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:

• Is the condition a long standing or newly onset condition?

• Is the condition progrossive or relatively static?

• Are there any associated systemic conditions such as heart problem or autoimmune disease?

• What is the serum CPK level?

Other helpful clinical information:

• History of myoglobinemia. If yes, when.

• Family history of neuromuscular disease.

• Sex and age.  

• Results of EMG studies.

• Muscle groups being affected.

• Presence of contracture.

• Muscle groups being involved.

• Site of biopsy.

• Therapy and medication at the time of biopsy.

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.

• High: (e.g. Dystrophinopathies) 200-300 times of normal.

• Intermediate: (e.g. Inflammatory myopathy) 20-30 times of normal.

• Low: (e.g. Neurogenic disorder) 2-5 times of normal.

Parameters to be evaluated:

Paraffin sections:

• Interstitial Inflammation                 

• Vasculitis (± fibrinoid necrosis)

• Endomysial and perimysial fibrosis/fatty infiltration

• Range of fiber caliber (very important in pediatric cases)

• Angulated fibers

• Rounding of fiber contour (better evaluated with frozen sections)

• Variation of fiber diameter (better evaluated with frozen sections)

• Increase in number of centrally located nuclei

• Central capillary migration

• Split fiber

• Group atrophy

• Necrotic/myopathic (degenerating) fibers

• Atrophic fibers

• Regenerating fibers

• Target fiber

• Whorl fibers

• Ring fibers

Histochemistry:

    Basic panel

•     HE frozen section

•     Modified Gomori Trichrome

•     ATPase

•     NADH-TR

•     Esterase

    Extended panel

•     Succinate dehydrogenase (SDH)

•     Cytochrome C oxidase (COX)

•     PAS with and without diastase

•     Adenylate deaminase

•     Myophosphorylase

•     Acid phosphatase

•     Oil red O

Electron microscopy

• Tissue preservation

• Myofibril architecture

• Plasma membrane

• Sacolemmal membrane

• Mitochondria (size, density, and shape)

• T-tubule

• Amount of lipid

• Nucleus

• Phagocytic granules

• Amount of glycogen

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:

                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.

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.  

·         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):

• This stain contains Gomori’s trichrome (containing chromotrope 2R, fast green, phosphotungstic acid, and glacial acetic acid) and hematoxylin. It is used for frozen sections in muscle biopsy.

• This stain stains nuclei red-purple, normal muscle myofibrils green with distinct A and I bands on longitudinal sections, intermyofibrillary network red, and interstitial collagen green.

• MGT is useful in demonstrating the intermyofibril network, rimmed vacuoles in inclusion body myopathies, and target fibers. These structures, however, can also be demonstrated well with well-performed hematoxylin-eosin stain on frozen muscle biopsy material.

• Very useful in detecting ragged fibers in mitochondrial myopathy. The muscle fibers are stained green and the ragged areas are stained red.

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.

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

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