Biopsy of Horse Muscle Tissue for Diagnosis

Biopsy of Horse Muscle Tissue for Diagnosis of Muscle Disorders – Submitting a Biopsy

Introduction 

Obtaining a Muscle Sample
Exertional Myopathies
Equine Motor Neuron Disease
Muscle Atrophy

Packing and Shipping Muscle Biopsy

Submission Forms  

Introduction:

It is important to select the most appropriate muscle for biopsy in order to get an accurate diagnosis. In general, we recommend open surgical biopsies of the semimembranous and tedinous muscle for Exertional Myopathies (ER) and Polysaccharide Storage Myopathy (PSSM), sacrocaudalis biopsies for Equine Motorneuron Disease and Immune-Mediated Myositis and biopsies of the semimembranous combined with formalin fixed TruCut samples of expaxial muscles for horses with atrophy of topline muscles. For evaluation of facial muscle atrophy, an open biopsy of the specific muscle group is most appropriate.

Please avoid crushing the muscle sample with forceps as it creates numerous artifacts. Hold the tissue in one location only

Upon arrival in the lab, we process the samples in a special fashion prior to freezing to prevent ice crystals from forming vacuoles in the tissue. Because muscle has such a large water component (~80% by mass), ice crystals can easily form, especially when the sample is frozen on dry ice.

The ice crystals create small holes in the sample (freeze artifacts) that can make histochemistry more difficult and less accurate.

We freeze the samples in a special fashion rather than using formalin-fixed samples because freezing allows a number of different stains to be used and better preserves the muscle architecture.

Obtaining a Muscle Sample:

Exertional Myopathies and Polysaccharide Storage Myopathy:

For Exertional Myopathies (ER) and Polysaccharide Storage Myopathy (PSSM), the best site for a biopsy is midway between the tuberiscni and the origin of the Achilles tendon at about the level of the vulvar lips. The semimembranosus muscle using open surgical technique provides the best information (see below).

• Tail hair hides scarring at this site
• Avoid tendinous insertions

Procedure:

• Tranquilize the horse and wrap the tail
• Lidocaine subcutaneously (10 ml) (not in muscle itself)
• 2 inch vertical incision
• Incise skin, subcutaneous tissue
• Vertical incision through fascia
• Undermine the fascia on one side
• Make two parallel vertical incisions, one-half inch apart
• hold muscle in one place with forcep then transect dorsally
• free biopsy one-half inch deep, then transect ventrally
• Close dead space with subcutaneous layers

Good subcutaneous closure is the most important technique to prevent wound dehiscence.

• Close skin with intradermal sutures or staples
• Small scar may remain

Equine Motor Neuron Disease:

For suspected cases of motor neuron disease, a biopsy of the sacrocaudalis dorsalis muscle is recommended (see below). This muscle is located above the tailhead on either side of the dorsal spinous processes of the sacrocoxygeal vertebra (see picture).

• Sacrocaudalis muscle has >20% slow twitch fibers which are most consistently atrophied.

Procedure:

• Lidocaine is injected under the skin but not into the muscle belly.
• A one and one half inch incision is made through the skin and subcutaneous fat and fascia.
• Parallel longitudinal incisions are made in the muscle one quarter inch apart.
• The cranial aspect of the muscle is grasped with forceps and the muscle is dissected out one quarter inch deep and one half inch long.

This area can have a lot of subcutaneous fat. Make sure the sample is deep enough to obtain muscle tissue.

Muscle Atrophy and Immune-Mediated Myositis :

Choose a muscle that appears atrophied on EMG.

• If muscle atrophy is present in the gluteal or epaxial (back) muscles, obtain two or three TruCut biopsies from the gluteals or epaxial muscles behind the saddle area, place them in formalin, and ship with the semimembranous chilled sample.
• To rule out PSSM or other myopathies, a fresh semimembranous muscle biopsy should be submitted also (see above for directions).

 Packing and Shipping Biopsy: 

• Do not squeeze, squish, or otherwise disturb the muscle sample, as that can damage cells.
• A 1" cube of fresh muscle should be wrapped in saline moistened gauze, placed in a hard, watertight container for protection, and shipped overnight on icepacks. The gauze should be moist and wrung out. Please do not ship samples floating in saline.
• Formalin fixed – If sample larger than TruCuts are submitted, they should sit in the air for five minutes before they are placed in formalin to prevent contraction bands from forming.
• Fresh samples are best, since frozen muscle samples for histochemical analysis require special processing prior to freezing. If circumstances are such that a biopsy cannot be submitted that day (e.g. death of animal on a weekend), samples can be placed in a freezer and submitted on Monday on ice packs, but this is not ideal and should be avoided if at all possible.
• Include a 10mL blood sample in an EDTA (purple top) tube with biopsy shipment. This sample is used to store DNA from cases for future research into the genetic basis of muscle disease. We request your horse's name for our records.

Submission Forms:

Include submission and invoice forms with all samples. Please print and enclose the appropriate submission form with all biopsies sent to the laboratory. These .pdf forms require Adobe Acrobat Reader. Results are usually available within ten days of submission.

Instructions for biopsy and shipment.

Submission Form and Invoice for all muscle biopsies- please include with all biopsies shipped.