A chromosome spread is a laboratory preparation where the chromosomes from a single cell are visually scattered across a glass slide for microscopic examination. This technique is fundamental to cytogenetics, the study of chromosome structure and number. The goal is to obtain a clear, unobstructed view of an organism’s entire set of chromosomes, allowing analysis for structural or numerical irregularities.
The Necessary Biological State: Why Metaphase is Key
A successful chromosome spread requires the cell to be captured during metaphase, a specific stage of the cell cycle. During cell division (mitosis), the genetic material must condense to be accurately separated into two new daughter cells. This condensation makes chromosomes visible as distinct, rod-like structures under a light microscope.
Chromatin, the loose DNA and protein complex in the non-dividing nucleus, is tightly coiled and packaged during metaphase. Analyzing cells at any other time results in an indiscernible tangle, making individual chromosome analysis impossible. To ensure maximal condensation, a chemical agent, such as Colcemid, is added to the cell culture. This agent disrupts the spindle fibers, halting the cells’ progress at the metaphase checkpoint.
Creating the Spread: Steps in Laboratory Preparation
The process begins by culturing living cells, often sourced from blood, amniotic fluid, or a tumor biopsy, to encourage active division. Once arrested in metaphase, the cells are treated with a hypotonic solution, typically a diluted salt solution like 0.075 M potassium chloride (KCl). Because this solution has a lower solute concentration than the cell, water rushes in via osmosis.
The influx of water causes the cells to swell dramatically, separating the chromosomes and making the outer membrane fragile. Following this swelling, the cells are preserved with a chemical fixative, usually a 3:1 mixture of methanol and glacial acetic acid. This fixative locks the chromosomes in their condensed state and hardens the cellular components.
The final step involves the “dropping” technique, where the fixed cell suspension is dropped from a controlled height (about 10-15 centimeters) onto a clean glass slide. The force of the drop and the rapid evaporation of the fixative cause the swollen cell membranes and nuclei to burst open upon impact. This action scatters the chromosomes across the slide, creating the characteristic “spread” ready for staining and analysis.
The Primary Application: Karyotyping and Diagnostic Use
Once the spread is prepared, the chromosomes are stained, photographed, and digitally arranged into a standardized format called a karyotype. Karyotyping involves sorting the chromosomes into homologous pairs based on size, centromere position, and unique banding patterns. In humans, the arrangement consists of 22 pairs of autosomal chromosomes and one pair of sex chromosomes (XX or XY).
The primary diagnostic use is detecting numerical abnormalities (aneuploidy), which involve an incorrect number of chromosomes. A common example is Trisomy 21 (Down syndrome), where an individual has three copies of chromosome 21. Karyotyping is routinely used in prenatal diagnosis, analyzing cells from amniotic fluid or the placenta to identify conditions like Trisomy 21 or sex chromosome variations such as Turner syndrome (monosomy X) and Klinefelter syndrome (XXY).
The technique is also employed in cancer diagnosis, as many malignancies are characterized by specific, large-scale chromosomal changes. For instance, the Philadelphia chromosome, a translocation between chromosomes 9 and 22, is a hallmark of chronic myeloid leukemia. Although newer molecular methods exist, karyotyping remains a standard tool because it provides a complete, genome-wide visual overview of both the number and large-scale structure of all chromosomes in a single cell.
Analyzing the Spread: Interpreting Structural Abnormalities
Beyond counting, a detailed analysis of the spread identifies structural defects within individual chromosomes. For this inspection, chromosomes are stained using Giemsa banding (G-banding). This process involves treating the chromosomes with an enzyme, such as trypsin, and then staining them with Giemsa dye, which produces a reproducible pattern of alternating dark and light bands along the length of each chromosome.
These distinct banding patterns act like a barcode, allowing geneticists to precisely map and identify chromosome segments. Structural abnormalities are detected by looking for disruptions in this expected pattern. Defects include translocations (a segment moved to another chromosome), deletions (a piece is missing), or inversions (a segment is flipped end-to-end). The resolution of standard G-banding is limited to structural changes larger than 5 to 10 megabases (Mb) of DNA. Observing these irregularities aids in diagnosing genetic syndromes and understanding changes in certain cancers.