The Principles of Clinical Cytogenetics - Extra Materials - Springer

Basic Laboratory Procedures 67
Preparation of Specimens for Culture
Amniotic fluid specimens, whole blood samples, and bone marrow samples arrive in the laboratory
as single cells in a fluid environment. Whole blood or bone marrow can be added directly to the
culture medium or the white blood cells can be separated from the other blood elements and used to
inoculate the culture medium. Separation of the white blood cells is easily accomplished by centrifuging
the sample or allowing it to rest undisturbed until the blood settles into three distinct layers.
The lowest layer consists of the heavier red blood cells, the top layer consists of plasma, and the
narrow middle layer, the buffy coat, consists of the desired white blood cells. The buffy coat can be
removed and used to establish the suspension culture.
Amniotic fluid contains a variety of cells that arise from the fetal skin, urinary and gastrointestinal
tracts, and the amnion. These are collectively referred to as amniocytes. Most of the cells in an
amniotic fluid sample are dead or dying and are not suitable for cytogenetic analysis. Amniotic fluids
are centrifuged at low speed (800–1000 rpm) to retrieve the small number of viable cells. The cell
pellet is then used to establish the cultures. The supernatant can be used for a variety of biochemical
tests including α-fetoprotein (AFP) and acetylcholinesterase (AChE) assays for open fetal defects.
Solid tissue samples received in the cytogenetics laboratory are usually too large to culture directly
and must be disaggregated before use. To obtain single cells, the sample must be finely minced using
sterile scissors or scalpels, or, alternately, cell dispersion can be achieved by enzymatic digestion of
the sample using collagenase or trypsin.
CULTURE MAINTENANCE
After cultures have been initiated, they are allowed to grow under specific conditions of temperature,
humidity, and pH until adequate numbers of dividing cells are present. The optimal temperature
for human cell growth is 37°C and it is essential that incubators be maintained at this temperature.
Cultures are maintained in either “open” or “closed” systems, depending on the type of incubator
used.
Open systems are those that allow the free exchange of gases between the atmosphere inside the
culture vessel and the surrounding environment of the incubator. To facilitate the exchange of gases,
the tops or caps of tissue culture vessels are loosely applied. A CO 2 incubator is required for open
systems to maintain the 5% CO 2 level necessary to sustain the ideal pH of 7.2–7.4. A humidity level
of 97% should be maintained to prevent cell death as a result of cultures drying out. This can be
accomplished by placing pans of sterile water in the bottom of the incubator. A major disadvantage of
open systems is that they are susceptible to microbial contamination, especially fungi, because of the
moist warm surfaces in the incubator. An open system is required for samples grown on cover slips
by the in situ method.
Closed systems are those in which the culture vessels are tightly capped to prevent exchange of
gases. Humidification is self-maintained, and CO 2 incubators are not required. Commercial media
are buffered to the appropriate pH necessary to sustain short-term cultures such as those from blood
and bone marrow samples. Long-term cultures from amniotic fluid and solid tissue specimens require
the use of additional buffering systems to maintain the proper pH over the longer culture period.
Microbial contamination is not as great a risk with closed systems.
In the final analysis, the decision to use an open or closed system or a combination of both involves
the type of sample being processed and the preference of the laboratory.
Culture Maintenance and Growth Interval
Once the culture requirements are met, the cells must be allowed time to grow and divide. The
time in culture varies depending on the cell type involved.
Peripheral blood cultures require little maintenance once the growth requirements have been met.
The culture vessels are placed in an incubator for a specified period of time, usually 72 hours.