A chromosome is formed from a single long DNA molecule that contains a series of genes. The complementary double-helix structure of DNA was established in 1953 by Watson and Crick.⁵ The backbone is a sugar-phosphate-sugar polymer. The sugar is deoxyribose. Attached to the backbone and filling the center of the helix are four nitrogen-containing bases. Two of these, adenine (A) and guanine (G), are purines; the other two, cytosine (C) and thymine (T), are pyrimidines (Figure 31-4).

Figure 31-4 The two DNA chains are held together by the pairing of bases on the interior of the helix. Hydrogen bonds unite a purine base with a pyrimidine base. A, Adenine; C, cytosine; G, guanine; T, thymine. (From Watson JD, Tooze J, Kurtz DT: *Recombinant DNA: a short course,* New York, 1983, WH Freeman, p 19. © 1983 James D. Watson, John Tooze, and David T. Kurtz. Used with the permission of WH Freeman and Company.)

The chromosomal DNA of the cell resides in the cell’s nucleus. This DNA and its accompanying proteins are referred to as chromatin. During the cell cycle, at mitosis, the nuclear chromatin condenses approximately 10,000-fold to form chromosomes.⁶ Each chromosome results from progressive folding and compaction of the entire nuclear chromatin. This condensation is achieved through multiple levels of helical coiling and supercoiling (Figure 31-5).

Figure 31-5 Chromosome structure. The folding and twisting of the DNA double helix. (From Gelehrter TD, Collins FS, Ginsberg D: *Principles of medical genetics,* ed 2, Philadelphia, 1998, Lippincott Williams & Wilkins.)

Metaphase Chromosomes

Metaphase is the stage of mitosis where the chromosomes align on the equatorial plate. Electron micrographs of metaphase chromosomes have provided models of chromosome structure. In the “beads-on-a-string” model of chromatin folding, the DNA helix is looped around a core of histone proteins.⁷ This packaging unit is known as a nucleosome and measures approximately 11 nm in diameter.⁸ Nucleosomes are coiled into twisted forms to create an approximately 30-nm chromatin fiber. This fiber, called a solenoid, is condensed further and bent into a loop configuration. These loops extend at an angle from the main chromosome axis.⁹

In 1956, Tijo and Levan ¹⁰ identified the correct number of human chromosomes as 46. This is the diploid chromosome number and is determined by counting the chromosomes in dividing somatic cells. The designation for the diploid number is 2n. Gametes (ova and sperm) have half the diploid number (23). This is called the haploid number of chromosomes and is designated as n. Different species have different numbers of chromosomes. The reindeer has a relatively high chromosome number for a mammal (2n = 76), whereas the Indian muntjac, or barking deer, has a very low chromosome number (2n = 7 in the male and 2n = 6 in the female).¹¹

Chromosome Size and Type

In the 1960s, before the discovery of banding, chromosomes were categorized by overall size and the location of the centromere (primary constriction) and were assigned to one of seven groups, A through G. Group A includes chromosome pairs 1, 2, and 3. These are the largest chromosomes, and their centromeres are located in the middle of the chromosome (i.e., they are metacentric). Group B chromosomes, pairs 4 and 5, are the next largest chromosomes; their centromeres are off center, or submetacentric. Group G consists of the smallest chromosomes, pairs 21 and 22, whose centromeres are located at the ends of the chromosomes and are designated as acrocentric (Figure 31-6).

Figure 31-6 Chromosome morphology. The three shapes of chromosomes are metacentric, submetacentric, and acrocentric. This figure also shows the position of the centromere and telomere as well as the two chromatids that comprise a chromosome.

Techniques for Chromosome Preparation and Analysis

Chromosome Preparation

Tissues used for chromosome analysis contain cells with an inherently high mitotic rate (bone marrow cells) or cells that can be stimulated to divide in culture (peripheral blood lymphocytes). Special harvesting procedures are established for each tissue. Mitogens such as phytohemagglutinin or pokeweed mitogen are added to peripheral blood cultures. Phytohemagglutinin primarily stimulates T cells to divide,¹² whereas pokeweed preferentially stimulates B lymphocytes.¹³

Chromosomes can be obtained from replicating cells by arresting the cell in metaphase. Cells from the peripheral blood or bone marrow are cultured in media for 24 to 72 hours. In standard peripheral blood cultures, a mitogen is added to stimulate cellular division. Neoplastic cells are spontaneously dividing and generally do not require stimulation with a mitogen. After the cell cultures have grown for the appropriate period, Colcemid, an analogue of colchicine, is added to disrupt the mitotic spindle fiber attachment to the chromosome. After culture, cells are exposed to a hypotonic (potassium chloride) solution that causes the chromosomes to spread apart from one another. A fixative of methanol and acetic acid is added that “hardens” cells and removes proteinaceous material. Cells are dropped onto cold, wet glass slides to achieve optimal dispersal of the chromosomes. The slides are then aged before banding.

Chromosome Banding

Analysis of each chromosome is made possible by staining with a dye. The name chromosome is derived from the Greek words chroma, meaning “color,” and soma, meaning “body.” Hence chromosome means “colored body.” In 1969, Caspersson, Zech, Modest, et al ¹⁴ were the first investigators to stain chromosomes successfully with a fluorochrome dye. Using quinacrine mustard, which binds to adenine-thymine–rich areas of the chromosome, they were able to distinguish a banding pattern unique to each chromosome. This banding pattern, called Q banding, differentiates the chromosome into bands of differing widths and relative brightnesses (Figure 31-7). The most brightly fluorescent bands of the 46 human chromosomes include the distal end of the Y chromosome, the centromeric regions of chromosomes 3 and 4, and the short arms of the acrocentric chromosomes (13, 14, 15, 21, and 22).

Figure 31-7 Q-banded preparation. Note the intense brilliance of Yq. (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, Ind.)

Other stains are used to identify chromosomes, but in contrast to Q banding, these methods normally necessitate some pretreatment of the slide to be analyzed. Giemsa (G) bands are obtained by pretreating the chromosomes with the proteolytic enzyme trypsin. GTG banding means “G banding by Giemsa with the use of trypsin.” Giemsa, like quinacrine mustard, stains AT-rich areas of the chromosome. The dark bands are called G-positive (+) bands. Guanine-cytosine–rich areas of the chromosome have little affinity for the dye and are referred to as G-negative (−) bands. G+ bands correspond with the brightly fluorescing bands of Q banding (Figures 31-8 and 31-9). G banding is the most common method used for staining chromosomes.

Figure 31-8 Normal male metaphase chromosomes.

Figure 31-9 Normal male karyogram, GTG-banded preparations. (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, Ind.)

C banding stains the centromere (primary constriction) of the chromosome and the surrounding condensed heterochromatin. Constitutive heterochromatin is a special type of late-replicating repetitive DNA that is located primarily at the centromere of the chromosome. In C banding, the chromosomes are treated first with an acid and then with an alkali (barium hydroxide) before Giemsa staining. C banding is most intense in human chromosomes 1, 9, and 16 and the Y chromosome. Polymorphisms are also observed in the C bands from different individuals. These polymorphisms have no clinical significance (Figure 31-10).

Figure 31-10 C-banded male metaphase chromosomes. (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, Ind.)

Specific chromosomal regions that are associated with the nucleoli in interphase cells are called nucleolar organizer regions (NORs). NORs contain tandemly repeated ribosomal ribonucleic acid (RNA) genes. NORs can be differentially stained in chromosomes by a silver stain in a method called AG-NOR banding.

Chromosome banding is visible after chromosome condensation, which occurs during mitosis. The banding pattern observed depends on the degree of condensation. By examination of human chromosomes early in mitosis, it has been possible to estimate a total haploid genome (23 chromosomes) with approximately 2000 AT-rich (G+) bands.¹⁵ The later the stage of mitosis, the more condensed the chromosome and the fewer total G+ bands observed.

Metaphase Analysis

After banding, prepared slides with dividing cells are scanned under a light microscope with a low-power objective lens (10×). When a metaphase cell has been selected for analysis, a 63× or 100× oil immersion objective lens is used. Each metaphase cell is analyzed first for chromosome number. Then each chromosome pair is analyzed for its banding pattern. A normal somatic cell contains 46 chromosomes, which includes 2 sex chromosomes and 22 pairs of autosomes (chromosomes 1 through 22). The technologist records his or her summary of the analysis using chromosome nomenclature. This summary is called a karyotype. Any variation in number and banding pattern is recorded by the technologist. At least 20 metaphase cells are analyzed from leukocyte cultures. If abnormalities are noted, the technologist may need to analyze additional cells. Computer imaging or photography is used to confirm and record the microscopic analysis. A picture of all the chromosomes aligned from 1 to 22 including the sex chromosomes is called a karyogram.

Nonradioactive in Situ Hybridization

The use of molecular methods coupled with standard karyotype analysis has improved chromosomal detection capability beyond that of the light microscope. DNA or RNA probes labeled with either fluorescent or enzymatic detection systems are hybridized directly to metaphase or interphase cells on a glass microscope slide. These probes usually belong to one of three classes: (1) probes for repetitive DNA sequences, primarily generated from centromeric DNA; (2) whole-chromosome probes that include segments of an entire chromosome; and (3) specific loci or single-copy probes.

Fluorescence in situ hybridization (FISH) is a molecular technique commonly used in cytogenetic laboratories. FISH studies are a valuable adjunct to the diagnostic workup. In FISH, the DNA or RNA probe is labeled with a fluorophore. Target DNA is treated with heat and formamide to denature the double-stranded DNA, which renders it single-stranded. The target DNA anneals to a similarly denatured, single-stranded, fluorescently labeled DNA or RNA probe with a complementary sequence. After hybridization, the unbound probe is removed through a series of stringent washes, and the cells are counterstained for visualization (Figure 31-11).

Figure 31-11 General protocol for fluorescence in situ hybridization. *PCR,* Polymerase chain reaction.

In situ hybridization with centromere or whole-chromosome painting probes can be used to identify individual chromosomes (Figure 31-12). Marker chromosomes represent chromatin material that has been structurally altered and cannot be identified by a G-band pattern. FISH using a centromere or paint probe, or both, is often helpful in identifying the chromosome of origin (Figure 31-13).¹⁶ Specific loci probes can be used to detect both structural and numerical abnormalities but are especially helpful in identifying chromosomal translocations or inversions.