Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.

The presence of donor-specific anti-HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long-term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement-dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10-year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10-year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10-year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

Cervicovaginal HPV infection in female renal transplant recipients: an observational, self-sampling based, cohort study.

Immunosuppressive treatment of organ transplant recipients is associated with an increase in the occurrence of human papillomavirus (HPV) related anogenital (pre)malignancies. This cohort study investigated the genotype-specific prevalence of HPV infections in a large cohort of female renal transplant recipients (RTRs). Participants self-collected a cervicovaginal sample for detection and genotyping of HPV. Besides, they completed a questionnaire regarding sociodemographic variables, medical data and sexual behavior. Anogenital screening was offered to all HPV-positive participants. A total number of 218 female RTRs was included. The prevalence of mucosal HPV infections was 27.1% and 17.4% for high risk HPV in particular. The studied cohort showed a broad range of HPV genotypes and multiple HPV genotypes were found in 27.1% of HPV-positive patients. Seven participants were identified with occult premalignant anogenital lesions. In conclusion, this study shows a high point-prevalence of HPV in female RTRs (age-matched West-European general population: 9-10%) with a shift in the distribution of genotypes as compared with the general population. Moreover, a substantial number of patients with occult premalignancies was identified. The introduction of self-sampling for HPV positivity can help in early detection of (pre)malignant anogenital lesions in this vulnerable population.

The PROCARE consortium: toward an improved allocation strategy for kidney allografts.

Kidney transplantation is the best treatment option for patients with end-stage renal failure. At present, approximately 800 Dutch patients are registered on the active waiting list of Eurotransplant. The waiting time in the Netherlands for a kidney from a deceased donor is on average between 3 and 4 years. During this period, patients are fully dependent on dialysis, which replaces only partly the renal function, whereas the quality of life is limited. Mortality among patients on the waiting list is high. In order to increase the number of kidney donors, several initiatives have been undertaken by the Dutch Kidney Foundation including national calls for donor registration and providing information on organ donation and kidney transplantation. The aim of the national PROCARE consortium is to develop improved matching algorithms that will lead to a prolonged survival of transplanted donor kidneys and a reduced HLA immunization. The latter will positively affect the waiting time for a retransplantation. The present algorithm for allocation is among others based on matching for HLA antigens, which were originally defined by antibodies using serological typing techniques. However, several studies suggest that this algorithm needs adaptation and that other immune parameters which are currently not included may assist in improving graft survival rates. We will employ a multicenter-based evaluation on 5429 patients transplanted between 1995 and 2005 in the Netherlands. The association between key clinical endpoints and selected laboratory defined parameters will be examined, including Luminex-defined HLA antibody specificities, T and B cell epitopes recognized on the mismatched HLA antigens, non-HLA antibodies, and also polymorphisms in complement and Fc receptors functionally associated with effector functions of anti-graft antibodies. From these data, key parameters determining the success of kidney transplantation will be identified which will lead to the identification of additional parameters to be included in future matching algorithms aiming to extend survival of transplanted kidneys and to diminish HLA immunization. Computer simulation studies will reveal the number of patients having a direct benefit from improved matching, the effect on shortening of the waiting list, and the decrease in waiting time.

The Maastricht Transplant Center: clinical setting and epitope searches in HLA class II molecules: does the structural localization of a polymorphic site contribute to its immunogenicity?

Our understanding of the immunological processes influencing the clinical outcome after kidney transplantation has advanced majorly over the last few decades. However, many factors still restrict graft and patient survival. Within the Maastricht transplant center we have successfully implemented an alternative immunosuppressive regimen involving Tacrolimus monotherapy in order to minimize the adverse effects associated with long-term use of immunosuppressive drugs. This clinical development has an impact on pre-transplant risk stratification which requires that patients are closely monitored immunologically. In this review we will elaborate on our strategy regarding the analysis of epitopes in HLA-DQ and HLA-DP molecules. In this respect we have also looked at the immunodominance of certain epitopes by assessing their structural localization, conformation and physiochemical properties.

Minor H antigen matches and mismatches are equally distributed among recipients with or without complications after HLA identical sibling renal transplantation.

Studies of the effect of minor H antigen mismatching on the outcome of renal transplantation are scarce and concern mainly single center studies. The International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 16 laboratories of the IHIW, the role of 15 autosomal, 10 Y-chromosome encoded minor H antigens and 3 CD31 polymorphisms, was investigated in relation to the incidence of renal graft rejection and graft loss in 444 human leukocyte antigens (HLA)-identical sibling renal transplantations. Recipient and donor DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, C19Orf48, LB-ECGF-1, CTSH, LRH-1, LB-ADIR and HY. The correlation between minor H antigen mismatch and the primary outcome graft rejection or graft loss was statistically analyzed. The incidence of rejection was very low and no correlation was observed between one or more minor H antigen mismatch(es) and a rejection episode (n = 36), of which only eight resulted in graft loss. In summary, in our study cohort of 444 renal transplants, mismatching for neither autosomal nor HY minor H antigens correlate with rejection episodes or with graft loss.

Preservation of kidneys from controlled donors after cardiac death.

BACKGROUND: Donation after cardiac death (DCD) expands the pool of donor kidneys, but is associated with warm ischaemic injury. Two methods are used to preserve kidneys from controlled DCD donors and reduce warm ischaemic injury: in situ preservation using a double-balloon triple-lumen catheter (DBTL) inserted via the femoral artery and direct cannulation of the aorta after rapid laparotomy. The aim of this study was to compare these two techniques. METHODS: This was a retrospective cohort study of 165 controlled DCD procedures in two regions in the Netherlands between 2000 and 2006. RESULTS: There were 102 donors in the DBTL group and 63 in the aortic group. In the aortic group the kidney discard rate was lower (4·8 versus 28·2 per cent; P < 0·001), and the warm (22 versus 27 min; P < 0·001) and the cold (19 versus 24 h; P < 0·001) ischaemia times were shorter than in the DBTL group. Risk factors for discard included preservation with the DBTL catheter (odds ratio (OR) 5·19, 95 per cent confidence interval 1·88 to 14·36; P = 0·001) and increasing donor age (1·05, 1·02 to 1·07; P < 0·001). Warm ischaemia time had a significant effect on graft failure (hazard ratio 1·04, 1·01 to 1·07; P = 0·009), and consequently graft survival was higher in the aortic cannulation group (86·2 per cent versus 76·8 per cent in the DBTL group at 1 year; P = 0·027). CONCLUSION: In this retrospective study, direct aortic cannulation appeared to be a better method to preserve controlled DCD kidneys.

HLA-DP antibodies before and after renal transplantation.

Human leukocyte antigen (HLA)-DP is considered a target for humoral immune response in clinical transplantation. This study analyses the incidence of HLA-DP antibodies in renal patients. Development and epitope specificity of donor-specific antibodies (DSA) and non-DSA (NDSA) were examined. Pre- and posttransplant sera of 338 patients were screened for HLA-DP antibodies using the luminex single antigen assay. Positive patients, partners and/or kidney donors were HLA-DP typed by sequence-specific oligonucleotides. Potential epitopes were mapped by comparing the amino acid sequences of HLA-DP hypervariable regions (HVR) A-F of recipient, partner and/or donor. Specificities in the sera were aligned to deduce the HVR motif responsible for the antibodies. HLA-DP antibodies were detected in 14% of the patients (48/338). Before transplantation, the antibodies were shown in 23% (10 females and 1 male) and 77% were found after transplantation (30 in patients after the first, 7 after the second graft). Specificities were never restricted to individual mismatched antigens; broad HLA-DP sensitization was found as a rule. A single HVR mismatch was present in 80% of the DSA and in 79% of the NDSA. No HLA-DPA specific antibodies were found. Our findings confirm that HLA-DP antibodies are specific for epitopes shared by different HLA-DP antigens, indicating that only a restricted number of mismatched epitopes are recognized by the recipients immune system. Matching for immunogenic HLA-DP epitopes for renal transplantation seems to be functionally more relevant than classical matching at the allelic level.

Clinical relevance of Luminex donor-specific crossmatches: data from 165 renal transplants.

The clinical significance of the presence of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA) prior to renal transplantation detected solely by solid-phase techniques remains unclear. This study was designed to determine the clinical relevance of the recently introduced bead-based Luminex donor-specific crossmatch (LumXm). A group of 165 patients transplanted between 1997 and 2001 were tested. Of 165 recipients transplanted with a negative complement-dependent cytotoxicity crossmatch, 32 proved to have a positive Luminex crossmatch. Sixteen were positive for class I, 15 were positive for class II, 1 was both class I and II positive and 133 recipients were negative. Acute rejection (AR)-free survival for all recipients was 77%, and there was no difference in AR-free survival between LumXm-positive and LumXm-negative recipients. Overall graft survival after a median follow-up time of 8 years was 56%. Recipients with a positive class I LumXm had worse long-term graft survival (P = 0.006). In recipients with a positive class I LumXm, 5-year graft survival was 41% vs 70% in negative patients and 10-year graft survival was 27% vs 56%. Positivity for class II LumXm was not a significant risk factor for graft failure (P = 0.7). In conclusion, pretransplant DSA detected by the LumXm had no impact on AR episodes. Class II LumXm positivity proved no significant risk factor for graft failure, but the value of the class II LumXm is questionable. A positive class I LumXm resulted in worse long-term graft survival compared with a negative one.

Therapeutic drug monitoring of everolimus using the dried blood spot method in combination with liquid chromatography-mass spectrometry.

An assay of everolimus based on finger prick sampling and consecutive application as a blood spot on sampling paper has been developed. We explored several methods [K. Hoogtanders, J. van der Heijden, M. Christiaans, P. Edelbroek, J. van Hooff, L. Stolk, J. Pharm. Biomed. Anal. 44 (2006) 658-664; A. Allanson, M. Cotton, J. Tettey, et al., J. Pharm. Biomed. Anal. 44 (2007) 963-969] and developed a new method, namely the impregnation of sampling paper with a solution of plasma-protein, formic acid and ammonium acetate, in combination with the extraction of the blood spot by filter filtration. This kind of sample preparation provides new possibilities for blood spot sampling especially if analytes are adsorbed to the paper. The dried blood spot was analysed using the HPLC-electrospray-tandem mass spectrometry method, with 32-desmethoxyrapamycin as the internal standard. The working range of our study was 2-30 microg/l. Within this range, intra-and inter-assay variability for precision and accuracy was <15%. Everolimus blood spot samples proved stable for 3 days at 60 degrees C and for 32 days at 4 degrees C. Everolimus concentrations of one stable out-patient were compared after both blood spot sampling and conventional venous sampling on various occasions. Results indicate that this new method is promising for therapeutic drug monitoring in stable renal transplant patients.

Histological assessment of preimplantation biopsies may improve selection of kidneys from old donors after cardiac death.

Kidneys from old donors after cardiac death (DCD) may increase the donor pool but the prognosis of these kidneys is unsatisfactory. To improve these results, we retrospectively evaluated the diagnostic utility of published selection algorithms for old donor kidneys. We studied all DCD kidney transplantations between January 1, 1994 and July 1, 2005 at our institution (n = 199). Selection algorithms were evaluated in the subset of kidney transplantations from donors aged 60 years or older (n = 52). For histological assessment of kidney biopsies, glomerulosclerosis, tubular atrophy, interstitial fibrosis and vascular narrowing were blindly scored. Functional kidney weight was calculated as renal mass multiplied by the fraction of nonsclerosed glomeruli. Graft function and survival of kidneys from DCD aged 60 years or older were inferior to those from younger DCD. Histological scores were associated with kidney function and graft survival of old DCD kidney transplantations. Functional kidney weight was associated with kidney function but not graft survival, while donor glomerular filtration rate (GFR), donor age and machine perfusion characteristics were associated with neither of the clinical outcomes of interest. We conclude that histological assessment of preimplantation biopsies may improve the selection of kidneys from old DCD and may therefore contribute to expansion of the donor pool.

Luminex donor-specific crossmatches.

In Luminex bead-based screening assays, color-coded microspheres coated with human leukocyte antigens (HLA) are used to identify both complement-binding and non-complement-binding HLA class I and II antibodies in recipient sera. Many laboratories rely on their specificity detection and use the information obtained for allocation of donor organs. A donor-specific crossmatch in the Luminex technique (LumXm) is for that reason desirable. A LumXm, in which the actual donor HLA are coated onto specific capture beads, was tested for 88 pre- and posttransplant sera of 18 recipients. The results were compared with previously published flow cytometric crossmatch (FCXm) results for the same donor-recipient combinations. All sera were also examined by Luminex single antigen (SA) tests. Class I LumXm detected 24 of 27 T-cell positive FCXm (89%) and class II 15 of 22 B-cell positive FCXm (68%). Sensitivity of LumXm for class I and II was 89% and 68% and specificity was 98% and 97%, respectively. Discrepant LumXm results were obtained in 13 sera of nine patients (15%). In general, based on SA testing, FCXm showed false-positive results for class I and LumXm gave false-negative and positive results for class II. The LumXm test was proven not to react with recipient sera containing DQ antibodies only, also DP detection was insufficient. The validity of the LumXm has been shown for class I, but its value for class II is uncertain. HLA-DR is most probably correctly identified, the validity for DQ and DP is doubtful.

Recipient hemodynamics during non-heart-beating donor kidney transplantation are major predictors of primary nonfunction.

Non-heart-beating donor (NHBD) kidneys may substantially expand the donor pool, but many transplant centers are reluctant to use these kidneys because of the relatively high incidence of primary nonfunction (PNF). In heart-beating donor kidneys, intravascular fluid depletion during transplant surgery is associated with delayed graft function (DGF). Therefore, we studied the effect of the recipients' hemodynamic status on the outcome of 177 NHBD kidney transplantations. Independent statistically significant predictors of PNF were average central venous pressure (CVP) below 6 cmH(2)O (adjusted odds ratio (AOR) 3.1 (95% CI: 1.4-7.1), p=0.007), average systolic blood pressure below 110 mmHg (AOR 2.6 (95% CI: 1.1-5.9), p=0.03) and pre-operative diastolic blood pressure below 80 mmHg (AOR 2.4 (95% CI: 1.0-5.9), p=0.05). Donor characteristics were not independently associated with PNF (p>0.10). In a subgroup analysis of 56 paired kidneys, 29% of the recipients with the lower CVP of the pair experienced PNF compared with 11% of their counterparts with higher CVP (p=0.09). Our study indicates that recipient hemodynamics during transplant surgery are major predictors of PNF. Therefore, improving recipient hemodynamics by expansion of the intravascular volume is expected to enhance the results of NHBD kidney transplantations and may enlarge the donor pool by increasing the acceptance of NHBD kidneys.

Therapeutic drug monitoring of tacrolimus with the dried blood spot method.

In a preliminary investigation an assay for tacrolimus based on fingerprick sampling and consecutive application as a blood spot on sampling paper has been developed. The dried blood spot was analysed by HPLC-tandem mass spectrometry. The validated range was 1-30 microg/l. Intra- and inter-assay variability for precision and accuracy was <7.5% and 15%, respectively. Tacrolimus concentrations of 24 stable out patients were compared after both blood spot sampling and conventional venous sampling. Method agreement was investigated with the methods of Passing and Bablok and Bland Altman and proved suitable for clinical use. The dried blood spot method for tacrolimus seems promising for patient monitoring.

Preservation of renal function and cardiovascular risk factors.

An update is given about some factors leading to loss of renal allograft, especially in relation to the use of tacrolimus and cyclosporine. We discuss both immunological, such as suboptimal immunosuppression, acute rejection, and noncompliance, as well as nonimmunological factor's such as hypertension, hyperlipidemia, chronic toxic effects of immunosuppressants, older donors, and delayed graft function.

Kidney transplantation using elderly non-heart-beating donors: a single-center experience.

Although acceptable outcomes have been reported in both non-heart-beating (NHB) and elderly donors individually, the large pool of elderly NHB donors has not yet been fully utilized. In 1994, we expanded our transplant protocol to include NHB donors aged over 65 years and this study compares the clinical outcomes with regular NHB transplantations. Up to June 2005, 24 patients were transplanted at our center with kidneys from NHB donors aged 65 years or more, whereas 176 patients received grafts from conventional NHB donors during the same period. Grafts from older donors were associated with inferior glomerular filtration rates (29 vs. 44 mL/min after 1 year, p=0.01) and graft survival (52% vs. 68% after 5 years, p=0.19) compared to younger NHB donor grafts, although the difference in graft survival was not statistically significant. Exclusion of older NHB donor kidneys with severe vascular pathology resulted in similar graft survival of older and younger NHB donor kidneys. We conclude that the use of elderly NHB donors in order to expand the donor pool was associated with unacceptable clinical outcomes and cannot be justified without further refinement in their selection, for example, by histological assessment of pretransplant biopsies.

Do blood pressure and arterial wall properties change after conversion from cyclosporine to tacrolimus?

Cardiovascular disease is the leading cause of death in renal transplant recipients. Arterial wall properties are surrogate markers for arteriosclerosis. Previous investigations have shown that the cardiovascular risk profile is better with tacrolimus compared to cyclosporine. Renal function, blood pressure, and lipid levels improve. The hypothesis is that arterial wall properties will improve after conversion from cyclosporine to tacrolimus. Thirty-four stable renal recipients were converted from cyclosporine microemulsion to tacrolimus without changing concomitant medication. Before and after conversion we performed wall track ultrasounds of the carotid and the brachial arteries; pulse wave velocity (PWV); laboratory investigations; 24-hour ABPM; estimates of renal function; and Framingham risk scores. After conversion the 24-hour ambulatory blood pressure monitoring (ABPM) did not change. Total cholesterol, LDL cholesterol, and triglycerides improved significantly. Renal function (Cockroft) improved. There were no significant changes in arterial wall properties, or in PWV. Framingham comparative risk scores improved only significantly in patients not receiving statins. In conclusion, 3 months after conversion from cyclosporine to tacrolimus total cholesterol, LDL cholesterol, and triglycerides were significantly decreased and renal function significantly improved. Contrary to expectation, ABPM did not change, probably due to prolonged use (>10 years) of cyclosporine. There was also no difference in arterial wall properties.

Restenosis after carotid angioplasty and stenting: a follow-up study with duplex ultrasonography.

OBJECTIVES: To prospectively document the incidence, location, risk factors for and clinical consequences of restenosis after carotid artery angioplasty and stenting (CAS). METHODS: Serial duplex and neurological examinations were performed in 217 patients one day (n = 216), 3 (n = 189), 12 (n = 129) and 24 (n = 48) months, after CAS. The relationship between patient, lesion and procedure variables and restenosis was determined at 12 months. RESULTS: The prevalence of restenosis > or = 50% was 14, 16, 18, and 21%, respectively, and was only significantly related with loss of proximal stent apposition (odds ratio 3.4, 95% confidence interval: 1.0-11.7, p < 0.05). Four restenoses were symptomatic. CONCLUSIONS: Restenosis after CAS is common, unpredictable but infrequently symptomatic.