The Principles of Clinical Cytogenetics - Extra Materials - Springer

66 Martha Keagle and Steven Gersen
Growth Factors
A variety of additional growth factors are commercially available and are used by some laboratories
to achieve optimal cell growth for different sample types. These include giant cell tumor extract
(GCT) for bone marrow culture and specially formulated amniotic fluid culture media.
Culture Vessels
The choice of culture vessel depends in part on the growth needs of the sample and in part on the
individual preference of the laboratory. Blood and bone marrow samples consist of single free-floating
cells. For such suspension cultures, sterile centrifuge tubes or tissue culture flasks (T-flasks) can
be used. The cells from samples such as amniotic fluid, chorionic villi, skin biopsies, and other solid
tissues need to attach to a surface to grow. Such samples can be grown in T-flasks or with an in situ
method.
Flask Method
Cells are grown on the inner surface of T-flasks until adequate numbers of dividing cells are
present. Cell growth is monitored using an inverted microscope. To remove the cells from the surface
of the culture flask where they have been growing, the cultures are treated with an enzyme such as
trypsin. This enzymatic treatment releases the individual cells into the fluid environment and permits
their collection, harvest, or subculture, as needed.
In Situ Method
Amniotic fluid, chorionic villus (CVS) and other tissue samples can be grown directly on cover
slips in small Petri dishes, in “flaskettes,” or in slide chambers. Growth of these cultures is also
monitored with an inverted microscope. They are harvested as “primary” cultures (those that have
not been subcultured) when adequate numbers of dividing cells are present, and cells do not have to
be enzymatically removed prior to harvest. Therefore, the cells can be analyzed as they grow in situ.
Advantages of the In Situ Method over the Flask Method
The primary advantage of using the in situ method is that it provides information about the colony
of origin of a cell. This is important when deciding whether an abnormality seen in some but not all
cells represents true mosaicism (constitutional mosaicism) or an artifact of tissue culture (pseudomosaicism).
True mosaicism is said to be present when there are multiple colonies from more than
one culture with the same chromosomal abnormality. Pseudomosaicism is suggested if a single colony
with all or some cells exhibiting a chromosomal abnormality is found. In such cases, all available
colonies should be studied to rule out the possibility of true mosaicism. If only a single colony with a
potentially viable abnormality is found, it might result in an equivocal diagnosis. Low-level mosaicism
cannot be completely ruled out in such cases. Clinical correlation can help clarify the picture. A
repeat amniocentesis might confirm the presence of true mosaicism, but cannot, of course, eliminate
the results of the first study. See also Chapter 12.
No inference can be made about the origin of cells when using the flask method, because cells
from all colonies are mixed together after they are released from the growing surface. It is impossible
to tell if multiple cells exhibiting the same chromosomal abnormality arose from one or multiple
colonies. Thus, two or more cells exhibiting the same structural abnormality or having the same extra
chromosome or three or more cells lacking the same chromosome must be treated as potential true
mosaics if the flask method is used. However, it should be noted that the presence of multiple abnormal
colonies in the same in situ culture might also represent an artifact. Guidelines for interpretation
of mosaicism are available for both methods.
Another advantage of the in situ method is that there is usually a shorter turnaround time (TAT)
because only primary cultures are harvested. Flask cultures are often subcultured, adding days to the
culture time.