Complement-dependent Cytotoxicity Crossmatch Research Articles

Treatment of early mixed cellular and humoral renal allograft rejection with tacrolimus and mycophenolate mofetil

This prospective study investigated the efficiency of the tacrolimus (Tac) combined with mycophenolate mofetil (MMF) alone without immunoadsorption (IA) or plasmapheresis (PPH) as treatment for early (within 2 weeks) acute humoral rejection (AHR) in non-sensitized renal allograft recipients. Of 160 patients enrolled in this prospective study, 11 patients had histologically and clinically confirmed early steroid-resistant acute rejection with an antibody response and received Tac-MMF therapy. No other aggressive rescue methods such as IA, PPH were used, according to the study design. Patients (n=11) were followed for 13.8+/-3.5 months; nine were females. The complement-dependent cytotoxicity crossmatch was negative before transplantation in all patients and only positive for panel-reactive antibody in one patient. Most of the rejection episodes were mixed with cellular rejection (four patients met Banff IIA criteria, five patients met Banff IIB, one patient met Banff IB, and one patient met Banff borderline). After 16.19+/-6.16 days of treatment, all rejection episodes were successfully reversed and all graft functions were stable, with a mean serum creatinine level of 1.12+/-0.32 mg/dl during follow-up. No patient suffered from severe infectious complications (except one case of urinary infection). Our investigation suggests that Tac combined with MMF alone is adequate to reverse early mixed cellular and humoral C4d-positive rejection in non-sensitized renal allograft recipients.

Relevance of Posttransplant Flow Cytometric T- and B-cell Crossmatches in Tacrolimus-Treated Renal Transplant Patients

De novo development of anti-human leukocyte antigen (HLA) antibodies after transplantation is associated with increased rejection and decreased graft survival. In this study, the effect of posttransplant HLA antibodies on clinical outcome was evaluated in patients treated with tacrolimus by means of flow cytometric crossmatches (FCXm). T- and B-cell FCXm were performed retrospectively on posttransplant sera of patients who received a graft between 1997 and 1999. Ninety-four kidney-only recipients were tested and all FCXm positive sera were investigated for the presence of HLA class I and II antibodies by Flow panel reactive antibodies. From 94 patients with a negative pretransplant complement-dependent cytotoxicity crossmatch, seven (7%) showed a positive pretransplant FCXm. After transplantation the FCXm became positive in five patients (6%). The predictive value of a positive FCXm after transplantation, and the log-transformed relative change in fluorescence ratio between pretransplant and posttransplant serum, were not significant to rejection within six months, nor to graft survival censored for death. The presence of HLA antibodies before rejection or graft failure could only be shown in a minority of patients; most antibodies were detected after graft failure, especially after transplantectomy. Monitoring through antibody testing after transplantation on the basis of our results has no added value with tacrolimus-based immunosuppression.

Osmotic nephrosis in a renal transplant recipient

CASE PRESENTATION A 59-year-old Hispanic male with a history of type II diabetes mellitus, hypertension, and end-stage renal disease (on hemodialysis for 2 years) was called in for a kidney transplant. He was in his usual health, having received dialysis earlier in the day. The kidney donor was from an expanded criteria pool and was a 59-year-old Caucasian male with diabetes mellitus who expired following a cerebrovascular accident. He had a terminal creatinine of 1.3mg/dl (114.92 μmol/l (nl range 0.6-1.2 mg/dl/53.04-106.08 μmol/l)) and no evidence of proteinuria. A donor renal biopsy revealed 10-15% tubular atrophy and interstitial fibrosis. There was a six-antigen mismatch, and cold ischemia time was 31 h. The final complement-dependent cytotoxic and flow cytometry crossmatch were negative. Renal transplantation was performed. At the time of transplantation, the patient received alemtuzumab (Campath; anti-CD52 monoclonal antibody) and solumedrol as induction therapy. The patient had an intra-operative episode of hypotension that resolved with a 500 ml bolus of normal saline and 250 ml of 6% hetastarch lactate electrolyte solution. At the completion of surgery, the patient was stable and was extubated and moved to recovery. On physical exam, he was normotensive with normal sinus rhythm, clear lungs, good dorsalis pedis pulses, and no peripheral edema. He had been anuric before surgery, but had made 10 ml of urine during the first hour. One hour after surgery, the patient's systolic blood pressure dropped to 80 mmHg. In an effort to sustain his blood pressure, he was given multiple boluses of normal saline, lactate Ringers solution, and an additional 11 of 6% hetastarch. He was eventually started on vasopressin and moved to the Intensive Care Unit. His hematocrit remained stable, but his potassium was elevated at 6.0 mM/I. Continuous renal replacement therapy was initiated. Renal ultrasound initially was unable to demonstrate arterial or venous blood flow. After 4 h, his blood pressure improved and he was weaned off vasopressin. A repeat ultrasound 24 h later showed restoration of arterial and venous blood flow. Maintenance immunosuppression was started on post-operative day 1 and included tacrolimus and mycophenolate mofetil. His tacrolimus trough level ranged between 9 and 13 ng/ml. The patient remained oliguric and hemodialysis-dependent during the first week. Due to delayed graft function and a creatinine of 6.2 mg/dl (548.08 μmol/l), renal allograft biopsy was performed on post-operative day 8.

Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure.

The detection of donor-specific anti-HLA antibodies by standard procedures such as complement-dependent cytotoxicity assay (CDC) or flow cytometric (FACS) analysis is limited by its low sensitivity and the quality of the donor cells. Therefore, an ELISA-based technique was employed using solid phase-immobilized monoclonal antibodies to capture HLA class I or class II molecules of the donor, respectively. In this HLA class I and class II antibody monitoring system (AMS) the donor-specific anti-HLA antibodies from the sera of recipients bind to the HLA molecules of the donor which have been immobilized by monoclonal antibodies (mAb) recognizing non-polymorphic epitopes. Upon binding of donor-specific anti-HLA antibodies they are recognized by secondary enzyme-conjugated anti-human immunoglobulin (Ig) antibodies. A newly established modification of the standard protocol allows the differentiation between bound antibodies of the IgG and IgM isotype. Furthermore, this assay was adapted for investigating small amounts of solid tissue of donors from whom no other cells (e.g. from blood) were available. We here provide an overview of the classical crossmatch methods with their advantages and limits. In addition, the design of the novel AMS-ELISA is described in terms of quality and sensitivity of the approach using exemplary cases of different application. The selected cases show that the AMS-ELISA represents a valuable tool for the post-transplantation monitoring of donor-specific anti-HLA antibodies during reaction crisis, after transfusion reactions and in particular cases of tissue transplantations lacking single cells.

Effect of Induction Therapy Protocols on Transplant Outcomes in Crossmatch Positive Renal Allograft Recipients Desensitized with IVIG

Here we retrospectively examine the efficacy of two antibody induction regimens using Zenapax or Thymoglobulin in patients with positive complement-dependent cytotoxicity crossmatches (CDC-CMXs) desensitized with IVIG (intravenous immunoglobulin). Between January 1999 and March 2005, 97 patients with (+) CDC-CMXs received kidney transplants (43 deceased donors/54 living donors). All patients received at least 2 g/kg IVIG (maximum four doses) until an acceptable CMX was obtained. Patients were divided into two groups: 1. IVIG + Zenapax (n = 58), 2. IVIG + Thymoglobulin (n = 39). A total of 94% of patients in Group 1 and 84% in G2 have at least 2 years of follow up. Patient and graft survival was 96%/84% in Group 1 and 100%/90% in Group 2, p = NS. The number and severity of AR episodes were similar (36% Group 1 vs. 31% Group 2, p = NS) as was the incidence of C4d (+) antibody-mediated rejection (AMR) (Banff Grade II/III) (22% Group 1 vs. 21% Group 2). Mean serum creatinines (SCrs) at 24 months were similar (Group 1: 1.4 +/- 0.7 vs. G2: 1.5 +/- 0.7 mg/dL). Induction therapy with Zenapax or Thymoglobulin results in excellent patient, graft survival and graft function at 2 years. There was no increased risk of viral infections or malignancies with either agent. Neither agent was effective in reducing the incidence of AMR.

Successful living donor kidney transplantation across HLA and ABO incompatibilities

A 56-year-old white female presented for evaluation for kidney transplantation in November 2003. Her end-stage renal disease (ESRD) was presumed due to medullary sponge kidney associated with stones and infection and she had started haemodialysis in April 2003. Other medical history included hypertension and anaemia. She was otherwise well. She had had four pregnancies and had received blood transfusions. Examination was unremarkable. Initial tests showed blood group ABO-O. Her current panel reactive antibody (PRA) was 64% by the complement-dependent cytotoxicity (CDC) method and the peak PRA, 5 months prior to that, was 100%. Flow PRA screening confirmed IgG antibodies against class I and II human leukocyte antigens (HLA). The cause of the sensitization to HLA antigens was presumed to be the pregnancies and transfusions. Her 37-year-old son was the only potential living donor but he was ABO-A1; furthermore, she had a positive CDC crossmatch against his T and B lymphocytes. The patient’s baseline anti-A antibody titre was 1:128 (Figure 1). Because of her high sensitization to HLA antigens, the idea of a transplant across the HLA and ABO barrier was discussed with the patient. She was advised to wait several months to see if her PRA or crossmatch titres fell with rituximab therapy. She was vaccinated against pneumococcus and meningococcus in the event that a splenectomy would be included in her immunosuppressive protocol. She received rituximab 375mg/m in March and April of 2004. However, the crossmatch remained strongly positive and the PRA remained high (Table 1). After detailed discussion of the risks vs benefits, the patient and son gave informed consent to proceed with transplantation of a kidney from the son to the mother. Before 4 weeks of the transplant, she began receiving thrice-weekly plasmapheresis and daily tacrolimus plus mycophenolate mofetil (MMF). She was given 10 g of IgG intravenously after each plasmapheresis session. The anti-microbial prophylaxis was sulfamethoxazole-trimethoprim (SMX-TMP) and acyclovir. On 20 December 2004, the T-cell and B-cell crossmatch against her donor were negative and the anti-A titre (IgG titer by enhanced antiglobulin assay) was 1:2 (Figure 1). A third dose of rituximab was given, and she underwent kidney transplantation the next day. A laparoscopic handassisted splenectomy was also performed. There were no intra-operative complications and the allograft produced urine immediately. She received five more sessions of plasmapheresis in the first 10 post-operative days, to maintain anti-A titres 1 : 4. The patient was discharged 1 week after surgery with a plasma creatinine of 63 mmol/l. Immunosuppression was now tacrolimus, MMF and prednisone. After 2 weeks of the transplant, the creatinine rose to 141 mmol/l, and an urgent allograft biopsy was performed. The anti-A titre was 1 : 4 and the crossmatch was positive against donor Tand B-cells by flow cytometry. The biopsy showed mild focal neutrophil capillaritis, acute tubular injury and intraluminal calcifications. There was diffuse staining for C4d in the peritubular capillaries. Interestingly, she had started p.o. phosphate 1–2 days prior to the elevation in plasma creatinine. The possibility of acute calcium—phosphate precipitation in the allograft [1] was, therefore, entertained and the phosphate was stopped. The presumed acute antibodymediated rejection was treated with four sessions of plasmapheresis over 10 days (followed by 10 g IgG) and pulse IV methylprednisolone. The plasma creatinine fell quickly to 71 mmol/l. A repeat flow cytometry crossmatch against donor Tand B-cells was negative. The patient was continued on immunosuppression with tacrolimus, MMF and prednisone. No further Correspondence and offprint requests to: Colm C. Magee, MD, Renal Division, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA. Email: cmagee@partners.org Nephrol Dial Transplant (2007) 22: 602–604

Successful Living Related Kidney Transplantation Across an Anti-Donor HLA Antibody

In preconditioning highly sensitized kidney transplant candidates, renal allograft outcomes have been better when the serum titer for class I anti-HLA donor-specific antibody (DSA) is low in the recipient at the time of transplantation. However, the ideal level to which the titer should be lowered is still controversial. We report a primary living related kidney transplant in a 34-year-old highly sensitized woman (pretransplant panel-reactive antibody = 70%) with end-stage renal disease secondary to chronic glomerulonephritis. We sought to desensitize by lowering the DSA titer specifically to 1:4 pretransplant. A standard complement-dependent cytotoxicity cross-match with her donor (sister) was repeatedly negative, although she was positive for class I antibody against her mismatched HLA antigen (A*2402) at a titer up to 1:16 by the single-antigen flowbead assay. The target DSA titer of 1:4 before transplant was achieved by 12 sessions of plasmapheresis (PP) over 7 weeks, plus two intravenous immune globulin infusions (IVIG) (500 mg/kg/infusion). The patient outcome was excellent. Neither IVIG nor PP was needed posttransplant. The serum creatinine ranged between 0.5 mg/dL and 1.2 mg/dL, and no rejection episode was documented at 28 weeks posttransplant. Therefore, we encourage the use of IVIG and PP to lower the DSA titer to at least 1:4 before kidney transplantation in highly sensitized patients. Large prospective trials are needed to establish a consensus for pretransplant risk assignment and to evaluate the need for desensitization.

Alloantibodies and the Outcome of Cadaver Kidney Allografts

The role of humoral immunity in causing antibody-mediated rejection (AMR) of organ allografts has been extensively documented. For this reason, negative complement-dependent cytotoxicity (CDC) cross-matches between recipient sera and donor T and B lymphocytes have become a mandatory requirement for cadaveric kidney transplantation. However, the significance of donor-specific antibodies (DSAs) detectable only by flow cytometry (FC) or solid phase assays (SPA) but not CDC is still controversial. We have performed a retrospective analysis of FC cross-matching results in 80 consecutive cadaver kidney allograft recipients. Antibodies against HLA class I and class II antigens were measured by CDC and SPA in sequential samples of sera obtained prior to transplantation. The preoperative cross-match was performed by CDC using magnetically sorted T and B cells from donor spleen. Sera obtained from each patient before and at the time of transplantation were included in the final cross-match. The sample of serum obtained at the time of transplantation was cross-matched retrospectively by FC and analyzed for anti-HLA antibody specificity on high resolution SPA. The actuarial kidney allograft survival at one year was 98%. Two of these eighty patients lost the graft, one due to AMR, the other for reasons unrelated to DSAs. Donor-specific antibodies were detected by FC in 17 of 80 patients, yet only 6 of 17 had an early episode of AMR. This episode was successfully reversed by desensitization therapy using intravenous immunoglobin (IVIG) and plasmapheresis. Flow cytomery cross-matching showed 95% specificity but only 35% sensitivity for prediction of AMR ( p = 0.002). There was a significant correlation between high panel reactive antibodies (PRA) and positive FC cross-matching ( p = 0 .0001), as well as high PRA and AMR ( p = 0.0004 by CDC and 0.0011 by Luminex). Reversible AMR occurred 12–30 days post-transplantation in 8 patients. Of these 8 patients, 3 had no detectable DSAs in spite of C4d positivity, 4 had C4d deposition in conjunction with anti-HLA antibodies, and 1 patient had DSAs (anti-MICA) yet no C4d deposition. We conclude that early initiation of desensitization protocols can prevent transplant failure and that retrospective FC cross-matches may facilitate the diagnosis of AMR. Extensive analysis of patients’ sera using a comprehensive set of tests may contribute to early treatment and better understanding of the mechanism underlying humoral rejection.

Detection of donor-specific antibodies using HLA-coated microspheres: another tool for kidney transplant risk stratification**Portions of this study were delivered as an oral presentation at the American Transplant Congress, Seattle, WA, 20–25 May 2005.

Sensitive techniques are able to detect low levels of circulating antibodies. For many newer techniques, the clinical consequences of these antibodies are unknown. We hoped to determine the significance of antibodies detected through the use of Luminex microsphere-based assay. Patients who received kidney transplants between March 2003 and May 2004 with negative anti-human globulin-augmented complement-dependent cytotoxicity (AHG-CDC) crossmatches were retested for pre-transplant panel reactive antibodies (PRA) using Luminex microspheres and stored sera. Patients were considered to have circulating antibodies if either class I or class II Luminex PRA was >or=15%. These patients were then analysed for pre-transplant donor-specific antibodies (DSA). Clinical outcomes were compared in patients with and without DSAs. Out of 136 patients who underwent transplantation, 55 had Luminex PRA >or=15%. Of these 55 patients, only 16 had a standard PRA >or=30% and 75% had a history of a sensitizing event. Twenty out of 55 patients were DSA+. Patients with DSA detected by Luminex had higher rates of primary non-function (PNF), delayed graft function, biopsy-proven acute rejection, and lower rates of graft survival at 6 months. A combined endpoint of immunological and clinical events was far more common in patients with DSA. The detection of DSAs by Luminex microspheres was associated with significantly higher rates of graft dysfunction and immunological events. Conversely, the presence of antibodies but no DSA by Luminex was associated with excellent outcomes. In patients with negative AHG-CDC crossmatches, the occurrence of low-level DSA by Luminex could assist in identifying patients that require more aggressive immune monitoring or immunosuppressive strategies.

Heart transplantation across antibodies against human leukocyte antigens and blood group A1 antigen. Post‐transplant follow‐up of donor reactive antibodies

We have successfully performed heart transplantation despite the most unfavourable risk factors for graft and patient survival: the presence of a high level of antibodies (Abs) against the donor's human leukocyte antigens (HLA) class I/II and blood group A1 antigens. The present study concerns post-transplant follow-up and characterization of donor reactive antibodies (DRA). Pre-transplant treatment consisted of mycophenolate mofetil (MMF), prednisolone, tacrolimus, intravenous immunoglobulin (IVIG), rituximab, protein-A immunoadsorption (PAIA) and per-operative plasma exchange. A standard triple-drug immunosuppressive protocol was used post-operatively. Abs were analyzed by the complement dependent cytotoxicity (CDC) test against donor and panel B/T cells and by flow cytometry (FlowPRA tests detecting isolated HLA class I/II antigens). Abs against the donor's erythrocytes were analyzed using a standard direct agglutination test for immunoglobulin M (IgM) Abs and a Bio-Rad AHG gel card test detecting IgG Abs and C3d. Pre-transplant treatment reduced Ab titers against the donor's lymphocytes from 128 to 16 and against the donor's blood group A1 antigen from 256 to 0. The patient was emergently transplanted with a heart from a blood group incompatible donor (A1 secretor to O). No hyperacute rejection was seen. DRA were present against all mismatched HLA class I and class II antigens at the time of transplantation; two of these DRA Abs disappeared within the first year post-transplant (anti-B62 and anti-DR4), one showed weakened reactivity (anti-A24) and one is still strongly reactive (anti-DQ3). The donor-specific CDC cross-match is still positive (titers 2 to 8). The level of panel reactive antibodies (PRA) remained unchanged from 6 months on post-transplant. Rising anti-A1 blood group Abs preceded the second rejection and were adsorbed by two blood group specific immunoadsorptions (Glycosorb)-ABO) and remained at a low level. IgM anti-A1 blood group Abs disappeared at 1 yr post-transplant and IgG Abs are still reactive with blood group A1 erythrocytes but at low titers (1 to 2). The patient is clinically well 2 years after heart transplantation despite the constant persistence of donor reactive IgG Abs against blood group A1 and HLA-DQ antigens. The reactivity of DRA against other mismatched HLA antigens disappeared or weakened during the follow-up period.

Is Positive Flow Cytometric Cross-Match a Risk Factor for Early Cadaveric Kidney Graft Dysfunction?

Introduction The final decision about transplantation is based primarily on a negative result of a complement-dependent cytotoxicity cross-match. The significance of a positive flow cytometric cross-match (FCXM) is unclear. Materials and methods From July 2002 to October 2004, FCXM was performed prior to cadaveric kidney transplantation in 63 patients aged 1.5 to 26 years (mean 13 ± 5). Immunosuppression (not adjusted to results of FCXM) was considered standard (prednisone + mycophenolate mofetil or azathioprine + cyclosporine or rapamycin) in 57%, or “enhanced” (+ monoclonal antibodies and/or tacrolimus) in 43% of patients. Results Immunoglobulin IgG and/or IgM antibodies against T and/or B cells were found in 14/63 patients (22.2%). The distribution of immunosuppressive regimens was similar for FCXM(+) and FCXM(−) patients. Deteriorated graft function (creatinine ≥1.5 mg/dL) or demand for dialysis was observed in 6/14 (42.9%) FCXM(+) group versus 6/49 (12.2%) in the FCXM(−) group. During the first month after kidney transplantation biopsy-proven rejection episodes occurred more frequently among the FCXM(+) than the FCXM(−) group: 21.4% versus 4.1%, respectively. During the first 3 months after transplantation two of four kidneys in the FCXM(+) group (14.3%) demonstrated histological evidence of rejection plus one case of immunological cause of graft failure later found to be associated with an extremely high panel-reactive antibodies that were absent before transplantation (altogether 21.4%). Only one kidney (2.0%) was lost due to rejection among the FCXM(−) group. Conclusion A positive flow cytometric cross-match should be considered an important risk factor for early kidney graft dysfunction.

Liver recipients harbouring anti-donor preformed lymphocytotoxic antibodies exhibit a poor allograft survival at the first year after transplantation: Experience of one centre

In this retrospective study, we analyzed the effect of the presence of anti-donor preformed alloantibodies in 268 liver allograft transplants. Crossmatches were performed by complement-dependent cytotoxicity (CDC) assay and HLA antibody screening by flow cytometry (FlowPRA™). Positive anti-donor crossmatch was detected in 5.2% of transplants. Acute rejection frequency in +CDC crossmatch patients was not different from that observed in −CDC crossmatch patients. None of the patients transplanted with +CDC crossmatch developed chronic rejection, but they showed a significantly lower allograft survival rate, and the majority of them had allograft failures before the end of the first post-transplant year, mainly within the 3 first months. Indeed, positive FlowPRA determination was concordant with data from the CDC assay. In conclusion, these findings show a direct correlation between the presence of anti-donor preformed antibodies and a poor allograft survival in liver transplant.

Peritransplant Immunoadsorption: A Strategy Enabling Transplantation in Highly Sensitized Crossmatch-Positive Cadaveric Kidney Allograft Recipients

Kidney transplant recipients with a current positive complement-dependent cytotoxicity crossmatch (CDCXM) are at high risk for hyperacute rejection and graft loss. Immunoadsorption (IA) represents an efficient strategy to remove donor-specific alloantibodies. In this analysis, we evaluated effectiveness of peritransplant IA as an anti-humoral strategy to overcome a current positive CDCXM in presensitized renal allograft recipients. Between 1999 and 2003, 40 high risk cadaveric kidney allograft recipients (median CDC panel reactive antibody [PRA] level, 77%; number of retransplants, n = 38) were subjected to peritransplant IA with protein A (one pretransplant IA session followed by a course of repeat posttransplant IA sessions) in addition to preemptive antilymphocyte antibody therapy. In nine of these patients, a current positive CDCXM was rendered negative by a single pretransplant IA session. Thirty-one recipients had a negative CDCXM already before pretransplant IA. No difference in graft survival was found between CDCXM-positive and CDCXM-negative recipients (3-year graft survival, 78% vs. 71%, P = 0.6). Comparable rates of immunological graft loss at 3 years were observed (11% vs. 13%, P = 0.8). Patient groups did not significantly differ with respect to median serum creatinine at 1 year (1.23 mg/dL [CDCXM-positive] vs. 1.57 mg/dl [CDCXM-negative], P = 0.07) and at the end of follow-up (median 32 months; 1.19 mg/dL vs. 1.63 mg/dL, P = 0.06). Moreover, patient groups showed similar rates of biopsy-proven cellular rejection (11% vs. 20%) or C4d-positive graft dysfunction (33% vs. 32%). Our results demonstrate that peritransplant IA enables successful cadaveric kidney transplantation in the context of a positive CDCXM.

New Crossmatch Technique Eliminates Interference by Humanized and Chimeric Monoclonal Antibodies

Humanized and chimeric antilymphocyte antibodies (Ab) are used to prevent and treat rejection and for treatment of human disease. Rituximab (RIT, anti-CD20), daclizumab (DAC; anti-CD25), alemtuzumab (ALE; anti-CD52), or infliximab (IFX) may interfere with Ab detection methods such as complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM). These agents are recognized as anti-human Ab or fix complement and are not differentiated from anti-allo-Ab. A new enzyme-linked immunosorbent assay crossmatch (XM) utilizing class I and II HLA antigens from donor cells called Transplant Monitoring System (TMS; GTI, Waukesha, Wisc) potentially precludes interference by eliminating non-major histocompatability complex antigens. To test this, normal sera (nonsensitized volunteers) were supplemented with 0.1 or 10 μg/mL of RIT, DAC, IFX or ALE, and were tested using three methods: the TMS T-cell CDCXM with antihuman globulin (AHG); and B-cell CDCXM without AHG; and FCXM with mean channel shifts of 45 and 150 indicating positive T-cell and B-cell crossmatch, respectively. No reactivity occurred with normal sera using any crossmatch technique. At 0.1 and 10 μg/mL, RIT interfered with CDC B-cell, but not T-cell crossmatch. RIT at 10, but not 0.1 μg/mL interfered with B-cell FCXM. No interference occurred with RIT in T-cell FCXM or TMS. ALE interfered with B-cell and T-cell CDC and FCXM but neither class I nor II TMS. DAC did not interfere with CDC or FCXM at 0.1 μg/mL, but gave false positive B-cell FCXM and CDCXM with some samples. No interference by DAC occurred using TMS. TMS may be useful to differentiate de novo donor-specific Ab after treatment with humanized or chimeric Ab.

The impact of pre-transplant flow cytometry crossmatches on renal allograft outcome: A pilot study

BACKgrOUND: Pre-Transplant Complement Dependent Cytotoxicity (CDC) Crossmatch is an established procedure known to influence renal allograft survival rates. The Flow Cytometry Crossmatch (FCXM) is considered more sensitive, and based on literature reports, is believed to enhance renal allograft outcome. Before utilizing this procedure, some programs are requiring center specific data. PURPOSE OF THE STUDY: The purpose of this study was to investigate the impact of a positive FCXM on renal allograft outcome in a single center. METHOD: Fifty-six patients who received renal transplants at The Transplant Institute of New Orleans were investigated. The pre-transplant workup included both a CDC and FCXM. A negative CDC crossmatch against donor T Cells was required for inclusion in the study. The results of the Flow Crossmatch were not used for excluding patients for getting a transplant. The clinical outcome in terms of successful versus failed transplants was recorded over a three year period. A failed graft was defined as return to dialysis. RESULTS: Nine out of fifty-six renal allografts failed due to immunologic rejections. An additional two grafts were lost due to other reasons. Amongst those who lost the renal grafts due to rejection, thirty-three percent had a positive pre-transplant FCXM. On the other hand, the FCXM was positive in sixteen percent of those who had functioning renal grafts. CONCLUSION: Although these numbers are small, the data suggest that positive FCXMS are related to lower renal allograft survival rates at our center. Further studies will be performed on a larger patient population.

Persistence of low levels of alloantibody after desensitization in crossmatch-positive living-donor kidney transplantation.

: Desensitization protocols have been developed to allow successful kidney transplantation in sensitized recipients. However, a detailed analysis of the impact of these protocols on alloantibody has not been performed. : We studied 12 living-donor kidney-transplant recipients with positive antihuman globulin-enhanced complement dependent cytotoxicity (AHG-CDC) crossmatches against their donors. Using a variety of crossmatch techniques and single-antigen flowbeads (SAFBs), we characterized the specificity and amount of alloantibody at baseline before desensitization, after desensitization (using plasmapheresis followed by 100 mg/kg intravenous immunoglobulin, and anti-CD20 antibody), and 4 months after transplantation (after splenectomy and on maintenance immunosuppression). : All 12 patients with a positive baseline AHG-CDC crossmatch were AHG-CDC crossmatch negative at the time of transplant (after desensitization). However, despite desensitization, the majority of patients had low-level donor-specific alloantibodies demonstrable on the day of transplantation by both flow crossmatch (FXM 8/12) and SAFBs (10/11). Four months after transplantation, no patient had a positive AHG-CDC crossmatch, but again the majority had persistent low levels of donor-specific alloantibodies by FXM (6/12) and SAFBs (9/11). No patient experienced hyperacute rejection, and the persistence of low levels of donor-specific alloantibodies did not correlate with the development of humoral rejection in the early posttransplant period. : Despite desensitization, a majority of positive crossmatch transplant recipients demonstrate low levels of donor-specific alloantibodies both on the day of transplant and 4 months after transplantation. The impact of these antibodies appears to be minimal early after transplant, but their long-term significance bears further study.

Will transplantation of kidneys from donors with blood group A2 into recipients with blood group B help British Indo-Asian patients with renal failure?

Despite a high incidence of renal failure, disproportionately fewer Indo-Asians in the United Kingdom receive a renal transplant, in part because of the high prevalence of blood group B. It is now clear that it is possible to safely transplant kidneys from donors with blood group A of the subtype A2 into recipients with blood group B if the latter have low titers of anti-A antibodies. We measured the anti-A titers in 25 Indo-Asian patients on dialysis being considered for transplantation and found stably low titers in all. Titers were comparable to those found in a control white population with blood group B. In addition, in a complement-dependent cytotoxicity crossmatch against group A lymphocytes, the only positive results were obtained in those with high preexisting panel reactivity (i.e., because of the presence of preformed anti-human leukocyte antigen antibodies). We conclude that there are grounds for investigating this approach further to solve the ethnic disparity in rates of transplantation.

Are preformed antibodies to biliary epithelial cells of clinical importance in liver transplantation?

During acute liver allograft rejection, most of the tissue damage to bile duct epithelium is thought to occur as a consequence of direct immunologic injury by T-cell-mediated immune effector mechanisms. However, the role of antibodies to biliary epithelial cells (BECs) in liver transplant rejection is not known. We therefore investigated cross-match sera obtained immediately before liver transplantation from 95 patients for the presence of BEC-reactive antibodies to determine their association with acute rejection. BECs were isolated from one normal healthy liver. Antibody binding was detected by using flow cytometric analysis. Donor lymphocyte-specific cross-matches using complement-dependent cytotoxicity (CDC) and flow cytometric assays also were performed. The 2-year patient survival rate in this study was 86.3%. Eleven patients were positive for either CDC or flow cytometric cross-matches. BEC antibodies were detected in 41 serum samples (43.2%). Patients with BEC antibodies experienced acute rejection more frequently (65.9%) compared with 42.5% without antibodies (P <.03). HLA specificity determinations indicated that in 5 of 41 cases, anti-BEC reactivity was caused by HLA antibodies. No correlations between the presence of BEC antibodies and patient survival and the occurrence of cholangitis and nonsurgical bile duct strictures were found within 2 years of follow-up. In conclusion, preformed antibodies to BECs are associated with acute rejection. Thus, the presence of these antibodies before transplantation may facilitate acute liver graft rejection.

Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients.

Hyperacute rejection (HAR) and acute humoral rejection (AHR) remain recalcitrant conditions without effective treatments, and usually result in graft loss. Plasmapheresis (PP) has been shown to remove HLA- specific antibody (Ab) in many different clinical settings. Intravenous gamma globulin (IVIG) has been used to suppress alloantibody and modulate immune responses. Our hypothesis was that a combination of PP and IVIG could effectively and durably remove donor-specific, anti-HLA antibody (Ab), rescuing patients with established AHR and preemptively desensitizing recipients who had positive crossmatches with a potential live donor. The study patients consisted of seven live donor kidney transplant recipients who experienced AHR and had donor-specific Ab (DSA) for one or more mismatched donor HLA antigens. The patients segregated into two groups: three patients were treated for established AHR (rescue group) and four cross-match-positive patients received therapy before transplantation (preemptive group). Using PP/IVIG we have successfully reversed established AHR in three patients. Four patients who were cross-match-positive (3 by flow cytometry and 1 by cytotoxic assay) and had DSA before treatment underwent successful renal transplantation utilizing their live donor. The overall mean creatinine for both treatment groups is 1.4+/-0.8 with a mean follow up of 58+/-40 weeks (range 17-116 weeks). In this study, we present seven patients for whom the combined therapies of PP/IVIG were successful in reversing AHR mediated by Ab specific for donor HLA antigens. Furthermore, this protocol shows promise for eliminating DSA preemptively among patients with low-titer positive antihuman globulin-enhanced, complement-dependent cytotoxicity (AHG-CDC) cross-matches, allowing the successful transplantation of these patients using a live donor without any cases of HAR.

Flow cytometry crossmatching as a predictor of acute rejection in sensitized recipients of cadaveric renal transplants

Flow cytometry crossmatching (FCXM) was developed as a more sensitive assay than the standard complement-dependent cytotoxicity crossmatch (CDCXM) for the detection of anti-donor antibodies, that mediate hyperacute rejection and graft loss in the early post-transplant period in renal transplant recipients. The role of FCXM in predicting long-term clinical outcome in renal allograft recipients is unclear. This study examines the role of FCXM in predicting long-term clinical outcome in highly sensitized recipients of cadaveric renal transplants. All patients (n = 100) with peak panel reactive antibody (PRA) levels > 30%, who received cadaveric renal transplants between 1/1/'90 and 12/31/'95 at our institution, were divided into FCXM + and FCXM - groups. The incidence of acute rejection was determined for each group during the first yr after transplant. Graft survival rates at 1, 2, and 3 yr, and creatinine levels were also compared between groups. FCXM + patients experienced a higher incidence of acute rejection during the first yr after transplant (69 vs. 45%), and a higher percentage of FCXM + patients had more than one episode of acute rejection during the first yr after transplant (34 vs. 8%) when compared to FCXM - patients. There was no statistically significant difference in 1-, 2-, or 3-yr graft survival between FCXM + and FCXM - patients (76 vs. 83, 62 vs. 80, 62 vs. 72%, respectively). These results suggest that sensitized FCXM + cadaveric renal transplant recipients have a higher incidence of acute rejection episodes in the first yr after transplant. Given the association of multiple rejection episodes with poor long-term allograft survival, FCXM may be a useful predictor of long-term clinical outcome in this sub-group of renal transplant recipients.