Muscle Biopsy

The diagnostic approach to a patient with suspected neuromuscular disease begins with a thorough history and complete neurologic examination, which will often yield information regarding the component of the motor unit affected. Performance of electrodiagnostic tests in patients with neuromuscular disease often provides important information pertaining to the specific localization and extent of the disease within the motor unit, however it is necessary in some cases to perform skeletal muscle biopsy, often in conjunction with peripheral nerve biopsy. Morphologic evaluation of biopsy specimens will confirm clinical and electrophysiologic findings and is required to diagnose and classify the underlying disease responsible for the clinical signs.

General clinical indications for muscle biopsy include generalized or focal muscle weakness, stiffness, contracture, atrophy, myalgia, or hypertrophy.^1,2 Less commonly encountered clinical abnormalities that are suggestive of underlying motor unit disease include muscle fasciculations, rippling, myokymia, and myotonia. Identification of biochemical alterations such as an elevated serum creatine kinase concentration, lactic acidemia, or myoglobinuria, in any animal with clinical signs compatible with myopathic disease is also an indication to perform muscle biopsy. It is recommended that at least two muscle samples from distant locations, such as the thoracic and pelvic limbs, be examined when attempting to confirm the presence of a generalized neuromuscular disorder.^1,2

Selection of Biopsy Sites

Several criteria should be considered prior to selection of the specific biopsy site. Primarily, there should be historical, clinical, and, ideally, electromyographic (EMG) evidence that the specific muscle is affected by the underlying disease.² Chronically affected, severely atrophied muscles are poor candidates for biopsy, as meaningful interpretation of biopsies sampled from such sites is often impeded by significant replacement of myofibers with adipose and fibrous tissues.¹ Muscles should also be evaluated for any previous disease, trauma, intramuscular injections, or surgery that could result in morphologic artifacts in the biopsy specimen. It is noteworthy that needle EMG evaluation can also induce focal muscle necrosis in areas of needle insertion.¹ Subsequently, when performing an EMG examination of a patient with a suspected generalized neuromuscular disease, it is preferred to electrophysiologically evaluate one side of the patient’s body, and then utilize the results of the EMG examination to obtain biopsy samples from affected muscles on the contralateral side.^1,2

The muscle selected for biopsy should be readily accessible and easily identified through a minimally invasive surgical approach; be able to be sampled with minimal resulting morbidity to the native muscle or surrounding soft-tissues; and ideally have previously published normative data regarding myofiber size, type, and distribution available for comparison.^1,2 Thoracic limb muscles commonly selected for biopsy include the distal thirds of the medial or long heads of the triceps brachii, or proximal portion of the superficial digital flexor. In the pelvic limb, the distal third of the biceps femoris or vastus lateralis, and proximal third of the lateral head of the gastrocnemius or cranial tibial muscles are frequently sampled. Reference data for both the dog and cat are available for each of these muscles.¹ If disease of the muscles of mastication is suspected, the temporalis muscle is the preferred biopsy site.

Additional factors to consider prior to selecting a biopsy site is the suspected localization of the disease within the motor unit, which is based on the differential diagnoses formulated following completion of the clinical examination. Biopsy of specific muscles or certain regions within a muscle may be required to provide the highest diagnostic yield. For example, when ultrastructural, immunohistochemical, or in vitro electrophysiological examination of the motor end plate is required, as would be necessary to confirm a diagnosis of congenital or seronegative, acquired myasthenia gravis, it is recommended that biopsy of a muscle, such as external intercostals, anconeus, or similar muscle that has high concentration of end plates and is able to be harvested intact from origin to insertion be performed.¹ In these circumstances, it is generally advised to discuss the proposed site and method of processing of muscle biopsy specimens with the laboratory or pathologist that will be charged with interpreting the biopsy before the procedure to facilitate collection of a diagnostic sample.

In situations where the specific location of the disease within the motor unit is unable to be determined following clinical examination and adjunctive electrophysiologic testing is unavailable, it is prudent to consider sampling anatomic sites that are amenable to simultaneous biopsy of muscle and peripheral nerve through a single surgical approach.² In the pelvic limb, the biceps femoris and lateral head of the gastrocnemius muscles, as well as the common peroneal nerve are all accessible through a single incision placed over the caudolateral aspect of the distal femur and proximal tibia. In the thoracic limb, performance of an oblique incision extending from the medial humeral condyle to the point of the olecranon provides satisfactory exposure to the distal third of the medial head of the triceps and superficial digital flexor muscle, as well as the ulnar nerve at the level of the elbow.

Skeletal Muscle Biopsy Procedures

Two muscle biopsy techniques have been described, the open and percutaneous needle-biopsy procedures.^1,3,4 The percutaneous method offers the advantages of not requiring general anesthesia for completion, being minimally invasive, and has been shown to be capable of providing diagnostic quality samples in dogs using readily available, inexpensive, commercial biopsy needles (Perfectum 11-gauge needle, Popper and Sons, Inc., New Hyde Park, NY) with minimal morbidity.^3,4 The primary limitations of the percutaneous procedure are the small sample size of tissue obtained using this method, and inability to prevent contraction of myofibers after sampling.^1,4

Although open muscle biopsy procedures can also be performed using local anesthetics, general anesthesia is usually indicated to facilitate completion of electrodiagnostic testing that often precedes performance of open muscle biopsy. If local anesthesia is considered for open biopsy, care must be taken not to infiltrate the anesthetic agent deep into the muscle that has been selected for biopsy.² Open muscle biopsy is readily performed with basic surgical instrumentation.

The skin overlying the biopsy site should undergo routine aseptic preparation, regardless of the type of biopsy procedure performed. When using the open technique, the skin and any superficial fascia are incised, carefully dissected, and retracted to facilitate visualization of the myofiber orientation of the muscle selected for biopsy. Manipulation of the muscle biopsy site with forceps should be avoided. Following identification of the intended biopsy site, there are three similar methods by which biopsies intended for routine histochemical analysis can be harvested: the stay suture procedure, the muscle clamp method, and the free hand technique. It is not necessary to maintain biopsy specimens that will be subjected to routine analyses in a stretched position.^1,4

To harvest the muscle biopsy using the stay suture procedure, a 0.5 cm diameter, 2 cm long strip of muscle is created by placement of two stay sutures. The stay sutures should be placed perpendicular to the longitudinal orientation of the myofibers, and be tied loosely so as not to excessively constrict the myofibers. After the stay sutures are in place, two 2 cm long incisions are made parallel to the direction of the myofibers and extending just beyond the proximal stay suture immediately distal to the other stay suture in order to further isolate the muscle (Figure 11-1). The two stay sutures can be used to manipulate the biopsy specimen atraumatically during the remainder of the procedure.
 

While holding the proximal stay suture, the isolated segment of muscle is undermined using a scalpel blade or sharp dissection scissors (Figure 11-2), with a desired final biopsy thickness of approximately 0.5 cm. Complete separation of the biopsy specimen from the native muscle belly is achieved by cutting of the ends of the biopsy sample proximal and distal to the stay sutures (Figure 11-3), in a fashion that permits removal of both stay sutures with the biopsy specimen.

When using a commercial clamp system (Price muscle biopsy clamp, V. Mueller Instrument, Chicago, IL) to perform open muscle biopsy, a cylinder of muscle is created by first making two 2 cm long incisions parallel to the direction of the myofibers, with the intent of separating an approximately 0.5 cm diameter segment of muscle between the two incisions. The muscle clamp system is then applied to the ends of the incised segment of muscle (Figure 11-4). Using the handle of the muscle clamp to manipulate the biopsy specimen, the isolated muscle cylinder within the clamp is undermined and collected in a manner identical to that described for the stay suture technique.
 

The procedure for the free hand, open biopsy is similar to that described for the stay suture and muscle clamp methods. The primary difference with the free hand technique is that instead of utilizing a stay suture or muscle clamp to manipulate the muscle specimen during procurement of the biopsy, the proximal end of the biopsy specimen is minimally but directly handled with microsurgical forceps. Following completion of the biopsy procedure, the end of muscle specimen that was manipulated with the forceps is trimmed using a sharp, fresh scalpel blade to remove any artifacts caused by direct handling of the muscle.²

If an open muscle biopsy procedure is planned, it is important to consider the potential need to obtain samples for electron microscopic evaluation. These samples are ideally collected first with minimal manipulation of the myofibers. A muscle biopsy clamp system should be considered if muscle is being harvested for ultrastructural evaluation, as these clamps prevent both handling artifacts and myofiber contracture after excision and immersion in fixative.¹ Alternatively, if a muscle biopsy clamp is not available, a 0.25 to 0.5 cm in diameter, 1.5 cm long cylinder of muscle can be created by performing a modification of the previously described stay suture technique. In order to maintain the muscle in a stretched position during completion of the biopsy, the isolated cylinder of muscle is secured to a 2 cm length of small diameter wooden dowel using the long ends of the stay sutures, prior to undermining and complete separation of the biopsy specimen. Biopsy samples destined for ultrastructural evaluation are typically fixed in glutaraldehyde, and muscle biopsy clamps applied to these samples can be removed without compromising sample quality after 24 hours of fixation.¹ Following collection of the biopsy for electron microscopic analysis, additional biopsies for routine histochemical examinations can be obtained from adjacent myofibers.

The degree of hemorrhage associated with muscle biopsy procedures is usually minimal, and can often be controlled with digital pressure after harvesting the biopsy. Suture ligation may be required if a larger intramuscular vessel is encountered. The use of electrocautery should be avoided until all muscle biopsy samples have been obtained. Closure of the superficial muscular fascia and subcutaneous tissues is performed with an absorbable suture, and the skin is closed with sutures or staples. Application of external wound dressings following open muscle biopsy is rarely necessary. Complications associated with both the open and percutaneous muscle biopsy techniques are uncommon, but can include hematoma formation, wound dehiscence, and infection.^1,3

Processing of Muscle Biopsy Specimens

Most freshly harvested muscle biopsy specimens are transported without delay to specialized diagnostic laboratories for processing.² Therefore, prior to obtaining the muscle biopsies, it is crucial to contact the individual laboratory to which the samples are being sent to obtain specific instructions regarding recommended handling of harvested tissue, and to coordinate timely shipping and receiving of tissue samples. The histochemical and cytochemical characteristics of freshly collected muscle biopsy specimens can be acceptably preserved for approximately 30 hours if biopsy specimens are placed on gauze pads lightly moistened with physiologic saline, subsequently sealed in an airtight container, and maintained at 4° C until processing occurs.² This method allows for appropriately handled and packaged samples to be safely transported overnight to the diagnostic laboratory.

Once harvested, proper processing of muscle biopsy specimens is necessary to prevent introduction of processing artifacts and prevent loss of metabolic substrates and tissue enzymes. Immersion of muscle biopsy specimens in formalin provides limited diagnostic information, but may allow for morphologic characterization of any cellular infiltrates present in the sample. The method of obtaining and preserving muscle in glutaraldehyde for ultrastructural analysis has been previously described and reviewed.^1,2 Routine histochemical analysis of muscle is ideally performed on biopsy specimens that are processed by fresh freezing using the gum tragacanth-isopentene-liquid nitrogen method.^1,3 Uncontrolled freezing of muscle biopsy specimens can result in massive artifact formation that can completely compromise the diagnostic quality of the sample.

Muscle biopsies are readily obtained, and when properly performed and processed, are capable of providing essential information regarding a specific etiology for, the underlying disease process occurring within the muscle. The normal morphologic and histochemical characteristics of skeletal muscle using a standard battery of stains have been reviewed extensively elsewhere.^1,2 Even in cases in which a specific etiological diagnosis is not obtained from the biopsy, certain pathologic abnormalities that can be identified in muscle biopsy samples often provide insight into the basic underlying mechanism of the disease. For example, visualization of any of the following changes in a biopsy specimen are consistent with denervation of the muscle, and thus are coined neuropathic lesions: angular myofiber atrophy, small grouped myofiber atrophy, fiber type grouping, pyknotic nuclear clumping, or large grouped myofiber atrophy.^1,2 Primary myopathies are usually divided into inflammatory and non-inflammatory types based on the presence or absence of inflammatory cell infiltrates in representative biopsy specimens, respectively. Morphologically, there are a variety of non-specific findings in muscle biopsies that are suggestive of myopathic disease. These include myofiber splitting, degeneration or regeneration, necrosis and phagocytosis, internalized nuclei, and vacuolization.^1,2 Increased amounts of fibrous or adipose tissues within muscle biopsy specimens can be a feature of both primary myopathic and neuropathic muscular disease.

References

1. Dickinson PJ, LeCouteur RA. Muscle and nerve biopsy. Vet Clin North America Sm Anim Pract 32: 63, 2002.
2. Braund KG. Diagnostic techniques- nerve and muscle biopsy evaluation. In: Braund KG, ed. Clinical syndromes in veterinary neurology. 2nd ed. St. Louis: Mosby, 1994, p 376.
3. Reynolds AJ, Fuhrer L, Valentine BA, Kallflez FA. New approach to percutaneous muscle biopsy in dogs. Am J Vet Res 56(8): 982, 1995.
4. Magistris MR, Kohler A, Pizzolato G, et al. Needle muscle biopsy in the investigation of neuromuscular disorders. Muscle Nerve 21: 194, 1998.