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Abstract

Objective

To determine whether a new desensitization protocol (mycophenolate mofetil [MMF], plasmapheresis and antithymocyte globulin [ATG], complemented with human leucocyte antigen [HLA] amino acid residue matching) could reduce panel-reactive antibody (PRA) levels in sensitized patients, to facilitate successful renal transplantation.

Methods

Patients awaiting transplantation with PRA levels >10% received treatment with MMF; those with PRA levels >30% were also treated with plasmapheresis. Patients whose PRA level was <20% after desensitization were eligible for transplantation. When a donor became available, traditional HLA matching and HLA amino acid residue matching were performed. All patients received ATG induction therapy postoperatively.

Results

Thirty-two sensitized patients were enrolled. Desensitization produced a significant decrease in PRA levels; 27 patients (84.4%) became eligible for transplantation and 26 (81.2%) subsequently underwent successful transplantation. Residue matching improved the proportion with a mismatch number of 0–1 from 7.7% to 65.4%, compared with traditional HLA matching. Postoperatively, all patients showed immediate graft function. Acute rejection occurred in three patients (11.5%) and infections in seven patients (25.9%); all were treated successfully.

Conclusion

The combination of a desensitization protocol (MMF, plasmapheresis and ATG) and residue matching appears to be an effective strategy for sensitized patients awaiting renal transplantation.

Keywords

Desensitization renal transplantation panel-reactive antibodies human leucocyte antigen amino acid residue matching

Introduction

Renal transplantation is the preferred treatment for patients with renal failure, with an improved survival rate, better health-related quality of life and lower costs than dialysis.¹ However, owing to the limited supply of organs available for transplantation, the number of patients waiting for a compatible donor has gradually increased worldwide, especially for human leucocyte antigen (HLA)-sensitized patients with an elevated panel-reactive antibody (PRA) level.^2–4 An increased PRA level can occur following blood transfusion, pregnancy or previous transplantation, and 25–30% of patients waiting for transplants are HLA-sensitized.^5–7 Sensitization poses many challenges for the transplant community: because matching is so difficult for these patients, they are more likely than unsensitized patients to die from the complications of dialysis before a donor is found.⁷ If patients with a high PRA level undergo renal transplantation without any pretreatment, they have a higher rate of acute rejection, delayed graft function and graft failure than those without antibodies.^2–4

A number of protocols have evolved to improve the transplantation rate in sensitized patients. Several approaches have been developed to remove preformed antibodies, including treatment with intravenous immunoglobulin, basiliximab, mycophenolate mofetil (MMF), sirolimus, antithymocyte globulin or rituximab and procedures such as plasmapheresis or protein A immunoadsorption.⁸ Combinations of therapies have also been used to increase treatment efficacy and reduce side-effects.⁸ Some treatments have been shown to decrease alloantibody levels before and after transplantation, but success has been limited: some patients did not respond, the acute rejection rate was high and protocol-related side-effects were common.⁴ The present prospective study investigated the effects of a new desensitization protocol consisting of mycophenolate mofetil, plasmapheresis and antithymocyte globulin, coupled with HLA amino acid residue matching, on PRA levels and transplantation rates among sensitized patients.

Patients and methods

Patients

Sensitized patients, defined as those having PRA to HLA class I (PRA I) and/or PRA to HLA class II (PRA II) levels >10%, awaiting transplant at the Department of Renal Transplantation, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China, between January 2009 and December 2010, were enrolled in this study. PRA I and PRA II levels were determined using the Lambda Antigen Tray™ (One Lambda, Canoga Park, CA, USA) according to the manufacturer’s instructions. PRA levels were measured every other month.

Written informed consent was obtained from all the study participants. The study protocol was approved by the institutional review board of the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.

Desensitization

After the patients were enrolled, MMF treatment was administered to all patients at a dose of 0.5 g orally twice a day. In patients with a PRA level >30%, plasmapheresis was performed at 1- or 2-week intervals using the Cell Saver® autologous blood recovery system (Haemonetics, Braintree, MA, USA); each session involved treatment for 3–4 h, removing 1 IU of plasma (approximately 2–3 l) for replacement with the same volume of fresh plasma and albumin. The PRA level was measured again after 1 month and if still >20%, treatment with MMF with or without plasmapheresis was continued. Patients whose PRA levels were <20% after desensitization treatment were eligible to receive a kidney from a living or deceased donor.

Unless the haemoglobin level was <5 g/l, blood transfusions were not permitted during the study. An additional session of plasmapheresis was performed 1 day before transplant surgery in patients whose PRA level had been >30%.

During the period of desensitization, any side-effects including fever, headache, gastrointestinal reactions and severe anaemia, were recorded.

HLA matching

When a donor became available, traditional six-antigen HLA-class typing and T-cell complement-dependent lymphocytotoxicity cross-matching were performed on the patient and donor. HLA I and II levels were determined using the Lambda Antigen Tray™ according to the manufacturer’s instructions. T-cell complement-dependent lymphocytotoxicity cross-matching was determined using a One Lambda microdroplet assay, according to the manufacturer’s instructions.

In addition, HLA amino acid residue matching (based on 10 amino acid residues for class I and seven DR/DQ supertypic groups for class II) was performed, as described previously.^9,10

An acceptable match between recipient and donor for transplantation was defined as a 0–3 mismatch, determined by residue matching and a negative (<5%) HLA cross-match detected on complement-dependent lymphocytotoxicity microdroplet assay.

Immunosuppressive therapy

All transplant patients received induction therapy consisting of 50–100 mg/day antithymocyte globulin (ATG) (ATG-Fresenius®, Fresenius Biotech, Munich, Germany) between day 0 and day 5; the ATG was diluted in an isotonic solution to a total volume of 500 ml and administered by slow continuous intravenous infusion over 5 h. Maintenance immunosuppressive treatment consisted of triple therapy with tacrolimus, MMF and prednisone. MMF was commenced immediately after surgery at a dose of 0.5–1 g, orally, twice daily. Tacrolimus was administered from 2 days post-transplantation at a dose of 0.12–0.15 mg/kg orally daily; the concentration in whole blood was monitored using the IMx Tacrolimus I assay (Abbott Laboratories, Abbott Park, IL, USA) and the dose was adjusted to give a trough concentration of 9–12 ng/ml during the first month, 7–10 ng/ml by month 3 and 5–8 ng/ml by month 6, then 4–6 ng/ml for the following 6 months. Prior to revascularization of the graft during the operation, 1.0 g intravenous methylprednisolone was administered, and daily boluses of 0.5 g intravenous methylprednisolone were given for 3 days post-transplantation. Oral prednisone was then given at a daily dose of 25 mg, which was gradually decreased to 5 mg daily after 3 months postoperation.

Assessment of rejection

Patients remained in hospital for 15 days postoperatively and were then followed up in the outpatient clinic once a week for 3 months, twice a month for 3–6 months and once a month after 6 months.

Delayed graft function was defined as urine output <40 ml/h or failure of the serum creatinine to fall to ≤115 mol/l within 7 days postoperatively. Any patient with ≥10% increase in serum creatinine per day was monitored using daily colour Doppler imaging (CDI) until acute rejection was confirmed or excluded. A real-time ultrasound-guided kidney biopsy was performed if CDI showed blood flow rarefaction in the transplanted kidney and a resistance index >0.8. Histopathology slides were evaluated and scored according to the Banff 2003 classification.¹¹

Acute rejection was treated with 0.5 g methylprednisolone intravenously daily for 3 days; rejection resistant to steroids was treated with anti-CD3 monoclonal antibody (OKT-3, Ortho Biotech, Bridgewater, NJ, USA).

Biopsy-proven episodes of acute rejection, infectious complications and serious adverse events were recorded.

Infection prophylaxis

All patients, regardless of their cytomegalovirus status, received intravenous ganciclovir (5 mg/kg twice a day) while in hospital and oral ganciclovir (500 mg three times a day) for 2 months as outpatients. Fungal prophylaxis was given in the form of 50 mg micafungin daily for 10 days. In addition, 80 mg trimethoprim and 400 mg sulphamethoxazole were given orally, daily, for 3 months as prophylaxis against Pneumocystis carinii and other bacteria.

Patients were monitored for viral infections for 6 months after transplantation using monthly polymerase chain reaction assays for cytomegalovirus, Epstein–Barr virus, parvovirus B-19 and polyomavirus (BK virus) performed on whole-blood specimens using standard procedures.

Statistical analyses

Data were presented as the mean ± SD for continuous variables and as the percentage frequency distribution for categorical variables. Student’s t-test was used to analyse PRA levels; χ² test was used to compare the HLA matching results. Reported P-values were two-sided and a P-value < 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS® software, version 15.0 (SPSS Inc., Chicago, IL, USA).

Results

A total of 39 (11.1%) out of the 351 patients registered in the Department of Renal Transplantation during the study period were found to be sensitized. Of these, 32 consented to receive the desensitization protocol. Baseline patient characteristics are shown in Table 1.

Table 1.

Baseline characteristics of 32 sensitized patients, defined as those having panel reactive antibody (PRA) levels >10%, awaiting kidney transplantation.

Characteristic	Value
Age, years
Mean ± SD	41.8 ± 11.5
Range	18–68
Sex, n (%)
Male	14 (43.8)
Female	18 (56.3)
Type of dialysis, n (%)
Haemodialysis	29 (90.6)
Peritoneal	3 (9.4)
Primary disease, n (%)
Chronic glomerulonephritis	22 (68.8)
Diabetic nephropathy	4 (12.5)
Hypertension	3 (9.4)
Polycystic disease	2 (6.3)
Immunoglobulin A nephropathy	1 (3.1)
Blood transfusions, n (%)
0	5 (15.6)
1	11 (34.4)
≥2	16 (50.0)
Previous transplants, n (%)
0	28 (87.5)
1	4 (12.5)

Desensitization

In the 32 patients who consented to receive the desensitization protocol, PRA levels ranged between 12% and 92% (mean ± SD 37.1 ± 21.4%); of these, 11 had raised levels of PRA I, 14 had raised levels of PRA II, and seven had raised levels of both PRA I and II. All 32 patients were treated with MMF for 1.5–13 months. Fifteen patients with a PRA level >30% underwent plasmapheresis between three and seven times (mean ± SD 3.81 ± 1.55 times).

Mean PRA levels after the desensitization protocol were 17.3 ± 12.4%, which represented a significant decrease from the pre-desensitization level (P < 0. 001). PRA levels pre- and post-desensitization are given in Table 2. The PRA level was<20% in 12 patients (37.5%) before desensitization and in 27 patients (84.4%) after desensitization (P < 0.01). No patients had any desensitization-related side-effects.

Table 2.

Panel-reactive antibody (PRA) levels pre- and post-desensitization in 32 sensitized patients awaiting kidney transplantation.

Time period	PRA level
Time period	0–10%	11–20%	21–40%	40–60%	>60%
Pre-desensitization, n	0	12	10	6	4
Post-desensitization, n	12	15	2	2	1

Of the 32 patients, 26 (81.3%) underwent successful renal transplantation; 15 received kidneys from cadaveric donors and 11 received kidneys from living donors. The cold ischaemia time for the transplanted organs was 1–12 h in 16 transplants and 12–24 h in 10 transplants. The remaining patient with a PRA level <20% was still waiting for a suitable transplant.

HLA matching

Mismatch numbers between recipient and donor using traditional HLA matching and HLA amino acid residue matching for the 26 transplant patients are shown in Table 3. The proportion of patients with a 0–1 mismatch between recipient and donor was 7.7% for traditional HLA matching and 65.4% for HLA amino acid residue matching (P < 0.001). Using residue matching, 25 of the 26 patients had a mismatch number of 0–3 and one had a mismatch number of 4.

Table 3.

Mismatch numbers using traditional human leucocyte antigen (HLA) matching and HLA amino acid residue matching in 26 sensitized patients who received a kidney transplant following desensitization.

Mismatch number	Traditional HLA matching	Residue matching
0	0 (0)	5 (19.2)
1	2 (7.7)	12 (46.2)
2	4 (15.4)	6 (23.1)
3	11 (42.3)	2 (7.7)
4	5 (19.2)	1 (3.8)
5	3 (11.5)	—
6	1 (3.8)	—

Data presented as n (%) of patients.

Rejection

After surgery, all 26 patients showed immediate graft function without hyperacute rejection. Biopsy-proven acute rejection episodes occurred in three (11.5%) patients. Two of these rejections occurred within the first month after transplantation; these were shown to be due to T-cell-mediated rejection and were reversed with methylprednisolone and OKT3 treatment. One patient developed C4d + antibody-mediated rejection 6 months post-transplant; he was treated successfully with methylprednisolone and ATG, and his creatinine level is currently 220–250 µmol/l.

Patient follow-up

All patients receiving transplants were followed for ≥12 months (mean ± SD 21.6 ± 6.5 months, range 12–33 months); changes in creatinine levels during this period are shown in Figure 1. Four of the 26 patients (15.4%) experienced a significant increase in creatinine levels. In two patients this was due to renal toxicity associated with the calcineurin inhibitor tacrolimus; in the other two patients it was due to a ureteral stone and a graft artery anastomosis, respectively. The creatinine level decreased to within the normal range after appropriate treatment.

Figure 1.

Creatinine levels in 26 patients following kidney transplantation; these patients were enrolled in a study undertaken in sensitized patients, defined as those having panel reactive antibody (PRA) levels >10%. Data presented as mean ± SD for continuous variables.

Infections developed in seven patients (25.9%): pulmonary infections occurred in four patients and urinary tract infection in three patients. All infections were successfully treated with antibiotics.

Discussion

Patients become sensitized after exposure to HLAs from another person. Approximately 15% of men are sensitized by transfusions and approximately 40% of women are sensitized by pregnancies or transfusions before their first graft.¹² The HLA-sensitized rate in the present study (39/351, 11.1%) was lower than that reported in other studies.^5,12 There are two possible reasons for this difference: first, few patients waiting for renal transplantation receive blood transfusions in our centre, and, secondly, the assay used in the present study to determine PRA levels was designed for Western patients, so may not be as accurate in a Chinese population.

Several protocols have been proposed to facilitate successful transplantation of sensitized renal allograft recipients; however, no single method has been shown to be superior. Intravenous gammaglobulin (IVIG) has been demonstrated to improve transplant rates and outcomes in highly HLA-sensitized patients.^3,13 However, IVIG is expensive and most patients, especially in China, cannot afford this treatment. Moreover, the use of high-dose IVIG has some limitations: approximately 10% of patients are non- or incomplete responders, and IVIG may interfere with PRA assays, making it difficult to measure the PRA level after its use.^14,15 Other techniques, including B-cell elimination with alemtuzumab, newer immunosuppressive agents, rituximab, protein A absorption, plasma exchange and MMF, have been investigated in clinical studies.^14–17

The relative safety and efficacy of MMF as a desensitization agent has been confirmed in clinical research. Kawase et al.¹⁸ reported that a third graft was successfully performed in a highly sensitized recipient (PRA level >90%) after desensitization using preoperative MMF and plasmapheresis. MMF has been shown to reduce the HLA alloimmune antibody response to valved allograft implantation and to achieve suppression of PRA to <20%.^19,20 A study involving 154 transplant recipients showed that MMF reduced anti-HLA class I and II antibody production after renal transplantation.²¹

Plasmapheresis can be used to decrease HLA antibodies in the pretransplant period, which may allow transplantation to occur. Its use has decreased the incidence of hyperacute rejection,²² but the reduction in PRA level seen with plasmapheresis alone is usually transient and is not effective for high-titre antibodies.¹⁴ Therefore, in the present study, preoperative plasmapheresis was combined with MMF to reduce the PRA level; PRA levels in 27 out of 32 patients (84.4%) were decreased to <20% and no desensitization-related side-effects were reported.

Before transplantation, the major goal of the desensitization protocols is to reduce the effective antibody levels to a point where hyperacute rejection is avoided. After transplantation, the main goal is to keep PRA levels low in order to prevent acute rejection. In the present study, ATG and HLA amino acid residue matching were used to decrease the production of new donor-specific antibodies. Both ATG and basiliximab induction therapy have been shown to achieve similar 1-year graft/patient survival rates; however, ATG was associated with a much lower acute rejection rate than basiliximab.²³ Donor–recipient HLA compatibility plays an important role in organ transplantation. However, only a small proportion of transplants are matched for the six antigens included in the traditional HLA matching process due to the high level of HLA polymorphism. The ultimate goal of a successful transplant is to distinguish acceptable from unacceptable HLA mismatches among potential donors. It has been suggested that compatibility for HLA amino acid residue matching is as advantageous as traditional six-antigen matching.²⁴ Residue matching also allows a greater number of patients access to well-matched grafts. In the present study, the proportion of patients with a 0–1 mismatch between recipient and donor was improved (from 7.7% to 65.4%) with residue matching, and 81.3% of the sensitized patients subsequently underwent successful transplantation.

In the present study, the use of a novel desensitization protocol using MMF plus ATG with or without plasmapheresis increased the proportion of sensitized patients eligible for kidney transplantation. There was a significant fall in PRA levels after the desensitization protocol, with 84.4% having levels <20%, and no desensitization-related side-effects were reported. This compares favourably with the results of Stegall et al.,¹⁴ who reported desensitization rates of 38% with high-dose IVIG, 84% with plasmapheresis, low-dose IVIG and anti-CD20, and 88% with plasmapheresis, low-dose IVIG thymoglobulin and post-transplant donor-specific alloantibody monitoring. In the present study, 81.3% of the patients subsequently underwent successful transplantation, which is higher than the transplantation rates reported using rituximab alone (11.1%),⁵ immunoadsorption alone (44.4%)¹⁷ or rituximab plus IVIG (80%).¹⁵ When HLA amino acid residue matching was used, the proportion of patients with a 0–1 mismatch between recipient and donor was improved from 7.7% to 65.4%. After surgery, all recipients showed immediate graft function and no hyperacute rejection occurred. Biopsy-proven acute rejection episodes occurred in 11.5% of patietns, and all such episodes were reversed with treatment. This is lower than acute rejection rates reported in patients using alemtuzumab or ATG (18.2% and 27.3%, respectively),²⁵ or IVIG plus daclizumab, or IVIG plus thymoglobulin (36% and 31%, respectively).²⁶ Pulmonary or urinary tract infections developed in seven patients, which were successfully treated with antibiotics. Therefore, the new protocol presented in this study effectively reduced the PRA level and was associated with a high rate of successful kidney transplantation in sensitized patients.

In conclusion, a desensitization protocol of MMF plus ATG with or without plasmapheresis, complemented with HLA amino acid residue matching, seems to be a promising strategy to increase the proportion of sensitized patients eligible for renal transplantation and was associated with a high rate of successful transplant procedures. The protocol was well tolerated and was associated with few infections. Larger and longer trials are needed, to assess the safety and efficacy of this approach in more detail.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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Panel-reactive antibody levels and renal transplantation rates in sensitized patients after desensitization and human leucocyte antigen amino acid residue matching

Abstract

Objective

Methods

Results

Conclusion

Keywords

Introduction

Patients and methods

Patients

Desensitization

HLA matching

Immunosuppressive therapy

Assessment of rejection

Infection prophylaxis

Statistical analyses

Results

Desensitization

HLA matching

Rejection

Patient follow-up

Discussion

Footnotes

Declaration of conflicting interest

Funding

References