Lymphocyte Crossmatch Research Articles

4-Color Flow Cytometric Crossmatch Using Whole Blood Lysis

BackgroundIn lymphocyte crossmatch using flow cytometry (flow cytometric crossmatch, FCXM), the conventional tricolor FCXM protocol requires a mononuclear cell isolation step. To develop a new, more streamlined protocol, we introduced whole blood lysis (WBL) and CD45 fluorescence-triggered acquisition using 4-color flow cytometry. MethodsA total of 186 donor/recipient pairs for transplantation were classified into donor-specific human leukocyte antigen (HLA) alloantibody-positive (DSA+, n = 78) and DSA-negative (DSA−, n = 108) groups. The latter group was reclassified into blood group ABO-incompatible (ABOi, n = 56) and ABO-compatible (n = 52) subgroups. The WBL FCXM protocol with CD45 V500-C was optimized using a FACSLyric cytometer (BD Biosciences) with 3 lasers. Measurements for T cells or B cells were calculated as a mean fluorescence intensity (MFI) ratio (test divided by control). WBL FCXM was compared with conventional FCXM in each group. ResultsWBL FCXM showed no difference quantitatively compared with conventional FCXM, except for the B cell FCXM in the DSA− group (B cell MFI ratio: 1.06 ± 0.44 and 0.92 ± 0.41, respectively [P = .0001]). There was no ABO antibody interference in the ABOi subgroup. Similar results were observed in the qualitative determinations of FCXM as follows: 1) In the DSA+ group, the sensitivity of B cell WBL FCXM (96.2%) showed no difference compared with that of conventional FCXM (91.0%, P = .2188) and 2) In the DSA− group, the specificity of T cell WBL FCXM (96.3%) showed no difference compared with that of conventional FCXM (98.1%, P = .6250). WBL FCXM reduced the turnaround time by 50 min compared with that by conventional FCXM. ConclusionsWBL FCXM demonstrated comparable assay performance to that of conventional FCXM. Because this new FCXM protocol is simple and does not compromise assay sensitivity, it has the potential to replace the conventional method in histocompatibility laboratory settings.

Lymphocyte Crossmatch Testing or Donor HLA-DP and -DQ Allele Typing Effectiveness in Single Cord Blood Transplantation for Patients With Anti-HLA Antibodies Other Than Against HLA-A, -B, -C, and -DRB1

Anti-human leukocyte antigen (HLA) antibodies other than those against HLA-A, -B, -C, and DRB1 are a risk factor for engraftment delay and failure, especially in cord blood transplantation (CBT). The primary objective of this study was to assess the impact of the presence of anti-HLA antibodies on CBT and to evaluate the utility of lymphocyte crossmatch testing or additional HLA-DP and -DQ typing of CB units in improving transplant outcomes. We retrospectively assessed the engraftment rates and transplant outcomes of 772 patients who underwent their first CBT at our hospital between 2012 and 2021. Donors were routinely typed for HLA-A, -B, -C, and-DRB1 alleles, and the anti-HLA antibodies of recipients were screened before donor selection in all cases. Among patients who had antibodies against other than HLA-A, -B, -C, and DRB1 (n = 58), lymphocyte crossmatch testing (n = 32) or additional HLA-DP/-DQ alleles typing of CB (n = 15) was performed to avoid the use of units with corresponding alleles. The median patient age was 57 years (16 to 77). Overall, 75.7% had a high-risk disease status at transplantation, 83.5% received myeloablative conditioning regimens, and >80% were heavily transfused. Two hundred twenty-nine of the 772 recipients (29.6%) were positive for anti-HLA antibodies. There were no statistical differences in the number of infused CD34-positive cells between the anti-HLA antibody-positive and the anti-HLA antibody-negative patients. Of the 229 patients with anti-HLA antibodies, 168 (73.3%) had antibodies against HLA-A, -B, -C, and-DRB1 (Group A), whereas 58 (25.3%) had antibodies against HLA-DP, HLA-DQ, or -DRB3/4/5 with or without antibodies against HLA-A, -B, -C, and -DRB1 (Group B). No patients in both Groups A and B exhibited donor-specific anti-HLA antibodies against HLA-A, -B, -C, and -DRB1. The neutrophil engraftment rate was lower in patients with anti-HLA antibodies than in those without antibodies (89.9% versus 94.1%), whereas nonrelapse mortality (NRM) before engraftment was higher in antibody-positive patients (9.6% versus 4.9%). In patients who received 2 or more HLA allele-mismatched CB in the host-versus-graft (HVG) direction (n = 685), the neutrophil engraftment rate was lower in the anti-HLA antibody-positive recipients than in the antibody-negative recipients with significant differences (88.8% versus 93.8%) (P = .049). Similarly, transplant outcomes were worse in the antibody-positive patients with respect to 2-year overall survival (OS) (43.1% versus 52.3%) and NRM (44.0% versus 30.7%) than in the antibody-negative patients. In contrast, the results of Group B were comparable to those of the antibody-negative patients, while those of Group A were statistically worse than the antibody-negative patients in terms of all engraftment rate (88.6%), OS (34.2%), and NRM (49.0%). The presence of anti-HLA antibodies negatively impacts engraftment, NRM, and OS in CBT. However, HLA-DP/-DQ allele typing of CB units or lymphocyte crossmatch testing could be a useful strategy to overcome poor engraftment rates and transplant outcomes, especially in patients with anti-HLA antibodies against HLA-DP, HLA-DQ, or -DRB3/4/5.

Living Donor Liver Transplantation in Patients with Preformed Donor-Specific Anti-Human Leukocyte Antigen Antibodies Using Preoperative Desensitization Therapy According to Intensity of Donor-Specific Antibodies: A Single-Center Study.

BACKGROUND In liver transplantation (LT), preoperative desensitization therapy is considered necessary in patients positive for donor-specific anti-human leukocyte antigen antibodies (DSAs). However, the relationship between DSA intensity and the necessary desensitization therapy is unclear. MATERIAL AND METHODS A total of 37 adult living donor (LD) LTs performed between January 2016 and March 2022 were examined. Mycophenolate mofetil (MMF) was administered preoperatively in DSA-positive cases with positive lymphocyte cross-matching who underwent LDLT. In those with strongly positive DSA (mean fluorescence intensity 10 000), rituximab was administered 2 weeks before LDLT in addition to MMF. Cross-reactive epitope group antigen (CREG)-alone-positive cases were also treated with preoperative MMF when lymphocyte cross-matching was positive. RESULTS Of the 37 patients, 9 were DSA-positive, 7 were CREG-alone-positive, and the others were double-negative. Of 9 DSA-positive cases, desensitization therapy was performed in 7, among which rituximab administration was performed in 3 strongly DSA-positive cases. Of 7 CREG-alone-positive cases, 2 were lymphocyte cross-match-positive and underwent desensitization therapy. The 1-year survival rate was 100% in both DSA- and CREG-alone-positive cases. The frequency of T-cell mediated rejection in DSA-positive, CREG-alone-positive, and double-negative cases was 22%, 43%, and 29%, respectively, with no significant difference. Antibody-mediated rejection occurred in only 1 patient, who was strongly DSA-positive and blood-group incompatible. There was also no significant difference among the 3 groups in terms of the frequency of biliary complications or 90-day mortality. CONCLUSIONS Satisfactory LDLT results were achieved in DSA- and CREG-alone-positive cases following desensitization therapy.

O048 Vote counting and sign testing methodology to assess the effect of tissue typing in liver transplantation

Abstract Introduction Liver grafts are currently matched by blood group and size. Due to immune privilege of transplanted livers, tissue typing (HLA matching) is not routinely undertaken. Emerging evidence suggests typing may be beneficial for specific patient groups. This systematic review aimed to determine patient and graft outcomes in HLA matched liver transplant patients. Methods Criteria included primary articles exploring effects of typing for liver transplantation, identified from PubMed/MEDLINE and Web of Science databases until January 2022, excluding articles published pre-2000. The Newcastle-Ottawa scale assessed risk of bias. Cochrane protocols for synthesis without meta-analysis (vote counting and sign testing) determined proportions of studies favouring typing. Results All 28 included articles were cohort studies with low-medium bias risk. Graft survival syntheses favoured typing across all patients in studies of both living (LDLT) and deceased donor liver transplantation (DDLT), where 1 study showed a beneficial effect, and 1 showed no effect. In LDLT-only studies, 2 showed benefit, 4 showed no effect, and 1 showed harm. Additionally, typing was favoured in adult-only DDLT recipients. Rejection syntheses favoured typing across all patients for studies of LDLT, DDLT, and both, and for subgroups including adult-only, paediatric-only, tacrolimus-treated, and negative lymphocyte crossmatch. Patient survival syntheses favoured typing across all patients, and for adult-only, PBC, and hepatitis/viral/cirrhosis diseases subgroups, in DDLT studies. Graft failure syntheses favoured typing across all patients in LDLT-only studies, and both LDLT & DDLT. Conclusion Findings evidenced disparate subgroup benefits of typing, and numerous studies reported no effect, or harmful effects.

The Assessment and Outcomes of Crossmatching in Lung Transplantation in Korean Patients.

BackgroundIn lung transplantation, human leukocyte antigen (HLA) compatibility is not included in the lung allocation score system or considered when placing donor allografts. However, HLA matching may affect the outcomes of lung transplantation. This study evaluated the current assessment status, prevalence, and effects of HLA crossmatching in lung transplantation in Korean patients using nationwide multicenter registry data.MethodsTwo hundred and twenty patients who received lung transplantation at six tertiary hospitals in South Korea between March 2015 and December 2019 were retrospectively reviewed. Clinical data, including general demographic characteristics, primary diagnosis, and pretransplant status of the recipients and donors registered by the Korean Organ Transplant Registry, were retrospectively analyzed. Survival analysis was performed using the Kaplan-Meier method with log-rank tests.ResultsComplement-dependent cytotoxic crossmatch (CDC-XM) was performed in 208 patients (94.5%) and flow cytometric crossmatch (flow-XM) was performed in 125 patients (56.8%). Among them, nine patients (4.1%) showed T cell- and/or B cell-positive crossmatches. The incidences of postoperative complications, including primary graft dysfunction, acute rejection, and chronic allograft dysfunction in positively crossmatched patients, were not significant compared with those in patients without mismatches. Moreover, Kaplan-Meier analyses showed poorer 1-year survival in patients with positive crossmatch according to CDC-XM (P < 0.001) and T lymphocyte XM (P = 0.002) than in patients without mismatches.ConclusionPositive CDC and T lymphocyte crossmatching results should be considered in the allocation of donor lungs. If unavailable, the result should be considered for postoperative management in lung transplantation.

Elucidating the role of cell-mediated inflammatory cytokines on allogeneic mouse-derived nucleus pulposus mesenchymal stem cells.

In this present study, our aim was to evaluate the cell-mediated specific anti-donor antibody and its associated inflammatory cytokine secretion along with its succeeding effects on Nucleus pulposus-derived mesenchymal stem cells (NPMSCs). Tissue from the NP compartment of 12 normal mice was isolated, expanded in cell culture, and the cell phenotypes were confirmed by flow cytometry. Multipotent differentiation and its specific gene expression analysis were confirmed by reverse transcriptase PCR. T and B cells were monitored for both donor and recipient mouse and further analysis of anti-donor antibody secretion was confirmed by lymphocyte crossmatch. In conjunction with anti-donor-specific antibody analysis, the associated inflammatory cytokine expression was analyzed by ELISA. In co-culture, cell-mediated antibody secretion was elevated in T and B cells positive mouse group, when compared to control mouse group. Allogeneic-derived donor NPMSCs were found to be stimulated the secretion of pro-inflammatory cytokines and the level of pro-inflammatory cytokines showed reduced expression in control mouse serum. In co-culture group the concentration of the cell-mediated pro- and anti-inflammatory cytokines found to be increased. PRACTICAL APPLICATIONS: Mesenchymal stem cell exhibit good regeneration capacity for many types of disease, and the mechanism belongs to regeneration is not clear. In intervertebral disc, the nucleus pulposus-derived mesenchymal stem cells showed a better regeneration capacity. On the contrary, the NP cells-based therapy, the Mesenchymal stem cells showed expanded anabolic and reduced catabolic activity together with induced anti-inflammatory effect. In this study, the T & B cells were used to evaluate the anti-donor antibody secretion and also to study how it stimulates the production of anti-donor antibodies against the donor cells. Finally, it was found that T & B cells lead the synthesis of inflammatory cytokines are IL-1, IL-6, and TNF-α. From this study, the results proved that the cell-mediated pro- and anti-inflammatory cytokines to be monitored in allogeneic stem cells-based therapy of intervertebral disc degeneration.

Double-Filtration Plasmapheresis Plus Low-Dose Anti-thymocyte Globulin and Tacrolimus in Asian Living-Donor Kidney Transplantation With Donor-Specific Anti-HLA Antibody

BackgroundPretransplant desensitization protocols, including plasmapheresis, intravenous immunoglobulin, induction antibody therapy, and intensive maintenance immunosuppression, are generally employed in kidney transplant recipients who have positive status for donor-specific anti-HLA antibody (DSA). To avoid serious infectious complications, the authors designed a novel low-dose protocol in Thai patients undergoing DSA+ living-related kidney transplantation (LRKT). MethodsA retrospective cohort study of the patients who underwent DSA+ LRKT was conducted. The novel protocol consisted of 3 to 5 sessions of pretransplant double-filtration plasmapheresis (DFPP) with or without low-dose intravenous immunoglobulin together with low-dose anti-thymocyte globulin (ATG) induction (1-1.5 mg/kg/d for 3-4 days) and low-dose tacrolimus (Tac) (trough level 5-10 ng/mL), mycophenolate, and prednisolone. ResultsThe study included 17 patients. The lymphocyte crossmatch via complement-dependent cytotoxicity was negative in 12 patients and positive for B cell immunoglobulin M in 5 patients. The novel desensitization protocol resulted in a decrease of at least 50% of DSA mean fluorescence intensity from baseline (from 4320 ± 549 before DFPP to 1601 ± 350 before transplantation, P < .005) and successful kidney transplantation with good allograft function in all cases. Early DSA rebound was observed in 3 patients after transplantation, and kidney biopsy revealed subclinical antibody-mediated rejection in 1 patient and diffuse C4d staining without cell infiltration in 2 patients. There were good long-term outcomes in patient and graft survival (100% and 94.1%, respectively). Only 1 allograft loss occurred because of nonadherence. The majority of patients have stable allograft function with serum creatinine less than 1.5 mg/dL. However, infections, including CMV and other organisms, were commonly observed. ConclusionsDesensitization protocol with DFPP, low-dose ATG, and Tac provides excellent outcomes in living donor kidney transplantation in highly sensitized Asian populations.

Case report - pre-transplant testing and post-transplant monitoring for an HLA-immunized recipient in heart transplantation

We report the workflow of immunogenetic pre-transplant testing and post-transplant monitoring in the case of a recipient immunized to human leucocyte antigens (HLA) who was waitlisted for heart transplantation. The recipient underwent heart transplantation across preformed HLA class I Donor Specific Antibodies (DSAs) detected by solid phase Luminex screening method but not by complement dependent cytotoxicity (CDC) screening method. The CDC lymphocyte crossmatch, which was performed retrospectively, was a weak positive. Post-transplant DSA monitoring by Luminex method revealed the decrease of HLA-A1, A25 and B57 DSAs with, at the same time, an increase of HLA-B8 DSA, as well as weak transient non-DSA HLA-DP antibodies. This case presents the importance of extensive immunogenetic testing and monitoring for identifying recipients with increased immunological risk for successful heart transplantation.

P024 Successful kidney transplantation in a HIV patient with strong positive T and B cell flow cytometry lymphocyte crossmatch: A case study

P024 Successful kidney transplantation in a HIV patient with strong positive T and B cell flow cytometry lymphocyte crossmatch: A case study

SP791The role of lymphocyte crossmatch testing in de novo HLA-DQ donor-specific antibody positive renal transplant recipients

SP791The role of lymphocyte crossmatch testing in de novo HLA-DQ donor-specific antibody positive renal transplant recipients

Image cytometry as an alternative to flow cytometry for the transplant histocompatibility crossmatch assay.

The lymphocyte crossmatch is currently the only cell-based compatibility assay performed by histocompatibility laboratories for transplant purposes. While in many transplant programs the complement-dependent cytotoxicity crossmatch (CDCXM) remains in use, when available, the flow cytometry crossmatch (FCXM) is the method of choice because of its superior sensitivity and specificity. Unfortunately, the maintenance and cost of a flow cytometer is a considerable limitation for small histocompatibility laboratories. Therefore, in this study, we evaluated the use of the Cellometer Vision CBA image cytometer (Nexcelom Bioscience LLC, Lawrence, Massachusetts) as an alternative instrument to perform the crossmatch assay. The 3-color FCXM protocol was modified into two separate 2-color panel image cytometry crossmatches (IXMs), one for T cells and one for B cells. After initial serum and cell incubation, a cocktail consisting of PE/Cy5-conjugated anti-human CD3 or CD19 and PE-conjugated anti-human IgG F(ab')2 was added to the T cell and B cell panels, respectively. The final cell preparation was added to a separate counting chamber. Images were captured using the Cellometer Vision CBA, an image cytometer designed for cell counting, size analysis and fluorescence intensity measurement. Thirty-nine IXMs were performed and compared with the FCXM. We obtained a concordance sensitivity of 94.1% and 100% and specificity of 100% and 88.9% for T cells and B cells, respectively. The linearity of the system was verified using dilutions of a sample containing known donor-specific anti-HLA antibodies (DSA) against the target cells. This feasibility study demonstrates that the FCXM test could be easily adapted to the Cellometer Vision CBA image cytometer without compromising specificity and sensitivity. The low instrumentation cost, minimal maintenance, and simple operation allow for efficient implementation or transition from the FCXM to the IXM method.

Improved flow cytometry crossmatching in kidney transplantation.

Flow cytometry crossmatching (FC-XM) assay is the most sensitive cell-based method for detecting donor-specific antibodies (DSAs). However, the use of FC-XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement-fixing and noncomplement-fixing antibodies. FC-XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20+ B-cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC-XM. Pronase treatment (0.5-1 mg/mL) prevents false-positive B-cell FC-XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B-cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti-rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin-D (7-AAD) or fluorochrome-conjugated C4d Ab, after complement incubation, allows complement-fixing antibodies to be distinguished from noncomplement-fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab-binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch.

P146 Variations in the expression of antibody epitopes associated with dqb1*03:01 alleles in B cells

Aim To evaluate variations of the expression of antibody epitopes associated with DQB1*03:01 alleles in B cells. Methods Antibody tests were performed using Luminex Single Antigen Bead (SAB) assay on serum samples treated with DTT. XMs were performed by flow cytometry, using lymphocytes from peripheral blood of healthy volunteers, which were treated with pronase. Results Four sera reacted strongly with beads carrying the following alleles: DQB1*03:01-DQA1*06:01, DQB1*03:01-DQA1*05:05, and DQB1*03:01-DQA1*05:03 (Figure 1). Sera JM and RH reacted strongly with B cells from all 6 donors who were positive for DQB1*03:01 indicating that these cells express adequate amount of DQ molecules (Figure 2A). However, XMs of sera CG and SR with the 4 heterozygous DQB1*03:01 B cells showed weak or no reactivity respectively (Figure 2B). In comparison to XM reactivity from sera JM and RH the XMs with sera SR and CG exhibited a 25-50-fold lower Δ MESF values (Figure 2A versus 2B). This unexpected low reactivity of sera SR and CG with B cells is striking since their reactivity with SAB is similar to that of serum JM except with the DQB1*03:01-DQA1*03:01 allele (Figure 1). Notably, SR and CG reacted with B cells that were homozygous for DQB1*03:01 (Figure 2B, Donor 1 and 2) indicating that the reactivity of these sera with the SAB was not due to denatured epitopes. Conclusions These results suggest that the observed variable expression of DQB1*03:01 epitope on B cells could be due to variation in the conformation of this epitope on the same DQ molecules with a potential impact on antibody affinity to this epitope. The discrepancy between SAB MFI values and XM reactivity is important to consider in assessing HLA compatibility and lymphocyte crossmatching.

P140Proper setting of cut-off value for the lymphocyte crossmatch by flow cytometry in kidney transplantation

Aim The flow cytometric lymphocyte crossmatch (FCXM) has become a routine test performed prior to kidney transplantation. Many studies have reported that a proportion of transplant recipients experienced early graft loss despite a negative complement-dependent cytotoxicity (CDC) crossmatch test. The FCXM is able to detect Human Leukocyte Antigen (HLA) antibodies of low titer or of a non-complement binding nature that may be responsible for graft loss. It is important to report positive and negative results using appropriate cut-off values. We reviewed the results of FCXM with CDC and panel reactive antibody (PRA) and attempted to set clinically useful cut-off values for FCXM. Methods From January 2015 to October 2017, 518 kidney recipients’ results of FCXM, CDC, and PRA (Screening test, Panel kit, and single antigen bead assay, LABScreen Luminex kits, One Lambda, Canoga Park, CA, USA) were analyzed. The PRA test was performed simultaneously from 403 patients, and 159 patients who were negative for HLA antibodies in the all PRA tests. Fifty-seven patients were positive for CDC test results and 37 patients were both positive in T cells and B cells. In 20 patients, only B cells CDC results were positive, and 13 patients were negative for FCXM test results. Results The final interpretation of FCXM result was decided through CDC, PRA result and existence of donor specific antibody (DSA). T cell FCXM result was interpreted as negative in 451 patients and positive in 67 patients according to DSA. The appropriate cut-off value of the Mean fluorescent intensity (MFI) ratio using the ROC curve for 518 patients was 1.86 (sensitivity 100%, specificity 98.2%, Youden’s index 0.982). However, patients who underwent desensitization treatment due to DSA or ABO blood type incompatibility were included. The cut-off value obtained using the ROC curve for 446 patients who have not experienced desensitization treatment was 2.45 (sensitivity 100%, specificity 99.3%, Youden’s index 0.993). Generally used, Median + 2 SD value of MFI ratio of patients judged as negative FCXM was 1.60. Conclusions Based on the statistical method used, various cut-off values could be obtained. It is important to determine the clinically significant cut-off value based on the data accumulated in each laboratory, not the simple median value from negative patients.

Prognostic tools to assess candidacy for and efficacy of antibody-removal therapy.

Currently, the ability to predict or monitor the efficacy of HLA antibody-removal therapies is deficient. We previously reported that titration studies are a consistent and accurate means of assessing antibody strength. To test whether titration studies can also predict which patients are better candidates for desensitization, we studied 38 patients from 3 centers (29 receiving plasmapheresis/low-dose intravenous immunoglobulin [IVIg]; 9 patients receiving high-dose IVIg). For patients undergoing plasmapheresis/low-dose IVIg, antibody titer reduction correlated with number of treatment cycles for both class I and II antibodies but only up to approximately 4 cycles. Reduction in titer slowed with additional cycles, suggesting a limit to the efficacy of this approach. Furthermore, initial titer (predesensitization) can guide the selection of candidates for successful antibody-removal treatment. In our experience, patients with antibodies at an initial titer>1:512 could not be reduced to the goal of a negative lymphocyte crossmatch, corresponding to a 1:16 titer, despite a significant increase in the number of treatment cycles. Change in mean fluorescence intensity (MFI) value did not correlate with success of treatment if initial MFI values were>10 000, likely due to single antigen bead saturation. Overall, we present a potential prognostic tool to predict candidacy and a monitoring tool to assess efficacy of desensitization treatment.

Human leukocyte antigen compatibility and lymphocyte cross-matching play no significant role in the current adult-to-adult living donor liver transplantation.

The impact of human leukocyte antigen (HLA) compatibility and positive lymphocyte cross-match (LCM) on organ transplantation is well-recognized particularly in kidney and heart transplantation; however, it is still debatable in liver transplantation (LT). So, the aim of this study was to evaluate the impact of HLA mismatch and positive LCM on the outcome of LT. We retrospectively analyzed the data of all adult recipients who underwent living donor LT at our institute between January 2010 and July 2016. We excluded all ABO blood group incompatible LDLT patients and patients with incomplete data regarding HLA genotyping (n=134). The type and degree of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ mismatch and LCM were assessed in each donor-recipient pair and their relationship to the occurrence of rejection, CMV infection and graft survival was evaluated. A higher percentage (>50%) of donor-recipient pairs had 1 HLA mismatch at each locus in the host-vs-graft direction and seventeen recipients (13%) had positive LCM. Human leukocyte antigen mismatch and positive LCM were not correlated with increased incidence of acute rejection (P=.37, P=.6, respectively), CMV infection post-transplant (P=.52, P=.76, respectively), or graft failure (HR 1.22, P=.68 and HR 1.73, P=.34, respectively). Positive LCM and HLA mismatches did not affect the overall graft survival after adult-to-adult LDLT and should not be considered as contraindications for liver transplantation.

P241 A study of immunocomplex capture fluorescence analysis using lymphocyte and monocyte fraction by freezing isolation

Aim Immunocomplex capture fluorescence analysis (ICFA) is a novel method to predict rejection/acceptance responses in organ transplantation. The method is less susceptible to the effect of drug compared with traditional methods. The method uses fluorescent beads to sensitively detect antibodies directed against HLA I/II antigens on blood cells.In this study, the changes in mean fluorescence intensity (MFI) control values of lymphocyte and monocyte fraction from donor cells before cryopreservation and donor cells after one month of cryopreservation were monitored using ICFA for a lymphocyte crossmatch. Methods The subjects were patients who were going to undergo living-donor kidney transplantation. ICFA method for a lymphocyte crossmatch was performed. The stability of MFI control values was monitored for donor cells before cryopreservation and those after one month of cryopreservation. Results The monitoring results showed that the MFI control value of lymphocyte and monocyte fraction from donor cells after one month of cryopreservation was 9.4% higher than that of lymphocyte and monocyte fraction from donor cells before cryopreservation. On the other hand, the MIF control value of leukocytes after one month of cryopreservation using the conventional method was 100%. Conclusions More reliable donor-specific HLA antibody test results can be obtained by lymphocyte crossmatch test using lymphocyte and monocyte fraction by freezing isolation from cryopreserved donor cells.

OR3 HLA-DPB1 single nucleotide polymorphism determines DP molecule expression and B lymphocyte crossmatch results

Aim HLA DSA are a major cause of antibody-mediated rejection (AMR) and allograft loss in solid organ transplantation. However, the role of DP DSA in solid organ transplantation is unclear. Recently, it has been shown that a single nucleotide polymorphism (SNP) at 3 ′ untranslated region (3 ′ UTR) of DPB1 genes upregulates DP expression and is associated with graft-versus-host disease in hematopoietic stem cell transplantation. The goals of this study are to 1) Investigate if the rs9277534G/A SNP at 3 ′ UTR affects the DP expression and correlates with the B cell flow crossmatch results. 2) Explore the expression of DP under inflammatory conditions to determine if DP DSA can be pathogenic and trigger AMR. Methods Peripheral blood mononuclear cells from 14 healthy donors and 19 human aortic endothelial cells (HAEC) were treated for 48 hours with/without IFN- γ /TNF- α before DP expression was measured by flow cytometry with a monoclonal antibody. The rs9277534G/A SNP of DP was typed using Real Time PCR. B lymphocyte flow crossmatch was performed with sera from patients sensitized to DP to study the impact of DP expression on crossmatch results. Results GG and GA genotypes had higher expression of DP molecules compared to AA genotypes in both B cells and HAEC. High DP expression in B cells correlated with stronger B lymphocyte flow crossmatch as compared to low DP expression (Table 1). DP expression was undetectable under basal conditions in HAEC. However, interferon- γ and TNF- α -stimulated HAEC upregulated DP. Conclusions These data demonstrate that the differential DP expression on B lymphocytes and HAEC is regulated by the SNP at 3 ′ UTR of the DPB1 gene and correlates with B lymphocyte flow crossmatch results. We also identified the 3 ′ UTR polymorphisms in yet-characterized alleles of DP. IFN- γ and TNF- α pre-treatment up-regulates DP expression in HAEC and thus increases DP DSA binding. Therefore, under inflammatory conditions, DP DSA might contribute to antibody-mediated allograft rejection.

Six years' experience of tolerance induction in renal transplantation using stem cell therapy

Tolerance induction (TI) has been attempted with chimerism/clonal deletion. We report results of TI protocol (TIP) using stem cell therapy (SCT) included adipose derived mesenchymal stem cells (AD-MSC) and hematopoietic stem cells (HSC) in 10 living-donor related renal transplantation (LDRT) patients under non-myeloablative conditioning with Bortezomib, Methylprednisone, rabbit-anti-thymoglobulin and Rituximab, without using conventional immunosuppression. Transplantation was performed following acceptable lymphocyte cross-match, flow cross-match, single antigen assay and negative mixed lymphocyte reaction (MLR). Monitoring included serum creatinine (SCr), donor specific antibodies (DSA) and MLR. Protocol biopsies were planned after 100days and yearly in willing patients. Rescue immunosuppression was planned for rejection/DSA/positive MLR. Over mean 6±0.37year follow-up patient survival was 80% and death-censored graft survival was 90%. Mean SCr was 1.44±0.41mg/dL. This is the first clinical report of sustained TI in LDRT for 6years using SCT.

Stem cell therapy facilitates tolerance in renal transplantation

Background: Strategies for tolerance induction (TI) include induction of chimerism and/or clonal deletion. We present 5-year experience of TI in living-donor related renal transplantation (LDRT) using adipose derived mesenchymal stem cell (AD-MSC) and hematopoietic SC (HSC) infusion with supportive therapy. Methods: Twenty patients divided in 2 equal, demographically balanced groups underwent LDRT under TI protocol (TIP) consisting of conditioning with Bortezomib, Methyl prednisone, rabbit-anti-thymoglobulin and Rituximab. Group-1 were administered in-vitro generated AD-MSC and HSC, group-2 were administered donor specific transfusion. Transplantation was carried out with acceptable lymphocyte cross-match, T and B-cell flow cross-match, single antigen assay and negative mixed lymphocyte reaction (MLR). No conventional immunosuppression was to be administered. Monitoring included serum creatinine (SCr-mg/dL), donor specific antibodies (DSA) and MLR. Protocol biopsies were planned after 100 days and yearly in willing patients. In event of rejection/ DSA/ MLR, rescue immunosuppression was planned. Results: Over mean 5.4 (range: 4.9-6.3) year follow-up patient survival was 80% in group-1 and 90% in group-2; deathcensored graft survival was 90% in group-1 and 70% in group-2. Mean SCr was 1.52 in group-1, and 1.97 in group-2. Five patients from group-1 and 3 from group-2 were on no conventional immunosuppression, 2 patients of group-1 and 1 of group-2 were on two immunosuppressants and 1 patient of group-2 was on 3 immunosuppressants. DSA appeared in 2 patients of group-1 without affecting graft function and 4 of group-2 causing graft dysfunction. MLR was negative in both groups. Conclusion: SCs facilitate TI in LDRT under non-myeloablative conditioning.