Muscle Biopsy

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MUSCLE BIOPSY

Methods
Selection of muscle to biopsy
Biopsy preparation & transport
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
- 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

Disease progression
- Chronic: Muscle with moderate, but not severe, weakness
- Acute: Muscle with severe or moderate weakness
Muscles
- Best specific muscles
  - Well established normal features
  - Deltoid; Biceps; Quadriceps
- Other muscles: More common features
  - Gastrocnemius: NMJs; Targets with denervation
  - Trunk: Type I predominance; Mitochondrial pathology
  - Extraocular: Small muscle fibers; Dark mitochondrial stains
  - Cardiac: Alkaline phosphatase+ capillaries; Acid phosphatase granules; No fiber type differentiation on ATPase
MRI analysis
- Can be used to select pathological muscle site in difficult cases
- May provide false positive muscle signals: Denervation; Fiber hypertrophy; Endomysial capillaries increased
Avoid
- Muscles that were site of: EMG, Injections, Trauma
- Myopathy: Muscles with normal strength

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

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 & Inclusions
- Internal nuclei
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 fiber atrophy
- Small angular fibers
- Distribution: Scattered individually
Spinal muscular atrophy
- Small rounded fibers
- Distribution: Large grouped atrophy

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 necrosis & regeneration
Neuropathy: Muscle fiber atrophy, Scattered angular or Diffuse

Chronic

Myopathy
- Endomysial connective tissue: Increased
- Muscle fiber hypertrophy or atrophy
Neuropathy
- Fiber type grouping
- Grouped atrophy
- 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
- Scattered muscle fibers: Acute myopathy; Acute neuropathy
Are there diagnostic features?
- Is there inflammation or excess cellularity?
  - What kind?
    - Lymphocytes
    - Macrophages
    - Eosinophils
    - Other: Neoplasm
  - Where?
    - Endomysial
    - Perimysial
    - Perivascular
    - Focal invasion of muscle fibers
- Metabolic: Is there storage or mitochondrial pathology?
  - Lipid
  - Glycogen
  - Amyloid
  - Mitochondrial
- Is there pathology in structures other than muscle fibers?
  - Vessels
  - Connective tissue: Endomysial; Perimysial
  - Intramuscular nerves

NORMAL MUSCLE Mean fiber diameter
Age (Years)	Diameter (μM)
Birth	12 to 15
1	18
3	20
6	27
8	35
10	40
12	45
15	50
Adult	35 to 85

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 Pathology patterns
	Cytochrome oxidase (COX)	Mitochondrial pathology Mitochondrial & Nuclear DNA encoded Pathology patterns
Glycolytic Enzymes	Phosphorylase	Phosphorylase deficiency
Glycolytic Enzymes	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

Mitochondrial Oxidative Enzyme Activities: Methods

MUSCLE BIOPSY RESULTS: Differential diagnosis

Muscle fiber
Aggregates
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
  - 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 + 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
- 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
- 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
  - Normal: 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
    - Fiber properties
      - Physiology: Slow
      - Oxidative capacity: High
    - 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
    - Abnormal fiber types
  - 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
      - Disorders: 2A fibers lost
        
        Artefact: 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)
      - Fiber properrties
        
        Physiology: Fast, Fatiguable fibers
        
        Highest force
        
        Inefficient: High myosin ATPase activity
        
        Oxidative capacity: Low
        Dependence on anaerobic metabolism
      - Ultrastructure
        
        Z-lines: Narrow
        Mitochondria: Few; Small
        Glycogen granules: More
      - Disorders
        
        Reduced # (2X/2A) with ¹⁸: ↑ Age; Female; ↓ Handgrip ; ↓ BMI *
        
        MYH1
    - 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 & Mitochondrial disorders
        
        Children < 12 months of age
        
        Muscle fiber morphology: Normal or Small size
  - 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
- Stains: Sudan black or Oil red O
  - Sudan black: Stains fat droplets & Phospholipids
  - Sudan method
- Normal
  - More lipid in Type 1 than Type 2 muscle fibers
- Lipid increased: Systemic disorders
  - Malnutrition
  - Cachexia
- 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
- Neutral lipid storage
  - Neutral lipid storage disease + Myopathy (NLSDM) (PNPLA2): Type 1 & 2 fibers
  - Neutral lipid storage disease + Ichthyosis (NLSDI) (CGI58): Type 1 & 2 fibers
  - Riboflavin-responsive MAD deficiency (ETFDH): Type 1 fibers
- Other
  - Congenital MG with DOK7 mutations
  - Sertraline
- Lipid pathology
- Laboratory evaluation: Other
  - Blood acylcarnitine profile
  - Plasma Carnitine: Low in
    - OCTN2 (SLC22A5) mutations
    - MCAD (ACADM) deficiency
    - ETFDH deficiency
- Lipofuscin
Vacuoles & Aggregates
- Lysosomal
  - Pathology
    - Histology: Stain for acid phosphatase
  - Disorders
    - Acid maltase deficiency
    - Ataxia, Retinal degeneration & Cognitive decline: CTSD
    - Batten's disease
    - Lipofuscin
- Autophagic
  - Pathology
    - Histology: May stain for esterase & sarcolemmal proteins
    - Ultrastructure: Vacuoles; AVSF
  - 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 + Vascular Pathology
        
        IM-VAMP (Inclusion body Myositis)
    - Autophagy: Other disorders
- Peroxisomes: Lafora disease
- Vacuoles with: Basophilic debris, Rims or Aggregates
  - Stain: Gomori trichrome; 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
      - TIA1: Welander
      - ZASP: Finnish-Markesbery
      - PLIN4: Distal dystrophy
      - Oculopharyngodistal
      - MATR3: MPD2
      - CTNS: Cystinosis
    - Hereditary inclusion body myopathies
      - HIBM: 1, 2, 3, 4
      - VCP: Myopathy + Paget's & Dementia (IBMPFD)
      - SQSTM1: DMRV
      - HNRNPA1: IBMPFD3
      - HNRNPDL: LGMD D3
    - LGMD: 1A, 1D, 1G, 2G, 2H, 2J
    - Myofibrillar: Desmin; Filamin C
    - Emery-Dreifuss, X-linked
    - Rimmed vacuoles: Young-onset (< 20 years)
      - SIL1: MSS
      - DNAJB6: LGMD D1
      - FHL1: SPM
      - HNRNPA2B1: IBMPFD2
      - Other occasional: GNE; OPDM
- 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
  - Danon disease
  - α-1 antitrypsin deficiency: Misfolded, polymerized proteins in hepatocytes
  - Also occur in: Myelinated axons
    - Intramuscular: Non-diagnostic
    - Peripheral nerve: Suggest Polyglucosan body disorders
  - Double athetosis (Bielschowsky bodies)
  - Aging: Corpora amylacea

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 in cells: Most around histiocyte nuclei
  - Esterase
    - Activated histiocytes
    - Stain location: Histiocyte cytoplasm
- Antibodies
  - CD68
    - Lysosomal marker
    - Most activated macrophages
  - CD163
    - M2 macrophages: Activated
    - Macrophage lineage marker
    - No association with lysosomae
  - HAM56
    - Resident + Activated macrophages
    - Present in normal muscle & Nerve
- Ultrastructure
Histiocyte pathology
- Activated Histiocytes
  - Stains: CD68, CD163, Acid phosphatase
  - Distribution patterns
    - Endomysium
      - Pattern: Scattered histiocytes; Near capillaries
      - Associations:
        
        Muscle fiber pathology
        
        Necrosis: Some disorders
        MHC1 upregulation
        
        Inclusion Body Myositis
    - Perimysium
      - Pattern: Scattered
      - Associations
        
        Connective tissue damage
        
        IMPP
    - 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 Endoneurial Histiocyte with Lipid debris

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 + Vascular Pathology
  - 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
- Morphologic features: Patterns of Capillary Pathology
  - Misoriented: Muscle denervation
  - Thick walls
  - Diabetes: Thick walls; Reduced numbers; C_5b-9 deposition
  - Inflammation in nerve: Ipilimumab
  - Humoral Immune Endoneurial Microvasculopathy: C_5b-9 deposition in nerve ± muscle
- Disorders with Capillary Pathology
  - Myopathies
  - Neuropathies
    - TS-HDS antibodies
    - POEMS
    - Humoral Immune Endoneurial Microvasculopathy: C_5b-9 deposition in nerve ± muscle
    - Diabetes

Molecular Pathology: Immunohistochemistry for Inherited Myopathies ¹

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
- 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

See: Pathology index

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References
1. J Neuropath Exp Neurol 2005;64:181-193
2. Neuromuscul Disord 2008;18:934-941
3. Molecular Aspects of Medicine 2015; Online Aug
4. J Surg Res 2019;243:27-32
5. J Neuroinflammation 2022;19:97, MedComm 2025;6:e70304

9/8/2025