A karyotype represents an organized profile of an individual’s chromosomes, which are the structures within the cell nucleus that carry genetic information. This profile provides a comprehensive view of the entire chromosome set, ordered and arranged by size and shape. The primary purpose of preparing a karyotype is to identify chromosomal abnormalities that may be linked to genetic disorders, developmental delays, or fertility issues. These abnormalities fall into two main categories: numerical changes, such as having an extra or missing chromosome (aneuploidy), and structural changes, which involve rearrangements like translocations or deletions. Analyzing the karyotype helps geneticists diagnose conditions like Down syndrome (Trisomy 21) or Turner syndrome (Monosomy X).
Sample Collection and Cell Culture
The entire process begins with obtaining a suitable cell sample that can be stimulated to grow and divide in a laboratory setting. For adults and children, the most common source is peripheral blood, specifically the white blood cells called lymphocytes. Prenatal diagnosis often relies on cells obtained from amniotic fluid through amniocentesis or from the chorionic villi. Other sources, such as bone marrow or skin fibroblasts, may be used depending on the specific reason for the analysis.
Once collected, the cells must be placed into a nutrient-rich growth medium and incubated in a controlled environment, typically at 37°C. This cell culture step is necessary because chromosomes can only be clearly visualized when a cell is actively dividing. Peripheral blood lymphocytes, which are normally non-dividing, must first be stimulated to enter the cell cycle using a mitogen like phytohemagglutinin. The cells are typically incubated for 48 to 72 hours, ensuring a sufficient population of actively dividing cells.
Arresting Cell Division and Spreading Chromosomes
The next phase involves chemically manipulating the dividing cells to make their chromosomes visible and spread out for observation. The first step is the addition of an agent like colchicine, which acts as a mitotic inhibitor. This chemical halts the cell cycle precisely at the metaphase stage, where the chromosomes are at their most condensed state. Metaphase is the ideal stage for chromosome analysis because the chromosomes are tightly coiled and clearly distinguishable from one another.
Following the mitotic arrest, the cells are treated with a hypotonic solution, often a weak potassium chloride (KCl) solution. This low-salt environment causes the cells to swell significantly. The swelling action physically separates the chromosomes and helps to rupture the cell membrane, allowing the chromosomes to spread out.
After this hypotonic treatment, the cells are fixed onto a microscope slide using a fixative solution, typically a mixture of methanol and glacial acetic acid in a 3:1 ratio. This fixative serves to stop all cellular activity and preserve the morphology of the chromosomes. The final step involves dropping the fixed cell suspension onto a clean, chilled slide from a controlled height, which uses the force of the drop to further scatter the chromosomes.
Staining Techniques for Banding Patterns
After the fixed cells are dried onto the slide, the chromosomes must be stained to reveal their unique structural details. The standard technique used in clinical cytogenetics is Giemsa banding, or G-banding, which provides a distinct pattern along the length of each chromosome. Before applying the Giemsa stain, the slides are briefly treated with an enzyme, usually trypsin, to partially digest the chromosome proteins.
This pretreatment allows the Giemsa dye to bind differentially to the chromosome arms, creating alternating dark and light segments. Dark bands correspond to regions of DNA that are rich in adenine and thymine, while the light bands are generally richer in guanine and cytosine. This reproducible banding pattern is unique for every one of the 23 pairs of human chromosomes.
The banding pattern allows a geneticist to identify not only the individual chromosomes but also subtle changes within them. While G-banding is the routine method, other specialized techniques exist for specific diagnostic needs, such as Q-banding using quinacrine or C-banding for centromere regions.
Final Analysis and Karyogram Construction
The prepared and stained slides are examined under a high-power microscope, and digital images are captured of well-spread metaphase cells. The goal is to find cells where the 46 chromosomes are maximally separated without overlapping. Modern cytogenetic laboratories use specialized imaging software to capture multiple images and enhance the contrast of the banding patterns.
The software facilitates the construction of the final karyogram, which is the standardized arrangement of the chromosomal images. Each chromosome is digitally “cut out” from the image and paired with its homologous partner. These pairs are then arranged on a chart in descending order of size, from the largest autosome, chromosome 1, down to chromosome 22. The two sex chromosomes (X and Y) are placed at the end of the arrangement.
Once the chromosomes are organized and aligned, the geneticist performs a detailed analysis, checking the total number of chromosomes for aneuploidy and meticulously comparing the banding patterns of the homologous pairs. This allows for the identification of structural anomalies, such as inversions, translocations, or duplications.