Since the 1 920s, when researchers began to study chromosomes, the analysis of human chromosomes has presented a particularly tough technological challenge, simply because there are so many of them. When displayed under a light microscope. the strands of human genetic material tend to bunch together maddeningly, overlapping and intertwining like so much spaghetti. For these reasons, it wasn’t until 1956 that the correct number of 46 human chromosomes (23 pairs) was clearly demonstrated. Since then, the field of cytogenetics—linking chromosome abnormalities with syndromes—has proceeded more or less in fits and starts, with key developments in chromosome preparation explaining more and more once mysterious medical conditions.
Key to connecting chromosomes to symptoms and traits is the karyotype, a size-order alignment of chromosome pairs in a chart. The first such efforts to align the pairs, however, were quite crude. By 1959, about all that could be discerned was an extra or missing chromosome (47 or 45 instead of 46), which might indicate Down syndrome or a sex chromosome anomaly, such as Turner’s or Klinefelter’s syndrome.
Throughout the 1960s, pioneering cytogeneticists amassed techniques for capturing, chromosomes at their most visible state. For most of a cell’s existence, the chromosomal material is unwound, and unable to absorb dyes. It is only during cell division that the chromosomes condense and become detectable, like a long piece of wire suddenly coiling into a spring. Researchers learned to treat cells with a hypotonic solution to swell them, spreading apart the tangle of chromosomes. A derivative of the autumn crocus plant, called colchicine, was found to arrest cell division when the chromosomes are at their most striking. A kidney bean extract, phytohemagglutinin, was found to entice lymphocytes—the blood cells most accessible for chromosomal study—to divide. With these tools, the art of karyotyping began to be transformed into a bona fide science.
But still, the chromosome pairs could not be distinguished very well, and researchers had to rely on such large-scale and subjective clues as chromosome size and position of the J centromere, a characteristically located constriction in each chromosome. Even staining the chromosomes with orcein, Feulgen, or Giemsa dyes distinguished unequivocally only four of the 23 chromosome pairs. The chromosome pairs were then grouped crudely by size. A child with a collection of symptoms and an abnormal karyotype could best be diagnosed as having “an anomaly of a G group chromosome.” Only large sections of extra or missing chromosomal material could be discerned.
By the 1970s, combining stains with digestive enzymes yielded far more subtle shadings, revealing a distinctive band pattern characteristic of each chromosome. Several different treatments could be used to further define each chromosome. Now, tiny inversions (reversals in the banding pattern), duplications, deficiencies, and translocations (chromosomes that swap parts) could be noted by trained eyes.
But building a karyotype required many hours from such skilled individuals. The karyotyping procedure involves obtaining blood or some other appropriate tissue, separating out dividing cells, culturing them until a workable number is present, fixing them, and then dropping them onto a microscope slide. A cell with all of the chromosomes untangled is located under the light microscope, and a photograph is taken. A print is developed, and the individual chromosomes are cut out and arranged in pairs by size order into a chart. This is the karyotype. It is literally a scissors-and-tape operation, archaic indeed in this age of sequencing the entire human genome. Yet cytogenetics laboratories, both clinical and research facilities, still depend chiefly on this method. But now an automatic chromosome analyzer—a system that includes a camera, a computer, and a microscope—may radically speed as well as improve the accuracy of our views of chromosomes.
An automatic karyotyper consists of a computerized video camera that digitizes, thereby enhancing and improving, images of chromosomes as seen in the field of a dissecting light microscope. The user can manipulate the chromosomes on a screen, without scissors and tape, and simply push a button to generate a hard copy. Minus darkroom time and the once-requisite arts and crafts, a lab’s output can increase by at least 100%. Of the 300 labs in the United States, about 50 have gone over to automatic karyotypers.
A leading automatic karyotyper is the Genetiscan system, from the Houston-based Perceptive Systems Inc. It is the brainchild of Kenneth Castleman, who developed the device in the 1970s, when he was director of biomedical image processing at the Jet Propulsion Laboratory in Pasadena, Calif. Another front-runner is the IBAS system from Carl Zeiss Inc. of Thornwood, N.Y Both allow the user to manipulate the image in ways not always possible with scissors—aligning chromosomes in the same orientation, straightening out bends without disturbing bands, delapping the overlaps, and, finally, pairing chromosomes by size. in the standard karyotype format. And they are much faster than the human eye in spotting cells that would yield meaningful karyotypes. Zeiss instrument, for example, can scan 16 microscope slides at a time and detect a metaphase (the stage of cell division when the chromosomes are at peak visibility) in a mere half a second; it then takes three more seconds to verfy the conclusion.
Karyotyping by computer can be performed in two modes. In automated karyotyping, the device is “pretrained” by a large sample of chromosome slides, in which the-researcher designates each chromosome according to conventional criteria, such as size, centromere position, and banding pattern. The computer “learns” these assignments, and then applies the rules to new cells. However, one director of a large cytogenetics facility points out that this mode allows perpetuation of human error if an inaccurate chromosome assignment is initially made. To quell such concerns, Genetiscan and IBAS also offer “interactive karyotyping,” in which the chromosomes can be highlighted one at a time and moved over to a standard karyotype sheet. In this mode, the user sorts the chromosomes a new each time. The Zeiss system also offers a compromise semiautomatic approach, in which the user classifies the different chromosomes, but the automatic mode finds each chromosome’s mate.
The basic Genetiscan setup costs $60,000, plus $14,000 for interactive karyotyping capability. Adding several workstations—a plus for the commercial cytogenetics lab—can bring the price tag to $170,000. Zeiss’ newest IBAS system sells for $79,000. Zeiss also offers a laser scan microscope. The use of a single, intense wavelength of light produces sparkling clear images of individual chromosomes. The laser scan microscope allows the user to select a particular chromosome in a field and zoom into it to spot minute breaks, inversions, or abnormal banding patterns.
Other automatic karyotype devices include the MiaMed MF/IK system, available for $150,000 from Leitz Medical Diagnostics of Erlanger, Ky. (a West German company), and the Cytascan RK-I, available for $96,000 from Image Recognition Systems of Pittsburgh (a British company). Leitz’ MiaMed does not offer as many chromosome manipulation capabilities, in response to the feeling of many cytogenetists that straightening chromosome images can disrupt banding patterns, leading to misclassification. Image Recognition Systems’ Cytascan, however, offers a range of chromosome manipulations. The Cytascan has done quite well in Europe in the past three years, and has recently been introduced in the U.S.
So far most users of automatic karyotypers have been clinical cytogenetics labs, teaching hospitals, and private reference labs. But the devices can work on any animal or plant chromosomes, so they should be suitable for research labs as well. An existing huge—and growing— market is prenatal diagnosis. Chromosome analysis is increasingly being used to track cancers linked to chromosomal anomalies, to investigate the cause of spontaneous abortion, and to assess the danger of chemical and radiation exposures. A device that not only improves existing technology for an expanding market, but also speeds it several-fold, is certain to be a success. Ricki Lewis teaches biology at the State University of New York, Albany.