Donor-directed human leukocyte antigen (HLA) antibodies (DDA) newly formed after transplantation have been shown to result in poor graft outcome (1–4). HLA antibodies have been demonstrated even before transplant rejection (5, 6). The results of these studies led to the suggestion that demonstrable HLA antibodies in recipients after transplantation are predictive of graft outcome and present a useful tool in monitoring of graft function.
DDA detected by Luminex bead-based assays were demonstrated in posttransplant sera and also on transplant eluates (7–10). DDA fixed to the transplant can lead to a possible underestimation of HLA antibodies in posttransplant sera of recipients with their graft in situ. The high number of recipients that remained antibody negative after transplantation, but turned positive after transplantectomy, arose our interest. We wondered whether in addition to posttransplant events, also patient and graft characteristics might be related to DDA positivity. The results on patients with a transplantectomy within 1 month after grafting were reported previously (11).
In contrast to many reports in the literature, we have not been able to demonstrate HLA antibodies before graft failure in earlier studies using conventional screening methods. The present study was designed with the hypothesis that antibodies before transplant failure would be detectable when using more advanced and sensitive methods. At present, the most sensitive antibody screening technique is the Luminex single antigen assay (LSA). Only patients with no DDA before their first transplantation were included. Production of DDA, time of appearance, and antibody class were analyzed. Antibody production was correlated with matching grade, donor and patient characteristics.
MATERIALS AND METHODS
From 541 patients transplanted with a first kidney graft between January 1992 and January 2005 at the University Hospital of Maastricht, 483 were HLA antibody negative in complement-dependent cytoxicity (CDC) and ELISA before transplantation. Ninety-six of them underwent transplantectomy before 2005. When tested by flow cytometric (FC) screening, another 27 proved HLA antibody positive before transplantation. From 13 recipients no serum samples were available. Therefore 56 patients were included in the study. They were analyzed taking into account the time of transplantectomy and divided into groups I (transplantectomy <1 month), II (1–6 months), and III (>6 months). Pretransplant patient characteristics are given in Table 1. Clinical follow-up was continued until 6 months after transplantectomy.
The immunosuppressive regimen was calcineurine inhibitor-based in all recipients; tacrolimus (TAC) in 38 recipients and cyclosporine A (CsA) in 18. Additional immunosuppression to TAC was prednisone (PRED) in 34, mycophenolate mofetil in 12, daclizumab in one, and sirolimus rapamycine (RAPA) in nine patients. In addition to CsA, PRED was added in 18 and azathioprine in two recipients. CsA levels were determined in whole blood by enzyme monoclonal immuno test (Dade Behring, Newark, DE) or high-performance liquid chromatography-MS/MS. In the first 3 months after transplantation, target CsA trough levels for CsA+PRED-treated recipients were 0.15 to 0.20 mg/L and for CsA+PRED+azathioprine-treated recipients 0.10 to 0.15 mg/L. TAC trough levels were measured in whole blood by Imx (Abbott) or high-performance liquid chromatography-MS/MS, target levels were 15 to 20 ng/mL for week 1 and 2, 10 to 15 ng/mL for week 3 and 4, thereafter tapering to 5 to 7 ng/mL. Immunosuppression was cessated after removal of the graft.
Clinical Outcome Parameters
A biopsy was taken during surgery 1 hr after reperfusion for every transplant. Rejection was defined as any rejection treatment within 3 months after grafting and was proven by needle core biopsy. Treatment consisted of three doses of methylprednisolone (0.5 to 1.0 g/dose) in 15 patients, two of which received additional doses of anti-thymocyte globulin (ATG). Sixteen of 56 recipients received rejection treatment or had positive rejection histology in the explanted graft according to the BANFF criteria. Because of the retrospective nature of the study, no material for C4d staining was available.
Nontransfused patients received one leukocyte-poor protocollary blood transfusion before being placed on the transplant waiting list. After transplantation, sera were drawn weekly during hospitalization, monthly in the first half year, and yearly thereafter. After transplantectomy, sera were collected at week 0, 2, 4, and 6 and at month 2, 3, 4, 5, and 6. Sera were stored at −30°C and centrifugated for 10 min at 10.000g before testing. Per patient, the pretransplant serum, a posttransplant but pretransplantectomy serum, and two posttransplantectomy sera drawn at week 6 and at months 2 to 6 were analyzed.
HLA Antibody Analysis by Luminex Single Antigen Assay
Specificity of HLA class I and II IgG antibodies in recipient sera was determined using the LABScreen SA assay (One Lambda, Canoga Park, CA) according to the manufacturer’s instructions. Microbeads coated with purified HLA were incubated with patient serum for 30 min. After washing to remove unbound antibody, the beads were incubated for 30 min with anti-human IgG-conjugated phyco-erythrine. All incubations were performed on a gently rotating platform in the dark at room temperature. The LABScan 100 flow analyzer (Luminex, Austin, TX) and HLA-Visual software (One Lambda) were used for data acquisition and analysis. All beads with a normalized median fluorescence intensity (MFI) value (i.e., raw MFI value of the test bead−MFI value of the negative control bead) more than 2000 were defined as positive. This cutoff point was chosen because MFI more than 2000 is associated with a positive FC crossmatch (9, 12).
Before transplantation, three complement-dependent crossmatches were performed: the standard NIH crossmatch, standard NIH with dithiothreitol to reduce IgM antibodies, and a two-color fluorescence crossmatch. Sera used for the final crossmatches were the pretransplant serum drawn at the time of transplantation, the last serum tested for anti-HLA antibodies in the quarterly screening, and all relevant positive historical samples. A negative class I dithiothreitol crossmatch was mandatory for transplantation.
Center Policy of Tissue Typing
During the investigation period, the patients and donors were typed for HLA-A, B, DR and DQ by serology and polymerase chain reaction using sequence-specific primers. HLA antigens were considered unacceptable for a patient if antibodies against the specificity had ever been demonstrated, at the time of transplantation or in the past. Mismatches from previous transplants were excluded, as were the paternally inherited antigens of children of female patients. All transplants were performed under the auspices of Eurotransplant, which means that only HLA A, B, and DR matches were included in the allocation algorithm.
Statistical analyses were performed by using Statistical Package for the Social Sciences software (SPSS, version 15.0 for Windows).
Nonparametric tests were performed when indicated. A P value less than 0.05 was considered to be statistically significant. The suspected risk factors for total HLA antibody positivity and for HLA class I and class II positivity separately, that were tested as independent variables in stepwise backward logistic regression were as follows: “mismatch HLA class I (0 vs. ≥1)”, “mismatch HLA class II (0 vs. 1 vs. ≥2)”, “recipient gender (male vs. female)”, “donor age (years)”, “recipient age (years)”, “acute rejection within 3 months (0 vs. 1)”, “donor type (living vs. postmortal)”, for “postmortal donor (heart beating vs. nonheart beating),” and “time of transplantectomy (group I–II–III)”. P value for removal in the model of the possible independent variables was set at P=0.05. All interaction factors were introduced separately into the final model.
Transplantectomy was performed in 56 first kidney graft recipients, who were all HLA antibody negative before transplantation based on screening by CDC, ELISA, and flow cytometry. Their sera were now analyzed with LSA. Three of 56 recipients proved to possess DDA in their pretransplant serum; one patient was class I and two patients were class II DDA positive. They were excluded from further analysis. The patient characteristics of the remaining 53 recipients are given in Table 1.
Non-DDA specificities were demonstrated in the pretransplant sera of 20 of 53 recipients (38%). They were directed against class I in 55%, class II in 5%, and both class I+II in 40% of the recipients. Specificities of non-DDA in pretransplant sera could be explained by transfusions and pregnancies. Possession of non-DDA was not an indication for exclusion from the analysis. After transplantation, non-DDA were demonstrated in 46 of 53 recipients (87%), class I in 65%, class II in 11%, and class I+II in 24% of the recipients.
After transplantation, 10 of 53 recipients (19%) stayed DDA negative after transplantation and transplantectomy. DDA positivity was demonstrated in 43 recipients (81%) (Table 2). Seven of 43 recipients (16%) showed DDA before transplantectomy (two class I and five class II), whereas in 36 (84%) they appeared thereafter. After transplantectomy, 37% of the patients turned positive in the 6-week serum, whereas 100% were found positive in the 2- to 6-month serum.
HLA DDA Antibodies
HLA class I and II antibody specificities were analyzed in the 43 patients, who became positive after transplantation. DDA antibodies directed against class I were detected in 23% of the recipients, against class II in 16%, and against both class I and II in 61% of the positive recipients. DDA after transplantectomy were directed against class I, II, and I+II in 25%, 14%, and 61%, respectively. The specificities of the DDA are shown in Table 3.
DDA and Rejection
Of the 16 recipients diagnosed with acute rejection, 15 (94%) had posttransplant DDA versus 28 of 43 (65%) patients without rejection (P=0.12). In rejectors, DDA were detected before transplantectomy in four and in 11 thereafter. Three nonrejectors had DDA before and 25 after transplantectomy. This difference in DDA detection before and after transplantectomy was again not statistically significant (P=0.09). The antibody class of the DDA detected in rejectors and nonrejectors is explained in Table 4. In rejectors, a higher incidence of combined class I+II DDA was found (P=0.16).
Time of Transplantectomy
Time of transplantectomy ranged from 1 day to more than 10 years after transplantation. Differences in demographic and clinical parameters depending on time of transplantectomy were explored by means of nonparametric statistical tests. Patients were divided into 3 groups: I transplantectomy less than 1 month (n=23), II 1 to 6 months (n=13), and III more than 6 months (n=17). The patient characteristics are given in Table 1. Group II proved to be significantly different for HLA class II mismatch (P=0.02) and occurrence of acute rejection (P<0.001). There was a trend toward a difference in the number of nonheart beating (NHB) donors (P=0.07). The number of patients becoming DDA positive in group I was 78%, in group II 100%, and in group III 71% (P=0.11) (Table 2).
Logistic Regression Analysis
To investigate the relationship between DDA positivity after transplantectomy and clinical parameters, such as matching grade, donor and patient characteristics, backward logistic regression analysis was performed. Outcome parameters were total DDA (class I or II or both) positivity, DDA class I positivity, and DDA class II positivity after transplantectomy. Outcome parameters tested for included the following: recipient age, recipient gender, donor age, donor type (living vs. cadaver), HLA class I or II mismatch, acute rejection within 3 months after transplantation, time of transplantectomy group I-II-III, and immunosuppressive regimen (TAC vs. CsA).
With total DDA positivity as outcome parameter, the only risk factor included in the final model (P=0.008, χ2=9.76, df=1, r2=0.27) was donor age (year) (Odds ratio=1.053, P=0.008). This can be interpreted as follows: each 1 year increase in donor age increases the risk of DDA positivity with 5.3%. The positive and negative predictive values of the model are 70%.
With class I DDA positivity as outcome parameter, the risk factors included in the final model (P<0.0001, χ2=23.05, df=3, r2=0.48) were donor age (Odds ratio=1.060, P=0.007) and donor type (NHB) (Odds ratio=32.080, P<0.0001). This means that each 1 year increase in donor age increases the risk of DDA positivity with 6.0% and patients receiving a kidney from a NHB donor are 32 times more at risk for DDA positivity than patients receiving a kidney from a heart beating donor.
With class II DDA positivity as outcome parameter, risk factors included in the final model (P=0.003, χ2=13.91, df=3, r2=0.31) were donor age (year) (Odds ratio=1.039, P=0.03) and HLA mismatch class II (0–1 vs. ≥2) (Odds ratio=5.307, P=0.01). Thus each 1 year increase in donor age increases the risk of DDA positivity with 3.9% and patients receiving a kidney with two or more HLA class II mismatches are five times more at risk for DDA positivity than patients receiving a kidney with less than two HLA class II mismatches.
The positive and negative predictive values of the models are given in Table 5.
Definition of HLA antibody specificities has become much more accurate by the use of increasingly sensitive antibody screening methods. After CDC and ELISA, flow cytometry proved to be the more sensitive method, equaled now by the Luminex bead based assays (12), particularly the single antigen bead assay. Although the clinical relevance of DDA detected by LSA is not yet clear (13), the pressure on laboratories to use it for clinical purposes and data presentation is growing. Testing by LSA screening demonstrated de novo DDA after transplantation in 81% of first transplant recipients, who were all DDA negative by LSA before transplantation. Antibodies were detected before transplantectomy in only 13% of the recipients; the majority was detected after transplantectomy. These findings are in contrast with several reports in the literature, where HLA antibodies have been shown to be associated with acute and chronic rejection (2, 14) and poor graft survival (15). The results of these studies led to the hypothesis that detection of HLA antibodies in patients after transplantation was a useful early predictor of graft outcome (16). The present findings could not confirm this view, which might be due to the selection of the patient group (transplantectomy of first graft patients only), the pretransplant antibody status of the recipients (DDA negative before transplantation by LSA), and the immunosuppressive protocol (calcineurine inhibitor-based). From 16 patients with rejection, 11 turned antibody-positive only after transplantectomy. Because only LSA DDA-negative first transplant recipients were studied, the appearance of DDA is considered to be related to the renal transplant.
DDA after transplantectomy were directed against HLA class I+II in 61%, class I only in 25%, and class II only in 14% of the recipients. The majority of the recipients showed the presence of combined class I and II antibodies. The subdivision in antibody class differed considerably between different studies; 32%, 21%, and 47% were found in a study of chronic rejection, which was performed using FC and Luminex methods (17). In two studies using ELISA for HLA antibody detection, 22%, 61%, and 17% (5) and 20%, 38%, and 43% (18) were found, respectively. The inclusion criteria for the patients were different in the articles mentioned.
Of the DDA positive recipients, 98% also showed non-DDA. Non-DDA pretransplant was shown in 38%, 62% demonstrated non-DDA only posttransplant. Of the recipients with non-DDA antibodies, 10 were female with a history of pregnancy. Two female recipients, who had been pregnant, showed the presence of non-DDA class I without specific DDA class I antibodies. Both women were proven DDA- negative after transplantation by LSA, the non-DDA were directed against a paternally inherited antigen from their children. Because these antigens were excluded in the kidney donor, this shows that antibody production against paternal antigens can be (re-) stimulated by a nonspecific stimulus (19, 20). For class II, the same phenomenon was observed in one female recipient. In two male recipients, the non-DDA were directed against antigens from a previous blood transfusion.
Mao et al. (21) reported that 77% of recipients with failed allografts had both DDA and non-DDA in their serum. They concluded that the majority of these non-DDA antibodies were directed against “donor-specific epitopes”. The immunogenicity of a kidney graft is related to differences in amino acid sequences of HLA molecules between donor and recipient, these differences or “epitopes” are considered to be the basic units of immunogenicity (22). In the study of Piazza et al. (23), only 9% of recipients developed class I alloantibodies with specificity restricted to the mismatched HLA class I antigens of the graft, in 90% of their study population a broad HLA sensitization was found, supporting the idea that non-DDA are elicited together with DDA in response to a donor-specific epitope.
The DDA appeared after transplantectomy in 84% of the positive recipients within 6 months, 37% was demonstrable at 6 weeks. Antibody detection after approximately 4 months was previously described for patients transplantectomised within 1 month after grafting (11). In the present group of recipients with a median time of transplantectomy of 50 days (range 2–3619), the findings were similar. Removal of the kidney transplant itself, probably in combination with cessation of immunosuppression after transplantectomy, is the probable event leading to antibody detection (24).
Recipients were divided into three groups according to the time of transplantectomy; within 1 month after grafting, between 1 and 6 months, and after more than 6 months. The first group predominantly consists of technical failures and primary nonfunction, in the second acute rejection is the major reason for graft loss, and in the third the number of acute rejections is comparable with the overall transplant group and graft loss is mainly due to chronic allograft nephropathy. When the characteristics of the groups are compared, HLA-DR mismatch and acute rejection episodes proved significantly different between the groups. The highest numbers of acute rejections and NHB donors were found in patients with early graft failure (groups I and II) and as a result early transplantectomy (<6 months after transplantation). In recent years, more NHB donors are transplanted in the Netherlands. Recipients of NHB donors experience more primary nonfunction, more rejection, and more early failures (25–27). The number of class II mismatches is highest in patients with transplantectomy less than 6 months (groups I and II). It is well known that class II mismatches are associated with a worse graft survival (28). The number of patients who turn DDA positive for HLA is also the highest in group II.
In an attempt to determine which factors play a role in the formation of HLA class I or II antibodies, we performed backward regression analysis. For total DDA positivity, an increase in donor age was related to an increase in DDA positivity. A higher donor age related to DDA positivity has also been shown for a group of recipients with transplantectomy within 1 month, as reported previously (11). Several studies have reported on the relation between higher donor age and increase in immunogenicity (29, 30). A mechanism called the “injury response” was suggested as explanation by Halloran et al. (31).
Factors related to DDA positivity could be different for class I and class II positivity and actual relationships might be obscured by the above analysis. Therefore the analysis was repeated for DDA class I positivity and DDA class II positivity separately.
For DDA class I positivity, donor type and donor age were statistically significant in the final model. Particularly, recipients of NHB donor grafts had a high risk for DDA positivity. The effect is most probably multifactorial, NHB grafts have a longer first warm ischemic time, and up-regulation of major histocompatibility complex and shedding of antigen might be higher in those grafts that often show primary nonfunction. But care should be taken in drawing firm conclusions from groups with small numbers, for example, the NHB donors were older and were transplanted into older recipients. No significant differences in DDA positivity were noticed between recipients of a heart-beating (HB) and NHB graft, who failed within 6 months. Because only few patients without class I mismatches were included, the factor “HLA class I mismatch” could not be analyzed. Others investigators, however, did find such a relationship with class I positivity (32).
For class II positivity, the related independent factors were donor age (year) and class II mismatches (DR and DQ). In an Italian study (2), no such correlation between DR mismatch and class II positivity was evidenced. The relation between class II mismatches and DDA class II positivity might be due to the fact that in our study, in addition to HLA-DR also DQ mismatches were included. It has been reported that HLA-DQ, rather than HLA-DR, is targeted by the alloantibody response, proving the importance of HLA-DQ antibodies (28, 33). For DDA class II positivity, matching for class II antigens might be of importance especially in high risk transplants, for example, from older donors and NHB grafts, as they have a higher failure rate.
In conclusion, our hypothesis that antibodies before transplant failure, which we were unable to demonstrate in earlier studies, would be detectable when using more advanced and more sensitive methods, proved not to be valid. De novo DDA HLA antibodies after transplantation were demonstrated in 81% of first transplant recipients, who were all DDA negative by LSA before transplantation. Antibodies were detected before transplantectomy in only 16% of the recipients; most of them were detected thereafter. The frequency of class I and II antibodies was approximately the same; the majority of the antibodies was found combined. NHB donor type and higher donor age is related to DDA class I positivity, whereas worse class II match and higher donor age are related to antibody class II positivity. These findings may have to be taken into consideration in the allocation of organs of marginal donors such as older or NHB kidneys.
The authors thank Mrs Diana van Bakel for her excellent assistance in preparing the manuscript and Dr F. Nieman for his advice with the statistical analysis.
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