Determinants of Poor Graft Outcome in Patients with Antibody-Mediated Acute Rejection

American Journal of Transplantation 2007; 7: 832–841Blackwell Munksgaard

C© 2007 The AuthorsJournal compilation C© 2006 The American Society of

Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2006.01686.x

Determinants of Poor Graft Outcome in Patientswith Antibody-Mediated Acute Rejection

C. Lefaucheura,∗, D. Nochyb, G. S. Hillb,C. Suberbielle-Boisselc, C. Antoined, D. Charronc

and D. Glotzd

aAP-HP, Georges Pompidou European Hospital,Nephrology, Paris, FrancebAP-HP, Georges Pompidou Hospital, Histopathology,Paris, FrancecAP-HP, Saint-Louis Hospital, Immunologyand Histocompatibility, Paris, FrancedAP-HP, Saint-Louis Hospital, Nephrology and KidneyTransplantation, Paris, France∗

Corresponding author: Carmen Lefaucheur,[email protected]

This study analyzes the incidence and course ofantibody-mediated rejection (AMR) in a cohort of 237renal transplant patients followed for 30 ± 20 months.Among these, 32 patients were considered to be atrisk for AMR and received intravenous immunoglobu-lin (IVIg), either as preconditioning (Group A, n = 18) orat the time of transplant (Group B, n = 14). The preva-lence of AMR was 27.8% in Group A, 57.1% in GroupB and 3.9% in the remainder of the population. Al-though graft loss remains greater among AMR than foracute cellular rejection (ACR) or the overall transplantpopulation, we have identified a good outcome group(GFR > 15 mL/min/1.73 m2) (n = 13), whose renalfunction at the end of follow-up was comparable tothat of the general transplant population. The factorsassociated with bad outcome are: (1) immunologic:presence and/or persistence of donor-specific anti-HLAantibodies post-transplantation and (2) histologic:neutrophilic glomerulitis, peritubular capillary dilata-tion with neutrophil infiltrates and interstitial edemaat the time of first biopsy; and at the time of late biopsy(3–6 months): lesions of vascular rejection, and mono-cyte/macrophage infiltrates in glomeruli and dilatedperitubular capillaries. Persistence of C4d does not pre-dict outcome. This study outlines for the first time theimmunologic and histologic profiles of AMR patientswith poor prognosis.

Key words: Antibody-mediated rejection, C4d, donor-specific anti-HLA antibodies, predictors of poor out-come

Received 18 June 2006, revised 27 August 2006 andaccepted for publication 20 November 2006

Introduction

Antibody-mediated rejection (AMR) is now widely recog-

nized as a major problem in organ transplantation. It does

not respond to conventional antirejection treatment, lead-

ing to frequent graft loss (1–7). The initiating event involves

fixation of various antibodies, particularly anti-HLA, to the

endothelium of graft vessels (8). It is now generally ac-

cepted that HLA antibodies are causally related to humoral

rejection (8–10) explaining its increased prevalence in pre-

sensitized patients (1), who represent a significant pro-

portion of potential transplant recipients (11). In the last

10 years, therapeutic strategies to prevent (12–17) and

treat (6,14,18–28) AMR have been developed. However,

since they involve prolonged heavy immunosuppression,

they are not without risk. It is thus important to define

strategies to reduce the risk of AMR, to identify risk fac-

tors linked to poor outcome, and to adjust treatment ac-

cordingly.

To address these questions, we present our 6-year experi-

ence in the long-term course (30 ± 20 months) of AMR

in a cohort of 237 renal transplant patients with a high

prevalence of pre-sensitized patients (35%). We correlate

the clinical course with histologic data and the presence

of donor-specific antibodies (DSA), as detected by sensi-

tive and specific ELISA techniques. We determine the inci-

dence of AMR in renal grafts, define optimal pre-transplant

assessment of rejection risk, and analyze the clinical, im-

munological and histological factors associated with a bad

outcome.

Patients and Methods

PatientsBetween January 1998 and August 2004, 237 consecutive renal transplants

(94F, 143M) were performed in our hospital, including 16 living donors

(6.8%) and 221 deceased donors. All donors and recipients were ABO-

compatible. Among them, 110 patients had at least one episode of clinical

acute graft dysfunction necessitating biopsy. Thirty-four patients (14.3%)

had biopsy-proven rejection: 11 patients (7M, 4F) had an acute cellular re-

jection (ACR) meeting the Banff 97 diagnostic criteria (29); 21 patients (15M,

6F) had AMR, as defined by the National Conference to Assess Antibody-

Mediated Rejection in Solid Organs (9) and by the Banff 97 addition (10).

Immunosuppression protocols: Three groups of patients were identified

according to their immunosuppressive treatment and their immunologic

risk. Group A consisted of 18 highly HLA-sensitized patients who were

832

Antibody-Mediated Rejection

desensitized by IVIg before transplantation according to a previously de-

scribed protocol (13). Patients were eligible for desensitization if their panel

reactive antibodies (PRA) of the IgG class directed against HLA Class I

molecules were equal to or greater than 50%. These patients received

three courses of IVIg (Baxter Gammagard, Baxter, Belgium) at 4-week in-

tervals, each course consisting of 2 g/kg of IVIg given over 48 h. The im-

munosuppressive regimen at transplantation consisted of another course

of IVIg (2 g/kg day 0 and 1), mycophenolate mofetil (CellCept®) (2 g daily),

steroids and Thymoglobulin (Sangstat, Menlo Park, CA) for 10 days at 1–1.5

mg/kg/day followed by tacrolimus (Prograf®). IVIg was given again at days

20–21 and 40–41, at the same dosage. The desensitization was considered

a success if the level of antibodies fell by at least 50%. The patients were

then transplanted with the first available ABO-matched kidney giving an

IgG T-cell negative cross-match (CXM) using the post-IVIg serum. Group

B consisted of 14 patients receiving IVIg at the time of transplantation as

prophylaxis against acute rejection (2 g/kg days 0–1, 20–21 and 40–41), be-

cause of the presence of remote positive IgG T- and B-cell CXM (11 patients)

or the presence of known historical DSA (3 patients). The remainder of the

immunosuppressive regime was similar to that of Group A. Group C was

composed of the remaining 205 patients, sensitized or not, who received

classic protocols of immunosuppression composed of an induction agent

(Thymoglobulin in 94.6% of cases or daclizumab [Zenapax®] in 5.4% of

cases), and maintenance therapy composed of tacrolimus or cyclosporine,

azathioprine or mycophenolate mofetil and steroids.

Clinical follow-up monitoringFor all patients, data were gathered after transplantation, and at the end of

follow-up. The following data were recorded: graft type (living or deceased),

sex and age, and serum creatinine (SCr) and glomerular filtration rate (GFR)

at the end of follow-up. GFR was estimated using the abbreviated MDRD

formula (30). For all patients who underwent renal biopsy, renal parame-

ters at the time of biopsy were recorded. The graft failure rate, defined as

need for hemodialysis or GFR ≤ 15 mL/min/1.73 m2, at the end of follow-up

was calculated. SCr values of non-dialyzed patients at the end of follow-up

were compared among AMR patients, ACR patients and the remaining pop-

ulation of transplant patients without biopsy-proven rejection episodes. All

results up through July 2005 were included, with a median follow-up of 30 ±20 months (8–78 months) for the entire population of transplant patients.

Immunologic testingFor all donors and recipients, HLA A, B and DR tissue-typing was per-

formed using the microlymphocytotoxicity technique with One Lambda

INC® tissue-typing trays. All pre-transplant sera were screened by ELISA

(LAT-M®, One Lambda, Canoga Park, CA) to determine the presence or

absence of anti-HLA Class I or Class II antibodies of the IgG isotype. In all

positive sera, PRA were measured using microlymphocytotoxicity in a panel

of 30 selected lymphocytes and 26 Epstein–Barr virus (EBV) transformed

cell lines, and their peak values were noted. All CXM were performed by two

techniques: direct complement dependent cytotoxicity (CDC) on T and B

donor lymphocytes and by antiglobulin-enhanced CDC (AHG-CDC) on nodal

lymphocytes of the donor. For all patients the CDC and AHG-CDC CXM were

performed on peak and on current sera. The patients were transplanted with

current negative IgG T-cell CDC CXM against their specific donors. No flow

cross-matches were performed.

For the 34 patients having acute rejection on graft biopsy, post-transplant

DSA (at the time of biopsy) were evaluated by specific ELISA techniques.

Positive anti-HLA Class II samples were reanalyzed by ELISA (LAT 240®,

One Lambda) to identify antibody specificity. Post-transplant donor specific

anti-HLA Class I antibodies were identified by ELISA high definition (LAT I

HD®, One Lambda). Pre-transplant peak PRA sera in these 34 patients were

retrospectively analyzed for the presence of DSA by CDC and ELISA.

Histology and immunohistologyAll specimens were evaluated by light microscopy and by immunofluores-

cence (IF) for C4d. Renal biopsies were fixed in alcoholic Bouin’s fixative and

stained by standard methods for routine microscopy. All biopsies contained

at least 10 glomeruli. All biopsies were evaluated and scored according to

the Banff 97 classification (29), and for AMR the criteria of Colvin et al.

(31) and the addition to Banff 97 classification (10). Biopsies were assessed

without knowledge of the clinical outcome. In a separate study, one of

the authors (GSH) counted the number of neutrophils (PMNs) and mono-

cyte/macrophagic cells (MNCs) in all glomeruli, with calculation of the mean

numbers of each/glomerulus. In addition, the number of PMNs and MNCs

in peritubular capillaries (PTC) was counted in 10 consecutive high power

fields for each biopsy and averaged.

Immunofluorescence: For C4d detection, we used a two-step indirect

immunofluorescence method with a monoclonal antibody specific for com-

plement fragment C4d on frozen tissue (Quidel, Santa Clara, CA). Biop-

sies were considered positive for C4d (C4d+) when the PTC were diffusely

and brightly linearly stained, excluding areas of necrosis. Six biopsies with

widespread necrosis were considered as uninterpretable on IF. These late

biopsies were also analyzed by an immunoperoxidase method on paraffin

sections using antihuman C4d polyclonal antibody (32).

Overall, 156 biopsies were performed on the 110 patients with clinical acute

graft dysfunction. All but 15 biopsies, in which there was inadequate ma-

terial, were stained by C4d. We had planned performing a control biopsy

in all patients, at 3–6 months after the episode of AMR. This was done in

14 patients (73.7% of those with grafts in place at 3 months). Eight patients

(5 patients with favorable evolution and 3 patients with poor outcome) had

more than two biopsies because of their clinical situation (persistent renal

insufficiency despite specific treatment). Four patients had their transplants

removed, at days 11 and 15, and at 6 months (2 patients).

Statistical analysisIn evaluating factors associated with outcome, sensitivity and specificity

and positive predictive values of serologic tests were calculated in stan-

dard fashion. Differences in various morphologic parameters between the

good outcome and bad outcome groups were compared using Student’s

t-test. The relationship of categorical variables, such as positive CXM, was

compared to outcome using v 2 tests.

Results

Immunologic characteristics of our populationof renal transplant recipientsAmong the 237 renal recipients between January 1998 and

August 2004, 83 patients (35%) were pre-sensitized, with

pre-transplant anti-HLA Class I and/or Class II antibodies

detected by ELISA: 23 patients (9.7%) anti-HLA Class I;

10 patients (4.2%) anti-HLA Class II; and 50 patients

(21.1%) both Class I and II antibodies. 64 patients (27%)

had a peak of PRA >0%, including 36 patients (15.2%) with

a peak of PRA >30%. Fourteen patients had a historic posi-

tive IgG T-cell CXM (11 patients with positive CDC CXM and

3 patients with positive AHG-CDC CXM) and 3 patients a

positive B-cell CXM. Three patients were transplanted with

a currently positive AHG-CDC CXM.

The prevalence of AMR among the 237 renal recipients

was 8.9%, representing 61.8% of the acute rejections

American Journal of Transplantation 2007; 7: 832–841 833

Lefaucheur et al.

Table 1: Predictive serologic factors for AMR

Sensitivity (%) Specificity (%) Positive predictive value (%)

Presence of Class I or Class II antibodies (by ELISA) 86 70 22

Historic positive CXM1 43 96 52

Historic PRA >0% 81 79 21

Historic PRA >30% 62 91 28

PRA = panel reactive antibodies.1Historic positive IgG T−, B-cell cross match.

diagnosed on biopsy. Among pre-sensitized patients,

21.7% developed AMR, significantly higher than among

non-pre-sensitized patients (2.0%) (p < 0.001). The preva-

lence of AMR was 27.8% (5/18 patients) in Group A, 57.1%

(8/14 patients) in Group B, and 3.9% (8/205 patients) in

Group C.

Table 1 shows the sensitivity and the specificity of the sero-

logic factors predictive of AMR.

Clinical characteristics of AMRIn 18 of 21 patients (85.7%), AMR occurred shortly

post-transplant, with a median onset of 16 days (range

3–42 days). In 3 patients AMR was recognized late at 24,

28 and 58 months. The mean age was 40.3 ± 12 years

(range 19–64). The sex ratio (M/F) was 2.5. Deceased donor

grafts were used in 19/21 patients, living donors in 2. In

19/21 patients (90.5%) there was at least one immuniz-

ing event, with 55.6% having multiple immunizing events

Table 2: Pre-transplant evaluation of immunologic risk factors in 21 patients with AMR

Days Sensitization Peak Remote DSA Class I DSA Class II

Group Sex to AMR history PRA (%) CXM J0 CXM (CDC/ELISA) (ELISA)

M 9 RG + BT 95 +IgGT/CDC IgGT/AHG +/CDC +M 50 RG 93 − − +/ELISA +

A1 M 9 RG + BT 93 +IgGT/CDC − +/ELISA −M 96 BT 90 +IgGT/AHG IgGT/AHG +/CDC −M 9 RG 80 +IgGT/CDC − +/CDC +M 18 RG (2) 90 +IgGT/AHG IgGT/AHG +/CDC +F 16 RG + P (3) 88 +IgGT/CDC − +/ELISA −F 7 RG + BT 85 − − +/ELISA −

B2 M 51 RG (2) + BT 80 +IgGT/CDC − − +M 10 RG + BT 66 +IgMT/CDC − +/ELISA +F 3 RG + BT + P (4) 13 +IgGB/CDC − +/ELISA +M 13 RG 36 − − +/ELISA −M 11 RG 30 +IgGB/CDC − +/ELISA −F 34 BT 40 − − − +M 15 RG + BT 17 − − − −F 58 BT + P (3) 7 − − +/ELISA −

C3 M 840 BT 5 − − − −F 8 BT + P 0 − − − −M 57 ND 0 − − − −M 730 BT 0 − − − −M 1750 0 − − − −

RG = renal graft; BT = blood transfusion; P = pregnancy; M = male; F = female; IVIg = intravenous immunoglobulins; Ig =immunoglobulins; CDC = complement dependent cytotoxicity; AHG = antiglobulin-enhanced CDC cross-match.1Group A: ‘highly HLA-sensitized’ patients undergoing desensitization by IVIg before transplant.2Group B: patients with an historical positive cross-match or a known historical antidonor antibody receiving IVIg for prophylaxie

at the time of transplantation.3Group C: all other patients receiving current immunosuppressive regimen.

(Table 2). Fifteen patients (71.4%) were Caucasian, includ-

ing 3 (14.3%) from North Africa; 6 patients (28.6%) were

Black.

All of the patients with AMR had delayed graft function (6

patients) or acute renal failure, with a median SCr at first

biopsy of 355 lmol/L ± 27 lmol/L (157–882 lmol/L), with

7 patients being dialyzed at the time of diagnosis.

Course and outcomeThe 21 patients with AMR were treated with specific pro-

tocols for antibody removal. 90.5% of patients received

boluses of steroids and IVIg (2 g/kg, monthly ×4 doses).

For certain patients this treatment was associated with:

plasma exchange in 6 (28.6%) cases; muromonab-CD3

(OKT3) in 3 (14.3%) cases; and anti-CD20 antibodies (Rit-

uximab) in 1 (4.8%) case. Those patients in Group C

not receiving tacrolimus or mycophenolate mofetil be-

fore their episode of AMR were switched to these drugs

834 American Journal of Transplantation 2007; 7: 832–841


Table 3: Outcome of patients with AMR and ACR

Final SCr Dialyzed Death

AMR (N = 21) 187 ± 70 6 (28.6%)1,2 1 (4.8%)

ACR (N = 11) 155 ± 77 0 0

Other transplanted patients (n = 203) 146 ± 67 16 (7.9%) 10 (4.6%)

1p < 0.001 in comparison with graft failure in patients without biopsy-proven rejection episode.2p = 0.05 in comparison with graft failure in patients with ACR.

Median follow-up: 30 ± 20 months (8–78 months).

SCr = serum creatinine levels (lmol/L) of non-dialyzed patients at the end of follow-up.

(4 patients from cyclosporine to tacrolimus and 2 patients

from azathioprine-cyclosporine to mycophenolate mofetil-

tacrolimus). At the end of follow-up (30 ± 20 months), graft

loss in AMR patients was significantly greater than in ei-

ther those patients with ACR or the other renal transplant

recipients without acute rejection (Table 3). One death (due

to lung cancer) occurred among the AMR patients, none

among the ACR patients, and there were 10 deaths among

the remaining 203 patients (p = NS).

The median SCr at the end of follow-up among the 15 non-

dialyzed patients with AMR was 187 ± 70 lmol/L (87–475

lmol/L) versus 155 ± 77 lmol/L (123–340 lmol/L) among

patients with ACR and 146 ± 67 lmol/L (59–477 lmol/L)

among the transplants without acute rejection. Thus, re-

nal function, even among non-dialyzed AMR patients, was

significantly worse (p = 0.023) than among those without

rejection, whereas those with ACR were not ultimately sig-

nificantly different from the non-rejection population.

Table 4: Renal biopsy findings in 21 patients with AMR

First biopsy (n = 21) Last biopsy1 (n = 14)

N % N %

g1-g3 2 21 100.0 8 57.1

Neutrophilic glomerulitis3 21 100.0 8 57.1

PTC dilatation 17 81.0 12 85.7

Neutrophils in PTC4 8 38.1 2 14.3

MNCs in PTC5 4 19.0 8 57.1

Acute tubular damage 13 61.9 8 57.1

Neutrophilic tubulitis6 4 19.0 0 0

t2, t3 2 5 23.8 4 28.6

Interstitial inflammation2 6 28.6 6 42.9

Interstitial edema 17 81.0 4 28.6

Vascular lesion (v1–v3)2 12 57.1 4 28.6

v1 7 33.3 1 7.1

v2 5 23.8 3 21.4

v3 0 0.0 0 0

Arteriolar TMA 2 9.5 4 28.6

Glomerular TMA 9 42.9 2 14.3

Cortical infarction 2 9.5 2 14.3

C4d+ 21 100 10 71.4

N = number of patients; %, percent; MNCs = mononuclear cells; TMA = thrombotic microangiopathy; PTC = peritubular capillary.1Biopsies/nephrectomies performed 3–6 months after the first biopsy.2Using standard diagnostic criteria by Banff 97 classification (29).3Cases with an average of ≥1 neutrophil per glomerulus.4Cases with an average of ≥2 neutrophils per high-power field in peritubular capillaries in ten 40 × fields.5Cases with an average of ≥2 MNCs per high-power field in peritubular capillaries in ten 40 × fields.6Cases with an average of ≥1 tubule with intraepithelial neutrophils in ten 40 × fields.

Histologic characteristics of AMRTable 4 shows the histologic characteristics of the first

and last graft biopsies. At initial biopsy 71.4% of patients

(14 patients) had histologic lesions of AMR exclusively. In

6 patients AMR was accompanied by lesions suggestive or

diagnostic of ACR (category IA in 4 patients and borderline

lesions in 2 patients). C4d was definitely positive in all of

the first biopsies and in 71.4% of last biopsies (57.1% on IF

and 14.3% by immunoperoxidase technique). With time,

the PTCs generally showed a diminution of PMNs and an

increase of monocyte/macrophages, sometimes showing

phagocytosed lymphocytes in their cytoplasm (Figures 1

and 2).

Four of the last biopsies were transplant nephrectomies. In

two cases, there was early (days 11 and 15) symptomatic

cortical necrosis, with lesions of thrombotic microangiopa-

thy (TMA), frank and developing hemorrhagic infarction and


Lefaucheur et al.

Figure 1: Antibody-mediated rejection. Biopsies at days 5and 7. (A) Infiltration of dilated peritubular capillaries by PMNs

and monocyte/macrophages. (B) Glomerulus with TMA with fo-

cal thrombosis (∗) and neutrophilic glomerulitis (→). Masson

trichrome stain; ×1000.

thromboses of the hilar arteries. Multiple foci of vascular

rejection (v2–v3) were observed. In these removed trans-

plants the C4d was positive on paraffin section.

Pre- and post-transplant HLA-specific antibodiesin AMR patientsPre-transplant circulating DSA: Eighteen of the 21 AMR

patients (85.7%) were pre-sensitized at transplantation: 5

were in Group A, 8 in Group B and 8 in Group C. Nine

of 21 patients (42.9%) had a historic positive IgG T- or B-

cell CXM: 5 patients with historic positive IgG T-cell CDC

CXM (3 patients from Group A and 2 patients from Group

B), 2 patients with historic positive AHG-CDC CXM (1 pa-

tient from Group A and 1 patient from Group B) and 2 pa-

Figure 2: Evolution of histologic lesions in 2 patients with-out improvement in renal function despite specific treatment.Biopsies performed 4 months after the diagnosis of AMR.(A) Markedly dilated peritubular capillary, as large as surround-

ing tubules, with numerous monocyte/macrophages sometimes

showing phagocytosed lymphocytes in their cytoplasm (→). (B)

Vascular rejection (v1) in artery; several dilated peritubular capil-

laries with monocyte/macrophages; only modest inflammation in

edematous interstitium. Masson trichrome stain; (A) ×1000, (B) ×400.

tients with IgG B-cell CXM (Group B). Three patients had

a current positive AHG-CDC CXM. Retrospective studies

for pre-transplant DSA on the peak sera were positive in

71.4% (15/21 patients): 28.6% had both Class I and II anti-

bodies, 33.3% had only Class I antibodies, and 9.5% Class

II only. In 5 patients with historic negative CXM, we iden-

tified pre-transplant Class I DSA by high definition ELISA.

Table 2 presents the immunologic risk factors for AMR

among our patients.



Table 5: DSA pre- and post-transplant in 21 patients with AMR

DSA pre-graft (n/%) DSA post-graft (n/%)

Class I Class II Class I + II Neg Class I Class II Class I + II Neg Persistent (+/+) De novo (−/+) Extinct (±)

7/33.3% 2/9.5% 6/28.6% 6/28.6% 3/14.3% 6/28.6% 5/23.8% 7/33.3% 10/47.6% 4/19% 5/23.8%

n/% = number and percent of patients; DSA = anti-HLA donor-specific antibodies.

Post-transplant circulating DSA: These were identi-

fied in 14 patients (66.7%): Classes I and II in 5 patients

(23.8%), Class I only 3 patients (14.3%), and Class II

in 6 patients (28.6%). Overall, in 90.5% (19/21) of pa-

tients, DSA were positive either pre- or post-transplant.

At the time of diagnosis, 14 patients with AMR (66.7%)

were C4d+/DSA+ and 7 patients (33.3%) were C4d+/DSA−

(Table 5).

Predictors of poor outcome in AMRAmong the 21 AMR patients, at the end of follow-up

8 patients (3 patients from Group A, 4 patients from

Group B and 1 from Group C) had a GFR ≤15 mL/

min/1.73m2 and constituted the bad outcome group, 6

of whom had to be dialyzed. Thirteen patients had a

good outcome, with renal function at the end of follow-up

160 lmol/L ± 44 (87–250 lmol/L), comparable to that of

patients with ACR and of the general population of renal

transplants without acute rejection (Table 3).

Histologic parameters: The histologic parameters asso-

ciated with poor outcome are detailed in Table 6.

Neutrophils and monocyte/macrophage count : At the

first biopsy, glomerular and capillary PMNs (Figure 1) were

higher in patients with bad outcome compared with those

with good outcome, as were glomerular and capillary

MNCs (Figure 2) at the last biopsy (Table 7).

Vascular rejection: The vascular rejection was much

more frequent among those with bad outcome (4/5 cases)

Table 6: Morphologic variables associated with bad outcome in 21 patients with AMR

Good outcome Bad outcome p

First biopsy

13 patients 8 patients

Glomerular PMNs1 3.2 ± 2.5 6.8 ± 4.1 0.02

Peritubular capillary PMNs1 1.0 ± 0.7 3.4 ± 2.5 0.004

Peritubular capillary dilatation1 1.1 ± 0.9 2.1 ± 0.9 0.03

Interstitial edema1 0.7 ± 0.8 1.7 ± 0.8 0.02

Last biopsy

9 patients 5 patients

Glomerular macrophages1 4.7 ± 3.5 9.3 ± 3.7 0.04

g1 – g3 1,2 1.8 ± 0.8 2.8 ± 0.4 0.03

Peritubular capillary macrophages1 5.8 ± 2.7 17.8 ± 16.5 0.04

Interstitial inflammation 0.5 ± 0.4 2.0 ± 1.8 0.03

v1 – v3 1,2 0.2 ± 0.7 1.7 ± 0.9 0.01

1Significantly associated with outcome on univariate analysis.2Using standard diagnostic criteria by Banff 97 classification (29).

than among those with good outcome (1/8 cases) (p =0.01). For the most part these lesions were graded v1 and

v2, but 1 patient had transmural fibrinoid necrosis (v3) at

transplant nephrectomy.

C4d staining: There is no useful correlation between pos-

itive C4d staining at last biopsy and outcome, since 6/9

patients with good outcome remained C4d-positive.

Immunologic parametersPre- and post-transplant anti-HLA DSA: Table 8 sum-

marizes the relationship of anti-HLA DSA status to

outcome in AMR patients. The identification of DSA

pre-transplant is not helpful in predicting the outcome

of AMR. Although seven of the 8 AMR patients with

bad-outcome had pre-transplant DSA, 8 of the 13 good-

outcome patients also had pre-transplant DSA (p = 0.20).

However, identification of DSA in the post-transplant pe-

riod was associated with a bad outcome for AMR. All

8 bad-outcome patients had post-transplant DSA at the

time of biopsy, compared with only 6 of the 13 with good

outcome (p = 0.011). In 7 of 8 bad-outcome patients with

pre-transplant DSA, the DSA persisted post-transplant de-

spite IVIg and immunosuppression, whereas only three

of 13 good-outcome patients had DSA persisting post-

transplant (p = 0.035). Our study supports a role for post-

transplant DSA Class I or Class I + II as a poor prognostic

factor (p = 0.006).

Serologic and histologic correlations: The DSA Class I

post-transplant were strongly correlated with glomerular


Lefaucheur et al.

Table 7: Evolution of neutrophils and monocyte/macrophages in AMR

Good outcome Bad outcome

First biopsy Last biopsy First biopsy Last biopsy

PMNs

Glomerular 3.2 ± 2.5 3.1 ± 3.2 6.8 ± 4.2 1 3.8 ± 3.9

Capillary 1.0 ± 0.7 0.8 ± 0.9 3.4 ± 2.5 1 9.7 ± 3.2

Monocyte/macrophages

Glomerular 6.9 ± 4.7 4.7 ± 2.7 3.4 ± 2.5 9.7 ± 3.2 1

Capillary 5.4 ± 5.3 5.8 ± 2.7 8.3 ± 7.0 18.2 ± 9.5 1

PMNs = neutrophils; MNCs = monocyte/macrophages.1Values significantly different (p <.05) between ‘bad outcome’ and ‘good outcome’ groups.

and PTC PMNs (p = 0.0014 and p = 0.022 respectively).

No correlation was noted between DSA Class II and mor-

phological parameters at this sample size.

Discussion

This retrospective study analyzes the incidence and course

of AMR in a population of 237 consecutive renal trans-

plants, 35% of them being pre-sensitized before transplan-

tation. It identifies risk factors for AMR and permits initial

immunologic and histologic profiles of patients with bad

outcome of AMR to be drawn, to be modified and fleshed

out with future experience.

The incidence of AMR in our population (8.9%), is compara-

ble to that previously reported (1,31,33), but its prevalence

among histologically confirmed acute rejections (61.8%)

is higher than earlier experience (1,2,19,31,34–37), and is

likely linked to our specific patient population. Our study

confirms an increased prevalence of AMR in pre-sensitized

patients. On the basis of immunologic risk and the specific

associated treatment, we have identified three groups of

patients. Group A consisted of highly HLA-sensitized pa-

tients receiving desensitization treatment before the graft

(n = 18). Group B was composed of patients at immuno-

Table 8: Relationship of anti-HLA DSA status to outcome in 21

patients with AMR

DSA p 1

Pre-transplant

Class I and/or Class II NS

Class I (± Class II) NS

Class II (± Class I) NS

Post-transplant

Class I and/or Class II 0.011

Class I (± Class II) 0.006

Class II (± Class I) 0.10 2

Persistence of Class I or Class II 0.035

Persistence of Class I 0.020

Persistence of Class II 0.10 2

DSA = anti-HLA donor-specific antibodies; NS = p > 0.05.1Values obtained using v 2 testing, with one degree of freedom.2NS, but retained for reference because of small sample size.

logic risk, that is, those with historic positive CXMs or DSA

recognized pre-transplant (n = 14). Group C was composed

of patients (n = 205) who received classic immunosup-

pression regimes which had no demonstrated impact on

the incidence of AMR (37). The prevalence of AMR was

highest in Group B (57.1%) versus 27.8% in Group A and

only 3.9% in Group C.

This study confirms the good results of transplantations

performed after desensitization. The identification of pa-

tients at risk for AMR should not prohibit their being con-

sidered for transplantation, given the good results we

achieved (83.3% graft survival at 3 years in Group A, and

71.4% in Group B) and the low predictive value of all of

the criteria studied (Table 1). Here we employed a de-

sensitization protocol described by Glotz et al. (12,13) us-

ing high-dose IVIg without plasmapheresis. In their study,

there was a mean 77% decrease of anti-HLA antibody titre

using this protocol of desensitization (12,13). Similar re-

sults, using one to four doses of 2 g/kg of IVIg have been

reported by Jordan et al. (15,38). Other techniques have

been employed for the pre-transplant suppression of anti-

bodies, using repeated plasmapheresis coupled with low-

dose IVIg (14), or rituximab and splenectomy associated

with plasmapheresis/IVIg (17). These protocols have en-

abled transplantation of patients at high risk immunologi-

cally who had previously been considered untransplantable

(12,14,16,39–41).

Pre-transplant DSA—a risk factor and post-transplantDSA—a prognostic factor : In our study, 71.4% of pa-

tients with AMR had DSA pre-transplantation. In 42.9%

of patients with AMR, the pre-transplant DSAs were iden-

tified by remote positive CDC CXMs. The presence of a

historic positive CXM is not always associated with AMR.

In our study, AMR occurred in 52.9% of patients with his-

toric positive CXM, and in 35.3% cases it was associated

with a bad outcome. Interestingly, retrospective study on

peak sera permitted the identification of Class I DSA by

high definition ELISA before transplantation in 5 patients

(23.8%) with remote negative CDC/AHG-CDC CXMs.

Further studies will be necessary to evaluate the predic-

tive value of pre-transplant DSAs identified by ELI0A.

Our study identifies the persistence of DSA post-

transplantation (by ELISA) as being associated with a bad



outcome for AMR. Even if the persistence of low levels

of alloantibody does not appear to be necessarily associ-

ated with the development of AMR (42), it appears to be

a prognostic factor in its evolution. Thus, our study em-

phasizes the importance of detection of DSA by sensitive

techniques and reinforces the importance of pre- or peri-

transplant suppression of antibodies. Identification of the

persistence of DSA post-transplantation despite IVIg be-

fore or at the time of transplantation would thus justify the

addition of plasma exchange to IVIg in the treatment of

episodes of AMR.

In our study, the AMR patients were treated using thera-

peutic strategies recognized as efficacious in the treatment

of AMR. 90.6% of patients received high-dose IVIg with or

without plasmapheresis and steroids (14,18–23) and all pa-

tients received mycophenolate mofetil-tacrolimus rescue

(6,27,28). Using this specific treatment, the percentage of

graft loss has significantly diminished, although it still re-

mains worse than that of ACR or of general transplant pa-

tients (2–4,31). However, in our series the course of the

good-outcome AMR patients was comparable to that of

ACR and of the general transplant population. Comparative

analysis of the good and bad outcome groups has permit-

ted the identification for the first time of several factors

associated with poor outcome of AMR.

PTC inflammatory cell accumulation scoring—histo-logic features of poor prognosis: A scoring system for

PTC capillaritis presented by Ian Gibson at the Eighth Banff

Conference on Allograft Pathology in 2005 (43) was found

to be predictive of chronic rejection and chronic allograft

nephropathy (44). In a parallel approach, we counted the in-

flammatory cells in the PTC, distinguishing between PMNs

and MNCs. We found that the cell counts have different

prognostic significance according to when they are made.

PMNs in PTC are an early histologic feature of poor prog-

nosis, whereas in late biopsies it is the MNCs in PTC that

are associated with poor prognosis. In our study, at first

biopsy, capillary PMNs were higher in patients with bad

outcome compared with those with good outcome and at

last biopsy, the number of MNCs differed significantly be-

tween the bad and good outcome groups (Table 7). Older

studies have also shown that glomerular or interstitial infil-

tration by monocyte/macrophages had a negative influence

on the survival of the graft, but without specific reference

to AMR (45–50).

C4d—not predictive for AMR outcome: Our study uses

C4d staining as a diagnostic marker for AMR, all AMR pa-

tients being C4d+ at the time of diagnosis. However, C4d

is not predictive of AMR outcome. In 25% of cases, the per-

sistence of C4d-positivity was not associated with the pres-

ence of post-graft DSA, a proportion substantially higher

than that reported previously (1). In the study of Herzen-

berg et al. (2), the deposition of C4d was a predictive factor

for graft survival, independent of the histologic type of re-

jection. In our study only three of the 9 AMR patients with

good outcomes were C4d- at last biopsy. Thus, we were

unable to find a correlation between C4d-positivity and out-

come of AMR, contrary to a retrospective study of 265 pa-

tients (34). Nickeleit et al. have found that the survival of

kidney grafts exhibiting PTC C4d staining is not different

from their C4d negative patients. Lastly, Salama et al. (51)

studied 4 patients with post-transplant positive cytotoxic

CXM despite pre-transplant immunoadsorption. Upregula-

tion of protective genes such as Bcl-xL was demonstrated

in three of those ‘accommodated’ grafts.

Conclusion

This study identifies a population at risk of AMR, consistingof patients with historic positive CDC CXMs and patientswith DSAs identified pre-transplantation. The identificationof these at-risk patients permits the administration of spe-cific prophylactic treatments aimed at suppression of anti-HLA antibodies (e.g. IVIg), associated with close post-graftmonitoring for early recognition and treatment of AMR.These measures have lead to a substantial increase in graftsurvival (over 60%) with good graft function. The identifi-cation of DSAs pre-transplant as a risk factor and post-graftDSAs as a prognostic factor for poor outcome underlinesthe importance of their detection by sensitive techniques.The persistence of antidonor antibodies, as well as spe-cific histological markers, is indicative of poor prognosisand might lead to further therapeutic modifications.

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Determinants of Poor Graft Outcome in Patients with Antibody-Mediated Acute Rejection

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