Costs

Since cytogenetic studies are expensive, they should be applied to autopsies in a frugal manner, but they certainly are indicated if chromosome analysis is the only means to obtain pertinent medical information. The cost of chromosome analysis varies among cytogenetic laboratories and ranges from a few hundred dollars to over $1,000, depending on the type of tissue studied.

SPECIMEN COLLECTION, TRANSPORT, AND PROCESSING

Most chromosome studies require living tissues to obtain successful cell culture for chromosome studies (18). For this reason, it is important to use sterile procedures to collect specimens. Whole blood and other tissues have been cultured successfully from mailed-in specimens for clinical purposes. Thus, it is not necessary for the autopsy pathologist to have ready access to a cytogenetic laboratory. Since living cells are involved, it is important to transport specimens to the cytogenetic laboratory in 1 or 2 d. Moreover, exposure of the specimen to temperature extremes (freezing or >30°C) can prevent a successful chromosome study. The specimens should not be frozen or packed on ice for delivery.

The cytogenetic laboratory is often used to culture cells from autopsies with evidence of a molecular or biochemical genetic disorder. In these cases, it is important that the prosector informs the cytogenetic laboratory to the need for molecular or biochemical genetic testing. This will assure that the cytogenetic laboratory processes the specimen correctly and forwards the cultured cells to another laboratory for appropriate genetic testing.

The following procedures may be used to prepare and mail specimens collected at autopsy for cytogenetic studies. When other tissues are needed, the collection procedure and mode of transportation should be discussed with personnel from the cyto-genetic laboratory to enhance chances of a successful result.

BLOOD Blood is generally the preferred specimen for chromosome analysis when a congenital disorder is suspected and it is possible to collect an appropriate specimen. Obtain 510 mL of unclotted, uncontaminated blood in a sterile fashion. Mix the blood sample with 1 mL of sodium heparin in a small sterile vial and send it to the cytogenetic laboratory.

In the cytogenetic laboratory, the cells are incubated for 6672 h at 37°C with a T-cell mitogen such as phytohemagglutinin. The cells are then harvested for chromosome analysis using ethidium bromide, colcemid, and hypotonic solution and then fixed with glacial acetic acid and methanol.

A few factors may interfere with processing blood for chromosome analysis. Cells may be lysed due to forcing the blood quickly through a needle. An improper anticoagulant (sodium heparin is best) or not mixing the blood with the anticoagulant can cause the blood cells to clot. Rare patients have blood T-lymphocytes that do not respond to mitogens used to cause cells to undergo mitosis.

FIBROBLASTS Specimens for fibroblast cultures should be collected at autopsy when a congenital disorder is suspected and blood is either unavailable or alternative tissues are needed to answer a medical question. Fibroblast cultures are generally more expensive than other chromosome studies because they require more time and culture maintenance. The prosector should make a longitudinal incision through the skin of the anterior thigh and dissect down to the fascia lata. A 5-15 mm2 sample of the fascia lata is then removed, together with about 2-3 mm thickness of underlying muscle. The tissue is wrapped in sterile gauze moistened with Hank's balanced salt solution (HBSS) and placed it in a small sterile vial for transportation to the chromosome laboratory.

Upon arrival in the cytogenetic laboratory, the specimen is cut into small pieces and treated with enzymes (19). The tissue is then placed into a culture flask with Chang and MEM-alpha-medium containing 20% fetal bovine serum (FBS) and antibiotics. After 5-14 d, the fibroblasts are processed for chromosome analysis with ethidium bromide, colcemid, hypotonic solution, and fixed with glacial acetic acid and methanol.

The most common problems with these specimens are lack of viable cells and bacterial contamination. These problems can interfere with attempts to establish fibroblast cultures.

BONE MARROW Bone marrow specimens may be required for chromosome studies at autopsy when a question of malignant hematological disorder is involved. Approximately 1 mL of bone marrow should be obtained in a sterile fashion and mixed with 1 mL of sodium heparin in a small sterile vial, and then sent to the cytogenetic laboratory.

In the cytogenetic laboratory, bone marrow specimens may either be processed for chromosome analysis directly or by a short-term (24-72 h) culture method (19,20). In either case, the bone marrow is harvested for chromosome analysis by using ethidium bromide, colcemid, and hypotonic solution and then fixed with glacial acetic acid and methanol.

PRODUCTS OF CONCEPTION OR STILLBIRTH These specimens should be collected when a congenital disorder is suspected and blood is unavailable. A 1-cm3 biopsy of muscle and fascia from the thigh, a 1-cm3 biopsy of lung, and 20-30 mg of chorionic villi should be obtained. Each biopsy sample is placed in a separate 15 mL sterile centrifuge tube with 10 mL of transfer culture media. In situations where the fetus is not identifiable, the specimen is placed in a single sterile container with 10 mL of HBSS or a similar solution.

Upon arrival in the cytogenetic laboratory, these specimens are cut into small pieces and treated with enzymes (19). The tissues are then placed into separate tissue flasks with Chang and MEM-alpha-medium containing 20% FBS and antibiotics to establish a fibroblast culture. After 5-14 d, the fibroblasts are processed with ethidium bromide, colcemid, hypotonic solution, and fixed with glacial acetic acid and methanol.

Chromosome analysis may be unsuccessful in some specimens because of a lack of viable cells or bacterial contamination. In our experience this occurs in up to 20% of cases and is usually due to a lack of viable cells. Sometimes maternal cells are cultured and analyzed rather than fetal cells.

SOLID TUMORS These specimens should be collected for chromosome studies only when the medical question relates to a solid tumor. The specimen should be representative of the solid tumor as the neoplastic chromosome abnormalities are rarely congenital or present in normal tissues. Using sterile procedures, a 5-mm3 or larger tumor biopsy is submitted. The specimen is placed in a transport container with 5 mL of HBSS or a similar solution.

In the cytogenetic laboratory, the tissue is dissociated using enzymes and/or mechanical means and then transferred to culture flasks (19). The cultures are incubated at 37°C with 5% CO2, 5% O2 and 90% N2 for 1-2 d depending on cell growth. The cells are harvested for chromosome analysis with ethidium bromide, colcemid, hypotonic solution, and fixed with glacial acetic acid and methanol.

Normal cells are often present in and around tumor tissue. In culture, these cells may grow better than neoplastic cells and result in the study of normal somatic cells.

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