Laboratory Considerations

Cytogenetics is used to detect gross abnormalities of the human chromosomes that are large enough to be seen with aid of the light microscope. To study human chromosomes, the cells must be dividing, as the individual chromosomes are only visible at this level during mitosis. Routine analyses are performed at metaphase, although techniques to collect cells earlier, in prophase or prometaphase, can be used, as mentioned previously. With the exception of bone marrows, most samples

From: Molecular Diagnostics: For the Clinical Laboratorian, Second Edition Edited by: W. B. Coleman and G. J. Tsongalis © Humana Press Inc., Totowa, NJ

received in the cytogenetics laboratory do not contain actively dividing cells. Therefore, the cells must be grown in tissue culture until sufficient numbers of dividing cells are present.

Cytogenetics laboratories receive four basic types of samples: (1) amniotic fluids, (2) bloods, (3) solid tissues, and (4) bone marrows. Although the individual details differ, the general steps of culture initiation or setup, culture maintenance, and cell harvest are basically the same for all sample types. To obtain dividing cells, the most critical requirement is that there be living cells present in the original sample. It is, therefore, crucial that cytogenetic samples be collected and handled using aseptic technique and that the samples be placed in a sterile growth or transport medium after collection, not in formalin or alcohol. Blood and bone marrow samples should be collected in tubes containing sodium heparin to prevent coagulation.

Once received in the laboratory, the cells are placed in sterile tissue culture vessels containing an appropriate complete growth medium. There are many commercial media available that can be used. Some are broad spectrum, suitable for many types of cells; others have been formulated for specific cell types. Microbial inhibitors, including antibiotics and fungicides, can be added to retard microbial growth in the event of contamination.

Blood, bone marrows, and amniotic fluids consist of single cells and can be used as they are received in the laboratory. Lymphocytes from blood and bone marrows are grown in suspension culture in sterile capped centrifuge tubes or T-flasks, whereas fibroblasts from amniotic fluids attach to the inner surface of a T-flask or directly on cover slips in small Petri dishes. The latter is referred to as the in situ method of tissue culture. Solid tissues usually must be disaggregated before they are added to the culture medium. This can be done mechanically using sterile scissors or scalpels, or enzymatically. Solid tissues can be grown by the flask or in situ method. A few solid tumors grow best in suspension culture.

Whereas most cell types will grow and divide spontaneously in tissue culture if given the proper growth requirements, peripheral lymphocytes must be stimulated to divide. This is done by adding phytohemagglutinin (PHA) to the medium. PHA is a mitogen derived from an extract of kidney beans. There are also B-cell mitogens that could be used when studying hematologic samples from patients suspected of having B-cell disorders. For a more complete discussion of culturing techniques, see refs. 17 and 18.

After step-up the cultures are placed in an incubator at 37°C and grown until there are sufficient cells in mitosis. The culture time varies depending on sample type. Actively dividing bone marrows cells are usually harvested directly without any time in tissue culture or after 24 h. Routine blood samples are usually allowed to grow for 72 h before harvest, and amniotic fluids can take from several days to a week or longer to produce adequate numbers of dividing cells. Solid tissues often need to culture for a week or longer. When adequate numbers of dividing cells are present, they are harvested.

The mitotic inhibitor, Colcemid, is used to collect dividing cells in metaphase. Colcemid is an analog of colchicine, an extract of the seeds of the autumn crocus. It acts by binding to tubulin and preventing formation of the spindle fibers (19). This prevents separation of the sister chromatids in anaphase, thereby collecting the cells in metaphase, the stage at which they are best observed by light microscopy. The dividing cells are then treated with a hypotonic solution such as potassium chloride. Hypotonic solution swells the cells and aids in the spreading of the chromosomes. Well-spread chromosomes are essential for an accurate cytogenetic evaluation. The cells are then fixed in that swollen state using a modified Carnoy's fixative consisting of three parts methanol to one part glacial acetic acid. This cytogenetic fixative has the added benefit of lysing any red blood cells present in the culture. The harvest procedure is completed by dropping a small amount of the fixed cell suspension onto clean glass microscope slides. There are many variables that influence the spreading and quality of the metaphase preparations. These include ambient temperature, relative humidity and the length of exposure of the cells to hypotonic solution. Good spreading is a function of slide drying, and technologists must be ready with a variety of techniques to compensate for less than optimal chromosome spreading. A longer evaporation time increases spreading, whereas a shorter evaporation time slows spreading. In general, increased temperature and humidity increases chromosome spreading by prolonging the evaporation time, and decreased temperatures and lowered humidity reduces spreading by shortening evaporation time.

The prepared slides are aged and then banded. Aging is essential to achieving good banding. Giemsa-banding (G-banding) is routinely used to study human chromosomes. This technique creates a unique series of light and dark bands along the length of the individual chromosomes (Fig. 1). There are a variety of G-banding techniques (see ref. 18 for sample protocols), but ones involving pretreatment with the enzyme trypsin are the mainstay of most cytogenetics laboratories. Giemsa light bands are rich in the nitrogenous bases CG and are early replicating, whereas the Giemsa dark bands are AT rich and late replicating. A higher proportion of active genes are located in the G-band light regions, making them biologically more significant.

G-banding allows for the positive identification of the individual chromosomes and for the characterization of many structural rearrangements. There are times when G-banding is not sufficient, however. In such cases, there are a wide variety of special stains and FISH probes that can be used to answer specific questions not answered by G-banding. Because a discussion of these is outside the scope of this chapter, the interested reader is referred to refs. 17, 18, and 20 for a more complete description of special stains.

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