Donor Specific Dna A Noninvasive Biomarker Of Renal Allograft Failure Sinescu Ea Si El [613858]

1133 nr. 3 / 2012 • vol 11 Revista Românæ de UrologieDonor-specific DNA – a noninvasive
biomarker of renal allograft failure
A. Moise1,2, I. Constantinescu1,2, R. D. Sinescu2,4, C. Gîngu3, I. Sinescu2,3
1Centrul de Imunogeneticæ øi Virusologie, Institutul Clinic Fundeni, Bucureøti
2Universitatea de Medicinæ øi Farmacie „Carol Davila”, Bucureøti
3Centrul de Uronefrologie øi Transplant Renal, Institutul Clinic Fundeni, Bucureøti
4Compartimentul de Chirurgie Plasticæ-Microchirurgie Reconstructivæ, Spitalul
Universitar de Urgenflæ Elias
Corespondenflæ: Dr. Ana Moise
Centrul de Imunogeneticæ øi Virusologie, Institutul Clinic Fundeni, Øos. Fundeni 258, Sect.2, Bucureøti Tel./Fax: 021 318 48 08, e-mail: [anonimizat]
Introduction: Renal transplantation is the most effective treatment for most patients with chronic renal failure,
significantly improving quality of life. However, about 15-20% of patients undergo at least one episode of acute
rejection, that can be fatal for allograft survival. Currently, accurate diagnosis is obtained by percutaneous renal
biopsy, an intervention with the potential for secondary complications.
Objectives: In our study we aimed to detect the presence of post-transplant donor-specific DNA in the serum of the
recipient and establish a possible correlation between this and episodes of rejection.
Material and methods: We selected a total of 40 kidney transplanted patients in 2011 in Fundeni Clinical Institute
(16 with cadaveric donor and 24 with living donor). Detection of donor-specific DNA was achieved by molecular
biology methods (SSP-sequence specific primers), following the presence / absence of donor specific HLA-DRB1.
Results: Post-transplant, on day three, donor-specific DNA was detected in nine (56%) of recipients with cadaveric
donor and only seven (29%) of recipients with living donor. One month postransplant, only three patients presented
donor-derived DNA in serum. Tests carried out during rejection episodes showed the presence of donor DNA in
seven of ten cases.
Conclusion: Because it is a noninvasive biomarker, accessible for laboratories of immunogenetics involved in post-
transplant monitoring, donor-specific DNA detection in recipient serum can be a biomarker for early diagnosis ofrenal allograft damage.
Key words: acute rejection, donor-specific DNA, post-transplant monitoring, renal transplantation
SSttuuddiiii cclliinniiccee

1144 Revista Românæ de Urologie nr. 3 / 2012 • vol 11SSttuuddiiii cclliinniiccee Introduction
Renal transplantation is the most effective treat-
ment for most patients with chronic renal failure, sig-
nificantly improving quality of life. However, about 15-
20% of patients undergo at least one episode of acuterejection, that can be fatal for allograft survival. Critical
period is the first month posttransplant and a special
issue represent subclinical rejection which may evolvein the absence of renal dysfunction and can damage
the allograft. Currently, accurate diagnosis of acute
rejection is obtained by percutaneous renal biopsy, an
intervention with the potential for secondary compli-cations (back pain, hematuria, perirenal hematoma,
renal infarction, infections, etc.). Furthermore, it takes
at least two days to obtain further histopathological
outcome and for the pathologist is often difficult todistinguish between histological features induced byrejection or due to immunosuppressive therapy side
effects. Therefore, identification of specific, noninva –
sive tests/markers, that can be widely used, representsan objective for research teams in the field of immuno-
logy of transplantation.
Since the 90s, Starzl and colleagues have reported
the presence of donor-derived cells as a chimera, in pa-
tients who underwent a kidney, liver and bowels trans –
plant [1-3]. Using molecular biology methods, the do-nor-derived DNA could be identified in the lymph-
nodes, dermis, jejunum or aorta of recipients with
long-term solid organ allograft survival [4].
In acute rejection, vascular and/or endothelial cells
of the transplanted kidney are target of donor
activated T-lymphocytes. Our study started from the
idea that following cellular destruction of the renal
allograft, donor-specific DNA is released into circula-tion of the recipient and therefore, by our tests we
tried to detect posttransplantation the presence of do-
nor specific DNA in the recipient serum and to estab-
lish a possible correlation between this and acute
rejection episodes, clinical or subclinical.
Material and Method
We have selected a total of 40 patients (26 men and
14 women, aged between 19 and 55 years) who under-
went a kidney transplant in the Fundeni Clinical Insti-
tute during 2011. Depending on the allograft source,recipients were divided into two groups: group A withallograft from living related donors (24 cases) andgroup B with allograft from cadaveric donors (16 ca-ses). The average time of cold ischemia was 8.3 hoursin cadaveric donors and 30 minutes for living related
donors. In all recipients, immunosuppressive induction
therapy was achieved with anti-CD25 monoclonal
antibody (Basiliximab, Simulect) and triple therapy wasused for maintenance – corticosteroids (Cs), tacrolimus
(T) and mycophenolate mofetil (MMF).
Pretransplant, in order to establish donor-recipient
histocompatibility, we performed HLA class I (HLA-A
and B) and class II (HLA-DRB1) genotyping. Patients
were selected so that between donor and recipient be
at least one allelic mismatch on HLA-DRB1 locus, ie
donor to have at least one specific allele, which is notfound in the recipient. Posttransplant, the presence ofdonor specific DNA in the recipient blood was basedon identifying the DRB1 donor specific allele.
In most patients, HLA matching for A, B, DRB1 loci
was 50%, ie there were three HLA mismatch (MM). In
three cases, all with cadaveric donor, there were 4MMand other six cases were with 2MM. Characteristics of
the two groups are represented in Table 1.
Table 1. Distribution and clinical characteristics of
patients in the 2 groups (M-Male, F-Female, CRF –
Chronic renal failure, CGN – Chronic Glomerulonephritis ,
CPN – chronic pyelonephritis , DN – diabetic nephropathy ,
CM –congenital malformation, GT-Grawitz tumor )
Recipients Recipients with Recipients with
profile living donors cadaveric donors
(n=24) (n=16)
Gender: M 13 13
F 11 3
Average age 35,1 (19 – 55) 37,4 (28 – 53)
(years)
CRF etiology:
– CGN 10 8
– CPN 4 2
– DN 76
– CM 2 0
– GT 1 0
Cold ischemia 8,3 (5 – 11) 0,5
time (hours)
HLA match:
– 2MM 4 2- 3MM 20 11
– 4MM 03
HLA typing and donor-specific DNA detection was
done by molecular biology methods (SSP-sequence
specific primers) using the ABDR AllSet Gold amplifica-
tion kit from Invitrogen and DRB1 group specific kits
from PROTRANS. Pretransplant, donor and recipient
DNA extraction was performed from whole blood,

1155 nr. 3 / 2012 • vol 11 Revista Românæ de Urologie
SSttuuddiiii cclliinniiccee collected in EDTA tubes and posttransplant DNA was
extracted from serum, using QIAamp DNA Blood Kit
(QIAGEN).
In principle, extracted DNA is placed in several wells
of a plate containing PCR amplification mixture. Each
well contains different primer pairs, which determinesthe amplification of a specific HLA allele. At the end ofthe amplification, the specific amplicons are visualized
by agarose gel electrophoresis (Fig. 1). The positivity
pattern is computer analyzed, using a dedicated
software that generates the final result of genotyping.
Fig 1. Electrophoresis gel – HLA-A, B øi DRB1 low resolution typing
(1 –DNA size marker, 2 – specific amplicons,
3 – amplification internal control, 4 – negative control)
Posttransplant, recipient blood samples were har –
vested on day three and one month after transplanta –
tion. In ten cases, additional samples were collected at
certain times when symptoms were suggestive of anacute rejection.
On day three, donor-specific DNA was detected in
nine (56%) of recipients with cadaver donor and only
seven (29%) of recipients with living related donor.
This result is consistent with our assumption that pro-
bably a large number of cells are affected as a result ofischemia reperfusion, and these cells are then removed
from circulation in the immediate post-transplant. One
month postransplant, only three patients presented
donor-specific DNA in the serum (Fig. 2).Fig. 2. Distribution of positive cases for donor-specific DNA
in the 2 groups. During rejection episodes, the donor DNA
was found in 83% (5/6) of recipients with cadaver donor and 50% (2/4)
of recipients with living donor (CD-cadaver donor, LD- living donor).
During follow-up, ten patients (four in group A and
six in group B) who had an initial stable outcome,
showed at a time (seven patients in the first month –
weeks two-three and three patients in weeks five, se-
ven and eight), gradual increases of serum creatinine,
moderate back pain, low grade fever, decreased uri-
nary excretion. This symptoms was attributed to acute
rejection episodes. Unfortunately, percutaneous renal
biopsy that confirmed the diagnosis was performed
only in one case. Instead of biopsy, in these patients we
have studied a triad of urinary cytokines (MIG – mono –
kine induced by interferon gamma, OPG – osteoprote –
gerin protein and IP-10 – interferon inducible protein
10). The latest studies have shown that this triple test is
a useful tool for diagnosis of subclinical renal disease
[5-8]. MIG was elevated in all cases, IP-10 protein was
increased in seven patients and OPG in four patients.
Study of cytotoxic antibodies, as markers for the
immune system hyperactivation, showed positive re –
sults only in three cases who had symptoms in the se-
cond month posttransplantation. In all cases it was the
female recipients probably sensitized before trans-
plantation by pregnancy and/or blood transfusions.
The fact that anti-HLA antibodies were not detected in
symptomatic patients in the first month after trans-
plantation does not invalidate the diagnosis of acuterejection, they may still be present, but at levels below
the detection limit of the screening method used (in
this case, ELISA). Quickly favorably response to methyl-
prednisolone therapy, support the diagnosis of acute
humoral rejection. In these patients, donor-specific
DNA was identified in serum in seven of the ten cases(70%) (Fig. 2).
41 2 3

1166 Revista Românæ de Urologie nr. 3 / 2012 • vol 11SSttuuddiiii cclliinniiccee Discussion
Identification of noninvasive biomarkers for
allograft monitoring in solid organ transplantation is a
major target of immunology of transplantation. In
renal transplantation, since the early 2000s, Zhang andcollaborators have shown the presence of donor
specific DNA in recipients urine. Their study showed a
positive correlation between the presence of donor-specific DNA in urine and acute rejection episodes, the
incidence being 88.9% in recipients with acute rejec-
tion and only 8.7% in recipients with stable renal func-
tion. Meanwhile, in patients with renal dysfunction,etiology other than rejection, donor DNA was absent in
the urine [9].
In subsequent years, researchers attention has
shifted to study the presence of donor specific DNA inrecipient plasma/serum. Using molecular biologymethods that allow quantitative measurements (Real-
Time PCR), some studies showed not only the pre –
sence, but also quantitative increasing of donor serumDNA during the rejection episode and decreasing as a
result of favorable response to therapy [10-12].
In our study, in the first week posttransplantation,
donor-specific serum DNA was detected in 56% of
recipients with cadaver donor and 29% of recipients
with living related donor. The higher incidence ingroup B is probably due to more intense cellular da-
mage as a result of prolonged ischemia (cold ischemia
time averaged 8.3 hours compared with 30min for
living donors).
The first month posttransplantation, especially
after the first week, is recognized as a high risk period
for acute rejection. Donor-specific DNA was identified
in a high percentage (70%.) of cases likely to be com-plicated with acute rejection. However, in the absence
of renal biopsy confirmation and taking into account
the small number of cases studied, we can not say to
what extent the presence of donor DNA in recipient
serum is specific marker of acute rejection versus othercauses of renal impairment. Although the presence of
donor specific DNA is not only a sign of acute rejection,
in our view it can be a potential marker on which to
base a decision to establish a renal biopsy or specific
therapy when biopsy is not possible. Therefore, an
algorithm for posttransplant immunological monitor-
ing may include, in addition to cytotoxic antibodiesscreening, quantification of urinary cytokines andstudy of the presence or the absence of donor specificDNA in the recipient blood.Conclusion
Detection of donor specific DNA in the recipient
serum is a useful biomarker for early diagnosis of renal
allograft damage. Since it is a noninvasive biomarker,
accessible for all immunogenetics laboratories in-volved in post-transplant monitoring, a careful,
prospective assessment, extended to other types of
transplantation, is justified. Quantification of donor-specific DNA can be useful, particularly, for the immu-
nosuppression management in case of pancreatic cells
transplantation , in which no organ is available for
biopsy.
Acknowledgement
This paper is supported by the Sectoral Operational
Programme Human Resources Development (SOP
HRD) 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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Rezumat
Introducere: Transplantul renal este tratamentul cel mai eficient pentru marea majoritate a pacienflilor cu insuficien-
flæ renalæ cronicæ, îmbunætæflind semnificativ calitatea vieflii. Totuøi, aproximativ 15-20% din pacienfli trec prin cel puflin
un episod de rejet acut care, netratat, poate fi fatal pentru supraviefluirea alogrefei. În prezent, diagnosticul de cer –
titudine se obfline prin biopsie renalæ percutanæ, intervenflie care însæ este grevatæ de o serie de posibile complicaflii.
Obiective: În studiul nostru am urmærit detectarea posttransplant a prezenflei ADN-ului specific donatorului în serul
primitorului øi stabilirea unei posibile corelaflii între aceasta øi episoadele de rejet.
Material øi metodæ: Au fost selectati un numar de 40 de pacienfli transplantafli renal în cursul anului 2011 (16 cu
donator cadavru øi 24 cu donator viu) în Institutul Clinic Fundeni. Detecflia ADN-ului specific donatorului s-a realizat
prin metode de biologie molecularæ (SSP-sequence specific primers), urmærind prezenfla/absenfla alelei HLA-DRB1
donor specificæ.
Rezultate: În ziua trei posttransplant, ADN-ul specific donatorului a fost detectat la nouæ (56%) din primitorii cu
donator cadavru øi la numai øapte (29%) din receptorii cu donator viu. La o lunæ postransplant, numai trei pacienfli
mai prezentau ADN seric specific donatorului. Testærile efectuate în timpul episoadelor de rejet au aratat prezenfla
ADN-ului donatorului în øapte din zece cazuri.
Concluzie: Deoarece este un biomarker noninvaziv, accesibil laboratoarelor de imunogeneticæ implicate în monito-
rizarea posttransplant, detecflia ADN-ului specific donatorului în serul primitorului poate fi biomarker de diagnostic
precoce al afectærii alogrefei renale.
Cuvinte cheie: ADN donor specific, monitorizare posttransplant, rejet acut, transplant renal