© 2002 Wiley-Liss, Inc.
Cytometry Part A 51A:46 –51 (2003)
A New Method for Improving Metaphase Chromosome Spreading Wen Deng,1 Sai Wah Tsao,1 Joe N. Lucas,2,3 C. S. Leung,4 and Annie L. M. Cheung1* 1
Department of Anatomy, The University of Hong Kong, Hong Kong, SAR, China 2 Lawrence Livermore National Laboratory, University of California, California 3 American Laboratory Technologies, Inc., Arlington, Virginia 4 Department of Paediatrics, The University of Hong Kong, Hong Kong, SAR, China Received 25 April 2002; Revision Received 8 July 2002; Accepted 10 July 2002
Background: The success of complex molecular cytogenetic studies depends on having properly spread chromosomes. However, inconsistency of optimum chromosome spreading remains a major problem in cytogenetic studies. Methods: The metaphase spreading process was carefully timed to identify the most critical phase of chromosome spreading. The effects of dropping height of cell suspension, slide condition, drying time, fixative ratio, and relative humidity on the quality of metaphase spreads were studied by quantitative examination of metaphase chromosome spreads. Normal and immortalized human epithelial ovarian cells, neuroblastoma cells, and normal lymphocytes were tested. Results: Humidity over the slide was the most important variable affecting the quality of chromosome spreads. Consistent improvement in chromosome spreading (larger metaphase area, less chromosome overlaps, or
Well-spread metaphase chromosomes are fundamental substrates for cytogenetic studies. However, inconsistency in obtaining optimum chromosome spreading remains a major problem in cytogenetic studies (1). Reports based on quantitative investigation on improving the quality of the metaphase spreads are scarce. Even in a few quantitative studies (2– 4) and a number of qualitative studies (5–9), there are apparently conflicting points regarding the speculated mechanisms and applied techniques. Spurbeck et al. (4) emphasized that chromosome spreading depends on the natural drying time of the fixative and that approximately 90 s of drying time is needed for best spreading under the ambient condition of 20°C and relative humidity of 55%. In contrast, Henegariu et al. (2) specified no ambient conditions and used hot steam to moisten the slides and then put them on a 65–75°C metal plate, which, according to our own tests, dries the slides in 10 s. Whereas some researchers dropped cells on dry slides (4,5), others suggested that having a thin layer of water on the slides immediately before adding the cells improves spreading (1,2,6). Dropping cells from a height reportedly enhanced chromosome spreading (1,3), but
lower frequencies of broken metaphases) was obtained for all cell types if dynamic cell rehydration, occurring as fixative absorbs moisture from air, was made to coincide with the prompt fixation of spread chromosomes to the slide. This was achieved by dropping cells on dry glass slides placed in a shallow metal tray and then quickly lowering the tray into a covered 50°C water bath for slide drying. Conclusions: A new and simple method for improving metaphase chromosome spreading was developed based on our study on the characteristics of chromosome spreading. Cytometry Part A 51A:46 –51, 2003. © 2002 Wiley-Liss, Inc.
Key terms: new method; dynamics; chromosome spreading; mechanism
others have disagreed (2,7). Aware of the “mysterious aspects” of chromosome spreading, Barch et al. (10) suggested that it is more of an “art” than science. To have a better understanding of the dynamics of metaphase chromosome spreading, we studied the effects of a number of variables, i.e., dropping height of cell suspension, wetness and angle of the slide, drying time, fixative ratio, slide temperature, and relative humidity, on metaphase chromosome spreading. The quality of metaphase chromosome spreads was assessed with the following quantitative parameters: (a) the metaphase area, (b) the number of chromosome overlaps per metaphase, and (c) the frequency of broken metaphases. To the best of our knowledge, this is the first systematic and quantitative study on the effects of these variables on metaphase chro*Correspondence to: Dr. Annie L. M. Cheung, Department of Anatomy, The University of Hong Kong, 1/F Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong. E-mail:
[email protected] Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cyto.a.10004
NEW METHOD FOR CHROMOSOME SPREADING
mosome spreading. With a better understanding of fundamental factors influencing chromosome spreading, we developed a new and simple method with which consistent improvement in the quality of metaphase chromosome spreads was obtained. MATERIALS AND METHODS Cell Harvesting and Fixation Primary cells and cell lines including normal lymphocytes after 72 h in culture, neuroblastoma cell line (HTB10) at population doublings (PD) 53, normal diploid human ovarian surface epithelial (HOSE) cells (HOSE 1112) harvested at PD 8 before cellular crisis, and an immortalized HOSE cell line, HOSE 6-3, at PD 22 and PD 150, were tested in this study. Neuroblastoma cells and lymphocytes were treated with a hypotonic solution of 0.075 M KCl for 20 –30 min at 37°C, and HOSE cells were treated with 0.8% sodium citrate for 15 min at 37°C. The choice of hypotonic solution for each cell type was determined by quantitative evaluation of chromosome spreads after testing different documented hypotonic treatments, i.e., 0.044 M KCl (8), 0.075 M KCl (1), 0.4 –1.2% of sodium citrate (1), or 0.3% NaCl (11) (manuscript in preparation). After hypotonic treatment, cells were prefixed with 5% (final concentration) 3:1 methanol:acetic acid for 5 min. The cells were collected by centrifugation and washed four times with 3:1 methanol:acetic acid. After the final centrifugation, the supernatant was removed completely, and the cell pellet was resuspended in 0.5 ml of final fixative (2:1⬃6:1 methanol:acetic acid). Cell Spreading In our improved new protocol, optimal cell spreading was accomplished with the aid of a warm water bath. The washed slide was wiped dry with Kimwipes tissue and then placed on the flat bottom of an 11- ⫻ 13-cm, 1.0-cmdeep, and 0.5-mm-thick stainless steel tray at room temperature (we turned over the metal cover of a Lipshaw staining jar and used it as a tray). Twelve microliters of cell suspension in 3:1 methanol:acetic acid final fixative was dropped onto the slide from a height of 1 cm. Then the steel tray was lowered into a 50°C water bath (with the outer surface of the tray touching the water), and the water bath was covered immediately. The area of water surface inside the water bath was 15.5 ⫻ 30.5 cm2, and the distance between the metal tray and the water bath cover was 3 cm. The water bath was used to provide moisture and suitable temperature for cell drying and chromosome spreading. Theoretically, at any given water temperature, the variable that influences the accumulation of moisture inside the covered water bath is the ratio (R) of the net area of water surface for evaporation (the area of the entire water surface minus the area occupied by the metal tray) to the volume of air inside the covered water bath. We also tested a larger water bath with a water surface area of 30 ⫻ 38.5 cm2. With the same metal tray placed 4 cm below the bath cover, the R of 0.22 was similar to that of our smaller water bath (R ⫽ 0.23). Both
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setups were equally effective in giving good chromosome spreading. After the slide dried in about 40 s, the steel tray was taken out and cooled under tap water for the next use. Quantitative Examination of Metaphase Spreading Quality The chromosome spreads were analyzed under a phasecontrast microscope connected to a video camera, and the signal was sent to a video terminal (VT). For each metaphase, the intervals between the left and right edges (X⬘) and between the upper and lower edges (Y⬘) of the metaphase area were measured on the VT screen. Coefficients were obtained to calculate the actual dimensions on the slide (X and Y) from the measurements obtained on the VT screen (X⬘ and Y⬘). The area of each metaphase on the slide was calculated as area ⫽ ⫻ (X/2) ⫻ (Y/2) according to Spurbeck et al. (4). Chromosome overlaps were counted in each metaphase. In addition, a metaphase was scored as broken if the chromosomes were overscattered and no longer spread within an approximately rounded area (Fig. 1D). The broken metaphases were rejected in the evaluation of spread area and overlaps. The two-tailed t test was used to compare the quality of chromosome spreading. RESULTS Multiple Dynamic Steps Are Involved in Metaphase Chromosome Spreading After dropping 12 l of cell suspension in 3:1 methanol: acetic acid from a height of 1 cm onto horizontally placed dry slides, detailed timing of various phases of drying and spreading was performed under a phase-contrast microscope at an ambient temperature of 20°C and 55% relative humidity. The following represents the average timing of 10 slides, with no apparent difference observed between cell types. (a) In the first ⬃10 s, the cells were first seen to float and move wildly in the fixative and then became immobilized as they touched the slide surface toward the end of this time-phase. (b) From ⬃10 to ⬃25 s, there was no visible change in the X and Y dimensions of the immobilized cells, and the chromosomes did not spread. However, at the end of this step the mitotic cells could be identified as their bunched chromosomes became visibly dark under the phase-contrast microscope. (c) From ⬃25 to ⬃50 s, as the fixative continued to evaporate, the mitotic cells increased in the X and Y dimensions, and chromosomes spread. During this process, there appeared to be a quicker phase lasting about 5 s, during which dramatic chromosome spreading occurred (Fig. 1A and 1B), followed by a slower phase of about 20 s with minor changes in chromosome spreading. At the end of this period, the chromosomes were not completely fixed to the slide surface. (d) From ⬃50 to ⬃90 s, uneven evaporation of the fixative caused some areas to dry faster, leaving fine streaks of fixative macroscopically visible on the slide. Two important phenomena happened in this time phase: (i) some cells and chromosomes became fixed
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FIG. 1. A–D: Dynamic images of metaphase chromosome spreading captured under a phase-contrast microscope (10⫻) after 12 l of human ovarian surface epithelial (HOSE) 6-3 cell suspension in 3:1 methanol:acetic acid was dropped from a height of 1 cm onto dry slides at the ambient temperature of 20°C and 55% relative humidity. The fast phase of dramatic chromosome spreading was observed from ⬃25 s (A) to ⬃30 s (B) after cell dropping. At the end of this period, the chromosomes were not completely fixed to the slide surface. From ⬃50 s (C) to ⬃90 s (D), the mitotic cell with fragile membrane was driven to rupture by the fixative currents, so that the chromosomes spread out and were flooded away until the chromosomes were tightly fixed to the slide. E, F: Typical metaphase spreads in a hypodiploid cell (E) and a hyperdiploid (F) HOSE 6-3 cell stained with 4,6-diamidino-2-phenylindole. Images were taken under a fluorescence microscope (100⫻).
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NEW METHOD FOR CHROMOSOME SPREADING
Table 1 Effects of Ambient Humidity on Quality of Chromosome Spreading†
Cell type HOSE 6-3 (PD 150)b
Lymphocytes
Relative humidity (%)
Volume of cell suspension (l)
65 55 45 30 65 55 45 30
12 6 12 25 12 6 12 25
Mode of drying
Drying time (s)
No. of metaphases examined
Metaphase area (m2)a
Overlaps/ metaphasea
% Broken metaphases
Under airflow Natural evaporation Natural evaporation Natural evaporation Under airflow Natural evaporation Natural evaporation Natural evaporation
70 70 70 70 70 70 70 70
68 100 100 100 100 100 100 100
2,099 ⫾ 75 2,264 ⫾ 38 1,864 ⫾ 61* 1,571 ⫾ 35* 1,724 ⫾ 38 1,702 ⫾ 40 1,501 ⫾ 29* 1,324 ⫾ 27*
1.3 ⫾ 0.2 1.5 ⫾ 0.2 2.7 ⫾ 0.3* 3.8 ⫾ 0.2* 2.2 ⫾ 0.2 2.0 ⫾ 0.2 3.3 ⫾ 0.3* 4.5 ⫾ 0.3*
7 2 0 0 3 0 0 0
†
Cell suspension in 3:1 methanol:acetic acid was dropped onto the slides from a height of 1 cm at ambient temperature of 20°C. Mean ⫾ standard error. For consistency, data from hyperdiploid cells were not included because very few hyperdiploid cells were analyzable due to clumping of chromosomes on slides dried at 45% and 30% relative humidities. HOSE, human ovarian surface epithelial cell line; PD population doubling. *P ⬍ 0.05 versus 6 l of cell suspension dried under natural evaporation at 55% relative humidity. a
b
on the slide surface without any visible change in spreading as the fixative evaporated; and (ii) where the fixative was slower to evaporate, there were some fixative currents that flowed around the cells, and the mitotic cell with fragile membrane at this time point was broken by the shearing forces of the fixative currents, and chromosomes spread out and were washed away (Fig. 1C and 1D). These chromosomes became overscattered or clumped together due to uneven currents. At the end of this step, the slide was dried completely and chromosomes were tightly fixed to the slide. When the chromosome spreads were made on wet slides, the first phase of “cell movement” shortened to ⬃5 s and the last drying step slowed down by ⬃15 s. The dramatic chromosome spreading, however, remained at 25–30 s after the cell suspension was dropped onto the slide. When cells were dropped from a height of 30 cm instead of 1 cm, the only difference in the drying process was observed in the first phase during which the duration of “cell movement” shortened to 3–5 s because the cell suspension splashed wider so that the layer of fixative was thinner and the cells became immobilized sooner. Effects of Dropping Height, Slide Angle, Slide Wetness, and Fixative Ratio on the Quality of Chromosome Spreads It is a common practice in cytogenetic laboratories to drop cell suspension onto slides from a height of 20 –30 cm. Inclining the slide at an angle of 20 –30 degrees and making a thin layer of icy water on the slide have been recommended by some investigators (1). In this study, HOSE cells and lymphocytes suspended in 12 l of 3:1 methanol:acetic acid fixative were dropped from a height of 30 or 1 cm onto slides or placed on the slide surface by touching the tip of the pipette to the slide surface. The slides were placed horizontally or tilted at an angle of 20 degrees to the bench, and allowed to dry at an ambient temperature of 20°C and relative humidity of 55%. Our
measurements showed that the quality of chromosome spreads, judged by the metaphase area, number of chromosome overlaps per metaphase, and percentage of broken metaphases, was not significantly affected by the dropping height of cell suspension, the angle of the slide, or the wetness of slide surface. We also tested the effects of different ratios of methanol:acetic acid (from 2:1 to 6:1) and found that metaphases fixed with higher ratios of the fixative were more resistant to cell breaking but had smaller metaphase areas. Effects of Slide Drying Time and Ambient Humidity To test whether the qualities of chromosome spreads depend on the speed at which the chromosomes dry on the slide (1,4), we used two methods to manipulate the drying time under the same ambient environmental condition (room temperature of 20°C and medium humidity of 55%). One method was to change the volume of the final drop of cell suspension. The other was to speed up the evaporation by drying the same volume of cell suspension under airflow in a hood, instead of drying by natural evaporation on the bench. We found that the chromosome spreading quality was not significantly influenced by slide drying time from 40 to 140 s under the same ambient condition. Slide drying time could be a confounding factor in the assessment of the contribution of ambient humidity to metaphase spreading, because a higher humidity results in a longer drying time for the same amount of cell suspension at the same ambient temperature. To test the effect of ambient humidity alone on the quality of metaphase spread, we changed the volume of the final drop of cell suspension so that an identical drying time was obtained for different relative humidities. Our results, shown in Table 1, clearly demonstrated that the sole factor of humidity played a major role in affecting the quality of chromosome spreads. With the same drying time of 70 s,
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Table 2 Improvement in Quality of Chromosome Spreads‡ No. of metaphases examined
Metaphase area (m2)a
Overlaps/ metaphasea
% Broken metaphases
114 (hypodiploid) 61 (hyperdiploid) 70 (hypodiploid) 30 (hyperdiploid) 69 (hypodiploid) 31 (hyperdiploid) 75 (hypodiploid) 30 (hyperdiploid)
2,166 ⫾ 67 3,237 ⫾ 110 2,296 ⫾ 125 2,974 ⫾ 200 2,696 ⫾ 90* 4,723 ⫾ 255* 2,641 ⫾ 102* 4,728 ⫾ 265*
1.8 ⫾ 0.2 4.7 ⫾ 0.4 1.4 ⫾ 0.3 3.6 ⫾ 0.6 0.7 ⫾ 0.2† 1.7 ⫾ 0.3† 0.5 ⫾ 0.2† 1.4 ⫾ 0.3†
3
Conventional Trial New
100 100 100
1,681 ⫾ 49 1,628 ⫾ 48 2,118 ⫾ 58*
1.9 ⫾ 0.3 2.2 ⫾ 0.2 1.0 ⫾ 0.2†
0 0 0
Conventional New
80 82
2,082 ⫾ 68 2,689 ⫾ 106*
1.5 ⫾ 0.3 0.5 ⫾ 0.2†
6 3
Conventional New
45 81
1,923 ⫾ 75 2,575 ⫾ 67*
1.9 ⫾ 0.3 0.6 ⫾ 0.2†
36 7
Cell type
Slide making method
HOSE 6-3 (PD 150)
Conventional Trialb New New (20°C, 45% RH)
Lymphocytes (diploid) HOSE 11–12 (PD 8, diploid) Neuroblastoma cells (HTB10)
0 6 5
‡ Twelve microliters of cell suspension in 3:1 methanol:acetic acid was dropped onto the slides from a height of 1 cm. Ambient condition was 20°C and 55% relative humidity unless specified. HOSE, human ovary surface epithelial cell line; PD, population doubling; RH, relative humidity. a Mean ⫾ standard error. b Trial method: same as in new method except the water bath was left uncovered. *P ⬍ 0.05 versus natural evaporation. † P ⬍ 0.01 versus natural evaporation.
the chromosome spreads of both immortalized HOSE cells and normal lymphocytes prepared at 45% and 30% relative humidities had significantly smaller (P ⬍ 0.05) metaphase areas and more (P ⬍ 0.05) overlaps per metaphase than did preparations made at 55% relative humidity. At a higher relative humidity of 65%, however, the quality of chromosome spreads was similar to that obtained at a relative humidity of 55%. A New and Simple Method Consistently Improves Chromosome Spreading in Three Different Types of Cells A comparison of chromosome spreading quality between our new method of cell drying and a “conventional” method, which allowed the cells to dry naturally at a temperature of 20°C and 55% relative humidity, is presented in Table 2. The new method significantly increased the mean metaphase areas and decreased the chromosome overlaps per metaphase (P ⬍ 0.05) in immortalized HOSE 6-3 cells, pre-immortal HOSE 6-3 cells (PD 22; data not shown), normal diploid HOSE cells (HOSE 11-12, PD 8), normal lymphocytes, and neuroblastoma cells. We also tested the role of slide temperature by putting the slide on the 50°C metal tray without covering the water bath (trial method), and we found no significant difference between the quality of metaphase spreads dried by this method and that by natural evaporation. Figure 1E and 1F show typical images of a hypodiploid and a hyperdiploid HOSE cell, respectively, with nice chromosome spreads obtained with the new method of cell spreading.
DISCUSSION In this study, we demonstrated that humidity alone plays a major role in affecting chromosome spreading, and our new method consistently improved chromosome spreading for a variety of cultured cells. The following is our interpretation of these findings. It is well known that methanol:acetic acid fixative is highly hygroscopic. As the fixative spreads over the surface of the slide, its contact area with air increases. While the fixative absorbs water from air, the water content in the fixative rises. This causes dynamic rehydration in cells. It has been shown that a dramatic release of free energy occurs when fixative mixes with water (12). We therefore speculate that it is this dynamic energy release from rehydration that adds energy for chromosome spreading and cytoplasm relaxation. The actual chromosome spreading takes place when the layer of fixative becomes sufficiently thin with evaporation, and the fixative meniscus pushes down on top of the cell and widens the metaphase (1). If this critical stage is coordinated with the dynamic rehydration in the cell, the added energy will make the metaphase chromosomes spread farther. The dynamic rehydration may decrease with time as the water content in the fixative increases. Therefore, if there is inadequate rehydration due to low ambient humidity, or if the chromosome spreading takes place after the dramatic dynamic rehydration, optimal chromosome spreading may not occur. The 50°C warm water bath in our new method served two critical functions: to gradually increase the air mois-
NEW METHOD FOR CHROMOSOME SPREADING
ture after the water bath was covered, and to provide a suitable temperature for the metal tray placed in contact with the warm water. The gradually warmed metal tray was needed to transmit heat to the slide for slide drying so that the spread chromosomes would become fixed to the slide promptly. We did not use higher temperature because we did not want the fixative to dry too quickly. We reasoned that, if drying is too quick, the chromosomes may not spread sufficiently because it takes time (⬃10 s) for the covered warm water bath to build up enough moisture to coincide with the time window of chromosome spreading as the fixative evaporates. It appears that, at a given humidity, there is a threshold for chromosome spreading, and manipulating drying time itself cannot break the threshold. However, a proper drying time serves to fix the spread metaphases to the slide promptly. Based on our careful observation of drying process under ambient temperature of 20°C and a relative humidity of 55%, the dramatic chromosome spreading (fast phase) occurs only within a few seconds, which is a short time compared with the full drying time. It is the few seconds of the fast phase that are crucial for chromosome spreading. This may explain the insensitivity of chromosome spreading quality to the entire drying time under ordinary ambient condition. Although there is dramatic energy release from mixing between the fixative and water (12), dropping cell suspension on a wet slide did not improve chromosome spreading. This can be explained by the fact that the initial energy release from mixing between the fixative and water did not coincide with the chromosome spreading that took place later (see first section under Results). A combination of a high humidity (65%) and natural evaporation did not result in a better quality of chromosome spreading compared with that at 55% relative humidity (Table 1). The reason may be that the higher humidity caused a faster increase in water content of the fixative, so that the dramatic dynamic rehydration took place earlier than the narrow time window of chromosome spreading in the
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relatively slow process of natural evaporation of the fixative. In conclusion, we have shown by carefully exploiting the characteristics of fixative (methanol:acetic acid), air moisture, heat transmission, and slide drying that consistent improvements in chromosome spreading can be obtained for a variety of cultured cells. ACKNOWLEDGMENTS We thank Jenny Cheung, Alla Li, Tony Chan, and Liang Hu for excellent technical assistance and Dr. Xin-Yuan Guan (Department of Clinical Oncology, The University of Hong Kong) for the use of his research facilities. LITERATURE CITED 1. Barch MJ, Knusten T, Spurbeck JL. The AGT cytogenetics laboratory manual. 3rd ed. New York: Raven Press; 1997. p 25–50. 2. Henegariu O, Heerema NA, Wright LL, Bray-Ward P, Ward DC, Vance GH. Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing. Cytometry 2001;43:101–109. 3. Hlics R, Muhlig P, Claussen U. The spreading of metaphases is a slow process which leads to a stretching of chromosomes. Cytogenet Cell Genet 1997;76:167–171. 4. Spurbeck JL, Zinsmeister AR, Meyer KJ, Jalal SM. Dynamics of chromosome spreading. Am J Med Genet 1996;61:387–393. 5. Barch MJ, Knusten T, Spurbeck JL. The AGT cytogenetics laboratory manual. 3rd ed. New York: Raven Press; 1997. p 63– 64. 6. McFee AF, Sayer AM, Saomaa SI, Lindhlm C, Littlefield LG. Methods for improving the yield and quality of metaphase preparations for FISH probing of human lymphocyte chromosomes. Environ Mol Mutagen 1997;29:98 –104. 7. Lambson B, Mendelow B, Berstein R. Metaphase spreading in chromosome preparations is minimally affected by mechanical disruption. Karyogram 1986;12:30 –32. 8. Sasai K, Tanaka R, Kawamura M, Honjo K, Matsunaga N, Nakada T, Homma K, Fujimura H. Chromosome spreading techniques for primary gastrointestinal tumors. J Gastroenterol 1996;31:505–511. 9. Chattopadhyay S, Marinduque B, Gilbert F. Ovarian cancer: Protocol for the preparation of banded metaphases from tumor tissue. Cancer Genet Cytogenet 1992;59:210 –212. 10. Barch MJ, Knusten T, Spurbeck JL. The AGT cytogenetics laboratory manual. 3rd ed. New York: Raven Press; 1997. p 51–52. 11. Pejovic T, Heim S, Mandahl N, Elmfors B, Flodrus UM, Furgyik S, Helm G, Willen H, Metelman F. Consistent occurrence of a 19p⫹ marker chromosome and loss of 11p material in ovarian seropapillary cystadenocarcinomas. Genes Chromosomes Cancer 1989;1:167–171. 12. Holmquist GP, Motara MA. The magic of cytogenetic technology. In: Obe G, Basler A, editors. Cytogenetics, basics and applied aspects. Berlin: Springer-Verlag; 1987. p 30 – 47.
