M. H. L. Christiaans

No advantage of flow cytometry crossmatch over complement-dependent cytotoxicity in immunologically well-documented renal allograft recipients.

The effect of flow cytometry crossmatches on clinical outcome was studied retrospectively in two groups of immunologically well-documented patients who had received transplants with a negative complement-dependent cytotoxicity crossmatch. The first group consisted of 114 consecutive renal allograft recipients, and the second group consisted of 76 immunologically at-risk recipients. Flow cytometry crossmatches were performed with current and historic sera. In group 1, positive flow cytometry (FC) crossmatches were shown in 15/114 (13%) recipients. Rejection occurred in 8/15 (53%) FC-positive versus 41/99 (41%) FC-negative recipients. The 1-year graft survival rate was 80% for FC-positive patients and 87% for FC-negative patients. Sixty-seven patients were nonsensitized patients; 4 of them had a positive FC crossmatch but no rejection episodes, graft loss, or patient loss. Of 47 retransplanted and/or sensitized recipients, 11 had a positive FC crossmatch. Rejection treatment was needed in 8/11 (73%) FC-positive patients compared with 19/36 (53%) FC-negative patients. Their 1-year graft survival rates were 73% and 81%. None of these differences reached statistical significance. Group 2 consisted of 76 at-risk recipients; 37 were retransplant patients and 39 were sensitized first-transplant patients. Twenty-one (28%) patients showed a positive FC crossmatch. Rejection episodes did not differ between the FC-positive (48%) and FC-negative patients (46%). There was no difference in 1-year graft survival rate (76% vs. 80%) or in 1-year patient survival rate (100% vs. 95%). We conclude that FC crossmatches in our patient group are not superior to the classical complement-dependent cytotoxicity crossmatches with regard to clinical outcome. On the contrary, transplantation with a mandatory negative FC crossmatch would have excluded 28% of the recipients from transplantation, who in fact are doing well.

Donor-specific antibodies after transplantation by flow cytometry: relative change in fluorescence ratio most sensitive risk factor for graft survival.

BACKGROUND There is no consensus on the role of donor-directed antibodies after renal transplantation detected by complement-dependent cytotoxicity (CDC) or by flow cytometry (FC). METHODS Therefore, antibody formation was studied by FC and correlated with clinical course in a group of patients who received transplants between 1983 and 1993. All had a negative current CDC crossmatch and were treated with cyclosporine. Current and posttransplant sera from 143 donor-recipient combinations were studied retrospectively. Antibodies were considered present in FC if the fluorescence ratio between serum and negative control was > 2.65. RESULTS Of 143 patients, 17 (11.9%) were found to be positive in the posttransplant FC crossmatch and 126 (88.1%) were negative. Of the positive patients, 3 were already positive in the current FC crossmatch, whereas 14 demonstrated a positive posttransplant FC crossmatch after a negative current FC crossmatch. It was noteworthy that, from 16 patients with a positive current FC crossmatch, 13 turned negative in the posttransplant crossmatch. In 113 recipients (79%), both pre- and posttransplant FC crossmatches were negative. The development of a positive FC crossmatch after transplantation was a significant risk factor for graft survival in Cox regression analysis (P = 0.01). The results were also studied as relative change in fluorescence ratio (RCFR). RCFR was determined by classifying the recipients in quartiles according to their change in flow cytometric value from current to posttransplant serum. Quartiles were defined as follows: quartile 1, decrease > 10%; quartile 2, decrease 0-10%; quartile 3, increase > 0-30%; and quartile 4, increase > 30%. RCFR proved to be the only significant risk factor for graft survival (odds ratio for quartile 4 vs. quartile 1, 3.27; P < 0.02). More rejections were shown for increasing quartile numbers (P < 0.001). CONCLUSIONS Classification of patients by RCFR detected more patients with unfavorable clinical outcome (25% vs. 11%) than by FC crossmatch.

RELATION BETWEEN STEROID DOSE, BODY COMPOSITION AND PHYSICAL ACTIVITY IN RENAL TRANSPLANT PATIENTS

BACKGROUND Fat mass is increased in renal transplant (RTx) patients, which may have untoward metabolic and cardiovascular effects. The influence of steroids on body composition (BC), resting energy expenditure (REE), and substrate oxidation rates was assessed in stable RTx patients in a cross-sectional design. Also, the relation between physical activity and nutrient intake, respectively, and body composition was studied. METHODS 77 RTx patients (42 males, 35 females) were studied. Twenty-one patients were on 10 mg and 27 patients on 5 mg maintenance steroid dose; 29 patients were receiving steroid-free immunosuppression. Assessed were BC (DEXA, anthropometry), REE and substrate oxidation (indirect calorimetry), physical activity (Baecke questionnaire), and nutrient intake (dietary records). RESULTS BC was not different between the 0-, 5-, and 10-mg steroid group, and no relationship existed between cumulative dose of steroids and BC. REE and substrate oxidation also did not differ between the various groups, apart from a small increase in glucose and decrease in lipid oxidation in female patients using 5-mg steroids. Especially in females, leisure time physical activity was positively related with the percentage lean body mass (r=0.571, P=0.004) and inversely related with fat mass (r= -0.588, P=0.003). Nutrient intake and BC (corrected for physical activity) were not related. CONCLUSIONS No relation was observed between daily and cumulative steroid dosage and BC and between daily steroid dose and REE and substrate oxidation in RTx patients. Especially in female patients, physical activity level and the percentage of lean body mass concluded and body fat were significantly related.

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.

Early detection of human cytomegalovirus infection after kidney transplantation by nucleic acid sequence-based amplification.

BACKGROUND The early detection of human cytomegalovirus infection after organ transplantation is a prerequisite for effective antiviral therapy. We evaluated the diagnostic value of monitoring the viral immediate-early (IE) 1 mRNA expression in blood leukocytes by nucleic acid sequence-based amplification (NASBA). METHODS Nucleic acids were isolated from 489 blood samples collected from 42 kidney transplant recipients and subjected to amplification by IE NASBA. The IE NASBA results were compared to those from pp67 NASBA, pp65 antigenemia, cell culture (DEAFF and CPE), and serology. RESULTS IE NASBA proved to be the most sensitive assay which detected the onset of both primary and secondary cytomegalovirus infection significantly earlier than the other assays. CONCLUSIONS The early detection of cytomegalovirus infection with IE NASBA would enable the start of effective antiviral therapy at an early state of infection to prevent cytomegalovirus disease in patients at risk.

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.

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.

Detection of HLA class I and II antibodies by ELISA and complement-dependent cytotoxicity before and after transplantation

BACKGROUND Anti-class I IgG can be detected by complement-dependent cytotoxicity (CDC) and by ELISA. We compared ELISA and CDC for both class I and class II antibodies on method agreement and relation to rejection-free and graft survival. METHODS Peak, current, and posttransplant sera (n=429) of 143 renal allograft patients were tested by National Institutes of Health technique (NIHT), two-color fluorescence (TCF), and ELISA. Method agreement was assessed by intraclass correlation coefficient (ICC). Rejection and graft survival were analyzed by uni- and multivariate techniques. The screening results for each serum were compared, as was the change in result of current to posttransplant serum. RESULTS The ICC of ELISA and NIHT was insufficient; it was lower for TCF than NIHT. Graft survival was not related to the result of any assay. Rejection-free survival was related to ELISA and NIHT in current and posttransplant serum. With the NIHT, the change in percent panel-reactive antibody (%PRA) correlated better with rejection than it did with ELISA. The combined antibody status of current and posttransplant serum was a risk factor for rejection in all assays, and for TCF also in multivariate analysis. The rejection rate was higher if the posttransplant serum was ELISA-negative/CDC-positive, rather than ELISA-positive/CDC-negative. For ELISA, class I specificities (and not %PRA) in peak and current sera were related to rejection, even if the antibodies were not donor-directed. In the case of the National Institutes of Health technique (NIHT), %PRA and not specificity was related to rejection. Class II antibodies were never related to rejection. CONCLUSIONS ELISA and NEIT are complementary screening techniques in this patient population. They are of equal predictive value for rejection. The optimal strategy in combining these techniques must be determined.

Risk factors for cytomegalovirus infection and disease in renal transplant recipients: HLA-DR7 and triple therapy

In a prospective study, an analysis of risk factors for the development of cytomegalovirus (CMV) infection and disease was performed on 77 renal allograft recipients. Twenty-five out of the 77 recipients (32%) had a CMV infection. Twenty-two of the recipients received triple immunosuppressive therapy (cyclosporin A, prednisolone, and azathioprine) while the remaining 55 received standard therapy (cyclosporin A and prednisolone). In 23 recipients (30%) acute rejection was diagnosed and the first positive parameter of infection occurred 22 days after rejection therapy. Infection occurred in 10 out of 18 HLA-DR7-positive recipients (56%) and in 15 out of 59 HLA-DR7-negative recipients (25%; P< 0.02). In multiple regression analysis, HLA-DR7 was found to be a significant predictor of CMV infection (P< 0.005). CMV disease was diagnosed in only 9 out of 25 recipients with an acute infection. Six recipients (67%) with CMV disease received triple therapy for maintenance immuuosuppression; this was significantly correlated to CMV disease (P< 0.05) as compared to three recipients (33%) with CMV disease maintained with standard therapy. Our data suggest that HLA-DR7-positive recipients are more susceptible to CMV infection and that CMV disease is associated with triple immunosuppressive therapy.

Use of tacrolimus in renal transplantation

Tacrolimus is a very potent drug for preventing all types of acute rejection after renal transplantation. The decrease of vascular rejection may have important long-term implications. This is demonstrated in the 3-year data of the U.S. multicentre trial, where a significant decrease of graft loss was observed in the tacrolimus patients compared to cyclosporine patients. Safety profiles of both drugs are the same, but there are some major differences in side effects. Hyperglycemia is more common during tacrolimus, but generally the induction of hyperglycemia is a dose-dependent reversible process, which leads generally to diabetes mellitus during high systemic exposure, in certain races and prediabetics. Gum hyperplasia and hirsutism are not seen during tacrolimus therapy and there is a more favorable cardiovascular risk profile during tacrolimus as compared to cyclosporine based immunosuppression.