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MUSCLE BIOPSY

Methods   Selection of muscle to biopsy   Biopsy freezing   Biopsy procedures   Stains   Biopsy request form: PDF Clinical   How to read a muscle biopsy   Indications   Results: Differential diagnosis   Pathology: Index   Unknowns Nerve biopsy

GENERAL INDICATIONS FOR MUSCLE BIOPSIES

• Presence of some evidence of muscle disease
  • Weakness
  • Muscle symptoms: Discomfort; Cramps; Fatigue with activity (r/o myasthenia)
  • Elevated (very high or high) creatine kinase (CK)
  • Myopathic EMG
• Presence of a neuropathy for which a nerve biopsy is indicated
• Presence of a systemic disorder that may have silent manifestations in muscle
  • Vasculitis
  • Sarcoidosis
• Not generally indicated in some disorders better diagnosed by electrodiagnostic methods
  • Myasthenia gravis (May be suggested on biopsy if esterase-stained NMJs are small)
  • Myotonias
• Wait: 1 month after episode of rhabdomyolysis

SELECTION OF MUSCLE TO BIOPSY

• Chronic disease: Muscle with moderate, but not severe, weakness
• Acute disease: Muscle with severe or moderate weakness
• Best specific muscles: Deltoid; Biceps; Quadriceps
• MRI can be used to select pathological muscle site in difficult cases
• Avoid: Muscles that were site of EMG, injections,or trauma

BIOPSY PROCEDURES

• Technique: We almost always use open biopsy procedure to obtain muscle.
  • Open biopsy strongly indicated for disorders with patchy pathology, e.g. polymyositis
  • Do not use cautery, sutures, or clamps.
  • Some laboratories use needle for many biopsies to minimize trauma.
    • May miss patchy or epimysial pathology
  • Obtain several samples from biopsy site: Especially with possible
    • Multifocal disease: Inflammatory myopathies
    • Need for metabolic analysis
• Transportation
  • Muscle may be saved in saline moistened gauze for several hours.
  • Keep specimen cool.
  • Muscle should NOT be immersed in saline, fixative or other liquids.
  • Frozen muscle may be safely shipped "overnight" with adequate dry ice
• Preservation
  • Freeze most tissue in isopentane precooled to -160^oC in liquid nitrogen.
    • Frozen muscle for histochemistry provides
      • Excellent muscle fiber morphology
      • Most diagnostic information with light microscopy
    • Freezing process should be rapid to prevent artifact.
    • Store frozen muscle at -80^oC.
  • Fix some muscle in 4% glutaraldehyde.
    • Embed most fixed muscle in plastic
      • For ultrastructural analysis, if necessary
      • Good visualization of muscle endomysial capillaries
    • Paraffin embedded material
      • Useful for surveys for inflammation & morphology of inflammatory cells
      • Gives poor muscle fiber morphology

HOW TO READ A MUSCLE BIOPSY

• Are muscle fibers abnormal?

  Normal muscle: Mean fiber diameter
  Age (Years)	Diameter (μM)
  Birth	15
  1	18
  3	20
  6	27
  8	35
  10	40
  12	50
  Adult	≥ 60

• 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 Type Enzymes	Myofibrillar ATPase	Muscle fiber type grouping or 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
Oxidative Enzymes	NADH-TR	Muscle fiber internal architecture; Tubular aggregates; Cores
	Succinate dehydrogenase	Mitochondrial pathology   Nuclear DNA encoded protein
	Cytochrome oxidase	Mitochondrial pathology   Mitochondrial DNA encoded protein
Glycolytic Enzymes	Phosphorylase	Phosphorylase deficiency
	Phosphofructokinase (PFK)	PFK deficiency
Hydrolytic Enzymes	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 disorders
	Alcian blue	Mucopolysaccharide
	Sudan black B	Lipid storage
	Oil red O	Lipid storage
Other	Congo red	Amyloid; Inflammation; Vacuoles
	Myoadenylate deaminase	AMPDA deficiency
	Methyl green pyronine	RNA
	Acridine orange	RNA
	Von Kossa	Calcium
	Alizarin red	Calcium
Fixed muscle	Toluidine blue	Muscle fibers; Capillaries

MUSCLE BIOPSY RESULTS: Differential diagnosis

Muscle fiber   Damage   Internal architecture   Mitochondrial   Necrosis   Size changes     Atrophy     Hypertrophy   Storage   Type disorders   Vacuoles

Amyloid Capillary pathology Complement Endomysial fibrosis Inflammation Inherited myopathies   Cytoplasmic proteins   Extracellular proteins   Nuclear proteins   Sarcolemmal proteins Nuclear 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 dystrophies
  • Rare: Inflammatory & immune myopathies, except IBM
• Muscle fiber damage
  • Necrosis
    • Rhabdomyolysis
    • Toxic
    • Hereditary with encephalopathy
    • Immune
      • Paraneoplastic necrotic myopathy
      • Myopathy with anti-SRP antibodies
      • Myopathy with pipestem capillaries
  • Regeneration
  • Focal invasion
    • Inclusion body myositis
    • Polymyositis with cytochrome oxidase negative muscle fibers
  • Myopathic grouping
    • Duchenne MD
    • Becker MD
    • LGMD: 2A; 2B; 2D
• 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
      • Fasciitis
      • Jo-1 myositis
      • Macrophagic myofasciitis
    • B-cells
      • Dermatomyositis, Childhood: Some patients
      • Nodular myositis
  • 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)
  • Type grouping
    • Chronic denervation with reinnervation or collateral sprouting
  • Type 1 muscle fibers
    • Predominance
      • All type 1
        • Central core; RYR1 mutations; Congenital myopathy; Large fiber type grouping; Demyelinating neuropathies
        • Most type 1: Bulbospinal muscular atrophy
    • Size: Small
      • Dystrophy: Emery-Dreifuss; ?Myotonic
      • Congenital myopathies: Centronuclear; Nemaline rod; Fiber type size disproportion
    • Size: Large
      • Spinal muscular atrophy 5q; Other chronic partial denervation
  • Type 2 muscle fibers
    • Size: Small
      • General causes: Disuse; Aging; Weight loss; Systemic disease; Paraneoplastic; Childhood myasthenia
      • Absent or small type 2A fibers: IBM3
      • Small 2B fibers: Early disuse
      • Small 2C fibers: Immature muscle fibers
    • Size: Large
      • Exercise training
  • Absent myosin ATPase staining at all pHs
    • Myosin loss myopathies
• 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
    • Ragged red fibers
    • Cytochrome b (MTCYB) mutation (Myalgias)
    • Dominant myopathy
    • Coenzyme Q₁₀ deficiency
  • Lipofuscin
• Vacuoles
  • Lysosomal: Acid maltase deficiency; Batten's disease
  • Autophagic
    • Histology: May stain for esterase & sarcolemmal proteins
    • Disorders
      • Autophagic vacuoles with sarcolemmal features
        • Danon disease
        • X-linked myopathy with excessive autophagy
        • Infantile autophagic vacuolar myopathy
        • Childhood autophagic vacuolar myopathy
        • Adult-onset autophagic vacuolar myopathy with multiorgan involvement
        • LGMD 1A
      • Autophagic vacuoles: Other
        • Toxic myopathy
        • Inflammatory myopathies
        • Hypokalemic myopathy
        • Acid maltase deficiency
  • Peroxisomes: Lafora disease
  • Rimmed
    • Inclusion body myositis
    • Oculopharyngeal Muscular Dystrophy (OPMD)
    • Distal myopathies
      • Welander
      • Finnish-Markesbery
      • Distal dystrophy
      • Oculopharyngodistal
      • MPD2
    • Hereditary inclusion body myopathies
      • HIBM: 1, 2, 3
      • Myopathy + Paget's & Dementia
    • LGMD: 1A, 1G, 2G
    • Emery-Dreifuss, X-linked
  • 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
  • PFK deficiency
  • Brancher enzyme deficiency (Glycogenosis IV)
  • Lafora disease
• Complement deposition: Patterns
  • Muscle capillaries
    • Dermatomyositis
    • Anti-SRP myopathy
    • Myopathy with pipestem capillaries
  • Within muscle fibers: Muscle fiber necrosis
  • Muscle fiber surface: Varying causes
  • Endomysial connective tissue: Brachio-cervical & other inflammatory myopathies
• Capillary pathology
  • Dermatomyositis
  • Anti-SRP myopathy
  • Myopathy with pipestem capillaries
• Immunohistochemistry: Inherited myopathies¹
  
  

  Cytoplasmic proteins Extracellular proteins Nuclear proteins Sarcolemmal proteins

  
  
  • Extracellular molecules
    • Laminins
      • Laminin γ1-chain: Control for assessment of basement membrane in basal lamina disorders
      • Laminin α2 (Merosin)
        • Normal: Diffusely present on basal lamina & IM nerves
        • Absent
          • Congenital muscular dystrophy
          • Associated with reduced laminin β2, α-dystroglycan & integrin α7
        • Reduced
          • Primary: Myopathy
          • Secondary: FKRP (LGMD 2I, CMD + muscle hypertrophy, MEB, Walker-Warburg syndrome
      • Laminin-β1: Reduced in Bethlem myopathy
    • Collagen
      • Bethlem myopathy: Collagen VI staining normal; Laminin-β1 reduced
      • Ullrich congenital muscular dystrophy: Collagen VI staining reduced on muscle & capillaries
    • Perlecan: Reduced or normal in Schwartz-Jampel syndrome
  • Sarcolemma-related proteins
  • Dystrophin
    • Utility of testing
      • Diagnosis: When gene testing is equivocal or negative
      • Prognosis: Better with more residual dystrophin
    • Testing methods & Results
      • Antibodies: 3 used, vs C-terminal, Rod & N-terminal domains of dystrophin
      • Immunocytochemistry
        • Absent dystrophin: High specificity for dystrophin gene defect
          • Has specificity for Duchenne MD
          • Often to all 3 antibodies
          • C-terminus always affected in Duchenne MD
        • Reduced dystrophin: Not specific for dystrophin gene defect
          • May occur in Becker MD
          • Patchy staining of sarcolemma of individual fibers may occur
          • C-terminus usually preserved in Becker MD
          • Selective loss of rod domain may produce Duchenne phenotype
          • Also found in some sarcoglycan & dysferlin disorders
        • Normal dystrophin: Does not rule out dystrophin gene defect
          • May occur in Becker MD
        • Revertant muscle fibers
          • Definition: Staining of isolated, or small groups of, fibers for more dystrophin than others
          • Due to 2nd mutation producing "in-frame" deletion: Small dystrophin molecule produced
      • Western blot: Useful for
        • Detecting reduced size of dystrophin
        • Detecting 2 different sizes of dystrophin in carriers
        • Confirming reduced amount of dystrophin
  • α-Dystroglycan: Secondarily reduced in some congenital muscular dystrophies (CMD) & LGMD
    • FKRP disorders
      • CMD with Muscle hypertrophy and Normal CNS (MDC1C)
      • LGMD 2I
    • Other congenital muscular dystrophies (CMD)
      • Fukuyama CMD
      • CMD with respiratory failure & muscle hypertrophy (CMD1B; MDC1B)
      • CMD with severe mental retardation (MDC1D)
      • Muscle-Eye-Brain Disease (MEB; Santavuori)
      • Walker-Warburg congenital MD
    • HIBM2 (Nonaka)
  • Sarcoglycans
    • Absent: Selective absence of 1 sarcoglycan has specificity for mutation in that gene
    • Reduced
      • Mutations in 1 sarcoglycan tend to reduce levels of other sarcoglycans
      • γ-sarcoglycan mutation: β- & δ-sarcoglycans may be normal
  • Dysferlin
    • Absent dysferlin on both IHC & Western blot: Has specificity for LGMD 2B
    • Reduced dysferlin: Non-specific; Dystrophin (50%), Sarcoglycan (20%), Caveolin-3 & Calpain-3 disorders
    • Western blot shows stronger correlation with gene defects
  • Caveolin-3
    • LGMD-1C: Reduced staining in membrane
  • Integrin-α7: Congenital muscular dystrophy
  • Cytoplasmic proteins: Soluble & Myofibrillar
    • Actin: May stain accumulations in
      • Nemaline myopathy: Actin disorders
      • Congenital myopathy with excess of thin filaments
    • Z-disk proteins
      • Nemaline rods: Stained by α-actinin; telethonin; myotilin
      • Myotilin: Abnormal accumulation with some mutations (Myofibrillar myopathy)
      • Telethonin: Absent in LGMD 2G
    • Myosin: Fiber typing
    • Titin: IHC not useful for diagnosis of LGMD 2J or Distal myopathy
    • Calpain-3
      • IHC: Not useful in testing for LGMD 2A
      • Western blot
        • Absent calpain-3: Has specificity for LGMD 2A
        • Reduced calpain-3: May occur in LGMD 2A, 2B, 2I, 2J
        • Also see: Other patterns of calpain-3 abnormality
    • Desmin
      • Intracellular accumulations: Marker for myofibrillar myopathies
      • Other associated proteins in accumulations: Dystrophin, CDC2 kinase, β-APP, Prion protein, NCAM & Myotilin
  • Nuclear proteins
    • Emerin: Absent staining in muscle & skin in EDMD 1
    • Lamin A/C: IHC not useful; Western blot may demonstrate reduced amount in EDMD 2