not exposed to extreme temperatures. There is one report of successful cell cultures after unfortunate delays of more than 2 weeks.668
Microbial contamination
Microbial contamination is a rare cause of culture failure in experienced laboratories and is largely preventable. As noted earlier, AF itself has bacteriocidal properties. If overwhelming microbial contamination is apparent within 24 hours after setup, it is probably due to improper handling of the specimen between amniocentesis and delivery to the laboratory (e.g. leakage from loose screw caps or poorly packaged syringes).
Approximately 10–20 percent of all samples are cell rich and their turbidity should not be a source of anxiety with regard to possible contamination. This also holds for brownish fluids containing cellular debris and granules in addition to erythrocytes. Seguin and Palmer669 measured cell growth from clear, cloudy (cell‐rich), bloody, and dark brown fluids. They showed that cloudy fluids yield better growth than clear ones. They confirmed earlier observations670 that very bloody fluids adversely affect the cloning efficiency. If bacterial or yeast contamination arises during the course of cell culture, it is by no means hopeless to attempt to salvage such cultures. Penicillin‐, streptomycin‐, or fungicide‐supplemented media are used to feed cultures daily after initial frequent washings. Increased chromosomal breakage rates and elevated rates of pseudomosaicism may be observed in such salvaged cultures but if the metaphase cells are analyzable, this rarely interferes with interpretation of the results.
Mycoplasma
Mycoplasma is not a significant problem in AFC culture, due mainly to better quality control by the serum manufacturers but also to the awareness of cell culturists that AFC cultures should not share incubator space with established cell lines. A shared water bath used for heating media and trypsin can be a source of mycoplasma contamination because permanent cell lines, frequently shipped from laboratory to laboratory, remain the prevailing source of mycoplasma contamination. As additional protection, many laboratories heat‐inactivate their sera before use. Commercial test kits are available for the detection of mycoplasma infections in cell cultures.671
Plastic ware and media storage
There have been occasional batches of cell culture‐grade plastic that do not, or barely, support cell attachment and growth. As with any component of the cell culture system, it is advisable to test new and old plastic ware in parallel for toxicity and ability to support growth in vitro. Hoehn's laboratory switched several times between Corning and Falcon and tested additional brands because of considerable fluctuations in quality.604
Incubator failure
Incubator failure is not a trivial cause of culture loss. The main threats are breakdown of the gas supply or equipment. AF cells cannot tolerate a pH close to or higher than 8 for more than 6–8 hours. On the other hand, a pH of less than 7.0 (for example, due to excess CO2 in the incubator) causes cells to stop dividing. A second danger is overheating of incubators due to mechanical failure or human error. Connection of incubators to emergency power sources is important. Temperature‐ and gas‐sensitive alarm systems are advisable.
Record keeping and quality control
With the advent of highly standardized cell culture methods, culture hazards have become a much rarer cause for concern in the prenatal diagnosis laboratory. Due to the greater number of specimens processed by the average laboratory, a variety of quality‐control measures need to be followed to avoid mistakes ranging from culture mixups to diagnostic errors. The most common and potentially serious laboratory errors are human errors in labeling and cross‐contamination of samples. Labeling errors can occur at any stage where cells are transferred between vessels: in the amniocentesis procedure room, at culture initiation, feeding and subculture, harvest, slide making, and even microscope analysis. Cross‐contamination of cells between patient samples is most common at the time of cell culture harvest, especially for suspension harvests. For these reasons, quality control and quality assurance programs must include a nonpunitive recording system for all laboratory events. A regular review of those events should seek patterns of error that can be eliminated by continuing education of laboratory staff or (often more effective) process improvement directed at reducing the opportunity for human error.
Laboratory directors and supervisors should be familiar with the College of American Pathologists Laboratory General and Cytogenetics checklists and the American College of Medical Genetics Standards and Guidelines.665 Laboratories should also participate in a peer review system such as the CAP proficiency surveys.
Safety in the laboratory
It is the responsibility of the laboratory director and all the laboratory staff to protect the rights, privacy and health of employees, ancillary staff and patients alike. AF specimens and all cultures up to the stage of fixation should be treated as potentially hazardous. Universal precautions are essential. Available resources include the CAP Safety Checklist and excellent reviews of laboratory safety and management.672–674
Mesenchymal stem cells in amniotic fluid
Multipotent mesenchymal stem cells (MSCs) can be obtained from several tissue sources and are of great interest for their potential uses in gene therapy and tissue repair. Those derived from adult bone marrow or other sources apart from AFCs have some drawbacks including their relative rarity and slow rate of proliferation in vitro. In contrast, MSCs derived from AFCs have distinct advantages.675–679 MSCs comprise about 1 percent of the cells in midtrimester AF and likely derive from fibroblastic F‐type cells.675 Recent advances in the isolation and culture of MSCs from AF are welcomed because these cells apparently do not form teratomas and are not tumorigenic even after many passages. Amniotic MSCs proliferate well and have stable normal telomeres, cytogenetics, and cell surface markers of pleuripotency, similar to embryonic stem cells. Amniotic MSCs also circumvent ethical objections associated with the use of embryonic stem cells.
Although more research is needed, amniotic MSCs appear to have immunogenic characteristics that are favorable for allogenic transplantation.680 They can be coaxed into differentiation along many lineages such as adipogenic, osteogenic, myogenic, endothelial, neurogenic, pancreatic, and hepatic, and including mesodermal, ectodermal, and endodermal lineages.480, 676, 678, 681–683 These cells are likely to find utility in a wide variety of cellular therapies,481, 675 including anticancer combination therapy.684 In a demonstration of whole‐tissue engineering for early repair of congenital malformations, heart valves have been fabricated using amniotic MSCs. The engineered heart valves exhibited normal endothelial surfaces and adequate opening and closing behavior.479 Under appropriate conditions, amniotic MSCs can form bone.676 The potential exists for amniotic MSCs to be employed for engineering tissues in time to be implanted shortly after birth to repair malformations of the heart, skin, bladder, or diaphragm.479, 685–687 This approach is unlikely, at least in the short term, to be helpful in the fetal therapy of spina bifida (see Chapters 29 and 30).
References
1 1. Parkin FM, Lind T, Cheyne GA. Biochemical and cytological changes in liquor amnii with advancing
