Chromosome Preparation from Cultured UNIT 4.

1
Peripheral Blood Cells
Chromosome preparations currently provide the only direct view of the genome as a
whole. Although molecular methods allow a more detailed analysis of specific regions of
the genome, the study of genetics is not complete without an appreciation of the metaphase
cell. Peripheral blood provides a simple and reproducible source for large quantities of
mitotic cells that may be used for clinical diagnosis (see UNITS 4.2 & 8.1) or studying
chromosome structure and organization (see UNIT 4.3).
From a clinical point of view, the metaphase cell reveals not only numerical and structural
abnormalities, but also provides some biochemical information useful in diagnosing
genetic disorders. For instance, the DNA instability associated with Fanconi anemia (UNIT
8.7) appears as increased chromosome breakage in metaphase cells; Blooms syndrome
(UNIT 8.6) is manifested as increased sister chromatid exchange; and ataxia telangiectasia
may be spotted in metaphase chromosome preparations as infidelity in somatic recombi-
nation. Chromosome preparations are essential for localizing genes and other DNA
sequences on the physical map. This mapping is done directly by chromosomal in situ
hybridization or indirectly by correlating the presence or absence of a marker (such as an
enzyme, a particular RNA, or a DNA sequence) with the presence or absence of a
chromosomal region.
The stimulated T cell system described here is the most widely used means of obtaining
large numbers of mitotic cells for genetic analyses. Synchronization of the cell cycle in
culture (basic protocol), combined with direct inhibition of chromosome condensation
(alternate protocol), yield longer high-resolution prophase or prometaphase preparations.
Such preparations are used for detailed analysis of microdeletions or subtle rearrange-
ments, fine breakpoint analysis, and refined mapping. Although chromosomes may be
obtained from other cells, human peripheral blood leukocytes are most amenable to
synchronization, and thus to high-resolution analysis of their chromosomes. Microscope
slide preparation of mitotic chromosomes from harvested cell culture suspensions is also
explained in the support protocol.
NOTE: All reagents and equipment coming into contact with live cells must be sterile.

CULTURE AND METAPHASE HARVEST OF PERIPHERAL BLOOD BASIC

PROTOCOL
T lymphocytes in whole blood are stimulated with the mitogenic plant lectin phytohe-
magglutinin (PHA). The T lymphocytes activate to blast-like cells within 12 to 24 hr
and continue to proliferate for 2 to 4 days. An optional cell-cycle synchronization step
can be performed (see Fig. 4.1.1), which increases the number of cells harvested in early
mitosis. (This synchronization is not as involved as the one described in the alternate
protocol, which results in longer chromosomes and higher resolution). Metaphase cells
are obtained by treating cultures with Colcemid, a colchicine analog that disrupts the
centriole/spindle-fiber complex by interfering with microtubule formation. This treatment
results in mitotic arrest, which in turn leads to an accumulation of cells in metaphase.
Mitotic arrest is followed by treatment with a hypotonic KCl solution (hypotonic shock)
to increase cellular volume. The cells are then fixed with methanol/acetic acid to remove
water and disrupt cell membranes before being spread onto slides (support protocol).

Cytogenetics

Contributed by Charles D. Bangs and Timothy A. Donlon 4.1.1

Current Protocols in Human Genetics (1994) 4.1.1-4.1.19
Copyright 2000 by John Wiley & Sons, Inc. CPHG
 Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
Heparinized whole blood obtained via Vacutainer (Becton Dickinson)
or syringe with preservative-free sodium heparin (25 U/ml)
Complete RPMI/10% FBS medium (APPENDIX 3G) containing 50 g/ml gentamycin
sulfate in place of penicillin and streptomycin
100 phytohemagglutinin-M (PHA) stock (GIBCO/BRL), reconstituted in
sterile deionized water (store at 4C)
10 M methotrexate (optional; see recipe)
1 mM thymidine (optional; see recipe)
10 g/ml Colcemid (GIBCO/BRL)
75 mM KCl (0.56 g in 100 ml H2O; store 2 weeks at room temperature)
Fixative: 3:1 (v/v) HPLC-grade absolute methanol/glacial acetic acid,
(prepare fresh)
15-ml sterile disposable conical polypropylene centrifuge tubes
TB syringe equipped with 21-G needle (VWR Scientific)
IEC HN-SII centrifuge with 958 rotor (or equivalent)
CAUTION: Human blood and methotrexate are hazardous; see APPENDIX 2A for guidelines on handling,
storage, and disposal.
NOTE: All incubations are performed in a humidified 37C, 5% CO2 incubator unless otherwise specified.

Collect sample and initiate cultures

1. Collect peripheral blood by venipuncture into a sodium heparin Vacutainer or a
syringe with 25 U preservative-free sodium heparin per milliliter of blood.
Other anticoagulants, such as lithium heparin or EDTA are toxic to cells and should never
be used. Samples should be shipped at room temperature. Blood in sodium heparin can be
held for 4 days and still be cultured successfully, but cultures are best initiated as soon
as possible. If necessary, the specimen can be stored at 4C.
2. Inoculate 0.25 ml of the whole blood obtained in step 1 (0.2 ml for newborns 3
weeks old) into a sterile 15-ml centrifuge tube containing 5 ml complete RPMI/10%
FBS medium, using a TB syringe equipped with a 21-G needle. Add 0.05 ml of
reconstituted 100 PHA solution.
Always replace the 25-G needle supplied with most TB syringes with a 21-G needle. Forcing
blood through the narrower needle can lyse leukocytes.
A single culture typically yields three to five full-slide preparations, or more if only part of
the slide is used. Multiple cultures may be set up to meet clinical or research needs.
3. Incubate 2 to 4 days with tubes tilted at 45.
Three-day incubations are optimal, but 2- or 4-day cultures can be used to accommodate
laboratory scheduling concerns. Cultures from newborns will usually work well at 2 days
but may also be harvested either directly or following a 1-day culture. Older patients
leukocytes require 3- or 4-day cultures because they do not seem to respond as quickly to
PHA.
Tilting culture tubes to 45 increases gas exchange and prevents cells from packing too
densely at the bottom. For these reasons it is best not to add more than 0.5 ml blood to any
tube.
Chromosome
Preparation
from Cultured
Peripheral
Blood Cells

4.1.2
Current Protocols in Human Genetics
Synchronize the cell cycle (optional)
Synchronization (Fig. 4.1.1) is optional for routine blood culture and harvest, but will
result in longer chromosomes on average and more mitotic cells. The synchronization
presented here (steps 4 and 5) will yield a greater number of mitotic cells; however, these
are not as long as those obtained using the high-resolution method (alternate protocol).
4. On the day before harvest (e.g., after 4:00 p.m.), add 0.05 ml of 10 M methotrexate
(107 M final) to block DNA replication. Incubate 16 to 18 hr (overnight).
5. On the following day (e.g., at 8:30 a.m.), add 0.05 ml of 1 mM thymidine (105 M
final) to release the methotrexate block. Incubate 4 hr (e.g., until 12:30 p.m.).
The exact hour of synchronization and release in steps 4 and 5 are not critical. However,
because of methotrexate toxicity, the culture should be blocked no longer than 18 hr. The
exact interval between release of block and harvest is critical, because cells will progress
quickly through G2, which lasts for 4 to 6 hr, to metaphase, which lasts 1 hr.

set up mixed cultures

(asynchronous cell cycles);
block with methotrexate

interphase: G1 mitosis interphase: G2

incubate
1618 hr

S phase

perform thymidine release

prophase

prepare slides from

synchronous cell cycles

metaphase

Figure 4.1.1 Cell culture synchronization. Two- or three-day asynchronous lymphocyte cultures
are blocked by addition of methotrexate, a folate antagonist that prevents thymidine synthesis.
Depletion of the thymidine pool prevents the cells from completing replication, and cells accumulate
in the synthetic, or S, phase of the cell cycle. Subsequent addition of thymidine releases a
synchronized wave of cells to complete replication and proceed through G2 and into mitosis.

Cytogenetics

4.1.3
Current Protocols in Human Genetics
 Harvest culture
6. Initiate harvest by adding 25 l of 10 g/ml Colcemid (0.05 g/ml final). Incubate
30 min.
For a 5-ml culture, this approximates two drops from a TB syringe equipped with a 25-G
needle.
If synchronization steps 4 and 5 have been omitted, the harvest can be initiated at any time
3 to 4 days following the culture described in step 3.
7. Centrifuge 8 min at 180 g (1000 rpm in IEC 958 rotor), room temperature. Discard
supernatant.
8. Add 6 ml of 75 mM KCl at room temperature and gently resuspend cells. Let stand
15 min at room temperature.
The amount of hypotonic solution to be added should be adjusted to the volume of the pellet.
Some laboratories vary the length of hypotonic treatment. Increasing the time will increase
chromosome spreading, but this treatment is a hypotonic shock, so that increasing the
amount of hypotonic solution will have more impact than increasing the time of treatment.
9. Add 10 to 12 drops of fixative with a Pasteur pipet and mix well. Centrifuge as in
step 7.
This treatment serves to reduce the pH of the cells gradually to precondition them for the
following fixation steps. It also lyses remaining red blood cells and begins the process of
clearing resulting cellular debris.
10. Remove all but 0.5 ml of the supernatant and resuspend pellet in remaining super-
natant by drawing it gently up and down with a Pasteur pipet. Add 1 ml fixative and
immediately mix gently. Adjust volume to 5 ml with fixative and mix thoroughly.
Centrifuge as in step 7.
The pellet after step 9 will be brown and clumpy because of erythrocyte debris. Resuspend
gently but thoroughly to avoid clumped lymphocytes which may complicate slide-making.
Do not draw too much volume into the pipet while resuspending because the cells will stick
permanently to glass. Do not press the pipet tip against the bottom of the tube when drawing
and delivering the suspension, as this will lyse cells.
The pellet after step 10 will be more homogeneous, and will usually have a light-brown to
white color. It may be 0.1 ml in volume.
11. Aspirate supernatant, resuspend pellet in 5 ml fixative, and centrifuge as in step 7.
12. Remove supernatant and resuspend pellet in a volume of fixative sufficient to produce
a light milky suspension. Allow to stand 30 min at room temperature or store
overnight at 4C.
Longer fixation will often improve chromosome spreading in difficult harvests. Keeping the
suspension overnight at 4C can improve the quality of the preparation or can be done for
scheduling reasons. Suspensions should be kept in polypropylene tubes containing plenty
of fixative (e.g., 5 ml). Polystyrene tubes will react with fixative and should not be used.
13. Prepare slides and analyze chromosome spreads as described in the support protocol.

Chromosome
Preparation
from Cultured
Peripheral
Blood Cells

4.1.4
Current Protocols in Human Genetics
CULTURE AND HARVEST FOR HIGH-RESOLUTION ALTERNATE
PROMETAPHASE CHROMOSOMES PROTOCOL
Longer chromosomes provide higher resolution in analyses based on chromosome
banding. Chromosomes condense and bands coalesce as cells progress through metaphase
(Fig. 4.1.2); chromosome length and hence resolution of chromosome analyses are
therefore functions of how early in mitosis fixation is performed. It is possible to obtain
high-resolution cell harvests (i.e., harvests enriched for mitotic cells at or above the level
of 750 G-bands per haploid karyotype) by a combination of cell-cycle synchronization
and inhibition of chromosome condensation.

In this protocol, synchronization is accomplished by blocking mitosis in cultures during

the synthetic, or S, phase of the cell cycle. Methotrexate, a competitive inhibitor of
dihydrofolate reductase, is used for this purpose and acts to reduce the levels of available
thymidine, which is required for DNA synthesis. The methotrexate block is released by
addition of thymidine and a series of sequential harvests are performed at short intervals
to capture the wave of late-prophase or prometaphase cells, which will have the longest
chromosomes. A short (10-min) mitotic arrest averts excessive chromosome condensa-
tion. Condensation may be reduced further by addition of the DNA-intercalating fluoro-
chrome ethidium bromide, just before harvest.
Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
1.25 mM ethidium bromide solution (see recipe)
CAUTION: Ethidium bromide is hazardous; see APPENDIX 2A for guidelines on handling, storage, and
disposal.
NOTE: All incubations are performed in a humidified 37C, 5% CO2 incubator unless otherwise specified.

1. Prepare blood and initiate four cultures as in steps 1 and 2 of the basic protocol.
Three cultures will be used for sequential harvest. The fourth culture is a backup in the
event that the sequential harvests do not work well. All cultures will be treated with ethidium

prophase prometaphase metaphase late metaphase

850 550 400 < 400

G-bands per haploid karyotype

Figure 4.1.2 A partial karyotype of G-banded chromosome 7 from cells at successive stages of
mitosis, illustrating the coalescing of subbands into larger, less defined, and less informative
landmark bands. Cells captured in late prophase may show 850 bands per haploid karyotype, i.e.,
high resolution. By mid-metaphase, fine band detail is lost as chromosomes condense and bands
fuse, and only 400 or fewer bands per haploid karyotype may be observed. Cytogenetics

4.1.5
Current Protocols in Human Genetics
 bromide in step 5. The backup culture will be treated with a 30-min Colcemid arrest as
described in the basic protocol and step 6 below.
2. Incubate cultures 3 or 4 days with tubes tilted to 45.
3. On the day prior to harvest (e.g., after 4:00 p.m.), add 0.05 ml of 10 M methotrexate
(107 M final) to block DNA replication. Incubate 16 to 18 hr.
The exact duration of the block is not critical but should be 16 to 18 hr to avoid methotrexate
toxicity.
4. The next morning (e.g., at 8:30 a.m.), release the cultures from the methotrexate block
by adding 0.05 ml of 1 mM thymidine (105 M final). Incubate 3 hr.
5. Add 0.05 ml of 1.25 mM ethidium bromide solution (1.25 105 M final) to each
culture. Incubate as described in steps 6 and 7.
6. After 50 min (e.g., at 12:20 p.m.) add 25 l of 10 g/ml Colcemid (0.05 g/ml final)
to the culture chosen for the first sequential harvest and the backup culture. Incubate
the first harvest tube 10 min, then perform steps 7 through 12 of the basic protocol.
7. After an additional 10 min (e.g., at 12:30 p.m.), repeat step 6 using the second harvest
culture.
IMPORTANT NOTE: It is critical to stay on time! To maintain the 10-min intervals, it will
be necessary to shorten all 8-min centrifugations described in the basic protocol (starting
with that in step 7) to 6 min, and the 15-min hypotonic treatment in step 8 of the basic
protocol to 12 min.
By doing these sequential harvests, a 30-min period is spanned by three 10-min mitotic
arrest intervals (Fig. 4.1.3). One of these arrest intervals should capture an early mitotic
cell population with chromosomes that are less condensed than the others, providing
optimal resolution. The backup culture spans the entire 30-min period.
The optimum length of time between addition of thymidine (step 4) and initiation of harvest
with Colcemid can vary between laboratories. It may be determined empirically by adding
several extra 10-min mitotic arrest intervals so that the overall harvest period extends for
a longer period (e.g., 50 min) and observing which interval results in the longest chromo-
somes.
9. Prepare slides and analyze chromosome spreads as described in the support protocol.

SUPPORT CHROMOSOME SLIDE PREPARATION

PROTOCOL
Slide-making is the least standardized and understood of cytogenetic protocols, about
which technologists have widely variable and sometimes contradictory ideas. In the end
what really matters is that slide preparations are consistent and appropriate for the desired
analysis. The protocol presented here is not the only approach to chromosome slide
preparation but it works under varied physical conditions (slide-making is very climate-
dependent) and for a wide range of cell cultures. It can be used for peripheral blood, bone
marrow (UNIT 10.2), ascites and pleural effusions, amniotic fluid (UNIT 8.4) and tissue flask
harvests (UNITS 8.3, 8.5 & 10.3), somatic cell or radiation hybrids (UNITS 3.2 & 3.3), lymphoblas-
toid cell lines, and nonhuman and hybridoma culturesin short, any culture harvest that
results in a fixed suspension of mitotic cells.
Harvested peripheral blood cultures suspended in methanol/acetic acid fixative (basic and
alternate protocols) are applied to wet microscope slides, flooded with fixative, and
Chromosome
Preparation air-dried. The drying process is adjusted according to ambient temperature and humidity
from Cultured to optimize spreading and morphology of chromosomes for subsequent banding and
Peripheral
Blood Cells analysis. The protocol described here produces preparations that are particularly suitable

4.1.6
Current Protocols in Human Genetics
 cultures
1 2 3 set up cultures and
perform thymidine release;
incubate 3 hr

add ethidium bromide;

incubate for specified time

50 min 60 min 70 min

30-min
sequential 10 min add Colcemid; harvest
cells sequentially
harvest
span (and 10 min
backup
culture
incubation) 10 min

prepare slides

slides

Figure 4.1.3 High-resolution harvest. Prometaphase chromosomes are obtained by sequential

harvests of synchronized cultures (1, 2, and 3), timed to capture a wave of cells early in mitosis,
when the chromosomes are longest. Staggered 10-min mitotic arrests span a 30-min harvest
window. The short mitotic arrest prevents excessive chromosome condensation. Addition of
ethidium bromide 1 hr before mitotic arrest directly inhibits chromosome condensation. In a typical
high-resolution harvest, culture 1 may yield little or no mitoses, culture 2 may capture an early mitotic
wave with long chromosomes, and culture 3 mitoses are shorter, having proceeded further into
metaphase.

Cytogenetics

4.1.7
Current Protocols in Human Genetics
 for analysis by G-banding (UNIT 4.2) or in situ hybridization (UNIT 4.3), although many other
staining techniques or procedures may be used.

Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
solutions, see APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.
Fixed cultures (basic or alternate protocols)
Fixative: 3:1 (v/v) methanol/acetic acid (use 100% methanol and glacial
acetic acid, both AR grade, from J.T. Baker)
Microscope slides (one end frosted) stored in 100% methanol (absolute AR
grade, J.T. Baker) in Coplin jars
Lint-free tissue (e.g., Kimwipe or gauze pad)
Zeiss Standard phase-contrast microscope with 16 Ph2 objective and
condenser ring (or equivalent)
1. Remove slide from methanol and polish with lint-free tissue, such as a folded
Kimwipe or gauze pad. Dip slide once in methanol and then several times in deionized
water until the methanol is gone and a thin, uniform film of water covers the slide.
Good slides have few pits and imperfections and will hold a thin film of water across the
entire slide, which reduces the surface tension prior to addition of the cell suspension.
Cleaning each slide is essential, as few precleaned slides are truly clean enough for
chromosome preparations.
2. Holding the frosted end between the thumb and finger, position the slide with the one
long edge parallel to the bench top, and blot the lower long edge on a paper towel to
draw off excess water. Keeping the lower long edge in contact with the paper towel,
lower the opposite edge until the slide forms a 30 angle with the bench top, with the
film of water facing up (Fig. 4.1.4).
3. From a Pasteur pipet held in a horizontal position 1 to 2 inches above the slide, place
3 drops of cell suspension, evenly spaced, onto the slide, moving successively toward
the frosted end. Drops should strike the tilted slide one-third of its width from the
elevated long edge (Fig. 4.1.4). The drops should burst on the water film and spread
out evenly as they strike.
Positioning and spacing of drops is critical. The goal is even dispersal of cells across the
entire surface of the slide. This contributes to consistent and uniform slide-drying, which
will optimize chromosome spreading. If discrete areas of cells are observed at the drop
sites, surrounded by areas with few cells, the slide should be held at a lower angle (i.e.,
<30) when the drops are applied. Applying drops in sequence toward the frosted end allows
excess water and fixative to flood onto the frosted end without pooling. Placing drops closer
to the elevated edge of the angled slide helps to disperse the cells in suspension uniformly
across the width of the slide. If amount of cell pellet is limited, slides should be made using
one or two drops.
4. Position the slide with one long edge parallel to the bench top and blot the lower long
edge to draw off excess fixative. Tilt the slide at a 30 angle as in step 2 and flood
with fresh 3:1 methanol/acetic acid fixative, dropwise, using a Pasteur pipet. Start at
the elevated corner of the nonfrosted end and move toward the frosted end, placing
drops on the upper edge of the slide.
This will uniformly displace any remaining water and allow the slide to dry evenly.
It is critical to flood the slide toward the frosted end so that excess fixative does not pool
Chromosome on the slide surface. As fixative is placed across the top of the slide, it will displace a front
Preparation of water and leave a uniform surface of fixative.
from Cultured
Peripheral
Blood Cells 5. Position the slide again so that one long edge is parallel to the bench top, blot the

4.1.8
Current Protocols in Human Genetics
 A
fixed cell suspension

1 2 3
1
3 ~30

apply fixative

~30

Figure 4.1.4 Chromosome slide preparation: (A) After blotting the long edge of the slide to obtain
a thin uniform layer of water, the slide is tilted to 30 and 3 separate drops of fixed cell suspension
are applied starting away from and proceeding toward the frosted end. This sequence allows excess
fixative and water to flood onto the frosted end without pooling on the slide. Application of the drops
1 of the distance from the top of the slide (indicated by Xs) counteracts the downhill dispersal
3
tendency of cells on the slide and promotes even dispersal across the slide width. (B) After
application of the cell suspension, the slide is flooded with fixative across the top edge, again
proceeding toward the frosted end. This displaces a front of remaining water across the slide and
onto the frosted end. It is important to avoid pooling of excess fluid on the surface of the slide, and
to obtain a thin, even film of fixative to ensure uniform drying.

lower long edge, and wipe off the back of the slide. Place so that the nonfrosted end
is elevated 30 with respect to the frosted end, with the cell side facing up. Air dry.
Correct drying, as indicated by chromosome spreading and contrast under phase micros-
copy, must be monitored on a slide-by-slide basis. It is a function of surface tension, which
in turn is related to relative humidity and ambient temperature. Simply placing a slide on
an angle to dry as suggested above may work well if ambient conditions are conducive (20
to 22C with a relative humidity of 50%). More often, additional manipulations to control
rate and duration of drying will be necessary to optimize quality of preparations. For a full
discussion of slide-making and drying, see Commentary.
6. Examine slides for good chromosome spreading and morphology by phase-contrast
microscopy (see Critical Parameters).
Storage of a slide preparation will depend on its intended use. Slides to be used for
fluorescence in situ hybridization (FISH) should be used within several weeks, but slides
for autoradiographic in situ hybridization will work well even after 1 year. Because the
chromosome preparations on slides are biodegradable, they should be stored in a clean,
dry container in the dark at room temperature (short-term storage), or frozen at 70C
(long-term storage).

Cytogenetics

4.1.9
Current Protocols in Human Genetics
 REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see SUPPLIERS APPENDIX.

Ethidium bromide solution, 1.25 mM stock

Dissolve 4.9 mg ethidium bromide in 10 ml Hanks balanced salt solution (HBSS;
APPENDIX 2). Transfer into a red-top Vacutainer (sterile, untreated tubes), wrap in foil
to protect from light, and store at 4C.
CAUTION: Ethidium bromide is hazardous; see APPENDIX 2A for guidelines on handling,
storage, and disposal.
Methotrexate, 10 M
Dissolve 0.5 mg methotrexate (amethopterin; Sigma) in 100 ml Hanks balanced salt
solution (HBSS; APPENDIX 2). Sterilize by filtration through a 0.22-m filter. Store
5-ml aliquots in red-top Vacutainers (untreated tubes) or any suitable sterile, un-
treated tube for 1 year at 20C. Store aliquot in use 1 month at 4C.
CAUTION: Methotrexate is hazardous; see APPENDIX 2A for guidelines on handling, storage,
and disposal.
Thymidine, 1 mM
Dissolve 25 mg thymidine (Sigma) in 100 ml Hanks balanced salt solution (HBSS;
APPENDIX 2). Sterilize by filtration through a 0.22-m filter. Aliquot 5 ml into red-top
Vacutainers (sterile untreated containers) and store 1 year at 20C. Store aliquot
in use 1 month at 4C.

COMMENTARY
Background Information
Mitosis is the only point at which the entire
genome may be viewed in an orderly and sys-
tematic manner. Culture and metaphase harvest
of peripheral blood has developed, therefore,
as a simple source of mitotic cells for an ever-
widening range of optically-based analytic
technologies.
Several developments in the late 1950s
and early 1960s led to the first explosion of
cytogenetics as a clinical science. The use of
colchicine to block mitosis (Levan, 1938) and
the accidental discovery of hypotonic shock
(Hsu, 1952) resulted in the correct determi-
nation of the human chromosome number
(Tijo and Levan, 1956) and to the identifica-
tion of trisomy 21 in Down syndrome as the
first known chromosomal abnormality (Leje-
une et al., 1959). Improvements in cell-cul-
ture technology and the observation that phy-
tohemagglutinin stimulates mitosis in leuko-
cytes (Nowell, 1960; Moorehead et al., 1960)
then provided a reliable source of mitotic
cells. This combination of improved culture
Chromosome and harvest techniques and consistent slide-
Preparation making methodology are the foundation
from Cultured u p o n w h ich cy tog en etics rests tod ay
Peripheral
Blood Cells (Hungerford, 1965).
As the understanding of chromosomes and
chromosomal disorders increased after the ad-
vent of banding techniques (UNIT 4.2), so, too,
did the need for more precise and detailed
structural analysis. Mitosis is a dynamic proc-
ess in which chromosomes become rapidly
more condensed. As this occurs, resolution is
lost by fusion of subbands into larger bands (see
Fig. 4.1.2). The odd late-prophase or prometa-
phase cell could occasionally be found in early
culture systems, but these cells, in which the
chromosomes are of maximum length, were not
abundant enough to facilitate reliable analysis.
In the late 1970s, the development of cell-cycle
synchronization yielded cultures enriched with
such early mitotic cells (Fig. 4.1.5A). Charac-
terization of the human karyotype at or above
the 800 band per haploid karyotype level
quickly followed (Yunis, 1976; Francke and
Oliver, 1978). Later, direct inhibition of chro-
mosome condensation by DNA-intercalating
chemicals, such as ethidium bromide and acti-
nomycin D, was introduced (Yunis, 1981;
Ikeuchi, 1984; Rnne, 1985). This may be used
alone or, more effectively, together with syn-
chronization to obtain preparations containing
longer chromosomes.
Chromosome preparations are used, in clin-

4.1.10
Current Protocols in Human Genetics
 A B

C D

E F
Figure 4.1.5 (A) A G-banded high resolution metaphase. Phase-contrast photomicrographs show
(B) low-power field (16 objective) with many polysegmented nuclei, small nuclei, and a lack of
metaphases associated with failure of PHA mitogenic stimulation; (C) poor spreading of sticky
chromosomes from two mitoses caused by excessive ethidium bromide exposure; (D) low-power
field showing an appropriate and moderate density of cells with two nicely spread metaphases; (E)
low-power field showing excessive cell density and resultant poor spreading of two metaphases;
and (F) category IV metaphase of poorly spread refractile chromosomes with encapsulation in
cellular debris due to improper regulation of the slide-drying regimen.

ical and research settings, for in situ hybridiza-
tion methods (UNIT 4.3) of detecting submicro-
scopic deletions (Ledbetter et al., 1992; Altherr
et al., 1991). They are also used for gene map-
ping by in situ hybridization with unique se-
quence probes (Harper and Saunders, 1981;
Donlon, 1986). Fluorescence in situ hybridiza-
tion using centromere-specific alpha satellite
probes can identify the centromeric origin of
rearranged or marker chromosomes. Similarly,
chromosome paint probescollections of
clones from chromosome-specific libraries
have become a tool for identifying cryptic chro-
mosomal translocations in otherwise normal
karyotypes. Paint probes also can be used to
measure induced chromosome damage as an Cytogenetics

4.1.11
Current Protocols in Human Genetics
 assay system for tumor radiosensitivity (Brown
et al., 1992).
Critical Parameters and
Troubleshooting
Peripheral blood culture, harvest, and slide
preparation are subunits of a single process.
This relationship is particularly evident in trou-
bleshooting (Table 4.1.1). Unless a gross failure
has occurred, such as a contaminated or clotted
culture, or a harvest with no visible pellet,
success in culturing or harvesting cannot be
determined until slides have been prepared.
Suboptimal metaphases resulting from prob-
lems in different areas of the process may be
very similar in appearance. For example, al-
though slide-making may be the obvious sus-
pect when chromosomes are poorly spread, the
problem may really lie with hypotonic treat-
ment or fixation. However, once mitotic cells
have been successfully isolated, slide-making
is the most likely source of problems, and
should be checked first.
It is critical to monitor the quality of the
metaphases before subsequent analytical tech-
niques are performed. Considerable time can
be wasted hybridizing or banding slides that are
poorly spread and therefore likely to be unin-
formative. This monitoring should be done on
a continual slide-by-slide basis using phase-
contrast microscopy.
Culture and harvest
A successful chromosome preparation must
start with a fresh peripheral blood sample in
good condition. Samples that are hemolyzed or
have been subjected to temperature extremes
will not grow well.
If a culture shows few or no mitotic cells,
few large blast-like nuclei, and many polyseg-
mented or small nuclei (Fig. 4.1.5B), the most
likely cause is insufficient or poor-quality mi-
togen. Starting over with the correct amount of
phytohemagglutinin (PHA), or with fresh
PHA, will correct the problem. Reconstituted
PHA is good for several weeks at 4C. It is
possible for a particular lot of PHA to be inef-
fective, in which case another lot or an alterna-
tive vendor (e.g., Burroughs Wellcome, HA-
15) may be necessary.
Slides with no mitotic cells may show many
large blast-like nuclei (a sign of good mitogenic
stimulation), but few or no polysegmented
Chromosome ones. This is most likely caused by failure to
Preparation achieve mitotic arrest during the harvest, and
from Cultured
Peripheral can be corrected by starting over with the ap-
Blood Cells propriate concentration of Colcemid, or with
4.1.12
fresh Colcemid. A final Colcemid concentra-
tion of 0.05 g/ml should be adequate. If arrest
failure seems likely, it is better to use fresh
Colcemid at the right concentration than to
increase the concentration, because too much
Colcemid will cause excessive chromosome
condensation. A failure of synchronization re-
lease can also lead to the absence of mitotic
cells. This is discussed in the section dealing
with high-resolution culture and harvest.
Complete cell culture medium typically has
four main components: a balanced salt solution,
growth factors (present in fetal bovine serum),
metabolic precursors (e.g., vitamins, amino ac-
ids, and glucose), and an antibiotic. Fetal bo-
vine serum (FBS) can vary considerably be-
tween manufacturers in ability to support
growth, and each lot should be pretested before
use. It should not be subjected to repeated
freeze-thaw cycles or to high temperatures
(e.g., heat inactivation), as either may inactivate
growth factors. It should be gently but com-
pletely mixed after thawing because it separates
when frozen. Culture medium must be able to
support cell proliferation for up to 4 days.
Supplemented medium older than 2 weeks or
with a pH 7.8 should be discarded (Barch,
1991). Excessively high pH is indicated by a
purplish color from the phenol red indicator
that is present in most media.
It is possible, although difficult, to contami-
nate antibiotic-treated short-term peripheral
blood cultures with bacteria although yeast
contamination can occur. Cultures should be
handled in a biosafety cabinet, and gloves
should be used to prevent contamination and as
a biosafety precaution.
Harvest failures are usually due to improper
hypotonic shock or cell fixation. Despite a
successful culture, if the salinity of the hypo-
tonic solution is too low, the mitotic cells will
burst, giving no resultant metaphases. Chromo-
some spreads that are knotted and encapsu-
lated in a visible envelope of debris may result
if the salinity of the hypotonic solution is nearly
isotonic, so that the cells are not adequately
swollen. Although this can also be a slide-mak-
ing problem (see below), the hypotonic shock
step should be repeated with fresh hypotonic
solution if there is any doubt.
Failures in fixation are usually due to the
use of either old fixative that is no longer an-
hydrous or of impure, low-grade methanol or
acetic acid. Fixative alters the cell membrane
by removing lipids and denaturing proteins,
leaving a fragile membrane remnant. It also
dehydrates the cell. If fixation is inadequate,
Current Protocols in Human Genetics
 Table 4.1.1 Troubleshooting Guide to Chromosome Preparation

Problem Possible cause Solution

General
No or few mitoses; cell Bad specimen
debris
Hemolysis Insufficient medium
Cultures contaminated
Few mitoses Poor gas/nutrient exchange
No mitoses; many Omission or failure of mitogen
polynucleated cells; few
T cell blasts
No mitoses; few Omission or failure of mitotic
polynucleated cells; arrest
many T cell blasts
Hypotonic solution too
hypoosmotic; mitoses lysed
Treatment too rough during
harvest; mitoses ruptured
Culture synchronization not
released
No mitoses; cell debris Fixative reagents are tainted
present
Obtain fresh specimen
Prepare fresh medium; start new
cultures; reduce amount of blood per
culture
Prepare fresh medium; obtain fresh
specimen; improve sterile technique
Tilt culture tubes slightly; mix cultures
daily
Add PHA at culture setup; use fresh
PHA
Add Colcemid before harvest; use fresh
Colcemid
Prepare hypotonic solution correctly
Resuspend and mix more gently
Release cultures with thymidine on
harvest day; make fresh thymidine;
omit synchronization
Use fresh, high-grade methanol and
acetic acid

High resolution
Few or no mitoses
Chromosomes not long
enough
Interval between release and
harvest not optimal; mitotic
wave missed
Interval between release and
harvest too long
Methotrexate block ineffective;
cultures asynchronous
Mitotic arrest too long
Ethidium bromide treatment
ineffective
Extend sequential harvest to determine
optimal harvest window
Shorten interval (usually not necessary
as it is already short)
Prepare fresh methotrexate
Maintain 10-min sequential harvest
cycle
Make fresh solution; increase
concentration

Slide-making
Poor spreading;
excessive contrast;
cytoplasm encapsulation
Poor spreading; cells
too dense
Slide-drying too fast
Cell suspension too concentrated
Slow drying; increase humidity by
breathing gently on slides; chill slides;
dry on wet paper towel
Dilute cell suspension with additional
fixative

continued

Cytogenetics

4.1.13
Current Protocols in Human Genetics
 Table 4.1.1 Troubleshooting Guide to Chromosome Preparation, continued
Problem Possible Cause
Poor spreading Hypotonic solution too saline;
cells not swelling
Incomplete fixation
Fixative reagents tainted
Poor spreading; Excessive ethidium bromide
chromosomes sticky exposure
Over-spreading; broken Slide-drying too slow
metaphases; low contrast
Hypotonic solution too
hypoosmotic; cells lysing
the membrane remnant/cytoskeletal structure
will not disintegrate completely, resulting in
encapsulated chromosomes that spread poorly.
Inferior or tainted reagents can render an oth-
erwise successful culture useless, and the dam-
age cannot be undone by repeated fixation.
Fixative should always be prepared using high-
quality, glass-bottled methanol and acetic acid
and should be used fresh because of its tendency
to absorb water over time (Lawce and Brown,
1991).
High-resolution culture and harvest
Both the methotrexate block and the thymid-
ine release must be carried out effectively to
produce high-resolution chromosomes by the
alternate protocol described above. If the
methotrexate block is ineffective, the result will
be a reduced mitotic index with little variation
in chromosome length between the sequential
harvests of the alternate protocol, because the
cultures will be essentially asynchronous. A
similar failure can result from too high a con-
centration of methotrexate. Very high concen-
trations of methotrexate (103 M, final) will
induce an alternate biochemical pathway for
thymidine synthesis, and the cells will proceed
through S phase. Block failure may be con-
firmed by skipping thymidine release and ob-
serving whether any mitotic cells are obtained.
If the block is effective, there should be no
mitotic cells when thymidine release is elimi-
nated. When problems are encountered, fresh
reagents should be used. The final concentra-
Chromosome
tion of thymidine must be 105 M. DNA syn-
Preparation thesis can be blocked by adding too much
from Cultured thymidine (103 M).
Peripheral In this protocol, all of the cultures are re-
Blood Cells
4.1.14
Solution
Make hypotonic solution correctly
Refix or fix overnight at 4C
Use new, high-grade methanol and
acetic acid
Make fresh ethidium bromide; reduce
concentration; reduce exposure time;
omit ethidium bromide
Warm slide on arm or slide warmer;
wave slide more gently
Make fresh hypotonic solution
leased simultaneously and harvested sequen-
tially. Empirical data (C.D.B. and T.A.D., un-
pub. observ.) suggest that this procedure works
better than performing a sequential release and
simultaneous harvest, perhaps because of cell
cycle and blockage variations. Ethidium bro-
mide is useful in controlling the effect of these
variations and in increasing the yield of early
mitotic cells. Added 1 hr before mitotic arrest,
it causes few problems with the harvest, but can
interfere with slide-making. Sometimes it
makes the chromosomes sticky and prevents
them from spreading effectively (Fig. 4.1.5C).
This problem is usually sporadic, but if it be-
comes chronic then the final concentration of
ethidium bromide should be reduced or its use
eliminated.
Slide preparation
A foolish consistency may be as Emerson
said, the hobgoblin of little minds, but con-
sistency is the essence of good slide prepara-
tion. It is impossible to fine-tune this process if
there is no framework of technical consistency.
This does not mean that flexibility and creativ-
ity are not important in dealing with variations
in laboratory climate. However, consistency
within the same slide, between slides of the
same preparation, and between slides of differ-
ent preparations is required if subsequent ana-
lytical techniques are to be applied reliably.
Without a uniform technical approach, it is
impossible to make the type of adjustments
necessary to yield consistent results.
Cell density. The first critical parameter in
preparation of slides is density and dispersal of
cells on the slide. A moderate and uniform cell
density (Fig. 4.1.5D) is important for subse-
Current Protocols in Human Genetics
quent drying and analysis. Excessive numbers
of cells on the slide can make drying erratic and
difficult to control. Dense preparations will
cause spatial problems in chromosome spread-
ing (Fig. 4.1.5E). On the other hand, it will be
difficult to find mitotic cells in excessively thin
preparations. Therefore, the harvest pellet
should be resuspended for slide-making so that
the resulting suspension is slightly opaque and
milky. Thick cell suspensions are easily cor-
rected by further dilution whereas dilute cul-
tures must be centrifuged and resuspended in
less fixative. A uniform dispersal of cells across
the slide will help avoid drying variations be-
tween areas of different density that would lead
to variations in banding. Careful application of
cell suspension and fixative as described in the
support protocol is the best course.
Degree of spreading. Proper spreading of
chromosomes is the second major concern in
slide preparation. The goal is to achieve uni-
form dispersal of chromosomes within a dis-
crete area that represents the genetic content of
one cell, with straight chromosomes and as few
overlaps as possible. Overlapping (i.e., poor
spreading) can be due to inadequate cell swel-
ling during hypotonic treatment or to poor tech-
nique (e.g., vigorous pipetting may lyse the
more swollen cells that may have otherwise
given the best spreads). Long, high-resolution
chromosomes will naturally show more over-
laps than shorter ones. There is no single correct
measure of spreading, and acceptable numbers
of overlaps will depend on the desired applica-
tion. In general, over-spread or broken meta-
phases will prove difficult to analyze. Under-
spread, tangled metaphases may prevent analy-
sis of the complete cell and obscure analysis of
specific chromosomal segments (Fig. 4.1.5E
and F).
Insight into the mechanics of cell spreading
can be gained by observing the slide-drying
process under phase-contrast microscopy. As
the cell suspension begins to dry, the metaphase
cell settles, contacting and adhering to the glass
slide. What happens next is variously inter-
preted as a rupturing of the membrane or an
attenuated flattening of the still-intact mem-
brane onto the slide. The best interpretation
may be that as the fixative evaporates and the
cell is no longer supported in suspension, the
membrane remnant (partly depleted of lipids
and composed of denatured proteins), col-
lapses, leaving the chromosomes to settle and
spread on the slide. If the disintegration is not
uniformi.e., the envelope ruptures in one
placechromosomes will spill out unevenly
Current Protocols in Human Genetics
and some may be lost, yielding a broken
metaphase preparation. If the envelope disinte-
grates before the metaphase cell has begun to
settle on the slide, the chromosomes disperse
in suspension and the metaphase is lost.
Chromosome morphology. The third major
concern is the morphology of a chromosome
preparationflatness of chromosomes and
sharpness of chromosome margins. Morphol-
ogy is best assessed by phase-contrast micros-
copy and is a determinant in the success of
subsequent banding or hybridization analyses.
The best mitoses will appear evenly spread with
minimal chromosome overlaps, and will show
a uniform dark and crisp contrast across the
spread. Chromosome preparations may be
sorted by phase-contrast morphology into four
categories (Donlon, 1986). Category I chromo-
somes (Fig. 4.1.6A and B) are very low con-
trast, gray and fuzzy. They can be thought of
as flat and firmly attached to the slide. On the
other extreme are category IV chromosomes
(Fig. 4.1.6G and H) that have so much contrast
as to appear refractile. These mitoses can be
thought of as raised and poorly attached to the
slide. They will be hypersensitive to the trypsin
pretreatment used for G-banding, and will not
withstand harsh treatments used in many spe-
cial staining methods such as C-banding (UNIT
4.2), or the denaturation process used for in situ
hybridization (UNIT 4.3). This type of preparation
will improve with age (i.e., storing slides for
6 months before using) as the chromosomes
desiccate.
As a rule, categories II and III are the most
useful for subsequent analysis. Medium-con-
trast, less gray chromosomes with sharp-edge
definition constitute category II (Fig. 4.1.6C
and D). This type of preparation will provide
stable chromosomes for in situ hybridization.
Category III (Fig. 4.1.6E and F) chromosomes
are very dark with sharp definition, and are
ideal for G-banding. Technical and intercellular
variabilities preclude a slide from having all of
its mitoses in the same, or even two, categories.
However, careful technique will produce slides
with the majority of metaphases in the desired
category.
Although the critical parameters of success-
ful slide preparation are complex and ill-de-
fined, there are a few rules that add some ob-
jectivity to this artful process. In general,
slow drying will yield well-spread or over-
spread category I chromosomes. Rapid drying
will yield poorly-spread category IV chromo-
somes. In very bad category IV metaphases,
there may even be encapsulation of the chro- Cytogenetics
4.1.15
 A B

C D

E F

G H

Figure 4.1.6 Sequential phase contrast and G-band photomicrographs of the different cate-
gories of metaphase. Category I metaphases (A, B) have a gray, fuzzy phase-contrast appear-
Chromosome ance and yield ragged, poor-quality G-bands. Categories II (C, D) and III (E, F) show increasingly
Preparation sharp and darkly defined phase contrast and are optimal for in situ hybridization and G-banding,
from Cultured respectively. Category IV metaphases (G, H) are refractile in appearance with excessive phase
Peripheral contrast. They yield G-bands with high contrast and a loss of subtle detail and are unable to
Blood Cells
withstand the in situ hybridization denaturation process unless aged.
4.1.16
Current Protocols in Human Genetics
mosomes within a visible envelope of debris
(Fig. 4.1.5F). High humidity contributes to
slow drying and low humidity to fast drying.
A hygrometer is recommended for monitor-
ing ambient humidity, and can be used as a
reference point at the start of each slide-making
session. It may be particularly useful to know
the actual humidity, because the above general
drying rules are not absolute. Category IV
spreads may be encountered under slow-drying
conditions and category I under rapid-drying
conditions, depending on the humidity. It is also
useful to visually monitor the drying process
by reflecting an overhead light source on the
drying surface. Drying should start at the edges
as the surface suspension thins and should pro-
ceed at a moderate and uniform pace toward the
center of the slide. Overly rapid drying will
produce drying marks on the slide and yield
category IV chromosomes. Slow, lingering dry-
ing will have the opposite effect. If the drying
surface breaks up into separate drying areas or
visible droplets, the surface was not uniform,
either because the suspension was not dropped
on a thin, even film of water (see support pro-
tocol, step 3), or the slide was not sufficiently
flooded with fixative (see support protocol, step
4) and carefully blotted before drying.
The drying process can be regulated in many
ways. The following suggestions are illustra-
tive, but there is no substitute for a creative and
empirical approach appropriate to a particular
laboratory environment. Slight changes in dry-
ing conditions can result in large changes in
spreading and contrast. With a relative humid-
ity of 50%, it is usually sufficient to prepare
the slide exactly as defined in this protocol. In
lower humidity, drying can be slowed by gently
breathing on the slide, or by holding it inverted
over a beaker of water momentarily before
setting it to dry. The added humidity on the slide
will slow the drying. Breathing too hard, how-
ever, may prematurely rupture the swollen
metaphases, spilling chromosomes across the
slide. In extremely low humidity, holding the
slide over a wet paper towel for a moment, or
even placing it directly on the towel, may be
helpful. The wet paper towel approach should
be used cautiously, however, because it slows
drying and diminishes the relationship between
spreading and contrast, producing category IV
chromosomes. Another common approach to
slow the drying times is to use chilled slides.
For higher humidities it may be necessary
to speed the drying process. This can be accom-
plished by warming the slide against a forearm
or a slide warmer, by waving the slide gently,
Current Protocols in Human Genetics
or by some combination thereof. The ideal
solution, beyond the means of most laborato-
ries, is a constant, environmentally controlled
room in which slide-making could be custo-
mized. Great improvements, however, may be
made by closely monitoring the laboratory mi-
croclimate for subtle differences in tempera-
ture, humidity, and air currentsto find that
sweet spot which is optimal for making
slides. An alternative that some laboratories
find useful is a temperature- and humidity-con-
trolled chamber.
Height for dropping. Cell suspensions are
dropped from a certain height onto slides. The
role that this height plays in chromosome
spreading is controversial. It is an article of faith
in some quarters that moderate to extreme
heights, measured in feet rather than inches, are
important for good spreading. This has not been
our experience or that of others (Lawce and
Brown, 1991; Lambson et al., 1986). Although
minor adjustments in the height from which the
suspension is dropped may be useful in some
cases, such as with difficult bone marrows or
tumors (UNITS 10.2 & 10.3), most spreading prob-
lems can be resolved by adjusting the drying
regimen.
Storage conditions. Cell suspensions may
be stored in fixative at 4C for an indefinite
length of time, although cell spreading and
contrast become more difficult to control after
several months. The shelf-life of a slide prepa-
ration will depend on its intended use. Slides
to be used for fluorescence in situ hybridization
(FISH) should be used within several weeks,
but those for autoradiographic in situ hybridi-
zation will work well even after 1 year in
storage. Because the chromosome preparations
on the slides are biodegradable, they should be
stored in a clean, dry container in the dark at
room temperature (for short-term storage), or
frozen at 70C (for long-term storage).
Consistency. Slide-making is truly the heart
and soul of good cytogenetics. It matters not a
bit that the best possible culture harvests are in
hand if the resulting slide preparations are un-
suitable for analysis. Nothing will facilitate
chromosome analysis more than the ability to
produce consistent, high-quality chromosome
spreads. Although it is something of an art form,
practice and a creative experimental approach
to troubleshooting will yield satisfactory re-
sults.
Anticipated Results
The success (or failure) of a peripheral blood
culture is often discussed abstractly in terms of Cytogenetics
4.1.17
 mitotic index, i.e., the number of mitotic cells
in a culture harvest as a percentage of all cells.
In practice, this index is seldom quantified, and
the cultures are judged subjectively based on
index and length of chromosomes. One or two
metaphases per low-power microscope field is
reasonable (see Fig. 4.1.5D). If the harvest is
done according to the basic protocol with the
optional cell-cycle synchronization, 50% of the
metaphases should contain chromosomes suf-
ficiently long to provide G-bands at 550 bands
per haploid karyotype (see APPENDIX 4B). With-
out the optional synchronization, the average
G-band number will be lower. For the alternate
high-resolution protocol the mitotic index
should be similar, but the average G-band num-
ber should be 750.
Time Considerations
Culture and harvest of metaphase or high-
resolution chromosomes is usually a 3-day
process, with culture initiation in the afternoon
of day 0, methotrexate synchronization (op-
tional for metaphase cultures) late on day 2, and
release and harvest in the morning and early
afternoon, respectively, of day 3. Four-day cul-
tures can be used to accommodate scheduling
concerns. These may be synchronized on day
3 and harvested on day 4. Longer cultures are
not recommended. Two-day metaphase cul-
tures generally work well in samples from new-
borns 1 month in age, although backup 3-day
cultures are advisable. One-day cultures can
even be performed for emergency chromosome
analysis of patients <1 day old by initiating and
synchronizing cultures on day 0 and harvesting
on day 1. These harvests take advantage of
circulating immature nucleated fetal erythro-
cytes and usually yield a very low mitotic index
and poor quality preparations suitable only for
diagnosis of numerical abnormalities (Tipton
et al., 1989). Three-day cultures work much
better with older patients, and 3- or 4-day cul-
tures are required for high-resolution prepara-
tions.
Slide preparations can be made on the day
of harvest, but it is best to allow cultures to sit
for 30 min after resuspending in fixative before
starting. It is highly recommended that slide
preparations be aged for further manipulation
such as banding and in situ hybridization be-
cause chromosomes are very fragile when
fresh. There are several approaches: oven-dry-
Chromosome ing overnight at 65C, 30 min at 90C, micro-
Preparation
wave 5 to 20 min, aging several days at room
from Cultured
Peripheral temperature, or freezing in liquid nitrogen for
Blood Cells
4.1.18
6 to 12 months. The preferred method will
depend on each investigators individual sched-
ule.
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Altherr, M. R., Bengtsson, U., Elder, F.F., Ledbetter,
D.H., Wasmuth, J.J., McDonald, M.E., Gusella,
J.F., and Greenberg, F. 1991. Molecular confir-
mation of Wolf-Hirschhorn syndrome with a
subtle translocation of chromosome 4. Am. J.
Hum. Genet. 49:1235-1242.
Barch, M.J. (ed.) 1991. The ACT Cytogenetics
Laboratory Manual, 2nd ed. Raven Press, New
York.
Barch, M.J., Lawce, H.J., and Arsham, M.S. 1991.
Peripheral Blood Culture. In The ACT Cytoge-
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prediction of human tumor radiosensitivity in
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detected by fluorescence in situ hybridization.
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Donlon, T.A. 1986. Practical approaches to in situ
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Francke, U., and Oliver, N. 1978. Quantitative
analysis of high resolution trypsin-Giemsa bands
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Genet. 45:137-165.
Harper, M.E., and Saunders, G.F. 1981. Localization
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Chromosoma 83:431-439.
Hsu, T.C. 1952. Mammalian chromosomes in vitro
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Hungerford, D.A. 1965. Leukocytes cultured from
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Ikeuchi, T. 1984. Inhibitory effect of ethidium bro-
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its application to high resolution chromosome
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Lambson, B., Mendelow, B., and Bernstein, R.
1986. Metaphase spreading in chromosome
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slide-making and chromosome elongation tech-
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Ledbetter, S.A., Kuwano, A., Dobyns, W.B., and
Ledbetter, D.H. 1992. Microdeletions of chro-
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Lejeune, J., Gautier, M., and Turpin, R. 1959. Etude
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Levan, A. 1938. The effect of colchicine on root
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Moorehead, P., Nowell, P., Mellman, W., Battips, D.,
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Yunis, J. 1976. High resolution of human chromo-
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Key References
Barch, M.J., 1991. See above.
Comprehensive treatment of the full range of cyto-
genetic technology, with an in-depth discussion of
chromosome harvest and slide preparation by the
Association of Cytogenetic Technologists.
Contributed by Charles D. Bangs
Stanford University Hospital
Stanford, California
Timothy A. Donlon
Kapiolani Medical Center
Honolulu, Hawaii
Cytogenetics
4.1.19