The Principles of Clinical Cytogenetics - Extra Materials - Springer

Basic Laboratory Procedures 77
Lack of viable cells or unsuitable cell type can compromise amniotic fluid samples. Samples from
patients with advanced gestational age (20 weeks or older) could consist primarily of mature nondividing
cells or dead cells. Some samples consist principally of epithelial cells, which typically produce
few metaphases of poorer quality than the desired fibroblasts.
Amniotic fluid samples are usually clear yellow in appearance. A brown fluid indicates prior
bleeding into the amniotic cavity, which could suggest fetal death or threatened miscarriage. In such
samples, there might be few, if any, viable cells present. Bloody taps containing large numbers of red
blood cells can be problematic. The physical presence of large numbers of red blood cells can prevent
the amniocytes from settling on and attaching to the growth surface of the culture vessel. In addition,
the red cells utilize nutrients in the culture medium, thereby competing with the amniocytes.
Patient factors can influence the success of peripheral blood and bone marrow samples. Disease
conditions, immunosupression, and use of other drugs can affect both the number of lymphocytes
present and their response to mitotic stimulants. The laboratory is not always made aware of these
confounding factors. Bone marrow samples that have been contaminated with blood might not have
adequate numbers of spontaneously dividing cells present. For this reason, it is important that the cytogenetics
laboratory receive the first few milliliters of the bone marrow tap. Bone marrow samples are
notorious for producing poor quality metaphases. There are sometimes adequate numbers of
metaphases, but the chromosomes are so short and so poorly spread that analysis is difficult or impossible.
In addition, metaphases of poor quality often represent an abnormal clone.
The failure rate of solid tissues may be quite high and is often the result of the samples themselves.
In the case of products of conception or stillbirths, the sample might not contain viable cells or the
wrong tissue type might have been collected. Additionally, microbial contamination is a frequent
contributing factor because many solid tissue samples are not sterile prior to collection.
PRESERVATION OF CELLS
Cells do not survive indefinitely in tissue culture. After a period of time, they become senescent
and eventually die. At times, a sample might need to be saved for future testing, to look at retrospectively,
or because it is unusual or interesting and might be of some value in the future. In such cases,
the cells need to be kept alive and capable of division long term or indefinitely.
Cultured cells can be kept alive by cryopreservation, the storage of cell in liquid nitrogen. The
freezing process is critical to cell survival. Rapid freezing will cause cell death due to formation of
ice crystals within the cells. Improper freezing can also denature proteins, alter the pH, and upset
electrolyte concentrations. The cells must be cooled slowly so that water is lost before the cells
freeze. The addition of 10% glycerol or dimethyl sulfoxide (DMSO) to the storage medium lowers
the freezing points and aids in this process. One milliliter aliquots of the sample in storage medium
are placed in cryogenic freezing tubes. The samples are then slowly frozen under controlled conditions
at a rate of 1°C per minute to a temperature of –40°C. The sample can then be rapidly frozen to
about –80°C. Alternately, the samples can be placed in a –70°C freezer for 1–4 hours. After this
initial freezing has been accomplished, the cells are stored in liquid nitrogen at about –190°C.
Thawing of the sample is also critical. Rapid thawing is necessary to prevent the formation of ice
crystals.
B-Lymphocytes can be transformed so that they will proliferate indefinitely in tissue culture by
exposing them to Epstein–Barr virus (EBV). These immortalized lymphoblastoid cell lines do not
become senescent and can, therefore, be maintained indefinitely in culture.
CHROMOSOME ANALYSIS
Selection of the correct specimen for chromosome analysis and additional tests is not always
straightforward, and the submission of an inappropriate sample to the laboratory can create frustration
for both patient and clinician.