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Orlando Paciello » 4.Muscle biopsy


When to carry out a muscle biopsy

Biopsy is generally indicated in the following cases:

  • Presence of symptoms relating to muscle pathologies:
    • Weakness.
    • Muscle symptoms: pain, cramps, fatigue after exertion.
    • High levels of serumcreatine kinase (CK).
    • Abnormal electromyogram.
  • Presence of type of neuropathy that may require nerve biopsy.
  • Presence of systemic disorder that may have silent effect on muscles:
    • Vasculitis.
    • Sarcoidosis.

A biopsy is not indicated, on the other hand, where the disease is better diagnosed through an electromyogram (EMG) like Myasthenia gravis or Myotonia.
You should wait 1 month after an attack of Rhabdomyolysis.

Muscle Biopsy

  • Critical for the diagnosis of muscle pathologies.
  • Easy to perform.
  • Collect fresh samples not formalin- fixed ones.
  • Assessment should be carried out by laboratories with experience in muscle pathologies.
Example of an open biopsy

Example of an open biopsy


Selection of Muscle Sample

  • Clinically-affected muscles.
  • Data supplied by electrophysiological results and clinical signs indicating muscle involvement.
  • Good idea to avoid muscles which are site of EMG tests, injections or trauma
Muscle biopsy collected following direction of fibres and gently laid on normal saline-soaked gauze

Muscle biopsy collected following direction of fibres and gently laid on normal saline-soaked gauze


Muscle biopsy

Biopsy of semitendinosus muscle: site of choice in cases of suspected polysaccharide storage myopathy

Biopsy of semitendinosus muscle: site of choice in cases of suspected polysaccharide storage myopathy


Muscle Biopsy (cont.)

In figure 1: Biopsy of sacrocaudal muscle: site of choice in suspected cases of motor neurone disease.
In figure 2: Gluteus: site used for biopsy in suspected cases of immune mediated inflammatory myopathy or in cases of gluteal atrophy.

Biopsy of sacrocaudal muscle: site of choice for suspected cases of motor neurone disease

Biopsy of sacrocaudal muscle: site of choice for suspected cases of motor neurone disease

Gluteus: site for performing biopsy in suspected cases of immune mediated inflammatory myopathy or with atrophy of gluts

Gluteus: site for performing biopsy in suspected cases of immune mediated inflammatory myopathy or with atrophy of gluts


Muscle Biopsy – Basic procedure

There are two basic procedures for carrying out a muscle biopsy: the surgical technique known as “open” and the technique using a percutaneous needle, also called punch.

The surgical method with incision into the cutaneous, subcutaneous and fascia layers is preferable to taking a sample using a transcutaneous punch because it allows for a sample which is the right size and direction.

Example of biopsy performed using biopsy needle. This technique is not recommended in veterinary medicine. Source: modified from Dubowitz V, Sewry CA: Muscle Biopsy (third edition), Saunders, Elsevier, 2007

Example of biopsy performed using biopsy needle. This technique is not recommended in veterinary medicine. Source: modified from Dubowitz V, Sewry CA: Muscle Biopsy (third edition), Saunders, Elsevier, 2007

Example of biopsy performed using biopsy needle. This technique is not recommended in veterinary medicine. Source: modified from Dubowitz V, Sewry CA: Muscle Biopsy (third edition), Saunders, Elsevier, 2007

Example of biopsy performed using biopsy needle. This technique is not recommended in veterinary medicine. Source: modified from Dubowitz V, Sewry CA: Muscle Biopsy (third edition), Saunders, Elsevier, 2007


Muscle Biopsy (cont.)

The direction of the muscle fibres in the sample is fundamental as it allows for the correct positioning of the sample during freezing and means that excellent transverse sections can be taken for histomorphological assessment of the muscle tissue.

The biopsy must be performed at the level of the muscle body, avoiding tendon insertion points and aponeuroses where the histology may be different.

Example of “open” biopsy on semitendinosus muscle of a horse

Example of "open" biopsy on semitendinosus muscle of a horse

Example of “open” biopsy. The sample is then placed on normal saline-soaked gauze

Example of "open" biopsy. The sample is then placed on normal saline-soaked gauze


Collecting the muscle sample

  • Treat sample gently.
  • Minimum size of sample 0.5X0.5X1.0 cm.
  • Place sample on gauze lightly soaked in 30GG of normal saline and keep it refrigerated.
  • The biopsy needs to reach the laboratory within 24-48 hours.
Muscle biopsy placed on gauze soaked in normal saline

Muscle biopsy placed on gauze soaked in normal saline

Glass test tube used to transport the biopsy. Rubber cap to prevent dehydration of sample

Glass test tube used to transport the biopsy. Rubber cap to prevent dehydration of sample


Collecting muscle sample (cont.)

The sample needs to be sent to the laboratory with an ice-pack and carefully labelled indicating clinical signs and history.

As an alternative to the containers shown in fig. 1, an ice-pack can be used which is pre-cooled in a freezer and then put in a bag for the transport of biological samples next to the muscle biopsy.

Fig.1 Containers for the test tubes with the muscle biopsies in them. These containers can be pre-refrigerated in a freezer and then used to transport the biopsies for long periods ( max. 48 hours)

Fig.1 Containers for the test tubes with the muscle biopsies in them. These containers can be pre-refrigerated in a freezer and then used to transport the biopsies for long periods ( max. 48 hours)


Collecting the muscle sample (cont.)

Before collecting the sample, the staff who are going to process the sample at the laboratory should be consulted to make sure what is the best way to collect the sample and to transport it.

Collecting the muscle sample (cont.)

  • Once at the laboratory, the fresh sample is frozen by plunging it into isopentane, pre-cooled in liquid nitrogen and kept at – 80° C.
  • Using a cryostat, a series of sections are taken which are stained according to routine histological and histochemical methods like hematoxylin and eosin and Engel’s Trichrome (to assess the shape and size of myofibres, endomysial and perimysial connective tissue and to identify any inflammatory cells), Periodic Acid Schiff (to see any accumulation of intramyofibral polysaccharides), histoenzymatic and immunohistochemical staining.

Staining techniques used in evaluation of biopsy samples


Staining techniques used in evaluation of biopsy samples (cont.)


Assessment of Muscle Biopsy: what are variations from normal?

  • Dimension of fibres (atrophy, hypertrophy) and their profile (polygonal, round, angular).
  • Proportion and distribution of fibre type (fibre type grouping, reduction or predominance).
  • Number and position of nuclei (peripheral, central, random).
  • Myonecrosis and regeneration.
  • Cellular infiltrate.
  • Connective tissue and morphology of vessel.
  • Morphology of intra-muscular nerves.
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