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MUSCLE BIOPSY

Methods   Selection of muscle to biopsy   Biopsy freezing   Biopsy procedures   Biochemistry     Glycogen pathways     Mitochondria   Stains     Histochemical     Immune     Inherited myopathies   Biopsy request form: PDF Clinical   How to read a muscle biopsy   Indications   Results: Differential diagnosis   Pathology: Index   Unknowns Nerve biopsy

Vesalius

Gowers

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
  • Gene testing: Guide or Validate ⁴
• Presence of a neuropathy for which a nerve biopsy is indicated
• Presence of a systemic disorder that may have silent manifestations in muscle
  • Immune or Inflammatory disorders
    • Vasculitis
    • Microvasculopathies
    • Sarcoidosis
  • Amyloid
  • Neoplasm: Angiotropic lymphoma; Adenocarcinoma
  • Mitochondrial disorders
• 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
• Also see: Nishino

HOW TO READ A MUSCLE BIOPSY

MUSCLE BIOPSY STAINS & PROCEDURES

Category	Method	Utility	Perimysial Vessels: Normal VvG stain
Morphology	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
Fiber Type   Enzymes (See MYH)	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 Blood vessels
Oxidative   Enzymes	NADH-TR	Muscle fiber internal architecture; Tubular aggregates; Cores
	Succinate dehydrogenase (SDH)	Mitochondrial pathology   Nuclear DNA encoded complex
	Cytochrome oxidase (COX)	Mitochondrial pathology   Mitochondrial & Nuclear DNA encoded
Glycolytic   Enzymes	Phosphorylase	Phosphorylase deficiency
	Phosphofructokinase (PFK)	PFK deficiency
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
Storage   material	PAS	Glycogen & Carbohydrate disorders
	Alcian blue	Mucopolysaccharide
	Sudan black B	Lipid storage
	Oil red O	Lipid storage
	Menadione-αGP	Reducing bodies; Dense bodies
Other	Congo red	Amyloid; Inflammation; Vacuoles
	Myoadenylate deaminase   (AMPDA)	AMPDA deficiency Cytoplasmic aggregates
	Methyl green pyronine	RNA
	Acridine orange	RNA
	Von Kossa	Calcium
	Alizarin red	Calcium
Fixed muscle	Toluidine blue	Muscle fibers; Capillaries

• General procedures
• Complex I
• Complex II
• Complex II+III
• Complex III
• Complex IV
• Coenzyme Q10
• Citrate synthase

MUSCLE BIOPSY RESULTS: Differential diagnosis

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

Amyloid Capillary pathology Complement Endomysial fibrosis Inflammation   MHC-I Inherited myopathies   Cytoplasmic proteins   Extracellular proteins   Nuclear proteins   Sarcolemmal proteins Nuclear pathology

Focal invasion of muscle fiber

Muscle Fiber Pathology

• Clustered
  • Necrosis
    • Fibers in clusters in similar stages of necrosis
      • Myopathic grouping: Dystrophies
      • Regional ischemic immune myopathy (RIIM)
    • Fibers in clusters in different stages of necrosis
      • Immune myopathies with Permysial Pathology
  • Atrophy
    • Inclusion body myopathies, Hereditary
    • Dermatomyositis + Vascular Pathology
• Atrophy (H&E; ATPase): Also see Fiber type disorders
  • Small fibers
    • Round shapes
      • Muscle fiber Regeneration
      • Myopathy: Chronic
      • Denervation: Diffuse
      • Type I smallness
    • Angular shapes
      • Neuropathy
      • Type 2 fiber atrophy
      • Inclusion body myopathies
    • Grouped
      • Neuropathy: Ongoing with Denervation & Reinnervation
        • Multiple small groups: Most commonly associated with ALS
        • Large groups: Chronic denervation; Motor neuron disease (SMA)
      • Myopathy
        • Perifascicular atrophy: Dermatomyositis with vascular pathology (DM-VP)
        • Clustered: Necrosis & Regeneration
          • Myopathic grouping: Often related to dystrophies producing fragile muscle fiber sarcolemma
          • Ischemia, focal: Regional Ischemic Immune Myopathy
• 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
  • Immaturity or Regeneration
  • Focal invasion
    • Inclusion body myositis
    • Polymyositis with cytochrome oxidase negative muscle fibers
  • Myopathic grouping
    • Duchenne MD
    • Becker MD
    • LGMD: 2A; 2B; 2D
  • MHC-I changes
• Nuclear pathology (H&E; Congo red)
  • Pyknotic clumps
    • Chronic denervation without reinnervation
    • Myotonic dystrophy, Type II
    • Myasthenia gravis, chronic untreated
  • Internal nuclei
    • Most abundant & > 60% of fibers: Myotonic dystrophy, Chronic
    • Single, centrally placed: Centronuclear myopathy
    • 10% to 30% of fibers: Chronic neuropathic or myopathic process
    • All or most in type 2 muscle fibers: Myotonic dystrophy, type 2
    • Nuclear chains: Myotonic dystrophy, Chronic
  • Large nuclei
    • Immature or Regenerating muscle fibers
    • Myosin-loss myopathy
  • Abnormal nuclear morphology
    • Inclusion body myositis: Empty; TDP-43 reduced
    • HMGCR myopathy
    • Age
    • Cachexia
    • Nuclear envelope
    • Rod myopathies
    • Other
  • Intranuclear rods (Actin aggregates)
    • IMPP
    • Nemaline rod myopathy
      • NEM3: ACTA1 mutations
      • MYH2
      • Sporadic, late-onset
    • Oculopharyngeal muscular dystrophy: Tubular filaments
    • PLEC1
    • LBD3
  • Absent staining
    • Muscle fiber necrosis
• 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 muscle fibers: Systemic connective tissue disorders; Chronic dystrophy
  • Rings
  • Linearization
  • Lobulated fibers: LGMD 2A; Ullrich CMD; Bethlem myopathy
  • Necrosis
• Fiber type disorders (ATPases pH 9.4, 4.6 & 4.3)
  • Fiber type
    • Properties
      • MYH contents
      • Patterns & Disorders
    • Staining patterns: Histochemistry
  • Type grouping
    • Chronic denervation with reinnervation or collateral sprouting
  • Type 1 muscle fibers
    • Properties
      • Staining
        • ATPase pH 9.4: Intermediate
        • ATPase pH 4.3: Dark
        • ATPase pH 4.6: Dark
      • Myosin Heavy Chain (MYH) type
        • MYH7 = Slow type 1
      • Physiology: Slow; Oxidative
      • Ultrastructure
        • Z-lines: Thicker; Intermediate width
        • Mitochondria: Many; Small
        • Lipid bodies: More
        • Glycogen granules: Fewer
        • Myofibrils: More compact
    • Predominance
      • All type 1
        • RYR1 mutations
          • Central core
          • Congenital myopathy
        • CACNA1S congenital myopathy
        • Fiber type grouping: Large
        • Demyelinating neuropathies
          • Especially CIDP with conduction block or temporal dispersion
      • Most type 1
        • ACTA1
        • Bulbospinal muscular atrophy
        • ACTN2
        • JPH1
        • LGMD 1A
        • MYBPC3
        • PTPLA/HACD1
        • SECISBP2
        • SCN4A
        • SEPN1
        • STAC3
        • ZAK
      • Trunk muscles
    • Size: Small
      • Dystrophy
        • Emery-Dreifuss
        • ? Myotonic
        • LGMD 1D
      • Congenital myopathies
        • Centronuclear
        • Nemaline rod
        • Fiber type size disproportion
        • RYR1 mutations: Few patients
    • Size: Large
      • Spinal muscular atrophy 5q
      • Chronic partial denervation, Other
  • Type 2 muscle fibers
    • Fiber subtypes
      • Type 2A
        • Staining pattern
          • ATPase pH 9.4: Dark
          • ATPase pH 4.3: Pale or Absent
          • ATPase pH 4.6: Pale or Absent
        • Myosin Heavy Chain (MYH) type
          • MYH2 = Fast, Type 2A
        • Physiology: Fast, Fatigue-Resistant fibers
        • Ultrastructure
          • Z-lines: Wide
          • Mitochondria: Many; Large
          • Glycogen granules: More
        • Histology: 2A fibers lost
          • Biopsies not frozen for > 6 to 12 hours
          • IBM3
      • Type 2B
        • Staining pattern
          • ATPase pH 9.4: Dark
          • ATPase pH 4.3: Pale or Absent
          • ATPase pH 4.6: Intermediate
        • Myosin Heavy Chain (MYH) types
          • MYH2 = Fast, Type 2A
          • MYH1 = Adult, Type 2X
          • No MYH4 (IIB)
        • Physiology: Fast, Fatiguable fibers
        • Ultrastructure
          • Z-lines: Narrow
          • Mitochondria: Few; Small
          • Glycogen granules: More
      • Type 2C
        • Staining patterns
          • ATPase pH 9.4: Dark to Intermediate
          • ATPase pH 4.3: Intermediate
          • ATPase pH 4.6: Intermediate
        • Myosin Heavy Chain (MYH) types
          • MYH2 = Fast, Type 2A
          • MYH7 = Slow, Type I
        • Histology
        • Clinical
          • Normal muscle: Few or None
          • 2C Muscle fibers increased (> 5%)
          • Fetal muscle
          • Regenerating muscle fibers
          • Immature muscle fibers
          • Coenzyme Q₁₀ deficiency
            • Children < 12 months of age
            • Muscle fiber morphology: Normal
    • Size: Small
      • Causes
        • Disuse
        • Aging
        • Weight loss
        • Systemic disease
        • Paraneoplastic
        • Childhood myasthenia gravis
      • Absent or small type 2A fibers: IBM3
      • 2B fibers: Early disuse
      • 2C fibers: Immaturity
    • Size: Large
      • Exercise training
    • Type 2 predominance
      • Shwachman–Diamond Syndrome
      • Chronic Partial Denervation
        • Incomplete Fiber Type Switching
      • Adult muscles: Uncertain significance
  • Abnormal expression of myosins in muscle fibers
    • TPM3: Congenital fiber type dysproportion (CFTD)
    • MYH7: Gowers distal myopathy
    • RYR1: Central core
      • MYH7 = Slow, Type I
      • MYH1 = Adult, Type 2X
      • ATPase pH 9.4 & 4.3 stains: All muscle fibers dark or intermediate
    • MYH3: Distal arthrogryposis 2A
      • MYH8: Increased
    • Shwachman–Diamond Syndrome
    • Early, Immature Regenerating muscle fibers
      • MYH4 = Fetal, Type 2B
      • Morphology: Often very small size
    • Chronic Partial Denervation
      • Incomplete Fiber Type Switching
    • Preparation artefact: Muscle not frozen for > 12 hours after biopsy
      • Type 2A muscle fibers: Absent
      • All muscle fibers are Type I or Type 2B
  • Absent myosin ATPase staining at all pHs
    • Myosin loss myopathies
• Mitochondrial (SDH & Cytochrome oxidase)
  • Pathology: Differential diagnosis
• Lipid storage (Sudan black or Oil red O)
  • Normal: More lipid in Type 1 muscle fibers
  • Malnutrition
  • Carnitine deficiency
    • OCTN2: Type 1 fibers
  • Fatty acid oxidation disorders
    • Multiple Acyl–CoA Dehydrogenase Deficiency: ETFA; ETFDH
    • FLAD1
    • SCAD deficiency
    • VLCAD: Type 1 fibers
  • Mitochondrial
    • Ragged red fibers
    • Cytochrome b (MTCYB) mutation (Myalgias)
    • Lipid myopathy
    • Coenzyme Q₁₀ deficiencies
  • Neutral lipid storage
    • Neutral lipid storage disease with myopathy (NLSDM) (PNPLA2): Type 1 & 2 fibers
    • Neutral lipid storage disease with ichthyosis (NLSDI) (CGI58): Type 1 & 2 fibers
    • Riboflavin-responsive MAD deficiency (ETFDH): Type 1 fibers
  • Other
    • Congenital MG with DOK7 mutations
  • Lipid pathology
  • Laboratory evaluation: Other
    • Blood acylcarnitine profile
    • Plasma Carnitine: Low in
      • OCTN2 (SLC22A5) mutations
      • MCAD (ACADM) deficiency
      • ETFDH deficiency
  • Lipofuscin
• Vacuoles & Aggregates
  • Lysosomal
    • Histology: Stain for acid phosphatase
    • Disorders
      • Acid maltase deficiency
      • Ataxia, Retinal degeneration & Cognitive decline: CTSD
      • Batten's disease
      • Lipofuscin
  • Autophagic
    • Histology: May stain for esterase & sarcolemmal proteins
    • Disorders
      • Vacuoles with Sarcolemmal & Autophagic features (AVSF)
        • Danon disease: LAMP-2
        • X-linked myopathy with excessive autophagy: VMA21
        • Infantile autophagic vacuolar myopathy
        • Childhood autophagic vacuolar myopathy
        • Adult-onset autophagic vacuolar myopathy with multiorgan involvement
        • LGMD 1G: HNRPDL
        • Myofibrillar myopathies: With other aggregates
          • LGMD 1A
          • Desmin mutations
        • Cardiomyopathy, Retinal degeneration & Epilepsy: CLN3
      • Autophagic vacuoles: Other
        • Toxic
          • Anti-microtubule drugs
          • 4-aminoquinolones
        • Hereditary
          • Hypokalemic myopathy
          • Acid maltase deficiency: GAA
          • Vici: EPG5
          • Congenital MG: GFPT1; DPAGT1
        • Inflammatory & Immune myopathies
          • Dermatomyositis with Vascular Pathology
          • IM-VAMP (Inclusion body Myositis)
      • Autophagy: Other disorders
  • Peroxisomes: Lafora disease
  • Vacuoles with: Basophilic debris ± Rims or Aggregates
    • Stain: Congo red
    • Stains for associated aggregates
      • SMI-31
      • TDP-43
      • LC3
      • Ubiquitin
      • VCP
      • β-amyloid
      • AMPDA
    • Disorders
      • Inclusion body myositis
      • Oculopharyngeal Muscular Dystrophy (OPMD)
      • Distal myopathies
        • Welander
        • Finnish-Markesbery
        • Distal dystrophy
        • Oculopharyngodistal
        • MPD2
        • Cystinosis
      • Hereditary inclusion body myopathies
        • HIBM: 1, 2, 3, 4
        • Myopathy + Paget's & Dementia (IBMPFD)
      • LGMD: 1A, 1D, 1G, 2G, 2H, 2J
      • Myofibrillar: Desmin; Filamin C
      • Emery-Dreifuss, X-linked
  • Subsarcolemmal vacuoles
    • Phosphorylase deficiency
  • Other vacuoles & aggregates
    • Periodic paralysis
    • Glycogen storage
    • Lipid storage
    • Centronuclear myopathy
    • Lipofuscin
    • Myofibrillar (Desmin storage)
    • Freezing artifact
• Glycogen storage (PAS stain)
  • Periodic acid-Schiff (PAS): Stains
    • Glycogen
    • Other molecules with carbohydrates + adjacent hydroxyl groups
      • Glycoproteins
      • Mucopolysaccharides
    • Type 2A & 2B muscle fibers: More glycogen & Darker stain
  • Glycogen reduced
    • GSD0: GYS1
    • GSD14: PGM1
    • GSD15: GYG1
  • Glycogen increased
    • Acid maltase (GSD 2)
    • McArdle (GSD 5): Some patients
  • Polyglucosan deposition
• Polyglucosan deposition ³
  • General
    • Polyglucosan bodies: Images
    • Polyglucosan Biochemistry
      • Glucose polymers
      • Amylopectin-like polysaccharide: Less branched than normal glycogen
      • May aggregate into polyglucosan bodies
    • Histochemistry
      • Stains
        • PAS positive
        • H&E: Basophilic
        • Toluidine blue: Metachromatic
        • VvG: Pale region within myelin sheath
      • Resistant to: Digestion by alpha-amylase (diastase)
    • Molecular
      • Polysaccharide
      • Contain abnormally long & poorly branched peripheral glucosyl chains
      • Increase in: Glycogen synthetase:Branching enzyme ratio
      • Similar to: Plant polysaccharide "amylopectin"
      • May contain: Ubiquitin; MuRF1 (TRIM63); Glycogenin; p62/SQSTM1
      • Resistant to α-amylase digestion.
    • Ultrastructure
      • Polyglucosan: Fibrillar
      • Bodies: Round with irregular branched filaments
      • Non-membrane bound
      • Cytoplasmic
      • May displace myofibrils: Lead to Z disk streaming
    • Locations
      • Common in myelinated axons & intramuscular nerves
      • Similar to: Corpora amylacea, Lafora bodies & Bielchowsky bodies
  • Disorders
    • PFKM deficiency (GSD 7; Tarui)
    • Brancher enzyme (GBE1) deficiency
      • Glycogenosis IV (GSD 4)
      • CNS + Polyneuropathy
    • Lafora disease
      • Laforin (EPM2A)
      • NHLRC1 (EPM2B)
      • PRDM8
    • PRKAG2 (AMP-activated protein kinase (AMPK)): Cardiomyopathy
    • RBCK1 (HOIL-1): Polyglucosan myopathy
      • Myopathy + Cardiomyopathy
      • Fatal immunodeficiency with Myopathy & Cardiomyopathy
    • Glycogen synthase 1 (GYS1)
      • Glycogen storage disease, Muscle, Type 0 (GSD0B)
      • Polyglucosan body myopathy in horses, Dominant (PSSM1)
    • Glycogenin-1 (GYG1; GSD 15): Polyglucosan body myopathy 2
    • Also occur in: Myelinated axons
      • Intramuscular: Non-diagnostic
      • Peripheral nerve: Suggest Polyglucosan body disorders
    • Double athetosis (Bielschowsky bodies)
    • Aging

Other Muscle Features

• Inflammation
  • General stains
    • H&E
    • Congo red
    • Acid phosphatase
    • Esterase
    • Immunocytochemistry
  • Immune myopathies: Immune cell patterns
    • Lymphocyte predominant: T-cells > Macrophages
      • Stains: T-cells (CD4, CD8); B-cells (CD20)
      • CD4 + CD8 cells: Often with focal invasion of muscle fibers
      • Associated disorders
        • IM-VAMP
          • Inclusion body myositis (IM-VAMP): CD4 + CD8 cells
          • Polymyositis with Mitochondrial Pathology (PM-Mito): CD4 + CD8 cells
        • BCIM: B-cell foci; Ectopic lymphoid structures
        • Vasculitis: Lymphocytes > Macrophages
        • Lymphorrhages (Myasthenia gravis): T-cells ± B-cells
        • Checkpoint inhibitors: B-cells & T-cells
        • FSH dystrophy: T-cells

      Macrophages (Histiocytes): Predominant or Prominent Stains & Visualization Histochemical Acid phosphatase Activated histiocytes Stain location: Most around histiocyte nuclei Esterase Activated histiocytes Stain location: Histiocyte cytoplasm Antibodies CD68: Most activated macrophages CD163: M2 macrophages HAM56: Resident + Activated macrophages Ultrastructure Histiocyte pathology Activated Histiocytes Stains: CD68, CD163, Acid phosphatase Distribution patterns Endomysium Pattern: Scattered histiocytic cells Associations: Muscle fiber pathology Necrosis: Some disorders MHC1 upregulation Inclusion Body Myositis Perimysium Pattern: Scattered Association: Connective tissue damage Endoneurium Distribution: Scattered Associations Wallerian degeneration Demyelination Epineurium Distribution: Focal or Scattered Associations: Vasculitis Histiocyte foci Histiocytes comprise most cells in foci Pattern: Cell clusters Granulomas Macrophage accumulations Distribution: Common locations Muscle: Endomysium, Perimysium Nerve: Epineurium Lymphocyte foci Distribution Histiocytes are scattered in foci Comprise 10% - 40% of cells Resident (Resting) Histiocytes Stains: HAM56+, Acid phosphatase- Cells near, or in, endomysial capillaries ? Relation to vascular pericytes M1 vs M2 Granulomatous disorders Sarcoid: Histiocytes in center of granulomatous cell foci Endomysium Active myopathies Especially with MHC1 upregulation by muscle fibers Location: Often near endomysial capillaries Connective tissue: Histiocytic cells scattered in perimysium Fasciitis Immune myopathies with perimysial pathology (IMPP) Jo-1 myositis Muscle fiber pathology: Necrosis or Focal invasion Muscle fiber necrosis Macrophage Pattern Invade & phagocytose necrotic muscle fibers Then migrate away Disorders Regional (clustered) fiber necrosis Myopathic grouping Myopathies with sarcolemma Δ RIIM Ischemia Borderzone vascular distribution Scattered fiber necrosis LHIM (Macrophage activation) Rhabdomyolysis & Necrosis Perifascicular-predominant fiber necrosis IMPP Focal invasion of non-necrotic muscle fibers Macrophage pattern Collections of cells including histiocytic & CD8 cells Focal invasion & replacement of alive muscle fibers Disorders Histiocytes migrate away after invading muscle fibers IM-VAMP BCIM Histiocytes remain after invading muscle fibers Granulomatous disorders Also see: Nerve, Wallerian degeneration

      Macrophage Types 5 Properties M1 M2 Activation Path Classical Alternative Actions Pro-inflammatory Microbicidal Tumorcidal Tissue damage Anti-inflammatory Angiogenesis Tumor progression Tissue repair Extracellular matrix ↑ Polarization LPS, IFNγ, TNFα. IL-4, IL-10, IL-13, IL-21 Secretion TNFα, IL-1α, IL-1β, IL-6, IL-12, IL-23, CXCL9, CXCL10, CXCL11, CXCL16, CCL5 IL-10, TGFβ, CCL1, CCL17, CCL18, CCL22, CCL24, CXCL13 Effectors, other ROS, iNO VEGF, Arg-1 Antigen presenting Yes No Cell chemotaxis Th1, Th17 Th2, Treg Surface markers HLA-DR, CD11c, CD80, CD86, pSTAT1, TLR2, TLR4, iNOS CD163, CD204, CD206, CD209, cMAF, FIZZ1, Ym1/2 Other features Proinflammatory markers   Nos2, Arg1, Ccl2,   Ccl7, Il1α, Alox15 Subtypes: Different inducers   M2a: IL-4, IL-13   M2b: TLR agonists, IL-1R   M2c: IL-10, Glucocorticoid   M2d: TLR antagonists Stimulants NF-κB PPARγ Endoneurial Histiocyte

    • B-cells
      • Stains: Foci contain
        • B-cells: CD20; CD19
        • T-cells: CD4; CD3
        • Vessels (Venules): MECA-79; ICAM-1; VCAM-1; MAdCAM-1; PNAd
      • Ectopic lymphoid structures
        • Dermatomyositis + Vascular pathology (DM-VP)
        • Brachio-cervical inflammatory myopathy (BCIM)
        • Orbital myositis
      • Immune checkpoint inhibitors
      • Nodular myositis
    • Eosinophils: Present in blood or muscle
      • Stains: H&E; Congo red
      • Eosinophilic fasciitis: Eosinophils in perimysial connective tissue
      • Eosinophilia-myalgia
      • Trichinosis
      • LGMD 2A: Eosinophils in muscle fiber necrosis-associated cells
      • Vasculitis: Churg-Strauss
      • Orbital myositis
      • Masticator myopathy
      • Hypereosinophilic syndrome
      • Inflammatory myopathy, Phenytoin-associated
  • Myofiber necrosis: Stains
    • H&E: Pale cytoplasm; Absent myofiber nuclei
    • NADH: Cytoplasm staining: Reduced
    • C_5b-9: Cytoplasm staining present
    • Acid phosphatase: Macrophages in & around muscle fibers
• 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
• Amyloid
  • Pathology (Congo red)
  • Differential diagnosis
• Complement (C_5b-9) deposition: Patterns
  • Muscle capillaries
    • Perifascicular: Dermatomyositis
    • Endomysial & Endoneurial: Immune neuropathy
    • Endomysial, scattered: Anti-SRP myopathy
    • Pipestem capillaries: Myopathy
  • Within muscle fibers: Muscle fiber necrosis
  • Muscle fiber surface: Varying causes
  • Endomysial connective tissue: Brachio-cervical & other inflammatory myopathies
  • Nerve: Microvessels
• Capillary pathology
  • Myopathies
    • Dermatomyositis
    • Anti-SRP myopathy
    • Myopathy with pipestem capillaries
    • Scleromyopathies
    • GvHD
    • IM-VAMP
  • Morphology
    • Misoriented: Muscle denervation
    • Thick walls
    • Diabetes: Thick walls; Reduced numbers; C_5b-9 deposition
    • Inflammation: Ipilimumab
    • Humoral Immune Endoneurial Microvasculopathy
  • TS-HDS antibodies

Molecular Pathology: Immunohistochemistry for Inherited Myopathies ¹

• 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
  • Antibodies: 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
  • Pathology in muscle: Reduced staining around muscle fibers with antibody to α-DG glycosylation moiety
  • Reduced in some Congenital muscular dystrophies (CMD) & LGMD
  • Disorders
• Sarcoglycans ²
  • Absent: Selective absence of 1 sarcoglycan may have specificity for mutation in that gene
  • Reduced
    • Mutations in one sarcoglycan gene often reduce levels of other sarcoglycans
    • Reductions in staining for all 4 sarcoglycans with preservation of other dystrophy-related proteins
      • Suggests mutations of a sarcoglycan gene
      • Only rarely enables accurate prediction of specific sarcoglycan genotype
    • γ-sarcoglycan mutation: β- & δ-sarcoglycans may be normal
    • γ-Sarcoglycan staining: Most often & most severely reduced in all sarcoglycanopathies
• 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 aggregates & accumulations: Marker for myofibrillar myopathies
    • Other associated proteins in accumulations: Dystrophin, CDC2 kinase, β-APP, Prion protein, NCAM & Myotilin
    • Cytoplasm staining: Immature, small muscle fibers
• Nuclear proteins
  • Emerin: Absent staining in muscle & skin in EDMD 1
  • Lamin A/C
    • IHC staining of Lamin A/C: Usually normal in nuclei
    • Western blot: May demonstrate reduced amount of Lamin A/C in EDMD 2
    • Staining for other nuclear envelope proteins: May show mislocalization