Apporach based seminar of ,yopathies

1. Myopathies ..

2. NORMAL MUSCLE

3. Basic structure and terminology

5. • Each muscle has a characteristic ratio of type 1 to type 2 myofibers.
• In normal muscle, the 2 myofiber types are interspersed in a random
interdigitating pattern.
• Information about changes in the myofiber types in a muscle biopsy often
provides significant diagnostic clues.
• The innervation of a particular muscle fiber determines whether it is type
1 or type 2

6. MUSCLE BIOPSY
STAINS
Category Method Utility
Morphology
Hematoxylin and eosin Muscle fiber pathology; Nuclei
Verhof van Giesson (VvG)
Connective tissue; Vessel structure
Intramuscular nerve
Gomori trichrome Connective tissue; Nemaline rods
Fiber Type Enzymes
Myofibrillar ATPase
Muscle fiber type grouping or
Atrophy
ATPase pH 9.4
Myosin loss; Type 1 or 2 fiber
atrophy
ATPase pH 4.6 Type 2B muscle fibers
ATPase pH 4.3
Type 2C (Immature) muscle fibers
Blood vessels

7. Category Method Utility
Oxidative Enzymes
NADH-TR
Muscle fiber internal architecture;
Tubular aggregates; Cores
Succinate dehydrogenase
Mitochondrial pathology
Nuclear DNA encoded complex
Cytochrome oxidase
Mitochondrial pathology
Mitochondrial & Nuclear DNA
encoded
Glycolytic Enzymes
Phosphorylase Phosphorylase deficiency
Phosphofructokinase (PFK) PFK deficiency
Hydrolytic Enzymes
Acid phosphatase Macrophages; Lysosomes; Lipofuscin
Non-specific esterase
Macrophages; Lysosomes;
Neuromuscular & Myotendinous
junctions
Denervated (small angular) muscle
fibers
Acetylcholinesterase
Neuromuscular & Myotendinous
junctions
Alkaline phosphatase
Regenerating or immature muscle
fibers;
Immune disease of connective tissue
or capillaries;

8. Category Method Utility
Storage material
PAS Glycogen & Carbohydrate disorders
Alcian blue Mucopolysaccharide
Sudan black B Lipid storage
Oil red O Lipid storage
Other
Congo red Amyloid; Inflammation; Vacuoles
Myoadenylate deaminase AMPDA deficiency
Methyl green pyronine RNA
Acridine orange RNA
Von Kossa Calcium
Fixed muscle Toluidine blue Muscle fibers; Capillaries

9. Histology of Normal
Skeletal Muscle
Frozen sections
1. Hematoxylin and eosin stain
Cross section shows several
fascicles surrounded by and
separated from each other by a
thin layer of perimysium. The
muscle fibers are of relatively
uniform size and shape, with
nuclei located at the periphery
of the cell.
High power view reveals the
thin, delicate endomysial
connective tissue, the
normal appearance of the
sarcoplasm, and the
peripherally placed nuclei.
Capillaries, normally
indistinct, are found at the
corners between myofibers.

10. 2. NADH stain (nicotinamide adenine dinucleotide tetrazolium reductase
stain)
• Low power view demonstrates 2
populations of myofibers.
• Type 1 myofibers stain more
darkly than type 2 myofibers
because of the greater use of
aerobic metabolism by type 1
fibers.
• The sarcoplasm stains fairly
uniformly across the cell.
HPV reveals the punctate
distribution of the stain
throughout the cell because of
its predominant colocalization
with mitochondria in the
intermyofibrillar network

11. 3. Myosin ATPase stain
• Myosin adenosine triphosphatase (ATPase) at pH 10.5 stains type
2 myofibers brown.
• Type 1 fibers are stained with an eosin counterstain so that they
are visible.

12. 4. Myosin heavy chain immunohistochemical stain
• IHC fiber-typing stain for myosin
heavy chain, slow type, in which
type 1 myofibers are brown.
• The eosin counterstain makes
the type 2 myofibers visible with
a pink color
• IHC stain for myosin heavy chain,
fast type.
• Type 2 myofibers are brown, and
the type 1 myofibers are pink due
to the eosin counterstain

13. 5. Periodic acid-Schiff stain
• It stains sugar moieties so that glycogen, mucopolysaccharides, and
glycoproteins are highlighted.
• Most useful for evaluating glycogen storage disease.
• Also provides information about vascular structure.
• It can highlight fibers that are degenerating or necrotic and demonstrate
some inclusions.

14. 6. Modified Gomori trichrome stain
• The modified Gomori
trichrome stain is valuable
in evaluating mitochondrial
myopathies, inclusion body
myositis, and some other
disorders with intracellular
inclusions.
• The nuclei and mitochondria stain red
• The cytoplasm is mostly blue-green
• The connective tissue is green.

15. 7. Sudan Black stain
• Sudan Black stain for lipid demonstrates slightly more staining of type 1 myofibers
because of their higher lipid content due to their greater dependence on aerobic
metabolism compared with type 2 myofibers.
• Hereditary and acquired disorders of lipid metabolism show excessive staining of
fibers.
• Some of the mitochondrial myopathies are associated with increased intracellular
lipid content.

16. Paraffin sections
• A paraffin section provides
more cytologic detail than
frozen material, so it improves
identification of cells involved
in inflammatory disorders.
• Detailed structure of vascular
walls can be seen in paraffin
sections.
• This section is usually larger
than a frozen section and
therefore offers more material
for examination
• LPV shows fibers aligned linearly in
longitudinal section.
• Most of the nuclei are myofiber nuclei, but
some are also capillary endothelial nuclei.
H & E
High power view
• Striations produced by the sarcomeres are
clearly visible.

17. When indicated, special stains can be performed on the paraffin
specimen.
These are :
1. Special stains for microorganisms, such as bacteria, fungi, and
parasites
2. Elastic stains to evaluate for disruption of the elastic lamina of
arteries in vasculitis
3. Immunohistochemical stains to determine the subtypes of
inflammatory cells within an infiltrate and a variety of other
purposes
4. In situ hybridization for identification of viruses
5. Congo red or thioflavin S staining for amyloid

18. MUSCLE PATHOLOGY

19. CLINICAL FEATURES
1. Weakness, which predominantly affects the proximal
muscle groups (eg, shoulder and limb girdles)
2. Myalgia, or muscle aching, which is present in some
patients with inflammatory myopathy
3. Relative preservation of muscle-stretch reflexes
4. Absence of abnormalities of somatosensation

20. Neurogenic Changes in
Muscle Biopsy
Neurogenic disorders have the following
characteristics on muscle biopsy:
1. Angulated atrophic fibers.
2. Group atrophy
3. Fiber-type grouping.
4. Target fibers - observed in active
denervation.
5. Nuclear clumps

21. Many biopsy samples with inflammation also demonstrate evidence of
neurogenic change. . A few possible mechanisms are :
1. Myogenic denervation, in which the sick muscle fibers lose their
innervation
2. Innocent bystander mechanism, in which the inflammatory process
overruns and entraps the intramuscular nerve twigs, resulting in
denervation
3. Concurrent inflammation of the nerves.

22. Muscle Biopsy in
Myopathy
Myopathy can have the following
characteristics on muscle biopsy:
1. Myofiber necrosis .
2. Myophagocytosis
3. Regeneration.
4. Rounded, atrophic fibers .
5. Myofiber hypertrophy and splitting

23. Muscle Biopsy in
Myopathy…
6. Increase in internal nuclei.
7. Fibrosis

24. Muscle Biopsy in
Myopathy…
Other ancillary findings that can be
found in myopathic muscle biopsy :
1. Nuclear chains .
2. Moth-eaten fibers
3. Ring fibers
4. Whorled fibers
5. Vacuoles

25. Muscle Biopsy in
Myopathy…
6. Inclusions
7. Inflammation

26. Segment of dense epimysial
connective tissue traverses the
field, forming a partial U shape.
Myofibers in its vicinity vary in size
and shape and have increased
numbers of internal nuclei. These
findings are expected at a normal
myotendinous junction and
therefore should not be
misinterpreted.
Myotendinous junction on hematoxylin and
eosin (H&E) paraffin section.

27. Muscle disorders..
Five important groups of disorders that can be diagnosed by muscle biopsy
include the following:
• Myositis
• Muscular dystrophies
• Glycogen storage diseases (metabolic myopathy)
• Mitochondrial myopathies (metabolic myopathies)
• Congenital myopathies

28. Myositis
The term myositis
refers to
inflammatory
disease of muscle

29. Polymyositis
• Polymyositis ("inflammation of many muscles") is a type of chronic
inflammation of the muscles (inflammatory myopathy) related to
dermatomyositis and inclusion body myositis.
• Strikes females with greater frequency than males.
• PM is an autoimmune disease in which lymphocytes (predominantly T cells)
have become sensitized to muscle antigens. A cell-mediated response leads
to the necrosis of muscle fibers and subsequent regeneration .
• Approximately 75% of cells in the endomysium are CD8+ cytotoxic
lymphocytes.

30. • Several autoantibodies can be demonstrated in the serum of patients with
PM. Many of these detect overlap syndromes.
• Anti-Jo-1, an anti-synthetase antibody, is a marker for coexistent
interstitial lung disease.
• Anti-PM-1 (anti-PM-scl) antibody is associated with PM and scleroderma.

31. Signs and symptoms
• Pain
• Marked weakness and/or loss of muscle mass in the proximal
musculature.
• Thickening of the skin on the fingers and hands (sclerodactyly)
• Dysphagia (difficulty swallowing) and/or other aspects of oesophageal
dysmotility occur in as many as 1/3 of patients.
• Foot drop in one or both feet in advanced polymyositis.
• Interstitial lung diseases.

32. The following are the key diagnostic
pathologic features of polymyositis :
1. Endomysial inflammation
2. Invasion of nonnecrotic myofibers
by autoaggressive lymphocytes.

33. • The following pathologic features are also often found in polymyositis, but
they are not diagnostically specific and can be found in a variety of
myopathic disorders:
1. Myofiber necrosis
2. Myophagocytosis
3. Internal nuclei
4. Myofiber atrophy
5. Regeneration
6. Fibrosis
In long-standing disease, fiber
necrosis and the inflammatory
reaction are generally absent.
Instead, the biopsy is likely to reveal
fiber atrophy and endoperimysial
fibrosis.

34. Dermatomyositis
• DM is essentially an angiopathy involving muscle and skin.
• Subcutaneous tissues, the intestine, and the peripheral nerves may also
be involved in children.
Epidemiology :
• Children (juvenile DM): most 10 to 15 years old at diagnosis
• Adults: peak onset between ages 45 and 60 years
• Women more likely affected than men

35. Site of Involvement :
» Face, especially periocular region
» Extensor surfaces of extremities, especially knuckles
Clinical Findings :
» Violaceous poikiloderma (hyper-/hypopigmentation, telangiectasia,
atrophy)
» Heliotrope sign (periocular discoloration)
» Gottron’s papules (violaceous discoloration over knuckles)
» Ragged cuticles (cuticular dystrophy)
» Nailfold telangiectasia
» Pruritus
» Calcinosis cutis (juvenile DM)

36. Pathologic features :
1. Chronic inflammation
2. Perifascicular atrophy
3. Myofiber necrosis and/or
regeneration
4. Complement deposition in
microvessel walls
5. Tubuloreticular inclusions
6. HLA class I antigen expression on
the surfaces of myofibers

37. Inclusion body myositis
• It is an inflammatory myopathy that causes a withering of acral muscles,
especially those in the extensor compartment of the arm.
• Affects persons between the ages of 50 and 70 years
• Men affected more than women, in whom the pace of deterioration is
slow.
• In contrast to PM and DM, inclusion body myositis does not respond to
steroid therapy.

38. Inclusion body myositis
Pathologic features of IBM:
• Chronic inflammation
• Invasion of nonnecrotic
myofibers by autoaggressive
lymphocytes
• Myofiber hypertrophy
• Atrophy
• Rimmed vacuoles
• Eosinophilic inclusions
• Tubulofilamentous
inclusions
• Occasional ragged red fibers

39. Two additional disorders
with inflammation
• Trichinosis
• Drug reaction

40. Muscular Dystrophies
• Duchenne and Becker dystrophies are now classified as
dystrophinopathies, because they are caused by mutations in the gene for
the protein dystrophin.
• Nowadays muscle biopsy for many muscular dystrophies, are usually done
in which immunohistochemistry for specific muscular dystrophy proteins
can narrow the diagnostic possibilities.

41. Duchenne muscular
dystrophy
• DMD gene is located in band Xp21 of the short arm of the X chromosome.
The gene has been identified as a 14-kb coding sequence within 2.5 to 3.0
Mb of DNA, and it is the largest known human gene.
• Presumably, the extraordinary size of the gene is responsible for the high
rate of mutations, accounting for approximately two-thirds of DMD cases.
• Patients with DMD lack the gene product dystrophin, a 427-kd, rod-shaped
protein that is found predominantly in skeletal muscle and also in smooth
and cardiac muscle

42. Signs and Symptoms..
• DMD almost exclusively affects boys .
• pelvifemoral and scapulohumeral weakness, sparing the muscles of facial
expression and swallowing.
• A paradoxical enlargement of selected muscles that also are weak and
flabby. This pseudohypertrophy, which is associated with fatty infiltration
and reactive fibrosis, is especially apparent in the calves and buttocks.
• Death is often hastened by an insidious cardiomyopathy leading to sinus
tachycardia, cardiac arrhythmias, and congestive heart failure.
• An extremely high level of serum creatine kinase is an early indicator of
this form of dystrophy, and it may actually precede the pathologic
alterations in muscle.

43. Biopsy features are :
• Endomysial and perimysial fibrosis
• Increased variability of myofiber size
caused by the presence of both
atrophy and hypertrophy with fiber
splitting
• Myofiber necrosis
• Increased internal nuclei
• Opaque fibers

44. • Dystrophin immunohistochemistry

45. Becker muscular
dystrophy
• Becker dystrophy, a mild form of X-linked dystrophyis allelic with DMD .
• The symptoms in Becker dystrophy are less severe than in DMD, and the
rate of progression is slower.
• Although patients with DMD lack dystrophin, the muscle of patients with
Becker dystrophy typically contains dystrophin, which may be sufficient in
quantity but is abnormal in molecular size or structure.
• The pathologic changes are similar to, but less severe than, those of DMD.

46. Biopsy features include :
• Myofiber necrosis
• Increased variability of myofiber size with myofiber atrophy and
hypertrophy
• Myofiber regeneration
• Increase in internal nuclei

47. • Dystrophin immunohistochemistry
Positive for antibody to the C-terminal
region of dystrophin
Negative for antibody to the N-terminal
region of dystrophin

48. Congenital muscular
dystrophy
• Most common is abnormality of laminin α-2, also known as merosin,
which is a component of the basal lamina of skeletal muscle, located just
external to the myofiber.
laminin α-2 (merosin) immunohistochemistry :
No labeling with an antibody to laminin
α-2
Normal

49. Glycogen Storage
Diseases
• Glycogenoses are inherited inborn errors of glycogen metabolism
• 9 of these disorders affect skeletal muscle.
• The 2 most commonly encountered by muscle pathologists are :
• type II glycogenosis (acid maltase or alpha glucosidase deficiency)
• type V glycogenosis (myophosphorylase deficiency).
• Type II glycogenosis is the only glycogen storage disease that is also a
lysosomal storage disease

50. Biopsy features..
• Clear vacuoles on hematoxylin and
eosin (H&E)
• Periodic acid Schiff (PAS)–positive
staining of these vacuoles, with
disappearance of staining following
digestion with diastase
• Acid phosphatase stain, which
demonstrates excessive staining for
lysosomes that correspond to the
vacuoles seen with the H&E stain

51. • Staining for myophosphorylase activity
Normal Absent in type V glycogenosis

52. Mitochondrial
Myopathies
• These include :
– Kearns-Sayre syndrome
– myoclonus epilepsy with ragged red fibers (MERRF)
– mitochondrial encephalomyopathy with lactic acidosis and stroke like episodes
(MELAS)
– Leber hereditary optic neuropathy (LHON).
• Many of these disorders present with a combination of central nervous
system (CNS) disease and myopathy and are referred to as
encephalomyopathies.
• The common etiology is the presence of mutations that affect mitochondrial
function

53. Characteristic findings..
• On trichrome stain, ragged red fibers
have a peripheral rim of red material
caused by the subsarcolemmal
aggregation of mitochondria
• Dense peripheral staining for the
activity of succinic dehydrogenase
(SDH)
• The presence of many fibers with
absence of the activity of cytochrome
oxidase (COX)
• Combined SDH/COX staining
demonstrates that many of the COX-
negative fibers are the ragged red
fibers.

54. Congenital Myopathies
• Congenital myopathies are a diverse group of disorders with the common
feature that each has its own characteristically distinctive morphologic-
pathologic finding.
• In central core disease, the central region of many myofibers has abnormal
structure.
• In nemaline myopathy, the fibers contain aggregates of rodlike material
seen on trichrome stain.
• In centronuclear (or myotubular) myopathy, the main pathologic finding is
centrally located nuclei in myofibers and fibers that appear immature.
• In congenital fiber type size disproportion, type 1 myofibers are small, and
type 2 myofibers are of normal size.

55. THANK YOU