Muscle and nerve biopsies are essential diagnostic tools for neuromuscular diseases. Unlike routine surgical pathology specimens, they require specialized handling — fresh tissue must be processed immediately for frozen sections and enzyme histochemistry, and parallel samples must be prepared for paraffin embedding and electron microscopy.
Muscle Biopsy: Indications and Selection
Muscle biopsy is indicated for: unexplained weakness (proximal > distal), elevated creatine kinase (CK), myopathic or neurogenic EMG findings, suspected inflammatory myopathy (dermatomyositis, polymyositis, inclusion body myositis), suspected muscular dystrophy, suspected metabolic myopathy (mitochondrial, glycogen storage, lipid storage), and congenital myopathy.
The biopsy site is selected based on clinical involvement and EMG findings. Vastus lateralis (quadriceps) is the most common site — easily accessible, representative of proximal muscle, and well-characterized for normal values. Deltoid is used for proximal upper extremity weakness. Biceps brachii is an alternative. The biopsy should be taken from a moderately affected muscle — end-stage muscle (severe atrophy, fatty replacement) provides little diagnostic information. The site must not have undergone recent EMG needling (artifact from needle trauma).
Muscle Biopsy Processing Protocol
The specimen is received fresh, kept moist with saline-moistened gauze. Under a dissecting microscope, the muscle fibers are oriented longitudinally. The specimen is divided: frozen section (the largest portion, 5-8 mm × 5 mm) — mounted on a cork disc in OCT compound, oriented for cross-sectioning, and snap-frozen in isopentane cooled to -160°C in liquid nitrogen. Paraffin (a 3-5 mm piece) — fixed in formalin for routine histology and IHC. Electron microscopy (a 1-2 mm piece, oriented longitudinally) — fixed in 2.5% glutaraldehyde. Biochemistry (optional, 10-20 mg) — snap-frozen for enzyme analysis or genetic testing.
The frozen block is sectioned at 8-10 µm in a cryostat at -25°C. A standard frozen section panel includes: H&E, modified Gomori trichrome, ATPase at pH 9.4, 4.6, and 4.3, NADH-TR, COX, SDH, acid phosphatase, Oil Red O (neutral lipid), PAS (glycogen), and non-specific esterase.
Frozen Section Enzyme Histochemistry
The ATPase reactions at different pH levels must be performed on unfixed frozen sections because fixation inactivates myosin ATPase. The reactions are: pre-incubation at pH 4.3 (type I dark, type II light), pH 4.6 (type I dark, IIA light, IIB intermediate), and pH 9.4 (type I light, type II dark). These differentiate type I (slow-twitch, oxidative) and type II (fast-twitch, glycolytic) fibers and their subtypes. The normal distribution is a checkerboard pattern with type I predominance in postural muscles and type II predominance in phasic muscles.
NADH-TR, COX, and SDH must be performed on frozen sections within 24-48 hours of sectioning — enzyme activity declines with storage. Positive controls (known normal muscle) are run with each batch.
Paraffin Sections and IHC
Formalin-fixed, paraffin-embedded muscle is sectioned at 4-5 µm and stained with H&E, LFB-PAS, and Congo Red. IHC for dystrophin (rod domain, N-terminus, C-terminus, and dropout controls) diagnoses Duchenne/Becker muscular dystrophy. Sarcoglycans (alpha, beta, gamma, delta) — mutations cause limb-girdle muscular dystrophy. Dysferlin — mutations cause Miyoshi myopathy and LGMD2B. MHC class I IHC — upregulated in inflammatory myopathies (dermatomyositis, polymyositis). MxA IHC — upregulated by type I interferon in dermatomyositis (perifascicular pattern).
Nerve Biopsy
Sural nerve biopsy is the most common nerve biopsy — the sural nerve is a pure sensory nerve, and its removal causes only minor sensory loss over the lateral foot. Indications include: suspected vasculitic neuropathy, hereditary neuropathy (Charcot-Marie-Tooth disease), amyloid neuropathy, inflammatory neuropathy (sarcoid, CIDP), and storage diseases.
The specimen (2-3 cm) is processed for: paraffin — H&E, LFB-PAS, Congo Red, IHC (neurofilament, MBP, CD68, CD3, CD20). Frozen — ATPase (for fiber type), NADH-TR, acid phosphatase. Teased fibers — 30-50 individual fibers separated and mounted for assessing axonal degeneration (myelin ovoids) and segmental demyelination (thin or absent myelin sheaths). Electron microscopy — quantifies myelinated fiber density, unmyelinated fiber loss, and identifies specific abnormalities (tomacula in hereditary neuropathy with pressure palsies, onion bulbs in demyelinating neuropathies, axonal atrophy).
Quality Control
All neuromuscular biopsy techniques require rigorous quality control. Frozen sections must be evaluated for freeze artifact (ice crystal vacuolation — indicates slow freezing; the tissue should be transparent when frozen). Enzyme histochemical reactions must show expected fiber type patterns on positive controls. IHC for dystrophin requires known normal and dystrophin-deficient controls. Participation in neuromuscular EQA programs is recommended for laboratories performing these specialized techniques.
