The Principles of Clinical Cytogenetics - Extra Materials - Springer

70 Martha Keagle and Steven Gersen
CHROMOSOME STAINING AND BANDING
Prior to the 1970s, human chromosomes were “solid” stained using orcein or other stains with an
affinity for chromatin. The chromosomes were classified according to their overall length, centromere
position, and the ratio of the short arm to long arm. Solid stains provided limited information. Simple
aneuploidies could be recognized, but structural aberrations were difficult to characterize, and in
some cases impossible to detect. In addition, it was not possible to specifically identify individual
chromosomes. See Chapter 1.
A large number of banding and staining techniques have since been developed. These can be
divided into two broad categories: those that produce specific alternating bands along the length of
each entire chromosome and those that stain only a specific region of some or all chromosomes.
Methods that produce specific alternating bands along the length of the chromosomes create unique
patterns for each individual chromosome pair. This property allows for the positive identification of
the individual chromosome pairs and permits characterization of structural abnormalities. These banding
techniques answer many questions by facilitating the numerical and structural examination of the
entire karyotype.
Those techniques that selectively stain specific regions of chromosomes are used in special circumstances
when a particular piece of information cannot be answered using a routine banding
method. These special stains are typically utilized to obtain such specific data.
Techniques That Create Bands Along the Length of the Chromosomes
An important measurement associated with these methods is the level of banding resolution
obtained. As chromosomes condense during mitosis, sub-bands begin to merge into larger landmarks
along the chromosome. Obviously, as this progresses, the ability to visualize subtle abnormalities is
reduced. Chromosomes with a greater number of visible bands and subbands (higher resolution) are,
therefore, more desirable. Laboratories accomplish this in two ways: by optimizing the banding and
staining procedures themselves so that a maximum number of sharp, crisp bands is produced, and by
choosing (and in some cases manipulating cultures to produce) cells with longer, less condensed
chromosomes.
Cytogenetic nomenclature (see Chapter 3) utilizes approximations of the number of bands present
per haploid set of chromosomes, estimates of the number of light and dark bands one would arrive at by
counting these in one of each chromosome (the definition of a haploid set). Minimum estimates usually
begin at approximately 400 bands. Well-banded, moderately high-resolution metaphases are usually in
the 500- to 550-band range, whereas prometaphase cells can achieve resolutions of 850 or more bands.
G-Banding (Giemsa Banding)
G-banding is the most widely used routine banding method in the United States. GTG banding
(G bands produced with trypsin and Giemsa) is one of several G-band techniques. With this
method, prepared and “aged” slides are treated with the enzyme trypsin and then stained with
Giemsa. This produces a series of light and dark bands that allows for the positive identification
of each chromosome (see Fig. 2). The dark bands are A-T-rich, late replicating, heterochromatic
regions of the chromosomes, whereas the light bands are C-G-rich, early replicating, euchromatic
regions. The G-light bands are biologically more significant, because they represent the
active regions of the chromosomes, whereas the G-dark bands contain relatively few active genes.
There are also G-banding techniques that actually utilize stains other than Giemsa, such as
Wright’s and Leishman’s stains.
Q-Banding (Quinacrine Banding)
Q-banding is a fluorescent technique and was the first banding method developed for human chromosomes.
Certain fluorochromes, such as quinacrine dihydrochloride, will bind to DNA and produce