We performed numerous tests aimed to improve the efficiency of cytogenetic slide preparation and to increase FISH signals. Several modifications of the general protocol resulted in better chromosome spreading, better chromosome morphology and shorter hybridization times, while yielding brighter FISH signals. Although the procedures presented use mostly known reagents or conditions (ethanol, fixative, acetic acid, heat, water vapors), these are used in a more "logical" way, based on step-by-step observations made at the microscope. The various observations or improvements are discussed below.

Improved cell recovery by micro centrifugation

The fixative solution (3:1 methanol: acetic acid), preserves the cells in their "swollen" state after hypotonic treatment, removes lipids and denatures proteins thus making the cell membrane very fragile, which helps chromosome spreading. Cells pelleted after hypotonic buffer treatment, can be re-suspended in 1 ml fixative, transferred into 1.5ml microfuge tubes, and all subsequent fixative washes can be performed in these small vials. The use of microfuge tubes increases speed of cell processing, as various centrifugation steps can be performed in a microfuge from 2 min/6,000 rpm to 10 seconds/14,000 rpm. When compared with a regular protocol, which uses 15ml tubes and 1000rpm centrifugation steps, there was no difference in cell or chromosome morphology (data not shown). Importantly, high speed centrifugation reduces or eliminates cell loss during harvesting. Cell suspensions can be stored in fixative in the same 1.5 ml vials for years at -20 C.

Slide storage

A common procedure in many clinical cytogenetic laboratories is to store the slides at room temperature, usually in a desiccators. We found that the quality of the biologic material on the slides is preserved better, especially for future FISH studies, if slides with metaphase spreads are stored at —20Ú C in jars with 100% ethanol. Slides stored at least 1-2 days in ethanol do not require aging prior to FISH. Slides used solely for G-banding can be stored dry at room temperature, but 18-24 month old slides did not yield FISH results. By comparison, six-year old slides stored in ethanol, yielded good quality FISH results.

"Controlled" chromosome spreading

In a "standard" laboratory procedure, to which our proposed protocol was compared, glass slides are washed and rinsed in triple distilled water, then kept in ice-cold water prior to preparing the chromosome spreads. Using a glass pipette, few drops of cell suspension are dropped on the wet, ice-cold slide from 30-40 cm distance. Excess liquid is drained on a paper towel and the slide is placed on a 60-65Ú C hot plate to dry. By comparison, our proposed procedure uses the same factors (water vapors, hot plate, acetic acid) in a more precise, step-by-step fashion, based upon the intimate processes taking place at the microscopic level. Chromosome spreading is superior to the "standard" protocol. In our protocol, precise timing is important. Initial slide exposure to hot water vapors, provides a uniform layer of moisture on the slide immediately prior to adding the cells. After the cell suspension is spread evenly on the slide, it is important to allow the fixative to partially evaporate, until the surface becomes "grainy" in aspect. Immediately, the slide is exposed again to the hot water vapors, so water is evenly distributed on the surface. Water arrives on the slide when most of the methanol from the fixative has evaporated and there is a resulting brief increase in acetic acid concentration. The mix of acid and water at this moment, followed by quick drying on a hot surface provides good chromosome spreading. To maximize spreading, after the slide surface becomes "grainy", 3-5 drops of glacial acetic acid are placed on the slide and allowed to cover the surface. Immediately, the slide is exposed 4-5 seconds to the water vapors, and then quickly dried on a hot metal surface. With good quality cell suspensions (for example peripheral blood), if overspreading occurs or metaphases "break", slides should be dried on the colder areas of the metal plate containing the heat gradient (Fig 1b). Thus, adjusting the drying temperature allows optimal spreading of any cell suspension, less influenced by initial cell quality.

Results of comparisons between the "standard" and the proposed slide preparation protocol are summarized in Table 1. We calculated the metaphase diameter of metaphases obtained from one peripheral blood culture and two to three different culture flasks (slightly modified culture conditions) of two bone marrow cultures (BM1 and BM2). In general, metaphase diameters were 20-33% larger using the procedure proposed in this study. This translated itself in metaphases with 45-70% increased surface. For the blood culture, where the chromosomes are longer, the number of all crossovers was also calculated. There were much fewer crossovers/metaphase when using the proposed procedure (1.4) versus the standard laboratory procedure (4.3). The standard deviation provided some interesting clues as well: for peripheral blood cells, our proposed protocol resulted in a lower standard deviation, meaning that most metaphases had similar sizes, and spreading was more homogenous. As peripheral blood metaphases usually spread better and easier than bone marrow metaphases, this result showed that our protocol increased metaphase spreading uniformly, on the entire slide surface. With bone marrow cultures, our protocol resulted in a higher standard deviation compared to the standard protocol. Because bone marrow metaphases are generally more difficult to spread, on any slide there will be a mixture of spread and non-spread metaphases. Using our protocol, more metaphases spread and acquire larger diameters compared with the standard protocol. However, because our slides still carried non-spread cells, the standard deviation increased.

Another interesting phenomena, is the consistency of the results. Data in Table 1 shows that, with both slide preparation protocols, the average diameters and standard deviations were consistent for the same cell suspension tested. For example, BM1 showed better spread metaphases (larger average diameter) and higher standard deviation than BM2. There was also consistency when the various culture flasks of the same tumor were compared. For example, regardless of the spreading procedure, GCT2 culture of BM1 yielded better spread metaphases than the GCT1 and the EB culture. This indicates that spreading is influenced by the way the culture was handled (hypotonic treatment and fixation). Nevertheless, when compared to the standard protocol, our spreading procedure resulted in most improved spreading of BM2. As the difference in metaphase diameter between BM1 and BM2 is smaller with our protocol and higher with the standard laboratory procedure, it appears that the technician working with BM1 was more successful than the technician working with BM2. In other words, human "errors" influence also the degree of metaphase spreading, even when, theoretically, the same standard protocol is in use in the laboratory.

Dropping cells from a height does not improve spreading.

If the drying process is observed at the phase microscope, five arbitrary steps can be described: [1] while the cell suspension spreads on the slide, cells float wildly in all directions but ultimately touch the glass surface and immediately become immobile. No chromosome spreading takes place. Cells look like small gray spheres (Fig. 3a,3aa). [2] Fixative starts drying. As its surface touches the cell surfaces, cells reflect the light and acquire a bright halo. Chromosomes are still not spread at all. Macroscopically, the surface of the slide becomes ‘grainy’ (Fig.3b,3bb). [3] The mitotic cells lose their halo and start flattening (chromosome spreading), faster than the non-mitotic cells. The chromosomes become dark and visible, most of the chromosome spreading being achieved at this step (Fig.3c,3cc). Non-mitotic cells still show the halo of light, indicating that the nuclear membrane/content is much more resistant to the flattening force of the drying fixative. [4] as the fixative is drying, non-mitotic cells continue to flatten, whereas chromosome spreading increases just a little more (less than 7% change in the diameter of various metaphase spreads) (Fig.3d,3dd). [5] tiny puddles of fixative surround each cell but quickly evaporate. Visible chromosome spreading stops, but minor changes may still take place (Fig.3e,3ee). The drying process is the same, regardless what height the cell suspension was dropped from, indicating that height does not influence chromosome spreading. It may only help distribute the cells more evenly on the slide surface. Actual spreading takes place later, when the surface of the slide becomes ‘grainy’. This is the critical step in which spreading can be helped by hot steam and acetic acid.

GTG-banding

GTG-banding on a variety of cell types tested worked well on slides prepared with our new procedure. Our slides had an increased number of analyzable metaphases, with larger diameters, and fewer chromosome cross-overs (Table 1). The more efficient spreading was accompanied by reduced cytoplasmic residua, which contributed to the good quality of G-banded preparations (Fig. 5b1,b2,b3) on an increased number of metaphases. The brief acetic acid exposure did not appear to alter in any noticeable way the sharpness of the Giemsa bands. On any slides, trypsin time can be varied. As a rule, longer trypsin incubation increases the thickness of the chromosomes and requires longer Giemsa-stain incubations in order to achieve banding.