1. Complete the Muscular Dystrophy Requisition. Questions about completing the requisition can be directed to: University of Iowa Diagnostic Laboratories (UIDL) at 319-384-7212 or toll free at 866-844-2522.
- a. A completed requisition must accompany all requests. It should contain: the patient name, identification number, date of biopsy, date of birth, male/female, tissue source, biopsy site, clinical history, question(s) to be answered and differential diagnosis.
b. Include other pertinent history and findings, such as radiographic findings, cancer history or treatment history.
2. Guidelines for Muscle Biopsy Preparation
- a. Before proceeding with distributing muscle for freezing and/or fixation, an assessment must be made of the following parameters: patient history, the quantity of tissue submitted, the quality of tissue submitted.
- i. If the biopsy is clearly intended to investigate inflammatory disease (e.g. dermatomyositis, polymyositis, inflammatory myopathy, vasculitis, PAN-polyarteritis nodosa, fasciitis), the tissue should be divided for freezing for enzyme histochemistry, formalin fixation for routine histology, and glutaraldehyde fixation for electron microscopy. When there is a scant amount tissue with this history, err on the side of fixing ample tissue in formalin.
ii. If the biopsy is clearly intended to investigate a muscular dystrophy (e.g. DMD – Duchenne’s dystrophy, BMD – Becker’s dystrophy, LGMD – limb girdle muscular dystrophy, SCARMD – severe childhood autosomal recessive muscular dystrophy, FSH – facioscapulohumeral dystrophy, MD – myotonic dystrophy, dystrophinopathy, sarcoglycanopathy), the tissue should be divided for glutaraldehyde fixation and for freezing for enzyme histochemistry, immunohistochemistry, and biochemistry.
iii. If the biopsy is clearly intended to investigate a metabolic abnormality or mitochondrial defect (e.g. glycogen storage disease, McArdle’s disease, carnitine deficiency, MELAS, Kearn-Sayre syndrome, MERRF, lipid storage disease, mitochondrial myopathy), the tissue should be divided for freezing for enzyme histochemistry, freezing for biochemistry, and glutaraldehyde fixation. When there is a scant amount tissue with this history, err on the side of freezing ample tissue for biochemistry.
iv. For histories that are vague or that are not clearly inflammatory, dystrophy, or metabolic/mitochondrial and for which there is ample tissue, the tissue should be divided for freezing for enzyme histochemistry, freezing for biochemistry, and glutaraldehyde fixation. If there is scant tissue, err on the side of freezing ample tissue for enzyme histochemistry.
FRESH TISSUE
- a. If you are within 2-3 hours travel time, you may prepare for delivery of fresh tissue; if not, then go to frozen tissue preparation.
b. At least one clamped biopsy is needed, measuring at least 0.5 cm in diameter and 0.8 cm in length.
c. Wrap the clamped muscle biopsy in gauze slightly moistened with normal saline. Do not immerse in saline, as this will produce an artifact, which may interfere with interpretation.
d. Immediately deliver to University of Iowa Hospitals and Clinics on wet ice.
FROZEN TISSUE
- a. The best results from enzyme histochemistry and immunohistochemistry studies are obtained when tissues are frozen rapidly and kept frozen at -60 to -80°C until sectioned. Any thawing which takes place and is followed by refreezing leads to ice crystal formation with loss of morphologic detail and cell membrane (hence antigenic) integrity. Enzymatic activity can also be lost.
b. The preferred methods of freezing are: - i. Liquid Nitrogen - Tissue for Biochemical studies
ii. Isopentane/liquid nitrogen - Tissue for Enzyme Histochemistry and Immunohistochemistry studies
Biochemical Studies
- Directions for Liquid Nitrogen are:
a. At least one piece of skeletal muscle tissue is required that measures at least 0.5 cm3 (about the size of a pencil eraser).
b. Cut the specimen from the clamp.
c. Wrap the tissue in aluminum foil and carefully immerse into the liquid nitrogen.
d. Leave the tissue in the liquid nitrogen for 1 minute. More time may be needed based on the size of the tissue.
e. Placed the wrapped tissue in a freezer or cryostat while you prepare the packing/shipping. A freezer must be used for storage of more than a few minutes.
f. When you are ready to ship, place the tissue in a small pre-cooled plastic Zip-lock bag to protect it from freeze-drying.
g. Always identify the specimen by writing with a waterproof marker on the foil and bag.
h. Label the specimen with the patient name, identification number, tissue source and date.
i. Write "Biochem" on the plastic bag.
Enzyme Histochemistry and Immunohistochemistry
- Directions for Isopentane/Liquid Nitrogen are:
a. At least one clamped biopsy is needed, measuring at least 0.5 cm in diameter and 0.8 cm in length.
b. Cut the specimen from the clamp.
c. Place a drop of O.C.T. embedding compound in the center of a small cork.
d. Place the biopsy in the O.C.T. with the muscle fibers perpendicular to the surface of the cork.
e. A plastic coverslip may be attached by slitting the cork to help support the biopsy (see diagram). However, do not cover the biopsy with OCT. This insulates the tissue causing artifact.
- f. Muscle tissue is prone to ice crystal artifact when frozen at temperatures quite adequate for other tissues. In order to consistently prevent this artifact and to preserve enzyme activity, it is important to follow the freezing protocol carefully.
g. Cool 50 ml of isopentane (2-Methyl Butane) in a small metal container to -155°C in a liquid nitrogen bath.
h. Stir continually to insure even cooling. If a cold temperature thermometer is not available, cool the isopentane until the outer area is frozen solid and the inner area is thick and slushy.
i. Insert a probe into the cork (to use as a handle) and plunge the specimen into the isopentane for 30 seconds. More time may be needed based on the size of the tissue.
j. Remove the specimen from the isopentane and place in a cryostat or freezer for several minutes.
k. Remove the specimen and dispose of the coverslip
l. Wrap in cold (pre-cooled) aluminum foil.
m. Leave the wrapped tissue in a freezer or cryostat while you prepare the packing/shipping. A freezer must be used for storage of more than a few minutes.
n. When you are ready to ship, place the tissue in a small pre-cooled plastic Zip-lock bag to protect it from freeze-drying.
o. Always identify the specimen by writing with a waterproof marker on the foil and bag.
p. Label the specimen with the patient name, identification number, tissue source and date.
q. Write "EH" on the plastic bag.
r. Ship tissue on dry ice. Tissue must be kept frozen.
- ROUTINE LIGHT MICROSCOPY
- a. Residual tissue not used in above protocols may be placed in formalin for routine histology.
b. Immerse the tissue in 10% neutral buffered formalin (NBF) and fix for four hours. Muscle may be fixed while still clamped.
c. Remove all of the tissue from the clamp and return to 10% NBF before delivery.
d. Ship at ambient temperature. DO NOT freeze.
- ELECTRON MICROSCOPY
- a. Tissue may be cut from clamped specimens prior to processing them for enzyme histochemistry or routine light microscopy. Cut longitudinally at 0.1 cm intervals. One fragment 0.1 x 0.1 x 0.5 cm is sufficient.
b. Immerse in pre-cooled electron microscopy (2.5% glutaraldehyde) fixative and refrigerate until delivery.
c. Ship at ambient temperature. DO NOT freeze.
- 4. Specimen Packaging
- Wet Tissue:
a. Double bag the specimen container with the requisition on the outside of the plastic bag.
b. Placed the double-bagged specimen into a styrofoam box (primary container) with adequate wet ice to keep specimen cool during transport.
c. Securely seal the primary container.
d. Place the primary container in a secondary shipping container, which should contain enough absorbent material to prevent any leakage from escaping outside the container.
e. These specimens can be sent by our Courier services within our service area.
Formalin-Fixed Tissue, Glutaraldehyde-Fixed Tissue:
a. Immerse the tissue in the appropriate fixative and container.
b. Label containers with "Formaldehyde precaution" or "Glutaraldehyde precaution".
c. Double bag the specimen containers and place into a box with adequate absorbent material in case of leakage.
d. Securely seal the box.
e. Indicate on the exterior of the box "Formaldehyde Precaution" and/or "Glutaraldehyde Precaution".
f. Ship at ambient temperature. DO NOT FREEZE.
g. This container can be taped to the dry ice box for shipping as a single unit.
Frozen Tissue:
a. Placed the double-bagged specimen into a Styrofoam container (primary container) with adequate dry ice.
b. Use at least 6-8 pounds dry ice. Use more in the summer months. DO NOT use wet ice, or coolants (i.e., Cool Packs).
c. Make certain the requisition is placed in the box, but on the outside of the double bag.
d. Securely seal the container and label with "Frozen Tissue- Do Not Thaw".
e. To avoid delivery problems due to prolonged transit time, please try to ship specimens on Monday, Tuesday, or Wednesday and never ship a frozen specimen over the weekend.
f. Call UIDL Client Services at 866-844-2522 for special situations such as weekend or holiday instructions.
g. These specimens can be sent by our Courier services within our service area or by overnight express airmail.
h. If using an overnight express service, make sure the box is labeled properly. Federal Express requires that all shipping labels be typed.
- 5. Specimen Transportation
- UI Diagnostic Laboratories can usually provide for specimen transport either through our own courier services or through other transportation providers such as overnight carriers. If you need assistance or information in transporting specimens to our laboratories, please contact UIDL Client Services and a representative will provide assistance in determining the most appropriate shipping arrangement.
- 319-384-7212 (local)
1-866-844-2522 (toll free)
- Shipping address:
University of Iowa Healthcare
UI Diagnostic Laboratories
200 Hawkins Drive, Room 6240 RCP
Iowa City, IA 52242-1087