Muscle biopsy is indicated for general conditions and specific conditions General ones include weakness of uncertain cause-generalized, proximal, floppy infant syndrome , in case of persistently increased muscle enzymes or in case of muscle pain cramps and stiffness Specific reasons include hereditary muscle disease connective tissue diseases metabolic diseases drug induced myopathy

1. Interpretation of
Muscle biopsy
Dr babita garg

2. Normal Muscle

3. EPIMYSIUM
•Loose CT
•Blood vessels
PERIMYSIUM
•Septa
•Nerve branches
•Muscle spindles
•Fat
•Blood vessels
ENDOMYSIUM
•Muscle fibers
•Capillaries
•Small nerve fibers

4. Skeletal muscle is composed of elongated, multinucleate
,unbranched contractile cell described as muscle fibre
Characteristic cross-striations seen on LM d/t arrangement
of contractile protein

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

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

8.  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.  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. The Weil-Blakelsley Cochotome with a 6mm bitting
tip

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

15.  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. Mitochondrial myopathies are types of myopathies associated
with mitochondrial disease.[1] On biopsy, the muscle tissue of patients
with these diseases usually demonstrate "ragged red" muscle fibers.
These ragged-red fibers contain mild accumulations of glycogen and
neutral lipids, and may show an increased reactivity for succinate
dehydrogenase and a decreased reactivity for cytochrome c oxidase
Nemaline myopathy (also called rod myopathy or nemaline rod
myopathy) is a congenital, often hereditary neuromuscular disorder
with many symptoms that can occur such as muscle
weakness, hypoventilation, swallowing dysfunction, and impaired
speech ability. The severity of these symptoms varies and can change
throughout one's life to some extent. The prevalence is estimated at 1
in 50,000 live births.[1] It is the most common non-dystrophic
myopathy.[2][3]
"Myopathy" means muscle disease. Muscle fibers from a person with
nemaline myopathy contains thread-like[4] rods, sometimes called
nemaline bodies.[5] While the rods are diagnostic of the disorder, they
are more likely a byproduct of the disease process rather than causing
any dysfunction on their own. People with nemaline myopathy (NM)
usually experience delayed motor development, or no motor

18. Frozen section stained for the
oxidative enzyme NADH-
tetrazolium reductase shows
darkly stained type 1 fibres.
High power of NADH-TR stained
frozen section shows positive
staining of both the
sarcoplasmic reticulum and
mitochondria, the latter more
numerous in type 1 fibres.

19. SDH stain
For mitochondrial
activity

20. Cytochrome c oxidas
•Mitochondrial activity
•Type 1 fibers are darker
than Type2

21. OIL RED O
Accumulation of lipid

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.  Assessed on scanner
 Note any adipose tissue infiltration and
fibrosis
Diffuse pattern
-dystrophy
Focal pattern
-neurogenic
Patchy
-inflammatory
myopathies

24. Atrophy or hypertrophy
Type 1 or type 2 fibres
Focal or diffuse

25. Atrophy
Type 1 fiber atrophy Type 2 fiber atrophy
•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)

26. Normal Atrophy

27. 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, myotonia congenita,
acromegaly
Normal Hypertrophy

28. dermatomyositis
denervation
chronic
denervation with
reinnervation

29.  Fibre type predominance is present when Type 1 fibres
constitute more than 55% of the total fibre population or
when more than 80% of fibres are Type 2.
 A predominance of Type 1 fibres is seen in Charcot-Marie
Tooth disease and Type 2 fibres are predominant in Motor
Neuron Disease.
 Fibre type deficiency is confirmed when less than 10% of
fibres constitute either group. A deficiency of Type 2 fibres
may be seen in limbgirdle dystrophy

30. Fiber shape
Normal muscle-polygonal
Angular Rounded

31. Fibre splitting
Limb girdle dystrophy
IBM

32. Nuclear changes
Normal Internalisation of nuclei

33.  Myotendinous insertion
 Centronuclear
myopathy
 Myotonic dystrophy
 Fiber regeneration
 Fiber atrophy
 Chronic neuropathies

34. Ring fibres
•Limb girdle
dystrophy
•Myotonic dystrophy

35. Hyaline Fibres
Duchene muscular dystrophy
Whorled fibres
•Limb girdle muscular dystrophy
•Chronic neuropathies

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

37. Fiber necrosis Perifascicular necrosis

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

39. 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

40. Inflammatory myopathy

41.  Granulomas tend not to cause significant
damage to adjacent myofibers.
Giant cell

42. Parasite ( Trichenella Spiralis)

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

44. Central cores Target fibres

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

46. Mottled fibres
FSHD

47. Mitochondrial Abnormalities
Ragged red fibres(RTC stain)

48. 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

49. Freezing artifact Rimmed Vacuole

50. Vacuoles in glycogen storage disease
Lipid storage myopathy. Numerous
osmiophilic, lipid-containing vacuoles are
evident in the sarcoplasm of the fiber at
the center (resin section, toluidine blue

51. Freezing artifact. Extensive vacuolar
change is caused by improper freezing.
Many of the vacuoles have linear,
noncircular geometric shapes.
Contraction artifact. Darker contraction
bands and disrupted lucent zones are
seen in several longitudinally oriented
fibers (periodic acid-Schiff stain).

52. Frozen section has partially lifted off the slide.
Tissue twists create artifact seen as fiber
curling with striped and ring structures in the
fibers (ATPase, pH 9.4, counterstained with
eosin).
Tendinous insertion. In this
location, the muscle fibers
normally vary in size, and they
are often surrounded by fibrous
tissue (Gomori trichrome).

53. Muscle specimen submitted in saline. Fluid
between fibers mimics edema. Several fibers
are damaged and disrupted and appear blown
out.
During the biopsy procedure, the
muscle has been roughly handled,
leading to a pseudovasculitis in the
perimysium. Neutrophils are
marginating in the vessel lumina and
beginning to traverse the vessel walls.

55. Neurogenic Neuromuscular
Disorder
Primary Myopathic
Changes
Inflammatory
Dystrophy
Congenital
Metabolic
Endocrinopathies
Toxic-Drug Induced
•Duchenne
•Becker
•FSHD
•Limb-Girdle
•OPMD
•Distal Myopathy
•Myototic
•Central Core
•Multicore
•Nemaline
•Centronuclear
•Fibre type Disproportion
•Myofibrillar
•PM
•DM
•IBM
•Sarcoidosis
•Viral
•Glycogenosis
•Lipid Storage
•Mitochondrial
•Malig Hyperpyrexia
•Myoglobinuria

56.  Most common dystrophy
 Most severe
 X-linked recessive- affects boys
 Neurologically intact at birth
 First sign when child attempts to walk/stand
 Weakness begins in pelvic girdle muscle then
extent to shoulder girdle sparing face muscle
and swallowing
 Psedohypertrophy of calves and buttock-
fatty infiltration and reactive fibrosis

57.  Elevated serum creatine kinase- first decade of
life
 Early death d/t cardiomyopathy
 Multiple exonic deletion DMD gene on chr Xp21
encoding dystrophin
 Bx- fiber necrosis and regeneration
- hyaline fibers
 Immunostain for membrane associated
dystrophin- absence of immunostaining
diagnostic of disease

59.  Less severe
 Rate of progression is slow
 Contains dystrophin but of abnormal size/structure
 Bx-variation in fibre size
- nuclear internalization
- necrosis, phagocytosis, regeneration
- endomysial connective ts proliferation

60.  Mild myopathy
 Involves face, shoulder & upper extrimities
 Onset in 2nd-3rd decade
 Bx- atrophic muscle clustered together
- moth eaten fibers
- perivascular lymphocytes
- absence of fiber necrosis/ regeneration

61.  Collection of myopathies
 Inv of proximal axial muscles
 Onset in young adult
 2B- Dysferlinopathy- most common
 Bx- nuclear internalization
- variability of fiber diameter
- fiber splitting

62.  Late onset- middle life
 Benign outcome
 Heralded by ptosis ,ophathalmopegia &
dysphagia
 Bx-mild dystrophic change (nuclear
internalization, atrophy & interstitial
fibrosis)
 EM- nuclear inclusion

63.  3rd-4th decade
 Begins with weakness of facial muscle and
acral muscle of extremities
 C/F- ptosis, expressionless visage, dysphagia
 Myotonia –inability of muscle to relax once
contracted
 A/W- cataracts, testicular atrophy, DM, CMP,
mild dementia

64.  AD- increased CTG trinucleotide repeat of gene at chr
19
 Bx-
 Early stage- pyknotic int nuclei
-selective atrophy of type 1 fiber
-ring fibers
 Chronic- fiber destruction, regeneration & fibrosis
A group of ‘ring’ fibres. This
abnormality may be a feature
in chronic myopathies e.g.
myotonic dystrophy

65.  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

66. 1.Central core disease
2.Multicore disease
3.Nemaline(Rod) myopathy
4.Centronuclear myopathy
5.Congenital fibre type disproportion
6.Myofibrillar myopathy

67.  Lack of muscular vitality
noted in infancy
 Mutation in RYR1 gene-
ryanodine receptor
protein that is a portion
Ca release channel of
sarcoplasmic reticulum l/t
Malignant hyperthermia
 Bx- muscle fiber show a
single centrally located
core
type 1 fibre
predominant
NADH-TR stain

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

69.  AR/AD
 Facial n proximal limb ms
 Facial dysmorphism- face
elongated, jaw prognathic,
high vaulted palate
 Aggregates of
subsarcolemmal spindle
shaped
particles(nemaline rods)
occuring predom. In
type 1 fiber best with
seen RTC stain
 Histochemical rxn-
selective atrophy of
oxidative fiber
Modified trichrome stain highlights rod bodies

70.  AD/AR/XL
 DNM2 gene/BIN1
gene/MTM 1 gene
 Age of onset not uniform:
infancy-7th decade
 Extraocular palsies & facial
asthenia with inv of
appendicular muscles
 Bx- central/paracentral
nucleus in most muscle
fibre resembling those
indeginious to fetal
myotube stage of
development
• Nuclei exceed the normal size and
have vesicular chromatin
• Sarcoplasm surrouding central nucleus
is disrupted ultrastructurally and appear
clear or vacuolated in frozen section
• few if any peripheral nuclei

71.  Atrophy of type 1 fibers
and hypertrophy of type
2 fibers
 Paucity of motor activity
& decreased muscle
tone at birth
 Deterioration throughout
1st decade then
cease/reversal
 Skeletal deformities-hip
dislocation,
kyphoscoliosis & joint
contracture
Congenital fiber:type disproportion
with hypertrophy of some type 2 fibers
and atrophy of type 1 fibers
(nicotinamide adenine dinucleotide,
reduced)

72.  Protein surplus myopathy
 Accumulation of
intermediate filaments-
desmin, actin, myosin,
æßcrystalline, myotilin
 Sarcoplasmic inclusions
 Adult onset, slowly
progressive
 Distal weakness, dysphagia
& cardiac involvement

73. Sarcoplasmic inclusion
- Myofibrilllar myopathy
Desmin myopathy. Two fibers contain slightly
basophilic smudged regions within the
sarcoplasm, which represent collections of
myofibrillar material (frozen section, rapid
Gomori trichrome).
Cytoplasmic body. Circumscribed inclusion
with three dense, red central foci surrounded
by green filamentous material (paraffin,
Gomori trichrome stain).
Hyaline body has distinct margins and a
subsarcolemmal location. The finely red granular
appearance of the mitochondria in the normal
sarcoplasm is absent from the more dense,
homogeneous look of the hyaline body (frozen
section, rapid Gomori trichrome).

74.  1.Dermatomyositis
 2.Polymyositis
 3.Inclusion body myositis

75.  Common myopathies of adult
 More prevalent in women, 20-40yrs
 Abrupt onset, rapidly progressive
 Remission & exacerbations
 Proximal muscle weakness
 DM- violaceous rash on eyelid, face and
extensor surface of digits
 DM- a/w ca lung, colon, breast

76.  ↑ ESR, creatine kinase
 Ab in serum- anti-Jo-1, anti-PM-1
 EMG- small, brief and polyphasic motor activity
 MHC class1 antigen expressed sarcolemmal
surface when examined by immunoperoxidase
 Bx- fiber necrosis & inflammatory rxn
- long standing ds- atrophy & endoperimysial
fibrosis
- DM- perifascicular atrophy is hallmark
-perivascular lymphocytic infiltrate

77. 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

78. Hematoxylin and eosin stain (20x) of a muscle biopsy from a
patient with dermatomyositis showing perivascular and
perimysial inflammation, as well as perifascicular necrosis.

79. Dermatomyositis. The
fibers at the edge of the
fascicle at the top are
atrophic.
Endomysial inflammation in H&E
paraffin section in a case of
polymyositis

80.  Withering of acral muscle esp extensor
compartment of arm
 Men, 50-70 yrs
 Does not respond to steroids
 Frozen section necessary for diagnosis
 Bx- small, angular & grouped fiber
- inflammation
- fiber hypertrophy & splitting
- variable necrosis/regeneration
- MHC class 1 expression
- rimmed vacuoles, inclusions, ragged red fiber

81. INCLUSION BODY MYOSITIS, “rimmed” vacuoles

82. 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%
malignancy 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

83. 1 ACID MALTASE DEFICIENCY
 Infants
 Prog weakness, hypotonia, macroglossia,
cardiomyopathy, organomegaly
 PAS positive, diastase labile vacuoles of
varying sizes
 EM: membrane bound glycogen filled
vacuoles
 Biochemical analyses of tissue is necessary
for diagnosis

84.  AR, in childhood/adolscence
 Muscle weakness, pain & stiffness
exacerbated by exercise
 Many crescentric PAS positive vacuoles in
sub sarcolemmal position.
 Histochemical reactions showing absence of
phosphorylase activity.

85.  AR, in childhood
 Muscular pain & stiffness induced by
exercise.
 Hemolytic anemia in few pts
 In frozen, PAS positive crescents adj to
sarcolemma
 PFK can be demons histochemically
unreliable.
 Confirmation by biochemical analysis.

86. 1 CARNITINE DEFICIENCY
 Infancy to middle age.
 Systemic / skeletal ms.
 Ac encephalopathy, heart failure,
 Diffuse vacuolization of ms fibres.
 Fat stains/ EM: dysmorphic, enlarged
mitochondria with paracrystalline inclusions
2 CARNITINE PALMITOYLTRANSFERASE
DEFICIENCY
 Weakness, myalgias ppt by exercise or fasting
 Lipid vacuoles may be normal or increased
 Detected by biochemical reactions

87.  Primary or secondary to lipid storage ds/
hypothyroidsm
1 Kearns-Sayre syndrome- ptosis, ext ophthalmoplegia,
pigmentary retinal degeneration, heart block, cerebellar ataxia
& short stature
2 Myopathy, encephalopathy, lactic acidosis,
strokes syndrome (MELAS)
3 Myoclonus Epilepsy with ragged red fiber
syndrome (MERRF)

88. Increased staining of mitochondria may be evident in H&E frozen
section in mitochondrial myopathy. Note basophilic stippling in
several fibres, particularly in sub-sarcolemmal zones.

89. increased red staining in subsarcolemmal zones
due to aggregates of abnormal mitochondria

90. Prominent subsarcolemmal
clumping of abnormal
mitochondria
Increased mitochondrial staining
associated with vacuolation at
periphery of muscle fibre.

91.  LMN ds- poliomyelitis, amylotrophic lateral
sclerosis,spinal muscular atrophy & peripheral
neuropathy
 Bx
- early denervation- random atrophy of both fibre
mainly type 2
- Angulated
- Small and later large groups of atrophied fibre
- Target fiber
- Denervation & renervation loss of checkerboard
pattern
- Motor unit territory enlarges newly recruited fibre
converted to single type fibre type grouping

92. H&E frozen section showing large group
atrophy
Small group atrophy seen in H&E stained
frozen section
The small angulated fibres
stain darkly in NADH-TR
reaction.

93. Reinnervation is evident
in fibre type grouping
A group of target fibres in NADH-TR
reaction. A clear central zone is
surrounded by a densely stained
intermediate zone
Chronic denervation with
reinnervation. Type grouping
replaces the normal
checkerboard staining pattern
(adenosine triphosphatase, pH
9.4).