2233 nr. 3 / 2013 • vol 12 Revista Românæ de UrologieMicrochimerism in renal transplantation – single centre experience A. Moise1,2, R. D…. [613853]

2233 nr. 3 / 2013 • vol 12 Revista Românæ de UrologieMicrochimerism in renal transplantation
– single centre experience
A. Moise1,2, R. D. Sinescu2,3, I. Constantinescu1,2, I. Sinescu2,4
1 Centrul de Imunogeneticæ øi Virusologie, Institutul Clinic „Fundeni”, Bucureøti
2 Universitatea de Medicinæ øi Farmacie „Carol Davila”, Bucureøti
3 Compartimentul de Chirurgie Plasticæ – Microchirurgie Reconstructivæ,
Spitalul Universitar de Urgenflæ „Elias”, Bucureøti
4 Centrul de Uronefrologie øi Transplant Renal, Institutul Clinic „Fundeni”, Bucureøti
Correspondence: Dr. Ileana Constantinescu
Centrul de Imunogeneticæ øi Virusologie, Institutul Clinic Fundeni Øos. Fundeni 258, Sect. 2, BucureøtiTel./Fax: 021 318 48 08e-mail: [anonimizat]
Background: a part of the solid organ transplant recipients develops spontaneously so-called microchimerism , due
to the persistence of the passanger donor cells or due to the presence of donor specific DNA, in the host sys-
temicblood flow. Microchimerism in renal transplant recipients is still a subject of debate and the possibility of its
detection depends by the methods used.
Material and methods: we have selected fifty patients who have underwent a kidney transplant in 2011 in Fundeni
Clinical Institute. Follow-up period was on average 22 months. The study of the peripheral blood microchimerism
was done in the first week posttransplant (between days 3-5), then one month, three months, six months and one
year post-transplantation, using microsatellite analysis methods.
Results: microchimerism was detected in 32% of cases, most frequently in the first month post-transplant and in
recipients of living donor allograft. HLA compatibility and the presence of microchimerism are independent vari –
ables. The presence of the microchimerism in the first month posttransplantation is associated with a statistically sig-
nificant increased incidence of acute rejection (57% vs. 12%, p = 0.042 ), but it not significantly influence the long-term
allograft outcome.
Conclusions: In renal transplantation, the incidence of a real, sustained microchimerism, as a result of donor pas –
sanger cells, is very low, especially in recipients with cadaveric donor. Detection of microchimerism is rather the con –
sequence of the presence of donor specific DNA and can be considered as a potential noninvasive biomarker that
allows early diagnosis of allograft damage.
Key words: chimerism, microchimerism, rejection, renal transplantation
SSttuuddiiii cclliinniiccee

2244 Revista Românæ de Urologie nr. 3 / 2013 • vol 12SSttuuddiiii cclliinniiccee Background
The chimerism is defined as the presence in an indi-
vidual of at least two genetically different cell popula-
tions. This status can be acquired by blood transfusion,
pregnancy or after the transplant procedure, eitherstem cells or solid organ transplantation.
A part of the solid organ transplant recipients devel-
ops spontaneously so-called
microchimerism , due to
the persistence of the passanger donor cells (hematopoi-etic cells, dendritic cells) or due to the presence of donorspecific DNA, in the host systemic blood flow.
In contrast to stem cell transplantation chimerism,
in kidney transplantation the number of these pas-sanger cells is much lower (about 1% or less), but they
can circulate through the blood flow and lymphoid
organs and sometimes can engraft an organ. Donor-
specific DNA sources can be these passanger cells or
dameged endothelial or parenchymal allograft cells,
due to ischemia, surgical trauma or as a result of the
cytotoxicity during rejection episodes. Furthermore,immunosuppressive therapy by its cytotoxic effect
could be the cause of the presence of donor specific
genetic material in the recipient’s blood stream.
Microchimerism theory was first issued by Starzl
who suggested that an allograft tolerance required the
continued presence of donor cells in the peripheral
lymphoid organs (namely the nodes) of the recopient[1]. Since the 90s, Starzl et al. reported the presence of
donor-derived cells, as a chimera, in patients who have
underwent a kidney, liver or bowel transplant [2-4].
Microchimerism in peripheral blood of renal trans-
plant recipients is still a subject for debate. Lo et al.
were the first who have reported detection of Y chro –
mosome-specific DNA in plasma of women whoreceived kidney or liver allografts from male donors [5].
Subsequently, Zhang, from the same working group,
has identified the presence of donor-specific DNA in
urine of the kidney recipients. The study showed a pos-
itive correlation between the presence of urinarydonor-derived DNA and acute rejection, the incidence
being 88.9% in recipients with acute rejection and only
8.7% in recipients with stable renal function.
Meanwhile, donor-derived DNA was absent in the
urine of patients with renal dysfunction, other than
rejection [6,7]. Some studies have shown not only the
presence but also serum quantitative increase ofdonor-DNA during the rejection episodes and thendecrease as a result of favorable response to treatment[8-10]. Two other studies have shown the presence ofmicrochimerism in recipients who received renal allo -graft from living donors and microchimerism absence
in patients who had cadaveric donors [11,12].
The possibility of microchimerism detection heavi-
ly depends by the sensitivity of tests used and from this
point of view, polymerase chain reaction (PCR) is apowerful, reliable tool for chimerism study, which
allows detection of minor donor cell populations
among the recipient cells.
The main objectives of the study are:
Kmicrochimerism assessment at different moments
post kidney transplantation and its impact on short
and long-term allograft outcome;
Kto establish possible correlations between the pres-
ence of microchimerism and degree of HLA com-
patibility;
Kto establish possible correlations between the pres-
ence of microchimerism and donor type (living orcadaveric donor) and thus, implicitly, to study the
influence of cold ischemia time and preservationmethods of the transplanted kidney on the
microchimerism occurrence.
Material and method
We have selected fifty patients who have under-
went a kidney transplant between march to november
2011 in Centre for Urology, Dialysis and Renal Trans –
plantation, Fundeni Clinical Institute. All immuno-
logical pre and posttransplant tests were performed in
Centre for Immunogenetics and Virology, Fundeni
Clinical Institute.
Recipients at the second kidney transplant and
recipients who have received allograft from donors
who were evaluated in other immunogenetics centers,were excluded. Patients were divided into 2 groups:
group 1 – recipients from related/unrelated living
donors (n = 32), group 2 – recipients from cadaveric
donors (n = 18).
Pretransplant, an immunological and virological
assessment was performed in all recipients and
donors: serological screening for viral hepatitis, her-
pesviruses and HIV, HLA genotyping for A, B and DRB1
loci, cytotoxic antibodies screening and, if necessary,
identification, crossmatching).
The study of the peripheral blood microchimerism
was done in the first week posttransplant (between
days 3-5), then one month, three months, six monthsand one year post-transplantation.
Virological screening was performed using Access2
automated analyzer (Beckman Coulter, USA) based on

2255 nr. 3 / 2013 • vol 12 Revista Românæ de Urologie
SSttuuddiiii cclliinniiccee chemiluminescence technique. HLA genotyping was
performed by molecular biology methods – SSP
(Sequence Specific Primers) using AllSet™ Gold HLA
ABDR Low Res kits (Invitrogen, USA). Cytotoxic antibod-ies screening and identification were performed by ELISA
method using Quick-screen and B-screen reagents (GTIDiagnostics, USA) and by Luminex technique usingLABScreen® Mixed, LABScreen® PRA, LABScreen® Single
Antigen reagents (One Lambda, USA). Crossmatch test
was also made by ELISA using AMS – Antibody
Monitoring System kit (GTI Diagnostics, USA).
Microchimerism assay was done using microsatellite
analysis methods. Mentype® Chimera® PCR Amplifi-cation Kit (Biotype Diagnostic GmbH, Germany) allowssimultaneous amplification, in one PCR reaction tube, of
twelve highly polymorphic autosomal loci and the gen-
der-specific locus – Amelogenin. The detection limit is
approx. 200 pg genomic DNA, but internal validationsdemonstrated reliable results with < 0.1 ng DNA.
Results and discussion
Clinical and demographic characteristics of the stud-
ied groups
Male recipients were predominant in both groups,
but with the relatively equal male: female ratio, 2.2:1 in
group 1 vs 2.6:1 in group 2. Overall, the age distribu-
tion was consistent and almost similar in both genders
(Fig. 1). Average and median age, percentage of
patients on dialysis, as well as the time of hemo- orperitoneal dialysis until transplantation, were elevated
in group 2 compared to group 1 (Table 1).
Table 1: Characteristics of the studied groups
Parameter Group 1 (n=32) Group 2 (n=18)
Gender: F 10 (31%) 5 (28%)
M 22 (69%) 13 (72%)
Age (years) 19 – 62 34 – 57
Average 34 44,4
Median 32,5 44
Hemodialysis (HD):
No. 12 (37,5%) 11 (61%)
Time of HD (months) 3 – 40 (median=6,5) 3 – 96 (median=12)
Peritoneal dialysis (PD):
No. 3 (9,4%) 4 (22%)
Time of PD (months) 1 – 8 (median=5) 10 – 24 (median=17)
Cold ischemia time (hours) 0,33 – 2 5 – 13
Median 0,5 10
Warm ischemia time (min) 20 – 30 25 – 35
Median 29 30
Follow-up (months) 16 – 24 17 – 24
Median 21,5 23Fig. 1 Recipients distribution by gender and age
The majority of cases (62%) in group 1 had a 50%
HLA compatibility (three HLA mismatches – 3MM). Intwo cases, when de recipients and the donors werebrothers, their compatibility was even 100% (0 MM),and in one case we have found four mismatches
(4MM). On the other hand, in group 2, where donor-
recipient pairs are unrelated, HLA histocompatibility is
lower, and 33% of cases had four HLA mismatches
(4MM) and there is no donor-recipient pair completly
HLA compatible for A, B and DRB1 loci (Fig. 2).
Fig. 2 The frequency of HLA mismatch in the two groups
(MM – mismatch)
Pretransplant, cytotoxic antibody screening was
negative in all recipients, as well as crossmatching.
After transplantation, all patients received anti-
CD25 monoclonal antibodies (20mg) and methylpred-
nisolone (500 mg) as induction treatment and then, inthe majority of cases, triple therapy with Tacrolimus
(T), mycophenolate mofetil (MMF), and prednisolone
(Pred) was adopted as the immunosuppressive main-tenance protocol. Only five patients, all of them with
living donors, have been received Cyclosporine –
Mycophenolate mofetil – steroids.
Microchimerism analysis
In our study, the microchimerism was detected
only in 16 recipients (32%), totally 19 records: in 14
patients once, in one patient for two times and in
another patient for three times (Fig. 3). From the graph

2266 Revista Românæ de Urologie nr. 3 / 2013 • vol 12SSttuuddiiii cclliinniiccee below it is clear that the highest frequency of microchi-
merism is recorded in the first week and first month
posttransplantation (seven and eight cases respective-
ly). Only four tests were positive for more than threemonths after transplant.
Fig. 3 The incidence and the moment of microchimerism detection (n=50)
The incidence of microchimerism is significantly
different depending on the type of donor. In patients
from group 2, microchimerism has been detected
rarely, only two of 18 recipients (11%): one patient in
the first week (day 3) and the other in the first month(day 30) after transplant.
Analyzing these two cases should be noted that in
the first patient cold ischemia time was extremely pro-longed (for 11 hours), and in the second case, the
recipient has made an early graft dysfunction with a
prolonged time up to normalization of serum creati-
nine level (5days). Furthermore, in the third week post-transplant, the recipient accused lumbar tenderness,
low grade fever and progressive increase in serum cre-
atinine. Based on clinical symptoms and favorable and
promptly clinical response to corticosteroid pulse ther –
apy, the episode was interpreted as an episode of
acute rejection.
In this context, we believe that microchimerism
detection is not the result of the presence of free donor
cells in the recipient systemic circulation, but rather is
the consequence of donor-derived DNA released from
the damaged endothelial or parenchymal cells of the
renal allograft, injuries caused, in the first case, proba-
bly by prolonged ischemia and in the second case due
to acute rejection episode.
The situation is different for recipients of group 1,
with living donors. The incidence of microchimerism is
43.8% (14/32), significantly higher than in group 2 (p =
0.026 for bilateral Fisher’s exact test, p = 0.008 for thenormal distribution Student t test). The number ofrecords was mostly positive in the first week (6 cases)and the first month (7 cases) posttransplantation (Fig.4). Perhaps also for this group, the microchimerism in
the first week has a component represented by the
donor-specific DNA, maybe as a result of surgical trau-
ma, but also includes a component represented by the
passanger donor’s hematopoietic cells.
Fig. 4 The incidence and the moment of microchimerism detection
depending by the type of donor
Studying a possible correlation between HLA com-
patibility degree and the probability of microchime-
rism detection we have found that these two variables
are independent and therefore, the knowledge of the
number of HLA mismatches is not a predictive factor
for microchimerism occurrence (Table 2).
Table 2. HLA compatibility in relation to the
presence/absence of the microchimerism
Microchimerism HLA compat. MM_A MM_B MM_DRB1 Total_MM
p=NS p=NS p=NS p=NS p=NS
Mean 58,11 ,83 ,94 ,72 2,50
Absent N 18 18 18 18 18
Std. Dev. 15,289 ,383 ,416 ,461 ,924
Mean 57,00 ,86 ,86 ,86 2,57
Present N 14 14 14 14 14
Std. Dev. 14,093 ,363 ,363 ,363 ,852
Mean 57,63 ,84 ,91 ,78 2,53
Total N 32 32 32 32 32
Std. Dev. 14,553 ,369 ,390 ,420 ,879
(NS – not statistically significant)
Four of the eight recipients with microchimerism in
the first month, have had in the first six weeks post-
transplantation, clinical and paraclinical symptoms
suggestive of acute rejection.
Statistical analysis using Pearson’s χ2 test, has shown
that the microchimerism in the first month posttrans-plant is associated with an increased incidence, statisti-
cally significant of acute rejection (57% vs. 12%,
p =
0.042 ). Odds ratio OR = 9.778 with a confidence interval
(1.43, 66.86) is significantly greater than 1, and this
means that the presence of microchimerism significant –
ly increases (five times) the risk of rejection – Table 3.

2277 nr. 3 / 2013 • vol 12 Revista Românæ de Urologie
SSttuuddiiii cclliinniiccee Table 3. Estimation of the acute rejection (AR) risk
based on the presence of microchimerism
Value 95% Confidence
Interval
Lower Upper
Odds Ratio for 9,778 1,430 66,860
Microchimerism_month1
For cohort AR_month1_4 = NO 2,053 ,862 4,889
For cohort AR_month1_4 = YES ,210 ,061 ,726
N of Valid Cases 32
After the first three months posttransplantation,
microchimerism was sporadically detected, in only three
patients: one patient at the beginning of the fourth
month, the second patient in the sixth month and the
third patient in the sixth month and the first year. In
these situations, the most likely, microchimerism couldbe the result of a transient and subclinical allograftinjury, due to drug nephrotoxicity but certainly, we can
not rule out the presence of donor-derived hematopoi –
etic cells which have persisted in the recipient systemic
blood flow and possibly also in lymphoid organs. In this
context it should be noted the case of a recipient who
has shown a sustained microchimerism detected in the
first month, sixth month and one year post-transplanta-
tion. Allograft was very well tolerated and at the end of
the 18 months follow-up, is functional.
The statistical analysis of correlation between
viral/bacterial infection and microchimerism has show
that these two variables are independent (
p = 0.377 ),
so, the microchimerism is not associated with the post-
transplant infections occurrence.
In this study, graft survival at one year was 94%
(47/50) and at the end of the follow-up 84% (42/50).
Three of 16 patients who had microchimerism lost
their graft – 18.8% (two in group 1 and one in group 2).
In the group of patients who had no detectablemicrochimerism, graft loss rate was slightly lower –
14.7% (four recipients from group 1 and one in group
2), but the difference is not statistically significant,
p =
0.358 (Table 4).Table 4. Graft functionality at the end of follow-up in rela-
tion to microchimerism status and type of donor
DONOR Graft functionality Total
Functional Non-functional
graft graft
Cadaveric Microchimerism Absent 5 11 16
Present 1 1 2
Total 6 12 18
Living Microchimerism Absent 0 18 18
Present 2 12 14
Total 2 30 32
Total Microchimerism Absent 5 (14,7%) 29 (85,3%) 34
Present 3 (18,8%) 13 (81,2%) 16
Total 8 42 50
Looking at table 4, it seems that the microchime-
rism would increase the percentage of graft failurecases. But having regard to the low frequencies, thesedifferences are not statistically significant so, the finalconclusion is that the microchimerism presence does
not significantly influence graft function at the end of
follow-up, regardless of the type of donor, either living
or cadaveric donor.
Conclusions
Although the other studies are stated the possibili-
ty of microchimerism occurrence in a part of solid
organ recipients, the results of this study have shown
that, in renal transplantation, the incidence of a real,
sustained microchimerism, as a result of donor pas –
sanger hematopoietic cells, is very low, especially inrecipients with cadaveric donor whose kidneys are well
washed of blood with different solutions, often even
in
situ.
In this context, we believe that microchimerism
detection is not the result of the presence of free donor
cells in the recipient systemic circulation, but rather is
the consequence of donor-derived DNA released from
the damaged endothelial or parenchymal cells of the
renal allograft, injuries due to in the first days post-
transplant probably prolonged ischemia, ischemiareperfusion or surgical trauma and later on due to
acute rejection or drug toxicity.
Moreover, a very important conclusion of this study
is that microchimerism, practically detection of donor-
derived DNA in the circulatory system of the receptor,
is well correlated with the occurrence of acute rejec –
tion, especially in the first three months posttransplan-
tation. The test is accessible to all immunogenetics lab –

2288 Revista Românæ de Urologie nr. 3 / 2013 • vol 12SSttuuddiiii cclliinniiccee oratories specialized in pre and post-transplant evalu-
ation and monitoring, and can be considered as an
important indicator, a potential noninvasive biomarker
that allows early diagnosis of allograft damage.
Acknowledgement
This paper is supported by the Sectoral Operational
Programme Human Resources Development
(SOPHRD) 2007-2013, financed from the European
Social Fund and by the Romanian Government under
the contract number POSDRU/107/1.5/S/82839“
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