Protocol

Protocol for a Patient Dying of Suspected UCD or Other Metabolic Disorder

It is important to obtain the proper samples for later diagnostic testing when a patient with a suspected UCD or other metabolic disease is dying. It may be helpful, especially if reimbursement may be problematic, to order the autopsy and sample collection as physician orders.

The Brain and Tissue Bank for Developmental Disorders, in contract to the National Institute of Child Health and Human Development, is a national tissue repository whose purpose is to drive research in the neurodevelopmental disorders. The Bank can be very helpful in coordinating sample collection from patients with developmental disorders involving the central nervous system. The Bank systematically collects, stores, catalogs and distributes human brain and other tissues for research dedicated to the improved understanding, care, and treatment of developmental disorders of the nervous system. Bank personnel are always available to answer questions and to coordinate tissue recovery.
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For further information, contact Philip H. Schwartz, PhD, Coordinator and Project Pathologist, or Stuart A. Stein, MD, Director, NICHD Brain and Tissue Bank for Developmental Disorders, (800) 418-1961 or at pschwartz@choc.org or see http://www.choc.com/btbmain.htm. Brain and Tissue Banks are also located in Miami and Baltimore. Information can be found at the above website.

The following protocol is recommended by the authors.

Inform pathologist of the impending autopsy. The pathologist or metabolic disease consultant should, ahead of time, label tubes and aluminum foil for tissues for sample collection with patient name, medical record number, tissue type, date, etc. For infants and children, call the newborn screening laboratory to ensure that they do not discard any excess newborn screening blood spots they might still be holding.
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Obtain a skin biopsy from any site (Achilles tendon, if postmortem). Place the sample in tissue culture medium and keep it at room temperature. If tissue culture transport medium is unavailable, sterile Ringer’s or sterile saline may be used. The specimen should not be subjected to extremes in temperature. If transport to laboratory is delayed, specimen may be stored in refrigerator overnight.

Transport the sample as soon as possible to a laboratory where the fibroblasts can be grown in culture. Most biochemical genetics and cytogenetics laboratories offer this service. Make sure any topical antibacterial agent used to clean the skin is fully removed with alcohol before the biopsy is taken. A skin biopsy is minimally invasive and is optimal if it can be taken before death. Skin fibroblasts from the Achilles tendon have been successfully grown from a biopsy taken as long as 4 days after death.
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In most instances permission for an autopsy must be requested. It is important to stress to the family the importance of the autopsy in making a diagnosis and how this may be invaluable for future genetic counseling and potential prenatal and postnatal diagnosis in siblings.

A complete autopsy is desirable and should be performed within 4 to 6 hours of death or earlier, if at all possible. Collect as much blood as possible from cardiac puncture; 20 mL total is a reasonable goal. The blood should be collected in the following tubes (5 mL in each tube, if possible): red top (small) serum separator tube (spin and store serum at ­70°C), 3 sodium hep-arin tubes (usually green top; caution: not all green tops contain sodium hep-arin), 2 green top tubes (spin and store plasma from 1 tube at ­70°C and leave second tube unspun on wet ice at 4°C for separation and isolation of white blood cells; transport the second tube to the biochemical genetics laboratory for separation, keep whole blood in the third tube at room temperature and send it to the cytogenetics lab immediately), 1 EDTA (usually lavender top); leave unspun on wet ice at 4°C and send to the biochemical genetics laboratory, and 1 ACD tube (usually yellow top—the only additive that usually will not be suitable for DNA sample is sodium heparin; keep at 4°C or in refrigerator for DNA and send it to the DNA diagnostics lab as soon as possible; call biochemical genetics, cytogenetics, and DNA diagnostic labs to ask for instructions on processing, shipping, and storage).
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Collect enough blood to fill 4 filter paper spots on a newborn screening card. This can have multiple uses. If a newborn screening card is not available, use any thick filter paper.

At the time of the autopsy, collect as much urine as possible from the bladder with a suprapubic tap. Even traces of urine swabbed from the posterior bladder wall with a cotton swab can be helpful. If unable to collect urine, collect vitreous humor from the eye with a syringe and freeze. These samples should be immediately frozen in liquid nitrogen or, if not available on dry ice, then kept at ­70°C.
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Collect several grams of muscle, cut into several 2-g pieces, place in labeled aluminum foil, and freeze in liquid nitrogen or dry ice or at ­70°C. Collect several 2-g pieces of liver, brain, kidney, and heart muscle with the same technique and place into separate labeled containers. Collect samples from each of these tissues for routine histology (formalin) and electron microscopy (glutaraldehyde). If not working directly with the pathologist, call to inquire about sample handling for histology and electron microscopy.

Collect bile by direct puncture of the gallbladder and store frozen (2 to 3 mL is usually sufficient). Smaller volumes can be spotted on filter paper and stored at room temperature.

In most instances the samples should be collected by a pathologist. All sample bags/foils/containers must be labeled carefully.

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