Article

Donor-Specific Antibodies after CeasingImmunosuppressive Therapy, with or without anAllograft Nephrectomy

Arnaud Del Bello,* Nicolas Congy-Jolivet,†‡ Federico Sallusto,§ Celine Guilbeau-Frugier,| Isabelle Cardeau-Desangles,*Marylise Fort,‡ Laure Esposito,* Joelle Guitard,* Olivier Cointault,* Laurence Lavayssiere,* Marie Beatrice Nogier,*Antoine Blancher,†‡ Lionel Rostaing,*¶ and Nassim Kamar*¶

SummaryBackground and objectives Within the last few years, anti–human leukocyte antigen detection assays have sig-nificantly improved. This study asked, using the Luminex single-antigen assay, whether an allograft nephrec-tomy allowed donor-specific alloantibodies to appear that were not previously detected in the serum when thefailed kidney was still in place.

Design, setting, participants, &measurementsAfter losing the kidney allograft and stopping immunosuppressivetherapy, the proportions of donor-specific alloantibodies and nondonor-specific alloantibodies were comparedin patients who had (n=48; group I) and had not (n=21; group II) undergone an allograft nephrectomy. Allograftnephrectomies were performed at 150 days after kidney allograft loss, and the time between allograft nephrec-tomy and last follow-up was 5386347 days.

ResultsAt kidney allograft loss, donor-specific alloantibodieswere detected in three group II patients (14.2%) andsix group I patients (12.5%). At last follow-up, donor-specific alloantibodies were detected in 11 patients (52.4%)without and 39 patients (81%) with an allograft nephrectomy (P=0.02). Anti–human leukocyte antigen class Idonor-specific alloantibodies were positive in 23.8% of group II and 77% of group I patients (P,0.001); anti–human leukocyte antigen class II donor-specific alloantibodies were positive in 42.8% of group II and 62.5% ofgroup I patients. Independent predictive factors for developing donor-specific alloantibodies after losing kidneyallograft and stopping immunosuppressants were number of anti–human leukocyte antigen A/B mismatchesat transplantation (zero versus one or more) and allograft nephrectomy.

Conclusions The development of donor-specific alloantibodies was significantly greater in patients with a failedkidney who had undergone an allograft nephrectomy compared with those patients who had not undergoneallograft nephrectomy.

Clin J Am Soc Nephrol 7: 1310–1319, 2012. doi: 10.2215/CJN.00260112

IntroductionAlthough improved immunosuppressive therapieshave increased kidney allograft survival within the lastdecade (1), the number of patients with a failed kidneytransplant that requires retransplantation has alsogrown (2). The majority of patients undergoing kid-ney retransplantation are sensitized against the hu-man leukocyte antigen (HLA) system. The risk ofantibody-mediated rejection (AMR) is increased inthese high-risk immunologic patients (3). Donor-specific anti-HLA antibodies (DSAs) are responsiblefor the acute and chronic AMR that reduces long-termkidney allograft survival (4). For this reason, anti-HLAantibodies are characterized before a first transplanta-tion or retransplantation to determine donor-mismatchacceptability.

In patients undergoing kidney retransplantation, ithas been suggested that allograft nephrectomy of the

failed kidney may promote anti-HLA antibodies (5–7);these antibodies may be attached to the kidney allo-graft and/or have a very low titer that cannot bedetected by standard immunologic assays. Indeed,DSAs have been previously detected in the eluatesof allograft nephrectomies obtained from patientswith DSAs not detected in the serum (8). However,within the last few years, anti-HLA detection assayshave significantly improved.The Luminex single-antigen assay is much more

sensitive than previous assays at detecting DSAs(9,10). Hence, we wondered whether an allograft ne-phrectomy allowed DSAs to appear that were notpreviously detected in the serum when the failed kid-ney was still in place. The aim of our study was tocompare the incidence of DSAs using the Luminexsingle-antigen assay in patients with a failed kidneyallograft after ceasing immunosuppressive therapy

*Department ofNephrology, Dialysisand OrganTransplantation, CentreHospitalier etUniversitaire Rangueil,Toulouse, France;†MolecularImmunogeneticsLaboratory, EA 3034,Faculte de MedecinePurpan, Universite PaulSabatier, IFR150 (InstitutNational de la Sante etde la RechercheMedicale), Toulouse,France; ‡Department ofImmunology, CentreHospitalier etUniversitaire deToulouse, Hopital deRangueil, Toulouse,France; §Department ofUrology and KidneyTransplantation, CentreHospitalier etUniversitaire Rangueil,Toulouse, France;|Department ofHistopathology, CentreHospitalier etUniversitaire Rangueil,Toulouse, France; and¶Institut National de laSante et de laRecherche MedicaleU1043, InstitutFederatif de Recherche–Recherche Bio-Medicale de Toulouse,Centre Hospitalier etUniversitaire Purpan,Toulouse, France

Correspondence:Dr. Nassim Kamar,Department ofNephrology, Dialysisand OrganTransplantation, CentreHospitalier etUniversitaire Rangueil,TSA 50032, 31059Toulouse Cedex 9,France. Email: kamar.n@chu-toulouse.fr

www.cjasn.org Vol 7 August, 20121310 Copyright © 2012 by the American Society of Nephrology

Tab

le1.

Comparisonsbetweenpatients

whohad

orhad

notundergo

nean

allograftnep

hrectomy

Parameters

Patien

tswithan

Allo

graftN

ephrectomy

(Group

I;n=

48)

Patien

tswithaClin

ically

Indicated

Allo

graft

Nep

hrectomy(n=31

)

Patien

tswitha

System

aticAllo

graft

Nep

hrectomy(n=17

)

Patien

tswitho

uta

nAllo

graftN

ephrectomy:

(Group

II;n

=21

)

Recipient

ageat

tran

splantation(years)

38.6616

40.3617

35.6614

34.6611

Recipient

sex:

male(%

)35

(72.9)

22(71)

13(76)

19(90)

Don

orag

e(years)

39.6618

.743

.4618

36617

40.3615

Typ

eof

don

or:d

eceased(%

)47

(98)

30(97)

17(100

)21

(100

)Num

berof

prev

ioustran

splantations

1.27

60.57

1.22

60.42

1.35

60.79

1.38

60.59

Nep

hrectomyforprev

iouskidne

ytran

splantation(%

)9/

11(81.8)

7/7(100

)2/

4(50)

4/7(57.1)

Num

berof

HLA

A/B/DR/DQ

mismatch

es3.98

61.83

4.03

62.02

3.88

61.45

3.56

1.8

Num

berof

HLA

A/Bmismatch

es2.45

61.05

2.38

61.11

2.59

60.94

2.16

1.27

Num

berof

HLA

DR/DQ

mismatch

es1.48

61.24

1.64

61.35

1.17

60.95

1.57

60.87

Patien

tswithDSA

sat

tran

splantation

00

00

Induc

tion

therap

y:ye

s(%

)39

(81.3)

25(81)

14(82)

18(85.7)

Polyclon

alab

/an

ti–IL-2Rinduc

tion

therap

y24

/15

a15

/10

9/5

17/1a

Ritux

imab

induc

tion

therap

y:ye

s(%

)1(2)

1(3)

00

Initialimmun

osupp

ressionaftertran

splantation:

CNIs

(%)

47(97.8)

30(97)

17(100

)20

(95)

Cyc

losp

orineA/tacrolim

us(%

)31

(66)/16

(34)

22(70)/8(30)

9(53)/8(47)

19(89.4)/2(10.6)

mTORinhibitors

(%)

4(8.9)

3(9.5)

1(6)

1(5)

MPA

(%)

32(66.7)

21(67.8)

11(60)

17(83)

steroids(%

)48

(100

)31

(100

)17

(100

)21

(100

)Im

mun

osupp

ressionat

graftloss

38(79.1)

CNIs

(%)

10(26.3)/28

(73.7)

25(80.6)

13(76.5)

16(75)

cyclospo

rine

A/tacrolim

us(%

)4(9)

6(24)/19

(76)

4(31)/9(69)

9(53)/7(47)

mTORinhibitors

(%)

36(75)

1(3)

3(17)

2(9)

MPA

(%)

43(89.6)

23(77.4)

13(82)

17(81)

steroids(%

)30

(93.5)

15(88)

21(100

)Rejection

episod

esduringthetran

splant

period

(%)

27(56)

18(58)

9(53)

12(55)

Num

berof

rejectionep

isod

espe

rpa

tien

t1(0–4)

1(0–4)

1(0–2)

1(0–2)

Steroid-sen

sitive

rejectionep

isod

es(%

)16

(33.5)

13(42)

3(18)

10(48)

Num

berof

steroid-sen

sitive

episod

espe

rpa

tien

t0(0–2)

0(0–2)

0(0–1)

0(0–2)

Steroid-resistant

rejectionep

isod

es(%

)15

(31)

9(29)

6(41)

4(20)

Hum

oral

rejectionep

isod

es(%

)6(12.5)

4(12.9)

2(11.7)

3(14.2)

Ritux

imab

useduringtran

splant

period

:yes

(%)

11(23)

8(26)

3(18)

6(28.5)

Num

berof

Rituximab

injections

/pa

tien

tb2(2–5)

3(2–5)

22

Tim

ebe

tweenlast

Rituximab

andgraftfailure

(day

s)41

0(20–

1740

)33

0(120

–87

0)59

6(20–

1740

)40

5(60–

2160

)Tim

ebe

tweenlast

Rituximab

therap

yan

dne

phrectomy

(day

s)58

1(95–

1800

)49

8(180

–10

50)

800(31–

1800

)—

Plasmaexch

ange

duringtran

splant

period

:yes

(%)

7(14.6)

6(19)

1(6)

6(28.5)

Num

berof

plasmaexch

ange

sduringthetran

splant

period

9(5–14

)a9(5–14

)12

6c

Tim

ebe

tweentran

splantationan

dgraftfailure

(day

s)17

68(81–

7785

)13

40(81–

7351

)33

30(80–

7785

)40

99(493

–10

013)

Tim

ebe

tweengraftfailure

andgraftne

phrectomy(day

s)15

0(100

–33

90)

169(100

–33

90)

143(110

–11

19)

—

Clin J Am Soc Nephrol 7: 1310–1319, August, 2012 DSA after Kidney Allograft Nephrectomy, Del Bello et al. 1311

and who had or had not undergone an allograft nephrec-tomy. We also assessed the incidence of DSAs in patientswho had a systematic or clinically indicated allograft ne-phrectomy.

Materials and MethodsBetween February of 2007 and November of 2010, 133

kidney allograft failures, defined as a permanent returnto dialysis, occurred in our department. However, onlypatients for whom a kidney retransplantation was plannedwere included in this study (n=95). After kidney allograftloss and reinitiation of dialysis, all immunosuppressivedrugs, except for steroids (if any), were stopped. Steroidswere then stopped at 6 months after initiation of dialysis.Between February of 2007 and July of 2008, no kidneyallograft nephrectomies were conducted unless clinicallyindicated (i.e., acute rejection after ceasing immunosup-pressants). Between July of 2008 and November of 2010,systematic allograft nephrectomies were performed beforepatients, who gave written informed consent, were regis-tered on the waiting list for a kidney retransplantation.Hence, 21 patients did not undergo an allograft nephrec-tomy (group II), and 74 patients underwent an allograftnephrectomy. Of the latter group, 26 patients underwentan allograft nephrectomy within 3 months of the initialtransplantation, mainly because of early vascular thrombo-sis, and therefore, they were excluded from this study; 48patients (group I) underwent an allograft nephrectomy ei-ther systematically (n=17) or as clinically indicated (n=31).Anti-HLA antibodies were assessed in both groups at

graft loss and 3- to 6-month intervals until the last follow-up of patients on a waiting list. Group I patients had ad-ditional analyses: before an allograft nephrectomy, on day5, and at months 3 and 9 after an allograft nephrectomy.

Immunologic AnalysisLuminex assays determined the specificity of HLA class I

and II IgG antibodies in the recipients’ sera (centrifuged at10,000 3 g for 10 minutes) using Labscreen single Ag HLAclass I and class II detection tests (One Lambda, CanogaPark, CA) according to the manufacturer’s instructions.The presence and specificity of antibodies were then detec-ted using a Labscan 100, and the mean fluorescence (base-line value) for each sample in each bead was evaluated. Thebaseline value was calculated as follows: (raw sample meanfluorescence intensity [MFI]2raw negative serum controlMFI)2(negative bead raw MFI with sample2negativebead raw MFI with negative serum control). A baselinevalue of .500 was considered positive. ImmunodominantDSA was defined as the DSA with the highest MFI.

HLA Matchmaker AnalysisWe used the HLA Matchmaker program version 2.1

(www.hlamatchmaker.net) to determine donor–recipientcompatibility at the structural level in class I HLA as de-scribed (11,12).

Pathologic Analysis of the Explanted AllograftForty-two of forty-eight explanted kidney allografts were

analyzed by light microscopy and scored according to 2009Banff criteria (13). C4d staining was also performed.

Tab

le1.(Continued

)

Parameters

Patien

tswithan

Allo

graftN

ephrectomy

(Group

I;n=

48)

Patien

tswithaClin

ically

Indicated

Allo

graft

Nep

hrectomy(n=31

)

Patien

tswitha

System

aticAllo

graft

Nep

hrectomy(n=17

)

Patien

tswitho

uta

nAllo

graftN

ephrectomy:

(Group

II;n

=21

)

Tim

ebe

tweengraftfailure

andlast

follo

w-up(day

s)68

0(111

–39

39)

696(111

–39

39)

673(137

–14

36)

836(33–

3018

)Num

berof

patien

tswithDSA

sat

graftfailure

(%)

6(12.5)

4(12.9)

2(11.7)

3(14.2)

Num

berof

patien

tswithDSA

sat

graftn

ephrectomy(%

)17

(35.4)

10(32.2)

7(41.2)

—Num

berof

patien

tswithDSA

sat

last

follo

w-up(%

)39

(81)

a25

(80.6)

14(82.3)

11(52.4)

c

Tim

ebe

tweenne

phrectom

yan

dde

novo

DSA

(day

s)5(5–10

97)

5(5–10

97)

5(5–27

0)—

a HLA,h

uman

leuko

cyte

antige

n;DSA

,don

or-specifican

tibo

dies;CNIs,calcine

urin

inhibitors;m

TOR,m

ammaliantarget

ofrapam

ycin;M

PA,m

ycop

heno

licacid.

bThe

Ritux

imab

doseof

each

injectionwas

375mg/

m2 .

c P,0.05

.

1312 Clinical Journal of the American Society of Nephrology

Figure 1. | The proportion of patient with donor-specific antibodies (DSAs) increases after an allograft nephrectomy. (A) Proportions ofpatients with DSAs, anti–human leukocyte antigen (anti-HLA) class I DSAs, and anti-HLA class II DSAs who did or did not have an allograft ne-phrectomy. (B) KineticsofDSAsafter anallograft nephrectomy. (C)Meanfluorescence intensity ofdenovo immunodominant anti-HLAclass IDSAs.(D) Mean fluorescence intensity of de novo immunodominant anti-HLA class II DSAs. FU, follow-up; GL, graft loss; GN, graft nephrectomy.

Clin J Am Soc Nephrol 7: 1310–1319, August, 2012 DSA after Kidney Allograft Nephrectomy, Del Bello et al. 1313

Statistical AnalysesReported values represent the means (6SD) or medians

(ranges). Proportions were compared using the Fisher ex-act test. Quantitative variables were compared using theMann–Whitney nonparametric or t test. The predictive fac-tors for developing DSA after graft failure were deter-mined by univariate and multivariate regression analyses.Factors associated by univariate analyses (at a significanceof P,0.10) with the detection of DSA after graft failurewere selected for multivariate analyses. A P value,0.05was considered statistically significant.

ResultsThe patients’ characteristics are presented in Table 1.

Emergence of DSAs after Graft Loss in Patients Who Didor Did Not Have an Allograft NephrectomyAt graft loss, DSAs were detected in three patients

(14.2%) from group II and six patients (12.5%) from group I.At last follow-up, DSAs were detected in 11 patients(52.4%) without an allograft nephrectomy and 39 patients(81%) with an allograft nephrectomy (P=0.02). Anti-HLAclass I DSAs were found to be positive in 23.8% of patients

Figure 2. | Proportion of patients with donor specific antibodies (DSAs) after a systematic or clinically indicated allograft nephrectomy. (A)Proportion of patients with anti–human leukocyte antigen (anti-HLA) class I + II DSAs regarding the indication of allograft nephrectomy. (B)Proportion of anti-HLA class I DSAs. (C) Proportion of anti-HLA class II DSAs. GN, graft nephrectomy.

1314 Clinical Journal of the American Society of Nephrology

from group II and 77% of patients from group I (P,0.001),and anti-HLA class II DSAs were found to be positive in42.8% of patients from group II and 62.5% for patients fromgroup I.De novo DSAs after graft loss were seen in 10 patients

(47.6%) from group II and 40 patients (83.3%) from group I(P=0.002). In one patient from each group, de novo DSA,which occurred after graft loss, disappeared during follow-up and was not detected at the last follow-up. De novo anti-HLA class I DSAs occurred in 23.8% of patients from groupII and 77.1% of patients from group I (P=0.001). De novoanti-HLA class II DSAs occurred in 38% of patients fromgroup II and 62.5% of patients from group I (P=0.06)(Figure 1A).

Emergence of DSAs in Patients Who Underwent aSystematic or Clinically Indicated Allograft NephrectomyThe patients’ characteristics and their induction thera-

pies, initial immunosuppressive therapies, immunosup-pressive therapies at graft loss, acute rejection rates, andnumbers, types, and treatments for acute rejection epi-sodes (if any) did not differ significantly between patientswho had undergone a systematic or clinically indicatedallograft nephrectomy (data not shown). With respect toDSAs, the proportions of patients that had at least oneDSA were similar in both groups at graft loss, the timeof an allograft nephrectomy, and last follow-up. The inci-dence of anti-HLA classes I and II antibodies was similarin the two groups (Figure 2).

Kinetics of the Appearance of DSAs in Patients after anAllograft NephrectomyThe kinetic appearance of DSAs after graft loss is

illustrated in Figure 1B. At graft loss, six patients(12.5%) had at least one DSA. At the time of allograftnephrectomy (i.e., 150 [100–3390] days after graft lossand cessation of immunosuppressive therapy), 35.4% ofpatients had developed at least one DSA. As early as5 days after an allograft nephrectomy, this percentage in-creased to 66% and thereafter, 81.4% and 84.6% at months3 and 9 postallograft nephrectomy, respectively. At lastfollow-up (i.e., at 5386347 days after allograft nephrec-tomy), 81% of patients had at least one DSA. The inci-dence of DSAs after allograft nephrectomy was 73%,

and 59% of patients who had a DSA before an allograftnephrectomy later developed another DSA. The incidenceof DSAs after graft loss was 83.3%. In one patient, a denovo DSA had disappeared at last follow-up. Interestingly,the incidence of DSAs after allograft nephrectomy did notdiffer between patients who did or did not receive a bloodtransfusion after an allograft nephrectomy: 71.4% versus71.8%.The MFI of immunodominant de novo anticlass I and/or

anticlass II DSAs remained stable from day 5 after an al-lograft nephrectomy until the last follow-up (Figure 1, Cand D).

Predictive Factors for the Occurrence of De Novo DSAafter Graft LossAll collected variables were analyzed. The statistically

significant results from the univariate and multivariateanalyses are presented in Table 2. The independent pre-dictive factors for the development of DSAs after graft lossand cessation of immunosuppressants were the number ofanti-HLA A/B mismatches (e.g., zero versus one or moremismatches) at transplantation and having undergone anallograft nephrectomy.

Pathologic Analysis of Explanted Kidney AllograftsAll elementary histologic lesions were scored according

to the Banff 2009 classification; they did not differ signif-icantly between patients with or without DSAs (Table 3).However, total interstitial fibrosis score and positive C4dstaining were significantly higher in patients who had de-veloped DSAs. No difference was observed between pa-tients who had undergone a systematic or clinicallyindicated allograft nephrectomy.

Emergence of Non-DSA Antibodies after Graft Loss inPatients Who Did or Did Not Have an AllograftNephrectomyAt graft loss, there was no significant difference in the

presence of non-DSA antibodies in patients with (sevenpatients; 15%) or without (one patient; 5%) an allograft ne-phrectomy (Figure 3). After graft loss, the proportion of pa-tients that had non-DSA antibodies increased in patients withand without an allograft nephrectomy; at last follow-up,no significant difference was observed between these two

Table 2. Predictive factors for the development of donor-specific antibodies after graft loss

Variables De Novo DSA afterGraft Loss (n=50)

No DSA afterGraft Loss (n=19) P Value

Univariate analysisanti-HLA A/B/DR/DQ MM (zero versus one or more MM) 4.361.63 2.8461.95 0.002anti-HLA A/B MM (zero versus one or more MM) 2.6460.96 1.6461.12 ,0.001transplantectomy (yes/no) 40/10 8/11 0.004anti–IL-2R induction therapy (yes/no) 15/35 1/18 0.05

Multivariate analysis OR 95% CIanti-HLA A/B MM (zero versus one or more MM) 2.32 1.32–4.08 0.004transplantectomy (yes/no) 5.56 1.56–4.08 0.008

DSA, donor-specific antibodies; HLA, human leukocyte antigen; MM, mismatches; CI, confidence interval.

Clin J Am Soc Nephrol 7: 1310–1319, August, 2012 DSA after Kidney Allograft Nephrectomy, Del Bello et al. 1315

groups (i.e., 41 patients in group I [85%] and 14 patients ingroup II [67%]).HLA Matchmaker analysis showed that the proportion

of de novo non-DSA anti-HLA antibodies that reacted tothe donors’ epitopes was 88.9%.

Complications from TransplantectomiesThirty percent of patients experienced a complication after

an allograft nephrectomy. The complication rate did not dif-fer significantly between patients who had a systematic orclinically indicated allograft nephrectomy (Table 4).

DiscussionThe harmful impact of AMR on kidney allograft survival

(3,4,14) has prompted transplant physicians to test recipi-ents’ sera for anti-HLA antibodies using the very sensitiveLuminex single-antigen assay to determine donor-acceptablemismatches. In patients considered for retransplantation,several previous studies tested for anti-HLA antibodies us-ing lymphocytotoxicity or ELISA techniques; they suggestedthat removal of the failed graft might allow the appearanceof previously undetected DSAs in serum (5,15,16). In thepresent study, we assessed the use of the Luminex single-antigen assay to determine the incidence of DSAs afterceasing immunosuppression in patients who had a failedkidney allograft with or without a subsequent allograft ne-phrectomy and were waiting for retransplantation.Our findings from this study are fivefold. (1) The ap-

pearance of DSAs was significantly greater in patientswho had undergone an allograft nephrectomy after ceas-ing immunosuppressive therapy compared with those pa-tients who had not undergone allograft nephrectomy. (2)De novo DSAs were detected in the sera as soon as 5 daysafter an allograft nephrectomy. (3) Donor-specific HLAclass I antibodies appeared more frequently than donor-specific HLA class II antibodies after an allograft nephrec-tomy. (4) No significant difference in the incidenceof DSAs was observed between patients who underwenta clinically indicated allograft nephrectomy and patientswho underwent a systematic allograft nephrectomy. (5) Theonly predictive factors for the appearance of DSAs after kid-ney allograft failure were the number of HLA class I mis-matches and having an allograft nephrectomy.The work by Billen et al. (7) used the Luminex assay to test

for anti-HLA antibodies and DSAs in 53 patients who had anallograft nephrectomy after a first kidney allograft failure. Allpatients were DSA-negative at transplantation; 16% of patientsshowed DSAs before the allograft nephrectomy, whereasDSAs appeared in 84% after an allograft nephrectomy.More recently, the work by Marrari and Duquesnoy (6)

tested for DSAs using the Luminex SA assay in 65 patientswho had undergone an allograft nephrectomy at .1 yearpost-transplantation. Sera were tested at 35 (1–306) daysbefore and 44 (14–337) days after transplantectomy. In pa-tients with HLA class I mismatches, the incidences ofDSAs before and after allograft nephrectomy were 64%and 87%, respectively (P=0.003). In patients with HLA-DRB1 mismatches, the incidence of DSAs was increasedfrom 57% before to 86% after an allograft nephrectomy(P=0.001). Surprisingly, in both studies, it was not men-tioned whether patients were receiving immunosuppressive

Tab

le3.

Histologican

alyses

ofallograftnep

hrectomies

Patien

tswithDSA

sat

LastF

ollow-U

p(n=34

)Pa

tien

tswitho

utD

SAsat

LastF

ollow-U

p(n=8)

PValue

Patien

tswithan

Allo

graft

Nep

hrectomyforClin

ical

Symptom

s(n=17

)

Patien

tswitha

System

atic

Allo

graft

Nep

hrectomy(n=17

)PValue

t0(0–3)

0(0–2)

0.4

0(0–3)

0(0–3)

0.60

i0(0–3)

0(0–1)

0.7

0(0–3)

0(0–1)

0.10

g0(0–3)

0(0–3)

0.9

0(0–3)

0(0–3)

0.25

v0(0–3)

0(0–3)

0.7

1(0–3)

0(0–3)

0.08

ah3(0–3)

3(3–3)

0.1

3(0–3)

3(2–3)

0.06

cg0(0–3)

0(0–3)

0.5

0(0–3)

0(0–3)

0.60

ci3(1–3)

3(0–3)

0.5

1(0–3)

3(0–3)

0.20

ct3(1–3)

3(1–3)

0.6

3(1–3)

3(1–3)

0.20

cv3(0–3)

3(0–3)

0.4

3(0–3)

3(0–3)

0.90

mm

2(0–3)

1(0–3)

0.9

1(0–3)

2(0–3)

0.80

ti3(0–3)

0(0–2)

0.00

22(0–3)

2(0–3)

0.95

PTc

0(0–3)

0(0–3)

0.8

0(0–3)

0(0–3)

0.74

Positive

C4d

staining

19(55.9%

)1(12.5%

)0.05

10(59%

)9(53%

)0.99

DSA

,don

or-specifican

tibo

dies;t,tubu

litis;i,m

onon

uclear

cellinterstitial

inflam

mation;

g,glom

erulitis;v

,intim

alarteritis;ah

,arteriolarhy

alinethicke

ning

;cg,

allograftg

lomerulop

athy

;ci,

interstitial

fibrosis;ct,tubu

laratroph

y;cv,v

ascu

larfibrou

sintimal

thicke

ning

;mm,m

esan

gial

matrixincrease,ti,totalinterstitialinfl

ammation;

PTc,pe

ritubu

larcapilla

ritis.

1316 Clinical Journal of the American Society of Nephrology

drugs before the allograft nephrectomy and if they were not,what the delay was between immunosuppressant cessationand allograft nephrectomy.Other studies have shown that the percentages of classes

I and II panel-reactive antibodies were significantly in-creased after an allograft nephrectomy (5,16,17). In thepresent study, we found that de novo DSAs appeared in47.6% of patients without allograft nephrectomy when im-munosuppressive therapy was stopped. In contrast, in pa-tients who had an allograft nephrectomy, the incidenceof de novo DSAs increased from 35.4% after cessation of im-munosuppressive therapy and before a transplantectomy to83.3% (P=0.002) at the last follow-up after an allograftnephrectomy. Both donor-specific anticlasses I and II anti-bodies were detected after an allograft nephrectomy.

However, after graft loss, the incidence of de novo DSAanti-HLA class I antibodies was significantly higher in pa-tients who had an allograft nephrectomy compared withpatients who had not had an allograft nephrectomy (77.1%versus 23.8%, P=0.001). The incidence of de novo DSA anti-HLA class II antibodies tended to be higher in patientswho had an allograft nephrectomy (62.5%) comparedwith patients who had not had an allograft nephrectomy(38%; P=0.07). Non-DSA anti-HLA antibodies, whichhave a negative impact on allograft survival (18), signifi-cantly increased after cessation of immunosuppressivetherapy. However, the proportion of patients with non-DSAs antibodies did not differ between patients with orwithout a nephrectomy. Interestingly, 89% of non-DSA an-tibodies reacted with the donors’ epitopes.

Figure 3. | The proportion of patient with non-DSA anti-HLA antibodies is not significantly different in both groups. (A) Proportions ofpatients with no donor-specific antibodies (DSAs) anti–human leukocyte antigens (anti-HLAs) antibodies either with or without allograft ne-phrectomy. (B) Proportions of patients with no DSAs anti-HLA class I antibodies with or without allograft nephrectomy. (C) Proportions ofpatients with no DSAs anti-HLA class II antibodies with or without allograft nephrectomy. GL, graft loss; GN, graft nephrectomy.

Clin J Am Soc Nephrol 7: 1310–1319, August, 2012 DSA after Kidney Allograft Nephrectomy, Del Bello et al. 1317

Allograft nephrectomy and the number of anti-HLA class Imismatches were the only two independent predictivefactors for de novo DSAs after graft loss and cessation ofimmunosuppressive therapy. In patients who had an allo-graft nephrectomy, the work by Billen et al. (7) found thatdonor age was a predictive factor for de novo DSAs,whereas the work by Knight et al. (17) found that boththe number of rejection episodes and having a ,10-monthinterval between graft failure and allograft nephrectomywere both significantly associated with greater percentagesof panel-reactive antibodies and mean DSA levels.How an allograft nephrectomy allows the appearance of

DSAs is not fully known. It has been suggested that DSAsare absorbed by the kidney allograft and that removal ofthe kidney allows DSAs to appear in the blood circulation(15,19). In contrast, it has been also suggested that an al-lograft nephrectomy may cause damage that results indanger signals that stimulate the immune system, leadingto the appearance of DSAs (20–22). The results of ourstudy support the first hypothesis, suggesting that DSAsare absorbed by the kidney. Indeed, DSAs appeared assoon as 5 days after allograft nephrectomy, suggestingthat antibodies were already preformed. Furthermore, al-though MFI is not quantitative and may have significantinterassay variability, the MFI of immunodominant anti-bodies remained stable or was decreased during follow-up, whereas if DSAs had appeared because of injurycaused by the nephrectomy, the MFI would have in-creased during follow-up.The improved ability to detect DSAs after an allograft

nephrectomymakes it more difficult to find a suitable kidneywith acceptable HLA mismatches for retransplantation.Nevertheless, if no allograft nephrectomy is performed, theacceptance of a kidney allograft when an HLA antigenagainst a DSA is present in the failed kidney allograft butnot in the sera may cause an AMR after retransplantation,

leading to reduced kidney allograft survival. Because of theshortage of transplant organs, it may be reasonable to pro-pose the use of a kidney when there are no DSAs in the seraafter an allograft nephrectomy. Although the time taken tofind a more suitable kidney is prolonged, this proposal maybe recommended rather than performing a kidney retrans-plantation without being sure of the absence of preformedDSAs in the failed kidney that are not detected in the sera.The removal of failed kidney allografts may reduce

chronic inflammation and be responsible for erythropoietinresistance (23). The work by Ayus et al. (24) showed thatallograft nephrectomy was associated with improved pa-tient survival and interestingly, a higher retransplantationrate compared with patients without an allograft nephrec-tomy. The work by Johnston et al. (25) found that, whengraft failure occurred at .12 months post-transplant, allo-graft nephrectomy was associated with a decreased riskof death. However, in the study by Johnston et al. (25),allograft nephrectomy significantly increased the risk ofrepeat transplant failure. The work by Schleicher et al.(16) showed that graft nephrectomy had a negative impacton graft function and survival after retransplantation,whereas the work by Ahmad et al. (26) found that nephrec-tomy of a failed allograft did not significantly influence thesurvival of the subsequent kidney transplant. However, itis not possible to draw any conclusions from these threestudies, because DSAs were not assessed routinely beforeor after transplantation using a sensitive assay.A mortality rate of between 0.7% and 7% has been re-

ported after graft nephrectomy (24–29). In the presentstudy, no patients died during or immediately after anallograft nephrectomy. However, the complication rate wasrelatively high at 30%. Hence, an allograft nephrectomyshould be proposed with caution.There are several limitations to the present study. It is

a nonrandomized study that included a relatively small

Table 4. Allograft nephrectomy-related complications

All Patients(n=48)

SystematicTransplantectomy

(n=17)

Clinically IndicatedTransplantectomy

(n=31)

P Value: SystematicVersus Clinically

IndicatedTransplantectomy

Duration of hospitalization(days)

6.5 (3–36) 6 (3–31) 7 (3–36) 0.91

Overall complications (%) 13 (30); 7 (15) 5 (30); 3 (18) 8 (30); 4 (13) 1Infectious complications (%) 4 (8) 2 (11) 2 (6) 0.71pulmonary infection (%) 1 (2) 1 (6) 0bacteremia (%) 1 (2) 0 1 (3)kidney allograft siteinfection (%)

1 (2) 0 1 (3)

cytomegalovirusreactivation (%)

Surgical complications (%) 9 (19) 3 (18) 6 (19)a 1hematoma 7 (15) 3 (18) 4 (13)cecal perforation (%) 1 (2) 0 1 (3)wound dehiscence (%) 1 (2) 0 1 (3)

Blood transfusion (%) 14 (30) 6 (35) 8 (26) 1Death (%) 0 0 0 1

aOne patient required additional surgery to drain a hematoma.

1318 Clinical Journal of the American Society of Nephrology

number of patients. We did not look for anti-HLA C andanti-HLA DP in our patients. However, the difference in denovo DSAs between patients with and without an allo-graft nephrectomy was statistically significant and mayhave been increased if these two HLA subtypes had beenassessed. We also did not assess inflammatory markers be-fore and after a nephrectomy. However, this assessmentwas not the purpose of our study.In conclusion, the incidence of DSAs is significantly

greater in patients with a failed kidney who have under-gone an allograft nephrectomy compared with thosepatients who have not undergone an allograft nephrec-tomy. Prospective studies are required to assess themechanism(s) responsible for the appearance of DSAs afterallograft nephrectomy and the impact of allograft nephrec-tomy on patient and graft survival rates and acute rejectionrates after retransplantation.

DisclosuresNone.

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Received: January 9, 2012 Accepted: April 30, 2012

Published online ahead of print. Publication date available at www.cjasn.org.

See related editorial, “Sequestration and Suppression of Anti-HLAAntibodies by a Failed Kidney Allograft,” on pages 1209–1210.

Clin J Am Soc Nephrol 7: 1310–1319, August, 2012 DSA after Kidney Allograft Nephrectomy, Del Bello et al. 1319