Fundamentals of Cytogenetic Techniques

Cytogenetics is the study of chromosome morphology, structure, pathology, function, and behavior. Chromosomes are studied by cytogenetic techniques and are best studied at mitotic or meiotic metaphase, although some studies, such as fluorescence in situ hybridization (FISH) methods, may utilize interphase cells. Once dividing cells are obtained, mitotic arresting agents (such as Colcemid, colchicine, or Velban) are used to collect the metaphase cells. Then the cells are harvested. During the harvest procedure, hypotonic solutions are used to increase cell volume, which spreads the chromosomes apart, and methanol–acetic acid is used to fix (preserve) them for study. Slides are prepared from fixed cells, cells are then stained with appropriate stains, observed, and analyzed. Computer‐generated images are now used to arrange the chromosomes in pairs on karyograms.

Workflow

Specimen

Specimens to be cultured must never be frozen or exposed to excessive heat, because live cells are required for cytogenetic preparations cell culture. The exception is cells cryopreserved in special freezing media to keep them viable. Refrigeration is helpful for some specimens, especially those, like products of conception, which may have a risk for contamination, but because the specimen could freeze by accident at only a few degrees colder than standard refrigerator temperatures, and some refrigerators are not well controlled, room temperature for specimen storage and shipping is often recommended.

Culture Techniques

The strictly aspetic technique of culturing is performed as microbes may lead to failure. Generally there exists to categories of tissue culture:

  • Suspension Culture, such as bone marrow and blood
  • Attached monolayer culture such as amniocytes, skin fibroblasts etc.

The cultures may be incubated in an open or closed system. Open cultures are able to exchange gases with the atmosphere surrounding the culture. Closed systems are sealed tighty and do not exchange gases with the atmosphere. The 5% CO2 adjusts the culture medium to pH 7.25–7.40 via the buffer in the medium, and the low‐oxygen formula of the 2–5% O2 mixture has been shown to increase the growth rate of many cell types.

Basal medium is supplemented with serum for protein and growth factors. Fetal bovine serum. (FBS) is widely used. Some cells, however, are sensitive to the presence of fetal bovine serum and may not divide as well with high concentrations (e.g., neuroblastoma cultures, lymphocytes), and others may arrive with serum in them (e.g., blood samples) and may not require as high a concentration of serum in the culture medium as other specimen types do. Other media supplements include antibiotics, l‐glutamine, and optional additions, such as selenium, insulin, and giant cell tumor conditioned medium. Once supplemented, the medium is called complete medium.

Cultures that are valuable or may be needed for future use may be frozen in medium with 5–10% dimethyl sulfoxide (DMSO) or glycerol to prevent ice crystal formation, and kept in liquid nitrogen until such time as they are needed.

Removal of attached cells and centrifugation

After mitotic arrest and just before the use of the hypotonic and fixative steps, an optional step for attached cells intended for a suspension harvest is the removal of the mitotic cells by mechanical methods or by enzymatic methods. Centrifugation is never complete, and a number of cells remain in the supernatant or are lost to damage from shear forces between cells or with the centrifuge tube walls. This is why the number of centrifugation steps should be kept to a minimum.

Mitotic arrest

The first step in most harvest procedures is to arrest cells in the mitotic stage required for standard cytogenetic analysis: metaphase. The effect of the most commonly used mitotic arrestant Colcemid is to prevent formation of the spindle fiber apparatus, which would normally pull the sister chromatids to opposite poles for incorporation into the two daughter cells. Colcemid also causes chromosome condensation, a process that becomes accentuated when increasing the time of exposure and the concentration.

Often the mitotic arrest is blamed for contracted chromosomes, but other factors may be involved. In blood and bone marrow cultures, over inoculation may result in depleted medium and increased metabolic byproducts that cause irreversible chromosome contraction.

Hypotonic treatment

The second major step in harvesting cells is treatment with a hypotonic saline solution (10-20 minutes) to increase cell volume so that the chromosomes have adequate space to spread out during slide preparation.

Prewarming the hypotonic solution to 37°C may increase effectiveness by speeding up water transport across the cell membrane and possibly by softening the cytoplasmic membrane, which has a lipid component, giving it more stretching capability. The type of salts used in the hypotonic can affect the width and sometimes the length of the chromatids, Sodium citrate, for example, often yields wider chromatids than KCl, and Ohnuki’s hypotonic usually yields longer chromatids than KCl. KCL concentration of 0.075 M  (0.56%) is commonly used.

Many methods call for addition of a few drops to several milliliters of fixative to the hypotonic at the end of the incubation period.

Fixation

The third constant feature of chromosome harvesting is fixation of the cells. This process removes water from the cells, killing and preserving them, hardening membranes and chromatin and preparing the chromosomes for the banding procedure. The first fixative may create turbulence at first when added to the remnant hypotonic solution. During this period of turbulence, the fixative is usually added slowly, or metaphase cells can be lost to breakage. Then the fixative is added more quickly. It is important to gently but thoroughly mix the cell pellet into suspension to prevent irreversible cell clumping. Once in the first fixative, the cells become stronger (are hardened), and subsequent fixations may be added much more quickly.

Cold fixative may improve chromosome morphology, and many protocols call for 1–24 hours for first fixative and/or final fixation in the refrigerator or freezer.

Chromosome anticontraction methods

As chromosomes progress through the stages of interphase and mitotic prophase toward metaphase, they condense from long, string‐like structures into shorter and shorter bodies. As they condense, the banding patterns coalesce, with sub‐bands merging into bands and major bands merging.

There are two basic methods of obtaining prometaphase and prophase chromosomes:

  • Cell synchrony and
  • Additives to prevent contraction.

They may be combined into still other permutations of synchrony and additive methods that may be synergistic. In theory, the synchrony method works by stopping cells in synthesis, collecting a large population of cells ready to begin division together. When the cells arrive at an early stage of metaphase together, they are harvested and put on slides. By timing the release period properly, one can obtain a large number of early metaphase and prophase cells.

The chemicals used to block synthesis include amethopterin (methotrexate), 5‐fluorodeoxyuridine (abbreviated FdU, FUdR, or FdUrd), 5‐bromo‐2′‐deoxyuridine excess (abbreviated as BrdU, BUdR, or BrdUrd), or thymidine excess. These chemicals are added to exponentially growing cells (e.g., 2–3‐day stimulated blood cultures, bone marrows the first or second day of culture, or young primary or early subcultured monolayer cultures.)

Numerous chemical agents bind to or intercalate into DNA or chromatin and, when added for periods before fixation, are capable of preventing normal chromosome contraction during metaphase. Some of these agents show a preference for certain areas of the chromosome. BrdU and 5‐azacytidine preferentially bind to G–C‐rich areas, and Hoechst 33258, DAPI, and distamycin A show a preference for A–T‐rich regions. These chemicals inhibit contraction differentially rather than lengthening the chromosome homogeneously. Agents may be added to the culture before or at the time of addition of the mitotic inhibitor or during the hypotonic period.

Chemical additives used to produce elongated chromosomes

Tissue typeChemicals usedConcentrationExposureMitotic arrest
Pleural effusionsActinomycin D5 µg/mL1 hourLast hour
LymphocytesActinomycin D5 µg/mL1 hour or moreColcemid, last 10 min
Human fibroblastsActinomycin D2 µg/mL1 hourVelban 0.012 g/mL, Last hour
LymphocytesEthidium Bromide10 µg/mL2.5 hourColcemid, last hour
AmniocytesEthidium Bromide5 µg/mL4.5 hourColcemid, 0.6 µg/mL, Last 1.5 hour
Chemical additives used to produce elongated chromosomes

Several successful methods have been developed for chromosome elongation using a combination of synchrony and anticontraction chemical additives.

Slide making

Once cells have been well‐fixed in 3:1 methanol–acetic acid, they are dropped onto glass slides and dried using specific conditions for optimal chromosome spreading and morphology. Air drying of cells that are fixed in 3:1 methanol–acetic acid is based on the theory that chromosomes, which are contained in cells that are much enlarged and have much thinner cell membranes than before the harvest, will be supported by the layer of fixative on the slide in the first few seconds after application to the slide. Then, as the fixative evaporates, the layer of fixative becomes thinner and the meniscus pushes down on the top of the cell, enlarging the area of the cell and pressing the metaphase chromosomes between the upper and lower membranes, spreading them out.

 Once the methanol–acetic acid‐fixed chromosomes are dried onto a glass slide, they stick fast until physically scraped off, and staining may be accomplished without losing cells from the slides.

If wet slides are used, the retreating water should be eliminated by immediate draining onto paper towels, blotting with KimWipes, and flooding with fixative. Residual water spots cause spreading and staining irregularities due to localized drying time variations. For coverslips in dishes, it is crucial to completely dry the edges of the coverslip by diligent withdrawal of residual fixative. Residual water spots cause spreading and staining irregularities due to localized drying time variations. For coverslips in dishes, it is crucial to completely dry the edges of the coverslip by diligent withdrawal of residual fixative. This ensures a consistent drying milieu across the coverslip and reduces cell breakage from waves of drying fixative. Wet slides may further facilitate spreading control by the use of different temperatures of water coating to speed up or slow down drying time. Cold, wet slides will slow drying, increasing spreading, whereas higher room temperature or warm wet slides will accelerate drying time.

Angle of the slide

Spreading may improve when cells travel and roll as the fixative level first becomes thin. The effect of dropping cells on slides angled along the long axis is that the water and fixative drain from the upper end of the slide to the lower end and pool there; this may cause faster evaporation at the upper end and drying may be uneven, both between cells at each end and within cells, giving different banding responses to trypsin from cell to cell and even within a metaphase.  Changing the angle of the slide and rapping the slide edge on the bench top are said to improve cell spreading.

Humidity and temperature

In dry climates, it is necessary to slow drying times, and in humid climates, to speed them up. Some ways to provide humidity are to dry slides in front of a humidifier, on top of wet paper towels, or over steam in a sink. The ideal relative humidity for slide‐making can vary owing to differences in temperature, hygrometer accuracy, technique, and specimen type. The ideal relative humidity for slide‐making can vary owing to differences in temperature, hygrometer accuracy, technique, and specimen type. Some suggest 20°C and 45% humidity, with the optimum range between 40% and 50%. Some other also found 50% humidity and 25°C to be the optimum setpoints, and they lower the humidity to 35% if chromosome scattering is a problem. Some recommend 25 seconds of drying time for high‐resolution preparations.

It is sometimes useful to flood the drops of cells with 6:1 methanol–acetic acid instead of the usual 3:1 ratio, and this accelerates evaporation. The temperatures which affect slide‐making results are that of the ambient air, that of the slides and water with which they are coated, and that of the surface on which they are dried.

Human Lymphocyte Chromosome cytogenetic techniques (Metaphase arrest)

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