How to work up a muscle biopsy

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Introduction   Evaluation   Normal Histological Parameter      Histochemistry and Immunohistochemistry   Organelle    Glossary

Introduction to Muscle Biopsy Head

Useful web sites:

Washington University Neuromuscular Disease Center Staining Protocol

Washington University Neuromuscular Disease Center

Mitochondrial myopathy link  

Muscular Dystrophy Association (MDA)

Negative result: A negative biopsy does not mean that the myopathy is not there. In some myopathies, only certain muscle groups are affected. The absence of evidence is not the evidence of absence..

Procuement: 1 piece for HE (formalin), histochemistry (fresh), EM (gluteraldehyde), and biochemistry/genetics (fresh) respectively.

For HE: both longitudinal and cross sections should be cut. The best way to do it is to process the whole piece of tissue (fixed in a clamp). After the tissue is infiltrated with wax, cut the longitudinal and cross sections respectively before embedding. Five levels should be obtained, especially for cases suspected for vasculitis.

Biopsy sites:

·        Take biopsy from muscle with recent involvment, partial weakness, tender. In muscles with abnormal EMG, take biopsy from the contralateral side to avoid articacts produced by the electrodes. 

·         Do not take biopsy from the side where electromyography was done.

·         Do not take biopsy from muscle that is not affected. You will see nothing.

·         Do not take biopsy from muscle that is very weak. You will see only end stage changes.

·         Ocular muscles have very small motor units and it may be difficult to tell if it is a neurogenic myopathy or myopathic myopathy.

Hint:

·    Many metabolic and congenital myopathies, particularly the milder form, have late juvenile or adult onset. The phenotype in metabolic myopathies is particularly complex. Vladutiu GD. Muscle Nerve 2000 Aug;23(8):1157-9.

·         A variety of muscle diseases may produce identical clinical symptoms.

·        A single disease may produce several different patterns of injury.

·        Finally, a single pattern of injury may be observed in several diseases.

·        The ocular muscles are frequently spared from many myopathies but they are affected in mitochondrial myopathy, myotonic dystrophy, and congenital myopathies.

Evaluation Head  

History:  

Four major pieces of clinical information are critical for the pathologist:

Other helpful clinical information:

Laboratory investigation:

Creatine kinase level: The pathologist can often use the creatine kinase level to rule out certainh categories of myoapthies because different myopathies tend to generate a different levels of elevation in CPK.

Parameters to be evaluated:

Paraffin sections:

Histochemistry:

    Basic panel

    Extended panel

Electron microscopy

Normal Histologic Parameters Head

Shape and Diameter    Muscle spindle    Satellite cells    Fiber typing    Miscellaneous

Shape and Diameter: Head

Shape: Adult muscle fibers should look a little polygonal. If they look round then some pathologic process is probably present. In contrast, normal muscle fibers in children can look round.

Diameter:  Muscle diameter is an information particularly in infant and children.

    Normal variation:

·         The normal range of muscle diameter is around 40-100 micron. Because of the shrinkage artifact introduced by paraffin embedding, it is more reliable to assess fiber diameter and variation in fiber diameter in frozen sections. Paraffin embedding will induce a 30% reduction in diameter. As high as up to 30% reduction in size has been claimed but I suspect that this is an over estimation. A 10-20% reduction is a better estimate.

·         Proximal muscles have larger mean fiber diameter (85-90 micron) for power generation.

·         Distal muscles have a smaller mean fiber diameter (20 micron) for fine coordination.

·         In general, fiber diameter is smaller in children and elderly and larger in active adults.

·         Muscle fibers in man are usually larger (may be up to 10 mm) than fibers in women.

·         In infants and children, type 1 and 2 fibers are equal in size and have small variation.

·         In adult male, type 2 fibers are usually larger than type 1 fibers.

·         In adult female, type 1 fibers are slightly larger than type 2 fibers.

Determination of muscle diameter: The diameter chosen is the shortest dimension bisecting the muscle fiber in a plan through the center of the fibers. Diameter of muscle fiber for children at different ages:

Age

Diameter (mm)- Frozen

Diameter (mm)- Paraffin(assuming  15% reduction)

Diameter (mm)- Paraffin(assuming 30% reduction)

 

 

 

 

Birth

15

12.8

10.5

3 months

17

14.5

11.9

1 year 

18

15.3

12.6

3 years

20

17.0

14.0

4 years

22

18.7

15.4

6 years

27

23.0

18.9

8 years

35

29.8

24.5

10 years

40

34.0

28.0

12 years

50

42.5

35.0

>13 years

60

51.0

42.0

 

 

 

 

                Data after Engel, A, 1998

Muscle spindles: Head

·         Muscle spindles are mechanoreceptors arranged in parallel with the muscle fibers. They are more often seen in biopsies of small muscles or in biopsies from children. For unknown reasons, muscle spindles are rarely affected in many myopathies.

·         Length varies from 2 mm in infants to 7-8 mm in adult males. The total number of fibers varies from 2 to 15.

·         Innervation: Each spindle is innervated by one large afferent (sensory) myelinated fiber at the equatorial zone. Axons from g motor neurons and b motor neurons provide efferent innervation and originate from the distal zone.

·         The equatorial zone contains nuclei but no myofibrils and are unable to contract; the distal parts contain striated fibers and are able to contract

·         The capsule: The intrafusal fibers are contained with a fluid filled capsule that is composed of multiple flattened lamellae. The structure is similar to the peirneurium and, in fact, the capsule joins the perineurium of nerve supplying the spindle.

·         Cell types: Three types of striated fibers are present. Each spindle contains up to 10 chain fibers and 1-4 bag fibers. As a common feature, their nuclei are located in the equatorial region of the spindle. The nuclei are concentrated in the equatorial zone.

·         Nuclear bag 1-fiber: smaller than bag-2 fiber.

·         Nuclear bag 2-fiber: they are the largest and may contain up to 50 nuclei and measures up to 50 mm in diameter.

·         Nuclear chain fiber: they are the thinnest and about 10-15 mm in diameter.

·         Fibroblasts are also present. They lack basal lamina and can be readily recognized under electron microscope.

Tendon organ (Golgi organ): They are found in tendons lies at the transition between the tendon and muscle. It is composed of collagen fibers of the tendon intermingled with the nerve endings of a myelinated fiber. The fibers that branches within the collagen bundles contain no myelin. When the muscle contracts, the pressure on the nerve endings will generate nerve impulses.

Satellite cells: Head

·        They are small mononuclear cells that are normally found underneath the basal lamina of muscle fibers and are considered as the reserve myogenic precursor cells. About 3.8% of the nuclei of extrafusal human adult fibers are satellite cells [Schmalbruch H and Hellhammer U, 1976]. They are probably more numerous in newborn and infants. They must be distinguished from other cells, such as plasma cells, that may be occasionally found under the basal lamina in pathologic conditions.

·        They can be well demonstrated by electron microscopy, sometimes in semithin sections. They can be easily demonstrated by immunostaining for N-CAM; they also express vimentin.

·        Satellite cells are evenly spaced along the length of muscle fibers but they are increased in number in the motor end plate and in intrafusal fibers.

·         Activated satellite cells express Myo-D and myogenin [Rantanen J et al., 1995].

Fiber Typing: Head

Although both oxidative enzymes and ATPase can be used for fiber typing, ATPase is more reliable than oxidative enzyme histochemistry. Atrophic change can induce an increase in oxdative enzyme staining because the mitochondria and T-tubles may be more densely packed.

·        Type 1 fiber (slow twitch): light staining in ATPase reaction, pH 9.4. They are rich in mitochondria and triglyceride lipid. They contain less glycoen, less ATPase, and enzymes that are associated with glycogen metabolism. Muscles that are responsible for continual or postural functions, such as the calf and gluteal muscles, contain more type 1 fibers. Variation also increase with depth and the general trend is an increase of type 1 fibers in locations closer to the bone. At the time of birth, some type 1 fibers have an unusually large diameter and are formerly called “Wohfart type B” fibers.

·        Type 2 (glycolytic) fiber (fast twitch): dark staining in ATPase reaction, pH 9.4. They are rich in glycogen, myophosphorylase, phosphofructokinase and ATPase but contain less mitochondria and triglyceride lipid. Type 2 fibers are further divided into 2A, 2B and 2C. ATPase reaction at pH 4.6, type 2c is the darkest, then 2b and then 2a; type 1 is dark but not as dark as 2c.

·         Embryology: differentiation into a checkerboard pattern of type 1 and 2 fibers is present after about 28 weeks of gestation. Type 2C fibers seem to be the precursors of type 1, 2A, and 2B fibers. Type 2C fibers are very rare in adults. At the time of birth, about 10% of the fibers are type 2C fibers.

·         Ratio: In children, type 1 and type 2 fibers are in equal proportions. At about 1 years of age, type 1 fibers comprise 60-65% of the fiber and 2A is still the predominant type of type 2 fibers. In adult the ratio of type 1 to type 2 fibers can vary but a typical muscle contains approximately twice as many type 2 (60-65%) than type 1 fibers (35-40%).

Miscellaneous: Head

Close to tendon: 

·         Increased in variation of muscle fiber diameter

·         Increased in the amount of connective tissue

·         Increased in the number of central nuclei

End plate: features include an array of nuclei with both parallel and longitudinal arranagement in reference to the fiber. Small capillaries is also a constant feature which may appear as a few red cells if the end plate is seen en face.

Capillaries: can be visualized by PAS, Gordon and Sweet reticulin, immunostaining against factor VIII related antigens and also histochemistry using Ulex europaeus lectin. 

Endomyseal connective tissue: they are best evaluated in frozen sections because tissue shrinkage in paraffin sections will make the evaluation less reliable.

Number of nuclei: There should be about 4-8 peripherally and subsarcolemmaly located nuclei in each fiber.

Central capillary migration: Capillaries are seen at the center of the fiber. This is a sign of chronic change.

Central nuclei: There should be about 3 central nuclei per 100 fibers. Too many central nuclei indicate a chronic condition.

Lipid: Increase of lipid can be detected by fat stain or by EM. Mitochondria diseases often have increase in amount of lipid.

Amount of glycogen: PAS stain with diastase digestion is not an accurate method to estimate the amount of glycogen nor is electron microscopy. The most reliable method is biochemical analysis.

Click thumbnail to see abnormal accumulation of glycogen in McArdle's disease.

 

Histochemistry and Immunohistochemistry Head  

ATPase          Oxidative Enzymes          Miscellaneous

ATPase: Head

·         ATPase at pH 9.4 (standard condition): type 1 with light staining and type 2 with dark staining are distinguished. No intermediately staining fiber is seen.

·         ATPase at pH 4.6 (incomplete acid reversal): also known as the reverse ATPase staining. Type 1 with extremely dark staining, type 2A with virtually no staining, type 2B with intermediate staining.

·         ATPase at pH 4.3 (complete acid reversal): type 1 with extremely dark staining, type 2A and 2B with no staining. Type 2C fibers have intermediate staining.

Oxidative Enzymes: Head

Oxidative enzymes (including NADH-TR, cytochrome C, succinic dehydrogenase) are present in muscle These histochemical reactions essentially distinguish fibers with low mitochondrial content (non-staining) to that with high mitochondrial content (darkly stained). They are useful in fiber typing and in investigating mitochondrial myopathy. Type 1 fiber is oxidative fiber (intensely stained), type 2A is intermediately stained (and intermediately oxidative) and type 2B is non-oxidative and lightly stained. In normal muscle, oxidative enzyme stains will give a “lattice network” pattern. Mitochondria in subsarcolemmal locations and around endplate will give a crescent shape staining.

·        NADH-tetrazolium reductase (NADH-TR): this reaction detects a reductase that is present in both mitochondria and sarcoplasmic reticulum, therefore, it is not a reliable marker for mitochondria. SDH and COX are better markers of mitochondria. Tubular aggregates accumulated in the sarcoplasmic reticulum can be stained by NADH-TR but not succinic dehydrogenase. NADH-TR can also demonstrate the intermyofibril network (T-tubules). A clear differentiation of fiber types is only seen in healthy muscle and this is often disturbed in pathological muscle.

·        Succinic dehydrogenase (SDH): stains for mitochondria. They give a staining pattern similar to NADH-TR. However, succinic dehydrogenase is code by nuclear DNA and is not usually deficient in mitochondrial myopathy. This is also a good stain to demonstrate abnormal subsarcolemmal accumulation of mitochondria in mitochondrial myopathies, see also ragged red fibers.

·         Cytochrome oxidase (COX): stains the inner mitochondrial membrane. More prominent staining in type 1 than type 2 fibers, reflecting the different number of mitochondria in these fibers. Cytochrome oxidase is coded by mitochondrial DNA and is most frequently deficient in mitochondrial myopathy.

·         Combined COX-SDH stain: This is a very efficient screening test for mitchondrial diseases.

·         Combined NADH-TR-SDH stain: This combination allows differentiation between tubular aggregates and mitochondrial accumulation.

Miscellaneous: Head

Acid phosphatase: Lysosomal vacuoles, autophagic or due to lysosomal storage disease, are acid phosphatase-positive.

Acetylcholinerase: detection of end plates and muscle spindles.

Myoadenylate deaminase: Myoadenylate deaminase is the muscle-specific isoform of adenylate deaminase.  Myoadenylate deaminase catalyses the deamination of AMP into IMP and ammonia. IMP can subsequently be re-aminated to AMP. In cases of myoadenylate deaminase deficiency, muscle biopsies are histologically normal but no myoadenylate deaminase can be demonstrated by staining.

Non-specific esterase: they have increased staining in sites of lysosomal and macrophage activity. They may also occur as small granules towards the edge of fibers in muscle from aged individuals or as larger deposits at the sites of neuromuscular junctions.

·         Esterase is a good stain to identify macrophages.

·         Neuromuscular and myotendinous junction are positive for esterase: The neuromuscular junctions appears as small enzymatic reactive areas at the rim of the fibers.  

    FNEWWU10-esterase-NMjunctio.gif (34722 bytes)

·         Esterase staining is often excessively dark in denervated fibers but not in other atrophic fibers.

·         A large amount of esterase can also be seen in muscle fibers undergoing necrosis.

Myphosphorylase: Type 2 fibers, particularly type 2b, are dark and type 1 fiber are pale. Myophosphorylase is deficient in McArdle’s disease.

PAS: can be used to detect glycogen storage. It can also be used to visualize the basement membrane of the capillaries. Glycogen dissolve out easily in paraffin section or cryostat section and give false negative results.

Modified Gomori’s Trichrome (MGT):

Oil red O or Sudan black: Stains for fat. Normal muscle fibers have evenly distributed deposits of fine droplets. Type 1 fibers have more lipid than type 2 fibers but such separation is not reliable for fiber typing. Fat accumulation is frequently seen in mitochondrial myopathy.

FNEWWU10-lipid-storage.gif (35383 bytes)    

Dystrophin: In normal muscle dystrophin appears as a thin continuous sarcolemmal staining that is present in every individual fiber.

Anti-neurofilament immunostaining or silver staining: detection of nerve fibers or end aborization.

Organelles  Head  

Myofibrils    Mitochondria    Vacuoles

Myofibrils: Head

Parameters:

·         Sarcomere unit: is the longitudinal portions of a myofibril between two Z-discs. Myofibrils in adults is 0.5 to 1.2 mm in diameter, 10% wider in the A-band than the I- band

·         I-band: is the light band (isotropic, non-birefrigent). The I-band is composed of the light chains that are 5-6 nm in diameter and roughly 1-1.25 mm in length. The length of the I band is variable.

·         A-band: (anisotropic, birefrigent) is the dark band and is composed of thick filaments that are 15-18 nm in diameter and a length of 1.6 mm. The length of A-band is constant 1.6 mm.

·         H-zone: (hell, means clear in german) is the region where the thick and thin filaments do not overlap; the pseudo-H zone is the narrow clear area flanking the M line.

·         M-line: is at the center of the thick filaments and contains fine interconnections between the thick filaments.

Regenerating muscle: Myofibril bundles are irregular in shape in regenerating fibers.

Mitochondria: Head

·        Volume: Mitochondria comprise about 4% of volume in normal skeletal muscle cells. [Kiessling KH et al., 1973].

·        Distribution: Type 1 and 2A fibers have more mitochondria than type 2B fibers. They are usually found in four locations: intermyofibrillar area adjacent to the Z-disc or I-band, beneath the sarcolemma, at the poles of nuclei, and at motor end plates.

·        Histochemistry: Distribution of mitochondria can be well demonstrated by oxidative enzyme reactivity. The intermyofibrillar mitochondria will give the characteristic “lattice network” appearance with oxidative enzymes. Mitochondria at motor endplates and subsarcolemmal regions will appear as crescents. Mitochondria are stained red with Giomori’s modified trichrome. See “Oxidative enzymes” for details.

·        Semithin sections: They appear as tiny black dots around myofibrils or as delicate lattice network. Subsarcolemal clusters of mitochondria large enough to appear as grayish cresents are a normal finding, especially in the muscle of children. [Carpenter and Karpati, 2nd edt., pp.162]

·         Electron microscopy: Intermyofibrillar mitochondria should lie adjacent to the I-band/Z-disc, they are round on cross section and elongated on longitudinal sections.

·         Mitochondrial myopathies: Features of mitochondrial dysfunction include

Ragged red fibers.

Subsarcolemmal accumulation of mitochondria- as demonstrated by modified Gomori’s trichrome or SDH stain.

Cytochrome oxidase (COX) negative fibers with or without subsarcolemmal accumulation of mitochondria.

Up regulation of antioxidants in mitochondrial myopathies: Antioxidants including MnSOD, CuZnSOD and reduced glutathione are up regulated in fibers with respiratory chain deficiency. The degree of MnSOD is usually more obvious than CuZnSOD. Pattern of expression of reduced glutathione (GSH) is similar to that of MnSOD. The degreee of antioxidant induction is correlated with the degree of mitochondrial proliferation but not with clinical phenotype, patients’ age, duration of disease, biochemical defects or mitochondrial DNA abnormalities. [Filosto et al., Acta Neuropathol 2002 103:215-220]

·         Glutathione: GSH is synthesized in the cytoplasm and transported into the mitochondria. Therefore, there is a pool of reduced glutathione in both the mitochondria and the cytoplasm.

·         Catalase (CAT): CAT is a peroxisomeal enzyme that converts hydrogen peroxide into water and is found in the cytoplasm. With the exception of heart muscle cells, hitochondria from most mammalian cells do not contain CAT. The GSH system is the only non-enzymatic system to reduce hydrogen peroxide. [Radi R et al., J Biol Chem 1991 Nov 15;266(32):22028-34]

Vacuoles: Head

Differential diagnosis on intracytoplasmic inclusions

Optically clear cytoplasmic vacuoles

·         Artifactual vacuoles: due to improper freezing.

·         Real vacuoles: In hypokalemic periodic paralysis (biopsy obtained at the time of attack).

Cytoplasmic spaces: They can be seen in non-lysosomal glycogen storage disease and contain glycogen or filamentous polyglucosan. They may also be seen in adult-onset centronuclear myopathy. 

T-tube vacuoles (can be seen with light and electron microscopy).

·        Artifactual: Often found as an artifact in muscle that has has been placed in isotonic saline for several minutes before fixation.

·        Non-artifactual: In stumps of muscle fibers that have undergone segmental necrosis. The best example in human is dermatomyositis. Can also be seen in Duchenne muscular dystrophy but they are far less common due to the extensive necrosis of fibers.

Sarcoplasmic reticulum vacuoles: They arise from the sarcoplasmic reticulum. They can be seen in sarcotubular myopathy which is a rare congenital myopathy. Must be distinguished from T-tube vacuoles.

Lysosomal vacuoles: They are acid phosphatase positive and can be autophagic vacuoles or associated with a lysosomal storage disease. Differential diagnosis.

·        Acid maltase deficiency: can be seen in all variants.

·        Batten’s disease: Large vacuoles in rare cases of juvenile Batten’s disease. Small vacuoles that can be demonstrated by autofluoresence or acid phosphatase in most cases of Batten’s disease.

·        X-linked vacuolar myopathy: Many of them are acid phosphatase positive and many stain positively for calcium.

·         Denervation atrophy: Acid phosphatase-positive vacuoles can be seen in the center of some muscle fibers.

·         Peroxisomal vacuoles: They can be seen in Lafora’s disease as multiple small clear vacuoles up to 8 mm in diameter.

Glossary on Muscle Biopsy Head

Angulated fiber: Muscle fibers in neurogenic atrophy often appear angulated and small. In the initial stage of neurogenic atrophy, they may be found in a scattered pattern within other fibers. Angulated fibers, however, is not specific and can be seen in many other myopathies.

FNEWWU10-angulated-fiber-AT.gif (33215 bytes)

Centrally located nucleus: In normal conditions, there should be about 3 central nuclei per 100 fibers. An increase in centrally located nuclei is a common but non-specific pathologic features and is often seen in chrnic myopathy. A substantial increase of centrally located nuclei shoud raise the question of myotubular (centronuclear) myopathy.

FNEWWU10-central-nucleus.gif (41215 bytes)

Central capillary migration: This is also a non-specific sign of chronic myopathy. They usually occur later than the appearance of centrally located nuclei.

 Centronuclear myopathy: a central or paracentral nucleus, which is frequently separated from the surrounding sarcoplasm by a halo, is identified within the majority of muscle fibers in transverse sections while there is an apparent nearly total absence of peripheral, subsarcolemmal nuclei.  

Cores: Cores are typically seen in central core disease and multicore disease. They are not seen in paraffin HE sections but best seen with Gomori’s modified trichrome. They are amorphous round structures in muscles and characteristically lack oxidative enzyme activity. The cores may be eccentric or central. Ultrastructurally, there are two types of cores- structured (myofibrils are contracted and retain their striated pattern and ATPase activity) and unstructured cores (severe disruption of myofibrils and disruption of myofibrils and a lack of ATPase activity). All cores show an absence of mitochondria although mitochondria may accumulate in the core and non-core interface. Cores do not express desmin but the surrounding non-core zones do. Target fibers and cores must be distinguished.  

FNN0IE08-NADH1.gif (39267 bytes)    FNN0IE08-NADH-Core-longitud.gif (47899 bytes)    Target fibers for comparison

Cytoplasmic bodies:

·         Histochemistry: They appear as round to irregular, 2-4 mm in diameter, cytoplasmic bodies with a halo on HE frozen sections and as purple or dark green bodies with a halo on Gomori’s trichrome. They have no enzymatic activities and may be restricted to type 1 fibers.

·         Electron microscopy: They appear as mesh works of thin filaments.

Delta lesion: This term is applied to wedge shaped optically empty areas on cross sections that are  resulted from torn or over stretched segments in fibers with hypercontraction. Particularly, this term implies necrosis of the fiber.

Disorganization of myofilament: This includes Z-disck streaming, central cores and nemaline bodies, cytoplasmic bodies, spheroid bodies and myofibrillar myopathy with desmin accumulation.

Destructive myopathy: characterized by segmental necrosis of muscle fibers followed by macrophage invasion and phagocytosis of muscle fiber debris. Recurrent episodes of muscle cell necrosis and regeneration are accompanied by fibrous scarring of the endomysium.  

Fiber grouping: Abnormally small fibers may appear in groups in neurogenic atrophy. On ATPase reactions, these groups are composed of the same type of fiber. Fiber grouping is resulted from nerogenic degeneration and regeneration of motor units.

FNEWWU10-neurogenic-atrophy.gif (38430 bytes)

Heteroplasmic: Mixture of normal and abnormal genomes in the mitochondria.

Hyaline fiber: they have an abnormal increase in diameter accompanied by a round configuration, a glassy and more deeply stained appearance. The hyaline change reflects hypercontraction. They are most frequently seen in Duchenne muscular dystrophy and, to a much lesser extent, other muscular dystrophy.

Lobulated fiber: they are fibers that has a lobulated cross section on HE sections and are best demonstrated by NADH staining. Although they are type 2 fibers, they may appear darker than usual because of the darker staining. Lobualted fibers are seen in most myopathic cases and do not carry a special meaning.  

Myositis: Myositis are characterized by inflammatory cell infiltration. The more common ones include dermatomyositis, polymyositis, and inclusion body myositis

 FNEWWU10-myositis.gif (44163 bytes)

Nemaline bodies (nemaline rods): Nemaline bodies have a longitudinal striation with a periodicity of 8 nm; they have biochemical and structural properties of Z-discs and arise as a product of polymerization or proliferation of the lattice of the Z-disc. Although nemaline bodies are the hallmark of nemaline body myopathy, they are also seen in fibers that have lost thick filaments, myopathy associated with certain types of HIV virus, some cases of dermatomyositis and polymyositis.

Ragged-red fibers and pre-ragged-red fibers:

 FNEWWU10-Ragged-Red-fiber.gif (44308 bytes)  FNEWWU10-Ragged-blue.gif (59564 bytes)

·        Age: They are not seen under the age of three.

·        Morphology: The characteristic histochemical picture of a mitochondrial myopathy is ragged red fibers, accumulation of lipid, and the absence of cytochrome oxidase staining in a proportion of muscle fibers in the biopsy. Ragged red fibers appear as blue muscle fibers with bright red irregular (moth eaten) subsarcolemmal depositions with Gomori trichrome on frozen sections. The lipid droplets appear as small vacuoles.

·        Histochemistry: Ragged red fibers are more specifically demonstrated by succinate dehydrogenase; these fibers are also known as ragged blue fibers (SDH stain is blue). Modified SDH reaction stain Ragged red fibers and “ragged red equivalents” (pre-ragged-red fibers that are not demonstrated by modified Gomori trichrome).

·        Ragged-red equivalent: The increase in mitochondria in these fibers can be demonstrated by SDH stain but not enough to be demonstrated by Gomori’s modified trichrome.

·        Differential diagnosis: Ragged red fibers must be distinguished from nemaline bodies which also stain red and tend to accumulate under the sarcolemma but have no enzyme reactivity. Histochemical demonstration of oxidative enzymes may show cresent shape reactivity in motor endplates where an increased number of mitochondria is present, they must not be confused with Ragged red fibers. The 3 most common mitochondrial syndromes associated with ragged-red fibers are:  

Regenerating fibers: Takes about three weeks to complete. Regenerating fibers express N-CAM, MyoD and myogenin, and also embryonic and neonatal isoforms of myosin heavy chain. In contrast to mature fibers, MCH class I histocompatibility complex is expressed in regenerating fibers.  

FNN0IE13-Reg-fiber.gif (73138 bytes)    

Rimmed vacuoles: On cryostat sections, rimmed vacuoles are popcorn like clear vacuoles with a densely blue rim in HE frozen sections. They are are best demonstrated in MGT. The vacuoles are often assoctiated with cytoplasmic and occasionally intranuclear eosinophilic inclusions. Rimmed vacuoles contain amyloid ABeta, ApoE, and ubiquitin. On paraffin sections the granules are dissolved so that only rather anonymous vacuoles remain. On semithin sections there are round osmiophillic granules of various sizes that often occur in clusters in subsarcolemmal locations. Differential diagnoses include the followins:

·       Inclusion body myositis (IBM): Inclusion bodies are most frequently seen in IBM but they are not pathogonomic and may also be seen in other conditions.

·        Oculopharyngeal muscular dystrophy.

·        Many cases of distal myopathy (probably a type of familial inclusion body myopathy).

·         Chloroquine myopathy (with acid phosphatase activity and are lysosomal).

·         X-linked vacuolar myopathy (positive for calcium and acid phosphatase).

·         As minor finding in other myopathies.

Ring fiber: the ring is formed by a bundle of peripheral myofibrils which are circumferentially oriented such that they encircle the internal portion of the sarcoplasm which is normal in structure and orientation. Rings are best seen with PTAH stains, semi-thin sections, or under phase contrast or polarizing microscopes. Under the electron microscope the pathologically oriented myofibrils are generally normal in structure except for hypercontraction of the sarcomeres. Ring fibers are seen in myotonic muscular dystrophy, limb girdle dystrophy and trauma.

Snake coils myofibrils: This is a non-specific change that can be seen in many situations incluing partial denervation and limb-girdle dystrophy). Snake coils fibers are best demonstrated in frozen sections and particularly well by Gomori’s trichrome and they arrpear as a swirling pattern of the myofibrils.

Target fiber:

·        General: Target fibers (as well as type grouping) are pathognomic of denervation but bone fide target fibers are present in less than 25% of cases of neurogenic atrophy. They are often strikingly abundant in subacute cases of Guillain-Barré syndrome. They difficult to be seen in paraffin sections but can be demonstrated nicely with HE stain or mododified trichrome in frozen sections.

·         Histochemistry: Most target fibers are type 1 fibers. Target fiber has a distinct three-zone architecture that is best demonstrated by NADH-TR histochemistry. Characteristically, there is a pale core with no NADH-TR activity, rimmed by a darkly stained ring and surrounded by a more normal staining peripheral zone. The core also lacks glycogen or phosphorylase activity. Demonstration of target fibers with ATPase is possible but unreliable. A fiber must have the three-zone architecture to be qualified as target fiber. Some two-zone fibers may be seen and they are called targetoid fibers. The distinction between target and targetoid fibers has no clinical significance.  

     FNEWWU10-target-NADH.gif (60760 bytes)  Cores for comparison

·        EM: The cores in target fibers contain myofibrils with various forms of dissolution and disorganization. Z-disc streaming is prominent.

·         Differential diagnosis: Although targets fibers and cores are morphologically similar, they are different and must be distinguished. Target fibers must be distinguished from “motheaten change” that is frequently seen in neurogenic atrophy.

Tubular aggregates: these are non-specific aggregates that can be seen in myopathies. They are aggregates derived from the sarcoplasmic reticulum and are, therefore, heavily stained by NADH-TR (present in both mitochondria and sarcoplasmic tubules) and not by succinic dehydrogenase technique (present only in mitochondria). A combination of NADH-TR and SDH allows differentiation between tubular aggregates and mitochondrial accumulation.

Z-disc loss: Selective Z-disc loss is probably a result of necrosis. This can only be recognized at the early stage and without contraction of the fibers. Once contraction is present, the lesion will appear only as disoriented fibers.

Z-disc streaming: Z-disc streaming is a non-specific features that can be encountered in many myopathies. They have also been described in normal control subjects. This is a result of disorientation of Z-disc.It is a common and non-specific lesion.   

·        Histochemistry: In Gomori’s trichome stain, they characteristically appear as poorly defined patches of increased staining; histochemically, these areas show considerable reduction in ATPase activity and often also as negative areas on oxidative enzyme reactions.

·        Semithin sections: Characteristically, they appear as flame-shaped structures under medium magnification. The number of Z-discs being affected is highly variable. On longitudinal sections, they varies from focal replacement of sarcomere by dense material that extends between Z-discs to long and longitudinal stretches of dense materials in advanced lesions. On higher magnification, they appear as collections of dark gray granular areas within a fiber.           

·        Electron microscopy: The streaming material is made up of electron dense granular substance. The streaming, particularly in the smaller streams, starts and ends at the Z-disc.