Objectives: The use of highly sensitive Luminex technology to assess the immunologic risk of renal transplant candidates have greatly added to the ability of stratifying patients and have paved the way to avoid hyperacute antibody-mediated rejection. Our aim was to understand how much the testing for pretransplant anti-HLA antibodies affects the decision for transplant and survival at 1 year posttransplant.
Materials and Methods: From January 2014 to June 2017, 336 transplant candidates were tested by complement-dependent cytotoxicity and by the Luminex platform for anti-HLA antibodies in our nephrology and renal transplant center (The Medical City, Baghdad, Iraq). All clinical and laboratory data were noted. Our program is a living-donor, blood group-compatible donor program. All transplant patients (sensitized or not) were followed for 1 year, and the Kaplan-Meier method was used to determine survival rate.
Results: Mean age of the study group was 34.07 ± 12.4 years. Of 336 transplant patients who were tested, there were 63 (18.75%) sensitized patients and 159 (47.35%) nonsensitized patients. Blood transfusion was the main cause of sensitization. Class I anti-HLA antibodies were detected in 54 of 63 sensitized patients (85.7%), and class II anti-HLA antibodies were detected in 39 of 63 sensitized patients (61.9%). Donor-specific antibodies were detected in 33/63 (52.3%). Thirteen sensitized patients (15.3%) underwent transplant. No incidences of hyperacute rejection were recorded. The 1-year survival of the nonsensitized patient group was 90%, whereas survival was 61.5% for the sensitized patient group.
Conclusions: Pretransplant testing for anti-HLA antibodies is undoubtedly useful for assessment of patient risk, to facilitate decisions regarding patient and donor selection, and to plan treatment strategies. The 1-year survival for sensitized patient was lower than for nonsensitized patients. More knowledge, experience, technology advancements, and support are needed to improve the Iraqi practice of trans-planting sensitized patients.
Key words : Anti-HLA antibodies, Iraq, Kidney transplant
Human leukocyte antigen (HLA) testing and immunology evaluation of organ recipients and their respective donors have been greatly developed since the 1960s work of Terasaki and coworkers.1,2
The microcytotoxicity test developed by Terasaki and McClelland in 1964 was the international standard test for HLA typing for more than 30 years. Although some laboratories still use serologic HLA typing as a supplemental technique, it is now more common to type individuals by DNA-based methods. Three basic methods are used in conjunction with polymerase chain reaction: sequence-specific oligo-nucleotide probes, sequence-specific primers, and sequencing-based typing.2,3
HLA typing is not the only tool for pretransplant immunology evaluation. The other tool is the test for pretransplant sensitization. This has been achieved through testing for anti-HLA antibodies and donor-specific antibodies (DSAs). Prior sensitization develops from exposure to allogeneic HLA antigens during the course of pregnancies, through exposure to blood transfusions, or, increasingly, because of a failed transplant. Patients who have circulating HLA antibodies are at high risk of hyperacute rejection or of accelerated acute rejection that is not easily controlled with immunosuppression. The clinical relevance of the humoral response was clearly demonstrated by the results of the first crossmatch by Patel and Terasaki in 1968 with the determination that early antibody-mediated tissue injury is caused by the binding of anti-HLA antibodies to the endothelial cells.4,5
This complement-dependent lymphocytotoxicity assay was the earliest method for HLA antibody screening. It has been confounded by issues of sensitivity and accuracy. The development of solid-phase antibody detection assays, including flow cytometric methods and the x-MAP multiple bead-based (Luminex, Austin, TX USA) technology, has revolutionized the detection and definition of HLA antibodies with a high degree of sensitivity and accuracy.1
The accurate and robust detection of such anti-bodies is of paramount importance. The presence of preformed HLA antibodies restricts the number of compatible donors for the sensitized patient to those who do not express the HLA antigens to which the patient is sensitized. Sensitized patients often must wait substantially longer for a crossmatch-compatible kidney. HLA antibodies have been shown to be strongly predictive of long-term graft failure with the risk of developing chronic antibody-mediated rejection, transplant glomerulopathy, and chronic allograft dysfunction.6,7
The Luminex antibody screening technology consists of a series of polystyrene microspheres (beads), which contain fluorochromes of differing intensity embedded within the bead. These give each group of beads with an HLA molecule or molecules derived from lymphoblastoid cell lines attached a unique signal. Patient serum is incubated with a mixture of beads, washed and bound antibody is detected by adding fluorescently labeled antihuman immunoglobulin G, and fluorescence is measured in a Luminex flow cytometer or a similar device. Interpretation of the test results is based on com-parisons of median fluorescence intensity measure-ments of the test serum to those of positive and negative serum controls. Neither viable lymphocytes nor complement fixation is required.8
There are 3 levels of bead attachment. The first level consists of beads bound with a large number of class 1 or class 2 molecules; these essentially provide a positive or negative result. At the second level, the bead is equivalent to a cell, with each bead containing 2 molecules derived from 2 alleles at each loci: HLA-A, HLA-B, and HLA-C in the case of class I and HLA-DR and HLA-DQ in the case of class II. The third level consists of beads with 1 HLA molecule attached (either class I or II), which is referred to as a single antigen bead. This third level is particularly useful for characterizing complex sera with high panel reactive antibodies, accurately defining the antibodies present.9
In Iraq, our unit was the first to start Luminex technology for evaluation of renal transplant candi-dates on January 2014. Here, we present the results of Luminex testing of 336 couples from January 2014 to June 2017. Our objective was to understand how much the testing for pretransplant anti-HLA antibody could affect the decision of transplant and the 1-year survival.
Materials and Methods
Settings and study design
A cross-sectional study was conducted at the nephrology and renal transplant center (The Medical City of Baghdad) for the period from January 2014 to June 2017.
We tested 336 couples consecutively with no exclusion in the tissue typing and immunology laboratory in our center. A living-donor program is the sole type of practice in Iraq, and there is no waiting list or historic sera to be tested.
Complement-dependent cytotoxicity testing
In our laboratory, patient samples are tested using a well-characterized selected panel of cells from 20 donors, which includes most HLA antigens. Our technique is consistent with the standard National Institutes of Health method,2 with 30 minutes with patient serum and target lymphocytes followed by 60 minutes with complement. Lymphocytes were isolated from fresh blood according to the procedure by Boyum and associates.10
Sera were tested with LIFECODES LifeScreen Deluxe-LMX (Gen-Probe-Immucor, Stanford, CT, USA). According to the manufacturer’s instructions, serum obtained by centrifugation of whole blood was incubated with a mixture of beads for 30 minutes to allow antigen-antibody binding. After samples were washed with phosphate-buffered saline to remove everything unbound, a secondary antibody-immunoglobulin G conjugated with a fluorochrome-phycoerythrin was added to allow detection of antigen-antibody complex by the Luminex laser system.
Flow cytometry for tissue typing and antibody testing is still not available in Iraq. One sensitized patient had flow cytometry testing, which occurred outside of Iraq. The detection of complement-fixing anti-HLA DSAs and the technology for detecting non-HLA antibodies are also not available.
Transplanted nonsensitized patients received basili-ximab as induction therapy and were maintained on triple therapy of calcineurin inhibitors, myco-phenolate mofetil, and steroids. Sensitized patients were defined as those with a sensitization history and positive immunologic testing (complement-dependent lymphocytotoxicity, Luminex platform, and DSA). Complement-dependent cytotoxicity-positive patients were not transplanted.
Those who had transplant procedures underwent desensitization by intravenous immunoglobulins, plasmapheresis, and rituximab (Figure 1). Antibody testing results were used to make decisions on whether to induce patients with antithymocyte globulin or basiliximab. The target tacrolimus levels were 8 to 11 ng/mL. Prednisone dose at end of month 1 was 5 to 10 mg/day. No corticosteroid withdrawal or avoidance was pursued.
Delayed graft function was defined as the need for dialysis during week 1 after transplant. Acute rejection was defined as an acute deterioration in allograft function associated with specific pathologic changes in the graft. All acute rejection episodes were biopsy proven using Banff 2007 criteria. Only 1 patient underwent surveillance biopsy at 3 months; all other patients refused, with allograft biopsies only performed at the time of acute graft dysfunction.
For treatment of acute antibody-mediated rejection, we used pulse steroid, intravenous immu-noglobulin, and plasmapheresis. All patients re-ceived cytomegalovirus prophylaxis for 100 days.
The transplant physician explained the steps of evaluation to the transplant couple. Formal informed consent and signatures were obtained before start of immunologic evaluation. All couples required approval by the central donor accepting committee at the Ministry of Health to exclude any evidence of commercial transplantation.
Data collection and analyses
All data regarding previous sensitizing events were documented, including the number of pregnancies, number and time of blood transfusions, and all previous transplant data for patients presenting for second transplant. Pretransplant and posttransplant clinical, immunologic, and laboratory data were noted, including results of allograft biopsies.
Transplant data at year 1 were collected through review of medical records and computer-based registry data, which included hospital records and transplant clinic follow-up data. Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 21, IBM Corporation, Armonk, NY, USA). The Kaplan-Meier method was used to determine the survival rate.
Baseline patient data are shown in Table 1. Clinical and immunologic data of sensitized patients are shown in Tables 2 and 3. Table 4 shows the transplant status of all patients, and Table 5 shows delayed graft function and rejection in all patients. The 1-year graft survival rate of nonsensitized patients was 90%, whereas the rate of sensitized patients was 61.1% (Figure 2).
This study showed that the information obtained through pretransplant antibody testing is essential for making transplant decisions and for facilitating transplant in sensitized patients through proper selection and well-designed immunosuppression protocols.
Fifty patients of the 63 sensitized patients were below the age of 40 years. This can be explained by the fact that we still have many young patients with end-stage renal disease, inadequate primary prevention, and a tendency in our society to save young beloved relatives through living donations. Our results are consistent with other data, which showed that only 1.2% of patients over a 5-year transplant period were older than 60 years.11 The characteristics of the immune system in younger patients may represent another challenge in tailoring immunosuppression and in planning immune monitoring. Indeed, the risk of antibody-mediated rejection in older patients with moderate sensitization is less compared with patients below 40 years.12
In our sensitized patient group, there were 38 female patients (60.3%). This can be explained by multiparity and probably by the common practice of blood transfusion with delivery. This is consistent with United Network for Organ Sharing (UNOS) data, which showed that 33% of causes of sensitization for panel reactive antibodies/calculated panel reactive antibodies ≥ 98% was due to pregnancy alone or pregnancy with transfusion. The UNOS data confirmed our finding that highly sensitized patients are younger and frequently female.13
When compared with the UNOS data, blood transfusion is a significant cause of sensitization in our practice (28% vs 5%). This can be explained by the practice of blood transfusion for uremic patients with mild to moderate anemia and partly due to irregular provision of erythropoiesis-stimulating agents in Iraq.13
Only 13 of 63 sensitized patients underwent renal transplant with desensitization. Two patients died waiting for transplant. This may indicate improve-ments in dialysis services but can also unfortunately indicate the difficulty in mitigating immune barriers in renal transplant in limited resource situations. This in turn may force patients to seek renal transplant through another way, mostly transplant tourism.
Living-donor transplant after desensitization provided a significant survival benefit for patients with HLA sensitization, compared with waiting for a compatible organ. Desensitization protocols may help overcome incompatibility barriers in living-donor renal transplant.14,15 The negative side of this is the significantly elevated risk for early acute antibody-mediated rejection and inferior graft outcomes associated with presence of DSAs-solid-phase antibodies even with negative pretransplant cytotoxicity and flow cytometric crossmatches.16,17 The 1-year graft survival analysis of our study was consistent with this fact, which showed more antibody-mediated rejection and less graft survival at 1 year (61.5% vs 90%).
In conclusion, pretransplant testing for anti-HLA antibodies is undoubtedly useful for the assessment of patient risk, to facilitate decisions regarding patient and donor selection, and to plan treatment strategies. The 1-year survival rate for sensitized patient was lower than for nonsensitized patients. More knowledge, experience, technologic advance-ments, and support are needed to improve the Iraqi practice of transplanting sensitized patients.
Volume : 17
Issue : 1
Pages : 164 - 168
DOI : 10.6002/ect.MESOT2018.P40
From the Nephrology and Renal Transplant Centre, The Medical City, Baghdad, Iraq
Acknowledgements: The authors have no sources of funding for this study and have no conflicts of interest to declare.
Corresponding author: Ala Ali, Nephrology and Renal Transplant Centre, The Medical City, Baghdad, Iraq
Phone: 96 477 06049163
Table 1. Baseline Characteristics of the Study Group (N = 336)
Table 2. Clinical Characteristics of Sensitized Patients (N = 63)
Table 3. Immunologic Data of Sensitized Patient Group
Table 4. Transplantation Status of the Study Group
Table 5. Delayed Graft Function, Hyperacute Rejection, and Biopsy Proven Acute Rejection in Nonsensitized and Sensitized Patients.
Figure 1. Desensitization Protocol
Figure 2. Kaplan-Meier Graft Survival Analyses at 12 Months in Sensitized and Nonsensitized Patients