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MUSCLE BIOPSY.pptx
- 1. MUSCLE BIOPSY PRESENTER :DR VIBHA NAIR MODERATOR : DR DEEPAK P J
- 2. OVERVIEW • Histology • Typesof muscle fibres • Indications of muscle biopsy • Procedure • Stains
- 3. • General abnormalitiesin response to injury • Diseases • Biochemical investigations • Pitfalls
- 9.
- 10. INDICATIONS FOR MUSCLEBIOPSY • General reasons • Weakness of uncertain cause • Generalized, proximal, floppy infant syndrome • Muscle pain , cramps ,stiffness • Persistently elevated muscle enzymes (CK) • Specific reasons • Hereditary muscle disease in other family members • Carrier detection
- 11. • Systemic connectivetissue disease and vasculitis • Certain metabolic diseases such as storage disease • Suspicion of steroid myopathy in treated myositis • Conflicting clinical ,EMG or laboratory findings • Confirm/ reinforce clinical diagnosis
- 12. CONTRAINDICATIONS • Electrolyte disturbance •Malignant hyperthermia • Periodic paralysis ,endocrine myopathies • Myasthenia syndromes • Poor nutrition ,prior trauma • Coagulation disorder
- 13. BIOPSY-PROCEDURE BIOPSY SITE Clinical historyis important • Representative of disease • Muscle with active disease process • Severely affected or previously traumatized muscle is avoided • From belly of muscle , away from tendon • Deltoid, biceps ,quadriceps
- 15. TECHNIQUE • Needle biopsy-Fast , simple , less traumatic But very small tissue • Open biopsy- Do not crush or overstretch Kept in saline moistened gauze
- 16.
- 17. • 2 unfixedspecimens in saline gauze • Clamped and unclamped Clamped • EM in glutaraldehyde • Frozen in isopentane Unclamped • Paraffin section - cross and longitudinal section • Additional frozen section for special study
- 18. Do not… • Donot directly immerse in ice • Do not directly immerse on saline • Do nor crush • Do not stretch
- 19. PROCESSING • Isometric state •Muscle clamp -isometric state • Fixed in 10 % buffered formalin • 1 x 0.5 cm • Resin ,EM or paraffin • Fresh sample • 1 x 0.5 x 0.5 cm • Frozen section
- 22. FREEZING MUSCLE BIOPSIES •Label and place in cryostat to cool • Pour liquid nitrogen into Dewar flask kept in freezer • Pour cold isopentane • White drops form on bottom of cup • Cut 5 -7 mm long sections • Powder the muscle with baby powder
- 23. • Place thetissue for freezing on a stiff paper • Vput a drop of freezing medium • Immerse in isopentane for 10 seconds • Store in -70 degree freezer • For biochemical studies –frozen en bloc in liquid nitrogen
- 24. STAINS Non enzymatic stains •Hematoxyline and eosin • Phospho tungstic acid hematoxylin (PTAH) – cross striations • Masson trichrome • Modified Gomori trichrome • Van gieson • PAS for glycogen • Fat stains – Oil red O
- 25. Enzymatic stains • Phosphorylase •Adenosine tri phosphatase at pH 9.4 • NADH – TR - mitochondria • SDH
- 27.
- 28. Observation in Hand E • Variation in fasicular architecture • Variation in fibre size,shape • Changes in sarcoplasm • Nuclear characteristics-position of nuclei
- 29. • Necrosis anddegeneration of muscle fibres • Fibre regeneration • Type and distribution of inflammatory infiltrate • Interstitial changes
- 30.
- 31.
- 32.
- 33. Nuclear changes • Normallyperipheral nuclei • Nuclear internalisation
- 35. Ring fibres • Ringformed by peripheral bundle of myofibrils directed circumferentially • Seen in • Limb girdle dystrophy • Myotonic dystrophy
- 36. Hyaline fibres • Deeplystained • Early stage of cell necrosis • Duchenne muscular dystrophy
- 37. Fibre necrosis • Moreintensely eosinophilic • Pyknotic nuclei • Sarcplasmic vacuoles • Phagocytosis • Seen in • Duchenne muscular dystrophy • Inflammatory myopathies
- 39. Fibre regeneration • Basophiliccytoplasm • Increased number of nuclei and larger • Arise from • Sprouts of sarcoplasm • Satellite cells
- 40. Inclusions • Seen in •Inclusion body myositis • Oculopharyngeal dystrophy • Inclusion is eosinophilic , smudged • EM-bundles of filaments
- 41. Inflammation • Chiefly lymphocytes •Seen in • Polymyositis • Dermatomyositis • Inclusion body myositis
- 43. Fibrosis and fattyinfiltration • Chronic Neuromuscular Disease
- 44. Observation in frozensection
- 45. Atrophy and hypertrophy Atrophy •MC-denervation atrophy • Disuse , ageing, ischemia, poor nutrition Hypertrophy • Exercise ,compensatory reaction
- 46. Atrophy • Selective process •Affects only one fibre type • Grouped atrophy –c/c neurogenic disorder • Pan fasicular atrophy – ISMA • Perifasicular atrophy – Dermatomyositis
- 51. Hypertrophy • Type 1hypertrophy • Infantile spinal muscular atrophy • Type 2 hypertrophy • Runners , sprinters • Both • LGD ,IBM , myotonia congenita
- 54. Fibre shape • Normal– polygonal • Muscular dystrophy – rounded fibres • Denervating diseases - angular fibres
- 55.
- 56. Fibre splitting • Splitinto smaller subunits • Seen in LGD, IBM, denervation
- 57. Mottled fibres • Minutezones of weak enzyme activity • Lack of mitochondria and destruction of myofilaments • Seen in • Fascioscapulohumeral dystrophy • LGD
- 58. Cores and Targets •Cores are • regions of depleted enzyme activity • Seen in neurogenic atrophy • Targets are • 3 zone structure • Seen in neurogenic atrophy • Greater diameter than cores,Only in few sarcomeres
- 60. Nemaline rods • Threadsor rods tend to cluster beneath sarcolemma • Visualised in RTC on frozen section • Oblong or rectangular structures with a greatest dimension 6-7 micrometre • Seen in • Nemaline myopathy • Muscular dystrophy • Polymyositis
- 62. Mitochondrial abnormalities • Raggedred fibres • Mitochondria seen as dark granular deposits
- 63. Vacuolar change • Vacuolescontain glycogen or lipid • Stain with PAS or Oil red O • Lipid storage diseases • Rimmed vacuole – oculopharyngeal dystrophy,IBM
- 66.
- 67. CLASSIFICATION Muscular dystrophies • Duchene, Becker muscular dystrophy • Emery Dreifuss muscular dystrophy • Fascioscapulohumeral dystrophy • Limb girdle dystrophy Myotonic disorders • Myotonic dystrophy • Myotonia congenita
- 68. Inflammatory myopathy • Dermatomyositis •Polymyositis • Inclusion body myositis • Infectious myositis Denervating diseases • Spinal muscular atrophy
- 69. Congenital myopathies • Centralcore disease • Nemaline rod myopathy • Centronuclear myopathy Storage diseases • Glycogen storage diseases • Lipid storage diseases
- 70. Mitochondrial myopathies Toxic myopathies Neuromusculardiseases • Myasthenia gravis • Lambert Eaton syndrome
- 71. DUCHENE MUSCULAR DYSTROPHY •X linked recessive inherited disorder • DMD gene located on Xp21. • Dystrophin gene mutation • Affects boys,difficulty to stand or walk • Pseudohypertrophy of muscles-calves, buttocks • Cardiomyopathy • Raised CK
- 72. • Fibre necrosis,regeneration • Atrophic myofibres • Hyaline fibres • Fatty infiltration BECKER MUSCULAR DYSTROPHY • Milder form of X linked dystrophy
- 74. EMERY DREIFUSS MUSCULARDYSTROPHY • Mutations in genes that encode lamina proteins – emerin and lamin • Humeroperoneal weakness , cardiomyopathy
- 75. FASCIOSCAPULOHUMERAL DYSTROPHY • ADinheritance • Overexpression of DUX4 • Face , shoulder , upper extremity • Atrophic muscle fibres, clustered or grouped together • Moth eaten fibres • Perivascular lymphocytes
- 76. LIMB GIRDLE DYSTROPHY •Collection of myopathies • Proximal muscle groups ,shoulder ,pelvic girdle • AD and AR inheritance • Mutations in genes encoding sarcoglycans • Young adults • Pseudohypertrophy
- 77. MYOTONIC DYSTROPHY • Autosomaldominant multisystem disorder • Expansion of CTG triple repeat in non coding region of DMPK • Facial muscle weakness, ptosis, dysphagia • Inability to relax after contraction • Cataract, testicular atrophy, diabetes mellitus ,cardiomyopathy
- 78. • Pyknotic internalnuclei • Type 1 fibre atrophy • Ring fibres • Reactive fibrosis
- 79. DERMATOMYOSITIS • Autoimmune disease,autoantibodies •Children and adults • Damage to small blood vessels cause muscle injury • C5b-9 • Heliotrope rash,gottron papule • Inflammatory infiltrate-lymphocytes and plasma cells • Anti Mi2,Anti Jo 1,Anti P155/P140
- 80. • Perimysial andperivascular inflammatory infiltrate • Perifasicular atrophy • Segmental fibre necrosis
- 82. POLYMYOSITIS • Adult onset •No cutaneous manifestation • Symmetric proximal muscle involvement • Involvement of heart , lungs • Endomysial mononuclear inflammatory infiltrate • Denerating , regenerating and atrophic muscle fibres
- 83. INCLUSION BODY MYOSITIS •Late adulthood • Slow progressive muscle weakness,esp quadriceps • Elevated CK • Endomysial mononuclear infiltrate • Cytoplasmic inclusions • Rimmed vacuoles • Endomysial fibrosis
- 85. INFECTIOUS MYOSITIS • Bacterial– Staph aureus • Viral • Influenza • Cox sackie • HIV • Parasitic • Trichinella spiralis • Cysticercosis • Toxoplasma
- 86. Parasitic myopathy • Mostcommon parasites • Trichinella spiralis • Cysticercosis • Undercooked pork • Encapsulated cyst • Calcification • Granulomatous reaction
- 87.
- 88. TOXIC MYOPATHIES • Statins •Chloroquine • Alcohol • Thyrotoxic myopathy • ICU Myopathy • Hypothyroidism
- 89. DISEASES OF LIPID/GLYCOGENMETABOLISM • Inborn errors of metabolism • Carnitine palmitoyl transferase 2 deficiency • Lipid metabolism • Episodic muscle damage with exercise or fasting • Mc Ardle disease-Myophosphorylase deficiency • Glycogen storage disease • Episodic muscle damage with exercise
- 90. • Acid maltasedeficiency • Glycogen storage deficiency • Impaired lysosomal conversion of glycogen to glucose • Phosphofructokinase deficiency • Glycogen storage deficiency
- 91. MITOCHONDRIAL MYOPATHY • Manyorgan systems involved • Weakness ,elevated CK, rhabdomyolysis • Ragged red fibres
- 92. SPINAL MUSCULAR ATROPHY •Neuropathic disorder in which loss of motor neurons leads to muscle weakness • FLOPPY INFANT - Generalized hypotonia in infants with neurologic disease .D/D are • Congenital myasthenic syndrome • Congenital myotonia • Congenital muscular dystrophies
- 94. ION CHANNEL MYOPATHIES(CHANNELOPATHIES) • Groupof inherited diseases caused by mutations affecting the function of ion channel proeins • Hypo / hypertonia • Hypo / hyperkalemia • Cerebellar dysfunction • KCNJ2 ,SCN4A , CACNA1S ,CLC1 ,RYR1 mutation
- 95. CONGENITAL MYOPATHIES • Centralcore disease • Nemaline myopathy • Centronuclear myopathy
- 96. Central core disease •Mild non progressive proximal muscle weakness • Muscle fibres show single centrally located defect or core • Predominant type 1 fibre
- 97. Nemaline (rod) myopathy •Females • Facial and proximal limb muscles, facial dysmorphism • Nemaline rods ,fibre atrophy • RTC stain
- 98. Centronuclear myopathy • Childhoodto 7th decade • Extraocular palsies • Central or paracentral nucleus within muscle fibres • Sarcoplasm appears vacuolated in frozen section
- 99. MYASTHENIA GRAVIS • Chronicautoimmune disease • Circulating antibodies against postsynaptic acetyl choline receptors • Females • Extraocular , facial muscles • Type 2 fibre atrophy
- 100. IMMUNOHISTOCHEMISTRY • Dystrophin –DMD • Emerin , laminin A/C – EDMD • Caveolin ,dysferlin ,calpain – LGMD • CD 4,CD8 –Dermatomyositis, polymyositis • Ubiquitin – IBM • Desmin – central core disease
- 101.
- 102. LAB FINDINGS • Creatininekinase levels – to rule out certain categories of myopathies • High – dystrophinopathies – 200-300 times of normal • Intermediate – inflammatory myopathies – 20-30 times of normal • Low – neurogenic disorder – 2-5 times of normal
- 103. PITFALLS • No properclinical data-thyroid ,statin myopathy • Freezing , transport, storage artifacts • Submitted in saline – blown out, vacuolated • Crush by teeth of clamps –appears atrophic • Aged people ,those on steroids – fibre atrophy • Roughly handled specimen –pseudovasculitis • Fatty infiltration in obese people resembles c/c muscle disease
- 104. Summary • Normal checkerboard pattern • Indications of muscle biopsy • Clinical history is important • 2 specimens –fresh and fixed • Paraffin, EM, frozen section • Stains-H and E,PTAH,MT,MGT, enzymatic stains
- 105. • Response toinjury • Atrophy, necrosis, hyaline change, inflammation,inclusion • Cores, targets,ring fibres ,nemaline rods • Diseases • DMD,DM,PM,IBM,LGD,SMA,congenital myopathies • Lab findings-CK
- 106. REFERENCES • Sternberg’s DiagnosticSurgical Pathology,5 th Edition • Anderson’s Pathology,10th Edition • Pathologic Basis of Disease, Robbins and Cotran, South Asia Edition • Rosai and Ackerman’s Surgical Pathology,11th Edition • Bancroft’s Histological Techniques,8th edition • Manual of Surgical Pathology, Susan C Lester,3rd Edition • Di Fiore’s Atlas of Histology,12th Edition