Introduction to Muscle Biopsy
Myopathies can be divided into two main categories:
The acquired myopathies are further classified to:
-Viral myositis (HIV, influenza, EBV)
– Bacterial (Staphylococcus Au, Streptococcus)
– Parasites (Trichinosis)
- Toxic myopathies
– Antiretroviral, protease inhibitors
- Myopathies related to systematic disorders
- Endocrine disorders
- – Thyroid, parathyroid, pituitary, adrenal glands
- Systematic inflammatory disorsers
- – SLE, RA, Scleroderma, Sjogren, Sarcoid
- Electrolytic disorders
- – Potassium, Magnesium, Phosphorus
- Critical illness myopathy
- Amyloid myopathy
- – Primary amyloidosis
– Secondary amyloidosis
The inherited-congenital myopathies on the other hand are further divided to:
- Muscular dystrophies
- Dystrophinopathies (Duchenne, Becker)
- Myotonic dystrophies type 1 and 2
- Fascio humeroscapular dystrophy
- Oculopharyngeal dystrophy
- Limb girdle muscular dystrophies
- Congenital myopathies
– nemaline bodies myopathy
– central core myopathies
- Metabolic myopathies
– acid maltase deficiency
– glycogen storage myopathies
– carnitine deficiency myopathy
– carnitine palmitransferase myopathy
– fatty acid metabolism myopathies
- Mitochondrial myopathies
- MERRF syndrome
- Progressive external ophtalmoplegia
Diagnosis of the muscle disease
Neuromuscular pathology must always be evaluated in a multispecialty context; clinical features (age of onset, pattern of involvement, course, family history, concurrent medical problems), electrophysiological studies (electromyography [EMG] and nerve conduction studies), laboratory results (e.g., creatine kinase levels, presence of a monoclonal gammopathy), and in some instances neuroimaging, provide information that permits the formulation of most likely clinical diagnostic hypotheses preceding the biopsy. The objective of the biopsy is then to confirm, extend, or perhaps disprove these initial hypotheses. Determining the need for a muscle or nerve biopsy, ensuring its performance, and formulating the interpretation can best be accomplished as a collaborative effort between the neurologist, the neurophysiologist and neuropathologist.
Muscle biopsy is an important tool for the evaluation and diagnosis of patients presenting to clinic with acute or progressive weakness who are suspected of having an underlying neuromuscular disorder. Alongside the clinical examination, electrodiagnostic, laboratory and molecular genetic testing, muscle biopsy has a critical role, providing diagnostic evidence that either establishes a disease etiology or focuses the differential diagnosis. The muscle biopsy itself is a fairly straight forward procedure with little risk. However, to get the full benefit of the procedure several experts need to be involved, including a surgeon, processing laboratory and pathologist which requires planning.
The objective of the biopsy is then to confirm, extend, or perhaps disprove these initial hypotheses. Determining the need for a muscle biopsy, ensuring its performance, and formulating the interpretation can best be accomplished as a collaborative eff ort between the neurologist and neuropathologist.
It is important to biopsy a muscle that is affected but not so severely that a biopsy will only reveal nondiagnostic end-stage changes. Thus, we advise biopsy
of a muscle that has a Medical Research Council (MRC) grade of around a 4/5. Occasionally, when there is only mild weakness (e.g., only in hip girdle, which is not typically biopsied), the choice of muscle to biopsy can be aided by doing an EMG on one side of the body and then to biopsy a muscle that was abnormal on EMG on the contralateral limb. Alternatively, skeletal muscle MRI scans can be used to guide the choice of muscle to be biopsied. It is important not to biopsy sites of prior EMG needle examination, where a focal necrotic inflammatory reaction might give misleading information.
We almost always use open biopsy procedure to obtain muscle. Open biopsy strongly indicated for disorders with patchy pathology, e.g. polymyositis. It is important not to use cautery, sutures, or clamps. Some laboratories use needle for many biopsies to minimize trauma, but with this technique patchy or epimysial pathology can be missed. It is important to obtain several samples from biopsy site, especially with possible multifocal disease, such as the inflammatory myopathies.
Transportation and preservation
Muscle may be saved in saline moistened gauze for several hours, and the specimen should remain cool. The muscle biopsy should not be immersed in saline, fixative or other liquids. Frozen muscle may be safely shipped “overnight” with adequate dry ice.
Frozen muscle for histochemistry provides excellent muscle fiber morphology and the most diagnostic information with light microscopy. The sample is frozen at -80oC and a small part is put in 4% glutaraldehyde to be used for EM.
Paraffin embedded material is only useful for surveys for inflammation and morphology of inflammatory cells and gives poor muscle fiber morphology.
Normal histology of muscle fibres
Striated skeletal muscles consist of bundles of fibres called fascicles, each of which is surrounded by connective tissue sheath termed perimysium. Perimysium contains blood vessels and nerve supply of the muscle fascicles. Each muscle fibre is enveloped by delicate fibrous stroma called endomysium. Individual muscle fibre is an elongated multinucleated syncytium-like cell about 100 μm in diameter and several centimeters in length. The muscle nuclei are spindle-shaped and lie at the periphery of fibre under the sarcolemma, the plasma membrane of muscle fibre. The cytoplasm of the muscle fibre contains myofilaments which are contractile elements.
Myofilaments are of 2 types—myosin comprising thick filaments and actin constituting thin filaments. These together produce cross-striations in muscle fibres seen in longitudinal sections on light microscopy. Sarcomeres are the partitions of
myofilaments into equal zones. Each sarcomere represents the distance between consecutive Z bands and contains the central A (anisotropic) band, and the lateral I (isotropic) bands. The major functions of striated skeletal muscle are to convert chemical energy into mechanical energy, to act as a store of energy and proteins, and to play a role in the metabolism of the body. The muscle, however, cannot function as a contractile organ without a nerve supply. For this purpose, there are motor units, each of which consists of the following:
- Motor neuron cell body located in the spinal cord anterior horn, or a cranial nerve nucleus.
- The axon of the motor neuron in the peripheral or cranial nerve.
- The neuromuscular junction.
- The muscle fibres innervated by the motor neuron.
The muscle fibre contraction occurs by action potential generated by chemical transmission of the impulse across the synaptic gap by acetylcholine.
Standard panel of stains
The main morphology stains in the standard panel are:
Hematoxylin & Eosin: Muscle fiber pathology; Nuclei
Verhoeff van Gieson (VvG): Connective tissue; Vessel structure; Intramuscular nerve (Myelinated axons)
Gomori trichrome: Connective tissue; Nemaline rods Cytoplasmic bodies
Standard stains for the differentiation between fibre types:
ATPase pH 9.4: Myosin loss; Type 1 or 2 fiber atrophy
ATPase pH 4.6: Type 2B muscle fibers
ATPase pH 4.3: Type 2C (Immature) muscle fibers; Blood vessels
The main oxidative enzymes that are usually checked with the routine methods are:
NADH-TR: Muscle fiber internal architecture; Tubular aggregates; Cores
Succinate dehydrogenase: Mitochondrial pathology – Nuclear DNA encoded complex
Cytochrome oxidase: Mitochondrial pathology – Mitochondrial & Nuclear DNA encoded
The main glycolytic enzymes are:
Phosphorylase: Phosphorylase deficiency
Phosphofructokinase (PFK): PFK deficiency
The main hydrolytic enzymes:
Acid phosphatase: Histiocytes; Lysosomes; Lipofuscin
Esterase, Non-specific: Histiocytes (Cytoplasm); Lysosomes; Neuromuscular & Myotendinous junctions; Denervated (small angular) muscle fibers
Acetylcholinesterase: Neuromuscular & Myotendinous junctions
Alkaline phosphatase: Regenerating or Immature muscle fibers
Immune disease: Connective tissue; Capillaries; Muscle fiber necrosis
The main stains for storage material are:
PAS: Glycogen & Carbohydrate disorders
Alcian blue: Mucopolysaccharide
Sudan black B: Lipid storage
Oil red O : Lipid storage
Menadione-αGP: Reducing bodies; Dense bodies
Additional stains that are usually performed are:
Congo red: Amyloid; Inflammation; Vacuoles
Mitochondrial Oxidative Enzyme Activities:
- General procedures
- Complex I
- Complex II
- Complex II+III
- Complex III
- Complex IV
- Coenzyme Q10
- Citrate synthase
How to read a muscle biopsy
The general questions to keep in mind are:
- Is the internal architecture preserved, disordered or lost?
- Are muscle fibers abnormal?
What is the size of the muscle fibres? Small or Large
- Shape: Rounded or Angular
In normal adult muscle, the muscle fibers are polygonal and in an infant the fibers are rounded. In infants and children there is very little endomysial connective tissue.
The fibers become rounded in muscular dystrophies and become angulated and atrophic in denervation.
Are the nuclei peripherally located or they are Internal?
In a normal muscle, nuclei are subsarcolemmal. They are small, oval and dark staining. 3-10% of fibers in transverse section may show internal nuclei. Large number of internal nuclei suggests a myopathy and transverse section is best to assess the same. Dystrophies show about 10-30% internal nuclei and myotonic dystrophy is characterized by profuse number of internal nuclei of about 60%. Myotubular/centronuclear myopathy shows more than 30% of fibers showing single centrally placed nuclei.
Is there fibre type Grouping? Fiber type predominance can be seen in congenital conditions or in chronic neuropathic processes.
Is the pathologic process Neurogenic or Myopathic?
- Shape of small muscle fibers
– Angular: Neurogenic
- Exceptions: Type 2 fibre atrophy with Small angular fibres
Question about the distribution of the atrophic fibres.
- Grouped: Denervation; Dystrophinopathies
- Scattered: Acute neuropathy or myopathy
Distribution of fibre types
– Type grouping usually seen in Chronic denervation
- Fibre type predominance
-Large fibre type grouping
- Fibre type smallness
- Type 1 small: Hereditary myopathies
- Type 2 small: Acquired disorders; Congenital MG
Is the pathologic process acute or chronic?
In acute myopathies there is muscle fibre necrosis and regeneration, while in neuropathic processes there is atrophy, and scattered angular or diffuse muscle fibres. In chronic myopathies there is increased endomysial connective tissue, with muscle fibre hypertrophy or atrophy, and in chronic neuropathic processes there is fibre type grouping, and grouped atrophy with occasional pyknotic nuclear clumps.
Additional diagnostic features
Is there inflammation or excess cellularity?
If yes, of what kind?
Is there infiltration by Lymphocytes, Macrophages, Eosinophils, or Other (neoplastic) cell types?
If there is inflammation, is that Endomysial, Perimysial, Perivascular, or Focal invasion of muscle fibers?
Normal muscle is devoid of any inflammatory cells. Cellular infiltrates are seen in inflammatory myopathies like dermatomyositis, inclusion body myositis (IBM).
A necrotic fiber is pale stained on H&E and infiltrated by phagocytes.
This is called myophagocytosis.
This is usually seen in myopathies especially dystrophies like Duchenne muscular dystrophy (DMD). These fibers are highlighted by acid phosphatase and esterase reactions.
Sometimes necrotic fibers are seen in inflammatory myopathies, paraneoplastic necrotizing myopathies, after rhabdomyolysis and in acute neuropathies.
Is there any Storage, or accumulated, material: Glycogen; Lipid; Mitochondria, rod bodies?
Rod bodies characteristically stain red with MGT. The rods are delicate and accumulate subsarcolemmaly. They stain negative with ATPase, NADH and SDH as they lack myosin and mitochondria. Seen in Nemaline myopathy but also seen in central core disease and various other diseases which include neurogenic disorders (amyotrophic lateral sclerosis, spinal muscular atrophy, undefined), inflammatory myopathies (dermatomyositis, polymyositis, periarteritis nodosa), metabolic myopathy (mitochondrial myopathy), muscular dystrophy (LGMD) and some undefined myopathies.
Are there Vacuoles & Inclusions?
Two types- one type contains some material within and the other appears as just empty spaces. The former are called as rimmed vacuoles and on H&E they contain basophilic granular material and on MGT they appear as having red granules. They appear in number of conditions like IBM, distal myopathies, oculopharyngeal muscular dystrophies, myofibrillar myopathies and others. In glycogen storage disease, vacuoles appear on H&E. The vacuoles are seen in acid maltase deficiency of childhood (Pompe’s disease) and adulthood (McArdle’s disease) and Glycogenses V.
Is there any pathology in structures other than muscle fibers?
- Vessels: Vasculitis
- Connective tissue: Endomysial; Perimysial
- Intramuscular nerves