&APTER 1
Mitotic Metaphase Chromosome
Preparation from Peripheral
Blood for High Resolution
George Spowart
1. Introduction
The advent of chromosome banding techniques some 20 years ago
(1,2) allowed the unequivocal identification of every chromosome in
the human metaphase and provided a mapping scheme along each chro-
mosome. Subsequently, a great deal of research has centered on prepar-
ing longer chromosomes with more bands visible. Chromosomes
condense as they move through mitosis, and adjacent bands close up and
appear to fuse. The earlier stages are longer with more bands recognized.
It is not always possible to define the mitotic stage of a particular cell.
International standards have been agreed for various numbers of bands
in the haploid set. Thus we have 400-, 550-, and 850-band sets (3). Other
workers report the use of even longer chromosomes (4,s). High-resolu-
tion banding has undoubted advantages in many fields. As well as allow-
ing greater accuracy in traditional karyotype analysis, there are many
reports of microdeletions and other abnormalities detected only on
extended chromosomes (6). Likewise, in situ hybridization and gene
localization techniques are taking advantage of the improved resolution.
The culture technique to prepare human chromosomes still follows
the basic scheme laid down by Hungerford (7). Lymphocytes from
peripheral blood are stimulated to divide in culture; cells are arrested
in mitosis, swollen with hypotonic solution, fixed in an acid-alcohol
fix, and spread on microscope slides by air-drying.
From Methods m Molecular B/ology, Vol. 29 Chromosome Analysis Protocols
Edlted by J R. Gosden Copynght 01994 Humana Press Inc , Totowa, NJ
2 Spowart
Published methods on the preparation of elongated chromosomes

are abundant. As with banding techniques, different laboratories have
preferences for particular methods and have developed their own varia-
tions. None of the methods is guaranteed to work with every speci-
men, being the nature of biological material. There is no doubt that it
is very difficult for one laboratory to reproduce exactly all the condi-
tions in another, and it is likely that published methods need some
experimentation to optimize them for local conditions. Methods of
preparation fall into three general categories. An individual protocol
may use one or more of these approaches.
1.1. Induction
of
Synchrony
Stimulated peripheral blood lymphocytes in culture grow and divide
asynchronously, and pass through the prometaphase and early metaphase
stages relatively rapidly. Cells are blocked around S-phase (8) and,
on release, will continue through mitosis in a wave of divisions, thus
enhancing the potential yield of early stages. Methotrexate (9) and excess
thymidine (10) are the most successful blocking agents. The timing of the
interval between release of the block and harvest is the critical stage
in the procedure and depends, in a complex way, on the various cul-
ture conditions, This is the main reason that many workers have found
it difficult to duplicate successfully published methods or to maintain a
high level of success with a particular scheme. To release the cells
from the block, the blocking agent is removed, or at least overcome,
and cells are encouraged to enter mitosis. The choice of release agent
may be determined by the banding technique to be used subsequently.
1.2. Use
of
Chemicals to Affect
the Condensation

of
the Chromosomes
Chromosomes progressively condense as the cell moves through
mitosis. A number of chemicals have been found to counter this (12-14).
Care must be taken to balance the reduction in contraction against
lowering of mitotic index and/or induction of chromosome aberrations.
1.3. Alteration
of
Arrest and/or Hypotonic
Treatments afZer Harvest (15-17)
Colcemid is used in most chromosome preparation techniques to
destroy spindle formation and arrest cells in metaphase. Wiley et al.
(17)
questioned the necessity for colcemid treatment, but most workers
Mitotic Metaphase Chromosome Preparation
3
continue to use it in a variety of concentrations and exposure times.
Hypotonic treatment with 0.075M KC1 still features in the majority
of protocols, but many other formulations have been advocated, some
with the specified aim of elongating chromosomes.
There arevarious reasons why a particular method will be preferred for a
line of research. However, in general, where time and material permit, it
will be good practice to run tandem methods on each specimen. I will
detail two protocols that have given good results.
2. Materials
Z.l.Methodl
2.1.1. Supplemented Culture Medium
This will usually be prepared in bulk and aliquoted to culture ves-
sels just before cultures are set up. The number of specimens to be pro-
cessed will determine batch size. A week’s supply is typical.

1. RPM1 1640 (Glbco, Galthersburg, MD), 340 mL.
2. Fetal bovine serum, 60 mL.
3. Phytohemagglutnun (HA15 Wellcome, Dartford, UK), 4 mL.
4. Penicillin and streptomycin (0.1 g and 100,000 U/mL) (Glaxo) mixed
solution, 0.4 mL.
Store at 4°C.
2.1.2. Blocking Agent
Methotrexate injection (Lederle #4587-24) is obtained as 25 mg/mL
(5
x
lo-%) solution. A working solution ( lc5m is prepared by diluting
20 pL with 9.980 mL sterile distilled water. This can be stored at 4°C
for several weeks. Methotrexate is a cytotoxic drug, and due care must be
taken in handling.
2.1.3. Release Agents
1. Thymidine (Srgma [St. Louis, MO] #T-9250) (10m3jV) is prepared by
dissolving 2.42 mg/mL rn sterile distilled water. Store at 4°C.
2. The alternative agent is 5bromo-2’-deoxyundine Bdu (Sigma #B-5002):
Working solution (10e2M) 1s prepared by dissolving 3 mg/rnL in distilled
water. Aliquots can be stored frozen for several months. Store vial in
use at 4°C. Care must be taken in handling this teratogen and mutagen.
2.1.4. Arresting Agent
Colcemid, 10 pg/rnL (Gibco). Store at 4°C.
4 Spowart
2.1.5. Hypotonic Solution
KC1 (0.075M) (1.4 g in 250 mL deionized water):
Make up fresh
for each
harvest, and heat
to 37°C.

2.1.6. Fix
Acetone-free methanol and glacial acetic acid are freshly mixed in
the proportion 3: 1.
2.2. Method 2
2.2.1. Supplemented Culture Medium
This will usually be prepared in bulk and aliquoted to culture ves-
sels just before cultures are set up. The number of specimens to be
processed will determine batch size. A week’s supply is typical.
1.
RPM1 1640 (Gibco), 340 rnL.
2. Fetal bovine serum, 60 mL.
3. Phytohemagglutmin (HA15 Wellcome), 4 mL.
4. Penicrllm and streptomycin (0.1 g and 100,000 U/mL) (Glaxo) mixed
solution, 0.4 mL
Store at 4°C.
2.2.2. Inducing Agent
Actinomycin D (Sigma #A- 1410) stock solution is prepared by
dissolving 10 mg in 1 mL dimethylsulfoxide. Small aliquots are stored
at -20°C. Working solution is 50 @n-J, made by diluting thawed stock
200-fold in distilled water. This can be stored at 4°C for up to 2 wk.
Actinomycin is poisonous, a known carcinogen and teratogen, and
due care must be taken to avoid all contact.
2.2.3. Arresting Agent
Colcemid (Gibco) 10 pg/mL. Store at 4°C.
2.2.4. Hypotonic Solution
KC1 (0.075M) 1.4 g in 250 mL deionized water: Make up fresh for
each harvest, and heat to 37°C.
2.2.5. Fix
Acetone-free methanol and glacial acetic acid are freshly mixed in
the proportion 3: 1.

Mitotic Metaphase Chromosome Preparation
5
3. Methods
Aseptic laboratory procedures must be observed to avoid micro-
bial contamination during the culture stages. All centrifugations are
carried out in centrifuge with swing-out buckets in the rotor.
3.1. Method 1
1. Dispense 9.5 mL of supplemented RPM1 1640 medium (see Notes l-4)
mto a sterrle culture vessel (see Note 5), and inoculate with 0.75 mL of
whole blood (see Note 6).
2. Incubate at 37°C for 72 h.
3. Inject the culture with 100 pL methotrexate solution (see Note 7), and
remcubate at 37°C.
4. After 17 h, carefully remove the supernatant above the cell layer, pref-
erably with a tube from a suction pump followmg the meniscus. Pipeting
can be used but 1s more laborious.
5. Flick the flask with the finger to distribute the cells, and resuspend in
9.5 mL supplemented medium.
6. Add 0.2 mL thymidine (see Note 8). Reincubate at 37°C for 3.75 h, or
add 0.2 mL 5-bromo-2’-deoxyuridine (see Note 8). Reincubate at 37°C
for 4.25 h.
7. Maintain the culture at 37’C while adding 60 pL colcemrd (see Note 9),
and incubate for a further 10 min.
8. Gently shake the flask, and transfer the culture to a conical-based
centrifuge tube.
9. Centrifuge at 15Og for 10 min.
10. Suck off the supernatant to around 3 mm above the cell pellet.
11. Flick the tube to distribute the cells and pipet m 10 mL of prewarmed
KC1 (see Note 10).
12. Incubate at 37°C for 10 min.

13. Centrifuge at 15Og for 10 min.
14. Three layers should be visible, a red cell pellet at the bottom, a slightly
opaque layer of white cells, and the supernatant. Suck off the superna-
tant to within 3 mm of the white cell layer.
15. Flick the tube to loosen the cells. Use a vortex mrxer to stir the cells
more thoroughly while carefully addmg a pipetful of frx dropwise to
the middle of the vortex (see Note 11). Add two more pipetfuls of fix,
and allow to stand for at least 30 mm.
16. Centrifuge at 15Og for 10 min, and remove most of the supernatant.
Flick tube to resuspend cells, and add 2 pipetfuls of fix.
6 Spowart
17. Centrifuge at 150g for 10 min, and remove most of the supernatant.
Flick tube to resuspend cells, and add 1 pipetful of fix.
18. Centrifuge at 150g for 10 mm, and remove supernatant to lust above
cell pellet. Flick tube to resuspend cells, and add fix to give about 0.5
mL suspension.
19. Mix the suspension gently with a pipet and place a drop on a clean
polished microscope slide (see Note 12). Allow to air-dry and examme
under microscope to check cell density, spreading of chromosomes, and
so forth. If cells are too densely packed, add more fix. If too sparse,
spin down and reduce volume. Different methods of spreading may have
to be adopted.
3.2. Method 2
1. Dispense 9.5 mL of supplemented RPM1 1640 medium (see Notes l-4)
into a sterile culture vessel (see Note 5), and maculate with 0.75 mL of
whole blood (see Note 6)
2. Incubate at 37OC for 68 h.
3. Inlect the culture with 100 pL actinomycm D solution (see Note 13),
followed by 60 FL colcemid solution (see Note 9), and reincubate at
37°C for 4 h.

4. Transfer to 15 mL conical-based centrifuge tube, and spm at 150g for
10 min.
5. Suck off the supernatant to around 3 mm above the cell pellet.
6. Flick the tube to distribute the cells, and pipet in 10 mL of prewarmed
KC1 (see Note 10).
7. Incubate at 37°C for 10 min.
8. Centrifuge at 150g for 10 min.
9. Three layers should be visible, a red cell pellet at the bottom, a slightly
opaque layer of white cells, and the supernatant. Suck off the superna-
tant to within 3 mm of the white cell layer.
10. Flick the tube to loosen the cells. Use a vortex mixer to stir the cells
more thoroughly while carefully adding a pipetful of fix dropwise (see
Note 11). Add two more pipetfuls of fix and allow to stand for at least
30 min.
11. Centrifuge at 150g for 10 mm and remove most of the supernatant.
Flick tube to resuspend cells, and add 2 pipetfuls of fix.
12. Centrifuge at 150g for 10 mm and remove most of the supernatant.
Flick tube to resuspend cells, and add 1 pipetful of fix.
13. Centrifuge at 150g for 10 min and remove supernatant to Just above the
cell pellet. Flick tube to resuspend cells, and add fix to give about 0.5 mL
suspension.
Mitotic Metaphase Chromosome Preparation 7
14. Mix the suspension gently with a pipet and place a drop on a clean
polished microscope slide (see Note 12). Allow to air-dry, and examine
under microscope to check cell density, spreading of chromosomes, and
so on. If cells are too dense, add more fix. If too sparse, spin down and
reduce volume. Different methods of spreading may have to be adopted.
4. Notes
1, Good results have been obtained with a number of culture media, includ-
ing RPM1 1640, RPM1 1603, TC 199, and McCoy’s 5A. I have chosen

the readily available RPM1 1640 (Gibco).
2. Published methods suggest a wide range of supplements. Most con-
sider bovine serum the appropriate source of necessary growth factors,
but pooled human serum and even artificial supplements have their advo-
cates. There is strong evidence that the serum chosen affects the timing
of the mitotic cycle, and since rt is difficult to maintain a source of
unchanging material, it must be anticipated that regular checks will have
to be kept to optimize timings, especially of the interval between release
and harvest 15% fetal bovine serum is chosen here.
3. Antibiotics are normally added to avoid microbial mfection. Pemcillm
and streptomycm are the usual choice. The relatively short culture time
means that mycoplasma infection is not a problem.
4. Phytohemagglutmm is unrivaled as mitogen to stimulate lymphocytes
to divide. Lyophiltzed HA15 (Wellcome) is reconstitued with distilled
water and added to the culture medium in the proportion 1: 100. With a
few hematological conditions, it may be necessary to use pokeweed
mitogen as well.
5. Heparinized peripheral venous blood is the most readily available and
convenient material to produce chromosome preparations. Some work-
ers prefer to enrich the proportion of leukocytes m the inoculum by
centrifugation and taking plasma and white cell layers along with some
of the red cell layer. I do not do this routinely, but if the medical history
of the donor suggests high red cell or low white cell count, it could be
advantageous. Specimens from patients on drug therapy or that have
taken several days to reach the laboratory often benefit from havmg
plasma replaced by pooled human serum, Blood from neonates nor-
mally contams a very high number of leukocytes, and a smaller mocu-
lum will suffice.
6. Several types of culture vessel give good results, and choice may be
determined by budget and availability. There is a complex relationship

among various dimenstons of culture vessel in whole blood culture. In
general, glass or plastic can be used, although it is safer to use plasticware
8
Spowart
that is designed for tissue culture. The ratio of cell volume of culture to
area of base of the vessel is important, and so is the volume of the gas
phase above the culture. Plastic or glass Universal contamers with a
base area 4-5 sq cm are excellent for the recommended 10 mL culture.
Tissue culture flasks with similar area of end wall can be used standmg
on end. If available specimen volume demands smaller culture, then 15
mL glass McCartney bottles give good results with 5-mL cultures usmg
0.5 mL blood. Plastic Universals with conical base are not suitable.
7. Methotrexate is the most widely used reagent in blocking cells to encour-
age synchronization to enrich the harvest of chromosomes in early
metaphase. Most reliable is Methotrexate injection (Lederle) equiva-
lent to 25 mg in 1 rnL. This ts diluted to a workmg solution of 10e5M
and used to give a final concentratron of 10e7M.
8. Cells are released from the S-phase block by removing the blockmg
agent and resuspending the cells in medium enriched in thymidine or
its analog 5-bromo-2’-deoxyuridine. Some published methods require
the blocking agent to be washed from the cells, but I prefer to minimize
the handling of the culture, which may result in cell loss or damage and
also requires more time and labor. The medium is stmply sucked from
above the cells, and the cell are resuspended m fresh medium with the
release agent. Thymidine is used as release agent for cultures that will
be banded to show G-bands, whereas bromodeoxyuridine incorpora-
tion is far better for staining of R-bands. Although different laborato-
ries may prefer one type of banding, most agree that it is better to have
preparations available from both for confirmatory analysis. I run two
sets of cultures, one to be released with thymidine and the other with

bromodeoxyuridine.
9. There is some debate over the role and efficacy of colcemid used to arrest
cells. However, there ts no doubt that if it is not used, then the mitotic
index drops dramatically, although the proportion of early stages is increased.
On balance, I prefer to use 60 pL of 10 pg/mL for a 10 mL culture.
10. The hypotonic stage of the harvest is crucial. Many formulations are
published, but the most popular is still 0.075M potassmm chloride. In
my experience, it is better if this solution is made up m deionrzed rather
than distilled water.
11. The first fixation stage is the most important. The red cells will fuse mto
insoluble clumps entrapping the lymphocytes if the cells are not vortexed
and the fix added dropwise to the middle of the vortex. Acetone-free
methanol may require to be filtered before use. Three parts methanol
and one part glacial acetic acid should be mixed in quantity required
just before use.
Mitotic Metaphase Chromosome Preparation
12. The spreading of cells on the slide depends on the size of the drop of
cell suspension, cell density, slide surface, ambient temperature, humidity,
and so on. If spreadmg is not satisfactory on a dry polished slide, the
following can be tried:
a. Breathing on the slide Just before placing the drop.
b. Suspending slide above an open 60°C water bath.
c. Chilling slide.
d. Reducing or Increasing size of drop.
e. Altering the height above the slide from which to drop the cells.
13. I agree with Wiley et al. (I 7) that actinomycin D and colcemld together
give a high mitotlc Index. Although actmomycm alone gives a higher
proportion of early stages, I prefer to have more cells of all stages to chose
from. I agree also that lower concentration of actinomycm is supenor.
References

1 Caspersson, T., Lomakka, G , and Zech, L (1971) The 24 fluorescence pat-
terns of the human metaphase chromosomes-dlstmguishmg characters and
varlablhty Hereditas 67,89-102
2. Sumner, A T., Evans, H. J , and Buckland, R. A. (1971) New technique for
dlstmguishing between human chromosomes. Nature New Biol 232,3 l-32
3. ISCN, (1985) An International System for Human Chromosome Nomencla-
ture (Harnden, D G. and Klinger, H P., eds.) S Karger, Base1
4 Yums, J J (198 1) Mid prophase human chromosomes; the attamment of 2000
bands Hum. Genet 56,295-298
5. Droum, R., Lemleux, N., and Richer, C -L. (1988) High-resolution R-banding
at the 1250-band level. II Schematic representation and nomenclature of human
RBG-banded chromosomes. Cytobios 56,425-439.
6. Schmzel, A. (1988) Microdeletion syndromes, balanced translocatlons, and
gene mapping. J Med Genet 25,454-462
7 Hungerford, D A. (1965) Leukocytes cultured from small inocula of whole
blood and the preparation of metaphase chromosomes by treatment with hypo-
tonic KC1 Stain Technol. 40,333-338.
8. Camargo, M. and Cervenka, J (1980) Pattern of chromosomal replication m synchro-
nised lymphocytes I. Evaluation of the methotrexate block Hum. Genet. 54,47-53
9. Yunis, J. J., (1976) High resolution of human chromosomes Science 191,
1268-1269.
10. Viegas-Peqmgnot, E and Dutrillaux, B. (1978) Une m&hode simple pour
obtemr des prophases et des promttaphases Ann. G&w?. 21, 122-125.
11 Schwartz S , and Palmer, C G. (1984) High-resolution chromosome analysis.
I. Apphcatlons and hmltations. Am J Med. Genet 19,291-299
12. Rybak, J., Tharapel, A., Robmett, S., Garcia, M., Ma&men, C , and Freeman,
M. (1982) A simple reproducible method for prometaphase chromosome analy-
sis. Hum Genet 60,328-333
10
Spowart

13 Matsubara, T. and Nakagone, Y. (1983) High-resolution banding by treating
cells with acridine orange before fixation. Cytogenet Cell Genet 35, 148-15 1
14 Ikeuchi, T. (1984) Inhibitory effect of ethidium bromide on mitotlc chromosome
condensation and its application to htgh-resolution chromosome banding
Cytogenet. Cell Genet. 38,56-61.
15 Bigger, T. R L and Savage, J. R. K. (1975) Mapping G-bands on human
prophase chromosomes Cytogenet Cell Genet 15, 112-121.
16 Ronne, M., Netlsen, K V , and Erlandsen, M (1979) Effect of controlled
colcemid exposure on human metaphase chromosome structure. Heredltus 91,
49-52.
17. Wiley, J. E , Sargent, L M., Inhorn, S. L., and Meisner, L. F. (1984) Compari-
son of prometaphase chromosome techniques wtth emphasis on the role of
colcemid In Vitro 20,937-941
CHAFFER
2
Chromosome Preparation
from Hematological Malignancies
Fiona M. Ross
1. Introduction
Hematological malignancies encompass a wide variety of diseases.
To a certain extent the same basic method can be used to prepare chro-
mosomes from all these diseases, largely because they all yield single-
cell cultures relatively easily. Nevertheless, the fact that the different
malignant cells have different properties is reflected in the differen-
tial success of their chromosome preparation; the acute myeloid leu-
kemias are now relatively well understood and, in general, give fairly
consistent results. The acute lymphoid leukemias still have major prob-
lems with the quality of the abnormal chromosomes, and some diseases,
such as Hodgkin’s disease, still do not have reliable methods to pro-
duce any abnormal metaphases.

The general standard of chromosome preparations from most
hematological malignancies has undoubtedly improved greatly dur-
ing the 1980s. It is not entirely clear, however, just why this improve-
ment has occurred. Certainly, many laboratories have found that the
use of synchronizing agents, such as methotrexate or FdU, improves
both the length of the chromosomes obtained and the quality of the sub-
sequent banding (1,2). However, this has not been a universal findmg,
and there is no other single technique available that can be proved to
have any significant effect. It seems probable that much of the improve-
ment is simply owing to accumulated experience and considerable
attention to detail.
From Methods fn Molecular Bology, Vol 29’ Chromosome Analysis Protocols
Edited by J. R Gosden Copyright 01994 Humana Press Inc., Totowa, NJ
11
12 Ross
The main problems with attempting to prepare chromosomes from
malignant tissue are the frequent low mitotic indices and the poor quality
of the chromosomes. A variety of conditioned media or potential
mitogens have been employed in an attempt to increase the mitotic
indices, but apart from the use of polyclonal B-cell mitogens, such as
TPA or EBV in B-cell CLL (3,4), nothing has been shown to have a
good enough effect to become generally accepted. Indeed, great care
must be taken when using any potential mitogen since several agents
can improve the mitotic index, but they do this by stimulating the
normal cells in preference to the abnormal cells (5).
An alternative approach to stimulating division is to remove cells
that are mcapable of dividing. Myeloproliferatrve disorders, particu-
larly CML, are characterized by the accumulation of large numbers
of mature neutrophils, cells that cannot divide under any circum-
stances. Thus, although these cells are part of the abnormal clone, it

makes sense to remove them before culturing the cells for chromo-
some analysis. Fortunately, a large reduction in neutrophil numbers
can be very simply achieved by standard lymphocyte separation tech-
niques. The quality of the chromosome preparations from such cul-
tures is often better than in conventional CML cultures, and it is thought
that this may be the result of the reduction in neutrophil enzymes
released into the culture by the dying cells.
The most commonly used tissue for the study of chromosomes in
hematological malignancies is the bone marrow, which is usually the
primary site of disease. However, peripheral blood can be treated as
if it were bone marrow in many leukemias, wherever there is a sig-
nificant proportion of malignant cells capable of spontaneous or stimu-
lated division present. The bone marrow is not the primary site of
disease in lymphomas, and is frequently not involved at all. Even
where there is lymphoma in the marrow, it is often localized to the
peritrabecular areas, and very few of the relevant cells will be found
in the aspirate. Lymph node, spleen, or other solid lymphoid deposits
give a much better yield of abnormal cells, at least in non-Hodgkin’s
lymphoma. The problems with these tissues tend to be more in the
logistics of getting them to the cytogenetics laboratory. The vast majority
of published cases are from single centers where there is good liaison
among the operating theater staff, the pathologists, and the cytoge-
neticists, so that very rapid processing is possible, I have had consid-
Hematological Cytogenetics 13
erable success, however, with samples sent in from outlying hospi-
tals. The viability of high-grade lymphomas does usually decrease
quite rapidly after removal from the body, so that the failure rate will
increase with time in transit, but there is little evidence for any such
effect in the low-grade lymphomas. Consistent results are not yet
appearing for Hodgkin’s disease, but it seems probable that such

samples will need very rapid processing if any abnormal mitoses are
to be found.
The abnormality rates for different leukemias, like the success rates,
vary. At one time, it was thought that improvements in techniques
would eventually lead to a 100% abnormality rate, at least in the acute
leukemias (6). It is now recognized, however, that in at least some
cases, all genetic changes in the malignant cells can occur at the sub-
microscopic level. The most notable example of this is in CML, where
the same molecular rearrangement can be achieved by a visible trans-
location between chromosomes 9 and 22 or a submicroscopic inser-
tion of part of the c-ABL gene from chromosome 9 into the BCR
gene on chromosome 22 (7). Whether similar events will be shown to
account for all the cases of acute leukemia where no abnormality can
be detected (presently around 20-30% in most centers [S]) or whether
there are also classes of disease where the relevant cells do not divide
under current culture conditions will have to await further advances
in our understanding of the molecular events in the leukemic process.
2. Materials
All hematological material should be considered potentially danger-
ous, and therefore, should be handled in sterile safety cabinets; gloves
and laboratory coats should be worn by the cytogeneticist. Sterile graduated
plastic pastets or Gilson automatic pipets with sterile tips can be used
in all situations in preference to needles.
1. Transport medium: Any medium containing heparin and serum can be
used if bone marrow or lymphoid tissue samples are to be sent through
the post. We use Leibovitz L-15 medium, because it retains a neutral
pH for longer than our standard culture medium. To each 500-mL bottle
of medium, add 100 mL serum FCS 30,000 IU penicillin + streptomy-
cm, 10 mL 200 IIN L-glutamine, and 6000 U preservative-free heparm.
The complete medium should be made up fresh every month and there-

after stored at 4OC. If only very short times are involved between col-
lection and processing of samples, a simple saline heparin solution is
14 Ross
adequate. This can be stored for long periods at 4°C. Peripheral blood
samples should be sent m lithium heparm tubes.
2. Culture medium: Although a variety of media have been used success-
fully to culture leukemic cells, the most popular medmm is RPM1 1640.
The levels of fetal calf serum supplements vary, but I find that 20%
is best since this allows some leeway for poorer batches of serum.
The medium should also be supplemented with 10 mM L-glutamine
and 5000 IU penicillm + streptomycin/ml. Make up the complete
medium as required, but use quantities that will ensure that it is all
used within 2 wk. Store at 4°C. Unseparated bone marrow and periph-
eral blood samples will do well in ungassed mcubators m this medmm.
If separated samples and lymph node tissue are being cultured m
ungassed mcubators, it is advisable to use the HEPES buffered version
of RPMI.
3. Culture tubes: The majority of laboratories use IO-mL culture volumes
m Universal tubes. I have found considerable benefit, however, from
moving down to 5-mL volumes and culturing in flat-sided plastic test
tubes. This gives improved gas exchange, which increases the mitotic
index, and it has the added advantage that more cultures can be set up
from small samples.
4. Mitotic arrest agent: colcemid at 10 ug/mL.
5. Synchronization and release agents: 10m5A4 FdU m PBS; 4 x lO-“M uri-
dine m PBS; 10m3M thymidme m PBS. All these solutions can be kept
for years at -20°C. Once thawed, store at 4”C, and use withm 1 mo.
Although the FdU and uridme are always used together, we have found
that they are more effective if stored separately.
6. Mitogens: TPA*-store frozen at 1 pg/mL in dimethyl sulfoxide. It is

helpful to store very small quantities m Eppendorf tubes to prevent exces-
sive thawing and refreezing. PHA-reagent grade. Store at 4’C after
reconstitution, and use within 2 wk.
7. Lymphocyte separation medium: Various commercial preparations are
available. The density should be 1.077 g/mL The method described m
Section 3. is for Lymphoprep. If any other preparation is used, follow
the manufacturer’s instructions for the ratio of separation medium to
sample and for optimum centrifuge speed.
8. Hypotomc solution: 0.075M potassium chloride. We prefer to make up
only small quantities and use within 2 d (See Note 2).
9. Fix: 3 parts methanol to 1 part glacial acetic acid. Fix should be made
up immediately before use.
*Potential carcmogen. Rinse all contamers with methanol
Hematological Cytogenetics 15
10. Wright’s stain: 2.5 g Wright’s stain in 1 L methanol. Stir for 1 h, and then
filter through a double thickness of Whatman no. 1 filter paper into a brown
bottle. Store for at least 2 wk before use. The bottle must be kept ttghtly
cappedbetweenuse; otherwise the staining time will become unpredictable.
11. Buffers: Wright’s buffer-0.06M Sorensen’s phosphate buffer, pH 6.8.
Add 0.06M disodium hydrogen orthophosphate to 0.06M potassium
dthydrogen orthophosphate to give requtred pH. The approximate quan-
tities will be 99 and 101 mL, respectively. (Ready prepared Sorensen’s
phosphate buffer is also available commercially.) Buffer for trypsm
staining is the same, but 10 times more dilute.
12. Destaining alcohols, (2 mm/alcohol):
a, 70% Ethanol.
b. 70% Ethanol.
c. 95% Ethanol containing 1% HCl.
d. Methanol. Slides need not be thoroughly dried between alcohols.
13. Trypsin: 5% Bacto-Trypsin in saline made up immediately before use.

14. Slides: Frosted end slides are easiest to label. Commercially precleaned
slides may be clean enough for direct use, but it is helpful to clean them
further either by rubbing with muslin (taking care not to transfer grease
from the hands to the muslin and thence to the slides) or by soaking in
acid alcohol (dilute HCl in ethanol).
15. Nigrosin: 0.45%. Once sterilized this will keep for prolonged periods
at room temperature.
16. Ammonium chloride: 1.5 rniV, sterile, stored at room temperature.
17. HzOz: 30% in tap water. Store at room temperature, and make up fresh
whenever it stops fizzing actively when put on the slides.
3. Method
The precise details of leukemic cytogenetic techniques tend to be labo-
ratory specific. I list here a general outline, but it will be necessary to
experiment with some of the steps to determine what is most effec-
tive under the given conditions. Certain steps, particularly the actual
slide making, may vary from day to day, because chromosome prepa-
ration is very sensitive to both ambient temperature and humidity.
3.1. Standard Bone Marrow Culture
3.1 .I. Counting and Setting Up
Bone Marrow Samples
The number of cells in a bone marrow sample may differ by three
orders of magnitude depending on the cellularity of the marrow, the
degree of fibrosis, the competence of the hematologist, and the propor-
16
Ross
tion of the sample required for other investigations. It is thus essential to
count the number of nucleated cells in the specimen if consistently
high success rates are to be achieved.
1. Mix a small quantity of marrow in transport medium (e.g., 0.1 mL)
with an equal quantity of 1% acetic acid containing a little methylene

blue. Count in a hemacytometer. If phase-contrast opttcs are available,
the methylene blue is unnecessary.
2. Meanwhile, spm down the marrow cells at 2OOg for 5 mm. Resuspend
m culture medium to give lo7 cells/ml.
3. Add 0.5 mL cell suspension to 4.5 mL culture medmm m a flat-sided
test tube for each culture required (i.e., final concentration lo6 cells/ml).
Incubate at 37°C. For the choice of cultures to set up, see Section 3.6.
3.1.2. Harvesting Standard Cultures
1. Add 0.05 mL colcemtd to the culture. Mix and remcubate at 37OC for
30 min.
2. Spin at 200g for 5 min.
3. Remove supernatant and resuspend in 8-10 mL hypotonic solution. Incu-
bate at 37°C for an appropriate length of time. Thts varies in different
laboratortes from the mmute or two that tt takes to resuspend all cul-
tures being harvested together up to 30 mm mcubatton. In general, leu-
kemic cells are likely to require a slightly longer time in hypotonic than
PHA-stimulated lymphocytes.
4. Spin at 200g for 5 min.
5. Resuspend in 8-10 mL fix, addmg the first 1 mL dropwise while agitat-
ing the suspension on a whirlimix. This step ts critical to successful
chromosome preparation.
6. Leave m refrigerator or freezer for a minimum of 1 h. It will often be
convenient to leave cultures m first fix overnight.
3.1.3. Slide Making
1. Change the ftx four times by spinning at 200g for 5 min each time.
2. At the final resuspension, add only enough ftx to make the suspension
slightly turbid.
3. Drop one or two drops of suspension onto a thoroughly clean slide from
about 2 cm above the slide. Allow to ax-dry.
4. If phase-contrast optics are available, check for suttable cell concentra-

tion and metaphase spreading under phase with a 10x lens. If this is not
possible, stain the slide for 30 s in 5% Getmsa, and examine with stan-
dard optics.
Hematological Cytogenetics 17
5. If necessary, adjust the cell concentration or method of making the slide
(see Note 1). When satisfied, make the requrred number of slides from
each culture. (We generally make 4 slides/culture to allow for prob-
lems with banding or for additional banding techniques. If the mitotrc
index is exceptronally low, more than four slides may be required.)
3.1.4. Banding
Two G-banding methods are described here. Wright’s banding has
two major advantages: It does not bloat the chromosomes as much as
trypsin, and therefore analysis of poorly spread metaphases is easier,
and poor quality banding from under- or overstaining can be rectified. It
is not always possible, however, to produce very sharp bands with good
contrast using Wright’s stain, and therefore it can be useful to have the
trypsin technique available. When this works well, it gives extremely
good definition, but the enzymatic treatment of the chromosomes
means that mistakes of timing in trypsin cannot be rectified.
3.1.4.1. G-BANDING
WITH WRIGHT’S STAIN
1. Place two slides horizontally on a rack over the sink.
2. Add 1 mL Wright’s stain to 3 mL buffer in a bilou. Mix rapidly with a
pastet, and pipet evenly onto the two slides.
3. Leave for 2-5 mm according to the batch of stain.
4. Rinse off the stain in gently running tap water for 5 s.
5. Dry rapidly in a warm air flow. Wright’s stain is water-soluble, so that
the drying must be rapid and consistent if a umform effect is to be
achieved.
6. Examme under a high dry lens (preferably 63x, but 40x is adequate with

practice). If the staining is underdone (chromosomes slightly bloated
and pale with only landmark bands visible), simply repeat the staining
procedure for an appropriate length of time. If rt is overdone (chromo-
somes dark and approachmg block staining), destam the slide by leaving it
m each of the destaining alcohols described m Section 2. Dry the slide
and stain it again for a shorter period.
7. Mount with DPX or Histomount.
3.1.4.2.
G-BANDING
WITH TRYFSIN
1. Pipet Hz02 onto a horizontal slide. Leave for 30 s to 2 min, depending
on the age of the slide (longer for younger slides), Meanwhile, prepare
the stain by adding 1 mL Leishman’s stam to 2 mL buffer m a Universal.
2. Wash peroxide off wtth buffer and blot dry.
18 Ross
3. Dip the slide m saline and then very briefly in trypsm.
4. Place the slide on a rack, and immediately flood with stain. Leave until
a goldrsh sheen covers the surface of the stain. Rinse off with buffer.
Dry and examine under a high dry lens. Understaunng can be corrected
by repeating the staining procedure. Overstaunng can be corrected by
further rmsmg with buffer.
5. Mount with DPX or Histomount.
3.1.5. Chromosome Analysis
Since it is usually necessary to analyze large numbers of cells to be
sure of detecting clones that form only a small proportion of the divid-
ing cells, it is not generally practicable to analyze fromphotokaryotypes.
With practice, it is possible to analyze most cases down the microscope,
the exceptions usually being those with very high chromosome counts
or very extensive rearrangement. Wherever possible, I would recom-
mend the construction of at least one hard copy karyotype from all

abnormal cases, but in many laboratories, time constraints mean that
this is only possible with the aid of an automatic karyotyper.
Care must be taken in the choice of cells to analyze from hema-
tological malignancies, since abnormal cells are often of poorer mor-
phology than their normal counterparts. In general, I recommend
scanning the slides very systematically and examining the first 30
metaphases that are found. In most cases, full analysis should be done
on at least 10 cells and preferably 15. The remaining 15 should be
counted and briefly examined for any obvious anomaly. The standard
of chromosome preparations should be such that even quite small unex-
pected abnormalities should be picked up while counting the cells in
this way. If this is not the case, then a higher proportion of the cells
must be fully analyzed. An abnormal clone is defined by ISCN (9) as
two cells with the same rearrangement or additional chromosome, or
three cells with loss of the same chromosome. Care needs to be taken
with the latter definition if many cells have been broken during prepa-
ration. Thirty cells are ridiculously few in terms of marrow turnover,
but the time involved in chromosome analysis usually precludes any-
thing more. Where patients are being followed up after therapy, how-
ever, when there was an abnormal clone found at diagnosis, it is
relatively quick just to scan 60-100 cells for that abnormality. Again
Hematological Cytogenetics 19
the quality of the banding should be such that all but the smallest
additional abnormality will also be detected.
The vast majority of cases can be adequately analyzed from G-banded
slides. Occasionally, more specialist staining techniques are required
in order to clarify particular rearrangements. Most staining techniques
for PHA-stimulated lymphocytes work in the same way on leukemic
chromosomes.
3.2. Alternative Cultures

Various modifications of the basic culture technique can help in
different situations. Here I list only those methods that I find most
useful for routine cultures in a busy diagnostic laboratory.
3.2.1. Synchronization
This may be attempted over any of the first three nights in culture.
The methods are identical except for the prior time in culture. If the
cells are to be blocked over the first night in culture, it tends to be
more successful if they have had 2-4 h to adjust to the culture medium
before the blocking agent is added. A recent paper (10) suggests that the
maximum number of abnormal metaphases are found in cultures har-
vested after 48 h. Thus, synchronization over the second night in culture
is probably the preferred option. In practice, the intervention of week-
ends often means that cultures have to be blocked over the first night.
1. Add 0.05 mL FdU + 0.05 mL uridine to the culture sometime
between 11
AM
and 4
PM.
Reincubate at 37OC
2. Add 0.05 mL thymidine 17-22 h later. Reincubate for 5 h 50 min.
3. Add 0.05 mL colcemid and reincubate for 10 mm.
4. Harvest as in Section 3.1.2. steps 2-6.
3.2.2. TPA Stimulation
1. Add 0.01 mL TPA to the culture at the time of settmg up.
2. Place in a box or wrap in silver foil to exclude light, and incubate at
37°C for either 72 or 120 h.
3. Harvest as in Section 3.1.2. steps l-6.
3.2.3. PHA Stimulation
1. Add 0.05 mL PHA to the culture at the time of setting up.
2. Incubate for 72 h.

3. Harvest as m Section 3.1.2. steps l-6.
20
Ross
3.2.4. Cell Separation
for High Neutrophil Count Samples
1, Pipet 10 mL Lymphoprep into a plastic Universal. Carefully layer 10 mL
marrow in medium on top of the lymphoprep.
2. Spin at 400g for 15 min.
3. Remove the rnterface layer into a test tube, and top up to 10 mL with m&urn.
4. Spin at 200g for 5 min.
5. Discard supematant, and resuspend in fresh medium. Spin again as before.
6. Resuspend in 5 mL culture medium. Count the number of cells in a
hemacytometer (see Section 3.1.1.).
7. Set up 5 x lo6 cells/culture, and treat exactly as unseparated cultures,
except that all resuspensions should be carried out by gentle flicking of
the tube with a finger rather than by whnhmixmg, since the cells tend
to be more fragile.
3.3. Leukemic Blood Samples
Many hematological malignancies will have significant numbers
of immature cells, normally only found in the bone marrow, in the periph-
eral circulation. If this is the case, they can be cultured in exactly the same
way as bone marrow cells. Although peripheral blood can be counted m a
hemacytometer, the vastly greater numbers of red cells compared to bone
marrow tend to cause problems. Full blood counts will nearly always
have been done by the hematology laboratory on the sample that is sent
for cytogenetic analysis; therefore, request that this information be sent
with the sample. If the white count is in the normal range (4-10
x
log/L),
add 0.4 mL/5 mL culture. Reduce the amount added if the count is higher.

If the white count is very low, it may be possible to obtain dividing cells,
but it is inadvisable to add more than 0.5 mL blood, since the increased
numbers of red cells cause problems with the fixation (see Note 6).
In CLL, there are vast numbers of relevant cells in the peripheral
blood, but these need to be stimulated to divide. I have had most consis-
tent success using TPA stimulation for B-CLL. If specialist studies of
CLL are contemplated, however, it may be helpful to use a variety of
polyclonal B-cell mitogens (3,4).
3.4. Lymph Nodes or Other Solid
Hematological Tissue
The basic techniques for dealing with solid lymphoid tissue are
very similar to those for blood and bone marrow once the cells have
been put into suspension culture. Lymph nodes should be placed in
Hematological Cytogenetics
21
medium containing serum as soon as possible after removal from the
body. This should be done in the pathology laboratory if the cytoge-
netics laboratory is located in a different hospital. In general, the appli-
cation of mitogens to lymph node cultures simply results in large
numbers of normal metaphases, and therefore, it is not worth setting
up such cultures. The exception to this is in well differentiated lympho-
cytic lymphoma, the solid tissue equivalent
of CLL, where TPA is
reasonably effective and the unstimulated mitotic index is often very low.
These
techniques can also be used for solid myeloid malignancies,
such as chloromas or spleen from patients with CML.
1. If a whole lymph node is received, cut through the sample with sterile
scissors. If the node is already cut, move straight to step 2.
2. Break up the node by macerating between two sterile defibrilating sticks

(see Note 7).
3. Add three drops of the cell suspension to three drops of rugrosin, and
perform a viable cell count using a hemocytometer.
4. Set up 5mL cultures similar to those in Section 3.1.1. with lo6 viable
cells/ml.
5. Harvest all lymph node cultures in the same way as marrows, except
that a whirlimrx should never be used. Do all resuspensrons by gentle
finger flicking.
6. Slide making is also the same as in Section 3.1.3., except that it may be
necessary to put the cells through one or two extra fix changes in order
to produce crisper chromosomes.
3.5. Pleural Effusions, As&tic Fluid,
and Other Exudates
These exudates may occur in many types of malignancy, and may
contain neoplastic cells or may simply be reactive. They often also
contain a lot of fat, dead cells, or otherwise undesirable material that
must be discarded before culture.
1, Spin down and resuspend in fresh medium two or three times until the
supematant appears clear.
2. Count the resulting suspension using nigrosin to obtain a viable cell
count and thereafter treat as lymph node tissue.
3.6. Choice
of
Cultures
In order to ensure success, more than one culture must be set up
wherever possible. With unlimited time and resources, it would prob-
ably be helpful to set up four or five cultures from each specimen, but
22
Table 1
Culture Prlorltles by Diagnosis

Ross
Diagnosis
Preferred
tissue Direct On FdU TPA PHA
ANLL, CMLa
MDS, MPD
ALL
B-CLL
T-CLL
NHL low-grade
NHL high-grade
HDd
MM
BM
BM
Blood
Blood
LN
LN
LN
BM
2 1
2 1 4 3 3
lb
1 1
1 2 3”
3 2
1
2 3
2 1 3

‘Blood often adequate m CML, particularly at dlagnosls.
bBoth 3 and 5 d
CPotential CLL-type lymphoma only.
dAdequate reliable methods not yet certam
in a busy routine laboratory, I can usually make do with 2 cultures/
sample (with a third set up, but not harvested unless there are prob-
lems with the other two). If there are enough cells available, there is
not usually any difficulty in deciding which cultures to set up, even
when the diagnosis is uncertain. The problems arise when there are
only enough cells for a single culture. In Table 1, I list my priorities
for the different diseases. Direct preps are not recommended for
ANLL, since the abnormal clone is often undetectable. This is thought
to be because the predominant dividing population in direct harvests
is erythroid, and these cells may not be part of the abnormal clone
(II). In contrast, a direct harvest is essential in samples whose cells
are likely to have poor viability in culture. Although synchronization
techniques work well in many disorders, such that they are my first
choice for the vast majority of specimens that I deal with, they are
not as good in ALL (I), where they tend to promote the normal cells
at the expense of the abnormal.
4. Notes
1. It is always worth putting more effort into the steps up to and including
making the slides (Sections 3.1 .l 3.1.3.). Once poor slides have been made,
nothing can rescue them. If the chromosomes appear fuzzy, it may help
to change the fix another two or three times before making more slides.
Hematological Cytogenetics
23
If the chromosomes do not spread adequately, check first that the slides
are clean enough; a drop of fixed matenal placed on a dry slide should
spread out absolutely evenly with a smooth edge. Beyond this, each lab

has its own methods of attempting to improve spreading. Some of these
include breathing on the shde immediately before dropping on the cell
suspension, breathing gently on the spreading drop, adding another drop
of fix before or after the drop of suspension, dropping from a greater
height above the slide, using slides fresh from the refrigerator, or using
warm slides. In childhood ALLs, which are notoriously difficult to
spread, there is considerable support for the edge-flaming techmque-
allowmg the fix to ignite by bringing the edge of the slide momentarily
in contact with a naked flame (13).
2. Some labs find that the water used to make up the hypotomc is critical;
deionized water works well, but distilled is disastrous. If distilled water
is a problem, the metaphases will fail to spread properly, and the qual-
ity will get worse with successive fix changes (Section 2. step 8).
3. In samples from
MPDs where there are too few cells to permit separa-
tion, it may be helpful to use a low dose of colcemid for a longer time,
e.g., 0.01 mL colcemid for 3-16 h. This technique may be useful in any
sample expected to have a low mitotic index. Because the chromosomes
can become excessively condensed under these conditions if the rate of
cell turnover 1s not as low as anticipated, it is advisable to use this tech-
nique on an extra culture rather than as a replacement for a standard
culture (Section 3.6).
4. If Wright’s stain does not produce enough contrast m the banding, a
brief pretreatment with H,O* as for trypsin banding may well help (Sec-
tion 3.1.4.).
5. If CLL samples arrive at a time that would be mconvenient for a har-
vest later, it is perfectly acceptable to set them up but leave them in the
refrigerator for up to 24 h before incubating. This may even improve
the mitotic index (Section 3.6.).
6. If the ratio of red cells to white cells is excesstvely high (e.g., blood

samples or marrow blood from pancytopenic patients), tt may be pos-
sible to improve the preparations by lysing the red cells with sterile
ammonium chloride (14) (Section 3.3.). Add ammonium chloride to
the marrow or blood sample (up to 5 mm) to make up to 14 mL. Stand
at room temperature for 30 min. Spin at 2OOg for 5 mm. Remove super-
natant and resuspend in fresh ammonium chloride for another 30 min.
Spur as before. Resuspend in culture medium, and set up as standard.
7. Macerating lymph nodes, spleens, and so forth, between’deftbrilating
sticks (Section 3.4.) produces a single cell suspension very easily if the
24 Ross
node has NHL involvement. If scalpels or scissors need to be used, the
diagnosis IS almost certainly not NHL. HD nodes can be quite difficult
and will often require fine chopping with scalpels.
Myelord malignan-
cies tend to grve firmer tissues, but they should stall break up relatively
easily without the use of scalpels.
6. Abbreviations
PHA, Phytohemagglutins; FdU, Fluorodeoxyuridine; TPA, 12-0-
Tetradecanoylphorbol- 13-acetate; EBV, Epstein-Barr virus; FCS, Fetal
calf serum; PBS, Phosphate buffered saline; CML, Chronic myeloid
leukemia; CLL, Chronic lymphocytic leukemia; ANLL, Acute
nonlymphocytic leukemia; ALL, Acute lymphocytic leukemia; MDS,
Myelodysplastic syndrome; MPD, Myeloproliferative disorder; NHL,
Non-Hodgkin’s lymphoma; HD, Hodgkin’s disease; MM, Multiple
myeloma.
References
1 Yunis, J. J (1981) New chromosome techmques in the study of human
neoplasia. Human Path01 12,540-549
2. Webber, L M. and Garson, 0. M (1983) Fluorodeoxyuridme synchronization
of bone marrow cultures. Cancer Genet. Cytogenet. 8,123-132.

3 Gahrton, G , Robert, K H., Friberg, K., Zech, L., and Bird, A. (1980) Nonran-
dom chromosomal aberrations m chronic lymphocytic leukaemta revealed by
polyclonal B-cell mttogen stimulation. Blood 56, 640-647
4 Morita, M., Minowada, J., and Sandberg, A. A. (1981) Chromosomes and cau-
sation of human cancer and leukaemia.
XLV
Chromosome patterns in stimu-
lated lymphocytes of chronic lymphocytic leukemia. Cancer Genet. Cytogenet
3,298-306
5. Sun, G , Koeffier, H P., Gale, R. P , Sparkes, R S., and Schreck, R. R. (1990)
Use of condtttoned media in cell culture can mask cytogenetic abnormalities
m acute leukaemia. Cancer Genet. Cyfogenet 46,107-l 13
6 Yums, J. J , Bloomfield, C D , and Ensrud, K. (1981) All patients with acute
nonlymphocytic leukaemia may have a chromosomal defect. New Engl. J Med
305135-139
7. Kurzrock, R , Gutterman, J. U , and Talpaz, M. (1988) Molecular genetics of
Philadelphia chromosome-positive leukemias. New Engl. J. Med. 319,990-998
8. Heim, S. and Mitelman, F. (1987) Cancer Cytogenetrcs. Ltss, New York
9. ISCN (1978) An international system for human cytogenetic nomenclature
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Foundation.
10 Li, Y S., Le Beau, M. M., Mmk, R., and Rowly, J D. (1991) The proportion
of abnormal karyotypes m acute leukaemia samples related to method of prepa-
ration. Cancer Genet Cytogenet. 52,92-100.
Hematological Cytogenetics 25
11. Keinanen, M., Knuutrla, S., Bloomfield, C. D., Elonen, E., and de la Chapelle,
A. (1986) The proportion of mitoses in different cell lineages changes during
short-term culture of normal human bone marrow. Blood 67, 1240-1243
12. Garipidou, V. and Seeker-Walker, L. M. (1991) The use of fluorodeoxyuridine
synchronization for cytogenetic mvestigatron of acute lymphobiasttc leukaemra

Cancer Genet. Cytogenet 52, 107-l 11
13 Williams, D. L., Harrrs, A , Williams, K J , Brosius, M J., and Lemonds, W
(1984) A direct bone marrow chromosome technique for acute lymphoblastic
leukaemia. Cancer Genet. Cytogenet. 13,239-257
14. Macera, M. J., Szabo, P , and Verma, R. S. (1989) A simple method for short
term culturmg of bone marrow and unstimulated blood from acute leukemras
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