Mutagenesis vol.16 no.6 pp.455–459, 2001
Lysis of whole blood in vitro causes DNA strand breaks in human
lymphocytes
S.Narayanan2, M.R.O’Donovan1 and S.J.Duthie2
2Rowett
Research Institute, Greenburn Road, Bucksburn,
Aberdeen AB21 9SB and 1Safety Assessment, AstraZeneca R&D,
Bakewell Road, Loughborough LE11 5RH, UK
DNA damage in lymphocytes, as measured by alkaline
single cell gel electrophoresis (pH 12.7), is greatly increased
by the concurrent lysis of whole blood in both freshly
isolated samples and in PHA-stimulated cultures over a
period of 7 days. Further, there is a marked progressive
increase in DNA damage with time in PHA-stimulated
lymphocytes cultured in whole blood even when the
lymphocytes are separated before analysis; no such increase
is seen in lymphocytes cultured alone. This indicates that
there are components in whole blood that can cause DNA
damage in lymphocytes, with granulocytes and lysis of red
blood cells likely candidates. The DNA damage is greatly
reduced in granulocyte-depleted whole blood cultures, but
even in these significant increases are seen at later sampling
times. Consequently, careful sample preparation is of paramount importance if the Comet assay is to be successfully
used to assess DNA damage in human peripheral blood
lymphocytes. Further, the progressive increase in DNA
damage in whole blood cultures may influence other
methods using lymphocytes for population biomonitoring
and may be significant for in vitro genotoxicity testing.
Introduction
Human peripheral blood lymphocytes (PBL) have been used
to monitor environmentally induced genetic damage by a
variety of methods, including cytogenetic end-points such as
micronuclei, chromosome aberrations, sister chromatid
exchanges (Perera and Whyatt, 1994) and somatic mutation
(Cole and Skopek, 1994). These involve monitoring lymphocytes after various times in culture, either as a separated
mononuclear cell fraction or as whole blood, with or without
lectin stimulation, depending on the particular end-point. Most
commonly, T cells stimulated to divide by phytohaemagglutinin
(PHA) have been used. Despite extensive use of lymphocytes
in these systems, to our knowledge little work has addressed
how time in culture or the system employed (whole blood or
separated lymphocytes) influences DNA damage. Time in
culture and cell division may influence pre-existing levels of
damage or response to genotoxic insult, as quiescent lymphocytes are functionally excision repair-deficient due to low
endogenous deoxyribonucleoside levels (Green et al., 1994).
Findings such as this and anecdotal evidence that contamination
of mononuclear cell fractions with red blood cells increases
DNA damage estimated by the Comet assay led to the present
study. Single cell gel electrophoresis (SCGE), known as the
Comet assay, is a rapid, sensitive, reliable and inexpensive
technique for measuring DNA strand breaks in individual cells
2To
(McKelvey-Martin et al., 1993). Cells are embedded in low
melting point agarose on a microscope slide, lysed with
detergent and treated with high salt. This results in the
formation of nucleoids containing non-nucleosomal but still
supercoiled DNA. The slides are then exposed to alkaline
electrophoresis. Breaks in the DNA cause local relaxation of
the supercoiled genetic material, enabling loops of DNA to be
pulled towards the anode during electrophoresis. Comets are
visualized by fluorescence microscopy after staining with the
fluorescent DNA-binding dye 4⬘,6-diamidine-2-phenylindole
dihydrochloride (DAPI). The relative intensity of fluorescence
in the tail is proportional to the frequency of DNA strand
breaks (Olive et al., 1990). The Comet assay is frequently
used in the field of genetic toxicology for the evaluation of
in vitro and/or in vivo genotoxicity. A wide range of cells
(normal and transformed) have been used, including human,
animal and plant cells. In addition, the Comet assay has
been shown to provide useful information in environmental
biomonitoring, assessing genetic hazards of pollutants. The
range, sensitivity and specificity of the alkaline Comet assay
have been increased by including bacterial DNA repair
enzymes which detect altered bases (Collins et al., 1993;
Duthie and McMillan, 1997).
Materials and methods
Histopaque-1077, L-glutamine, penicillin G, sodium pyruvate and streptomycin
sulphate were obtained from Sigma (Poole, UK). Dutch modified RPMI 1640
medium was obtained from ICN Flow Laboratories (Irvine, UK). Fetal calf
serum (heat-inactivated) was obtained from Globepharm Ltd (Surrey, UK).
Low melting point (LMP) and high melting point (HMP) agarose, together
with Nunc sterile tissue culture plastics, were supplied by Gibco Life
Technologies Inc. (Paisley, UK). LymphoPrep was supplied by Nycomed UK
(Birmingham, UK). Murex Diagnostics Ltd (Dartford, UK) provided HA15
PHA. Recombinant interleukin (Aldesleukin, 18⫻106 IU) was purchased from
Euro-Cetus UK Ltd (Harefield, UK). DAPI was obtained from Boehringer
Mannheim (Lewes, UK). Frosted microscope slides were from Richardson
Supply Co. (London, UK). Dynabeads M-450 CD 15 were from Dynal Ltd
(Wirral, UK).
The effect of storage and red blood cell contamination on DNA strand
breakage in human lymphocytes
DNA damage: isolated lymphocytes versus lymphocytes in whole blood
Isolated human lymphocytes were prepared from a ‘finger prick’ sample from
eight volunteers as follows. Whole blood (30 µl) was mixed with 1 ml of RPMI
1640 medium supplemented with 10% fetal calf serum (FCS), underlayed with
100 µl Histopaque and centrifuged at 200 g for 3 min at 4°C. The lymphocytecontaining ‘buffy coat’ (100 µl) was washed once in phosphate-buffered
saline (PBS), pH 7.4, centrifuged as before and resuspended in LMP agarose
(85 µl) for Comet analysis. Lymphocytes in whole blood were obtained by
mixing 30 µl of whole blood with 1 ml of RPMI 1640 medium supplemented
with 10% FCS and centrifuging the sample for 3 min at 200 g at 4°C. The
pellet was washed once in PBS, pH 7.4, centrifuged as before and resuspended
in LMP agarose for Comet analysis (Duthie and McMillan, 1997).
The effect of storage on DNA strand breakage in human lymphocytes
Venous blood samples (2⫻10 ml) were collected from the same eight
volunteers. Fresh blood was immediately taken for Comet analysis as described
above. One vacutainer from each volunteer was stored in a polystyrene box
at either 4°C or room temperature. Comet analysis was carried out on blood
after 24 and 48 h storage.
whom correspondence should be addressed. Tel: ⫹44 1224 712751; Fax: ⫹44 1224 716629; Email: [email protected]
© UK Environmental Mutagen Society/Oxford University Press 2001
455
S.Narayanan, M.R.O’Donovan and S.J. Duthie
DNA damage in PHA-stimulated lymphocytes grown as a separated
mononuclear fraction, in granulocyte-depleted blood culture and in whole
blood cultures
Lymphocytes grown as a separated mononuclear fraction
Venous blood (1⫻10 ml) was collected from the same eight subjects.
Lymphocytes were immediately isolated from 5 ml of whole blood. The blood
was centrifuged at 1500 g for 15 min at 4°C. The ‘buffy coat’ was removed
and diluted 1:1 with RPMI medium before layering onto an equal volume of
LymphoPrep lymphocyte separation medium (specific gravity 1.077 ⫾ 0.001
g/ml) and centrifugation at 700 g for 30 min at 20°C. The lymphocytes were
removed to a fresh centrifuge tube, washed using medium and spun for a
further 15 min under the same conditions. The supernatant was decanted and
the cells resuspended in RPMI medium containing 10% FCS before being
counted using a Neubauer Improved Haemocytometer. Isolated lymphocytes
were resuspended at 1⫻105 cells/ml and stimulated to divide in medium
containing 100 µg/ml pyruvic acid, 2 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 20% (v/v) FCS, 100 U/ml interleukin and 1% PHA.
All cell cultures were maintained at 37°C in a humidified atmosphere of 95%
air/5% CO2. Comet analysis was performed on days 0, 1, 2, 3, 4 and 7.
Samples (1 ml) were spun at 200 g for 3 min at 4°C. The supernatant was
discarded and the pellet washed in 1 ml of PBS, centrifuged as before and
resuspended in LMP (85 µl) agarose for Comet analysis as described.
Lymphocytes grown in granulocyte-depleted whole blood cultures
Depleted whole blood cultures were prepared by removing granulocytes from
2.5 ml of venous blood sample using Dynabeads M-450 with a covalently
bound mouse anti-CD15 monoclonal antibody. Dynabeads were washed in
washing buffer (PBS with 2% FCS), separated on a Dynal magnetic particle
concentrator and resuspended in buffer. Dynabeads (12⫻107) were added to
2.5 ml of whole blood, previously diluted 1:1 with washing buffer, mixed
gently and incubated for 20 min at 4°C on a rolling mixer. After incubation,
the sample was placed in a Dynal magnetic particle concentrator to remove
Dynabead-bound granulocytes and the supernatant transferred to a fresh tube.
These samples were centrifuged at 700 g for 20 min, resuspended in 5 ml of
RPMI 1640 medium with 20% (v/v) FCS, 100 µg/ml pyruvic acid, 2 mM Lglutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 100 U/ml interleukin
and 1% PHA. The cell suspension was added to 20 ml of medium and grown
at 37°C in a humidified atmosphere of 95% air/5% CO2. Comet analysis was
carried out on days 0, 1, 2, 3, 4 and 7 following lymphocyte isolation from
1 ml of culture, i.e. underlaying the sample with 100 µl Histopaque,
centrifuging at 200 g for 3 min at 4°C, washing the pellet in 1 ml of PBS
and resuspending in LMP.
Lymphocytes grown in whole blood cultures
Cultures were established by mixing 2.5 ml of whole blood with 22.5 ml of
RPMI 1640 medium containing 20% (v/v) FCS, 100 µg/ml pyruvic acid,
2 mM L-glutamine, 100 µg/ml streptomycin and 100 U/ml penicillin. The
cells were stimulated to divide by adding interleukin (100 U/ml) and PHA
(1%) to the medium. Whole blood cultures from each volunteer were
maintained at 37°C in a humidified atmosphere of 95% air/5% CO2. Comet
analysis was carried out on days 0, 1, 2, 3, 4 and 7. Lymphocytes were
sampled in two different ways. (i) Blood culture samples (1 ml) were
removed from the flasks and spun at 200 g for 3 min at 4°C. The supernatant was discarded, the pellet washed in 1 ml of PBS, centrifuged and
resuspended in LMP agarose for Comet analysis. (ii) Lymphocytes were
isolated before Comet analysis from 1 ml of whole blood culture by underlaying
with 100 µl of Histopaque and centrifuging at 200 g for 3 min at 4°C. The
supernatant was discarded, the pellet washed in 1 ml of PBS and centrifuged
before resuspension in LMP agarose for Comet analysis.
DNA strand breakage measured using the Comet assay
Cells were suspended in 85 µl of 1% (w/v) LMP agarose in PBS, pH 7.4, at
37°C and immediately pipetted onto a frosted glass microscope slide precoated
with a layer of 1% (w/v) HMP agarose similarly prepared in PBS. The
agarose was allowed to set for 5–10 min at 4°C and the slide incubated in
lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA, NaOH to
pH 10.0 and 1% v/v Triton X-100) at 4°C for 1 h to remove cellular proteins.
After lysis, the slides were aligned in a 260 mm wide horizontal electrophoresis
tank containing buffer (1 mM Na2EDTA and 0.3 M NaOH, pH 12.7) for
40 min before electrophoresis at 25 V for 30 min (at an ambient temperature
of 4°C with the temperature of the running buffer not exceeding 15°C). The
slides were washed three times at 4°C for 5 min each with neutralizing buffer
(0.4 M Tris–HCl, pH 7.5) before staining with 20 µl of DAPI (5 µg/ml)
(Duthie and McMillan, 1997).
Quantitation of the Comet assay
Nucleoids were scored visually using a Zeiss Axioskop fluorescence microscope. One hundred comets from each slide (scored at random) were classified
456
Fig. 1. The effect of whole blood contamination on DNA breakage in
human lymphocytes. DNA damage was measured in isolated human
lymphocytes and in lymphocytes from whole blood. Results are means ⫾
SEM for n ⫽ 8. A significant increase (*P ⬍ 1.06e–10) in DNA strand
breakage was observed in whole blood lymphocytes compared with isolated
lymphocytes.
into one of five classes according to the relative intensity of fluorescence in
the tail and given a value of 0–4 (from undamaged, 0, to maximally damaged,
4). The total score for 100 comets can range from 0 (all undamaged) to 400
(all maximally damaged). This method of visual classification has been
extensively validated by comparison with comets selected using computerized
image analysis. Representative images of comet classes were analysed (Komet
3.0; Kinetic Imaging Ltd, Liverpool, UK) and the percentage of fluorescence
in the comet tail (representing the fraction of DNA in the tail) plotted against
the total score for 100 comets in each class. There is a clear linear relationship
(r ⫽ 0.987) between visual classification and the percentage of DNA measured
in the tail (Duthie and McMillan, 1997).
Flow cytometer analysis
The granulocyte-depleted whole blood cultures were analysed on a FACScan
(Becton-Dickinson, Oxford, UK) flow cytometer, enabling quantitation of
independent cell groups. Briefly, 1 ml of sample was added to 1.4 ml of 1⫻
lysing solution (FACS Lysing Solution; Becton Dickinson), mixed and
incubated for 10 min at room temperature in the dark. After incubation, the
sample was centrifuged (300 g for 5 min), the supernatant discarded,
the pellet resuspended in 1 ml of PBS and centrifuged (200 g for 5 min). The
supernatant was discarded and the pellet resuspended in 0.5% formaldehyde in
Sheath fluid (Becton Dickinson) for flow cytometer analysis. The lymphocyte,
monocyte and granulocyte populations in each sample were identified and
gated manually by visual inspection of the size (forward scatter) versus
complexity/granularity (side scatter) analyses.
Statistical analysis
Student’s t-test and ANOVA, together with Tukey’s honestly significant
difference test, were carried out as appropriate using SPSS 8.0 for Windows.
Results
The presence of whole blood induced a significant increase in
DNA damage (single-strand DNA breaks and/or alkali-labile
sites measured using the Comet assay) in lymphocytes
obtained from a finger prick sample. DNA damage was
elevated 10-fold in lymphocytes analysed in the presence of
red blood cells compared with isolated lymphocytes (Figure 1).
A significant increase in DNA damage was observed after
24 h storage at both 4°C and room temperature. A further
increase in DNA damage after 48 h was only observed after
storage at room temperature (Figure 2).
Whole blood lysis causes lymphocyte DNA damage
Fig. 2. The effect of storage on DNA damage in human lymphocytes.
Whole blood samples were stored either at 4°C or at room temperature
(RT). Lymphocyte DNA strand breakage was measured on fresh blood or
after 24 and 48 h storage. Results are means ⫾ SEM for n ⫽ 8. *P ⬍ 0.05,
significant differences in DNA damage between fresh blood and after 24 h
storage. **P ⬍ 0.05, significant differences in DNA breakage between
24 and 48 h storage.
Fig. 4. FACScan analyses of white blood cells in whole blood cultures (A)
and granulocyte-depleted blood cultures (B). Size (forward scatter) versus
complexity/granularity (side scatter) are shown. Populations identified are
lymphocytes (L) and granulocytes (G). 60% granulocytes and 30%
lymphocytes present in whole blood cultures (A) compared with 1.4%
granulocytes and 87% lymphocytes present in granulocyte-depleted blood
cultures (B).
Fig. 3. DNA damage in human lymphocytes grown as a separated
mononuclear fraction compared with damage in human lymphocytes
grown in whole blood cultures. Results are means ⫾ SEM for
n 艌 4. **P ⬍ 0.005, differences in DNA breakage in lymphocytes grown
in mononuclear cell culture (black bars) or lymphocytes isolated from whole
blood culture (hatched bars). *P ⬍ 0.005, differences in DNA breakage in
lymphocytes isolated from whole blood cultures (hatched bars) compared
with lymphocytes sampled from whole blood cultures (white bars).
This increase in DNA damage might have been due to
release of components from other cell types present in whole
blood during the lysis step of the Comet assay. To test this
hypothesis, PHA-stimulated lymphocytes were grown either
as a separate mononuclear fraction or as whole blood cultures.
Samples were obtained on days 0, 1, 2, 3, 4 and 7 for Comet
analysis. There was a significant increase (P ⬍ 0.0005)
in endogenous DNA strand breakage from day 0 between
lymphocytes grown in whole blood cultures, compared with
those grown as a mononuclear fraction or with those isolated
from a whole blood culture. There was no significant difference
between lymphocytes grown as a mononuclear fraction and
lymphocytes isolated from whole blood culture on days 0 and
1. However, there was a linear increase in DNA damage in
lymphocytes isolated from whole blood cultures after 2 days
(Figure 3).
Two cell types are potentially responsible for induction of
DNA damage in whole blood cultures; granulocytes and
erythrocytes. To determine which caused induction of DNA
damage, whole blood cultures were compared with whole blood
cultures depleted of granulocytes. To ensure that Dynabead
treatment in fact depleted granulocytes, the samples were
analysed on a FACScan flow cytometer. The granulocytes
comprised ~60% of total white cells in whole blood cultures, as
expected, compared with 1.4% in Dynabead-depleted cultures
(Figure 4). Samples were obtained on days 0, 1, 2, 3, 4 and 7
for Comet assay analyses. Lymphocyte DNA strand breakage
was significantly higher after culture for 1 day in whole blood
compared with lymphocytes grown in granulocyte-depleted
blood (Figure 5). Similarly, lymphocytes grown for 2 days
in granulocyte-depleted blood showed higher DNA damage
compared with those grown as a mononuclear fraction (Figure
5). There was no increase in DNA strand breakage in lymphocytes grown for 7 days in isolated culture. Conversely, DNA
damage increased linearly in lymphocytes from both whole
blood and granulocyte-depleted blood. Strand breakage was
significantly higher in lymphocytes from whole blood
(Figure 5).
Discussion
Our results clearly show that endogenous DNA strand breakage
(measured using the alkaline Comet assay) is significantly
increased in lymphocytes upon storage at either 4°C or room
temperature when compared with freshly isolated cells. This
457
S.Narayanan, M.R.O’Donovan and S.J. Duthie
Fig. 5. DNA damage in human lymphocytes grown in whole blood
compared with damage in human lymphocytes grown in granulocytedepleted blood or as a mononuclear fraction. Results are means ⫾ SEM for
n 艌 4. *P ⬍ 0.005, differences in DNA breakage in lymphocytes grown in
whole blood cultures (black bars) compared with lymphocytes sampled from
granulocyte-depleted blood cultures (hatched bars).**P ⬍ 0.005, differences
in DNA breakage in lymphocytes grown in mononuclear cell culture (white
bars) or lymphocytes from granulocyte-depleted whole blood.
is in contrast to a previous study using the Comet assay to
detect both background and chemically induced DNA damage
in lymphocytes that indicated that human blood samples could
be stored under these conditions for up to 4 days (Anderson
et al., 1997). Subtle differences in assay conditions may partly
explain this discrepancy. In the present study nucleoids were
allowed to unwind for 40 min, compared with 20 min in the
earlier study, which may allow greater relaxation of the DNA.
Similarly, slides were electrophoresed for 30 min, compared
with 20 min in the experiment by Anderson et al., presumably
resulting in different rates of migration through the agarose.
Finally, four times as many images per gel were analysed in
the work presented here, when compared with the previous
study reported by Anderson and co-workers (Anderson et al.,
1997). Our results suggest that human blood samples cannot
be stored for more than 24 h, even at 4°C. Rather, lymphocytes
should be used fresh or cryopreserved and stored at –196°C
for future analysis.
DNA damage in lymphocytes is greatly increased by the
concurrent lysis of whole blood and this level of damage is
very similar in freshly isolated samples and PHA-stimulated
cultures over a period of 7 days. Further, although the level
of damage declines slightly with time in PHA-stimulated
separated lymphocyte cultures, there is a marked progressive
increase with time in whole blood cultures even when the
lymphocytes are separated before analysis.
Although it would seem most likely that we are observing
DNA damage in lymphocytes caused by some factor(s) present
in whole blood, it is also possible that the differences may be
due to different cell populations being compared in the various
preparations used in these experiments. Whole blood contains
~5⫻109 red blood cells (RBC) and 4–10⫻106 white blood
cells (WBC) per ml; of the WBC, 25–40% are lymphocytes,
with neutrophils (the major granulocyte type) comprising the
majority of the rest. Mononuclear cell preparations separated
on Ficoll gradients should contain at least 90% lymphocytes
(Gurtoo et al., 1975), so it is possible that the differences in
458
response between separated lymphocytes and whole blood
seen on day 0 represent differences in DNA damage in
lymphocytes and the other WBC present. However, background
damage assessed by the Comet assay appears to be very
similar in freshly isolated lymphocytes, phagocytes and monocytes from normal individuals (Vijayalaxmi et al., 1993;
Hannon-Fletcher et al., 2000). The available evidence, therefore, indicates that different responses of the various WBC
types do not account for the findings in freshly isolated cells.
The WBC populations in PHA-stimulated whole blood
cultures are complex (O’Donovan et al., 1995b), with the
monocytes and granulocytes disappearing within the first 2
days; by ~4 days the majority of the living cells are probably
dividing T cells, as would be expected. The behaviour of T
cells with respect to DNA damage is also complex. Resting
T cells are effectively excision repair deficient through
having low intracellular deoxyribonucleotide pools, but this is
up-regulated after mitogen stimulation (Green et al., 1994).
Although complex, there is no evidence to suggest that
background levels of DNA damage increase 4–5 days after
PHA stimulation in comparison with the levels in unstimulated
cells (Green et al., 1992, 1994). This agrees with our findings
with separated lymphocytes and would appear to reflect DNA
damage in lymphocytes alone; immediately after preparation
the population comprises a mixture of T and B cells, but 7
days later ⬎99% are T cells (O’Donovan et al., 1995a). The
most obvious conclusion is that the increases in damage seen
with time of culture in whole blood reflects DNA damage in
lymphocytes caused by some factor(s) in whole blood, not
differences in the cell populations being examined.
There are two obvious candidates for the components in
whole blood that may be capable of causing the observed
DNA damage: neutrophils and lysis of RBCs. Both appear to
increase lymphocyte DNA strand breakage in this study.
Neutrophils, the most numerous WBC type in the peripheral
blood of healthy individuals, undergo an oxidative burst when
activated, releasing various reactive oxygen species, including
the superoxide anion and H2O2 into the extracellular environment. In addition, they have myeloperoxidase that can also
generate reactive nitrogen species (Byun et al., 1999).
Activated mouse neutrophils produce DNA damage in adjacent
cells in vitro which is longer lasting than that produced by
H2O2 alone (Schacter et al., 1988). Moreover, granulocytes
incubated at a 1:1 ratio with plasmocytoma cells produced
DNA single-strand breaks equivalent to a 1 min incubation
with 20 µmol/l H2O2 (Schacter et al., 1988). Similar results
were obtained when activated mouse neutrophils were cocultivated with B cells using unscheduled DNA synthesis as
the end-point (Janz and Schachter, 1993). Therefore, lysis of
neutrophils during comet preparation from whole blood or
prolonged incubation in whole blood could contribute to DNA
damage seen in lymphocytes, although their activation status
in these culture conditions has not been determined.
The overwhelming majority of blood cells are RBC, so even
relatively small amounts of DNA-damaging material released
from them could have marked effects on the WBC population.
Conversion of oxyhaemoglobin to methaemoglobin generates
superoxide and H2O2 and, consequently, RBCs have a range
of protective mechanisms, including superoxide dismutase,
catalase and glutathione peroxidase (Halliwell and Gutteridge,
1985). During lysis, either suddenly during preparation of
samples for Comet analysis or with time in culture, large
amounts of haemoglobin will be released into the medium.
Whole blood lysis causes lymphocyte DNA damage
Perhaps surprisingly, we could find no published work on
possible oxidative damage generated by haemolysis in vitro.
Overall, it appears that both granulocytes, particularly
neutrophils, and RBC lysis contribute to the increased DNA
damage seen in lymphocytes in the present study and that the
effects could be additive. Since granulocyte numbers are
known to decline rapidly with time in whole blood culture, it
is possible that they contribute more to the DNA damage seen
during the first few days, with progressive red blood cell lysis
responsible for the increases at later sampling times. Platelets,
the third major cellular component of blood, are not likely to
contribute to the DNA damage, since it has been reported that
they can act as scavengers of neutrophil-derived oxidants
(Dallegri et al., 1989) and that this may represent a natural
defence mechanism against neutrophil-mediated oxidative
stress.
In conclusion, the findings in the present study confirm
the anecdotal evidence that contamination of lymphocyte
preparations with whole blood significantly affects the levels
of DNA damage as measured by the Comet assay. In practice,
this means that lymphocytes must be prepared extremely
carefully if the Comet assay is to be used as an accurate tool
for human population biomonitoring. Further, methods for
measuring DNA damage in human peripheral blood lymphocytes such as cytogenetics and assays for point mutations may
be significantly influenced by the culture conditions employed
before the end-points are measured. In this context, it has been
reported that both chromosome aberrations (Ivanov et al.,
1973) and micronucleus frequency (Lee et al., 1999) are
increased in lymphocytes in heparinized blood samples after
3 and 5 days storage, respectively. Finally, for in vitro tests
for genotoxicity the possibility must exist that agents
causing cell lysis may cause DNA damage through indirect
mechanisms.
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Received on April 10, 2001; accepted on June 14, 2001
Acknowledgement
This work was funded by SERAD.
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