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Muscle biopsy & interpretationMuscle biopsy & interpretation
Dr. Rashmi Gujarathi
IndexIndex
 Structure & histology of muscle
 Types of muscles
 Indications & Contraindications of muscle Bx
 Procedure...
Normal structure & histology of muscleNormal structure & histology of muscle
Types of muscle fibresTypes of muscle fibres
Type - 1 Type -2
Red /oxidative/slow glycolytic/fast/white
o ATPase Low High
...
Types of muscle fibresTypes of muscle fibres
Type - 1 Type -2
Inc.Myoglobin. Dec myoglobin
 Postural activity Sudden
inte...
Diff. Between These types done byDiff. Between These types done by
histochemical stain on frozen sectionhistochemical stai...
Normal histochemistry of MuscleNormal histochemistry of Muscle
NADH-TR
II
I
Muscle Biopsy - IntroductionMuscle Biopsy - Introduction
 Introduced by Duchenne – 1868
 Simple but invasive procedure
...
Indications of Muscle Bx –Indications of Muscle Bx –
Kakulas &Adams- 1985Kakulas &Adams- 1985
 Investigation of muscle we...
Indications of Muscle Bx –Indications of Muscle Bx –
Kakulas &Adams- 1985Kakulas &Adams- 1985
 Suspected drug induced myo...
Diseases ass. with weakness in whichDiseases ass. with weakness in which
muscle Bx is low-yieldmuscle Bx is low-yield
 El...
Muscle Bx - TechniqueMuscle Bx - Technique
Selection of Muscle :
1) Moderately affected or muscle in which disease is
evol...
ProcedureProcedure
1) Open Bx
2) Needle Bx
Open Bx –
 Use for diseases with patchy involvement e.g PM
 Is obtained throu...
ProcedureProcedure
Needle biopsy
 Less traumatic , simple
Uses-
 Particularly for biochemical investigation and
 Follow...
ProcedureProcedure
 Muscle is maintained in isometric state by using
muscle clamp in situ
e.g Price muscle clamp
Wooden s...
ProcedureProcedure
 Fresh specimen for histochemistry & fixed specimen for
LM & EM
 3rd
specimen is taken if required fo...
ProcedureProcedure
Precautions:
 Do not crush the muscle.
 Do not stretch the muscle.
 Do not tie any stick.
 Do not p...
Processing of fresh sampleProcessing of fresh sample
 Flash freezing by isopentane cooled in
liquid nitrogen at –160 deg ...
Various stain used areVarious stain used are
 Congo red Detection of amyloid
deposition
 Myophosphorylase McArdle’s dise...
Processing of fixed specimenProcessing of fixed specimen
 Clamped specimen fixed in
10 % buffered neutral formalin – LM
3...
Abnormalities seen on Paraffin /Abnormalities seen on Paraffin /
frozen sectionsfrozen sections
Paraffin section
 Nuclear...
General abnormalities of muscleGeneral abnormalities of muscle
in response to injuryin response to injury
Nuclear changesNuclear changes
 Normal nuclei- peripheral
 Large no. of myofibres with central / paracentral
nuclei - s/...
Nuclear changesNuclear changes
Internal nuclei are seen in:-
 Myotendinous insertion
 Fibre atrophy - multiple pyknotic
...
Hyaline fibres ( Degeneration)Hyaline fibres ( Degeneration)
Degenerating rounded and enlarged
More deeply stained than ...
Fibre necrosisFibre necrosis
Presence of degeneration & necrosis – definite sign of
myopathy
Best seen in H&E section
Loss...
Fibre NercosisFibre Nercosis
Necrosis seen in:
 Small groups of necrotic fibre - DMD
 Perifascicular necrosis - DM
 Ran...
Fibre regenerationFibre regeneration
Degeneration/ Necrosis
Compensatory regeneration
Increased basophilia
Source of regen...
Fibre splittingFibre splitting
 Hypertrophic fibres split into 2/>2 subunits.
 A spilt like space from invagination indi...
AtrophyAtrophy
 MC histological change
 Interpretation better in cross section & in frozen
section.
General causes of at...
Selective atrophySelective atrophy
Type 1 atrophy
 Myotonic dystrophy
 Nemaline myopathy
 Distal myopathy
 Centronucle...
Morphometric analysis of atrophyMorphometric analysis of atrophy
 Done either manually / computer assisted image
analyzer...
Fiber hypertrophyFiber hypertrophy
 Type 1:
 ISMA
 Type 2:
 Runners sprinters
 Congenital fiber type
disproportion
•L...
Changes in histochemical profile:Changes in histochemical profile:
Denervation
Renervation of different Nv.
Causes change ...
Type groupingType grouping
ATPase
I
II
InflammationInflammation
PM
1)Inflammatory myopathies-PM, DM &
IBM
2)Immunologically mediated
myopathies SLE & RA.
PM –
1....
InflammationInflammation
 DM – Infiltration of vascular walls by mononuclear
cells.
 RA :Nodular infiltration of plasma ...
 SLE- Small vessels are
affected
Fibrinoid necrosis present with
neutrophils
 Granulomatous inflammation:
1. Sarcoidosis...
Core & TargetsCore & Targets
Better seen in oxidative enzyme stain
Targets -
Appear as central pallor s/by darkly stained ...
Core & TargetsCore & Targets
Target Cores
Always single Single/multiple &
eccentric
Extends upto few sarcomere Extends thr...
Ring fibresRing fibres
F
Formed by peripheral bundle of myofibril.
Directed circumferentially, encircling inner portion of...
Mitochondrial ABNRMitochondrial ABNR
Ragged Red fibres
 Normally- mitochondria scattered within
sarcoplasm.
 Redistribut...
Mottled fibersMottled fibers
 Seen in oxidative preparations NADHTR
 Seen as unevenly staining fibres.
 D/t minute, irr...
Vacuolar changeVacuolar change
In the center arranged in size
gradient
Subsarcolemmal PAS +ve.
In scattered fibers, small,...
Nemaline rodsNemaline rods
 Nemaline – thread like
 Seen as clusters of threads
beneath sarcolemma.
 Rods are osmiophil...
Fibrosis and Fatty InfiltrationFibrosis and Fatty Infiltration
 End stage disease :
 Previous Angiocentric – DM, Connect...
WorkingWorking Classification of neuromuscularClassification of neuromuscular
diseasesdiseases
Neurogenic atrophy
Neurom...
Neurogenic atrophyNeurogenic atrophy
Diseases affecting lower motor neurons or
their axons are-
-Amyotrophic lateral scler...
Histological changesHistological changes
Denervation
randomly scattered atrophic fibers
Predominantly atrophy of type 2 fi...
Histological changesHistological changes
Denervated muscle fibers
Reinnervated by collateral sprouting. ,
all of the reinn...
NADH-TR stain-
Reinnervation
Target fiber-central pallor with darkly stained rim
MC in polyneuropathies,
Long standing de...
Inflammatory / immune mediatedInflammatory / immune mediated
myopathymyopathy
 Polymyositis
 Dermatomyositis
 Inclusion...
Inflammatory / immune mediatedInflammatory / immune mediated
myopathymyopathyPM
 Insidious onset
 Affects females 20-40 ...
Histological featuresHistological features
•Myonecrosis
•Intrafascicular- endomysial
mononuclear cells infiltration of T
c...
DermatomyositisDermatomyositis
 Present as Adult & Juvenile form
 Purple (heliotrope) discolorarion of the
upper eyelids...
DermatomyositisDermatomyositis
 Circulating anti-endothelial antibodies.
Activation of complement,
Endothelial swelling/ ...
DermatomyositisDermatomyositis
H & E
IBMIBM
 50 -70yrs
 M > F
 nonresponsive to steriod
 Asymetrical weakness of acral muscle esp. in extensor
compartment ...
IBMIBM
 Small group atrophy with angular fibres
Hypertrophic & split fibres
 Endomysial chronic inflammatory infiltrate
...
IBMIBM
Rimmed
vacuoles
H & E
Resin
Muscular dystrophiesMuscular dystrophies
 Duchenne muscular dystrophy
 Becker’s muscular dystrophy
 Limb girdle muscula...
Duchene muscular dystrophyDuchene muscular dystrophy
 MC muscular
dystrophy
 XR
 Age- 2-4 yrs
 Exclusively male
 Gene...
Duchene muscular dystrophyDuchene muscular dystrophy
 C/F -Difficulty in running , jumping getting
up steps
 Proximal > ...
Duchene muscular dystrophyDuchene muscular dystrophyBx-
 Excessive variation in size shape of fibers
 Large rounded fibe...
DMD
IHCIHC
normal DMD
Dystrophin in normal & in DMD
Becker’s MDBecker’s MD
X- linked
Less severe than DMD
Late onset
Pathogenesis – contains dystrophin in
normal amt. but...
Becker’s MDBecker’s MD
Microscopy
Endomysial fibrosis
Scattered large hypercontracted
Fibers of variable size with necr...
FSHDFSHD
 AD
 3-4 th
decade
 Involves voluntary muscles of face shoulder's , UL
 Bx –
- Atrophic fibers in absence of ...
Limb girdle dystrophyLimb girdle dystrophy
 AR
 Young adult
 Sarcoglycan deficiency – 17q12 (4types)
 Consists of myop...
Myotonic DystrophyMyotonic Dystrophy
Myotonia—Failure to relax after contraction d/t
membrane defect.
AD
Defective gene...
Myotonic DystrophyMyotonic Dystrophy
Bx
 Multiple pyknotic internal nuclei
 Selective atrophy of type 1 fibers
 Type II...
Distal myopathyDistal myopathy
1.Welander myopathy
2.Miyoshi myopathy
3.Hereditary variant of IBM
4.Non Scandinavian myopa...
OPMDOPMD
 Late onset myopathy
 Ptosis, Opthalmoplegia & dysphagia
 Benign course.
 Bx- -
-Mild dystrophic change with ...
Developmental disorders ofDevelopmental disorders of
skeletal musclesskeletal muscles
Floppy infant with delayed motor
de...
Centronuclear MyopathyCentronuclear Myopathy
AD/ AR/ X linked
CF- Presents from in infancy till 7 th decade.
Extraocula...
Centronuclear MyopathyCentronuclear Myopathy
Bx –
High proportion of fibres
with central nuclei,
+nce of perinuclear hal...
Congenital fibre type disproportionCongenital fibre type disproportion
 Atrophy of type 1 and hypertrophy of type 2 fiber...
Myofibrillar myopathiesMyofibrillar myopathies
Group of disoders with genetic defects
manifesting myofibrillar & cytoskel...
Central core diseaseCentral core disease
 AD
 Chr.19q13.1
 Affects RYR1gene – inv. Ryanodine receptor prot.
 Type 1 fi...
Multi core diseaseMulti core disease
 Congenital, non progressive myopathy
 Generalised weakness and hypotonia.
 Type 1...
Nemaline rod myopathyNemaline rod myopathy
 More prevalent in females.
 AD/AR
 Mutation of 5 thin filament genes- TPM3 ...
Desmin MyopathyDesmin Myopathy
 AD
 Childhood
 Missense mutation/ del. – located on 2q35 encodes
desmin..
 Distal weak...
Neuromuscular junction disorderNeuromuscular junction disorder
 Immune mediated
 Slowly progressive
 EMG- diagnostic
 ...
Metabolic diseasesMetabolic diseases
CHO storage -
 Acid maltase deficiency
 Mc-Ardle
 PFK deficiency
Lipid storage d...
Metabolic diseasesMetabolic diseases
1) Acid maltase deficiency
•Type -2 glycogenosis, AR
•mutation at chr. 17 - Mc at exo...
Acid maltase deficiencyAcid maltase deficiency
Acid maltase deficiency-H & E PAS
•BX - PAS+ve diastase labile vacuoles of ...
Metabolic diseasesMetabolic diseases
2) CHO storage -Mc-Ardle disease.
 Type 5 glycogenosis-myophosphorylase deff.
 AR p...
Metabolic diseasesMetabolic diseases
3) PFK deficiency
Type 7 glycogenosis / Tarui disease
AR, mut.12q13
 Present in c...
Lipid storage diseasesLipid storage diseases
Cytosol Inner mito. Mitochondria
membrane
Acyl- Carnitine Carnitine Acyl
coA ...
Lipid storage diseaseLipid storage disease
Deletion of either I.e. CPT 1 & CPT 2 lead
to myopathy
Histology – Myofibrils...
Mitochondrial MyopathiesMitochondrial Myopathies
1) Kearns Sayre Syndrome
2) MELAS
3) MERRF
Deficiencies of enzymes of the...
Mitochondrial MyopathyMitochondrial Myopathy
The red color -due to large numbers of
abnormal mitochondria that represent ...
ChannelopathiesChannelopathies
Myopathies with periodic paralysis-
Due to abnormality in Na/Ca channels
C/F myotonia , ...
Toxic & Drug induced myopathyToxic & Drug induced myopathy
Chloroquine - vacuolar myopathy – proximal
myopathy
Procamami...
Biochemical investigations in muscleBiochemical investigations in muscle
diseasedisease
 Always done before biopsy.
CK-
...
Biochemical investigations inBiochemical investigations in
muscle diseasesmuscle diseases
D/D of elevation of CK-
1. Motor...
Biochemical investigations in muscleBiochemical investigations in muscle
diseasesdiseases
3. CK isoenzymes are not advocat...
SummarySummary
What to be seen on
1. Transverse
 Most of abnormalities are
seen on TS
 Size, shape, position of
nuclei, ...
Atrophy
Angular
Random atrophy+ Necrosis
1.Neurogenic-
Group
atrophy,targets
Type grouping
2. LGD-
hypertrophy,
fibresplit...
Perifascicular atropy
Normal histology of MuscleNormal histology of Muscle
Muscle biopsy
Muscle biopsy
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Muscle biopsy

  1. 1. Muscle biopsy & interpretationMuscle biopsy & interpretation Dr. Rashmi Gujarathi
  2. 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. 3. Normal structure & histology of muscleNormal structure & histology of muscle
  4. 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. 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. 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
  7. 7. Normal histochemistry of MuscleNormal histochemistry of Muscle NADH-TR II I
  8. 8. Muscle Biopsy - IntroductionMuscle Biopsy - Introduction  Introduced by Duchenne – 1868  Simple but invasive procedure  Interpretation requires detailed clinical history , physical examination, biochemical studies.
  9. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
  21. 21. Abnormalities seen on Paraffin /Abnormalities seen on Paraffin / frozen sectionsfrozen sections Paraffin section  Nuclear changes  Fiber regeneration  Fiber necrosis  Hyaline fibers  Inflammation  Ring fibers  Fibrosis & fatty infiltration Frozen section  Fiber shape  Changes in histochemical profile  Fiber spiltting  Mottled fibers  Cores & targets  Nemaline rods  Mitochondrial abnormalities  Vacuolar change
  22. 22. General abnormalities of muscleGeneral abnormalities of muscle in response to injuryin response to injury
  23. 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. 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. 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. 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. 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. 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. 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. 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.
  31. 31. Selective atrophySelective atrophy Type 1 atrophy  Myotonic dystrophy  Nemaline myopathy  Distal myopathy  Centronuclear myopathy  Congenital fiber type disproportion. Type 2 atrophy  Corticosteroid therapy  MG  Disuse atrophy  Acute denervation  Paraneoplatic myopathy.
  32. 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. 33. Fiber hypertrophyFiber hypertrophy  Type 1:  ISMA  Type 2:  Runners sprinters  Congenital fiber type disproportion •Limb – girdle dystrophy IBM Myotonia congenital Acromegaly
  34. 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
  35. 35. Type groupingType grouping ATPase I II
  36. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
  52. 52. Inflammatory / immune mediatedInflammatory / immune mediated myopathymyopathy  Polymyositis  Dermatomyositis  Inclusion body myositis  Viral myositis-Influenza, Cox-sackie A & B  Pyomyositis -Drug addicts and immunocompromised  Granulomatous inflammation -TB & sarcoidosis  Parasitic infestation-Trichinella & cysticercosis
  53. 53. Inflammatory / immune mediatedInflammatory / immune mediated myopathymyopathyPM  Insidious onset  Affects females 20-40 yrs.  S/S-Symmetrical proximal muscle weakness, - Abrupt in onset, rapidly progressive period of  Increased ESR, CPK.  Cell-mediated autoimmune disorder in which cytotoxic T-cells and macrophages invade and destroy myofibers.
  54. 54. Histological featuresHistological features •Myonecrosis •Intrafascicular- endomysial mononuclear cells infiltration of T cells surrounding non-necrotic fibres. •Early phase- necrotic fibers- hypereosinophilic, granular, nuclear pyknosis •Pale vacuolated fibers with myophagocytosis.
  55. 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. 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
  57. 57. DermatomyositisDermatomyositis H & E
  58. 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. 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
  60. 60. IBMIBM Rimmed vacuoles H & E Resin
  61. 61. Muscular dystrophiesMuscular dystrophies  Duchenne muscular dystrophy  Becker’s muscular dystrophy  Limb girdle muscular dystrophy  Fascioscapulohumeral dystrophy  Myotonic dystrophy  Distal myopathy  Oculopharyngeal muscular dystrophy
  62. 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. 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. 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
  65. 65. DMD
  66. 66. IHCIHC normal DMD Dystrophin in normal & in DMD
  67. 67. Becker’s MDBecker’s MD X- linked Less severe than DMD Late onset Pathogenesis – contains dystrophin in normal amt. but ABNR structure/ size. C/F -Pelvic girdle & thigh muscles involved first
  68. 68. Becker’s MDBecker’s MD Microscopy Endomysial fibrosis Scattered large hypercontracted Fibers of variable size with necrosis & regeneration ATPase
  69. 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. 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. 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. 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. 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.
  74. 74. OPMDOPMD  Late onset myopathy  Ptosis, Opthalmoplegia & dysphagia  Benign course.  Bx- - -Mild dystrophic change with internalization of nuclei. -Fiber atrophy. -Interstitial fibrosis -Sometimes rimmed vacuoles present
  75. 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. 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. 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. 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. 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. 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. 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. 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. 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
  84. 84. Neuromuscular junction disorderNeuromuscular junction disorder  Immune mediated  Slowly progressive  EMG- diagnostic  1) MG  2) Lambert- Eaton syndrome  Bx- often normal/ nonspecific pattern of atrophy MG- mod.- severe type-2 fibre atrophy
  85. 85. Metabolic diseasesMetabolic diseases CHO storage -  Acid maltase deficiency  Mc-Ardle  PFK deficiency Lipid storage diseases  Carnitine deficiency  Carnitine Palmitoyl transferase deficiency Channelopathies
  86. 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. 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. 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. 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. 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. 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. 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.
  93. 93. Mitochondrial MyopathyMitochondrial Myopathy The red color -due to large numbers of abnormal mitochondria that represent a compensatory proliferation. Ragged- Coarse and disorganized fibers
  94. 94. ChannelopathiesChannelopathies Myopathies with periodic paralysis- Due to abnormality in Na/Ca channels C/F myotonia , relapsing episodes of hypotonic paralysis EM -Vacuoles represent dilated sacs of sarcoplasmic reticulum Bx- non specific findings- -Variation in fibre size -Fibre regeneration & degeneration -Inc.in internal nuclei -Endomysial fibrosis -Vacuolar change
  95. 95. Toxic & Drug induced myopathyToxic & Drug induced myopathy Chloroquine - vacuolar myopathy – proximal myopathy Procamamide & Penicillamine - inflammatory myopathy Steriod – type 2 Atrophy Ethanol,AZT - Subacute necrotising myopathy Hypothyroidism –Atrophy internalization of nuclei Tyrotoxicosis- Necrosis degeneration, regeneration, lymphocytic infiltrate
  96. 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. 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. 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. 99. 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
  100. 100. Atrophy Angular Random atrophy+ Necrosis 1.Neurogenic- Group atrophy,targets Type grouping 2. LGD- hypertrophy, fibresplitting Myopathic Inflammation Endomysial fibrosis Nuclear changes Sarcoplasmic changes1.PM, IBM- Endomysial 2.DM- Perivascular 3.FSHD -Perivascular - Dysrophy 1, MD-Multiple nuclei 2. Centonuclear-central nuclei in each fibre 1.Ring fib-MD 2.Core- Centralcore/multicore 3.Nemaline rods- Nemaline myo. 4.Vacuole-Storage dis. 5.RRF- Mitochond.myo.
  101. 101. Perifascicular atropy
  102. 102. Normal histology of MuscleNormal histology of Muscle

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