Chromosome spreads

In order to evaluate the frequency and quality of chromosome spreads, stain one of the air-dried slides following the instructions that accompany the Diff Quik stain set (total time about 1 min) or stain with Giemsa stain (3). 

The applications of FISH for chromosome-based studies involve the use of chromosome spreads, chromosomes of tissue sections, and interphase nuclei. Other applications include the study of adventitious organisms (2) and messenger RNA synthesis (3). The applications, especially to chromosome spreads, are sometimes enhanced by exploiting the opportunity presented by FISH for double or triple labeling. 

A 19-year-old woman has been referred to an endocrinologist by her gynecologist because of delay in the initiation of her menstrual periods. Physical examination reveals underdeveloped breasts, an enlarged clitoris (rudimentary penis), and the presence of small masses within the labia majora. Blood testosterone is in the normal range for males and a chromosome spread indicates a karyotype of 46,XY. 

Labeled gene probes can also be used to detect specific genes or mRNA in tissue sections or in chromosome spreads. This technique is known as in situ hybridization. The detection of specific genes or gene... 

Gene Framer (Advanced Biotechnologies, Surrey, UK) This is an adhesive frame that forms a seal around the section or chromosomal spread, and prevents loss of solutions as well as forming a boundary around the area of interest... 

Denver the mixture to the chromosome spreads under a cover slip, and seal with Gene Frame ... 

A chromosome spread is a collection of at least two longitudinal proximate covers its size must lie within a square of certain dimensions which is determined by the magnification. The center of the spread is determined as the arithmetic means of the minimum and maximum coordinates of all the covers. 

Figure 12. (A) Relative magnification of metaphase cells under a low and a high objective lens (B) a partial chromosome spread under a low objective lens digitized in two grey-level and low resolution (C) the same partial chromosome spread under 10Ox objective (D) a chromosome spread digitized in 16 levels.

Figure 13. (A) A field showing two metaphase chromosome spreads (M) and some blood cells (C) (B) search algorithm locating the two spreads based on the hierarchal structure of objects, covers, and spreads (C) another metaphase spread with a higher magnification (D) horizontal lines showing the covers found during each line scan.

Figure 5. FISH mapping of ISYNA1 gene. Each dot represents the double FISH signals detected on chromosome 19 and 4, after analysis of 100 metaphase chromosomal spreads. Chromosome 19p 13.1 harbors the expressed gene and chromosome 4pl5, the pseudogene.

Fig. 1, Human chromosome spread probed with a biotinylated p-globin probe. There is a signal at band pl.5 (arrow) on chromosome 11.

There is no reason why chromosome spreads should not be prepared from cells other than PHA-activated lymphocytes (whole blood cultures). In practice, however, unless there is a specific reason for using other sources of cells, such as mapping translocations, deletions, and so on, blood cultures are easy to set up, and provide an ample source of metaphase spreads. 

Among the variables found in procedures for making chromosome spreads are the concentration of colchicine, the duration of treatment, the composition of hypotonic salt solution, the approaches to splattering cells... 

Table 3 lists the major steps involved in in situ hybridization of labeled probes to chromosomal targets in cultured cells. The first and second steps entail the preparation of chromosome spreads on slides. The third and... 

Quality of chromosome preparation The quality of the chromosome spreads are very important for REVISH. You may need to make several batches and test them to drop. In order to gain maximum information from the hybridizations, the... 

Demyelination may be confused with neoplastic disease because it produces abundant gliosis. Large cells with short chromosomes spread apart in their cytoplasm mimic mitotic activity in a glioma.If numerous lipidladen KPl-positive macrophages are encountered within parenchyma and around vessels, demyelinating disease should be considered. Appropriate stains for myelin, NF stain for axons (see Box 20.2), and features described in this section should be considered in the interpretation (see Figs. 20.3A, 20.11 A and B, and 20.60). 

A FIGURE 1-12 Chromosomes can be "painted" for easy identification. A normal human has 23 pairs of morphologically distinct chromosomes one member of each pair is inherited from the mother and the other member from the father. (Left) A chromosome spread from a human body cell midway through mitosis, when the chromosomes are fully condensed. This preparation was treated with fluorescent-labeled staining reagents that allow each of the 22 pairs and the X and Y... 

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