This document provides information about muscle biopsy procedures and findings. It describes the normal anatomy and histology of skeletal muscle. Common indications for muscle biopsy include weakness, elevated enzymes, and suspected hereditary or acquired myopathies. Proper biopsy technique and sample processing are outlined. A variety of stains are used to examine muscle fiber morphology, inclusions, inflammation, vacuoles, and other abnormalities. Common myopathic patterns are presented along with the diseases they may indicate.

1. Muscle
biopsy
Dr Neha Mahajan

2. Normal Muscle

3. Organization of Skeletal Muscle Including
Connective Tissue (CT) Compartments
EPIMYSIUM
•Loose CT
•Blood vessels
PERIMYSIUM
•Septa
•Nerve branches
•Muscle spindles
•Fat
•Blood vessels
ENDOMYSIUM
•Muscle fibers
•Capillaries
•Small nerve fibers

4. Normal H&E-stained frozen cross-section of skeletal muscle
Perimysial
connective tissue
Endomysial
connective tissue
Note uniform sizes, polygonal shapes, and eccentric nuclei.

5. Normal muscle Adult Normal Muscle child

7. Ultrastructure of a Sarcomere
Myosin
I band I band
H band
Actin
Z M Z
 Extends from Z-band to Z-band.
A band
 A band includes overlap of actin & myosin.
 Note arrangement of thick and thin filaments.

8. Indications of Muscle biopsy
General reasons:
Weakness of uncertain cause-generalised, proximal
,floppy infant syndrome
Muscle pain ,cramps, stiffness
Persistently elevated muscle enzymes(CK)
Specific reasons:
Hereditary muscle disease in other family members
Carrier detection
Systemic connective tissue disease & vasculitis
Certain metabolic diseases such as storage disease
Suspicion of steroid myopathy in treated myositis
Exclude drug induced myopathy
Conflicting clinical ,EMG or lab findings
Confirm/reinforce clinical diagnosis

9. Contraindications
1.Electrolyte disturbance
2.Most endocrine
3.Malignant hyperthermia
4.Periodic paralysis
5.Poor nutrition
6.Prior Trauma

10. Site of biopsy
• Muscle with moderate disease, NOT severe
• Muscle belly, not from tendon
• Proximal myopathies/generalised systemic
disease- Vastus Lateralis
• Other sites-Biceps,gatronemius
• Avoid Deltoid,muscle that are site of
EMG,injections/trauma
• Imaging used to select pathological muscle
site in difficult cases.

11. Bergston Needle
Technique
1.Needle Biopsy
2.Open Biopsy- indicated for
disorders with patchy pathology

12. Processing
Transportation:
Muscle may be saved in saline moistened guage for several hrs
Keep the specimen cool
Do NOT immerse in saline ,fixative or other liquid
Fresh Fixed
Glutaraldehyde
RESIN section/EM
( 1mm x 0.5 cm)
Formalin
PARAFFIN
(0.5x0.5cm)
Snap freeze
HISTOCHEMISTRY
(0.5x0.5cm)

13. Sample size: 0.5 cm diameter
& 1 1cm length
Biopsy is processed:
1.Paraffin embedding
2.Histochemistry
3.For electron microscopy
4.For molecular biology

14. Stains
1. H&E- gen architecture & morphology
2.Masson`s trichome-Collagen,fibrosis
3.Mod Gomori`s Trichome- red ragged fibres,nemaline
bodies,nuclei,myelinated fibres
4.PAS-glycogen
5.Oil red O-neutral lipid
6.Acid phosphatase-lysosomal enzymes,necrotic fibres
7.Crystal voilet, Congo Red-amyloid
8.ATPase PH 9.4 Type 1 fibres pale
Type 2 fibres dark
9. ATPase PH 4.6 Type 1 fibres dark
Type 2 fibres pale
10. NADH- Sarcoplasmic structural details
11.SDH- oxidative enzyme activity
12.Cytochrome C Oxidase- mitochondrial enzyme activity
13.NSE-lysosomal &macrophage activity

15. Normal ATPase pH 9.4
 Type I fibers are light
 Type II fibers are dark

16. Rapid gomori`s trichome
stain
Red ragged fibers,nemaline
bodies,myelinated fibers
Nuclei :red purple
Normal muscle myofibrils:Blue
Green
Intermyofibril muscle
membrane: Red
Interstitial collagen: Green

17. NADH-TR stain
Sarcoplasmic structural
details

18. SDH stain
For mitochondrial activity

19. Cytochrome c oxidase
•Mitochondrial activity
•Type 1 fibers are darker
than Type2

20. OIL RED O
Accumulation of lipid

21. Esterase stain
Lysosomal & phagocytic activity
 Macrophages are ingesting the remnants of a degenerating fiber. This is a non-specific
myopathic finding.
Neuromuscular junction

22. Observations in routine paraffin sections
H & E
Used to evaluate gen architecture of muscle and variation in
morphology of individual fibres
•Variation in fascicular architecture
•Variation in fiber size
•Necrosis and degeneration of muscle fibres
•Nuclear characteristics
•Type & distribution of inflammatory infiltrate
•Interstitial changes

23. Architecture of muscle fascicles -scanner view
PATCHY Inflammatory myopathies
FOCAL Neurogenic DIFFUSE Dystrophy

24. Nuclear changes
Normal Internalisation of nuclei

25. Internalisation of nuclei
• Myotendinous insertion
• Centronuclear myopathy
• Myotonic dystrophy
• Fiber regeneration
• Fiber atrophy
• Chronic neuropathies

26. Ring fibres
•Limb girdle dystrophy
•Myotonic dystrophy

27. Hyaline Fibres
Duchene muscular
dystrophy

28. Whorled fibres
•Limb girdle muscular dystrophy
•Chronic neuropathies

29. Fibre necrosis seen in biopsy specimen
Pathologic features Disease
Small groups of necrotic
DMD
fibres
Perifascicular necrosis Dermatomyositis
Random fibre necrosis PM,IBM
Infarcts with large areas of
PAN
necrosis
Extensive,diffuse Rhabdomyolysis

30. Fiber necrosis Perifascicular necrosis

31. INCLUSIONS
Nuclear inclusions:
Oculopharyngeal dystrophy
Sarcoplasmic Inclusions:
Myofibrillar myopathy
Inclusion body myositis

32. Inflammation
Pathologic feature Disease
Perivascular, angiocentric DM, connective tissue
disease ,FSHD
Endomysial, around
fibres
PM, IBM,viral
Nodular Rheumatoid arthritis,
granuloma
Polymorphs with
eosinophils
PAN, drug reactions,
trichinosis, eosinophilic
fascitis

33. Inflammatory myopathy

34. Atrophy
Type 1 fiber atophy Type 2 fiber atophy
•Myotonic dystrophy
•Nemaline myopathy
•Centronuclear myopathy
•Congenital fibre type
disproportion
Corticosteroid therapy
Myasthenia Gravis
Disuse Atrophy
Acute denervation
Paraneoplastic myopathy
Pattern of atrophy:
Grouped atrophy Chronic neurogenic disorders
Panfascicular ISMA(Infantile spinal muscular atrophy)
Perifascicular DM(Dermatomyositis)

35. Normal Atrophy

36. Hypertrophy
Type 1 fiber
hypertrophy
Type 2 fiber
hypertrophy
Type 1&2 fiber
hypertrophy
ISMA
Normal
Atheletes
Sprinters
Congenital type
disproportion
Limb girdle
dystrophy,IBM,myotoni
a congenita,acromegaly
Normal Hypertrophy

37. Fiber shape
Normal muscle-polygonal
Angular-neurogenic Rounded-myopathic

38. Fibre splitting
Limb girdle dystrophy
IBM

39. Cores and targets
Oxidative enzymes are ideal to assess depleted enzyme
activity
Cores : Neurogenic atrophy, central core disease
Target fibres: Chronic neuropathies
CORE TARGET

40. Central cores Target fibres

41. Mottled fibres
FSHD

42. Nemaline rods
Detected by RTC(Rapid gomori trichome)
Seen in Nemaline myopathy

43. Mitochondrial Abnormalities
Ragged red fibres(RTC stain)

44. Sarcoplasmic vacuoles seen in biopsy specimen
Pathologic feature Disease
In centre arranged in size
gradient
Freezing artifact
In scattered fibres ,small
round osmiophilic ,oil
red O positive
Lipid storage disorders,
Mitochondrial
myopathies
Often subsarcolemmal
PAS +
Glycogen storage
Rimmed, ubiquitin + IBM ,Distal myopathy,
OPMD

45. Freezing artifact Rimmed Vacuole

46. Vacuoles in glycogen storage disease

47. Parasite ( Trichenella Spiralis)

48. Granulomatous Myositis
in a Patient with Sarciodosis
 Granulomas tend not to cause significant
damage to adjacent myofibers.
Giant cell

49. Pyomyositis Gram Positive Cocci

51. History & examination
History:
• Four major pieces of clinical information are critical for the pathologist:
• Is the condition a long standing or newly onset condition?
• Is the condition progressive or relatively static?
• Are there any associated systemic conditions such as heart problem or
autoimmune disease?
• What is the serum CPK level?
• Other helpful clinical information:
• History of myoglobinemia.
• Family history of neuromuscular disease.
• Sex and age.
• Results of EMG studies.
• Muscle groups being affected.
• Presence of contracture.
• Muscle groups being involved.
• Site of biopsy.
• Therapy and medication at the time of biopsy.

52. LAB Investigations:
Creatine kinase level: To rule out certain categories of myopathies because
different myopathies tend to generate a different levels of elevation in CPK.
•High: (e.g. Dystrophinopathies) 200-300 times of normal.
•Intermediate: (e.g. Inflammatory myopathy) 20-30 times of normal.
•Low: (e.g. Neurogenic disorder) 2-5 times of normal.

55. Myopathies
INHERITED
Muscular dystrophies
DMD,BMD
FSHD
LGD
Distal myopathy
OPMD
Myotonic muscular dystrophy
Congenital myopathy
Central core disease
Multicore disease
Centronuclear
Cong fibre type disproportion
Myofibtillar myopathy
Metabolic diseases
Glycogen storage diseases
Mitochondrial diseases
Myoadenylate deficiency
Channelopathies
ACQUIRED
Inflammatory-PM,DM,IBM,granulomatous
Infections
Trichinosis, cyticercosis,toxoplasma,HIV,Coxackie A&B
Toxic
Alcohol, Vit E, OPP, snake venoms
Drugs
Steroids,statins, b blocker, zidovudin,
amiodarone,Chloroquine, chlofibrate,
vincristine,cyclosporine,opiates
Endocrine & metabolic
Hypo/Hyperthyroidism
Acromegaly
Cushing`s syndrome
Conn`s disease
Osteomalacia
Hypercalcemia
Hypokalemia
NEOPLASTIC
Benign-Rhabdomyoma Malignant-RMS

56. Muscular dystrophies
•Usually not congenital
•Onset in childhood ,young adults
•Hereditary diseases ,often with a family history
•Weakness frequently severe ,variable distribution
•Proximal in DMD,BMD,LGD
•Facial in fascioscapulohumeral dystrophy
•Distal in distal myopathy MD
•Progressive course
•Fiber destruction pathological, damage is random ;not all
fibers are affected

57. Duchenne muscular dystrophy
• Inheritance- X- linked recessive disorder
• Defective gene- Dystrophin
• Onset- usually b/n 3-5yrs age
• C/F – progressive weakness of the girdle muscles
- difficulty running , jumping, hopping, unable to get up from the
floor (Gower’s maneuver)
-enlargement of muscles of lower leg a/w weakness-
Pseudohypertrophy
- contractures( hip, knee, elbow, wrist) with chest deformities
→severe pulmonary infections → death at age 16-18yrs

58. Others : cardiomyopathy , mental retardation
Lab. – Serum CK : elevated 20-100x normal
- EMG : myopathic features
- Muscle biopsy- Fiber necrosis & regeneration
Variation in fiber size,internalisation of nuclei
proliferation of endomysial connective tissue.
Deficiency of dystrophin seen on western blot analysis &
immunohistochemical staining.
- DNA analysis : mutation of gene that encodes dystrophin

60. Becker muscular dystrophy
• Inheritance – X- linked recessive disorder
• Defective gene – dystrophin
• Onset- experience difficulty b/n 5- 15yrs of age
• C/F – proximal muscles especially of lower extremities are prominently
involved.
- hypertrophy of muscles , particularly the calves, is an early & prominent
finding.
- cardiomyopathy may occur , MR is less common
• Lab. – CK : elevated
- EMG : myopathic
- muscle biopsy : similar to DMD
: reduced amount or abnormality of dystrophin( Dx)
- DNA analysis : deletions or duplications( Dx)
• Treatment – supportive
• Survival : survive in to the 4th to 5th decade

61. Duchene muscular dystrophy
Western Blot

62. DMD
End stages-extensive
replacement
by adipose tissue and
fibrosis

63. NORMAL DMD

64. Myotonic dystrophy
• Inheritance : AD
• Defective gene: two types with distinct molecular genetic defects
-DM1 : expansion CTG repeat
- DM2 ( proximal myotonic myopathy – PROMM ): CCTG repeat
• C/F – myotonia : usually appears by age 5 yrs
- Hatchet- faced appearance: temporalis , masseter , facial muscle
atrophy & weakness
- frontal baldness in men
- foot drop : ankle dorsiflexor weakness
- weakness of wrist extensors , finger extensors, & intrinsic hand muscles
- early involvement of neck muscle flexors, sternocleidomastoids
- dysarthritic speech, nasal voice, swallowing problems due to palatal ,
pharyngeal, and tongue involvement
- respiratory insufficiency : diaphragm & intercostal muscle involvement
- cardiac disturbances : conduction block with sudden death
: CHF from cor pulmonale 2ry to respiratory failure

65. Cont,d
• - other system manifestations : intellectual impairment, hypersomnia,
cataract, gonadal atrophy, insulin resistance, reduced esophageal & colonic
motility
• Lab. – Dx ; usually based on clinical findings
- CK : N or mildly elevated
- EMG : evidence of myotonia
Biopsy : variation in fiber size,
increase in internal nuclei(chains)
ring fibers, atrophy which
selectively involves type – 1 fibers in 50%
Myotonic dystrophy

66. Oculopharyngeal dystrophy
• Inheritance: AD with complete penetrance
• Defective gene: expansion, poly-A-RNA binding protein
• Onset – usually late onset ( 4th – 5th decade )
• C/F – progressive external ophthalmoplegia ( slowly progressive ptosis,
limitation of eye movements with sparing of pupillary rxns.
- dysphagia : can be life threatening
: may result in repeated episodes of aspiration
- mild weakness of the neck and extremities
• Lab. – EMG: myopathic features
- CK : 2-3x N
- BIOPSY : distinct features – presence of tubular filaments in muscle
cell nuclei, mild dystrophic changes with
nuclear internalisation ,fiber atrophy,
interstital fibrosis, rimmed vacuoles in type 1 fibres

67. Fascioscapulohumeral /FSH/ muscular dystrophy
• Inheritance: AD
• Onset : childhood or young adulthood
• Defective gene: deletion, distal 4q
• C/F- facial weakness: initial manifestation
- weakness of shoulder girdle muscles : weak arm elevation
: scapular winging
- weak wrist extension > wrist flexion
- foot drop : weakness of anterior compartment muscles of the legs
- weakness of the pelvic girdle muscles : 20%
- other organ ( rarely) : labile HTN, nerve deafness
• Lab. – CK : N or elevated
- EMG: myopathic pattern
- BIOPSY: Atrophic muscle fibres in clusters/groups in absence of necrosis,
Moth eaten fibres and perivascular inflammatory infiltrate

68. Distal myopathies
• Notable for their preferential distal distribution of muscle weakness in contrast to
most muscle conditions associated with proximal weakness
• Four types : mode of inheritance, age of onset, pattern of weakness
1. Welander DM : AD
2. Tibial MD : AD
- late onset, usually after age 40; start in the hands
3. Nonanka DM: AR
4. Miyoshi myopathy : AR
- early onset in late teens or twenties; start in the lower limbs
• Lab. – CK : only slightly elevated except in Miyoshi myopathy-
EMG : myopathic
Biopsy : non- specific dystrophic changes
selective type1 fiber atrophy in welander DM

69. Inflammatory and immune
mediate myopathies
1.Dermatomyositis
2.Polymyositis
3.Inclusion body myositis

70. Dermatomyositis
• Inflammatory
myopathy
– Prevalence: 1:100,000 in
general population
– Female to male prevalence
of 2:1
– peak incidence ages 40-50
– Immune complex deposition
in the vessels considered to
be part of a complement-mediated
vasculopathy
Hematoxylin and eosin stain
(20x) of a muscle biopsy from a
patient with dermatomyositis
showing perivascular and
perimysial inflammation, as well
as perifascicular necrosis.

71. Diagnostic Criteria
• Bohan and Peter Criteria:
– Symmetric proximal muscle weakness
• most common symptom
– typical rash
– elevated serum muscle enzymes
– myopathic changes on EMG
– characteristic muscle biopsy abnormalities and
absence of histopathologic signs of other
myopathies

72. Grotton’ s Sign:
An erythematous, scaly eruption over the
extensor surfaces of the
metacarpophalangeal joints and digits
Heliotrope rash:
A reddish-purple eruption on the
upper eyelid
accompanied by swelling of the eyelid
Most specific rash in DM

73. Dermatomyositis
 Perifascicular atrophy
 Degeneration
 Inflammatory cells in the perimysium
surrounding a blood vessel
 Inflammatory cells tend to be B-cells.

74. Polymyositis
Symmetrical proximal muscle involvement similar to DM
Lack of cutaneous involvement,endomyseal inflammatory
involvement
Inclusion body myositis
Involvement of distal muscles,esp extensors of knee and
flexors of wrist,ASYMMETRICAL

75. PM DM IBM
Age at onset >18yrs Adulthood, childhood >50yrs
sex M=F F>M M>F
Weakness proximal proximal Proximal, early distal
involvement
Familial association No No Yes, in some cases
/familial inflammatory
myopathies /
Response to treatment good better poor
CTDs yes yes Yes, in up to 20%
malignacy No yes, in up to 15% of
cases
No
Rash Absent Present Absent
Biopsy “primary” inflammation
with the CD8/MHC-I
complex & vacuoles
Perifascicular,
perymysial, or
privascular infiltrates,
perifascicular atrophy
Primary inflammation with
CD8/MHC-I complex;
vacuolated fibers with
b-amyloid deposits ,
cytochrome oxygenase-negative
fibers ; signs of
chronic myopathy

76. POLYMYOSITIS, usually endo-myseal

77. INCLUSION BODY MYOSITIS, “rimmed” vacuoles

78. Congenital myopathy
• Rare disorders distinguished from muscular dystrophies by the presence of specific
histochemical & structural abnormalities in muscle fibers.
• Onset : infancy or childhood
• C/F - progressive muscle weakness ( proximal> distal, legs> arms) &
limpness, hypotonia & delayed milestones
- skeletal deformities (kyphoscoliosis, club foot, hip dislocation)
• Lab. - CK: usually N or slightly elevated
- EMG : myopathic/ mostly/; positive sharp waves, myotonic discharges
- Biopsy : features specific to each type
Congenital myopathies
1.Central core disease
2.Multicore disease
3.Nemaline(Rod) myopathy
4.Centronuclear myopathy
5.Congenital fibre type disproportion
6.Myofibrillar myopathy

79. Central core disease
•AD Ryanodine receptor(RYR1)
Gene 19q13.1
•Early onset hypotonia, floppy infant,
associated skeletal abnormalities,
malignant hyperthermia
•Biopsy- cytoplasmic cores in type 1
Fibres(NADH-TR stain)

80. Multicore disease
•Cong non progressive myopathy(gen
weakness,hypotonia)
•Biopsy –type 1 fiber predominance&
minute core like structures in
majority of fibres

81. Nemaline (Rod)myopathy
•AR/AD
•Childhood weakness
•Ebbing of strength is more in facial &
proximal limb muscle
•Facial dysmorphism
•Aggregates of subsarcolemmal spindle
shaped particles(nemaline rods) occuring
predominantly
In type 1 fiber best seen RTC stain

82. Myotubular/Centronuclear
myopathy
•AD/AR/XL
•MTM 1 gene(Xq27-28) XL
•Clinical findings-severe congenital hypotonia
Floppy infants, poor prognosis, extraocular
palsies & facial asthenia occur
simultaneously with involvement of
appendicular muscle
•BIOPSY- Abundance of centrally located
nuclei involving majority of muscle fibres
(mostly in type1 fibres)

83. Congenital fibre type
disproportion
•Atrophy of type 1 fibres,
hypertrophy of type 2 fibres
•Detectable at birth, paucity of
motor activity & diminished tone
•Muscle deterioration tends to
continue throughout first decade
& then ceases or undergo reversal
•Skeletal deformities-hip dislocation
,kyphoscoliosis ,joint contractures

84. Myofibrillar myopathies
•Heterogenous group of
disorders(protein surplus
myopathies)
•Accumulation of intermediate
filaments including
desmin,actin,myosin,ab crystalline
& myomotilin within fibres
•Adult onset
•Distal weakness,dysphagia &
cardiac involvement
Desmin myopathy (RTC)

85. General questions to keep in mind during
reporting muscle biopsy
• Are the muscle fibres abnormal??
• Is the pathologic process-neurogenic/
myopathic??
• What is the distribution of pathology??
• Are there any diagnostic features??

86. Are the muscle fibers abnormal??
Size-small or large
Shape-rounded or angular
Type-grouping, fiber predominance
Internal architecture-disordered/lost, vacuoles,Internal nuclei,
inclusions
Storage/acumulated material-glycogen, lipid, mitochondria
Is the pathologic process
 NEUROGENIC/MYOGENIC
• Shape of muscle fibers
ROUND Myopathic
ANGULAR Neurogenic

87. • Distribution of atophic fibers
Grouped-Denervation ,dystrophinopathies
Scattered-Acute neuropathy or myopathy
 ACUTE OR CHRONIC
Acute:
Myopathy-Muscle fiber regeneration &denevation
Neuropathy-Small angular muscle fibers
Chronic:
Myopathy-increased endomysial connective tissue, muscle fiber
hypertrophy
Neuropathy-fiber type grouping ,pyknotic nuclear clumps

88. • What is the distribution of the pathology??
UNIFORM-Dystrophy, fiber type predominance
REGIONAL
Patchy fascicular changes-inflammatory myopathies,focal denervation
Group of muscle fibers
Neuropathy -Progressive denervation with reinnervation
Myopathy- Myopathic grouping, perifascicular atrophy
SCATTERED muscle fibers: Acute myopathy, Acute neuropathy
• Are there diagnostic feaures??
INFLAMMATION
SITE
STORAGE MATERIAL
PATHOLOGY other than muscle fibers

89. References
 Vinay Kumar,Abul.Kabbas,Nelson Fausto. Robbins & Cotran
Pathological Basis of Disease.8th ed. Chicago,Illinois:Elsevier.2010
.p. 905-969
 Stephen S sternburg, Donald A.A,Daryl. Carter,Stacey.E, Oberman
H.A. Diagnostic Surgicl Pathology.3rd ed. Newyork: Lippincort
Williams &Wilkins;1999.p.1701-1784.
 C. Sundaram and Megha S.Uppin,Approach to the interpretation
of muscle biopsy ,Nizam institute of medical sciences, Hyderabad.
 Ivan Damjanov,Anderson`s Pathology.10th
ed.Kansas:Elsevier;2009.Vol2.p2653-2692.
 WellerR.O.Systemic Pathology,Nervous system,muscle & eyes.3rd
ed/vol4.Chuechill Livingston;1990.p.580-665.