Applications of Cytogenetics in Modern Pathology

Specimens

Virtually any viable, nucleated cell sample can be used for cytogenetic analysis. However, certain types of cells are easier to obtain and culture and are therefore favored for chromosomal preparations. For routine cytogenetic studies of adults and children, heparinized peripheral blood is the preferred specimen. Obtaining a sample via standard phlebotomy is usually easy to arrange and relatively painless for the patient. In hematologic disorders, the best results are obtained from bone marrow samples because this is the origin of the disease. Fibroblast cultures from skin biopsies or skin punches provide an adequate source of metaphase cells. Tissues such as liver, kidney, lung, and muscle are not routinely used because of the invasive nature of acquiring such specimens; however, these tissues can be useful to evaluate a fetal loss or an autopsy. Products-of-conception samples may contain a mix of maternal and fetal tissue and so require extra care in establishing a culture ( ).

The most common specimen for prenatal analysis is amniotic fluid collected by amniocentesis. Under ultrasound guidance, the physician passes a needle through the mother’s abdomen and uterus into the amniotic sac. This is typically done between 16 and 18 weeks of gestation, at which time 20 to 30 mL of amniotic fluid (fetal urine) can be withdrawn without endangering the fetus. Cells present in the sample are derived from the fetus and provide a source of material for cytogenetic, molecular genetic, and biochemical assays. The fluid itself contains alpha fetoprotein (AFP), as well as other proteins and enzymes, and forms the substrate for other prenatal assays. The risk of fetal loss due to amniocentesis is approximately 0.2% to 0.3%.

Another prenatal procedure is chorionic villus sampling (CVS), which provides tissue from the developing placenta (chorionic villi) ( ; ). The transabdominal or transvaginal procedure is performed at 10 to 13 weeks of gestation and has a risk of fetal loss of approximately 1%. Because no amniotic fluid is collected, no AFP or related testing can be done, although standard cytogenetic, biochemical, and molecular analyses are possible.

Cordocentesis, or percutaneous umbilical blood sampling (PUBS), results in a fetal blood specimen that can be used for rapid karyotyping or molecular studies. This procedure is done at later gestational ages (≥20 weeks) and carries a higher risk of fetal loss (2–5%).

All clinical samples for cytogenetic analysis must be collected in a sterile manner. The presence of bacteria or fungi severely compromises the study because prokaryotic cells usually outcompete and overgrow any human cells that are present. To maximize the number of viable cells, specimens should be transported to the laboratory as soon as possible after collection. Blood, bone marrow, amniotic fluid, and chorionic villi should be maintained at room temperature, whereas solid tissue is transported on wet ice. The difference in temperature of transport is due to the native conditions of the sample. Blood, bone marrow, and amniotic fluid cells exist as individual cells in a fluid substrate, and the integrity of the sample will not be compromised as long as its temperature is maintained close to body temperature. However, collection of tissues such as skin requires cells to be excised from the body, leaving broken and dying cells at the periphery of the sample. The resultant release of lysosomal enzymes facilitates degradation of the dead cells, but the enzymes will also attack and destroy adjacent living cells. If the temperature is dropped to near 4°C, enzyme activity will be inhibited, and the viability of the sample is maintained.

Cell Culture Technique

Depending on the cell type, either a suspension (floating) or monolayer (fixed to a surface) culture technique may be employed. Blood and bone marrow cells are grown in suspension, so cells from the sample can be aliquoted directly into an appropriate culture medium. Bone marrow is typically cultured for 24 to 48 hours, whereas lymphocytes require 3 to 4 days in culture for maximum yield. Furthermore, because lymphocytes do not normally divide in culture, they must be induced using a mitogen, usually phytohemagglutinin. The resultant metaphase cells can then be collected by the use of a mitotic inhibitor, such as colcemid. Amniotic fluid cells, chorionic villi, and solid tissue are all grown as a monolayer in situ. Tissue and chorionic villi are first disaggregated using a mild collagenase treatment, and the individual cells are then seeded onto glass coverslips and covered with culture medium or placed into culture flasks. In an amniotic fluid sample, the cells must first be separated from the fluid by centrifugation before being plated in dishes or flasks and allowed to form in situ colonies. Typical culture times are 5 to 7 days for chorionic villi and amniocytes and up to 2 weeks for solid tissue culture.

Once maximum growth has been achieved, all cell types are harvested using similar techniques. The cells are swelled hypotonically (to the point that the cell membrane is stretched but not broken) and are then fixed, typically using a methanol:acetic acid fixative. Gentle blowing on the coverslip containing the fixed cells from in situ cultures results in the rupture of the cell membrane and a spreading of the metaphase chromosomes. For suspension cultures, the fixed cells must be dropped onto a clean microscope slide, which mechanically breaks the membranes and leaves the chromosomes separated slightly from each other but in a discrete region occupied by a single cell ( Fig. 71.3 ). After air drying, the slides are suitable for staining. In some cases, artificial aging of the cells at approximately 65°C for 30 to 60 minutes improves the quality of the staining. Although most laboratories still perform these steps manually, an automated robotic harvester has been developed that can process large numbers of cultures quite efficiently.

Computer-Assisted Imaging

To capture the images, cytogenetics laboratories now use computer-assisted imaging. A CCD (charge-coupled device) video camera is used in combination with specially designed software to capture an image of a metaphase cell, which is digitized and displayed on the computer monitor. The image can be modified by lightening, darkening, or changing the contrast, and a variety of manipulations of the chromosomes are possible, including straightening and importing chromosomes from other fields. A karyogram can then be generated using a pattern-recognition subroutine that will automatically identify the chromosomes and place them in their proper places on a karyogram form. Depending on the software and the quality of the image, the accuracy of this process can vary from 10% to 85%. After the computer has taken its “best guess,” a trained cytogenetic technologist must make the appropriate corrections, and the final version of the karyogram can be printed out by a high-resolution printer and then archived. A routine karyogram from capture to printing should take a trained technologist on average 15 to 20 minutes.

Technique

FISH can be performed on either metaphase or interphase cells. For metaphase FISH, cells are cultured and harvested as for a routine karyotype analysis, but no culture is required for interphase FISH. Fixed cells are collected, and slides are made as described for karyotyping. The DNA on the slides is then denatured, and a fluorescently labeled single-stranded molecular probe is allowed to hybridize to the chromosomal DNA using an annealing temperature that favors hybridization of homologous regions of DNA ( Fig. 71.7A ) . After an appropriate period of hybridization, excess probe is washed away and the nonhybridized DNA is counterstained with another fluorochrome to allow visualization of the entire chromosome complement. A fluorescent microscope with light from a 100-watt mercury bulb in combination with appropriate sets of exciter filters allows evaluation of the cells. Due to the optical limitations of the glass filters, a maximum of three colors can be visualized on a typical fluorescent microscope. For documentation, images are captured using a computer-assisted system.

One of the most critical elements in FISH is the selection of a specific probe(s) that will help to answer a clinical question. For example, one of the most useful clinical applications of FISH is the detection of microdeletions too small to be seen using classical cytogenetics (see Fig. 71.7B-D ). The gene must be known, and the probe used must be homologous to the critical region of the gene that is usually deleted. Hybridization with the probe should result in a fluorescent signal only at the target locus. If a signal is present, DNA complementary to the probe is present, so there is no deletion (see Fig. 71.7B ). However, the absence of signal indicates that a deletion exists (i.e., there is no DNA sequence present on the chromosome that is complementary to the probe, so no hybridization can occur) (see Fig. 71.7D ). A control probe to a different region of the chromosome being tested is usually used as a hybridization control (see Fig. 71.7C ). Therefore, when evaluating a disease-related probe, an unaffected individual should have two signals per cell for each autosomal gene: one signal for each chromosome of the pair ( Fig. 71.8A ). An affected individual with a deletion should have a single signal per cell, showing one normal and one deleted chromosome (see Fig. 71.8B ). For both affected and unaffected individuals, the control probe should show two signals per cell in all cells that are scored. In a metaphase cell, the chromosomes may be short and condensed, with each chromatid individually visible, resulting in a discrete FISH signal on each chromatid. In these cells, there may appear to be two signals per chromosome, for a total of four signals, but in actual scoring only complete chromosomes are considered so a signal on one or both chromatids would be counted as just one positive signal.

One of the problems with this detection system is that the lack of signal is the positive indication of a deletion. Technical failure of hybridization may also result in absence of a signal. To eliminate this as a source of error, a minimum of 20 cells must be evaluated, and all cells must agree in signal count. If mosaicism is suspected, additional cells may be surveyed. In addition, unique sequence probes are currently used in combination with a second probe—a control probe—that is localized to a different locus on the same arm of the target chromosome (see Fig. 71.7C ). The two probes are usually labeled with fluorochromes of different colors. In all cells evaluated, two clear control signals must be seen, and then the corresponding signals for the disease locus can be recorded. This provides a hybridization control as well as a marker for the chromosome of interest (see Fig. 71.7D ). If a dual-color system is used, results may be obtained in interphase as well as metaphase cells.

Chromosome painting probes are most useful in identifying complex rearrangements or marker chromosomes. If an individual has an abnormal chromosome with extra material of unknown origin, it may be possible to use chromosome painting to identify the source of the extra DNA. This may help in either diagnosis or prognosis of the individual. For example, in the case of a patient with only one identifiable X chromosome and a small, unidentified marker chromosome, it will be important to determine if the origin of the marker is an X or a Y chromosome. This can be accomplished using chromosome paints for the X and Y chromosomes labeled with different fluorochromes.

FISH is a targeted assay. The probes will only provide information about the locus from which they are derived. Thus when interpreting results, it is critical to know which probe was used. When using metaphase cells, the necessary data can usually be obtained from viewing about 20 cells. A larger sample of interphase cells (200–500) can yield statistically significant data about the frequencies of disease clones.