Abstract

Background. ABO-incompatible living donor kidney transplantation based on specific conditioning has been successfully adopted by transplant centres worldwide. Excellent short-term results have been reported in small cohorts. However, long-term data and comparative analyses are still sparse. We report on the outcome of 40 consecutive ABO-incompatible living donor kidney transplant recipients and compare their clinical course to a control group of 43 ABO-compatible living donor transplant patients transplanted during the same time period.

Methods. This is an observational single-centre analysis of 40 consecutive patients undergoing ABO-incompatible kidney grafting between April 2004 and April 2009, using a protocol of rituximab, antigen-specific immunoadsorption, intravenous immunoglobulin, basiliximab induction and oral triple immunosuppression with tacrolimus, mycophenolic acid and prednisone. Forty-three ABO-compatible kidney transplant recipients served as controls. The control group had also received basiliximab induction and an identical initial maintenance immunosuppression. The two groups were observed for an average of 39 and 19 months, respectively.

Results. There was a significantly higher incidence of lymphoceles requiring surgical revisions in the ABO-incompatible group. However, this surgical complication did not affect patient or graft survival. Mean serum creatinine, estimated glomerular filtration rate and proteinuria did not differ between the two groups. Furthermore, ABO-incompatible and ABO-compatible patients had the same incidence of humoral and cellular rejections. Despite a more aggressive induction therapy, no differences in infectious or malignant complications were observed.

Conclusions. ABO-incompatible living donor kidney transplantation utilizing a combination of rituximab and antigen-specific immunoadsorption yielded results identical to ABO-compatible transplantation despite a significantly higher number of human leukocyte antigen mismatches.

Introduction

Despite intensive efforts to promote post-mortem organ donation, the transplant community persistently faces a worldwide shortage of kidney grafts. Throughout the European Community, post-mortem donor rates averaged at 16.2 per million population in 2007 [1]; 7% of patients within the 27 member states of the European Community died while waiting for a suitable graft in 2007 [1]. This dilemma has recently sparked considerable interest in ABO-incompatible living donor kidney transplantation. It is estimated that an additional 10–20% of living donor kidney transplantations can be performed through the implementation of such programmes [2,3], reducing morbidity and mortality of patients on the waiting list. Desensitization protocols in the USA and Japan mostly rely on single- or double-filtration plasmapheresis for reduction of blood-group specific antibodies [3,4]. Antigen-specific immunoadsorption combined with anti-CD20 treatment was first introduced by Tydén [5]. It has become the most widely used desensitization strategy in Europe and the number of transplant centres adopting this technique is rapidly growing. Several short-term or uncontrolled series proved that this approach can successfully circumvent the blood group barrier [5–10] but comprehensive long-term and comparative studies are still sparse [11]. Several important questions, particularly the incidence of humoral rejections, long-term graft survival and the potential impact of rituximab and IVIG on the incidence of infectious complications and malignancies, remain to be carefully addressed. Therefore, the aim of this study was to analyse the long-term outcome of one of the largest ABO-incompatible kidney transplant cohorts exposed to the combination of antigen-specific immunoadsorption and rituximab.

Materials and methods

Patients and study design

This is an ad hoc analysis of two prospectively maintained living donor kidney transplantation cohorts at a single centre. Complement-dependent cytotoxic (CDC) T-cell crossmatch-negative adult ABO-incompatible and ABO-compatible living donor kidney recipients transplanted between April 2004 and April 2009, who had received induction therapy with basiliximab and an initial oral immunosuppression based on tacrolimus, mycophenolic acid and prednisone, were included and repetitively evaluated, based on clinical data derived from inpatient and outpatient visits. A total of 41 adult ABO-incompatible patients and 124 adult ABO-compatible patients received an allograft during the inclusion period. One patient in the ABO-incompatible group received cyclosporine A and was therefore excluded. Eighty ABO-compatible patients were excluded because they did either not receive basiliximab induction or tacrolimus. One patient in the ABO-compatible group was lost to follow-up and therefore excluded. Thus 40 ABO-incompatible and 43 ABO-compatible kidney transplant recipients were analysed. The ethics committee of the Freiburg University Hospital approved the ABO-incompatible kidney transplantation protocol. All patients enrolled in the living donor kidney transplantation programme at the Freiburg University Hospital, gave written consent to store and analyse their electronic health care records and follow-up data. Three previous original studies [8,12,13] included data gathered from the ABO-incompatible cohort described here.

Kidney graft preparation

Donor nephrectomy was performed via an open extraperitoneal minimal incision anterior approach. After removal, the kidney was flushed using histidine-tryptophane-ketoglutarate (HTK) solution in ABO-compatible patients. ABO-incompatible kidneys were prepared by injecting 20,000 units heparin dissolved in 50 mL of isotonic sodium chloride into the renal artery, followed by flushing with 4 L of HTK solution. Postoperatively, recipients received 20,000 units of heparin/day, starting 6 h after surgery, through postoperative days 4–5.

Antigen-specific immunoadsorptions for ABO-incompatible patients

Immunoadsorptions were performed using a commercially available apheresis device (Octo Nova®, Diamed Medizintechnik, Germany). Hollow-fibre plasma separators (P2®, Fresenius Medical Care, Germany or Microplas MPS 07®, Bellco, Italy) were used for plasma separation. Plasma was passed through the antigen-specific carbohydrate column (Glycosorb A/B®, Glycorex Transplantation AB, Sweden). A blood flow of 120 mL/min and a plasma flow of 35–40 mL/min were used. Extracorporeal treatments in the preoperative period were performed every other day. Target plasma volume was estimated using the Kaplan formula [(0.065 × kg) × (1 − haematocrit)] [14]. During each session, 2.5–3 plasma volumes were processed. Anticoagulation was maintained by a combined citrate–heparin protocol. Immunoadsorptions were performed until IgG anti-A/B titers were 1:4 or less on the day of surgery. Postoperatively, at least 2 plasma volumes were processed per treatment. Postoperative immunoadsorptions were performed only if anti-A/B IgG titers exceeded 1:8 in the first postoperative week and 1:16 in the second postoperative week [12].

Isoagglutinin titer measurements, crossmatching and alloantibody detection

Initially, anti-donor isoagglutinin titers were measured by the tube centrifugation test [15]. In May 2007, this method was replaced by a microtube column agglutination technique, using the Diamed-Coombs-Anti-IgG® and Diamed-ID-NaCl® systems (DiaMed Diagnostika Deutschland, Germany). Donor EDTA full-blood samples, resuspended in Diamed cell stab® solution to a final concentration of 0.8–1% and stored at 4°C for up to 4 weeks, were used as target cells. Equal volumes of donor red blood cells and dilutions of recipient serum were incubated in the Diamed-Coombs-Anti-IgG® card at 37°C and the Diamed-ID-NaCl® card at room temperature for 15 min, respectively. After centrifugation, the titer endpoint was considered to be the reciprocal of the highest dilution demonstrating agglutination, i.e. the last dilution displaying reactivity was defined as IgG and IgM titer, respectively.

Human leukocyte antigen (HLA) antibodies and crossmatches were determined in accordance with the guidelines of the European Federation for Immunogenetics using a commercially available ELISA test (Lambda Antigen Tray®, One Lambda, Canoga Park, California, US) and commercially available microplates preloaded with frozen viable lymphocyte cell panels (Biotest, Dreieich, Germany).

Immunosuppression and anti-infective prophylaxis in ABO-incompatible patients

A single dose of rituximab (375 mg/m2) was administered ∼4 weeks before the anticipated transplantation. Seven days before presumed surgery, triple oral immunosuppression consisting of tacrolimus (trough level 12–15 ng/mL), mycophenolate mofetil (2 g/day) or mycophenolate sodium (1440 mg/day) and prednisone (30 mg/day) was initiated. A single dose of intravenous immunoglobulin (IVIG, 0.5 g/kg) was applied between 5 and 1 day before surgery. Basiliximab induction (20 mg) was applied on the day of surgery and on postoperative day 4. Prednisone was administered at a dose of 500 mg, 250 mg and 100 mg on the day of surgery, postoperative day 1 and postoperative day 2, respectively, and then tapered to a dose of 15–20 mg at discharge. Three months after allografting, maintenance immunosuppression included tacrolimus (trough level 4–8 ng/mL), mycophenolate mofetil (2 g/day) or mycophenolate sodium (1440 mg/day) and prednisone (5 mg/day). Later adjustments of maintenance immunosuppressants occurred during the follow-up period and were based on biopsy data or clinical events. All patients received cytomegalovirus (CMV) prophylaxis (valganciclovir) for 3 months postoperatively, regardless of CMV serostatus. Oral PcP prophylaxis (trimethoprim/sulfamethoxazole 80 mg/400 mg/day) was administered 3–4 months postoperatively. All patients received fluconazole (100 mg/day) through postoperative day 21.

Immunosuppression and anti-infective prophylaxis in ABO-compatible patients

ABO-compatible patients received induction therapy with basiliximab (20 mg) on the day of surgery and on postoperative day 4. Prednisone was administered at a dose of 250 mg, 125 mg and 50 mg on the day of surgery, postoperative day 1 and postoperative day 2, respectively, and was further tapered to 15–20 mg at the day of discharge. Three months after allografting, maintenance immunosuppression included tacrolimus (trough level 4–8 ng/mL), mycophenolate mofetil (2 g/day) or mycophenolate sodium (1440 mg/day) and prednisone (5 mg/day). Further adjustments of maintenance immunosuppressants occurred during the follow-up period and were based on biopsy data or clinical events. CMV prophylaxis (valganciclovir) was administered in cases of D+/R− CMV serostatus for 3 months. PcP prophylaxis (trimethoprim/sulfamethoxazole 80 mg/400 mg/day) was administered for 3–4 months postoperatively.

Clinical data, histological data and statistical analysis

Parameters analysed included: recipient/donor age and gender, related versus unrelated donor–recipient status, HLA mismatches, preemptive versus non-preemptive transplantation, number of previous transplants, time on dialysis, diabetes mellitus, prior malignant disease, anti-neutrophil cytoplasmic antibody-associated vasculitis or lupus nephritis as underlying kidney disease and actively replicating hepatitis at the time of transplantation. Graft function was assessed by recording serum creatinine at the time of discharge from hospital and 1, 2, 3, 4 and 5 years after transplantation, respectively. Estimated glomerular filtration rate (eGFR) was calculated using the modification of diet in renal disease formula. Proteinuria was determined in spot urine collections.

Infectious complications monitored for comprised: sepsis, all infections requiring hospitalization, CMV disease, BK-virus associated nephropathy, pneumocystis pneumonia and recurrent urinary tract infections requiring antibiotic prophylaxis. CMV disease was defined as proof of active CMV replication plus clinical symptoms. BK-virus associated nephropathy was defined as histologically proven BK virus infection. Non-infectious complications recorded included: post-transplant lymphoproliferative disease, other malignancies, surgical revisions and complications related to immunoadsorption or the administration of rituximab. Delayed graft function was defined as the need for at least one dialysis postoperatively, irrespective of the underlying reason. Graft failure was defined as an irreversible loss of graft function with the need to maintain or resume dialysis. All indication or protocol biopsies were scored according to BANFF’07 criteria [16].

Data are expressed as means ± standard deviation [range] unless otherwise stated. Group comparisons for continuous data were performed using the Student’s t-test. Comparisons of discrete data were calculated using Fisher’s exact test. Kaplan–Meier survival analysis was calculated via the logrank test (GraphPrism V4.03). Overall survival was defined as time from transplantation to death or graft failure; death with a functioning graft was referred to as censored. Grafts functioning at the last follow-up were censored at date of last contact. Statistical significance was assumed at a P-value < 0.05.

Results

Patient characteristics

All transplantations were performed in patients with a current negative CDC T-cell crossmatch. Donor and recipient characteristics were similar in both groups except for the number of HLA mismatches (Table 1). ABO-incompatible patients had a significantly higher number of mismatches in A/B/DR antigens (4.0 ± 1.3 mismatches in ABO-incompatible versus 3.1 ± 1.7 mismatches in ABO-compatible patients, P < 0.005, Table 1). While there were no HLA-identical siblings serving as donors in the group of ABO-incompatible patients, 3/43 (7%) donors for ABO-compatible recipients were HLA-identical siblings, P = 0.24. All ABO-incompatible patients and 40/43 (93%) ABO-compatible patients had a panel reactivity of 0–19% prior to transplantation, P = 0.24. No patient in the ABO-incompatible group had a panel reactivity of 20–79%, while 3/43 (7%) in the ABO-compatible cohort did, P = 0.24. There were no highly sensitized patients (as defined by a panel reactivity >80%) in both cohorts. Donor specific antibodies were detected in two patients in the ABO-compatible group: One patient had an isolated anti-DR8 antibody not resulting in a positive CDC crossmatch. Another patient had an isolated anti-DR9 antibody that produced a persistently positive CDC B-cell crossmatch. The percentage of female recipients with a history of pregnancies prior to transplantation and a spousal donor non-significantly tended to be higher in the ABO-incompatible group [7/12 (58%)] than in the ABO-compatible cohort [3/9 (33%)], P = 0.39.

Table 1

Patient and donor characteristics and patient comorbidity

 ABO-incompatible ABO-compatible P-value 
(n = 40) (n = 43) 
Patient and donor characteristics 
Recipient age at Tx (years) 46 ± 11 [18–67] 44 ± 12 [19–64] P = 0.37 
Donor age at Tx (years) 52 ± 9 [38–75] 50 ± 10 [29–73] P = 0.43 
Recipient gender female/male (%) 12/28 (30/70) 9/34 (21/79) P = 0.45 
Donor gender female/male (%) 27/13 (68/32) 27/16 (63/37) P = 0.82 
Related donors (%) 14 (35) 21 (49) P = 0.27 
Unrelated donors (%) 26 (65) 22 (51) P = 0.27 
 
Immunological risk factors and comorbidity 
HLA mismatches (A/B/DR) 4.0 ± 1.3 3.1 ± 1.7 P < 0.005 
First Tx (%) 37 (92) 33 (77) P = 0.07 
Second or third Tx (%) 3 (8) 10 (23) P = 0.07 
HLA-identical sibling as donor (%) 0 (0) 3 (7) P = 0.24 
Panel reactive antibody    
 0–19% (%) 40 (100) 40 (93) P = 0.24 
 20–79% (%) 0 (0) 3 (7%) P = 0.24 
 >80% (%) 0 (0) 0 (0) P = 1.00 
Recipients with donor specific antibodies (%) 0 (0) 2 (5) P = 0.50 
Pregnancy prior to Tx and spousal donor (%) 7/12 (58) 3/9 (33) P = 0.39 
Preemptive Tx (%) 7 (18) 11 (26) P = 0.43 
Time on dialysis before Tx (months) 32 ± 31 [0–140] 24 ± 35 [0–145] P = 0.26 
Diabetes mellitus at Tx (%) 3 (8) 1 (2) P = 0.35 
Prior malignancy (%) 3 (8) 2 (5) P = 0.67 
ANCA ass. vasculitis or lupus nephritis (%) 2 (5) 4 (9) P = 0.68 
Active hepatitis B/C infection at Tx (%) 1 (3) 1 (2) P = 1.00 
 ABO-incompatible ABO-compatible P-value 
(n = 40) (n = 43) 
Patient and donor characteristics 
Recipient age at Tx (years) 46 ± 11 [18–67] 44 ± 12 [19–64] P = 0.37 
Donor age at Tx (years) 52 ± 9 [38–75] 50 ± 10 [29–73] P = 0.43 
Recipient gender female/male (%) 12/28 (30/70) 9/34 (21/79) P = 0.45 
Donor gender female/male (%) 27/13 (68/32) 27/16 (63/37) P = 0.82 
Related donors (%) 14 (35) 21 (49) P = 0.27 
Unrelated donors (%) 26 (65) 22 (51) P = 0.27 
 
Immunological risk factors and comorbidity 
HLA mismatches (A/B/DR) 4.0 ± 1.3 3.1 ± 1.7 P < 0.005 
First Tx (%) 37 (92) 33 (77) P = 0.07 
Second or third Tx (%) 3 (8) 10 (23) P = 0.07 
HLA-identical sibling as donor (%) 0 (0) 3 (7) P = 0.24 
Panel reactive antibody    
 0–19% (%) 40 (100) 40 (93) P = 0.24 
 20–79% (%) 0 (0) 3 (7%) P = 0.24 
 >80% (%) 0 (0) 0 (0) P = 1.00 
Recipients with donor specific antibodies (%) 0 (0) 2 (5) P = 0.50 
Pregnancy prior to Tx and spousal donor (%) 7/12 (58) 3/9 (33) P = 0.39 
Preemptive Tx (%) 7 (18) 11 (26) P = 0.43 
Time on dialysis before Tx (months) 32 ± 31 [0–140] 24 ± 35 [0–145] P = 0.26 
Diabetes mellitus at Tx (%) 3 (8) 1 (2) P = 0.35 
Prior malignancy (%) 3 (8) 2 (5) P = 0.67 
ANCA ass. vasculitis or lupus nephritis (%) 2 (5) 4 (9) P = 0.68 
Active hepatitis B/C infection at Tx (%) 1 (3) 1 (2) P = 1.00 

All values represent means ± SD [range], unless otherwise stated.

Abbreviations: Tx, transplantation; HLA, human leukocyte antigen; PRA, panel reactive antibody; ANCA, anti-neutrophil cytoplasmic antibody.

Furthermore, there were non-significant trends towards more preemptive transplantations, more related-donor transplantations and more second or third transplantations in the ABO-compatible control group. ABO-incompatible patients non-significantly tended towards longer pre-transplantation dialysis times (32 months for ABO-incompatible versus 24 months for ABO-compatible patients), P = 0.26.

Blood groups and isoagglutinins in ABO-incompatible patients

Seventy-three percent of recipients in the ABO-incompatible cohort were blood group O recipients (Table 2), with the most common donor–recipient blood group combination being A1→O (16/40, 40%). Thirty percent were high-titer patients with initial IgG anti-A/B titers >1:128. In order to reach the preoperative target IgG anti-A/B titer, a mean of 5.9 ± 3.4 [1–17] immunoadsorptions per patient was required. Postoperatively, 11 patients (28%) underwent immunoadsorptions to maintain isoagglutinin titers within the predefined target (Table 2). Two patients needed additional plasmapheresis to lower the isoagglutinin titers to the target range preoperatively (mean of 3.5 treatments).

Table 2

Immunohaematological characteristics in ABO-incompatible patients (n = 40)

Blood group combinations (donor → recipient) 
A1 → 0 (%) 16 (40.0) 
A2 → 0 (%) 6 (15.0) 
B → 0 (%) 6 (15.0) 
AB → 0 (%) 1 (2.5) 
B → A (%) 3 (7.5) 
A1 → B (%) 1 (2.5) 
A2 → B (%) 2 (5.0) 
A1B → A (%) 2 (5.0) 
AB → B (%) 3 (7.5) 
 
Isoagglutinin titers 
Initial IgG anti-A/B titer before first IA (median) 1:128 [1:1–1:1024] 
Initial IgM anti-A/B titer before first IA (median) 1: 32 [1:0–1:256] 
High-titer patients (initial IgG anti-A/B titer > 1: 128) (%) 12 (30) 
 
Immunoadsorptions 
Total number of IA performed in 40 ABO-incompatible patients 275 
Number of preoperative IA to reach target titer 5.9 ± 3.4 [1–17] 
Patients requiring postoperative IA (%) 11 (28) 
 Number of postoperative IA if required 3.1 ± 1.8 [1–6] 
Patients not requiring postoperative IA (%) 29 (73) 
Patients receiving additional plasmaphereses preoperatively (%) 2 (5) 
 Number of preoperative plasmaphereses if administered 3.5 ± 0.5 [3–4] 
 
Anti CD-20 treatment 
Rituximab dosage applied (mg) 721 ± 82 [540–851] 
Days from rituximab administration to Tx (median) 31 ± 12 [6–79] 
Blood group combinations (donor → recipient) 
A1 → 0 (%) 16 (40.0) 
A2 → 0 (%) 6 (15.0) 
B → 0 (%) 6 (15.0) 
AB → 0 (%) 1 (2.5) 
B → A (%) 3 (7.5) 
A1 → B (%) 1 (2.5) 
A2 → B (%) 2 (5.0) 
A1B → A (%) 2 (5.0) 
AB → B (%) 3 (7.5) 
 
Isoagglutinin titers 
Initial IgG anti-A/B titer before first IA (median) 1:128 [1:1–1:1024] 
Initial IgM anti-A/B titer before first IA (median) 1: 32 [1:0–1:256] 
High-titer patients (initial IgG anti-A/B titer > 1: 128) (%) 12 (30) 
 
Immunoadsorptions 
Total number of IA performed in 40 ABO-incompatible patients 275 
Number of preoperative IA to reach target titer 5.9 ± 3.4 [1–17] 
Patients requiring postoperative IA (%) 11 (28) 
 Number of postoperative IA if required 3.1 ± 1.8 [1–6] 
Patients not requiring postoperative IA (%) 29 (73) 
Patients receiving additional plasmaphereses preoperatively (%) 2 (5) 
 Number of preoperative plasmaphereses if administered 3.5 ± 0.5 [3–4] 
 
Anti CD-20 treatment 
Rituximab dosage applied (mg) 721 ± 82 [540–851] 
Days from rituximab administration to Tx (median) 31 ± 12 [6–79] 

All values represent means ± SD [range], unless otherwise stated.

Abbreviations: IA, immunoadsorption; Tx, transplantation.

Patient and graft survival

The median follow-up period in the ABO-incompatible group was 39 months ± 16 [8–66] versus 19 months ± 12 [6–60] in the ABO-compatible group (Table 3). Patient survival was 98% in ABO-incompatible patients and 98% in ABO-compatible control patients (Table 3). One ABO-incompatible patient died with a functioning graft due to Clostridium difficile sepsis on postoperative day 115. One ABO-compatible patient died with a functioning graft due to oesophageal cancer on postoperative day 276.

Table 3

Patient survival, graft outcome and rejections

 ABO-incompatible ABO-compatible P-value 
(n = 40) (n = 43) 
Median follow-up, months 39 ± 16 [8–66] 19 ± 12 [6–60] P < 0.0001 
 
Patient survival and graft outcome 
Patient survival (%) 98 98 P = 1.00 
Death-censored graft survival (%) 100 93 P = 0.24 
Graft loss (%) 0 (0) 3 (7) P = 0.24 
Delayed graft function (%) 1 (2) 4 (9) P = 0.36 
Serum creatinine at discharge from hospital (mL/min/1.73 m21.57 ± 0.4 [0.8–2.8] 1.49 ± 0.4 [0.7–2.8] P = 0.42 
eGFR (MDRD) at discharge from hospital (mg/dL) 50 ± 13 [24–85] 57 ± 17 [30–101] P = 0.046 
Tacrolimus trough level at discharge from hospital (ng/mL) 11.2 ± 2.6 [7.1–17.6] 10.1 ± 2.6 [4.9–19.1] P = 0.06 
Serum creatinine at last follow-up (mg/dL) 1.54 ± 0.5 [0.8–3.3] 1.55 ± 0.5 [0.9–4.0] P = 0.99 
eGFR (MDRD) at last follow-up (mL/min/1.73 m251 ± 14 [16–88] 53 ± 12 [14–84] P = 0.77 
Proteinuria at last follow-up (g/g creatinine) 0.21 ± 0.3 [0.03–1.7] 0.16 ± 0.2 [0.04–1.3] P = 0.44 
 
Rejections 
Patients with biopsies (per protocol or indication) (%) 38 (95) 37 (86) P = 0.27 
    
Biopsy results according to Banff’07 classification    
Patients with borderline changes (%) 5 (13) 1 (2) P = 0.10 
Patients with acute T-cell-mediated rejections (%) 9 (23) 10 (23) P = 1.00 
 IA 4 (10) 3 (7) P = 0.71 
 IB 1 (3) 0 (0) P = 0.48 
 IIA 3 (8) 5 (12) P = 0.71 
 IIB 1 (3) 0 (0) P = 0.48 
 III 0 (0) 2 (5) P = 0.50 
Patients with acute antibody-mediated rejections (%) 2 (5) 2 (5) P = 1.00 
 I 0 (0) 0 (0) P = 1.00 
 II 2 (5) 2 (5) P = 1.00 
 III 0 (0) 0 (0) P = 1.00 
Patients with rejections or borderline changes (%) 16 (40) 13 (30) P = 0.37 
 ABO-incompatible ABO-compatible P-value 
(n = 40) (n = 43) 
Median follow-up, months 39 ± 16 [8–66] 19 ± 12 [6–60] P < 0.0001 
 
Patient survival and graft outcome 
Patient survival (%) 98 98 P = 1.00 
Death-censored graft survival (%) 100 93 P = 0.24 
Graft loss (%) 0 (0) 3 (7) P = 0.24 
Delayed graft function (%) 1 (2) 4 (9) P = 0.36 
Serum creatinine at discharge from hospital (mL/min/1.73 m21.57 ± 0.4 [0.8–2.8] 1.49 ± 0.4 [0.7–2.8] P = 0.42 
eGFR (MDRD) at discharge from hospital (mg/dL) 50 ± 13 [24–85] 57 ± 17 [30–101] P = 0.046 
Tacrolimus trough level at discharge from hospital (ng/mL) 11.2 ± 2.6 [7.1–17.6] 10.1 ± 2.6 [4.9–19.1] P = 0.06 
Serum creatinine at last follow-up (mg/dL) 1.54 ± 0.5 [0.8–3.3] 1.55 ± 0.5 [0.9–4.0] P = 0.99 
eGFR (MDRD) at last follow-up (mL/min/1.73 m251 ± 14 [16–88] 53 ± 12 [14–84] P = 0.77 
Proteinuria at last follow-up (g/g creatinine) 0.21 ± 0.3 [0.03–1.7] 0.16 ± 0.2 [0.04–1.3] P = 0.44 
 
Rejections 
Patients with biopsies (per protocol or indication) (%) 38 (95) 37 (86) P = 0.27 
    
Biopsy results according to Banff’07 classification    
Patients with borderline changes (%) 5 (13) 1 (2) P = 0.10 
Patients with acute T-cell-mediated rejections (%) 9 (23) 10 (23) P = 1.00 
 IA 4 (10) 3 (7) P = 0.71 
 IB 1 (3) 0 (0) P = 0.48 
 IIA 3 (8) 5 (12) P = 0.71 
 IIB 1 (3) 0 (0) P = 0.48 
 III 0 (0) 2 (5) P = 0.50 
Patients with acute antibody-mediated rejections (%) 2 (5) 2 (5) P = 1.00 
 I 0 (0) 0 (0) P = 1.00 
 II 2 (5) 2 (5) P = 1.00 
 III 0 (0) 0 (0) P = 1.00 
Patients with rejections or borderline changes (%) 16 (40) 13 (30) P = 0.37 

All values represent means ± SD [range], unless otherwise stated.

Abbreviations: eGFR, estimated glomerular filtration rate; MDRD, modification of diet in renal disease.

Death-censored graft survival was 100% in the ABO-incompatible group and 93% in the ABO-compatible control group (Figure 1). Graft losses in the ABO-compatible group occurred within the immediate postoperative period and were due to hyperacute rejection in one case and refractory humoral rejection with renal artery thrombosis in another. The cause of graft failure in the third ABO-compatible patient could not be resolved because the patient refused further diagnostic workup. Two of the graft losses occurred in patients who had received a second renal allograft.

Fig. 1

Death-censored kidney graft survival rate (Kaplan–Meier) in ABO-incompatible (grey line) (n = 40) and ABO-compatible (black line) (n = 43) living donor kidney transplant recipients.

Fig. 1

Death-censored kidney graft survival rate (Kaplan–Meier) in ABO-incompatible (grey line) (n = 40) and ABO-compatible (black line) (n = 43) living donor kidney transplant recipients.

We therefore performed a subgroup analysis of patients receiving a ‘first transplant only’. In this subset of cohorts (37 ABO-incompatible and 33 ABO-compatible patients), overall patient (97% versus 97%) and graft survival (100% versus 97%, P = 0.49) was also observed to be similar (data not shown).

In a separate analysis, all adult living donor transplantations performed in the index period irrespective of the immunosuppressive protocols were analysed (41 ABO-incompatible transplant recipients versus 123 ABO-compatible transplant recipients). Median follow-up in both groups was similar (38 months for ABO-incompatible and 37 months for ABO-compatible patients). Patient and graft survival in the ABO-incompatible group at 3 years was 98 and 100%, respectively, and patient and graft survival in the ABO-compatible group at 3 years was 97 and 94%, respectively (data not shown).

Graft function

The incidence of delayed graft function was the same in both groups (Table 3). eGFR and serum creatinine during the follow-up period are depicted in Figure 2. ABO-compatible patients had a significantly higher eGFR at the time of discharge from hospital than ABO-incompatible patients (eGFR 57 mL/min/1.73 m2 ± 17 [30–101] versus 50 mL/min/1.73 m2 ± 13 [24–85], P = 0.046), Figure 2. At that point, tacrolimus trough levels showed a non-significant trend towards lower levels in the ABO-compatible control group (Table 3). At later intervals of the follow-up period, this difference in eGFR could not be observed anymore. Both groups maintained good renal function over time; mean serum creatinine at the last follow-up was 1.54 mg/dL ± 0.5 [0.8–3.3] in the ABO-incompatible group and 1.55 mg/dL ± 0.5 [0.9–4.0] in the ABO-compatible control group (P = 0.99), corresponding to an eGFR of 51 mL/min/1.73 m2 ± 14 [16–88] in ABO-incompatible and 53 mL/min/1.73 m2 ± 12 [14–84] in ABO-compatible patients, P = 0.77 (Table 3).

Fig. 2

Mean eGFR (mL/min/1.73 m2) ± SEM in ABO-compatible patients (open circles) and ABO-incompatible patients (black squares) during the follow-up period. There was a significant difference in eGFR between the two groups at the time of discharge (*P = 0.046). From 1 year on, GFR in both groups did not differ at any of the follow-up intervals.

Fig. 2

Mean eGFR (mL/min/1.73 m2) ± SEM in ABO-compatible patients (open circles) and ABO-incompatible patients (black squares) during the follow-up period. There was a significant difference in eGFR between the two groups at the time of discharge (*P = 0.046). From 1 year on, GFR in both groups did not differ at any of the follow-up intervals.

Proteinuria at the last follow-up did not differ significantly (0.21 ± 0.3 [0.03–1.7] g protein/g creatinine in ABO-incompatible and 0.16 ± 0.2 [0.04–1.3] g protein/g creatinine in ABO-compatible controls, P = 0.44), Table 3.

Rejections

Despite the difference in the length of the observation periods, allograft rejections in all categories of the Banff classification occurred at the same frequency in both groups (Table 3).

Acute T-cell-mediated rejections (Banff IA-III) occurred in 9/40 (23%) of ABO-incompatible patients and in 10/43 (23%) of ABO-compatible patients, respectively (P = 1.00).

Acute antibody-mediated rejections (Banff I–III) were observed in 2/40 (5%) of ABO-incompatible patients and in 2/43 (5%) of ABO-compatible patients, respectively (P = 1.00).

Complications

The percentage of patients experiencing infectious and malignant complications did not differ between the groups (Table 4). However, surgical complications tended to occur more often in the ABO-incompatible group. Overall surgical revisions were required in 17/40 (43%) of ABO-incompatible cases versus 11/43 (26%) of ABO-compatible cases (P = 0.11). There was a non-significant trend to a higher incidence of immediate postoperative bleeding complications [10/40 (25%)] in the ABO-incompatible group versus the control group [5/43 (11%)] (P = 0.16). Strikingly, the incidence of lymphoceles requiring revision surgery in the ABO-incompatible group was significantly elevated. While only 3/43 (7%) of patients underwent surgical revision for lymphoceles in the ABO-compatible control group, 11/40 (28%) of patients in the ABO-incompatible group needed surgery to remove lymphoceles (P < 0.05). Immediate adverse events related to the infusion of rituximab encompassed diaphoresis in one patient and diarrhoea and fever in another. Both events were self-limited and required no specific intervention. Two hundred seventy-five antigen-specific immunoadsorptions were performed in the ABO-incompatible cohort and were well tolerated. The only significant adverse event related to immunoadsorption was an inadvertently punctured carotid artery upon central venous line placement in one patient, resulting in a cervical haematoma without the need for further intervention.

Table 4

Complications in living donor kidney transplant recipients

 ABO-incompatible ABO-compatible P-value 
(n = 40) (n = 43) 
Median follow-up, months 39 ± 16 [8–66] 19 ± 12 [6–60] P < 0.0001 
 
Infectious complications 
Sepsis (%) 1 (3) 1 (2) P = 1.00 
Infectious complication requiring hospitalization (%) 9 (23) 13 (30) P = 0.62 
CMV disease (%) 3 (8) 3 (7) P = 1.00 
BKVAN (%) 3 (8) 1 (2) P = 0.35 
PcP (%) 1 (3) 0 (0) P = 0.48 
Recurrent UTI requiring antibiotic prophylaxis (%) 3 (8) 4 (9) P = 1.00 
 
Malignant complications 
Skin tumours 1 (3) 0 (0) P = 0.48 
Other carcinoma 0 (0) 1 (2) P = 1.00 
PTLD 0 (0) 1 (2) P = 1.00 
 
Surgical complications 
Patients requiring surgical revision (%) 17 (43) 11 (26) P = 0.11 
 Revision for bleeding complication (%) 10 (25) 5 (11) P = 0.16 
 Revision for lymphocele (%) 11 (28) 3 (7) P < 0.05 
Time from Tx to discharge from hospital (days) 21 ± 7 [12–45] 16 ± 9 [8–70] P < 0.02 
 
Procedural complications related to desensitization for ABO-incompatible transplantation 
Adverse events related to immunoadsorption (minor/major) 0/1 Not applicable  
 Type of adverse event n = 1 accidental puncture of carotid artery during central line placement 
Adverse events related to rituximab infusion (minor/major) 2/0 Not applicable  
 Type of adverse event n = 1 diaphoresis 
n = 1 elevated temperature and diarrhoea 
 ABO-incompatible ABO-compatible P-value 
(n = 40) (n = 43) 
Median follow-up, months 39 ± 16 [8–66] 19 ± 12 [6–60] P < 0.0001 
 
Infectious complications 
Sepsis (%) 1 (3) 1 (2) P = 1.00 
Infectious complication requiring hospitalization (%) 9 (23) 13 (30) P = 0.62 
CMV disease (%) 3 (8) 3 (7) P = 1.00 
BKVAN (%) 3 (8) 1 (2) P = 0.35 
PcP (%) 1 (3) 0 (0) P = 0.48 
Recurrent UTI requiring antibiotic prophylaxis (%) 3 (8) 4 (9) P = 1.00 
 
Malignant complications 
Skin tumours 1 (3) 0 (0) P = 0.48 
Other carcinoma 0 (0) 1 (2) P = 1.00 
PTLD 0 (0) 1 (2) P = 1.00 
 
Surgical complications 
Patients requiring surgical revision (%) 17 (43) 11 (26) P = 0.11 
 Revision for bleeding complication (%) 10 (25) 5 (11) P = 0.16 
 Revision for lymphocele (%) 11 (28) 3 (7) P < 0.05 
Time from Tx to discharge from hospital (days) 21 ± 7 [12–45] 16 ± 9 [8–70] P < 0.02 
 
Procedural complications related to desensitization for ABO-incompatible transplantation 
Adverse events related to immunoadsorption (minor/major) 0/1 Not applicable  
 Type of adverse event n = 1 accidental puncture of carotid artery during central line placement 
Adverse events related to rituximab infusion (minor/major) 2/0 Not applicable  
 Type of adverse event n = 1 diaphoresis 
n = 1 elevated temperature and diarrhoea 

All values represent means ± SD [range], unless otherwise stated.

Abbreviations: Tx, transplantation; CMV, cytomegalovirus; BKVAN, BK virus allograft nephropathy; PcP, pneumocystis pneumonia; UTI, urinary tract infection; PTLD, post-transplant lymphoproliferative disease.

The time between transplantation and discharge from hospital was significantly longer in the ABO-incompatible group [21 days versus 16 days, (P < 0.02)], (Table 4).

Discussion

Living donor kidney transplantation across the blood group barrier has become an alternative to ABO-compatible kidney transplantation, particularly for those end-stage renal disease patients facing long waiting times. While early protocols relied on aggressive measures to prevent hyperacute, isoagglutinin-induced rejection, one current strategy employs highly selective procedures to remove the potentially damaging blood group antibodies and slow their production during a critical postoperative period. Several reports have documented the short-term successes of ABO-desensitization using antigen-specific immunoadsorption in combination with rituximab [5–10] but most of these studies lacked a control group and only one analysis addressed long-term results [11]. In their landmark publication [11], Genberg and colleagues thoroughly investigated the outcome of 15 adult ABO-incompatible living donor transplant recipients and a well-matched control group of 30 ABO-compatible patients and reported similar patient and graft survival rates after a mean follow-up of 3 years. However, to this day, the small number of patients analysed in the Genberg study remains to be the only comprehensive set of data in the field of specific conditioning in adults.

With 40 patients and a median follow-up of more than 3 years, the present study therefore represents the so far largest single-centre analysis delineating the long-term outcome of ABO-incompatible kidney transplantation after single-dose rituximab followed by antigen-specific immunoadsorption and tacrolimus-based oral maintenance immunosuppression. Our data strongly suggest that this approach achieves identical outcomes compared to patients undergoing ABO-compatible kidney transplantation who received basiliximab induction followed by tacrolimus-based maintenance immunosuppression, substantiating the results of the Stockholm group [11].

However, the present study must also be interpreted with caution and carries important limitations owing to its non-interventional design and composition of cohorts. One major limitation resides in the difference in follow-up periods of the groups analysed. The follow-up period in the ABO-compatible control group was significantly shorter than that of the ABO-incompatible group. This is explained by the fact that we did not routinely use basiliximab induction or tacrolimus for ABO-compatible living donor transplantations until 2007 at our institution. However, since the results of the ELITE-Symphony trial argued in favour of a tacrolimus-based immunosuppressive regimen [17], we reasoned that a control group which included patients maintained on cyclosporine A or sirolimus would potentially lead to an impermissible bias against the control group and therefore insisted on exclusively analysing patients who were on an immunosuppressive regimen comprising tacrolimus and basiliximab induction. It could be argued that the longer follow-up time of the ABO-incompatible patients would go in line with a higher risk of patient and/or graft losses and higher numbers of rejections. However this was not observed, supporting the conclusion that ABO-incompatible kidney transplantation was non-inferior to standard blood group compatible grafting. Interestingly, this non-inferiority occurred despite significantly more HLA mismatches, non-significant trends towards longer pre-transplant dialysis times and less preemptive transplantations in the group of ABO-incompatible patients.

Three early graft losses occurred in the ABO-compatible cohort, two of which were observed in patients receiving a second allograft. Furthermore, the rate of second or third transplants in the ABO-compatible group tended to be higher resulting in three patients being moderately sensitized. Despite the fact that this difference did not reach statistical significance, we believe that it represents a clinically highly significant difference that also explains the higher frequency of delayed graft function in this group. Our subgroup analysis of patients only receiving a ‘first graft’ performed in order to assess the outcome independently of the number of previous transplantations and the level of presensitization revealed identical outcomes, further substantiating the findings in the original cohorts.

How do these results compare to published data on the outcome of ABO-compatible living donor kidney transplantation or latest reports on ABO-incompatible grafting using other desensitization strategies? In the 2009 annual report of the United States Renal Data System, the overall graft survival rate in living donor renal transplant recipients was 89% at 3 years after transplantation [18]. Data from the Collaborative Transplant Study demonstrate 3-year graft survival rates of ∼93% in living donor kidney transplantations performed between 2000 and 2007 for tacrolimus-based immunosuppressive regimes (n = 6686) [19]. Tanabe et al. [20] reported on a 3-year graft survival rate of 95.8% in 24 ABO-incompatible transplant recipients preconditioned with rituximab and double-filtration plasmapheresis. A recent comprehensive single-centre study summarizing the outcome of 60 ABO-incompatible living donor kidney transplantations performed by the Johns Hopkins group in the USA[3] reported on 3-year graft survival rates of 92.9% in patients desensitized using different strategies (plasmapheresis/CMVIg in combination with either splenectomy or rituximab or no splenectomy/rituximab).

Thus the 3-year death-censored graft survival of 100% in this specifically desensitized ABO-incompatible cohort does compare favourably to published reports on ABO-compatible living donor transplantation as well as other internationally applied ABO-desensitization strategies.

One of our major concerns was that the routine administration of the anti-CD20 antibody rituximab would lead to an increased rate of infectious complications and/or malignancies. In fact, two recent retrospective analyses of renal transplant patients receiving rituximab for various indications suggested an increased risk of infectious disease [21,22] and death related to infectious disease [21] in patients receiving rituximab in combination with anti-thymocyte globulins. Our current analysis did not reveal an increased risk of such adverse events, reaching the same conclusion as the Genberg study [11] albeit in a larger patient population. This finding is consistent with the results of a randomized, double-blind, placebo-controlled multi-centre study that was recently initiated to compare the effects of rituximab in 68 patients after renal transplantation with 68 patients who received placebo in combination with standard immunosuppression [23]. At 6 months, no difference in the incidence of infections was reported; a 3-year follow-up of this study is still pending. Despite the reassuring results of this randomized controlled trial, we believe that caution is warranted in patients requiring additional anti-thymocyte antibody treatment in the postoperative course.

We observed a non-significant trend towards a higher incidence of postoperative bleedings. Since ABO-incompatible grafts were submitted to a heparin flush prior to rinsing with HTK solution, we investigated whether ABO-incompatible transplant recipients had higher activated partial thromboplastin times (aPTT) after leaving the operating room. The mean of the first aPTT after surgery in the ABO-incompatible group was 39.9 s (SD ± 10.7 s) versus 45.0 s (SD ± 16.6 s) in the ABO-compatible group, indicating that the heparin flush as such did not result in an aptness to bleeding through direct effects on aPTT (P = 0.11) (data not shown). Registry data from Japan, where 60% of transplant centres used anticoagulation strategies in patients undergoing ABO-incompatible grafting between 1989 and 2001, showed that graft survival rates were significantly better in patients receiving anticoagulation therapy with nafamostat mesilate (a drug not available in Europe) than in those ABO-incompatible patients not receiving anticoagulation therapy [4]. Based on these findings, we have not abandoned the anticoagulation strategy from our protocol but maintain a high suspicion for bleedings and perform regular ultrasound examinations postoperatively to rule out haematoma.

One unexplained finding in our study was an increased incidence of lymphoceles requiring surgical revision in ABO-incompatible patients, a phenomenon that has previously been described in a small series of ABO-incompatible kidney recipients [10]. Perhaps, wound healing and repair of the lymphatic system is more significantly compromised if oral immunosuppression is initiated several days before surgery. It is likely that the higher rate of post-transplant surgical interventions, for example to remove lymphoceles, contributed to the significantly longer postoperative hospital stay in the ABO-incompatible group. In addition, it is our current practice to monitor isoagglutinin titers in ABO-incompatible patients for 2 weeks after grafting precluding discharge from hospital prior to postoperative day 14.

Conclusion

In conclusion, this report represents the so far largest in-depth analysis describing the long-term outcome in a cohort of ABO-incompatible kidney transplant patients desensitized with antigen-specific immunoadsorption and rituximab. Graft survival, patient survival and risk of rejection are identical to standard ABO-compatible living donor transplantation while the risk of infectious complications is not increased. These findings further establish ABO-incompatible kidney transplantation as a safe and effective means to combat the shortage of kidney donors.

The authors would like to acknowledge the work of Dr U. Wisniewski who established and refined the isoagglutinin assay techniques and thank Katharina Wolf for her excellent assistance in renal biopsy analyses. We would also like to acknowledge Prof. G. Kirste, Dr E. Schwertfeger and Dr J. Donauer who initiated the ABO-incompatible transplantation programme in Freiburg.

Conflict of interest statement. None declared.

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Author notes

5
Present address: HBH-Kliniken Singen, Renal Division, Germany.
*
Both authors contributed equally to this work.

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