Lymphocyte Crossmatch Research Articles

International kidney paired donation transplantations to increase kidney transplant of O group and highly sensitized patient: First report from India.

AIMTo report the first international living related two way kidney paired donation (KPD) transplantation from India which occurred on 17th February 2015 after legal permission from authorization committee.METHODSDonor recipient pairs were from Portugal and India who were highly sensitized and ABO incompatible with their spouse respectively. The two donor recipient pairs had negative lymphocyte cross-matching, flow cross-match and donor specific antibody in two way kidney exchange with the intended KPD donor. Local KPD options were fully explored for Indian patient prior to embarking on international KPD.RESULTSBoth pairs underwent simultaneous uneventful kidney transplant surgeries and creatinine was 1 mg/dL on tacrolimus based immunosuppression at 11 mo follow up. The uniqueness of these transplantations was that they are first international KPD transplantations in our center.CONCLUSIONInternational KPD will increases quality and quantity of living donor kidney transplantation. This could be an important step to solving the kidney shortage with additional benefit of reduced costs, improved quality and increased access for difficult to match incompatible pairs like O blood group patient with non-O donor and sensitized patient. To the best of our knowledge this is first international KPD transplantation from India.

Comparison of Clinical Outcomes in Hepatitis B Virus–Positive and –Negative Renal Transplant Recipients

Our aim was to compare the short- and long-term clinical outcomes of hepatitis B surface antigen–positive (HbsAg+) renal transplant recipients with HbsAg− recipients. A total of 204 patients who underwent renal transplantation in our center between 2001 and 2014 were included in the study. The patients were divided into 2 groups. Group 1 was the HbsAg− group (n = 136), and group 2 was the HbsAg+ group (n = 68). There was no significant difference between the groups in terms of lymphocyte crossmatches, numbers of mismatches, immunosuppressive treatment protocols, and induction treatments. In the HbsAg+ group, 51 patients were hepatitis B virus DNA+, 64 patients were HbeAg−, and 4 patients were HbeAg+. A total of 57 patients (83.8%) were treated with lamivudine, 4 patients (5.9%) with entecavir, and 7 patients (10.3%) with tenofovir for hepatitis B infection. Graft and patient survival rates, graft functions, acute hepatitis rates, acute rejection rates, and other clinical outcomes of the groups were compared. Demographic data and immunologic risk profiles of the groups were similar. Acute rejection rates, graft survival rates, and patient survival rates were similar. Acute hepatitis rates, glomerular filtration rates on the last controls, and delayed graft function rates were higher in group 2, whereas chronic allograft dysfunction and new-onset diabetes mellitus after transplantation rates were similar between the groups. Our study revealed that graft and patient survival, and acute rejection rates were similar between HbsAg+ and HbsAg− recipients, whereas acute hepatitis rate was higher in HbsAg+ recipients.

HLA compatibility assessment and management of highly sensitized patients under the new kidney allocation system (KAS): A 2016 status report from twelve HLA laboratories across the U.S.

Twelve HLA laboratories were surveyed to assess the methods and operational issues involved to define highly sensitized patients and to assess HLA compatibility under the new kidney allocation system (KAS) in the U.S. All laboratories used single antigen bead assays both pre- and post-KAS to define both broad and allele-specific HLA antibodies. The methods and threshold used to list HLA unacceptable antigens in UNet for virtual crossmatch (vXM) and the criteria used for determining HLA compatibility varied among laboratories. Laboratories reported several limitations of the current assays including the accuracy of quantifiable antibody fluorescence values, inadequate coverage of common alleles on the bead panels, and challenges in calibrating the vXM. The new KAS has resulted in a significant surge of deceased donor organ offers requiring vXM evaluation under tight time constraints. In the post-KAS period, eight of twelve laboratories (67%) indicated that their center did not proceed to transplant based on vXM without a prospective lymphocyte crossmatch. In conclusion, HLA laboratories play a critical role in deceased donor allocation for highly sensitized patients under the new KAS. Significant opportunities exist to improve the methods used in the assessment of HLA compatibility to safely transplant highly sensitized patients.

Therapy Modalities for Antibody Mediated Rejection in Renal Transplant Patients

ABSTRACTIntroduction: The aim of our study was to determine the effectiveness of immunoglobulin, rituximab and plasmapheresis in renal transplant patients with antibody mediated rejection (AMR). Patients and Methods: Fourteen renal transplant patients with AMR were included in this study. The mean age of the patients was 33.9 ± 10.3 years and 10 (71.4%) of them were male. Lymphocyte cross match was negative for all patients and 10 (71.4%) of them were living donor transplants. Six patients were administered tacrolimus, three patients cyclosporine, two patients everolimus, and three patients sirolimus for immunosuppression. The patients with AMR were administered IVIG, rituximab and plasmapheresis. Results: Patient survival rate was 100%, graft survival rate after AMR was 50% in the first year and 33% in the 2nd and third years. AMR developed 31.9 ± 25.9 months after transplantation. Seven (50%) patients lost their grafts. Delayed graft function was observed in 28.6%, chronic allograft dysfunction in 78.5%, diabetes after transplantation in 14.3%, and cytomegalovirus infection in 7.1% of the patients. At the last follow-up, the mean blood creatinine was 3.1 ± 1.4, the mean proteinuria was 2300 (1300–3300) mg/day and the mean GFR was 34.5 ± 17.6 ml/min. C4d was positive in peritubullar capillaries in all patients, while neutrophil accumulation in peritubular and glomerular capillaries was observed in 8 patients. Chronic allograft vasculopathy was observed in 12 patients. Conclusion: AMR leads to progressive loss of renal function and has low graft survival. More effective treatment alternatives are needed for this clinical issue.

Effects of hepatitis B surface antigen (HBsAg) positivity of donors in HBsAg(+) renal transplant recipients: comparison of outcomes with HBsAg(+) and HBsAg(-) donors.

The aim of this study was to determine the effects of hepatitis B surface antigen (HBsAg) positivity of the donors on graft survival and liver complications in HBsAg(+) renal transplant recipients. A group of 55 patients who underwent renal transplantation (RTx) in our hospital between 2001 and 2012 were included in the study. Patients were divided into 2 groups. Group 1 (n = 50) consisted of HBsAg(+) renal transplant recipients (RTR) whose donors were HBsAg(-). In Group 2 (n = 5), RTR and donors were both HBsAg(+). Lymphocyte cross matches, number of mismatches, donor types, renal replacement treatment modalities, drugs of induction treatment, and preoperative hepatitis B virus DNA titers of the groups were similar. In Group 1, 42 patients were taking lamivudine, 3 patients were taking entecavir, and 5 patients were taking tenofovir. All of the patients in Group 2 were taking lamivudine. Patient and graft survival rates, graft functions, acute hepatitis rates, acute rejection rates, and other clinical outcomes of the groups were compared. Demographic data of the groups were similar. Acute rejection rates (P = 0.458), graft survival rates (P = 0.515), and patient survival rates (P = 0.803) were also similar. No significant difference was found between the groups in terms of acute hepatitis rate (P = 0.511), glomerular filtration rate (calculated by Modification of Diet in Renal Disease formula) in the last follow-up (P = 0.988), alanine aminotransferase levels (P = 0.069), or delayed graft function rate (P = 0.973). Rates of chronic allograft dysfunction and new onset diabetes mellitus after transplantation were similar. Our study revealed that, RTx from HBsAg(+) donors to HBsAg(+) recipients is safe with antiviral treatment.

A novel procedure of flow cytometry lymphocyte crossmatch test with high sensitivity using human lymphocytes in renal transplantation

A novel procedure of flow cytometry lymphocyte crossmatch test with high sensitivity using human lymphocytes in renal transplantation

Graft Failure after Hematopoietic Cell Transplantation for Hemoglobin Disorders: Successful Application of a Rescue Regimen

BackgroundAlthough patients with clinically significant hemoglobinopathies can be supported with blood transfusions and iron chelation therapy, the only curative option is allogeneic hematopoietic stem cell transplantation (HCT). Unfortunately, there is a risk of graft rejection as a consequence of donor sensitization and alloimmunization related to transfusion exposures and a hyperproliferative marrow niche. Typically, graft rejection after HCT for hemoglobinopathies is accompanied by autologous hematopoietic recovery, but there is also a risk of graft failure and marrow aplasia which can cause a fatal outcome. To accommodate individuals who lack a suitable sibling donor, transplant options are being expanded to include alternative sources of hematopoietic donors which carry higher risks of graft rejection, graft-versus-host disease and transplant-related mortality. Given the significant risk of graft failure after alternative donor HCT, it is important to develop a plan for stem cell rescue when these complications occur.Methods and ResultsWe reviewed 3 patients with hemoglobin disorders who were successfully rescued by alternative transplantation regimens following primary or secondary graft failures. Two patients with sickle cell disease and one with β-thalassemia major were treated at our institution with a reduced-intensity regimen. All 3 patients had graft rejection accompanied by marrow aplasia within 2 months after HCT. Patient 1 was successfully rescued with mobilized peripheral blood stem cells from the same HLA mismatched sibling donor after a minimal toxicity preparative regimen. Patients 2 and 3 were successfully rescued from HLA-haploidentical donors after a minimal toxicity preparative regimen that included in vivo T-cell depletion by post-grafting high dose cyclophosphamide. Refer to Table 1 for patient and transplant characteristics.ConclusionThere are 3 methods to rescue patients from graft rejection: donor lymphocyte infusions, the intensification of immunosuppression or a rescue allogeneic transplant. Here we report our local experience with graft failure and its successful management in 3 patients with hemoglobin disorders. Although graft rejection remains a major obstacle to successful transplantation for hemoglobinopathies, better donor selection and screening by lymphocyte cross matching, optimized conditioning regimens and improved supportive care should improve outcomes after alternate donor HCT. We speculate that it is also possible now to rescue from graft failure in most cases and that an allogeneic HCT rescue might be utilized for a curative outcome.Table 1Patient characteristics, rescue transplant regimen, engraftment, graft-versus-host-disease (GVHD), and duration of immune suppression post-rescue HCT. BM, Bone Marrow; UCB, Umbilical Cord Blood; ATG, Anti-thymocyte Globulin; HLA, Human Leukocyte Antigen; MMF, Mycophenolate Mofetil; TBI, Total Body IrradiationPatient 1Patient 2Patient 3Age, Gender14 yo Male3 ½ yo Male14 yo MaleDiagnosisHemoglobin SSβ-Thalassemia majorHemoglobin SSIndication for TransplantSubarachnoid Hemorrhage Veno-occlusive crisesAlternative to life-long transfusions and iron chelation therapyMultiple Cerebral infarcts Acute Chest Syndrome Veno-occlusive crisesTransplant #1 Conditioning RegimenBusulfan Thiotepa Cyclophosphamide Rabbit ATGBusulfan Thiotepa Fludarabine Rabbit ATGBusulfan Fludarabine Rabbit ATGTransplant #1 Cell SourceBM, 9/10 HLA mismatch at A locusSingle 6/6 HLA matched UCBBM, 10/10 HLA matched siblingGVHD Prophylaxis for Transplant #1Cyclosporine MMFCyclosporine MethotrexateType of Graft FailureSecondaryPrimarySecondaryRescue Transplant Conditioning RegimenFludarabine 30mg/m2 on days -4 through -2; TBI 400cGy on day 0Fludarabine 30 mg/m2 on days -6 through -2; TBI 400cGy on day 0; Cyclophosphamide 14.5 mg/kg/day on days -6, -5; and 50 mg/kg/day on days +3,+4Recue Transplant Cell SourceSame donor, Mobilized PBSCHaploidentical motherHaploidentical fatherRescue GVHD ProphylaxisTacrolimus MMFTime to Neutrophil Engraftment (days)141415Time to Platelet Engraftment (days)182522Day 28 Percent Donor Cells98%100%99%Acute GVHDNoneSkinNoneChronic GVHDNoneOral and Integument (resolved)Pericardial effusion (resolved)Duration of Post-Rescue Transplant Immune Suppression17 months37 months16 months DisclosuresNo relevant conflicts of interest to declare.

DSA and C1q assay in kidney post-transplant monitoring

Aim Predicting the risk of graft loss using C1q assay in the post-transplant DSA monitoring. Methods Retrospective review of post-kidney transplant patients who were tested for DSA after ABO compatible kidney transplantation with negative T and B lymphocyte crossmatch. Patients in post-transplant follow-up since 2007 were tested for donor specific HLA antibody (DSA) IgG class according to schedule by Labscreen phenotype beads/Single Antigen beads (SAB, One Lambda), and if detected, reflexed to C1q assay (One Lambda) since 2011. Results Of the 437 post-kidney transplant patients (range: 1–27 years post-transplant, mean 9.1 years), de novo DSA was detected in 74 (16.9%). C1q assay was performed in 50/74 DSA positive patients: Positive at least once N = 35 (70%) and Negative N = 15. DSA in 26 patients were tested for > 1 year; persistently positive C1q, N = 17, eventually became C1q negative N = 6, or became DSA negative N = 3. Overall, C1q positive DSA presented higher maximum MFI detected by both phenotype beads and SAB, and resulted in higher graft loss. C1q MFI was similar between graft lost and functional (defined by not on dialysis). Regardless of C1q status, graft loss was observed after month-to-years with DSA, indicating chronic antibody mediated rejection. Conclusions Patients with C1q positive DSA had higher MFI (IgG) levels and resulted in higher graft loss. Download : Download full-size image

A Successful Transplantation from Maastricht Category-4 Non-Heart Beating Donor

The shortage of available organs is the limiting factor for kidney transplantation (Ktx). One of the strategies to increase access to Ktx is the use of non-heart beating (NHB) donors. We herein present a successful Ktx from an NHB (Maastricht Category-4) donor. The donor was a 21-year-old woman who had brain death due to cerebrovascular accident. She had severe hypotension for three hours despite 30 μg/kg/min dopamine infusion, followed by cardio-pulmonary arrest just before the operation began. Cardio-pulmonary resuscitation was consequently initiated and continued during the operation. Donor organ harvesting (hepatectomy and bilateral nephrectomy) was performed after direct aortic cannulation. The warm ischemia time was 35 minutes. The recipient was a 54-year-old man who had undergone hemodialysis for four years. There were 3/6 mismatches, and the lymphocyte cross-match was negative. The induction immunosuppressive was anti-thymocyte globulin, followed by tacrolimus/ MMF/steroid after the 12th day. He had delayed graft function and hemodialysis was required at the third and sixth days. The urine output gradually increased and sCr slowly declined afterwards and he discharged with sCr 1.5 mg/dl on the 24th day. At the first year of transplantation, he is in good clinical condition with a stable baseline sCr of 1.5 mg/dl. NHB donors can be an alternative and important source to expand the kidney donation pool, and successful long-term outcomes favor this modality.

Preoperative selective desensitization of live donor liver transplant recipients considering the degree of T lymphocyte cross-match titer, model for end-stage liver disease score, and graft liver volume.

Several studies have suggested that a positive lymphocyte cross-matching (XM) is associated with low graft survival rates and a high prevalence of acute rejection after adult living donor liver transplantations (ALDLTs) using a small-for-size graft. However, there is still no consensus on preoperative desensitization. We adopted the desensitization protocol from ABO-incompatible LDLT. We performed desensitization for the selected patients according to the degree of T lymphocyte cross-match titer, model for end-stage liver disease (MELD) score, and graft liver volume. We retrospectively evaluated 230 consecutive ALDLT recipients for 5 yr. Eleven recipients (4.8%) showed a positive XM. Among them, five patients with the high titer (> 1:16) by antihuman globulin-augmented method (T-AHG) and one with a low titer but a high MELD score of 36 were selected for desensitization: rituximab injection and plasmapheresis before the transplantation. There were no major side effects of desensitization. Four of the patients showed successful depletion of the T-AHG titer. There was no mortality and hyperacute rejection in lymphocyte XM-positive patients, showing no significant difference in survival outcome between two groups (P=1.000). In conclusion, this desensitization protocol for the selected recipients considering the degree of T lymphocyte cross-match titer, MELD score, and graft liver volume is feasible and safe.

Flow Cytometry–Based Cytotoxicity and Antibody Binding Assay

Human leukocyte antigen (HLA) antibodies with the ability to activate complement are associated with an increased risk of early antibody-mediated graft rejection in kidney transplantation (KTx). Detection of these potentially harmful complement-fixing HLA antibodies is commonly performed via the complement-dependent cytotoxicity (CDC) assay according to protocols that were developed as early as 40 years ago. The read-out for this assay is based on manual scoring by visual inspection of cells under a fluorescence microscope. CDC is often used in combination with the flow cytometry-based lymphocyte crossmatch assay (FCXM), which, with high sensitivity, detects HLA antibody binding. Here we describe a new approach wherein both cytotoxicity and antibody binding can be simultaneously assessed with flow cytometry. Two strategies are described, using either magnetic bead-enriched T and B lymphocytes or bulk peripheral blood mononuclear cells (PBMC) as donor target cells.

The outcome of the endothelial precursor cell crossmatch test in lymphocyte crossmatch positive and negative patients evaluated for living donor kidney transplantation

The presence of human leukocyte antigen (HLA) and non-HLA antibodies (Abs) in kidney transplant recipients is associated with graft rejections. This study reports the results of an endothelial precursor cell crossmatch (EPCXM) test for detection of non-HLA Abs and its correlation to lymphocyte crossmatch (LXM) test results, the degree and type of sensitization, and transplantation (Tx) outcome in patients evaluated for living donor (LD) kidney transplantation (KTx). Patients were tested before any pre-transplantation (pre-Tx) treatment and at Tx. Pre-Tx treatments included B cell depletion and Ab removal. Patient records were reviewed for assessment of renal graft function, results of biopsies, and identification of complications affecting the graft. Pre-Tx sera from 32% of the LD patients had IgG and/or IgM-binding donor EPCs. Twenty-five percent of the patients were EPCXM IgM+. Of the patients with negative LXM tests, 25% had EPC Abs mainly of IgM class not reactive with HLA. There was no difference in rejection frequency or serum creatinine levels between the EPCXM+ and EPCXM- groups. The pre-Tx EPCXM+ group had significantly more patients with delayed graft function. Prospective studies with appropriate control groups are needed to establish whether pre-treatments aiming at removing anti-endothelial cell antibodies, as detected by the EPCXM pre-Tx, have a beneficial effect on short-term and long-term graft survival.

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Abstract Aim It is generally accepted that a positive cytotoxic lymphocyte crossmatch (CDC-XM) is a contraindication for transplantation. However the assay is insensitive for detecting low levels of alloantibodies. The Flow cytometry crossmatch (IgG-FXM) is very sensitive but cannot differentiate HLA complement fixing antibody (CFAb) from non-CFAb and its clinical significance is still controversial. Based on the clinical relevance of CDC, we have successfully developed a new Flow Cytometry C1q crossmatch (C1q-FXM) to specifically detect Methods Donor PBMCs (0.1x 10 6 ) were incubated with 30 ul serum and CD3-PerCPCy5.5, CD19-FITC and SAPE-Bio-C1q for 30 minutes. After washing, the cells were collected and analyzed on a Canto-II flow cytometer. The PE fluorescence intensity on the T and B lymphocytes is proportionate to the amount of C1q fixed on the cell surface and reflects the complement fixing capability of DSA. CFAb with high sensitivity. Results A total of 120 XMs (66 Tc-XM, 54 Bc-XM) were performed in parallel using CDC-XM, IgG-FXM, and C1q-FXM. C1q-FXM correlated well with CDC-XM but has higher sensitivity. The concordance of two crossmatches was 88% (P Conclusion The newly developed C1q-FXM combines the advantage of the CDC-XM for detecting CFAb with the high sensitivity of the IgG-FXM assay. It can replace the traditional CDC-XM.

Kidney Transplantation in Immunologically High-Risk Patients

An increased number of sensitized patients await kidney transplantation (KTx). Sensitization has a major impact on patient mortality and morbidity due to prolonged waiting time and may preclude live donor transplantation. However, recent reports have shown that KTx can be performed successfully using novel immunosuppressive protocols. This study presents our experience with patients displaying donor-specific antibody (DSA) (+). We enrolled 5 lymphocyte cross-match (LCM) negative (complement-dependent cytotoxicity) and panel-reactive antibody (PRA) plus DSA-positive patients mean fluorescein intensity [MFI] > 1000) who underwent living kidney donor procedures. All subjects were females and their mean age was 36.7 years. In our protocol, we started mycophenolate mofetil (2 g/d), tacrolimus (0.01 mg/kg) and prednisolone (0.5 mg/kg) on day −6. We performed 2 sessions of total plasma exchange (TPE) with albumin replacement and administered 2 doses of IVIG (5 g/d). On day −1, we added rituximab (200 mg). On the operation day and on day +4, the patients received doses of basiliximab. Serum samples were taken on days −6, 0, and 30 as well as at 1 year after transplantation. All patients displayed immediate graft function. Mean basal DSA titer was 5624 MFI. After desensitization, the MFI titers decreased at the time of transplantation to 2753 MFI, and were 2564 MFI at the 1st month and 802 MFI at 1st year. Three patients experienced acute rejection episodes (60%). After treatment for rejection, the average follow-up was 17 months and last creatinine levels were 0.6–0.8 mg/dL (minimum-maximum). In conclusion, KTx can be succesfully performed in sensitized patients displaying DSA. However, there seems to be a greater acute rejection risk. There is no consensus regarding adequate doses of IVIG or plasmapheresis treatments; furthermore, more studies are needed to clarify the safe MFI titer of the DSA.

Mesenchymal stem cell transfusion for desensitization of positive lymphocyte cross‐match before kidney transplantation: outcome of 3 cases

Donor specific antibodies (DSA) and a positive cross-match are contraindications for kidney transplantation. Trials of allograft transplantation across the HLA barrier have employed desensitization strategies, including the use of plasmapheresis, intravenous immunoglobulins, anti-B-cell monoclonal antibodies and splenectomy, associated with high-intensity immunosuppressive regimens. Our case 1 report suffered from repeatedly positive lymphocyte cross match after 1st renal transplantation. Graft nephrectomy could not correct the state of sensitization. Splenectomy was done in a trial to get rid of the antibody producing clone. Furthermore plasmapheresis with low dose IVIG could not as well revert the state of sensitization for the patient. About 50 millions donor specific MSCs were injected to the patient. MSCs transfusion proved to be the only procedure which could achieve successful desensitization before performing the second transplantation owing to their immunosuppressive properties. This case indicates that DS-MSCs is a potential option for anti-HLA desensitization. In cases 2 and 3 IV DS-MSCs transfusion was selected from the start as a successful line of treatment for pre renal transplantation desensitization to save other unnecessary lines of treatment that were tried in case 1.

Outcome of second kidney transplant: A single center experience

Nowadays, a repeat transplantation is considered to confer a better survival advantage to patients over dialysis. The cost-effectiveness of transplantation for end-stage renal disease patients shows benefits over dialysis even for re-transplanted patients. This retrospective single center ten-year study was undertaken to evaluate patient/graft survival, function vis-à-vis serum creatinine (SCr) and rejection episodes in 62 re-transplanted patients. Sixty-two patients underwent a second renal transplant (24 living related, 38 deceased donors) at our center between 2000 to 2009. The mean recipient age was 41.9 ± 12.27 years. Fifty-three recipients were male and nine recipients were female. Recipients had negative acceptable lymphocyte cross-matching using anti-human globulin complement-dependent cytotoxicity tests and flow cytometric cross-match before transplant. All recipients except those who were hepatitis C virus or hepatitis B surface antigen positive received single-dose rabbit-anti-thymocyte globulin induction and steroids, calcineurin inhibitor ± mycophenolate mofetil/azathioprine for maintenance immunosuppression. Of the 62 patients, 38 patients received kidneys from deceased donors and 24 patients received kidneys from live donors. Over the mean follow-up of 4.03 ± 2.93 years, the 1-year, 5-year and 10-year patient survival rates were 85.33%, 66.7% and 66.7%, respectively, and the graft survival rates were 96.7%, 79.7% and 79.7%, respectively. The acute rejection rates were 17.6%, with a mean SCr of 1.92 ± 0.5 mg/dL. There was unexplained interstitial fibrosis with tubular atrophy in 11.2% patients (n = 7), all leading to graft loss eventually. Overall, 25% (n = 16) of the patients were lost, mainly to infectious complications. Re-transplantation has acceptable graft and patient survival over a ten-year follow-up period and should be encouraged for better quality of life as compared with dialysis.

Donor Specific Mesenchymal Stem Cells MSCs Transfusion for Desensitization of Resistant Positive Donor Specific Lymphocyte Cross Match and Reduction of Panel Reactive Antibodies PRA. Report of 4 Cases.

Sensitization to HLA antigens may be induced by previously administered blood products, pregnancy, or prior transplantation. It represents an important barrier to renal transplantation. Procedures adopted to achieve desensitization include single high dose IVIG or low dose IVIG in combination with antibody reduction via plasmapheresis with or without splenectomy. Mesenchymal stem cells (MSCs) among their properties have an important imuno-modulatory effect and have been shown to inhibit anti-HLA allo-antibody production in vitro. Four cases were given 7 - 10 million donor specific MSCs infused intravenously in 2 divided doses 1 week apart. After initial increase in the positivity of the cross match, it turned negative in 2 to 4 months period with reduction of the percent of PRA and were subsequently successfully transplanted. Donor specific MSCs represents a therapeutic option for desensitization of positive lymphocyte cross match.

Donor Specific Mesenchymal Stem Cells MSCs Transfusion for Desensitization of Resistant Positive Donor Specific Lymphocyte Cross Match and Reduction of Panel Reactive Antibodies PRA. Report of 4 Cases.

Sensitization to HLA antigens may be induced by previously administered blood products, pregnancy, or prior transplantation. It represents an important barrier to renal transplantation. Procedures adopted to achieve desensitization include single high dose IVIG or low dose IVIG in combination with antibody reduction via plasmapheresis with or without splenectomy. Mesenchymal stem cells (MSCs) among their properties have an important imuno-modulatory effect and have been shown to inhibit anti-HLA allo-antibody production in vitro. Four cases were given 7 - 10 million donor specific MSCs infused intravenously in 2 divided doses 1 week apart. After initial increase in the positivity of the cross match, it turned negative in 2 to 4 months period with reduction of the percent of PRA and were subsequently successfully transplanted. Donor specific MSCs represents a therapeutic option for desensitization of positive lymphocyte cross match.

Evaluation of Histocompatibility after SLA-Matched Kidney Allo-Transplantation in Cloned Miniature Pigs

Purpose: Immunological and histological compatibility in kidney transplantation between genetically identical somatic cell-cloned miniature pigs. Materials and methods: Eight out-bred domestic swine and eight genetically identical cloned miniature pigs were used. Donor-recipient pairs were chosen according to RBC or lymphocyte cross match by flow cytometry, and swine leukocyte antigen (SLA) genotyping. We examined hypervariable region 1 (HV1) of mitochondrial DNA (mtDNA). Kidney transplantation was performed in four different groups as followings: auto-transplantation, out-bred allo-transplantation, different somatic cell cloned allo-transplantation, and same somatic cell cloned allo-transplantation. Kidney function was daily evaluated after transplantation and in the case of rejection renal grafts were harvested for histology prior to euthanasia. Results: The mtDNA sequence of the HV1 region was found to be variant among genetically identical cloned miniature pigs. Accelerate acute rejection was observed in all three allograft pairs. Although delayed increase in plasma creatinine and urea nitrogen, and less characteristic lesions of mononuclear cell infiltration were observed, the general pattern of all-rejection episode was shown in the same somatic cell-cloned pigs. Conclusions: The results suggest that genetically identical somatic cell nuclear transfer (SCNT) cloned miniature pigs were immunologically incompatible with one another in renal allo-transplantation despite matched SLA. Other allo-antigens including minor histocompatibility antigen encoded by mt DNA might be related to the outcome. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No.PJ008032), from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

Co-Transplantation of Donor Stem Cells with Kidney Leads to Clinical Tolerance Associated with Clonal Deletion and T-Regulatory Cell Generation

Introduction: The acid test for clinical tolerance in transplantation (Tx) is normal functioning graft with intact recipient immune status in absence of immunosuppression (IS). Tolerance has been achieved anecdotally in renal transplantation (RT) recipients by minimizing IS however there is no reported series of successful RT without IS. Tolerance is believed to be achieved by clonal T/B cell deletion accompanied by active immune regulation. We present our experience of clinical tolerance in live-donor renal transplantation (LDRT) using co-transplantation of donor adiposetissue-derived mesenchymal stem cells (AD-MSC) and hematopoietic stem cells (HSC) with clonal deletion, associated with generation of peripheral T-regulatory cells (CD4+/25 High/127+) (T-regs). Material: Totally 44 patients (41 males, 3 females) with mean age 28.7 years and HLA-A/B/DR match 3.4/6, underwent LDRT sans conventional IS between March′08 and July,′09 using pretransplant stem cell transplantation (SCT) (group 1) with Institutional Review Board approval. Stimulation was done with AD-MSC and HSC ± donor specific transfusions, and deletion with total lymphoid irradiation (TLI)/Bortezomib as the principal agent. Other adjuvants were Cyclophosphamide, rabbitantithymocyte globulin (r-ATG) and Rituximab. Every stimulation and deletion was monitored by lymphocyte cross-match, donor specific antibodies (DSA), and T and B-cell flow-cross-match. Graft biopsy was performed in event of dysfunction and managed as per standard guidelines. Rescue IS was started with rise of SCr/DSA/positive mixed lymphocyte reaction (MLR). Protocol biopsies were performed in subset of patients with their consent. Thirty two contemporary controls underwent LDRT with standard triple immunosuppressants. Their mean age was 36.6 years and HLA-A/B/DR match 2.6/6 (group 2). The commonest original disease was chronic glomerulonephritis in both groups. Post-transplant monitoring for both groups was done for serum creatinine (SCr), DSA, MLR and T-regs at regular intervals. Results: Clinical tolerance was successfully induced and maintained in 27 (61.4%) group 1 patients with mean follow-up, 3.31 years. No untoward effect of protocol was registered and no patient/graft loss was observed. Twenty seven (61.4%) patients are doing well with mean SCr, 1.39 mg/dL, no rejections/DSA, negative MLR and mean T-regs, 3.61%. Graft biopsies were performed in 4 patients at 1 year post-transplant and were normal. Mean nucleated cell count of infused HSC (n×107 cells/kgBW) was 3.16, and of MSC (n×104 cells/kgBW) was 3.02 vs 3.12 and 1.42 respectively in remaining 17 (38.6%) who were rescued with IS. Their mean SCr is 1.71 mg/dL. Acute rejections (AR) were noted in 5 (29.4%) patients out of whom 4 developed chronic changes. DSA were observed in 4 patients. MLR was negative in all and mean T-regs were 2.93%. All responded to anti-rejection management. In group 2, over mean 3 years follow-up, mean SCr is 1.37 mg/dL and T-regs, 2%. AR were noted in 3 (9.4 %) patients out of whom 2 developed chronic changes. There was no graft loss. Two patients were lost; 1 to cerebrovascular accident and 1 to chronic graft dysfunction-related complications. Conclusion: Clinical tolerance is feasible in LDRT with combined SCT associated with clonal deletion and T-regs generation.