2. IndexIndex
Structure & histology of muscle
Types of muscles
Indications & Contraindications of muscle Bx
Procedure & Practical aspects of muscle Bx
Sample preparation & Staining
General abnormalities seen in response to
injury.
Neuromuscular diseases
Biochemical investigations
3. Normal structure & histology of muscleNormal structure & histology of muscle
4. Types of muscle fibresTypes of muscle fibres
Type - 1 Type -2
Red /oxidative/slow glycolytic/fast/white
o ATPase Low High
Oxidative High Low
enzyme cont.
Glycogen Low High
Phosphorylase Low High
Lipid cont. High Low
5. Types of muscle fibresTypes of muscle fibres
Type - 1 Type -2
Inc.Myoglobin. Dec myoglobin
Postural activity Sudden
intermittent activity
Aerobic Anareobic
35- 40% 60-65%
6. Diff. Between These types done byDiff. Between These types done by
histochemical stain on frozen sectionhistochemical stain on frozen section
Type-1 Type-2A Type 2B
1)ATPase reaction Light Dark Intermediate
at 9.4 pH
2)ATPase reaction Dark Light Intermediate
At acidic pH
3)NADH-TR reac. Dark Intermediate Light
Depend on conc.of
mitochondria
4)Antibodies
Slow +nt -nt -nt
Fast - nt +nt +nt
8. Muscle Biopsy - IntroductionMuscle Biopsy - Introduction
Introduced by Duchenne – 1868
Simple but invasive procedure
Interpretation requires detailed clinical history ,
physical examination, biochemical studies.
9. Indications of Muscle Bx –Indications of Muscle Bx –
Kakulas &Adams- 1985Kakulas &Adams- 1985
Investigation of muscle weakness, static &
progressive
To diff bet. myogenic /neurogenic cause of
weakness.
Patients suspected of having inflammatory
myopathy & collagen vascular diseases
Metabolic diseases where muscle is likely to
be affected.
10. Indications of Muscle Bx –Indications of Muscle Bx –
Kakulas &Adams- 1985Kakulas &Adams- 1985
Suspected drug induced myopathy.
In asymptomatic carrier of genetic diseases.
To access the disease progress and
prognosis
For research
11. Diseases ass. with weakness in whichDiseases ass. with weakness in which
muscle Bx is low-yieldmuscle Bx is low-yield
Electrolyte disturbances
Endocrine diseases
Malignant hyperthermia
Myasthenic syndromes
Old age
Periodic paralysis
Poor nutrition
12. Muscle Bx - TechniqueMuscle Bx - Technique
Selection of Muscle :
1) Moderately affected or muscle in which disease is
evolving should be selected.
2) Ideal site- Quadriceps / deltoid /biceps.
3) Bx should be taken from belly of muscle, away from
tendinous insertion.
4) In autopsy cases, Bx can be taken upto 12 hrs.
Avoid-
1) Severely / minimally affected muscle
2) Previously injured site- by EMG, intramuscular
injection
3) If rhabdomyolysis occurred, 3 mths back.
13. ProcedureProcedure
1) Open Bx
2) Needle Bx
Open Bx –
Use for diseases with patchy involvement e.g PM
Is obtained through a skin incision under local
anesthesia
Infiltration of local anaesthesia into muscle is avoided.
Avoid use of dithermy , cautery.
14. ProcedureProcedure
Needle biopsy
Less traumatic , simple
Uses-
Particularly for biochemical investigation and
Follow up Bx to monitor the progress of treatment.
Disadvantage:
Small size of Bx
Difficulty in orientation of tissue.
15. ProcedureProcedure
Muscle is maintained in isometric state by using
muscle clamp in situ
e.g Price muscle clamp
Wooden swab stick
Use- to prevent contraction artefact
Orientation of specimen
16. ProcedureProcedure
Fresh specimen for histochemistry & fixed specimen for
LM & EM
3rd
specimen is taken if required for biochemical analysis
& genetic analysis
Size of specimen should be 1x0.5xo.5 cm.
2 pieces of muscle should be taken
17. ProcedureProcedure
Precautions:
Do not crush the muscle.
Do not stretch the muscle.
Do not tie any stick.
Do not put directly into ice ( to prevent freezing
artefact)
Do not directly immerse in saline
Transportation
Moisten the specimen with saline dampened gauze
and send it to laboratory.
If sent in a flask containing ice cubes , this can be
delayed for up to 4-6 hrs.
18. Processing of fresh sampleProcessing of fresh sample
Flash freezing by isopentane cooled in
liquid nitrogen at –160 deg C
Frozen cryostat section of 10 micron are cut
at –18 to – 20 deg C
19. Various stain used areVarious stain used are
Congo red Detection of amyloid
deposition
Myophosphorylase McArdle’s disease
PFK PFK deficiency
Myoadenylate Myoadenylate deaminase
deaminase deficiency
Dystrophin DMD & BMD
immunostain
Dysferin immunostain LGMD 2B
Membrane attack Dermatomyositis
complex immunostain
20. Processing of fixed specimenProcessing of fixed specimen
Clamped specimen fixed in
10 % buffered neutral formalin – LM
3% glutaraaldehyde – EM ( sp. Size0.5x 0.5 x 0.2
mm)
2 sections are taken - longitudinal
- Transverse
Each of 6 micron thick
Stains used – H&E, Retic ,Trichrome, PAS
22. General abnormalities of muscleGeneral abnormalities of muscle
in response to injuryin response to injury
23. Nuclear changesNuclear changes
Normal nuclei- peripheral
Large no. of myofibres with central / paracentral
nuclei - s/o Myopathic diseases.
Increase in internal nuclei –MC abnormality
24. Nuclear changesNuclear changes
Internal nuclei are seen in:-
Myotendinous insertion
Fibre atrophy - multiple pyknotic
nuclei forming clusters.
Fibre regeneration – vesicular
nuclei with prominent nucleoli.
Centronuclear myopathy- Single
central/ paracentral nucleus in
almost all fibers-
Myotonic dystrophy- randomly
distributed nuclei with active
appearing nuclei (dispersed
chromatin)
25. Hyaline fibres ( Degeneration)Hyaline fibres ( Degeneration)
Degenerating rounded and enlarged
More deeply stained than normal
Sarcoplasm – smudged, homogenous
Nuclei – Pyknotic in centre / periphery
DMD- Large no. of Hyaline fibres
26. Fibre necrosisFibre necrosis
Presence of degeneration & necrosis – definite sign of
myopathy
Best seen in H&E section
Loss of striation
Swelling of myofober eosinophilia
(acute necrotic fibers)
Later pale color
Sarcoplasm striated to coarsely granular
Nucleus –Pyknotic,fragmented ,absent
Macrophage seen in surrounding
fibres
27. Fibre NercosisFibre Nercosis
Necrosis seen in:
Small groups of necrotic fibre - DMD
Perifascicular necrosis - DM
Random fibre necrosis - PM,IBM
Infarcts & large areas of necrosis - PAN
Extensive, diffuse necrosis -
Rhabdmyolysis, in patient with canitine pamitoyl
transferase deficiency, alcoholics, military recruits.
28. Fibre regenerationFibre regeneration
Degeneration/ Necrosis
Compensatory regeneration
Increased basophilia
Source of regeneration:
1.Sprouts of remaining
sarcoplasm
2.Satellite cells – more
capacity to regenerate.
Regenerating fibres – increased basophilia
Nuclei increased in number, larger than normal
With vesicular chromatin, prominent nuclei.
29. Fibre splittingFibre splitting
Hypertrophic fibres split into 2/>2 subunits.
A spilt like space from invagination individual
segment.
-Limb girdle dystrophy
-IBM
Mechanism- (A form of
Regeneration)-failure
to unite to form a
single fibre
30. AtrophyAtrophy
MC histological change
Interpretation better in cross section & in frozen
section.
General causes of atrophy( non selective )-
1. Denervation- MC
2. Disuse
3. Ischemia
4. Aging
5. Poor nutrition.
32. Morphometric analysis of atrophyMorphometric analysis of atrophy
Done either manually / computer assisted image
analyzer.
Lesser diameter of each muscle fiber should be
measured.
At least 200 fibers should be present in the sample.
Interpretation:
Grouped atrophy- 5/>5 angular fibers –
pathognomonic for chronic neurogenic disease
Panfascicular Atrophy – ISMA
Perifascicular atropy – DM
Atrophic fibers randomly situated in the section is
non specific.
33. Fiber hypertrophyFiber hypertrophy
Type 1:
ISMA
Type 2:
Runners sprinters
Congenital fiber type
disproportion
•Limb – girdle
dystrophy
IBM
Myotonia
congenital
Acromegaly
34. Changes in histochemical profile:Changes in histochemical profile:
Denervation
Renervation of different Nv.
Causes change in muscle
Loss of checkerboard type
pattern - Type grouping
Motor unit
36. InflammationInflammation
PM
1)Inflammatory myopathies-PM, DM &
IBM
2)Immunologically mediated
myopathies SLE & RA.
PM –
1.Inflammatory cells invade the
endomysium, enveloping necrotic
fibres.
2. Sheets of inflamm. cells expand
endomysial spaces in acute severe
disease.
37. InflammationInflammation
DM – Infiltration of vascular walls by mononuclear
cells.
RA :Nodular infiltration of plasma cells
PAN – affinity for large vessels – arteries within
epimysium and perimysium.
Entire wall shows infiltration
by inflammatory cells
including eosinophils.
DM
38. SLE- Small vessels are
affected
Fibrinoid necrosis present with
neutrophils
Granulomatous inflammation:
1. Sarcoidosis-non necrotizing
granulomas
2. Idiopathic granulomatous
myositis. – Chronic
progressive myopathy of
middle aged person.
InflammationInflammation
39. Core & TargetsCore & Targets
Better seen in oxidative enzyme stain
Targets -
Appear as central pallor s/by darkly stained rim, in turn s/by
normal appearing area.
Cores-
Appear as a central pallor s/by normal appearing area
without dark zone.
Target Cores
Central -pale
area: -nce of
oxidative
enzyme
Central-amorphous
electron dense material
Intermed-Mitochondria
Outer- normal
40. Core & TargetsCore & Targets
Target Cores
Always single Single/multiple &
eccentric
Extends upto few sarcomere Extends throughout
the length
>diameter. <diameter
Pathognomonic for Neurogenic Central core disease
atrophy
41. Ring fibresRing fibres
F
Formed by peripheral bundle of myofibril.
Directed circumferentially, encircling inner portion of
myofibre.
Seen in transverse section.
Striation are visible under PAS, Resin & EM
Normally seen in extraocular muscles.
Seen in LGD & MD
Large no. of ring fibres – S/O MD
42. Mitochondrial ABNRMitochondrial ABNR
Ragged Red fibres
Normally- mitochondria scattered within
sarcoplasm.
Redistribution of mitochondria beneath
sarcolemma-forming aggregates.
Aggregates app as red with trichrome.
Margins of involved fibres- irregular, ragged
appearance
EM- enlarged, deformed
mitochondria due to inclusion
of creatine kinase.
43. Mottled fibersMottled fibers
Seen in oxidative preparations NADHTR
Seen as unevenly staining fibres.
D/t minute, irregular zones of weak enzyme
activity- scattered in sarcoplasm.
Mechanism- Lack of mitochondria &
destruction of myofibrils.
Fascioscapular and limb girdle dystrophy.
44. Vacuolar changeVacuolar change
In the center arranged in size
gradient
Subsarcolemmal PAS +ve.
In scattered fibers, small,
round, osmiophilic, ORO
+ve
Rimmed ubiquitin +ve
Diseases
Freezing artifact
Glycogen Storage dis., Lipid
storage disease
Mitochondrial myopathy.
IBM, distal myopathy, OPD
Sarcoplasmic vacuoles
Rimmed Vacuole – sharply demarcated vacuole
containing granular material that forms red rim in frozen
section. Supposed to be autophagic vacuole – membrane
bound.
45. Nemaline rodsNemaline rods
Nemaline – thread like
Seen as clusters of threads
beneath sarcolemma.
Rods are osmiophilic
oblong structures of 6-7
micrometer.
Resembling Z band
Seen in nemaline myopathy
– congenital nonprogresive
muscle dis. of childhood
Trichrome
46. Fibrosis and Fatty InfiltrationFibrosis and Fatty Infiltration
End stage disease :
Previous Angiocentric – DM, Connective
tissue disease, FSHD
Endomysial around fibres – PM, IBM, Viral
myositis
47. WorkingWorking Classification of neuromuscularClassification of neuromuscular
diseasesdiseases
Neurogenic atrophy
Neuromuscular Junction disorders
Primary myopathic diseases
- Inflammatory myopathies
- Non inflammatory myopathies
1) Muscular dystrophies
2) Developmental disorders of skeletal muscle
3) Myofibrillary myopathies
4) Metabolic myopathies
5) Toxic & Drug induced myopathies
48. Neurogenic atrophyNeurogenic atrophy
Diseases affecting lower motor neurons or
their axons are-
-Amyotrophic lateral sclerosis in adults
-Spinal muscular atrophy usually in children
-Poliomyelitis - now rare
-Peripheral neuropathy of various types
49. Histological changesHistological changes
Denervation
randomly scattered atrophic fibers
Predominantly atrophy of type 2 fibers.
smaller and more angular, (Round in childhood
spinal muscular atrophy)
atrophy of adjacent
motor units groups of fibers
Group atrophy.
50. Histological changesHistological changes
Denervated muscle fibers
Reinnervated by collateral sprouting. ,
all of the reinnervated fibers are converted
to a single histochemical fiber type
loss of the normal checkerboard pattern
”Type grouping."
51. NADH-TR stain-
Reinnervation
Target fiber-central pallor with darkly stained rim
MC in polyneuropathies,
Long standing denerveation
Hypertropyhy/ fiber splitting
& Small atrophic fibers
Fibroadipose tissue
Histological changes
55. DermatomyositisDermatomyositis
Present as Adult & Juvenile form
Purple (heliotrope) discolorarion of the
upper eyelids
Dusky red patches over extensor surfaces
of the extremities
B/L symmeterical proximal weakness , slow
in onset.
Extramuscular manifestations frequent in
children.
Asso. With Ca lung ,colon & breast
56. DermatomyositisDermatomyositis
Circulating anti-endothelial antibodies.
Activation of complement,
Endothelial swelling/ necrosis capillary loss
Ischemic infarction
Myofiber necrosis
Histology-
Inflammation around
endomysial and perimysial
capillaries and arterioles.
perifascicular atrophy
58. IBMIBM
50 -70yrs
M > F
nonresponsive to steriod
Asymetrical weakness of acral muscle esp. in extensor
compartment of arm
S/o viral origin
59. IBMIBM
Small group atrophy with angular fibres
Hypertrophic & split fibres
Endomysial chronic inflammatory infiltrate
Rimmed vacuoles- slit like vacuoles in
cytoplasm surrounded by haematoxyphilic
granules
inclusion bodies in vacuoles
Inclusions contain beta amyloid,
hyperphosphorylated tau protein,
apolipoprotein E
62. Duchene muscular dystrophyDuchene muscular dystrophy
MC muscular
dystrophy
XR
Age- 2-4 yrs
Exclusively male
Genetics- Def. Of
dystrophin
Gene for dystrophin
located on Xp21
63. Duchene muscular dystrophyDuchene muscular dystrophy
C/F -Difficulty in running , jumping getting
up steps
Proximal > distal & LL> UL
Weakness of pelvifemoral &
scapulohumeral muscles, cardiac muscle
Pseudohypertrophy of quadriceps gluteal
deltoid
Cardiac involvement +nt
64. Duchene muscular dystrophyDuchene muscular dystrophyBx-
Excessive variation in size shape of fibers
Large rounded fibers with small atrophic fibers
Inc. hyaline fibers
Necrosis & phagocytosis in both fibers type with
small basophilic regeneration fibers.
Patchy endomysial fibrosis
Fiber splitting is seen in
Hypertrophic fibers
69. FSHDFSHD
AD
3-4 th
decade
Involves voluntary muscles of face shoulder's , UL
Bx –
- Atrophic fibers in absence of necrosis/ regeneration.
- Numerous moth eaten fibers
- Perivascular Lymphocytes +nt in early stages.
70. Limb girdle dystrophyLimb girdle dystrophy
AR
Young adult
Sarcoglycan deficiency – 17q12 (4types)
Consists of myopathies involving proximal axial muscles.
Gradual onset, progressive weakness
Bx-
Marked nuclear internalization variability in fiber size.
Hypertrophic fibres with fiber splitting.
71. Myotonic DystrophyMyotonic Dystrophy
Myotonia—Failure to relax after contraction d/t
membrane defect.
AD
Defective gene – 50/>50 repeats of CTG
trinucleotide
Two forms- Congenital & adult form.
Muscles of face, jaw & eyelids-involved.
Ptosis, expression less face, dysphagia
Myotonia present in earlier stages
Other manifestation – cataract, COM, Mild
Dementia, DM, testicular Atrophy.
72. Myotonic DystrophyMyotonic Dystrophy
Bx
Multiple pyknotic internal nuclei
Selective atrophy of type 1 fibers
Type II hypertrophy
Ring fibers & sarcoplasmic masses
In chronic cond.– fibrosis, degeneration,
regeneration, fibrosis.
73. Distal myopathyDistal myopathy
1.Welander myopathy
2.Miyoshi myopathy
3.Hereditary variant of IBM
4.Non Scandinavian myopathy
Bx –
-Abnormal no. of internalized nuclei
-Variable fiber diameters
-Selective atrophy at type 1- Welander
-Rimmed vacuoles in non Scadinavian myopathies.
75. Developmental disorders ofDevelopmental disorders of
skeletal musclesskeletal muscles
Floppy infant with delayed motor
development
Nonprogressive myopathies
Persistent proximal muscle atrophy with
weakness
1. Centronuclear myopathy
2. Congenital fiber type disproportion
76. Centronuclear MyopathyCentronuclear Myopathy
AD/ AR/ X linked
CF- Presents from in infancy till 7 th decade.
Extraocular palsies and facial asthenia with
inv. of appendicular muscles.
Theory - arrest in maturation at myotube stage
– hence name myotubular myopathy.
77. Centronuclear MyopathyCentronuclear Myopathy
Bx –
High proportion of fibres
with central nuclei,
+nce of perinuclear halos
Small diameter of fibres
Pred. Of type 1 fibres,
Nuclei exceeds normal
size vesicular chromatin.
78. Congenital fibre type disproportionCongenital fibre type disproportion
Atrophy of type 1 and hypertrophy of type 2 fibers
May occur in families
Clinically at birth, paucity of motor activity and
diminished muscle tone.
Deformities – hip dislocation , kyphoscoliosis, joint
contracturs.
Bx-
Uniformly small
type 1fibres.
Type 2 fibres- large
79. Myofibrillar myopathiesMyofibrillar myopathies
Group of disoders with genetic defects
manifesting myofibrillar & cytoskeletal
abnormality.
1. Central core disease
2. Multicore disease.
3. Nemaline rod myopathy
4. Desmin myopathy.
80. Central core diseaseCentral core disease
AD
Chr.19q13.1
Affects RYR1gene – inv. Ryanodine receptor prot.
Type 1 fibres are affected
Mild , proximal, non progressive muscle weakness.
Bx –
1. Many fibers show single
centrally located defect or core.
2. More than one core per fiber
may be
encountered.
81. Multi core diseaseMulti core disease
Congenital, non progressive myopathy
Generalised weakness and hypotonia.
Type 1 fibre predominance.
Bx-
Numerous , multiple core like structure in majority of
muscle fibres.
Multicore tend to be
numerous within each fibre.
PAS/ Trichrome/ NADH-TR –
Stains pale
82. Nemaline rod myopathyNemaline rod myopathy
More prevalent in females.
AD/AR
Mutation of 5 thin filament genes- TPM3 &
Nebulin
Affects facial and proximal limb muscles.
CF- Facial dysmorphism
Bx-
Rods in majority of fibers
and in numerous number.
83. Desmin MyopathyDesmin Myopathy
AD
Childhood
Missense mutation/ del. – located on 2q35 encodes
desmin..
Distal weakness, dysphagia, cardiac muscle
involvement.
In frozen section – with RTC-
smuged area on peripheral
sarcoplasm.
NADH-TR- Unstained
86. Metabolic diseasesMetabolic diseases
1) Acid maltase deficiency
•Type -2 glycogenosis, AR
•mutation at chr. 17 - Mc at exon 18
•Fatal systemic disease of infant
•Adult form is also +nt
•Progressive weakness hypotonia, macroglossia COM &
organomegaly
•Biochemical analysis is must
87. Acid maltase deficiencyAcid maltase deficiency
Acid maltase deficiency-H & E PAS
•BX - PAS+ve diastase labile vacuoles of varying sizes
that replace much of sarcoplasm of the fibres.
•E/M memb bound glycogen filled vacuoles are seen.
88. Metabolic diseasesMetabolic diseases
2) CHO storage -Mc-Ardle disease.
Type 5 glycogenosis-myophosphorylase deff.
AR point mut. 11q13
Childhood / adolescence
C/F muscle weakness , exercise intolerance
Bx- Vacuoles located subsarcolemmaly.
Control-
phophorylase
Patient.
89. Metabolic diseasesMetabolic diseases
3) PFK deficiency
Type 7 glycogenosis / Tarui disease
AR, mut.12q13
Present in childhood
Muscle pain, exercise intolerance
Prolonged exercise leads to nausea, vomitting ,
myoglobinuria HA d/t RBC PFK def.
Frozen section - PAS +ve crescents seen adjacent
to sarcolemma of muscle fibres
Biochemical analysis must
90. Lipid storage diseasesLipid storage diseases
Cytosol Inner mito. Mitochondria
membrane
Acyl- Carnitine Carnitine Acyl
coA Co-A
CPTI CPT- 2
Co-A Acyl Acyl COASH
carnitine carnitine
Deletion of either I.e. CPT 1 & CPT 2 lead to myopathy
Histology – Myofibrils are seperated by vacuoles that stain with ORO /
SBB with in muscle fibre
91. Lipid storage diseaseLipid storage disease
Deletion of either I.e. CPT 1 & CPT 2 lead
to myopathy
Histology – Myofibrils are seperated by
vacuoles that stain with ORO / SBB with in
muscle fibre
Predominantly type-1 fibres are affected
92. Mitochondrial MyopathiesMitochondrial Myopathies
1) Kearns Sayre Syndrome
2) MELAS
3) MERRF
Deficiencies of enzymes of the respiratory chain
affect more severely muscle and brain
Impairment of intramitochondrial protein synthesis
Deformed enlarged mitochondria forming aggregates
Ragged red fibres.
Muscle involvement is characterized by weakness and
ophthalmoplegia.
96. Biochemical investigations in muscleBiochemical investigations in muscle
diseasedisease
Always done before biopsy.
CK-
used as a single most useful marker
Elevated in pt. With muscle disease but it may be normal in
slowly progressive disease.
Main use-
1. To differentiate between types of dystrophies
In DMD, 10-100 times normal
Raised before 1 yr of s/s, Peak at 3 yrs.
Other dystrophies- elevation is lower except in LGD
2. Floppy infant syndrome-Only congenital myopathy whr CK
levels are raised.
97. Biochemical investigations inBiochemical investigations in
muscle diseasesmuscle diseases
D/D of elevation of CK-
1. Motor neuron diseases.
2. GBS
3. Drugs- Steroids
4. Hypothyroidism/Hypopartathyroidism
5. Trauma
Remember-
1. CK levels are not raised in neurogenic atrophy.
2. Elevation of CK without weakness –No
myopathy.
98. Biochemical investigations in muscleBiochemical investigations in muscle
diseasesdiseases
3. CK isoenzymes are not advocated, as there is
elevation of CKMM, CKMB.
Other enzymes used are- Aldolase, AST, ALT, LDH.
1. Aldolase –more sensitive than CK in PM.
2. AST, ALT, LDH –measured as screening
procedures.
Elevation of either advocate prompt CK
measurement.
99.
100. SummarySummary
What to be seen on
1. Transverse
Most of abnormalities are
seen on TS
Size, shape, position of
nuclei, inflammation,
endomysial fibrosis, .Ring
fibers ,cores, Targets ,
2. Longitudinal
Striations
Position of
nuclei,inflammation
segmental necrosis,
Fiber splitting
Neurogenic cause Primary myopathic cause
1.Atrophy – angular
- group
2.Type grouping
3.Target fibre
1.variation in fibre size
2.Fibre degeneration &
Necrosis