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 (59), 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 20C
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 6575C 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 50C 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: lmcheung@hkucc.hku.hk
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 37C, and HOSE cells were treated
with 0.8% sodium citrate for 15 min at 37C. 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 (2:16:1 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 50C 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

47

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 20C 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

48

DENG ET AL.

FIG. 1. AD: 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 20C 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).

49

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 20C.
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

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
2530 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 35 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 20C 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 20C 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,

50

DENG ET AL.

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

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 (20C, 45% RH)

Lymphocytes
(diploid)
HOSE 1112
(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 20C 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 20C 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 50C 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 50C 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 20C 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

51

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.
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