HOW TO READ A MUSCLE BIOPSY

General questions to keep in mind

Are muscle fibers abnormal?
- Size: Small or Large
- Shape: Rounded or Angular
- Type: Grouping; Fiber type predominance
- Internal architecture: Disordered or lost; Vacuoles; Internal nuclei; Inclusions
- Storage, or accumulated, material: Glycogen; Lipid; Mitochondria
Is the pathologic process
- Neurogenic or Myopathic?
  - Shape of small muscle fibers
    - Round: Myopathic
    - Angular: Neurogenic
    - Exceptions
      - Type 2 atrophy ® Small angular fibers
      - Spinal muscular atrophy ® Small rounded fibers
  - Distribution of atrophic fibers
    - Grouped: Denervation; Dystrophinopathies
    - Scattered: Acute neuropathy or myopathy
  - Distribution of fiber types
    - Type grouping: Chronic denervation
    - Fiber type predominance
      - Congenital disorder
      - Demyelinating neuropathy
      - Large fiber type grouping
    - Fiber type smallness
      - Type 1 small: Hereditary myopathies
      - Type 2 small: Acquired disorders; Congenital MG
- Acute or Chronic?
  - Acute
    - Myopathy: Muscle fiber degeneration & regeneration
    - Neuropathy: Small angular muscle fibers
  - Chronic
    - Myopathy: Increased endomysial connective tissue; Muscle fiber hypertrophy
    - Neuropathy: Fiber type grouping; Pyknotic nuclear clumps
- Producing specific diagnostic features?
What is the distribution of the pathology?
- Uniform (Similar in all parts of the biopsy): Dystrophy; Fiber type atrophy
- Regional
  - Patchy fascicular changes: Inflammatory myopathy; Focal denervation
  - Groups of muscle fibers
    - Neuropathy: Progressive denervation with reinnervation
    - Myopathy: Myopathic grouping; Perifascicular atrophy
- Scattered muscle fibers: Acute myopathy; Acute neuropathy
Are there diagnostic features?
- Is there inflammation or excess cellularity?
  - What kind?
    - Lymphocytes
    - Macrophages
    - Other: Neoplasm; Eosinophils
  - Where?
    - Endomysial
    - Perimysial
    - Perivascular
    - Focal invasion of muscle fibers
- Is there storage material?
  - Lipid
  - Glycogen
  - Amyloid
- Is there pathology in structures other than muscle fibers?
  - Vessels
  - Connective tissue: Endomysial; Perimysial
  - Intramuscular nerves

MUSCLE BIOPSY STAINS

Category	Method	Utility
Morphology	Hematoxylin and eosin	Muscle fiber pathology; Nuclei
	Verhof van Giesson (VvG)	Connective tissue; Vessel structure Intramuscular nerve
	Gomori trichrome	Connective tissue; Nemaline rods
Fiber Typing	Myofibrillar ATPase, any pH	Muscle fiber type grouping & atrophy
	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
Enzymes: Oxidative	NADH-TR	Muscle fiber internal architecture; Tubular aggregates; Cores
	Succinate dehydrogenase	Mitochondrial pathology
	Cytochrome oxidase	Mitochondrial pathology
Enzymes: Glycolytic	Phosphorylase	Enzyme deficiency
Enzymes: Glycolytic	Phosphofructokinase	Enzyme deficiency
Enzymes: Hydrolytic	Acid phosphatase	Macrophages; Lysosomes; Lipofuscin
	Non-specific esterase	Macrophages; Lysosomes; Neuromuscular & myotendinous junctions Denervated (small angular) muscle fibers
	Acetylcholinesterase	Neuromuscular & myotendinous junctions
	Alkaline phosphatase	Regenerating muscle fibers; Immune disease of connective tissue
Storage material	PAS	Glycogen; Carbohydrate
	Alcian blue	Mucopolysaccharide
	Sudan black B	Lipid
	Oil red O	Lipid
Other	Congo red	Amyloid; Inflammation; Vacuoles
	Myoadenylate deaminase	Enzyme level
	Methyl green pyronine	RNA
	Acridine orange	RNA
	Von Kossa	Calcium
Fixed muscle	Toluidine blue	Muscle fibers; Capillaries

MUSCLE BIOPSY RESULTS: Differential diagnosis

Amyloid
Complement
Endomysial fibrosis
Inflammation
Muscle fiber
Damage
Internal architecture
Mitochondrial
Necrosis
Size changes
Atrophy
Hypertrophy
Storage
Type disorders
Vacuoles
Nuclear changes
Capillary pathology

Focal invasion of muscle fiber

Atrophy (H&E; ATPase): Also see Fiber type disorders
- Small rounded fibers: Myopathy (Regeneration; Chronic); Type I smallness
- Small angular fibers: Neuropathy; Type 2 atrophy; Inclusion body myopathies
- Grouped: Neuropathy with denervation
  - Multiple small groups: Most commonly associated with ALS
  - Large groups: Chronic denervation; Motor neuron disease
  - Rule out: Myopathic grouping
- Perifascicular: Dermatomyositis
Hypertrophy
- Type I fibers: Spinal muscular atrophy
- Type II muscle fibers: Exercise
- Mixed fiber types
  - Partial denervation: Very chronic
  - Chronic myopathies: Muscular dystrophy
Muscle fiber damage
Nuclear pathology (H&E)
- Pyknotic clumps
  - Chronic denervation without reinnervation
  - Myotonic dystrophy, Type II
  - Myasthenia gravis, chronic untreated
- Internal nuclei
  - Most abundant & > 60% of fibers: Chronic myotonic dystrophy
  - Single, centrally placed: Centronuclear myopathy
  - 10% to 30% of fibers: Chronic neuropathic or myopathic process
- Abnormal nuclei: Inclusion body myositis
- Large nuclei: Regenerating muscle fibers; Myosin-loss myopathy
Inflammation (H&E; Congo red; Acid phosphates; Esterase; Immunocytochemistry)
- Immune myopathies
  - T-cells > Macrophages
    - Often with focal invasion of muscle fibers
    - Specific disorders: Inclusion body myositis; Myositis with mitochondrial disorder
  - Macrophage predominant
- Vasculitis: T-cells > Macrophages
- FSH dystrophy: T-cells
- Sarcoid: Macrophages & histiocytes in center of granulomatous inflammation
- Fiber necrosis: Macrophages in muscle fibers
- Lymphorrhages (Myasthenia gravis): T-cells
Endomysial fibrosis (H&E; GT & VvG)
- Diffuse & abundant
  - Dystrophy
  - Endstage muscle
  - Anti-SRP myopathy
- Patchy: Acquired myopathy
- Increased endomysial connective tissue between muscle fibers is normal in regions with NMJs
Abnormal internal fiber architecture (NADH stain)
- Targets: Denervation & reinnervation; Tenotomy
- Cores: Central core syndromes
- Coarse: Immature or regenerating fibers
- Rods (GT stain): Rod myopathy; Reinnervation with targets
- Tubular aggregates
- Moth-eaten fibers: Systemic connective tissue disorders; Chronic dystrophy
- Rings
- Lobulated fibers: LGMD 2A; Ullrich CMD; Bethlem myopathy
- Necrosis
Fiber type disorders (ATPases pH 9.4, 4.6 & 4.3)
- Absent myosin ATPase staining at all pHs: Myosin deficiency myopathies
- Type grouping: Chronic denervation with reinnervation or collateral sprouting
- Type 1
  - Type 1 predominance
    - All type 1: Central core; Large fiber type grouping; Demyelinating neuropathies
    - Most type 1: Bulbospinal muscular atrophy
  - Small type 1 fibers:
    - Dystrophy: Emery-Dreifuss; ?Myotonic
    - Congenital myopathies: Centronuclear; Nemaline rod; Fiber type disproportion
  - Large type 1: Spinal muscular atrophy 5q
- Type 2
  - Small type 2 fibers: Disuse; Aging; Weight loss; Systemic disease; Paraneoplastic
  - Large type 2 fibers: Exercise training
  - Absent or small type 2A fibers: IBM3
Mitochondrial (SDH & Cytochrome oxidase)
- Pathology: Differential diagnosis
Glycogen storage (PAS)
Lipid storage (Sudan black or Oil red O)
- Carnitine deficiency
- Malnutrition
- Fatty acid oxidation disorders
  - Multiple acyl–CoA dehydrogenase deficiency
  - SCAD deficiency
- Mitochondrial
- Lipofuscin
Vacuoles
- Lysosomal: Acid maltase deficiency; Batten's disease
- Autophagic
  - 2° lysosome: Stains + for acid phosphatase
  - Acid maltase; Toxic; Inflammatory myopathy; Hypokalemic myopathy;
    X-linked myopathy with excessive autophagia
- Peroxisomes: Lafora disease
- Rimmed
  - Inclusion body myositis
  - OPD
  - Distal myopathies: Welander; Finnish-Markesbery; Distal dystrophy;
    Oculopharyngodistal; MPD2; Myopathy + Paget's
  - Hereditary inclusion body myopathies (HIBM): 1, 2, 3
  - LGMD: 1A, 2G
- Subsarcolemmal: Phosphorylase deficiency
- Other: Periodic paralysis; Glycogen storage; Lipid storage; Centronuclear myopathy;
  Lipofuscin (aging); Freezing artifact; Desmin storage
Amyloid (Congo red)
Polyglucosan deposition: Increase in glycogen synthetase:branching enzyme ratio
Complement deposition: Patterns
- Muscle capillaries
- Within muscle fibers: Muscle fiber necrosis
- Around muscle fibers: Varying causes
Capillary pathology