Sampling Instructions

Skin fibroblasts – skin biopsy

The Skin Biopsy must be obtained under sterile conditions, be sure to remove all betadine prior to procedure then wipe with alcohol allowing the alcohol to dry. Use a 3 or 4 mm punch biopsy kit or take from the site of incision during a muscle biopsy procedure.

Place biopsy in a container of sterile Hanks Balanced Salt Solution (HBSS) [1x; + calcium, + magnesium] with gentamicin reagent.

NOTE: To make Hanks Balanced Salt Solution (HBSS) with gentamicin reagent, add 0.5 ml of gentamicin reagent to 100 ml of Hanks Balanced Salt Solution (HBSS).

If HBSS with gentamicin is unavailable, sterile HBSS or sterile physiological saline may also be used. You may also request that we send a tube of HBSS with gentamicin to your physician.

Frozen plugs of cultured skin fibroblasts are maintained in liquid nitrogen for future use.

Send sample at room temperature using overnight courier.


Skin Fibroblasts – cultured cells

When assay of PDC, PC, PEPCK, KDC, MCC, or PCC in cultured skin fibroblasts is requested, the sender is asked to provide two small flasks (T-25) filled with media for subculture at the CIDEM Tissue Culture facility. Flasks should have non-vented caps and parafilmed. These will be subcultured concurrently with an appropriate control to provide confluent (T-75) flask(s) for the patient and control

Cultured skin fibroblasts must be pre-tested for mycoplasma contamination, and documentation of such testing must be forwarded with the each sample. When received, fibroblasts are stored and subcultured in a 37°C incubator until harvested for assay.

Cells which are received without documentation that mycoplasma testing is negative must be kept in isolation until such testing can be done by the CIDEM Tissue Culture facility. Cells which test positive for mycoplasma will be immediately discarded and the sender notified. (Note: Cells which are assayed with mycoplasma, fungal, or bacterial contamination can produce unsatisfactory results, e.g., low specific activity, poor replicates.)

Frozen plugs of cultured skin fibroblasts are maintained in liquid nitrogen for future use.

Send sample at room temperature using overnight courier.


Frozen Tissue (Muscle, Liver, Heart)

Tissues obtained at biopsy or autopsy should be immediately frozen in liquid nitrogen or dry ice, and then stored at -70°C until shipment to CIDEM. Tissues must be packed in dry ice and shipped overnight. Samples received at CIDEM are stored at -70°C. Ordinarily, at least 100 mg should be provided; in special circumstances, the assay can be performed with as little as 20 mg.

Samples, which have thawed during shipment due to insufficient dry ice, or samples which have not been quick frozen or stored at –70°C may produce unsatisfactory results due to deterioration. Samples obtained at autopsy done more than ~8 hours after death may also produce unsatisfactory results due to autolysis. Submitters of such samples will be informed of the uncertainty of obtaining adequate results prior to performing the assay.

Send sample on dry ice using overnight courier.


Blood Lymphocytes

Collection and shipment of whole blood for lymphocyte assays is arranged in advance by calling the CIDEM laboratory. Lymphocytes must be isolated within 48-72 hours of collection, and prior arrangements are needed to reserve sufficient laboratory time to complete the assays.

The volume of blood required for PDC, KDC or PC assay varies according to the patient’s age, as follows (multiple assays may be possible but will require additional volume):

5 ml – patients <6 months old
10 ml – patients 6 months – 6 years old
20 ml – patients >6 years old
20 ml from an unrelated adult control

Sterile technique is used to collect blood from the patient and a volunteer adult control in ACD. The blood is shipped to CIDEM in a styrofoam box by overnight delivery at room temperature, with the outside of the shipping container marked: Room temperature: Do not freeze or refrigerate. Upon receipt, the blood is stored at room temperature until the lymphocytes are isolated (48-72 hours after blood is obtained).

*Note: ACD is available in yellow stoppered Vacutainer tubes. Yellow stoppered vacutainers containing SPS for use in microbiology are not suitable for enzymes assays.

Send sample at room temperature using overnight courier.


Deproteinized blood or CSF (cerebrospinal fluid)

Collect venous or arterial blood without prolonged stress to the patient with use of tourniquet, if needed. As soon as the blood or CSF is obtained, measure exactly 1 ml (with tuberculin syringe or a pipette) and transfer immediately to 2 ml of 8% w/v perchloric acid*. Stopper tube and shake vigorously for at least 15 seconds. The sample is now stable for local transport to a laboratory where it can be centrifuged. Freeze clear supernatant in a fresh tube.

For infants measure exactly 0.5 ml (with tuberculin syringe or a pipette) and transfer immediately to 1 ml of 8% w/v perchloric acid*. Stopper tube and shake vigorously for at least 15 seconds.

Send sample on dry ice using overnight courier.


Plasma

Collect 0.5 – 1 ml blood in an EDTA tube (lavender top), mix gently, and centrifuge as soon as possible after collection. Then transfer the plasma into a polypropylene tube and freeze.

The specimen of choice for this laboratory is EDTA plasma (plasma gel tube is NOT acceptable). Serum is acceptable if from a plain red top, no gel tube.

Send sample on dry ice using overnight courier.

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