A karyotype is a standardized visual profile of an individual’s chromosomes, representing the complete set of genetic material organized by size and shape. This image, also called a karyogram, is generated in a laboratory setting to provide a snapshot of the person’s genome. Its primary medical purpose is to identify large-scale chromosomal abnormalities, such as changes in chromosome number (aneuploidy) or significant structural defects like translocations or deletions. The karyotype is a powerful diagnostic tool in cytogenetics for disorders like Down syndrome and certain cancers. Creating this profile is a multi-step process involving biological culturing, chemical manipulation, and advanced digital analysis.
Obtaining and Preparing the Sample
The process begins with obtaining a biological sample containing actively dividing cells with nuclei. For adults and children, the most common source is a peripheral blood sample, which provides lymphocytes that can be stimulated to divide. Prenatal testing may use cells from amniotic fluid (amniocytes) or a chorionic villus sample (CVS) from the placenta. For cancer diagnosis, samples might be taken from bone marrow or a solid tumor.
Since chromosomes are only visible during cell division, the collected cells must be placed into a rich nutrient medium containing a mitogen, such as phytohemagglutinin. This stimulates them to begin multiplying. This process, called cell culture, is performed in a laboratory incubator for 48 to 72 hours. The goal of this incubation is to increase the total number of cells and ensure a high percentage are actively progressing toward division.
Arresting and Staining the Chromosomes
Arresting the Cells
Once the cells are actively dividing, the next step is to chemically halt the cell cycle when the chromosomes are most visible. This is achieved by adding a mitotic inhibitor, such as colchicine or Colcemid, to the culture. This chemical disrupts the spindle fibers, effectively arresting the cells in metaphase. Chromosomes are at their most compact form during metaphase, which is necessary for clear visualization.
Hypotonic Treatment and Fixing
The arrested cells are then subjected to a hypotonic solution, typically warm potassium chloride (KCl). The low salt concentration causes water to rush into the cells, making them swell. This swelling disperses the chromosomes inside the nucleus, preventing them from clumping together once the cell is broken open. The cells are then fixed using a mixture of methanol and acetic acid, which hardens the cellular components and preserves the chromosome structure.
Staining and Banding
The fixed cell suspension is dropped onto a clean glass slide, causing the swollen cells to burst and spread the chromosomes across the surface. To make the chromosomes visible and identifiable under a microscope, a specialized dye, most commonly Giemsa stain, is applied. This G-banding technique creates a characteristic pattern of light and dark bands along the length of each chromosome. These unique banding patterns act like a barcode, allowing laboratory technicians to distinguish between the 23 different pairs of chromosomes.
Digital Analysis and Final Assembly
After the slides are stained, they are examined under a high-powered light microscope to locate suitable metaphase spreads. A suitable spread is one where the chromosomes are well-separated, not overlapping, and appear clearly banded. High-resolution digital images are then captured of the best metaphase plates.
Specialized computer software performs the digital analysis, which is the modern equivalent of manually cutting out chromosomes from a photograph. The software isolates and segments the individual chromosomes from the image based on their shape and banding pattern. The final step involves arranging these digitized chromosomes into a standardized format called a karyogram.
The chromosomes are organized into homologous pairs, starting with the largest (chromosome 1) and proceeding down to the smallest (chromosome 22), with the sex chromosomes (X and Y) placed last. This arrangement is based on three main criteria: descending size, the position of the centromere, and the unique G-banding pattern. The resulting karyogram is then interpreted by a cytogeneticist to detect any numerical or structural abnormalities.