Laboratory Diagnosis in Neurology, 1 Ed.

9 Biopsy

A. Rosenbohm, A. Sperfeld, H. Tumani

Brain Biopsy


Brain biopsy is indicated when:

• Neither analysis of CSF and blood nor imaging procedures have enabled clarification as to the etiology of a circumscribed or diffuse disease of the CNS (autoimmune, viral, or bacterial inflammation, parasitic disease, brain neoplasm or brain metastases, degenerative or metabolic disease).

• A precise diagnosis is of prime importance for treatment.

• Staging is required in case of brain neoplasm or brain metastasis.

There are a few diseases (primary cerebral lymphoma is one) for which brain biopsy, as the gold standard, is required.


Tissue may be removed by stereotactic methods or via an open approach. The risks involved with stereotactic biopsy are much lower than those associated with open neurosurgery.

Procedure for Brain Biopsy

• The operation is planned using the data obtained by cranial CT and/or MRI or nuclear medicine imaging procedures.

• The best approach is determined using three-dimensional image processing.

• Leptomeningeal tissue or tissue directly from the lesion of interest is removed.

• The removed tissue is fixed or the unfixed tissue transferred to liquid nitrogen, as required.

• The tissue is subjected to neuropathological evaluation.


Possible complications include hemorrhage into the biopsy region, local swelling of the brain, and—very rarely—infection of the skin incision (disturbed wound healing). If any complications occur, new neurological symptoms may arise or those already present may be enhanced.

Nerve Biopsy

The classification of disorders of the peripheral nerves by the Research Group on Neuromuscular Diseases of the World Federation of Neurology is organized by etiology. Nerve biopsy results, on the other hand, are organized by structure, in the sense of organ, cell, and organelle pathology (Schröder, 1987).


The indications for nerve biopsy are well defined (Schaumburg et al., 1992; Midroni and Bilbao, 1995). Apart from knowledge of specific pathognomonic changes in certain diseases, however, some diseases are easier to ascertain by other procedures (e. g., determination of blood metabolites). Nerve biopsy may be particularly helpful in the diagnosis of systemic diseases. These include:

• Neuropathies of the multiplex type (e. g., due to vasculitis).

• Lysosomal metabolic disorders that are not fully accessible to biochemical blood analysis (e. g., juvenile or adult metachromatic leukodystrophies).

Biopsies are also justified when thorough clinical evaluation of a generalized neuropathy fails to clarify its cause. In this situation it is often necessary to exclude concomitant disease: for example, when a sensory component is demonstrated in a purely motor syndrome. A number of molecular biological tests are now available for hereditary neuropathies. However, neuropathological findings can help with narrowing down the number of genes to be examined.

It should be noted that, frequently, only selective fiber deficiencies or changes specific to whole disease groups (inflammatory, axonal, myelinopathic) are found. However, selective fiber deficiencies do provide some information about the degree of severity, stage of damage, progression of the disease, and extent of regeneration.


Techniques for morphological examination. Morphological examination of a nerve biopsy includes the following:

• Preparation of nerve tissue.

• Embedding of tissue in plastic for semi-thin sections and for electron microscopy.

• Embedding of tissue in paraffin.

• Storage of unfixed tissue in liquid nitrogen for possible enzyme histochemical, immunohistochemical, and molecular genetic studies.

Morphometric methods. Specific morphometric methods to determine the number and size of myelinated and nonmyelinated nerve fibers are carried out as part of the neuropathological evaluation.

Procedure for Nerve Biopsy

• Nerve biopsy can be performed as an outpatient procedure.

• Select the nerve: the sural nerve is the first choice (sufficient control data are available); other possibilities are the superficial peroneal nerve and cutaneous branches of the deep peroneal nerve, saphenous nerve, superficial radial nerve, and great auricular nerve. In special cases, a mixed nerve may be used, such as the musculocutaneous nerve or the lateral part of the terminal branch of the deep peroneal nerve (Hall et al., 1992).

• Excise a nerve segment 2–3 cm in length (depending on the tests planned).

• Fix one part in a suitable solution (e. g., 3.9% glutaraldehyde solution in 0.1 mol phosphate buffer) and freeze the other part in liquid nitrogen, as required (see under Methods).

Pitfalls in Nerve Biopsy

Detailed morphological examination requires flawless techniques of excision, preparation, and fixation, and also optimal embedding or freezing conditions. Squeezing and tearing of the tissue should be avoided, since peripheral nerves are extremely prone to artifacts.


Symptoms experienced during and after biopsy of the sural nerve have been well studied (Neundörfer et al., 1990; Dyck et al., 1993). About 10% of the persons interviewed reported persistent disturbing complaints. In addition to wound infections, neuroma formation, and persistent dysesthesia, local thrombophlebitis was among the most common complications.

Muscle Biopsy



The patient's medical history, laboratory diagnosis, and clinical data are of prime importance when a muscle disease is suspected. In addition, electrophysiological tests, metabolic function tests, and possibly also imaging procedures are used in diagnosing myopathy. However, it is usually the histological findings that finally confirm the diagnosis.

For most neuromuscular diseases, despite the rapid development of molecular genetic testing, the muscle biopsy is the decisive diagnostic test.


Myositis. Of the acquired myopathies, the various forms of myositis (polymyositis, dermatomyositis, inclusion body myositis) absolutely require muscle biopsy. In these immunological inflammatory diseases, biopsy reveals not only characteristic inflammatory changes but also differences in the composition and distribution of infiltrates within the muscle. In addition, some other pathognomonic changes may also be present in the biopsy tissue. To confirm the diagnosis, however, the clinical presentation often needs to be considered as well. Vasculitis or other forms of connective tissue disease can give rise to concomitant myositis. Infectious myositis caused by bacteria or viruses is rare; for this reason, detection of pathogens has no role to play in muscle biopsy.

Muscular dystrophies. In this heterogeneous group of hereditary myopathies, with different ages of onset and phenotypes, the indications for muscle biopsy vary:

• Most can be diagnosed by specific immunohistochemical examination of the biopsy tissue. This is the case for the Duchenne and Becker dystrophies (see Fig. 9.5 b), autosomal dominant and autosomal recessive forms of limb girdle muscular dystrophy (12 of the 15 forms known can be examined by immunohistochemistry), distal myopathies, and several forms of congenital myopathies.

• By comparison, facioscapulohumeral muscular dystrophy and oculopharyngeal muscular dystrophy can be diagnosed by molecular genetic tests on blood cells when these diseases are suspected on a clinical basis. Muscle biopsy therefore has no advantage other than confirming myopathic changes. Similarly, in myopathies with myotonia the typical clinical electrophysiological findings decide the diagnosis: muscle biopsy has nothing to add.

Lower motoneuron diseases. Occasionally, muscle biopsy may be required for the differential diagnosis in a patient in whom lower motoneuron disease is postulated, to rule out inclusion body myositis, which takes a different clinical course and is associated with different therapeutic options.


Procedure for Muscle Biopsy

• Apply local anesthesia (but not into the muscle itself).

• Remove a piece of tissue from the skeletal muscle (about the size of a hazelnut) without damaging the muscle fibers with the instruments.

• Separate the biopsy tissue into several pieces (about 5 × 5 × 8 mm in size).

• Freeze the biopsy material and prepare frozen sections which can then be used for all subsequent tests.

• Fix selected pieces in 2% glutaraldehyde and embed for electron microscopic examination.

Pitfalls in Muscle Biopsy

Fixation in formalin and embedding in paraffin are obsolete for muscle biopsies: firstly, they can obliterate histological changes, and, secondly, they make further analysis by immunohistochemical or enzyme histochemical methods impossible.

Preparation of Frozen Sections

Fixation, deep freezing. The muscle biopsy material is first attached with pins to a cork lamella (in the longitudinal direction of the muscle fibers), and then frozen in isopentane (2-methylbutane) cooled in liquid nitrogen or on dry ice. Several pieces of frozen tissue are stored in labeled plastic vials at −80°C. This makes it possible to re-examine the biopsy many years later.

Frozen sections. Using a cryostat, the frozen tissue pieces are processed into frozen sections 20 μm in thickness. Normally, four serial sections are placed on a microscopic slide; about 10–12 slides are required for a routine diagnosis. For further diagnostic tests, additional slides with frozen sections are prepared during the same session, or new frozen sections are prepared at a later time.

Serial sections. If myositis is specifically suspected, serial sections are prepared and every fifth section on the slide is marked, to ensure that the deeper regions of the biopsy are also screened for infiltrates. The sections between the marked sections are used for other staining procedures or are discarded.

Range of Tests

The following tests may be performed:

• Routine staining, enzyme histochemical and immunohistochemical staining.

• Electron microscopic examination.

• Immunoblots (verification of abnormal immunohistochemical results by Western blot).

• Biochemical analysis (for suspected metabolic myopathy).

• Molecular genetic tests (e. g., for mitochondrial myopathy).

Routine staining. Routine staining procedures (Table 9.1) include staining with hematoxylin–eosin (HE, Figs. Gomori trichrome (Fig. 9.2), oil red O (Fig. 9.4), periodic acid–Schiff (PAS), and Congo red (for amyloid). Also used are specific staining procedures for acid phosphatase, cytochrome C oxidase (COX, Fig. 9.6), and succinate dehydrogenase (SDH). To distinguish between different types of muscle fibers, ATPase staining is carried out at alkaline and acidic pH.

Enzyme histochemical staining. Routine enzyme histochemical examination includes staining for myoadenylate deaminase (MADA). Specific questions regarding glucose metabolism require staining for phosphorylase and phosphofructokinase.

Immunohistochemical staining. For immunohistochemical examination, antibodies are available against specific types of leukocytes (T lymphocytes, B lymphocytes, macrophages) or complement; they are required for classification and differential diagnosis of different forms of myositis. Commercial antibodies are available for diagnosing muscular dystrophies (Duchenne's, Becker's, and limb–girdle dystrophies) and various other hereditary myopathies. Immunohistochemical staining of cell membranes, cells, or organelles is then evaluated under the fluorescence microscope (Fig. 9.5).

Biochemical analysis. In a more detailed diagnosis, various enzyme activities (specific for glycogen metabolism, respiratory chain complexes, glucose metabolism, and lipid metabolism) are determined using biochemical methods. For reliable detection of defective surface proteins that show conspicuous features in immunohistochemical staining, Western blots distinguish between protein defects and secondary changes due to myopathies.

Table 9.1 Detection of pathological features in routinely stained sections

Staining method


Hematoxylin–eosin (HE)

Evaluation of muscle fiber size and shape, vascular and nerve structures, infiltrates, general histology (Fig. 9.1)

Gomori trichrome

Detection of ragged red fibers (mitochondrial dysfunction, Fig. 9.2), rimmed vacuoles (inclusion bodies, Fig. 9.3)

Oil red O

Evaluation of lipid deposition (Fig. 9.4)

Periodic acid–Schiff (PAS)

Glycogen deposition

Acid phosphatase

Lysosomal enzyme, indicative of degenerative changes of muscle fibers

Cytochrome C oxidase (COX)

Mitochondrial enzyme (Fig. 9.6)

Succinate dehydrogenase (SDH)

Mitochondrial enzyme


Differentiation between type I and type II muscle fibers;.ber type grouping (Fig. 9.7)

NADH-tetrazolium reductase (NADH-TR)

Enzyme in mitochondria, T tubules and sarcoplasmic reticulum, target fibers


Fig. 9.1 Histology of normal muscle (HE stain). The section shows muscle fibers of normal size and shape, and normal vascular and nerve structures. Infiltrates are absent.


Fig. 9.2 Ragged red fiber (Gomori trichrome stain).


Fig. 9.3 Inclusion body, or rimmed vacuole (Gomori trichrome stain).


Fig. 9.4 Fine lipid droplets in type I muscle fibers (oil red O stain).


Fig. 9.5 a, b Staining with dystrophin antibody.

a Normal muscle.

b Duchenne's dystrophy.


Fig. 9.6 COX-negative muscle fibers in mitochondrial myopathy with respiratory chain defect.


Fig. 9.7 ATPase staining helps distinguish between type I and type II muscle fibers. Muscle fibers of mixed types create a checkered pattern, whereas successful reinnervation leads to fiber type grouping.


Fig. 9.8 Myopathic changes (HE stain).

Molecular genetics. As a last resort, molecular genetic tests are available; however, they may also be performed with EDTA-blood from the patient.

Typical Muscle Biopsy Findings

Basically three different groups of changes are found in the biopsy material:

• Myopathic changes.

• Neurogenic changes.

• Myositic changes.

Myopathic Changes

The biopsy contains round, atrophic muscles fibers, individually or in groups, and also some hypertrophic muscle fibers (Figs. 9.8 and 9.5 b). There is an increase in connective tissue up to the point of fatty atrophy. Internal nuclei, necrotic muscle fibers, and clefts may also be present.

Neurogenic Changes

The biopsy contains individual atrophic fibers that look lacerated, angular, or stretched, and there is also advanced denervation atrophy (Fig. 9.9). Oxidative activity in atrophic muscle fibers is increased. Successful reinnervation leads to fiber type grouping because the muscle fibers—which are normally arranged in a mixed, checkered pattern—are innervated by neighboring neurons so that all muscle fibers nearby become the same fiber type. Some muscle fibers become target fibers for reinnervation.

Myositic Changes

Infiltrates that consist of lymphocytes, monocytes, and macrophages are found directly around blood vessels, between muscle fibers, with invasion of and between the fascicles (Fig. 9.10). In addition, the typical myopathic changes are seen. Muscle fibers may also show conspicuous structural features. Subforms of myositis can be distinguished by immunohistochemical methods, the composition of infiltrates, and HLA expression on the surface of muscle fibers.


Fig. 9.9 Neurogenic changes (HE stain).


Fig. 9.10 Myositic changes (HE stain).


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