Habib Kashi

Blood levels of donor-specific human leukocyte antigen antibodies after renal transplantation : Resolution of rejection in the presence of circulating donor-specific antibody

Background. Accommodation to antibody is an important mechanism in successful ABO-incompatible transplantation, but its importance in human leukocyte antigen (HLA) antibody-incompatible transplantation is less clear, as sensitive techniques facilitating daily measurement of donor-specific HLA antibodies (DSAs) have only recently been developed. Methods. We report 24 patients who had HLA antibody-incompatible kidney transplantation (21 living donors, 3 deceased), 21 of whom had pretransplant plasmapheresis. Eight had positive complement-dependent cytotoxic (CDC) crossmatch (XM) pretransplant plasmapheresis, nine had positive flow cytometric (FC) XM, and seven had DSA detectable by microbead analysis only. After transplant, DSA levels were monitored closely with microbead assays. Results. Rejection occurred in five of eight (62.5%) CDC-positive cases, in three of nine (33%) FC-positive cases, and in two of seven (29%) of microbead-only cases at a median of 6.5 days after transplantation. Resolution occurred at a median of 15 days after transplantation, in 8 of 10 cases when the microbead level of DSA had median fluorescence intensity (MFI) >2000 U, in 6 of 10 when the microbead MFI >4000 U. In 8 of 10 cases, the microbead MFI at the time of resolution was greater than at the onset. DSA did not always cause clinical rejection. In five cases with a posttransplant DSA peaking at MFI >2000 U on microbead assay, rejection did not occur. Conclusion. These data suggest that the dominant method of successful transplantation was function of the transplant in the presence of circulating DSA, and they also define the period during which this occurred.

Human leukocyte antigen antibody-incompatible renal transplantation: excellent medium-term outcomes with negative cytotoxic crossmatch.

Background. Human leukocyte antigen (HLA) antibody-incompatible renal transplantation has been increasingly performed since 2000 but with few data on the medium-term outcomes. Methods. Between 2003 and 2011, 84 patients received renal transplants with a pretreatment donor-specific antibody (DSA) level of more than 500 in a microbead assay. Seventeen patients had positive complement-dependent cytotoxic (CDC) crossmatch (XM), 44 had negative CDC XM and positive flow cytometric XM, and 23 had DSA detectable by microbead only. We also reviewed 28 patients with HLA antibodies but no DSA at transplant. DSAs were removed with plasmapheresis pretransplant, and patients did not routinely receive antithymocyte globulin posttransplant. Results. Mean follow-up posttransplantation was 39.6 (range 2–91) months. Patient survival after the first year was 93.8%. Death-censored graft survival at 1, 3, and 5 years was 97.5%, 94.2%, and 80.4%, respectively, in all DSA+ve patients, worse at 5 years in the CDC+ve than in the CDC−ve/DSA+ve group at 45.6% and 88.6%, respectively (P<0.03). Five-year graft survival in the DSA−ve group was 82.1%. Rejection occurred in 53.1% of DSA+ve patients in the first year compared with 22% in the DSA−ve patients (P<0.003). Conclusions. HLA antibody-incompatible renal transplantation had a high success rate if the CDC XM was negative. Further work is required to predict which CDC+ve XM grafts will be successful and to treat slowly progressive graft damage because of DSA in the first few years after transplantation.

Rises and falls in donor-specific and third-party HLA antibody levels after antibody incompatible transplantation

Background. After human leukocyte antigen (HLA) antibody-incompatible transplantation, donor specific and third party HLA antibodies may be found, and their levels fall in a donor-specific manner during the first month. However, these changes have not been previously described in detail. Methods. Donor-specific HLA antibody (DSA) and third-party HLA antibody (TPA) levels were measured using the microbead method in 44 presensitized patients who had renal transplantation. Results. DSA+TPA fell in the first 4 days after transplantation, and greater falls in DSA indicated absorption by the graft. This occurred for class I (57.8% fall compared with 20.2% for TPA, P<0.0005), HLA DR (63.0% vs. 24.3%, P<0.0004), and for HLA DP/DQ/DRB3–4 (34% vs. 17.5%, P=0.014). Peak DSA levels occurred at a mean of 13 days posttransplant, and they were higher than pretreatment in 25 (57%) patients and lower in 19 (43%) patients (P=ns). The risk of rejection was associated with peak DSA levels; 15 of 25 (60%) patients with DSA at median fluorescence intensity (MFI) more than 7000U experienced rejection, compared with 4 of 7 (57%) patients with peak DSA MFI 2000 to 7000U, and 2 of 12 (17%) patients with peak DSA MFI less than 2000U (P<0.02). DSA levels subsequently fell in a donor specific manner compared to TPA. Conclusion. DSA levels may change markedly in the first month after antibody incompatible transplantation, and the risk of rejection was associated with higher pretreatment and peak levels.

Application of flow cytometry to monitor antibody levels in ABO incompatible kidney transplantation.

Current methods of measuring ABO antibody levels based on the hemagglutination (HA) titers have the disadvantages of relatively poor reproducibility and do not offer fine discrimination of antibody concentration. We therefore developed a simple and rapid method of measuring ABO antibody levels using flow cytometry (FC). For validation, we analyzed plasma samples from 79 blood donors. Both IgM and IgG were detected and measured with IgG essentially restricted blood group O donors. Forty-two successive samples were collected from a patient with blood group O undergoing antibody removal and subsequent transplantation from a group A2 donor and tested by both HA and FC. Changes in IgG measured by FC (relative median fluorescence) correlated well with HA titers and importantly rejection episodes were preempted by a rising relative median fluorescence. The method allowed quantitative discrimination in the range of antibody levels relevant to ABO incompatible transplantation and has the advantages over HA of objective measurement and reproducibility.

Double Filtration Plasmapheresis in Antibody-Incompatible Kidney Transplantation

Double filtration plasmapheresis (DFPP) was used in preference to plasma exchange in our program of antibody‐incompatible transplantation, to treat higher volumes of plasma. Forty‐two patients had 259 sessions of DFPP, 201 pre‐transplant and 58 post‐transplant. At the first treatment session, the mean plasma volume treated was 3.81 L (range 3–6 L), 55.5 mL/kg (range 36.2–83.6 mL/kg). Serum IgG fell by mean 59.4% (SD 10.2%), and IgM by 69.3% (SD 16.1%). Nine patients did not require increases in plasma volumes treated, and six did not tolerate higher plasma volumes. In the remaining patients, the mean maximum plasma volume treated pre‐transplant was 6.67 L (range 4–15 L), 96.1 mL/kg (range 60.2–208.9 mL/kg). The complement dependent cytotoxic crossmatch was positive in 14 cases pre‐treatment, and remained positive in six (42.8%) cases. The flow cytometric crossmatch was positive in 29 cases pre‐treatment, and in 21 (72.4%) after DFPP. Post‐transplant, DFPP was ineffective at reducing donor specific antibody levels during periods of rapid donor specific antibody synthesis. Post‐transplant, the one year graft survival rate was 94%, although there was a high rate of early rejection. In summary, DFPP enabled the treatment of plasma volumes that were almost double those that would have been feasible with plasma exchange. Despite this, most patients were transplanted with a positive crossmatch, and DFPP post‐transplant was unable to control rising antibody levels.

Conversion from tacrolimus to cyclosporine in stable renal transplant patients: safety, metabolic changes, and pharmacokinetic comparison.

BACKGROUND Although conversion between tacrolimus and cyclosporine has been performed when indicated for rejection or adverse effects, the safety and metabolic outcome of elective conversion from tacrolimus to cyclosporine has not previously been examined. METHODS Conversion from tacrolimus to cyclosporine was performed in 19 recipients of cadaver renal transplants at 3-6 months after transplantation. Pharmacokinetic profiles and biochemical studies were performed three times, in steady state, before, and after conversion. RESULTS Patient and graft survival was 100% at 3 months after conversion, with no rejection episodes. Three patients have been subsequently converted back to tacrolimus, two for rejection and one for hirsutism. There were no significant changes in creatinine, urate, or blood sugar levels after conversion, but the mean plasma magnesium rose from 0.73 (0.63-0.97) to 0.82 (0.65-1) mmol/L (P=0.037), and the mean plasma cholesterol rose from 5.2 (3.4-6.8) to 5.5 (3.8-7.6) mmol/L (P=0.033). Pharmacokinetic profiles were measured before and after conversion, and showed that cyclosporine (Neoral) exhibited significantly less interpatient and intrapatient variability than tacrolimus, for area under the curve (AUC), maximum concentration after dose (Cmax), minimum concentration after dose (Cmin), and time to maximum concentration (Tmax). CONCLUSION This is the first study that has examined the outcome of conversion from tacrolimus- to cyclosporine-based immunosuppression in stable patients after renal transplantation. This conversion was performed without early immunological hazard, but there was a small rise in blood cholesterol levels after conversion. Pharmacokinetic studies showed that cyclosporine in the form of Neoral exhibited less inter- and intrapatient variability than tacrolimus, although this is of uncertain clinical significance.

The histological development of acute antibody-mediated rejection in HLA antibody-incompatible renal transplantation

BACKGROUND The aim of this study was to examine the development of acute antibody-mediated rejection in HLA antibody-incompatible renal transplantation in relation to the Banff 07 histological classification. METHODS Renal biopsies were scored using the Banff 07 diagnostic criteria, and paraffin-embedded sections were stained with the pan-leucocyte marker CD45. RESULTS Thirty-six patients had 72 renal biopsies. In biopsies performed 30 min after graft reperfusion, the mean number of CD45+ cells per glomerulus was higher than in control grafts (P < 0.04) and was associated with the donor-specific antibody (DSA) level at transplantation measured by microbeads (P < 0.01), and eight out of nine patients with greater than five CD45+ cells per glomerulus had early post-transplant rejection or oliguria, compared to 11 out of 20 with less than five cells per glomerulus (P < 0.01). In the first 10 days post-transplant, although peritubular capillary (PTC) leucocyte margination grade 3 and C4d deposition were specific for rejection, their sensitivities were low. PTC C4d staining was only seen in two out of 11 biopsies taken in the first 5 days after transplant, even in the presence of rejection, but was present in the majority of later biopsies with rejection. In biopsies stained for CD3, CD68 and CD20, it was notable that CD20+ cells were not seen during acute rejection, the infiltrates comprising CD3+ and CD68+ leucocytes. CONCLUSIONS Glomerular margination of leucocytes occurred early after transplantation and was associated with DSA level and early graft dysfunction. The Banff 07 PTC margination scoring system was easy to apply, especially when CD45 staining was used, and PTC margination grade 3 was always associated with clinical rejection.

Human leukocyte antigen-specific antibodies and gamma-interferon stimulate human microvascular and glomerular endothelial cells to produce complement factor C4

Background The role of the complement system in antibody-mediated rejection has been investigated in relation to circulating complement interacting with renal microvascular endothelium, resulting in the formation of peritubular capillary C4d. However, the possible importance of local complement synthesis is less clear. The aim of this study was to determine whether human vascular endothelium could produce C4 in response to stimulation in vitro. Methods Human microvascular endothelial cells and glomerular endothelial cells were stimulated with endotoxins, cytokines, and human leukocyte antigen-specific antibodies. Synthesis of complement was investigated using western blotting and indirect immunofluorescence. De novo C4 synthesis was confirmed by using C4 small interfering RNA. Results Glomerular and microvascular endothelium, both produce C3 and C4 complement protein. Complement synthesis was stimulant-specific—C3 was produced mainly after stimulation with lipopolysaccharide whereas C4 synthesis occurred on treatment with gamma interferon. Culture with human leukocyte antigen-specific antibodies resulted in a significant increase of C4 protein synthesis by both cell lines. Conclusions We have shown for the first time that human microvascular endothelium can be stimulated to synthesize C4 in vitro. The implications of this for clinical transplantation, especially in the context of antibody-mediated rejection, its histological interpretation and as a potential target for therapy would have to be determined by further studies.

Pregnancy-induced HLA antibodies respond more vigorously after renal transplantation than antibodies induced by prior transplantation

Acute antibody mediated rejection after HLA-specific antibody incompatible renal transplantation is related to donor specific HLA antibody (DSA) levels. DSA levels may rise sharply after transplant, and aim of this study was to examine changes in DSA levels, particularly according to the primary sensitising event. Changes in 220 HLA specificities in 64 patients over the first 30days after transplantation were evaluated using microbead assays. The greatest increase from pre-treatment to peak DSA levels was seen in pregnancy-stimulated specificities, median (IQR) increase in MFI of 1981 (94-5870). The next highest increase was for those sensitised by transplant with repeat HLA epitope mismatch, at 546 (-308-2698) (p<0.01). The difference was especially marked when the pre-treatment antibody level was low; with pre-treatment MFI <1000, peak level was >1000 in 19/26 (73%) of pregnancy stimulated specificities, compared with 9/29 (31%) for all others (p<0.001). DSA production to specificities stimulated by previous pregnancy was marked, even from very low pre-transplant levels. By contrast, there was a lower rate of antibody resynthesis to specificities repeated from previous transplants, both at antigen and epitope levels.

Acute rejection after renal transplantation is reduced by approximately 50% by prior therapeutic blood transfusions, even in tacrolimus-treated patients.

Background. The authors investigated the relationship between therapeutic blood transfusion before renal transplantation and rejection rates in cyclosporine- and tacrolimus-treated patients. Methods. In one center, 265 consecutive recipients were studied. Protocol induction was with azathioprine, prednisolone, and cyclosporine or tacrolimus; 37% had biopsy-proven acute rejection in the first 6 months and 46% had received zero to two units of blood before transplantation. Results. Lower risk of rejection was associated with tacrolimus induction (odds ratio [OR], 0.53; 95% confidence interval [CI], 0.29–0.95; P =0.049), prior transfusion of three or more units of blood (OR, 0.54; 95% CI, 0.33–0.90; P =0.024), and older age at transplantation (mean, 44.23±12.56 [±SD] years vs. 38.96±12.37 years; P =0.001). Multiple logistic regression modeling showed the effect of three or more prior transfusions on acute rejection was as follows: OR, 0.49; 95% CI, 0.29 to 0.83; P =0.008. Conclusions. Induction immunosuppression should take account of the higher risk of rejection in patients coming to transplantation who have previously received zero to two units of blood.