The Principles of Clinical Cytogenetics - Extra Materials - Springer

68 Martha Keagle and Steven Gersen
Likewise, bone marrow cultures need little attention once the culture has been initiated. Bone
marrow contains actively dividing cells and, therefore, can be harvested directly, without any time in
culture, or a 24- to 48-hour culture time can be used to increase the mitotic index. Longer culture
periods are generally not advised because the abnormal cancerous cells might be lost over time or be
diluted out by normal precursor cells that might be present. A short growth period usually provides a
more accurate reflection of makeup of the tumor; however, there are exceptions, as some tumor cells
are slow growing. Certain B-cell mitogens require increased culture times.
Amniotic fluid and solid tissue specimens require longer culture periods and do not grow at predictable
rates. Cell growth is monitored periodically until there are sufficient numbers of dividing
cells present, indicating that the culture is ready for harvest. An inverted phase-contrast microscope
is used to visualize the mitotic cells, which appear as small, refractile spheres. In situ amniotic fluid
cultures are generally harvested at 6–10 days, sometimes earlier. For amniotic fluid and solid tissue
specimens grown using the flask method, the culture interval might be 2 weeks or more.
Amniotic fluid and solid tissue specimens cultured with either the in situ or flask method become
depleted of required nutrients and additives during the culture period. Depleted medium must be
removed and replenished with fresh medium. This process is called “feeding” the culture and is done
on a regular basis throughout the culture maintenance period dependent on the number of cells growing,
the length of time in culture, and the protocol of the laboratory. Exhausted medium becomes
acidic and will appear yellow if the medium contains a pH indicator such as phenol red.
CELL HARVEST
After the cell cultures have grown for the appropriate period of time and there is a sufficient
number of dividing cells, the cells are harvested. Harvest is the procedure of collecting the dividing
cells at metaphase, their subsequent hypotonic treatment and fixation, and the placement of
the chromosomes on glass slides so they can be stained and microscopically examined. The
basic steps of cell harvest are the same for all specimen types, with minor variation. An example
is shown in Fig. 1.
Mitotic Inhibitor
A mitotic inhibitor must be used to obtain adequate numbers of cells in the metaphase. Colcemid ® ,
an analog of colchicine, is used in most cytogenetics laboratories. Colcemid binds to the protein
tubulin, obstructing formation of the spindle fibers or destroying those already present. This prevents
separation of the sister chromatids in anaphase, thus collecting the cells in the metaphase. Exposure
time to Colcemid is a trade-off between quantity and quality. A longer exposure results in more
metaphases being collected, but they will be shorter because chromosomes condense as they progress
through metaphase. Longer chromosomes are generally preferred for cytogenetic studies. Exposure
time to colcemid varies by specimen type.
Hypotonic Solution
A hypotonic solution is added to the cells after exposure to Colcemid. The hypotonic solution
has a lower salt concentration than the cell cytoplasm, allowing water to move into the cell by
osmosis. This swells the cells and is critical for adequate spreading of the chromosomes on the
microscope slide. Timing is crucial, as too long an exposure will cause the cells to burst. Too short
an exposure to hypotonic solution will not swell the cells sufficiently, which results in poor spreading
of the chromosomes.
There are a variety of acceptable hypotonic solutions, including 0.075M potassium chloride (KCl),
0.8% sodium citrate, dilute balanced salt solutions, dilute serum, and mixtures of KCl and sodium
citrate. Morphology of the chromosomes is affected by the hypotonic solution used. The choice of
hypotonic solution is based on specimen type and laboratory protocol.