histopathological features of myopathies and muscular dystrophy along with approach to interpretaion of muscle biopsy

1. Interpretation of
Muscle biopsy with
special reference
to Muscular
Dystrophy and
Myopathies Dr. Adrija Pathak

2. The Weil-Blakelsley Cochotome with a 6mm bitting
tip

3. Collection & Preparation of
Biopsy Specimen
 Clinical information
 Appropriate muscle-representative of
ds
-ds process is active and evolving
 Before excision muscle maintained in
isometric state by introducing it into
clamp

5. Normal Muscle
Skeletal mucle is composed of
elongated, multinucleate ,unbranched
contractile cell described as mucle fibre
Characteristic cross-striations seen on
LM d/t arrangement of contractile protein
Normal muscle (transverse
section). The fibers are typically
polygonal, and the sarcolemmal
nuclei are located peripherally.

6. • Individual muscle fibers are surrounded
by endomysium and are grouped
in fascicles which are surrounded by a small
amount of connective tissue known
as perimysium.
• Epimysium, in turn, is the connective
tissue which surrounds multiple muscle
fascicles
• In normal muscle, the endomysium is so
inconspicuous that individual muscle fibers
appear to abut one another.

8. Normal muscle. In the alkaline
adenosine triphosphatase
(ATPase) reaction, type 1
fibers are light, and type 2
fibers are dark because of
their high content of ATPase
for use
in the glycolytic pathway.
(ATPase, pH 9.4,
counterstained with eosin).
‘Reverse’ ATPase ph 4.3 shows
the normal distribution of dark
type 1 fibres, pale type 2A fibres
and also
intermediate type 2B fibres.
ATPase at ph 9.4 shows a normal
‘checkerboard’ or ‘mosaic’ distribution of
fibre types 1 and 2. Type 2 fibres stain
darkly.

9. 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.

10. Stain for succinic
dehydrogenase is paler and
has a particulate appearance
due to selective staining of
mitochondria.
Staining for cytochrome
oxidase (COX) shows a
similar distribution to
SDH staining (more
prominent in Type 1
fibres) but in this stain
the end product is
golden brown.

11. Frozen section stained for phosphorylase. Type 2 fibres
are stained darkly but this reaction is not used routinely to
demonstrate fibre type differentiation. Complete absence
of staining is typical of McArdle’s disease (Type V
Glycogenosis).

12. A modified PAS stain to
demonstrate glycogen. Type 2
fibres which are dependent on
intrinsic glycogen stain darkly.
Verhoeff Van-Gieson (VVG)
stain of frozen tissue to
show fibrous tissue, elastin
and myelinated nerve
fibres. The fine black dots
represent
mitochondria (hence the
darker staining of type 1
fibres) and the
intermyofibrillary network.

13. Oil Red-O in frozen section
demonstrates normal
distribution of fine lipid droplets
within muscle fibres, more
prominent in type 1 fibres
(arrow).
The modified Gomori
trichrome stain identifies
mitochondria as small red
dots within the muscle
fibre, most numerous in
type 1 fibres and at the
fibre periphery, in the
subsarcolemmal zone
(arrow). This biopsy
contains
a normal number of
mitochondria in usual
distribution.

14. Working Classification of Muscular
Diseases
Neurogenic Neuromuscular
Disorder
Primary Myopathic
Changes
Inflammatory
Dystroph
y
Endocrinopathies
Toxic-Drug Induced
Metabolic Congenital
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

15. Pathological Reactions of
Muscle
Nuclear internalization.
Many fibers contain one or
more internal, often
pyknotic nuclei.
Nuclear Internalization

16. Ring Fibres
Circumferential orientation of the peripheral
myofibrils produces a striated ring that
encircles a transversely sectioned fiber in the
center of the field (periodic acid-Schiff stain).
Hyaline Fibres
The fiber in the center of the
photograph is rounded, ellarged and
darkly stained, opaque sarcoplasm.
- Myotonic dystrophy
- Limb-Girdle dystrophy
-Early stage of necrosis
- Duchenne muscular dystrophy

17. Fiber Necrosis
The necrotic process in the fiber at
the centre of this longitudinal
section is recognized by a loss of
cross striations and early
phagocytosis.
Fiber Regeneration
Sarcolemmal nuclei are large and
vesicular, and they contain prominent
nucleoli. The sarcoplasm is basophilic.

18. Inclusions
Nuclear inclusion
- Oculopharyngeal dystrophy- EM
- Inclusion Body Myositis- LM
Oculopharyngeal muscular dystrophy.
High-power electron micrograph showing
intranuclear inclusion composed of 8.5-
nm tubulofilamentous material.
Inclusion body myositis. An
intranuclear inclusion is shown
at the center of the picture. The
inclusion is eosinophilic and
smudged; it is located within a
sarcolemmal nucleus.
Sarcoplasmic inclusion
- Myofibrilllar myopathy

19. Inflammation
Inflammatory myopathy. Sheets of
lymphocytes expand the
endomysial spaces and surround
the fibers.

20. Frozen section in inflammatory myopathy illustrating perivascular
and
endomysial inflammation (black arrow), individual necrotic fibres
(yellow arrow) and fibres undergoing phagocytosis (blue arrow).

21. Multiple discrete granulomas are seen in this case of
sarcoidosis.

22. •Fibrosis and Fatty infiltration
•Atrophy and Hypertrophy
Most common form of atrophy-denervation
Type 1 fibre hypertrophy- specific for infantile spinal muscular atrophy
(ISMA). Also seen in athletes undergoing endurance training
Type 2 fibre hypertrophy- sprinters& congenital fibre type
disproportion
Hypertrophy involving both fibres- limb-girdle dytrophy, IBM, myotonia
congenita & acromegaly

23. ATPase ph 9.4 shows diffuse
selective atrophy of type 2 fibres.
This was a common finding in
biopsies from patients attending
the Rheumatology clinic.
Type 2 atrophy in a patient with
malignancy and cachexia
(immunostain for fast myosin).

24. denervation
dermatomyositis
chronic
denervation with
reinnervation

25.  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

26. Fiber Shape
Chronic neurogenic atrophy.
Grouping of many small
angular fibers is evident.
Neurogenic atrophy. Many atrophic fibers are
angular (adenosine triphosphatase, pH 9.4).
Infantile spinal muscular atrophy.
Most of the fibers in the fascicle
are atrophic and rounded.

27. Mottled Fibers
Fiber Splitting
Several hypertrophic fibers are seen. The fiber at the
bottom and center is divided into two smaller subunits
(frozen section, rapid Gomori trichrome).
The sarcoplasm appears moth
eaten as a result of the presence
of patchy areas of poor staining
due to lack of mitochondria&
destruction of myofilament (NADH-TR).
- Limb-Girdle dystrophy
- Denervation
- IBM
-FSHD
-Limb-Girdle
-Denervation

28. Cores & Targets
The focal areas of reduced enzyme
activity are single, and they are
centrally positioned within many
fibers (NADH-TR).
Neurogenic atrophy. In target fibers, an inner,
unstained zone is surrounded by a rim of
increased enzyme activity (NADH-TR).
Cores- in variety of diseases, most prevalent in neurogenic atrophy
Target- pathognomic for neurogenic atrophy

29. Nemaline Rods
Collections of dark, rod-shaped structures are
evident in many of the fibers (frozen section, rapid
Gomori trichrome).
Ultrastructurally, rods are osmiophilic and elongated or
rectangular, resembling Z-bands.
-Nemaline myopathy
-Muscular dystrophy
-PM

30. Mitochondrial Abnormalities
Ragged red fiber. Collections of
mitochondria appear as red-stained,
irregular, subsarcolemmal areas within the
involved fiber (frozen section, rapid Gomori
trichrome).
Ragged red fiber is seen with abnormally
large mitochondria, several of which contain
paracrystalline inclusions.

31. Vacuolar changes
Lipid storage myopathy. Numerous osmiophilic,
lipid-containing vacuoles are evident in the
sarcoplasm of the fiber at the center (resin section,
toluidine blue).
A rimmed vacuole contains abundant
red, granular material (frozen section,
rapid Gomori trichrome).

32. Artifact & Pitfall
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).

33. 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).

34. 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.

35. Non-specific features- thyroid ds, statin therapy
Prior trauma during EMG, i/m inj – necrosis, regeneration,
inflamm, endomysial fibrosis
Not all muscle look alike. Ex. Paraspinal muscle- internal nuclei,
grps of fasicles seperated by abundant conn ts resembling fibrosis
Crush artifact- fibres appear atrophic
Fatty infiltration seen in obese

37. Neurogenic Atrophy
 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

38. 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.

39. 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).

40. Duchenne Muscular
Dystrophy
 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

41.  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

42. Absent staining for Dystrophin
in the majority of fibres in a
case of
Duchenne dystrophy
Normal immunostaining pattern for
dystrophin. The sarcolemmal regions of
the fibers are outlined

43. Becker Muscular Dystrophy
 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

44. Facioscapulohumeral
Dystrophy
 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

45. Limb-Girdle Dystrophy
 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

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

47. Myotonic Muscular Dystrophy
 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

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

49. Central Core/ Multicore
Disease
 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- cores
type 1 fibre
predominant
Multicore disease. Numerous small globular-shaped
cores are seen in the fibers. Cores appear unstained
with oxidative enzyme reactions (nicotinamide adenine
dinucleotide, reduced).

50. Nemaline (Rod) Myopathy
 Female
 Facial dysmorphism-face
elongated, jaw
prognathic, high
vaulted palate
 Histochemical rxn-selective
atrophy of
oxidative fiber
This is an example of nemaline myopathy seen in a
biopsy of
paravertebral muscle taken during spinal surgery for
kyphoscoliosis in a young girl.
Frozen section H&E shows numerous rods in most
fibres with many grouped in peripheral clusters.
Modified trichrome stain highlights rod bodies

51. Centronuclear Myopathy/
Myotubular Myopathy
 Age of onset not
uniform: infancy-7th
decade
 Extaocular 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

52. Congenital Fiber-Type
Disproportion
 Atrophy of type 1
fibers and
hypertrophy of type 2
fibers
 Paucity of motor
activity & decresed
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)

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

54. Sarcoplasmic inclusion
- Myofibrilllar myopathy
Cytoplasmic body. Circumscribed inclusion with
three dense, red central foci surrounded by
green filamentous material (paraffin, Gomori
trichrome stain).
Desmin myopathy. Two fibers contain slightly
basophilic smudged regions within the
sarcoplasm, which represent collections of
myofibrillar material (frozen section, rapid
Gomori trichrome).
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).

55. Polymyositis and
Dermatomyositis
 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

56.  ↑ 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

57. 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

58. Inclusion Body Myositis
 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

59. Many fibres contain ‘rimmed vacuoles’ in this
biopsy from a patient with Inclusion Body
Myositis (IBM). Myopathic features such as
increased variability in muscle fibre diameters
and increased central nuclei are present and
neurogenic features may also be identified in
such biopsies. Congo Red staining may reveal
deposits of beta amyloid within the fibres.
‘Rimmed vacuoles’ contain basophilic
granular inclusions and have a
basophilic rim. Electron microscopy
will show membranous whorls
(‘myeloid bodies’) within the vacuoles.

60. Carbohydrate Storage Ds
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
2 MCARDLE’S DISEASE
 AR, in childhood/adolscence
 Muscle weakness, pain & stiffness exacerbated by excercise
 Many crescentric PAS positive vacuoles in sub sarcolemmal position.
 Histochemical reactions showing absence of phosphorylase activity.
3 PFK DEFICIENCY
 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.

61. Lipid Storage Ds
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

62. Mitochondrial Myopathy
 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)

63. 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.
Prominent subsarcolemmal
clumping of abnormal
mitochondria.
Increased mitochondrial staining
associated with vacuolation at
periphery of muscle fibre.
increased red staining in subsarcolemmal
zones
due to aggregates of abnormal mitochondria

64. References
 Sternberg’s diagnostic surgical
pathology, 5th edition
 Dubowitz V, Sewry CA, Muscle
Biopsy: a Practical approach, 3rd
edition, Saunders, 2006
 Robbins & Cotran pathological basis
of disease, 8th edn
 Theory and practice of Histological
techniques: John D Bancroft, 6th edn
 Wheater’s funtional histology, 5th edn