specifically, in chronic disease it should be taken from a muscle with moderate but not severe weakness, while in acute disease a principally involved muscle should be preferred.

In some cases it could be useful to perform sonographically guided percutaneous muscle biopsy or magnetic resonance imaging to assess muscle composition, especially in myositis where the distribution of inflammation inside the muscle may be heterogeneous (patchy) [9, 10].

Usually in case of proximal muscle involvement the quadriceps (rectus femoris or vastus lateralis) and the biceps are biopsied. Moreover, the quadriceps is considered relatively safe for needle biopsy because major nerves and blood vessels are far from the biopsy site. In contrast, needle biopsy of the biceps may be more dangerous, therefore some centers prefer to practice open biopsy of the biceps. Another muscle usually biopsied is the deltoid, especially under suspicion of mitochondrial disorder. Finally, to investigate distal myopathy, it may be useful to perform biopsy of the tibialis anterior muscle, which is also easy to perform by needle technique.

Specimen preparation

Diagnostic muscle biopsy consists of two types of fresh specimens: some for histopathological studies, and others for biochemical or genetic analysis. Sometimes a sample is required to be fixed.

The samples must be immediately transported to the pathology laboratory for processing to prevent loss of enzymatic reactivity, DNA depletion or RNA degradation. Since it is an invasive investigation, it is important that the biopsy procedure and subsequent laboratory processing of the specimen are well performed to obtain an optimal sample for diagnosis. Ideally the specimens should be frozen immediately after excision, and the correct methods of freezing as well as the storage of frozen biopsies are crucial for histopathological or biomolecular diagnosis [11]. They can be sent to the laboratory on saline-moistened gauze and kept cool in a sealed container on ice. Rapid freezing at ultracold temperature and good storage of muscle specimens are essential to avoid morphologic alterations and degradation of nucleic acids. Indeed, slow tissue freezing can yield a riddled sample as a consequence of formation of ice crystal artifacts. When the specimens arrive in the laboratory, fresh samples are mounted in the appropriate orientation on cork by using a cryostat embedding medium, such as OCT compound, with fibers oriented longitudinally in the vertical plane and snap-frozen by immersing it into isopentane prechilled in liquid nitrogen. Frozen cryostat cross-sections (5–8 μm) are cut from this sample for histological, histochemical, and immunohistochemical analysis. The other fresh samples are snap-frozen in prechilled isopentane without using mounting medium which could interfere with the results of biochemical or genetic analysis.

Sometimes a fixed specimen may be useful for routine light microscopy or for electron microscopy (EM). For light microscopy, the preferred fixative is 4% paraformaldehyde; however, if it is not available, a good alternative is 10% neutral buffered formalin. For EM, a small piece of muscle can be fixed in 3% glutaraldehyde.

A battery of stainings is performed on every frozen muscle biopsy arriving in the pathology laboratory. These stainings are important in the evaluation of fiber atrophy, morphological and structural changes, and metabolic disorders [8]. The most common stainings which are useful for diagnosis of myopathies will be described here.

Hematoxylin and eosin (H&E) and the modified Gomori trichrome stain [12] are routine histological stains for evaluation of basic tissue organization and cellular structure. Moreover, Gomori trichrome is important for diagnosis of mitochondrial disorders, inclusion body myositis, and nemaline myopathy [13–15] (Fig. 1a, b).

Fig. 1
figure 1

Histological and histochemical stainings of transverse sections of frozen muscle biopsy. a Myotonic dystrophy type 1: hematoxylin and eosin (H&E). High fiber size variability with several atrophic (arrow) and hypertrophic (asterisk) fibers. An increase in internal nuclei is also evident. Bar 80 μm. b Hypokalemic periodic paralysis: Gomori trichrome. Note the presence of several vacuoles (arrows) in myofibers. Bar 80 μm. c Inflammatory myopathy: hematoxylin and eosin (H&E). Endomysial lymphocytic inflammatory infiltrate among myofibers that show increased size variability. Bar 80 μm. d HyperCKemia: hematoxylin and eosin (H&E). In this cross-section myofiber hypertrophy, atrophy (arrow), and splitting (1) are present. Bar 80 μm. e Neurogenic disorder: myosin adenosine triphosphatase (ATPase) pH 4.3. Cluster of myofibers composed exclusively of type 1 fibers stained brown. Bar 80 μm. f HyperCKemia (same patient as in d): myosin adenosine triphosphatase (ATPase) pH 4.3. Predominance of type 2 fibers (unstained). Only a few myofibers are of type 1 (brown). Bar 80 μm. g–i Mitochondrial myopathy: combined staining for activities of cytochrome oxidase (COX) and succinic dehydrogenase (SDH) (g), COX activity stain (h), and nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) stain (i). In g several blue fibers have a subsarcolemmal dense blue stain representing aggregates of mitochondria (1, 2, and 3). All of the fibers negative for COX activity (h) are blue fibers in the COX/SDH preparation (i). These fibers correspond to the ragged red fibers on Gomori trichrome stain. The dark-blue rim is also evident in COX/SDH stain (i). Bar 80 μm

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Since muscle is composed of two main fiber types, type 1 and type 2, a myosin adenosine triphosphatase (ATPase) stain at multiple pH levels is performed to demonstrate the different fiber types [16]. Indeed, several muscular diseases affect one type or the other, causing atrophy of either type 1 or type 2 fibers (Fig. 1d). Moreover, neurogenic disorders can alter the distribution of both fiber types.

Periodic acid-Schiff (PAS) reaction is used to stain glycogen and other polysaccharides, and it is useful for diagnosis of glycogen storage diseases. Oil-red-O or Sudan Black are used for fat staining, which is normally present in muscle fibers. However, an abnormal amount or distribution of lipid is present in carnitine deficiency and in some mitochondrial disorders.

The activity of a group of enzymes which are present in mitochondria and endoplasmic reticulum is demonstrated by nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR). This staining is useful for neurogenic atrophy, mitochondrial disorders, central core disease, and for detecting alterations of myofiber intracellular structure. Moreover, the activities of two mitochondrial enzymes, succinic dehydrogenase (SDH) and cytochrome oxidase (COX), can be assessed. These histochemical stainings are often combined, and fibers that are positive for both COX and SDH appear taupe, resulting from the combination of the brown color of the stain for COX with the blue color of the SDH stain. Blue fibers are those that stain for SDH activity alone because of absence of COX activity (ragged red fibers on trichrome stain) (Fig. 1h). Lysosomal activity is evaluated by acid phosphatase staining since it is important for certain metabolic and some toxic disorders.

When the clinical history and the results of the evaluation of the routine stainings require, further stainings may be carried out on the frozen sample.

The increasing availability of antibodies against muscular proteins allows pathologists to use immunohistochemistry as a diagnostic tool for many muscle diseases (Table 2). Indeed, immunohistochemistry may be used for both identification of normal antigens in skeletal muscle and their over- or underexpression or mislocalization in corresponding myopathies. Currently, three categories of muscle diseases are characterized by using diagnostic antibodies: dystrophic, congenital/structural, and inflammatory myopathies [17].

Table 2 Primary and secondary protein defects in muscle disorders detectable by immunoanalysis
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Elevated levels of serum creatine kinase (CK; >1,000 IU/L) often indicate muscle disease with a primary problem in the muscle membrane. A number of sarcolemmal proteins have been identified whose deficiency causes different forms of limb-girdle dystrophy, including dysferlin [18], sarcoglycan [19, 20], calpain [21], and caveolin [22] (Figs. 2a and 3). For these muscular dystrophies, immunohistochemical studies using antibody against specific antigens can be performed to verify the expression of these proteins. In addiction to or alternatively to immunohistochemistry, biochemical analysis, i.e., Western blotting analysis, may lead to a specific diagnosis. Inflammatory myopathies are the largest group of acquired muscle diseases. To identify autoimmune or inflammatory myopathies, immunohistochemistry against major histocompatibility class I, also known as human leukocyte antigens (HLA I), is often used, since it has been demonstrated that this protein is strongly upregulated in inflammatory myopathies [23–25]. In particular, since in polymyositis the inflammatory infiltrates are typically dominated by CD8+ T lymphocytes whereas in dermatomyositis CD4+ T lymphocytes are predominant, immunohistochemistry can be used to distinguish between these two inflammatory disorders (Fig. 2b). In sporadic inclusion body myositis (s-IBM), a chronic inflammatory myopathy, abnormal muscle fibers containing characteristic filamentous inclusions are present in addition to lymphocytic inflammation. The abnormal fibers contain vacuoles well visualized by Gomori trichrome stain and amyloid foci positive with Congo red, thioflavin-s, and crystal violet. However, these amyloid deposits can often be missed since they are very small and present in only a few muscle fibers. Thus, for s-IBM diagnosis, immunocytochemical staining for the presence of phosphorylated tau in the form of paired helical filaments utilizing SMI-31 monoclonal antibody is recommended (Fig. 2c) [26, 27]. Immunostaining of type 1 and type 2 myofibers using antibodies against different myosin heavy chains is an alternative and more sensitive method than ATPase for myofiber typing [28]. Recently, myotonic dystrophy type 2/proximal myotonic myopathy (PROMM) has been defined as “a disease of type 2 fibers” since both fiber atrophy and central nucleation selectively affect type 2 fibers [29, 30] (Fig. 2d). It appears that it is of particular importance for definitive diagnosis of DM2 to distinguish between the two types of fibers.

Fig. 2
figure 2

Immunohistochemical and fluorescence in situ hybridization (FISH) stainings of transverse sections of frozen muscle biopsy. a Normal muscle: dystrophin immunohistochemistry. Sample of normal muscle stained with an antibody to C-terminal region of dystrophin shows the normal subsarcolemmal localization of this protein demonstrated by the linear peripheral brown staining of every myofiber. Bar 80 μm. b Dermatomyositis: immunohistochemistry for CD4 (T lymphocytes) with hematoxylin counterstain. T lymphocytes (brown cells) infiltrate the endomysial connective tissue. Bar 80 μm. c Inclusion body myositis: immunohistochemistry for SMI-31. Characteristic inclusion stained with SMI-31 antibody (arrow) in an abnormal myofiber. Bar 80 μm. d Myotonic dystrophy type 2: fast myosin immunohistochemistry. The immunostaining (brown) demonstrates the presence of very atrophic fibers which are fast positive, i.e., type 2 fibers (arrows). Bar 100 μm. e Myotonic dystrophy type 2: FISH. Direct visualization of mutant messenger RNA (mRNA) containing CCUG repeat (red spots) in myonuclei [4’,6-diamidino-2-phenylindole (DAPI), blue]

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Fig. 3
figure 3

Simplified scheme of the extracellular matrix and sarcolemmal proteins

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It is noteworthy that most of the analysis described can be performed on frozen but not on fixed material. However, fixed and paraffin-embedded specimens maintain more cytological details and are more useful to detect myofiber necrosis, to determine the type of inflammatory infiltrate, and to examine the structure of vessel walls, making these specimens perfect for evaluating, for example, inflammatory myopathies or vasculitis. EM on muscle biopsy samples is not a routine procedure, but it is performed when the pathologist decides that EM might contribute significantly to determining a specific diagnosis on the basis of clinical history and the results of light microscopy evaluations [31].

The development of molecular biology and its application to muscle diseases has led to the identification of gene defects in many inherited neuromuscular diseases and therefore to accurate and specific diagnosis (Table 3). The best example of this is Duchenne muscular dystrophy (DMD) and the discovery in the late 1980s of the gene at locus Xp21 whose mutation causes deficiency of dystrophin, an essential protein in muscle fibers (Fig. 3) [32]. Some investigators give first preference to noninvasive molecular analysis versus microscopic study of invasive muscle biopsy [33], for example in cases where clinical and genetic history is highly suggestive for DMD but confirmation is necessary for differential diagnosis from Becker or other forms of muscular dystrophy. Others investigators choose demonstration of dystrophin deficiency by immunohistochemistry or Western blot analysis on muscle biopsy. In selected cases it is possible to demonstrate the genetic alteration directly on muscle section using in situ hybridization (ISH). An example of this is represented by myotonic dystrophy type 2, a muscular dystrophy caused by a CCTG repeat expansion in intron 1 of the zinc finger protein 9 gene on chromosome 3 [34, 35]. Due to the extremely large size of the CCTG expansion and the extensive somatic instability [36, 37], standard molecular analysis such as Southern analysis and polymerase chain reaction (PCR) fail to detect the DM2 expansion. To date, a three-step method based on repeat primed (RP) PCR with 99% accuracy is recommended to determine the presence of DM2 mutation [38]. Several alternative highly specific and sensitive methods have been developed in addition to RP-PCR; however, they are expensive, not available as routine diagnostic test, and very time-consuming. For these reasons, muscle biopsy represents an essential tool for definitive biomolecular diagnosis of DM2 which may be obtained in few hours by ISH using a specific probe which allows direct visualization of mutant mRNA containing CCUG repeat on muscle sections [39, 40] (Fig. 2e). However, there are myopathies that do not need muscle biopsy for definitive diagnosis, such as some congenital and limb-girdle dystrophies, myotonic dystrophy type 1, certain mitochondrial disorders, and periodic paralyses. In these cases, genetic tests are performed on a blood sample. 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 DMPK gene on chromosome 19 [41–43].

Table 3 Classification of some muscle diseases according to the molecular defect
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It should be noted that, from a skeletal muscle biopsy, it is possible to isolate the progenitors of muscle fibers, the satellite cells. Skeletal muscle satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally differentiated muscle fibers [44]. These are normally quiescent in adult muscle, but act as a reserve population of cells, able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells [45]. Isolated satellite cells are very useful for in vitro studies. Indeed, muscle cell cultures provide a system for studying the growth and differentiation of muscle cells in a controlled environment.

Conclusions

In addition to the well-defined pathological conditions that occur in various neuromuscular disorders, skeletal muscle may also be directly or indirectly involved in many acute or chronic diseases. Muscle biopsy may reveal striking and unexpected changes in situations where involvement of the muscle is not clinically apparent.

The molecular era has led to the identification of many gene defects and proteins responsible for neuromuscular disorders. Several proteins of muscle fibers, the extracellular matrix, the plasma membrane, the cytoskeleton, the Golgi apparatus, the internal membrane systems, nuclei, myofibrils, neuromuscular junction, and the cytosol have been found. The wide spectrum of defective proteins that have been identified and the interactions between them have challenged the traditional classification of neuromuscular disorders based on clinical features. Neuromuscular diseases are now referred to on the basis of the protein defect, such as dystrophinopathy, sarcoglycanopathy, and actinopathy, and according to the pathogenic mechanisms.

When the pathologist is asked to evaluate a muscle biopsy, much of the interpretation is based on his previous experience and the recognition of similarities between the biopsy and muscle from known diseases. In such instances, such as an advances dystrophy or spinal muscular atrophy, the changes may be striking and unequivocal. In others, the changes may be more subtle and a systematic approach is required in the evaluation and interpretation. Once the pathology is defined, correlation of the clinical features is essential and the pathology must be interpreted in light of this. Muscle biopsy is a useful technique which can make an enormous contribution in the field of neuromuscular disorders but that should be considered and interpreted together with family history, clinical history and presentation, and the result of any other investigation. There are considerable advantages if the clinician assessing the patients can also review the biopsy with the pathologist and then provide a comprehensive diagnosis of the patient. This is our policy and gives continuity between the clinical diagnosis and other investigations.