RESEARCH Open Access
Mitochondrial DNA alterations of peripheral
lymphocytes in acute lymphoblastic leukemia
patients undergoing total body irradiation therapy
Quan Wen
1
, Yide Hu
1*
, Fuyun Ji
2
and Guisheng Qian
2
Abstract
Background: Mitochondrial DNA (mtDNA) alterations, including mtDNA copy nu mber and mtDNA 4977 bp
common deletion (CD), are key indicators of irradiation-induced damage. The relationship between total body
irradiation (TBI) treatment and mtDNA alterations in vivo, however, has not been postulated yet. The aim of this
study is to analyze mtDNA alterations in irradiated human peripheral lymphocytes from acute lymphoblastic
leukemia (ALL) patients as well as to take them as predictors for radiation toxicity.
Methods: Peripheral blood lymphocytes were isolated from 26 ALL patients 24 hours after TBI preconditioning (4.5
and 9 Gy, respectively). Extracted DNA was analyzed by real-time PCR method.
Results: Average 2.31 times mtDNA and 0.53 fold CD levels were observed after 4.5 Gy exposure compared to
their basal levels. 9 Gy TBI produced a greater response of both mtDNA and CD levels than 4.5 Gy. Significant
inverse correlation was found between mtDNA content and CD level at 4.5 and 9 Gy (P = 0.037 and 0.048).
Moreover, mtDNA content of lymphocytes without irradiation was found to be correlated to age.
Conclusions: mtDNA and CD content may be considered as predictive factors to radiation toxicity.
Keywords: mtDNA, 4977-bp Common deletion, Total body irradiation, Real-time-PCR, Acute lymphoblastic leukemia
Background
Breakage of cellular DNA following radiation is a dose
dependent phenomenon a nd occurs in both the nuclear
and extra-nuclear DNA. Thus, besides nuclear nDNA,
mitochondrial DNA (mtDNA) is equally affected as an
only extra-nuclear genome [1,2]. Numerous investigations
showed that mtDNA can be an easily av ailable target for
endogenous reactive oxygen species (ROS) and free radi-
cals ca used by ion izing radiation ( IR), which resulted in
mtDNA copy number alteration and mtDNA damage
(such as mutation and depletion) [3,4].
The mechanisms of cellular response to radiation with
regard to mtDNA alterations were mainly involved in the
following two ways. On one hand, mtDNA has few repair
mechanisms and continued mitochondrial function is pre-
served primarily due to its high copy number. One of
possible radio-protective m echanism is that enha nced
replication of mtDNA reduces the mutation frequency of
total mtDNA and delays the onset of lethal radiation
damage to the mitochondria [5,6]. This hypothesis has
been recently supported by Zhang et al with exhibiting
increased mtDNA copy number in gut and bone marrow
of total body irradiated rats [7]. On the other hand, IR
usually prompts cell apoptosis by displaying an accumula-
tion of large scale mtDNA deletions, especially the specific
4977 bp deletion, referred to as the “common deletion
(CD)" [8]. The site of CD is flanked by two13 bp direct
repeats (ACCTCCCTCACCA) at mtDNA nucleotide site
8470 and 13447 respectively, and easy to make deletion
for its unique formation mechanism [9]. Studies have
shown that CD can be as a sensitive marker of oxidativ e
damage to mtDNA [10-12]. Unfortunately, only few
experiments have evaluated the association between
CD and I R till now. For example, accumulation of CD
has been identifie d by qualitative PCR method on several
irradiated cell lines (such as human skin fibroblasts,
* Correspondence: huyide_mit@yahoo.com.cn
1
Third Department of Oncology, The second affiliated hospital, Third Military
Medical University, Chongqing 400037, China
Full list of author information is available at the end of the article
Wen et al. Radiation Oncology 2011, 6:133
http://www.ro-journal.com/content/6/1/133
© 2011 Wen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creative commons.org/licenses/by/2.0), whi ch permits unrestricted use, di stri bution, and reproduct ion in
any medium , provided the original work is properly cited.
glioblastoma and colon carcinoma lines) and primary lym-
phocytes [13-15]. Furthermore, CD was induced by IR in
human hepatoblastoma cell line performing on real-time
PCR with nonspecific dsDNA-binding dye SYBR Green.
However, their conclusions were largely controversial. The
inconsistency may be due, in part, to the use of non-quan-
titative PCR strategies. Additionally, none of these studies
have assayed mtDNA or CD level in peripheral blood lym-
phocytes (PBLs) after in vivo irradiation exposure for lack
of appropriate human beings radiation model.
In this study, we performed real-time PCR t echnique
with a specific fluorogenic TaqMan probe conjugated with
minor groove binder (MGB) groups, which is more sensi-
tive and appropriate than nonspecific dsDNA-binding dye
PCR methods previously used [16 ]. Besides, we ta ken the
acute lymphoblastic leukemia (ALL) patients undergoging
total body irradiation (TBI) precondionting as human
beings in vivo irradiation model. The advantage of using
this model lies in full view of in vivo microenvironment,
and without need for irradiating hea lthy i ndividuals. We
attempted to address t he mtDNA status in irradiated
human peripheral blood lymphocytes in vivo to elucidate
whether alterations in mtDNA can be li nked to exposure
to total body irradiation.
Materials and methods
Study participants
This study comprised peripheral blood (PB) samples from
26 high risked ALL patients undergoing TBI as pre-trans-
plantation treatment in their first complete remission
(CR1) at hematology department of our institution. The
diagnoses were. according to world healthy organization
(WHO) classification and high risk factors were measured
on Ribeca’ s report [17]. The patients age from 19 to 56
years with a mean of 39.4 ± 10.5. Of these, 10 are females
and 16 males. Besides, a total of 39 healthy volunteer indi-
viduals without IR were included in this study for compar-
ing the difference of basal mtDNA and CD levels between
ALL patients and normal donors before IR. The donors
age from 18 to 55 years with a mean of 37. 2 ± 9.4. 19 are
females and 20 males. All tested subjects signed an
informed consent to the use of blood samples in accor-
dance with the Declaration of Helsinki and with the
approval from our Institutional Review Board. The
amount of CD in skeletal muscle under physiological con-
ditions is relatively high (up to 1-2% from total mtDNA
content) [18]. Therefore, DNA isolated from skeletal mus-
cle of a 75-year-old male at autopsy was used as positive
control in the present study.
In vivo irradiation and peripheral blood lymphocyte
isolation
All patients were treated with two 4.5 Gy TBI sessions
daily using an Elekta SLi 8 MV linear accelerator (Elekta
Co., Stockholm, Sweden) set to deliver a dose rate of
4.5-4.9 cGy/min over two successive days. None of t he
patients had p rior exposure to any cytotoxic treatment
for at least 2 weeks before the start of radiotherapy. All
patients had 7 ml of PB collected prior to and 24 h fol-
lowing exposure for each radiation treatment. Besides,
39 healthy do nors had the sam e volume of PB collected
without ionizing radiation. Preparation of PBLs followed
standard methods, using human lymphocyte isolation
reagent (TBD Biological Technology Co., Tianjin,
China) for separation of mononuclear cells.
DNA extraction
DNA from lymphocytes in vivo and the skeletal muscle
was obtained with the TIANamp Genomic DNA Kit
(Tiangen Ltd, Beijing, China), and stored at -70°C until
further study.
Analysis of amount of mtDNA and CD by real-time PCR
TaqMan probes with conjugated MGB groups were per-
formed to ensure maximal specificity in real-time PCR
reaction. Nuclear DNA content was estimated by mea-
suring the human ß-actin gene. The hypervariable region
2 (HVR2) in the mitochondrial D-Loop was used to
represent the total amount of mtDNA since this region is
relatively conserved in Han Chinese [19]. The forward
primer (ß-actin: 5’-AGGACCCTGGATGTGACAGC-3’;
HVR2: 5’ -GCTTTCCACACAGACATCATAACAA- 3’;
CD: 5’ -CTTACACTATTCC TCATCACCCAACTAA
AAA-3’), reverse primer (ß-actin: 5’-TGGCATTGCCGA-
CAGGAT-3’;HVR2:5’-GTTTAAGTGCTGTGG CCA-
GAAG-3’ ;CD:5’ -GGAGTAGAAACCTGTGAGGAA
AGG-3’ ) and Taq Man MGB hybridization probes
(ß-actin: 5’ -AAAGACACCCACCTTGAT-3’ ;HVR2:5’ -
AATTTCCACCAAACCCC-3; CD: 5’-CATTGGCAGCC
TAGC ATT-3’ ) were synthesized by GeneCore Bio Tech-
nologies Co. Ltd., Shanghai, China. Dose-depende nt plas-
mid-constructed ß-actin, HVR2 and CD standards were
used in each run of real-time PCR. Of these, both plas-
mids containing the CD breakpoint and the HVR2 region
were kindly provided by Professor E. Kirches [20]. All
TaqMan reactions were carried out in 96-well plates on
an ABI 7500 Real-Time PCR instrument (Applied Biosys-
tems, Fost er City, CA, USA) using the R eal-Time PCR
Master Mix kit from Toyobo Co. (Osaka , Japan). Each
reaction was carried out in total volume of 25 μlwith
50 ng total DNA template, 300 nM each primer, and
100 nM TaqMan-MGB probe. After an initial denatura-
tion step at 94°C for t hree minutes, 40-45 PCR cycles of
15 s at 94°C, 20 s at 60°C, and 30 s at 72°C were per-
formed. Real-time PCR of all samples and standards were
carried out in quadruplicate. The data from a PCR
run were rejected if the correlation coefficient was less
than 0.98.
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Statistical analysis
All statistical computations were done using the SPSS
v15.0 (SPSS, Chicago, IL). Logarithmi c transformation of
data was essential for further parameter statistical analysis
since the original values of the mtDNA and CD copy
number in lymphocytes showed a nonnormal distribution.
Univariate analysis of variance and Stude nt-Newman-
Keuls post hoc tests were used to analyze the difference in
mtDNA and CD level with IR exposure. The relative
change of mtDNA and CD levels after different dosage
exposure were tested by nonparametric Friedman test.
The Pearson’s correlation test was used to explore associa-
tion between mtDNA and CD levels. The correlation
between mtDNA, CD level and gen der, age wa s analyzed
by the nonparametric Spearman’s rho correlation test and
the Pearson’s correlation test individually. P values < 0.05
are considered statistically significant. All reported
P values are two sided.
Results
Reliability and reproducibility of the TaqMan-MGB PCR
assay
The level of mtDNA and CD from lymphocytes was deter-
mined in a set of independent ex peri ments. First, a Taq-
Man reaction targeting the house keeping gene ß-actin
was used to measure the amount of genomic DNA present
in cells. A second TaqMan assay was designed to the
HVR2 region to quantitate the total amount of mitochon-
drial DNA. The mt DNA content was normalized to the
amount of genomic DNA in a lymphocyte and expressed
as a ratio of mtDNA molecules relative to total genomic
DNA molecules per cell. A third TaqMan assay targeted
the CD breakpoint and measured the abundance of the
CD in the samples. The level of CD was normalized using
mtDNA amount and was expressed as a ratio to the mito-
chondrial DNA amounts. In other words, the CD ratio
was expressed as a percentage of deleted mtDNA mole-
cules relative to total mtDNA molecules in per genomic
DNA molecules. These primer sets have been used exten-
sively for measuring the CD and mtDNA in tissues con-
taining low CD and give reliable results [20,21]. Figure 1
showed the standard curve for the mtDNA common dele-
tion and the CD amplification plots for the samples exam-
ined. It demonstrated that employed TaqMan assay was
sensitive enough to detect single molecule of CD and high
linearity was found (y =3E
-12e-0.6358x
) in the range of stan-
dard samples. CD levels in most of the samples were
detected between Ct 35 and 39. In all samples examined,
PCR products were amplified within the linear range of
assays (r
2
> 0.98). Positive control DNA from a 75 year
old male skeletal muscle contained about 0.729% CD ratio
and most of the lymphocytes samples contained from
0.003% to 0.04% CD ratio, consistent with other measure-
ments [18,22]. These results suggest that the TaqMan-
MGB PCR approach produces high sensitivity, and could
give reliable and corroborating data in our study.
Basal level of mtDNA content and CD ratio from healthy
donors and ALL patients
We first quantified the mtDNA co ntent (median = 197,
minimum = 65, maximum = 1124 in ALL; median = 398,
minimum = 39, maximum = 1283 in healthy donors) and
CD ratio (median = 0.0116%, minimum = 0.0019%, maxi-
mum = 0.085% in ALL; median = 0.0193%, m inimum =
0.0027%, maximum = 0.121%) per cell in PBLs from ALL
patients and healthy donors before irradiation to deter-
mine the distribution pattern. Since both variables did not
show normal distribution (P < 0.01, Kolmogorov-Smirnov
test), a logarithm of the mtDNA content and CD ratio was
made for normal distributions (see details in additional file
1, Figure S1). Data of mtDNA content and CD ratio after
logar ithm in the three study groups (0, 4.5 and 9 Gy TBI
respectively) were given in Table 1 as mean ± SD, median
and range. Mean ± SD values of initial mtDNA and CD
level in healthy donors cohort were at 2.507 ± 0.28 1 and
-3.683 ± 0.414. No statistically significant difference was
found for logarithm of basal mtDNA and CD level
between healthy donors and patients with ALL.
Changes of mtDNA content and CD ratio after TBI in
patients
Next, we investigated whether the irradiation dose has an
effect on mtDNA and CD level with lymphocytes. Signifi-
cant differences were found between IR status and
mtDNA alteration among lymphocytes 24 h after the irra-
diation (P = 0.038 for mtDNA content, 0.027 for CD ratio,
Univariate analysis of variance). Furthermore, Student
Newman-Keuls post-hoc tests were used to compare the
difference among the three groups. mtDNA content was
significantly increased in 4. 5 and 9 Gy irradiation groups
compared with 0 Gy group (mean value of mtDNA con-
tent 2.526 and 2.711 compared with 2.360), as well as CD
ratio reduced in 4.5 and 9 Gy irradiation groups compared
with 0 Gy group (mean value of CD ratio -4.148 and
-4.233 compared with -3.935).
Relative change of mtDNA and CD in lymphocytes from
each patient after TBI
The results above obtained from in vivo lymphocytes iso-
lated from patients suggest a correlation of increased
mtDNA and decreased CD level with dosage (4.5, 9 Gy)
irradiation in cohort study. To better examine the associa-
tion bet ween mtD NA alterations and IR in individuals,
relative changes of mtDNA and CD levels after different
dose TBI were compared for each patient. As shown in
Figure 2, the increase in mtNDA content was average 1.87
and 2.13 times individually after 4.5 and 9 Gy TBI ( P <
0.001, Friedman test). Meanwhile, decrease in CD was
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0.78 and 0.61 when 4.5, 9 vs. 0 Gy cohorts respectively (P
< 0.001, Friedman test). Moreover, significant difference
was observed in mtDNA copy (P = 0.041) and CD ratio (P
< 0.00 1) in each patient when comparing 9 Gy vs. 4.5 Gy
exposure. Besides, proportions of increase d mtDNA con-
tent in lymphocytes was found to be 80.8% (21 /26) and
decreased CD ratio to be 84.6% (22/26) after 4.5 Gy o f
TBI. Similar trends occurred aft er 9 Gy expo sure, where
84.6% of increased m tDNA content (2 2/26) and 88.5% of
decreased CD ratio (23/26) observed.
Relation between mtDNA and CD level after irradiation
No relation was found between the level of mtDNA and
CD at 0, 4.5 and 9 Gy, when they were analyzed as
Figure 1 TaqMan PCR assay for measuring the common mitochondrial deletion in DNA extracted from lymphocytes.Thetoppanel
shows the amplification plot for the standard curve whereas the bottom panel shows the amplification plot for the lymphocyte samples. The
level of the common mitochondrial deletion in the lymphocyte samples is within the linear range of the standard curve.
Wen et al. Radiation Oncology 2011, 6:133
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continuous variables (Pearson test used in all corre la-
tions). However, when CD values were segregated in
two p opulations (the lower third against the two upper
thirds of the distribution), a modest inverse correlation
was found reaching signi ficant level for mtDNA content
at different dosage (P = 0.037 for 4.5 Gy, 0.048 for 9 Gy,
shown in Figure 3). Besides, significant elevate d mtDNA
content was observed not in high but in low CD popula-
tion (P = 0.021) after 4.5 Gy TBI exposure.
Effect of age and gender
Finally, the correlations betwee n age, gender, mtDNA
and CD level were analyzed individually. No relationship
was found between mtDNA, CD level and gender. How-
ever, a significant positive effect of age was found for
basal logarithm mtDNA content in PBLs. A regression
analysis allowed quantification of the effect of age on
basal mtDNA content (regression coefficient = 0.0085 y-
1; r2 = 0.251; P = 0.011). The corresponding graphs are
presented in Figure 4 . These results suggest that older
people contained higher mtDNA content in general in
the age range of 19-56.
Discussion
In this paper, we described a sensitive and reliable real-
time PCR assay o f identifying the mtDNA and common
deletion levels. As expected, employ ed TaqMa n-MGB
probe was sensitive enough to det ect singl e molecule of
CD in our experiment. The sensitivity increased at least 5
fold compared with non specific SYBR Green dye real-
time PCR experiment [23]. Besides the improvement of
PCR method, we used human tissues and in vivo irradia-
tion model, whereas the cell s trains and ex vivo irradia-
tion model was exclusively used in other studies. As we
known, the ex vivo cultured cells is unlikely to reflect full
view of in vivo microenvironment. What is more, a lots
of apoptotic cell occurs after IR, which is hardly to isolate
from the whole cell population of strain, and will extre-
mely affect the accurate q uantification of mtDNA and
CD level for cell heterogeneity [23]. In contract, lympho-
cytes in vivo mostly consist of survival cells (> 95%) and
could avert the effect of apoptosis [24]. No doubt, it had
integrity advantage and is a big step u p compared to ex
vivo model. Based on t hese evidences above mentioned,
we can declare that direct analysis of lymphocytes
Table 1 Logarithm of mtDNA and CD levels in peripheral blood lymphocytes from patients before and after
irradiation
Group Log (mtDNA content) Log (CD ratio)
Median (range) Mean ± SD Median (range) Mean ± SD
0 Gy 2.294 (1.811~ 3.051) 2.360 ± 0. 320 -3.934 (-4.730 ~ -3.071) -3.935 ± 0.459
4.5 Gy 2.566 (1.950 ~ 3.069) 2.526 ± 0. 384 -4.069 (-4.857 ~ -3.063) -4.148 ± 0. 531
9 Gy 2.715 (1.956 ~ 3.186) 2.711 ± 0. 363 -4.437 (-4.952 ~ -3.255) -4.233 ± 0.527
P
a
0.038 0.027
Abbreviations: SD, standard devitation; CD, common deletion;
P value was demonstrated by univariate analysis of variance.
Figure 2 Relative change of mtDNA content (A) and CD ratio
(B) from patients’ PBLs (n = 26) after different dose of total
body irradiation therapy. Significant difference was observed in
relative mtDNA (*P = 0.041) and CD (*P < 0.001) change of every
patient when comparing 9 Gy vs. 4.5 Gy exposure. A circle
represents mean value of relative change level from each patient
undergoing irradiation compared to their basal levels. The lines
connect the mean values of relative change level from all cases.
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isolated from human bodies who received TBI would
greatly improve specificity and reliability. These techni-
que refinements take us closer to a methodology that is
likely to produce reliable and quantitative results.
The role of mtDNA content has been investigated in
relation to TBI th erapy for the first time in ALL patients.
The number of mtDNA copies was elevated in lympho-
cytes from above 80% of cases after TBI. Besides, mtDNA
content of irradiated PBLs elevated consisting with a dose
response. This phenomenon has been explained as a com-
pensatory replication of mtDNA to replace damaged
mtDNA
7
.
Our statistic al analysis showed induce d levels of CD
after TBI in PBLs, compared to some other reports that
IR-induced oxidative stress may cause increase of CD
ratio [13,14]. Considering that h igh deletion level of
mtDNA increases the susceptibility of human cell to
apoptosi s[25], the difference is most likely due to the fact
that IR exposure causes lymphocyt es differentiat e into
two major populations immediately: apoptotic population
usually containing relative high CD level a nd thus being
sensitive to apoptosis, while surviving population con-
taining relatively low CD level and more resistant to IR.
The cell source in other studies is li kely mixed w ith
many apoptotic cells, which may resulted in relatively
high CD level detection.
Here, we report a statistical inversely association
between these two predictive values (mtDNA content and
CD ratio) for radiation toxicity. That is to say , lowest
values of CD ratio were related to higher values of mtDNA
content, at the same radiation dose in our experiment. Cell
response to IR is individual, and the amount of initial
mtDNA and CD levels depend on each patient. The
mechanism behind the relationship remai ns unclear. One
possible reason is that lymphocytes containing lower level
of deleted mtDNA h ave stronger ability to replicate wild
mtDNA than cells with high CD level, in order to resist the
irradiation induced mitochondrial damage [26]. Besides,
abundant mtDNA replication only occurred in low CD
population after moderate dosage t reatmen t, which sug-
gests the stronger replication ability of low CD population
and a mass of mtDNA copy number production. However,
the strong replication ability was not shown in high CD
population after modest dosage treatment.
Cellular oxidative stress is thoug ht to play a role in the
aging process and may affect mtDNA replication. In the
present study, we found that mtDNA copy num ber is
increased with age by lineal regression in our limited
cohorts. Similar results has been described that individuals
after middle age may be attributed to the enhanced oxida-
tive stress than young adults [27], suggesting age factor
should be considered when measuring mtDNA content
from both nonirradiated and irradiated lymphocytes.
Conclusion
This study describes the development of a rapid, sensitive,
and p ractical real-time PCR method to quantify the
mtDNA copy number and common deletion in PBL sam-
ples. Our results suggest that radiation increased mtDNA
content and declined common deletion ratio in peripheral
lymphocytes of ALL patients, and an inverse association
was observed bet ween both parameters after irradiation,
Figure 3 Box plot shows an association between CD ratio and
mtDNA content. The lines connect the medians, the boxes cover
the 25
th
to 75
th
percentiles, and the minimal and maximal values
are shown by the ends of the bars. Patients with lower amount of
CD ratio suffered higher levels of mtDNA.
Figure 4 Regression analysi s of the relationship between age
and basal mtDNA content from patients’ lymphocytes (n = 26).
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which may be considered as predictive factors to radiation
toxicity.
Additional material
Additional file 1: Figure S1. The histograms show the frequency
distribution of logarithm of both mtDNA content (A) and CD ratio (B)
from patients (n = 26) after different dose of irradiation. Both population
showed normal distributions (P = 0.488 and P = 0.753 respectively,
Kolmogorov-Smirnov test).
List of abbreviations
mtDNA: mitochondrial DNA; CD: common deletion; PB: peripheral blood; PBLs:
peripheral blood lymphocytes; TBI: total body irradiation; IR: ionizing radiation;
HVR2: hypervariable region 2; nDNA: nuclear DNA; ALL: acute lymphoblastic
leukemia; MGB: minor groove binder; ROS: reactive oxygen species.
Acknowledgements
We would like to acknowledge Professor E. Kirches for his assistance with
plasmids donation. We are indebted to associate Professor Jieqiong Lei
(Mathematics department, College of biotechnology, TMMU University) for
statistical assistance, to the patients and donors who donated blood for this
study. This study was supported by grants from the Keystone Project of the
“Eleventh Five-year Plan” for Medical Science Development of PLA (No.06G068)
and the National Natural Science Foundation of China (No.30772144).
Author details
1
Third Department of Oncology, The second affiliated hospital, Third Military
Medical University, Chongqing 400037, China.
2
Institute of Human
Respiratory Disease, The second affiliated hospital, Third Military Medical
University, Chongqing 400037, China.
Authors’ contributions
QW and YH designed the study, FJ provided real-time PCR assay, QW
analyzed the data and written the paper, GQ contributed to revising the
paper. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 26 May 2011 Accepted: 6 October 2011
Published: 6 October 2011
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doi:10.1186/1748-717X-6-133
Cite this article as: Wen et al.: Mitochondrial DNA alterations of
peripheral lymphocytes in acute lymphoblastic leukemia patients
undergoing total body irradiation therapy. Radiation Oncology 2011 6:133.
Wen et al. Radiation Oncology 2011, 6:133
http://www.ro-journal.com/content/6/1/133
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