DUKE UNIVERSITY MEDICAL CENTER AND HEALTH SYSTEM
Department of Pathology
Box 3712
Durham, North Carolina 27710

INSTRUCTIONS ON HOW TO SEND A MUSCLE BIOPSY

What to do with a muscle biopsy?:

1. Get history. Fill in patient information form or send your patient information form.
2. Obtain one piece fresh and one in gluteraldehyde, preferably in a muscle clamp or sutured to an applicator stick in situ. You can also put a piece in formalin if you like. I do not, but you might want to, process a piece to see if further studies are likely to be helpful.
3. If you decide to send the muscle to us,
a. Delivery accepted Monday-Friday. Please avoid sending specimens over the weekend. You need to freeze a specimen if it is going to be kept more than 24 hours before being sent to us.
b. If you can get it to us in a few hours or by the next day, you can keep it fresh, cold, and very, very slightly damp (it should not dry out but it should not be soaked). Pack it in ice or, better yet, use freezer packs (the kind you put in ice chests to keep things cold).
c. If you send it to us frozen, see instructions below and attached. Some people send muscle in immunofixative (Michel's; Zeus), but this is difficult to stain, so avoid it.
d. Send it air express to:

DUHS Clinical Laboratories/Histology
4425 Ben Franklin Boulevard
Durham, NC 27704
Phone: (919) 684-2332 (office) or 684-3648 (Histochemistry Lab); FAX (919) 684-5916

e. Current charges are attached. Stains beyond my usual battery will be charged extra.
f. If you send glutaraldehyde-fixed tissue, send it in a container separate from the frozen tissue. ( It can be sent with fresh tissue in ice).

We assume that receipt of glutaraldehyde-fixed tissue gives us permission to perform full EM or EM thick sections if we think it is necessary. Possible inclusion body myositis, metabolic myopathies, and congenital myopathies warrant EM studies
.
You may prefer to retain the glutaraldehydefixed tissue and await the results of the histochemical studies. Formalin fixed tissue can be used for EM if no glutaraldehyde is available.

How to freeze the muscle?:
1. A fancy method is attached.
2. You can freeze a piece of muscle in dry ice and acetone, liquid nitrogen, or, believe it or not, slowly in a regular freezer.
3. Ideally, it should be frozen on cork or a chuck so we do not have to remount it, but we will take what we can get.
4. Send it in dry ice.

PROCEDURE FOR FREEZING OF A MUSCLE BIOPSY

Principle
Enzyme histochemistry has provided a means through which muscle fiber types can be determined in the classification of neuromuscular disorders. Tissue fixation is an important phase of enzyme histochemistry. The use of fresh unfixed snap-frozen tissue is employed to preserve muscle and cellular detail without compromising or sacrificing enzyme activity.

Equipment/Supplies
Liquid nitrogen
Isopentane (2 methyl butane)
Long-handled forceps
Two metal bowl containers, one larger than the other
Gloves
Disposable blocks
Low temperature thermometer

Safety Concerns
CAUTION: Isopentane is extremely flammable. Do not allow material to reach room temperature. Use goggles or safety glasses with liquid nitrogen.

Quality Assurance
Slides should be checked for the presence of ice crystal artifact.

Reagent Preparation
7% Gum Tragacanth
1. Using a Mettler balance, weigh 7 gm Gum Tragacanth and place into plastic storage container with lid.
2. Using a graduated cylinder, measure 100 ml distilled water and add to plastic container containing 7 gm Gum Tragacanth.
3. Add 5 grains Thymol and dissolve by manually stirring at room temperature.

NOTE: The Gum Tragacanth may take several hours to dissolve into a smooth consistency. Therefore, the solution may sit at room temperature. Stir at frequent intervals.

Label 7% Gum Tragacanth with an expiration of 6 months and store in refrigerator (4° C).

Procedure
1. Place appropriate amount of Gum Tragacanth on disposable block. Patient name and pathology accession number should be written on the block and container in which the specimen is to be stored.

2. Place the tissue to be frozen onto the mound of Gum Tragacanth. (Tissue should be on top of Gum Tragacanth, not surrounded or sunk within.) Muscle biopsies should be oriented transversely (for cross section).

3. Arrange containers so that a small container with Isopentane is placed into a larger container of liquid nitrogen. Allow the temperature of the Isopentane to drop to -140° C.

4. Using long-handled forceps to hold the block, invert block with tissue, oriented on top of Gum Tragacanth into pre-cooled Isopentane so that only the tissue is immersed in Isopentane.

5. Hold block steady in this position for 10 seconds (5 seconds if biopsy is smaller than 0.3 cm in width). Do not remove biopsy from Isopentane until freezing is complete.

6. Slowly lower inverted block deeper into Isopentane to allow freezing of the Gum Tragacanth only for 7 seconds. Do not lower the entire block into Isopentane.

7. Carefully remove frozen biopsy and quickly place into pre-chilled storage container that is airtight, or transfer to cryostat for immediate sectioning. Can be stored frozen indefinitely at -40° C or below.

References
1. Goss, Gwendolyn R., HT(ASCP). Muscle Biopsy: Role of the Histotechnologist in Optimum Freezing. Enzyme Histochemistry and Diagnosis. Cleveland OH 1992.

2. Muscle Biopsy Procedure, Department of Pathology, Duke University Medical Center.

Revised July 2006