Metaphase spread

In situ hybridization (ISH) permits examination of a wide range of materials, ranging from cells and tissues to metaphase spreads of chromosomes affixed to a slide. Hybridization with specific probes can thus be performed directly on the slide, with the hybridized signal viewed under a microscope. Briefly, the steps involved in ISH include pretreatment of the slide, application of the sample, fixing of the sample, hybridization with probe, and visualization of the probe signal. 

Fig. 5. Schematic representation of signals illustrative of chromosomal abnormalities in metaphase spreads and interphase nuclei.

Preparation of metaphase spreads from whole blood cultures. 

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.

Analysing karyotypes by hand is extremely tedious and the increasing demands of the health service have led to the development of several computer operated systems which automatically locate and analyse metaphase spreads (Zeidler, 1988). A manual of chromosome techniques has recently been published (Verma and Babu, 1989). 

Figure 2. Metaphase spread of human lymphocyte stained with Telomere PNA FISH Kit/Cy3, Dako Code K5326.

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. 

After Giemsa staining, the numbers of chromosomes and their normal or abnormal appearance in metaphase spreads are determined. In the micronucleus test, femoral bone marrow smears are stained with Giemsa or a fluorescent dye such as acridine orange, and the numbers of polychromatic erythrocytes containing micronuclei are determined. Normal polychromatic erythrocytes contain no nuclei. In the process of red blood cell maturation, the nucleus is expelled just prior to the polychromatic erythrocyte stage. Micronuclei represent chromosomes or chromosome fragments that are left behind in the cell after expulsion of the nucleus. 

Figure 39-9 Fluorescence in situ hybridization for the t(l4 l8) anomaly on metaphase spreads of a case of follicular lymphoma. The immunoglobulin heavy chain sequences on I4q32 (red) when juxtaposed to the bd-2 sequences on I8q2l (green) yield a yellow fusion signal indicative of the presence of the t(l4 l8). (See Color Plate 2.)...

With recent publication of the genome sequences of man, mouse and rat, we have learned that mammalian genomes are exceptionally dynamic due to the presence of repetitive sequences, remnants of retroviral genomes and transposable elements. This phenomenon can be termed sequence mobility . Mammalian cells - particularly immortalized cells - have an even more intrinsic genomic fluidity. This becomes evident when studying chromosome numbers in metaphase spreads [99]. It appears that immortalized cell populations will diverge, even when established as clonal cell lines, in the number and structure of their... 

In summary, when cultivating these cell lines in the absence of MTX, unique and characteristic integrations were found in 95-99% of metaphase spreads. We continued to cultivate these cell lines for extended periods in the absence of MTX, and occasionally performed FISH analyses. We found that the chromosomal structures described above were stable within the observation period (a minimum of 60 days, and in one case of 160 days). Cytogenetically, these observations indicate a high degree of genetic stability of chromosomally amplified sequences in the absence of MTX. Equivalent observations as those presented above have been made recently by Kim and Lee [106]. 

Figure 5. Subcellular immunolocalization of PARP-1 and PARP-2 (a, e, f) interphas (b, gj metaphase spreads (c, c , h) metaphase (i) anaphase (d) eady telophase (j) late telophase. In b, g, h and i the image was merged with the CREST (centromeres) stainii (gteen). The polyclonal antibodies anti-PARP-1 (a to e) and anti-PARP-2 (f to j) were labelled with an Alexa Fluor 568 goat anti-rabbit. Bars indicate 10 )im. A color version of this figure is available online at www.Eurekah.com.

Examine the preparation under a phase contrast microscope and retain if at least five good metaphase spreads can be seen. Mark the position of the good spreads and label the slide with a diamond pencil. 

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