Protocol step 1.
After usual hypotonic treatment, cells are pelleted, resuspended in 1 ml fixative and transferred into 1.5ml vials, in which all subsequent fixative washes are performed. All centricentrifugations performed in a microfuge.
Comments. After usual hypotonic incubation, cell suspensions are usually washed several times in fresh fixative solution (3:1 methanol : acetic acid). To make these steps faster, the cells can be transferred to 1.5 microfuge vials and centrifugations performed at 6-7,000 rpm for 1-2 minutes each. This minimizes cell loss while shortening the procedure, without affecting cell quality. Cell suspensions can be stored at -20° C in the same vials (in fixative). Prior to slide preparation, cells were resuspended in fresh fixative only when stored longer than 2-3 days in a refrigerator or more than two weeks in a freezer. For quality preservation, slides with metaphase spreads can be conveniently stored at -20? C in jars with 100% ethanol.
Protocol step 2.
With an automatic pipette, evenly distribute 25-35µl cell suspension on several locations on the slide and spread the liquid by gently moving the pipette tip parallel to the surface. Spreading should be very gentle and applied only when there is sufficient liquid on the slide, so that the cells are not scratched by the tip. Dropping from any height is not important (see below). As the fixative gradually evaporates, the surface of the slide becomes ‘grainy’ (cells visible). At this moment, place the slide face down into the steam of the hot water bath for 1-3 seconds, then dry by placing the slide on the metal plate (carrying a gradient of temperature across its surface, Fig. 1a). The degree of spreading is adjusted using the different temperatures of the heated plate, with higher temperatures increasing chromosome spreading.
Protocol step 2 supplement. For very hard to spread cell suspensions (as often the case with bone marrow cultures) increased spreading can be obtained using acetic acid (95% in water or 100% glacial). Pass the slide through the hot steam 2-3 seconds to moisturize the surface (numerous fine water droplets become visible). Place 30ul cell suspension on the slide, as described before, and pay attention not to let the liquid dry. After the surface becomes ‘grainy’, pass the slide briefly through the water vapors, then with an automatic pipette place 15-25µl (4-6 droplets) of acetic acid on the slide. (Warning: The acid should be placed on the surface only after the slide acquires the ‘grainy’ appearance, otherwise the cells may be washed away). After the acetic acid slowly spreads and covers the surface evenly, hold the slide for 3-5 seconds in the steam of the waterbath, then quickly dry on the hottest area of the metal plate or the metal block.
Comments and observations for chromosome spreading. Appropriate hypotonic treatment is vital to the quality of cytogenetic preparations. Too long or too short hypotonic incubations result in poor quality cells which do not spread, and show heavy cytoplasmic residua around nuclei and chromosomes. Good quality cell suspensions spread easily with most common laboratory protocols and yield clean metaphases, with little proteinaceous residua. Simultaneous presence of acetic acid and water on the slide during drying seems to be the most important factor for chromosome spreading. Our proposed slide preparation protocol is based on the intimate processes taking place at the microscopic level. Although based on the same known factors (water vapors, hot plate, acetic acid), our procedure uses them in a characteristic, step-by-step fashion, which allows better chromosome spreading when compared with the "standard" protocol (see "methods") used in our clinical laboratory. One of the most important features of the proposed protocol for slide preparation is its precise timing. Brief slide exposure to hot water vapors in the beginning, provides a very 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. It is at this precise moment that the slide is passed again through the hot vapors, so water is evenly distributed onto the slide surface. It is likely that water arrives on the slide right when most of the methanol from the fixative has evaporated and there is a resulting brief increase in acetic acid concentration. Adding a uniform layer of water at this moment, followed by quick drying on the hot area of the metal plate provides good chromosome spreading. Summary of steps: water vapors - cell suspension - water vapors - dry. To increase spreading even more, after the cell suspension is placed on the slide and the slide is passed through the hot water vapors, 3-5 drops of glacial acetic acid are placed on the slide. The acid spreads slowly on the surface until it covers it uniformly, and slows the drying process. At this moment, the slide is passed again through the water vapors, adding another layer of water to the acid. Then the slide is quickly dried on the hot area of the metal plate. Summary of steps: water vapors - cell suspension - water vapors - acetic acid - water vapors - dry. It is this combination of acid/water and fast drying that spreads the chromosomes the most. With good quality preparations (particularly peripheral blood culture) adding the layer of acetic acid is not necessary, as the procedure may result in overspread chromosomes and disruption of the metaphases. If this phenomenon occurs, it can be easily rectified by drying the slide on the colder areas of the metal plate containing the heat gradient (a "perfect" recipe is not possible, as spreading depends on the initial quality of the cell suspension). This is, in fact, the reason why the heat gradient of the plate is useful. With most cell suspensions, drying in the hottest area of the metal plate works well (therefore, if it is not available, the metal plate with the heat gradient can be replaced with a simple heat block or hot plate). However, if a cell suspension spreads too extensively, the slide must be dried in the colder areas of the plate (this can be replaced by placing the slide directly on a room-temperature surface in the lab (bench top). In our hands, the best visual indicator for optimal spreading is the presence of numerous, very small liquid droplets on the slide after it is placed on the hot metal plate, indicating that an optimal amount of water was mixed with the fixative. Few, larger droplets, indicate the presence of too much water. "Lack" of droplets, or the presence of a "sheet" of liquid, drying non-uniformly, indicates too much acetic acid or fixative and not enough water.
Also important is the observation that 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: (s1) 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). (s2) 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). (s3) 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. (s4) 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). (s5) 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.
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% longer using the procedure proposed in this study. This translates itself in metaphases with 45-70% increased surface. For the blood culture, where the chromosomes are longer, the number of all crossovers were 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 provides some interesting clues as well. For the peripheral blood cells, the protocol proposed resulted in a lower standard deviation compared with the standard protocol, which means that most metaphases had closer sizes, so the spreading was more homogenous. This is not unexpected, considering that peripheral blood metaphases usually spread better and easier than bone marrow metaphases. With the bone marrow cultures, the proposed protocol resulted in a higher standard deviation compared to the standard protocol. This can be easily explained, based on the fact that bone marrow metaphases are generally more difficult to spread. On a bone-marrow slide, there are always non-spread metaphases as well as spread ones. When using the protocol proposed in this study, more metaphases spread compared with the standard protocol, and to larger diameters. However, because the 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,regardless what slide preparation protocol was used, the average diameters and standard deviations, we both obtained the same "pattern" for the same culture. This is visible by comparing BM1 and BM2. With both procedures, BM1 showed better spread metaphases (larger average diameter) and higher standard deviation. There was also consistency when the various cultures of the same tumor were compared. For example, regardless of the spreading procedure, GCT2 culture of BM1 yielded better spread metaphases than the EB culture and than the GCT1 culture. This indicates that spreading is influenced by the way the culture was handled (hypotonic treatment and fixation). Nevertheless, the proposed spreading protocol yielded the best results on BM2, when compared to the standard protocol. As the difference in metaphase diameter between BM1 and BM2 is smaller with the proposed protocol and higher with the standard laboratory procedure, it appears that the technicians 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.