Donor Antigens Research Articles

Circulating Immune Complexes and Complement Activation in Sensitized Kidney Transplant Recipients.

Chronic antibody-mediated rejection in kidney transplantation is a common cause of graft loss in the late post-transplant period. In this process, the role of the classical complement activation pathway is crucial due to the formation of immune complexes between donor-specific antibodies (DSAs) and donor antigens and the attachment of the C1q complement fragment. This study aimed to determine the levels of circulating C1q immunocomplexes (CIC-C1q) and complement activation (CH50), in sensitized kidney transplant recipients (KTRs). In this cross-sectional study we used serum samples from KTRs with de novo or preformed DSAs (n = 14), KTRs without DSAs (n = 28), and 22 subjects with no history of chronic kidney disease (controls). C1q immunocomplexes and CH50 concentration in serum were measured with the enzyme immunoassay (EIA) kit MicroVue CIC-C1q (Quidel, Athens, OH, USA) and EIA kit MicroVue CH50 (Quidel, OH, USA), respectively. Higher concentrations of CIC-C1q was observed in KTRs with DSAs in comparison with controls and with KTRs with no DSAs (6.8 ± 2.7 and 4.8 ± 1.9 vs. 5.0 ± 1.2 μg Eq/mL, respectively, p < 0.01). We found no difference in CIC-C1q between KTRs with no DSAs and controls. CIC-C1q levels were positively correlated with DSA titer. CH50 levels were decreased in KTRs with DSAs in comparison with controls and KTRs with no DSAs (39 ± 15 vs. 68 ± 40 and 71 ± 34 U Eq/mL, respectively, p < 0.01). There was no difference in CH50 between DSA-negative KTRs and controls. Kidney transplant recipients with DSAs had increased serum levels of C1q immunocomplexes and increased classical pathway complement activation.

Successful A2 to O Simultaneous Liver and Kidney Transplantation in the Setting of Pre-operative Positive HLA Crossmatch: A Case Report

Simultaneous liver and kidney transplantation (SLKT) is possible for patients with high donor-specific HLA antibodies or with A2 donors to O recipients with high A2 titers. We report the first case of SLKT in a highly sensitized O recipient with organs from an A2 donor. The recipient is a 59-year-old woman with chronic kidney disease and liver failure due to autoimmune hepatitis and drug-induced liver injury. Immune work-up 8 days pre-transplant demonstrated a negative crossmatch and no HLA antibody (calculated panel reactive antibodies = 0%). Anti-A2 IgG levels were 512. The donor was a deceased 24-year-old man. One day before transplantation, serum from the recipient showed a significant increase in antibody reactivity (calculated panel reactive antibodies = 100%) attributable to blood product transfusion and memory response from previous pregnancies. Consequently, a crossmatch was positive for T and B cells with two newly detected HLA antibodies against the donor's antigens. On the day of surgery, the liver was transplanted first. Six hours and 37 minutes later, a repeat flow crossmatch was negative; donor-specific antibodies (DSAs) fell below the positive threshold, and anti-A2 IgG titer fell to 256. Thus, the kidney was transplanted after basiliximab induction therapy. Seven days post-transplant, non-donor-specific HLA antibodies were present but DSAs remain negative. The patient was discharged on postoperative day 57 with no signs of rejection at 4 months. This case illustrates a rapid and prolonged reduction in antibody titers (HLA and ABO) after SLKT. SLKT is feasible in patients with both DSA and high anti-A2 titer.

#2314 Circulating immune complexes concentration and complement activation in sensitized kidney transplant recipients

Abstract Background and Aims Chronic antibody mediated rejection of the kidney transplant is the most common cause of graft loss in the late posttransplant period. Crucial in this process seems to be the role of classical pathway complement activation due to the formation of immune complexes. The formation of immune complexes is a benign process of a normally functioning immune system. In kidney transplant recipients (KTRs), the production of donor specific IgG/IgM antibodies (DSAs) against donor antigens (MHC molecules, ABO blood group antigens) leads to the attachment of immune complexes to cell membranes and subsequent tissue damage through complement activation. The attachment of C1q complement fragment to the immune complexes holds an important part in this process. This study aims to determine the levels of immune complexes as well as classical pathway complement activation in sensitized kidney transplant recipients. Method In this cross-sectional study 3 groups of KTRs were compared as to the levels of immunocomplexes concentration and classical pathway complement activation. Fourteen KTRs with preformed or de novo donor specific antibodies were compared to 28 kidney transplant recipients with no DSAs and to 22 subjects with no history of chronic kidney disease (controls). We used MicroVue Complement CIC-C1q EIA that is designed to detect immunocomplexes that bind to C1q in human serum or plasma. In order to estimate total classical pathway complement activation in serum, we used MicroVue Complement CH50 Eq EIA. Results Kidney function (eGFR) of KTRs with and without DSAs was similar (53.1 ± vs. 60.8 ± ml/min/1.73 m2, p = 0.54) but lower in comparison to controls (106.4 ml/min/1.73 m2, p &amp;lt; 0.001). We found a significantly higher concentration of C1q-immunocomplexes in the serum of KTRs with donor specific antibodies, in comparison to controls, as well as to the group of kidney transplant recipients with no DSAs (6.78 ± 2.7 vs. 4.83 ± 1.9 vs. 5.04 ± 1.2 μgEq/ml respectively, p = 0.0078). Furthermore, there was no significant difference of C1q-immunocomplexes between kidney transplant recipients with no DSAs and controls. Finally, a significant reduction of CH50 levels was observed in KTRs with DSAs as compared to controls and KTRs with no DSAs (39.2 ± 14.9 vs. 70.6 ± 34.3 vs. 67.9 ± 39.9 U/ml respectively, p = 0.003), which suggests higher levels of classical pathway complement activation. CH50 levels in KTRs with DSAs, who were further subcategorized in those with preformed and those with de novo DSAs were similar (39.68 ± 15.6 vs 39.08 ± 16.04 U/ml). Conclusion Kidney transplant recipients with donor specific antibodies have increased serum levels of immune complexes and increased classical pathway complement activation.

Histocompatibility Testing: A Fundamental Aspect of Renal Transplant Workup

Histocompatibility testing is pivotal in any renal transplantation workup, aimed at enhancing prospective donor recipient compatibility and improving transplant outcomes. The evolution and advancement of histocompatibility testing, particularly HLA typing, have significantly improved its precision. This study outlines the historical progression from serologic to DNA-based HLA typing, emphasizing the role of HLA proteins in immune response. Anti-HLA antibodies, targeting HLA proteins, pose challenges in renal transplantation. Monitoring and managing these antibodies are critical for renal transplant success. Complement-dependent cytotoxicity crossmatch and flow cytometry crossmatch are essential techniques for assessing donor–recipient compatibility. Panel-reactive antibody assesses antibodies against a panel of donor antigens, often HLA. Higher PRA levels (percentage) complicate donor matching, requiring specialized protocols. Virtual crossmatch evaluates recipient anti-HLA antibodies against potential donors through synthetic beads. This approach predicts crossmatch outcomes by comparing antibody profiles, offering a valuable tool for the risk assessment of renal transplantation. Despite advancements, a comprehensive understanding of alloreactive immune responses requires a combination of assays, emphasizing the importance of a multifaceted approach in histocompatibility testing. This is an attempt to compile the relevant information, providing a basis for comparison in a clear and foundational format for histocompatibility testing laboratories.

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

Pretransplant, Th17 dominant alloreactivity in highly sensitized kidney transplant candidates.

Sensitization to donor human leukocyte antigen (HLA) molecules prior to transplantation is a significant risk factor for delayed access to transplantation and to long-term outcomes. Memory T cells and their cytokines play a pivotal role in shaping immune responses, thereby increasing the risk of allograft rejection among highly sensitized patients. This study aims to elucidate the precise contribution of different CD4+ memory T cell subsets to alloreactivity in highly sensitized (HS) kidney transplant recipients. Stimulation of peripheral blood mononuclear cells (PBMC) with various polyclonal stimulating agents to assess non-specific immune responses revealed that HS patients exhibit elevated immune reactivity even before kidney transplantation, compared to non-sensitized (NS) patients. HS patients' PBMC displayed higher frequencies of CD4+ T cells expressing IFNγ, IL4, IL6, IL17A, and TNFα and secreted relatively higher levels of IL17A and IL21 upon stimulation with PMA/ionomycin. Additionally, PBMC from HS patients stimulated with T cell stimulating agent phytohemagglutinin (PHA) exhibited elevated expression levels of IFNγ, IL4 and, IL21. On the other hand, stimulation with a combination of resiquimod (R848) and IL2 for the activation of memory B cells demonstrated higher expression of IL17A, TNFα and IL21, as determined by quantitative real-time PCR. A mixed leukocyte reaction (MLR) assay, employing third-party donor antigen presenting cells (APCs), was implemented to evaluate the direct alloreactive response. HS patients demonstrated notably higher frequencies of CD4+ T cells expressing IL4, IL6 and IL17A. Interestingly, APCs expressing recall HLA antigens triggered a stronger Th17 response compared to APCs lacking recall HLA antigens in sensitized patients. Furthermore, donor APCs induced higher activation of effector memory T cells in HS patients as compared to NS patients. These results provide an assessment of pretransplant alloreactive T cell subsets in highly sensitized patients and emphasize the significance of Th17 cells in alloimmune responses. These findings hold promise for the development of treatment strategies tailored to sensitized kidney transplant recipients, with potential clinical implications.

Pre-transfusion compatibility tests in Malagasy sickle cell patients

Introduction: Blood transfusion is a key component of sickle cell disease management. However, iterative transfusions of labile blood products for sickle cell patients increase the risk of anti-erythrocyte alloimmunization. It is usual to perform pre-transfusion compatibility tests in order to check ABO compatibility and to detect the possible existence of irregular antibodies in patients against the donor's antigens. Methods: This is a prospective and descriptive study dealing with pre-transfusion compatibility tests over a 3-month period, from September 2018 to November 2018, at the Regional Blood Transfusion Centre (CRTS) of Analamanga Region, the capital of Madagascar. Results: We included 33 out of 113 patients during the study period. Compatibility test was positive in 21.21%, of all tested samples. Major and minor compatibility tests were positive alone respectively in 6.25% and 12.50%. And both were positive in 3.12%. The self-test was positive in 3.03% with a positive direct antiglobulin test. Positive compatibility tests were more frequent in patients who received more than two transfusions. Compatible blood bag for the patient was found after testing 2 donor pockets in 27.27% of cases, and after testing 3 pockets in 3.03%. Conclusion: Pre-transfusion compatibility tests are essential and have to be performed systematically on each blood bag before any transfusion. They are used to identify the presence of alloantibodies and provide compatible blood products to patients.

Favorable outcome of non-myeloablative allogeneic transplantation in adult patients with severe sickle cell disease: A single center experience of 200 patients.

Allogeneic hematopoietic stem cell transplant (HSCT) for adults with severe sickle cell disease (SCD) is potentially curative but not commonly utilized therapy due to complications such as graft failure (GF) and organ toxicity. Herein, we are reporting our long-term outcome data of non-myeloablative (NMA) HSCT in adults with severe SCD with emphasis on factors predicting event free survival (EFS). Adults with severe SCD undergoing NMA match-related donor allogeneic HSCT from 2015 to 2021 with at least 12 months of follow-up were included. A total of 200 patients were included with a median age of 26 years (14-43) and 56% were male. The median infused CD34 dose was 13.7 (5.07-25.8), respectively. Median absolute neutrophil count engraftment was 19 (13-39) days with 51% of patients receiving GCSF to expedite recovery. A total of 17 patients experienced GF; 3 as primary and 14 as secondary within a median time of 204 days (40-905). A 76% successfully discontinued sirolimus at the last follow-up. Median follow-up for the cohort is 29.2 (2.1-71.4) months. Estimated 3-year EFS and OS were 88.2% (81.9-92.5) and 94.6% (89.2-97.3). At multivariable analysis, minor ABC incompatibility hazard ratio (HR) 4 (1.3-12.1; 0.014) and allo-antibody against non-ABO donor antigens HR 4.3 (1.3-14.1; 0.016) were significant for EFS. No clonal evolution or myeloid malignancies were seen. This largest single-center report of NMA HSCT in adults with severe SCD further delineated its feasibility, potential toxicities, and fertility outcomes. GF remains a major impediment and appears dependent on ABO matching and non-ABO antibodies.

Enhanced Donor Antigen Presentation by B Cells Predicts Acute Cellular Rejection and Late Outcomes After Transplantation.

Enhanced B-cell presentation of donor alloantigen relative to presentation of HLA-mismatched reference alloantigen is associated with acute cellular rejection (ACR), when expressed as a ratio called the antigen presenting index (API) in an exploratory cohort of liver and intestine transplant (LT and IT) recipients. To test clinical performance, we measured the API using the previously described 6-h assay in 84 LT and 54 IT recipients with median age 3.3 y (0.05-23.96). Recipients experiencing ACR within 60 d after testing were termed rejectors. We first confirmed that B-cell uptake and presentation of alloantigen induced and thus reflected the alloresponse of T-helper cells, which were incubated without and with cytochalasin and primaquine to inhibit antigen uptake and presentation, respectively. Transplant recipients included 76 males and 62 females. Rejectors were tested at median 3.6 d before diagnosis. The API was higher among rejectors compared with nonrejectors (2.2 ± 0.2 versus 0.6 ± 0.04, P value = 1.7E-09). In logistic regression and receiver-operating-characteristic analysis, API ≥1.1 achieved sensitivity, specificity, and positive and negative predictive values for predicting ACR in 99 training set samples. Corresponding metrics ranged from 80% to 88% in 32 independent posttransplant samples, and 73% to 100% in 20 independent pretransplant samples. In time-to-event analysis, API ≥1.1 predicted higher incidence of late donor-specific anti-HLA antibodies after API measurements in LT recipients (P = 0.011) and graft loss in IT recipients (P = 0.008), compared with recipients with API <1.1, respectively. Enhanced donor antigen presentation by circulating B cells predicts rejection after liver or intestine transplantation as well as higher incidence of DSA and graft loss late after transplantation.

Negative Vaccination Strategies for Promotion of Transplant Tolerance.

Organ transplantation requires the use of immunosuppressive medications that lack antigen specificity, have many adverse side effects, and fail to induce immunological tolerance to the graft. The safe induction of tolerance to allogeneic tissue without compromising host responses to infection or enhancing the risk of malignant disease is a major goal in transplantation. One promising approach to achieve this goal is based on the concept of "negative vaccination." Vaccination (or actively acquired immunity) involves the presentation of both a foreign antigen and immunostimulatory adjuvant to the immune system to induce antigen-specific immunity. By contrast, negative vaccination, in the context of transplantation, involves the delivery of donor antigen before or after transplantation, together with a "negative adjuvant" to selectively inhibit the alloimmune response. This review will explore established and emerging negative vaccination strategies for promotion of organ or pancreatic islet transplant tolerance. These include donor regulatory myeloid cell infusion, which has progressed to early-phase clinical trials, apoptotic donor cell infusion that has advanced to nonhuman primate models, and novel nanoparticle antigen-delivery systems.

Blood Transfusion cross match on the basis of Phenotype &amp; Rh Antigen in Donors and Receivers and Analysis of Variations

Background: In brief, knowing the local Rh blood group system frequency helps develop a donor pool for patients who need numerous transfusions and alloantibody-compatible antigen negative blood. Patients have been matched for component blood transfusions using ABO and Rh phenotypes since 2020. Our detection of all blood transfusion-needy patients began. Aim: Our department began To detect C, E, c, and e Rh-specific antigens in multi-transfused patients in 2020. Methods: For the aim of this investigation, 2300 patient samples were obtained from patients who required clinical blood transfusions at our hospital between March 2020 and October 2022. These samples were gathered for the purpose of this investigation. One patient was counted as a single sample even though they required repeated blood transfusions. 1900blood donor samples were provided by the Blood Centre of (duplicated samples were removed based on the identification numbers that were provided by the Blood Centre once they were identified). Results: The study obtained 4200 samples, including 1900donor and 2300patient samples. The allele frequency distribution of blood group antigens in the studied population was compared with the prevalence observed in the present study. The D antigen exhibited a frequency of 99.34% in the studied population, slightly lower than the prevalence observed in the present study at 98.9% (95% CI: 98.5 - 99.0). Conversely, the C antigen was found in 93.1% of the studied population, with a slightly higher prevalence observed in the present study at 99.1% (95% CI: 92.0 - 99.2). Implication: The study suggests serological testing is a cost-effective method for blood transfusion management, identifying Rh phenotypes. Compiling a database of donor Rh genotypes simplifies transfusion selection, reducing unfavorable responses. Pre-transfusion Rh phenotype examination is crucial for matching patient blood type with donor blood, reducing adverse reactions, and improving patient safety. Conclusion: In transfusion applications, serological testing are cost-effective for Rh phenotype identification but not genotypes. Creating a database of blood donors' Rh phenotypes and evaluating each patient before their initial transfusion should lessen adverse reactions and speed up antigen-negative blood availability, saving more patients. Keywords: Blood transfusion, phenotype, Rh antigen, donors, receivers, analysis of variations

Establish a Graded Method to Avoid HLA Class I Antibodies Corresponding Antigen and Combining HLAMatchmaker Application in Improving the CCI Value after Platelet Transfusion for Patients with IPTR

To establish a graded method to avoid mean fluorescence intensity (MFI) threshold of HLA Class I antibodies corresponding antigen, and the HLAMatchmaker program has been used to select the minimum mismatch value of donor-patient epitopes. Evaluate the application value of combining both methods in selecting HLA compatible platelets (PTL) for patients with immune platelet transfusion failure (IPTR) in improving platelet the corrected count increment (CCI). A total 7 807 PLT cross-matching compatible were performed by the solid-phase red cell adherence (SPRCA) method for 51 IPTR patients. The Luminex single antigen flow cytometry was used to detect HLA Class I antibodies in patients, and detected the MFI value for different specificity antigens of HLA Class I antibodies, was graded into strong positive group (MFI>4 000, level 1), medium positive group (1 000< MFI≤4 000, 2), weak positive group (500< MFI≤1 000, 3), and one negative control group (MFI≤500). The results of 7 807 SPRCA their negative/positive reaction wells were enrolled and statistically analyzed in different grades and the four groups, the statistical differences between the four groups were compared. Multiple applications for the select HLA Class I compatible donor events were made for patients in two cases, and HLAMatchmaker program was used to calculate the number of HLA Class I epitopes mismatches between the donors and patients. The donor with the minimum number of epitopes mismatches was selected, while avoiding the corresponding antigens of HLA Class I antibodies in levels 1 and 2, the provision of HLA compatible platelets for IPTR. After the transfusions, the CCI value of the platelet transfusion efficacy evaluation index was calculated, and the clinical evaluation of the transfusion effect was obtained through statistical analysis. There were statistically significant differences in the positive results of SPRCA immunoassay among the strong positive group, medium positive group, and weak positive group of 51 IPTR patients with different specific of HLA -I class antibodies and corresponding antigens(all P <0.001). The positive results showed a range from high to low, with strong positive group>medium positive group>weak positive group. There were a statistical difference among between the strongly positive or moderately positive groups and the negative control group(P <0.001). There was no statistical difference between the weakly positive group and the negative control group(P >0.05). The strong positive group was set as the corresponding specific HLA Class I site corresponding antigen grade 1 avoidance threshold, the medium positive group as the grade 2 avoidance thresholds, and the weak positive group as the grade 3 avoidance threshold. In the case of donor platelet shortage, it is not necessary to avoid the weak positive group. Avoiding the strategy of donor antigens and HLAMatchmaker program scores ≤7 corresponding to HLA Class I antibodies of levels 1 and 2, with CCI values>4.5×109/L within 24 hours, can obtain effective clinical platelet transfusion conclusions. When selecting HLA Class I compatible donors for IPTR patients, the grading avoids HLA Class I antibodies corresponding to donor antigens, and the donor selection strategy with the minimum scores of HLAMatchmaker program is comprehensively selected. The negative result confirmed by platelet cross-matching experiments has certain practical application value for improving platelet count in IPTR patients.

Immunology Status of Patients During Kidney Transplant.

The immunology status of a patient has a crucial role in kidney transplant. We investigated the effectiveness of a desensitization protocol, guided by the immunology status of patients, for kidney transplant candidates. Antibody screening for human leukocyte antigens was conducted with the Luminex single-antigen microsphere bead assay method for 34 patients from June 2021 to June 2022. Donor human leukocyte antigen genotypes at 8 loci (A*, B*, С*, DRB1*, DQA1*, DQB1*, DPA1*, and DPB1*) were determined, to correlate the specificities of recipient human leukocyte antigen antibodies with donor antigens and identify unacceptable donor antigen combinations. Specialized immunology studies measured panel reactive antibody levels and human leukocyte antigen class I and class II antibodies. A crossmatch compatibility test using complementdependent cytotoxicity was conducted. Of the 34 patients, 10 completed all 3 stages of the desensitization therapy. Most patients experienced decreased sensitization to human leukocyte antigen class I and class II antibodies. Two patients achieved complete clearance of A1 and DQ5 antibodies, respectively, whereas 1 patient exhibited an increase in donor-specific antibody mean fluorescence intensity. Prior to desensitization therapy, the crossmatch compatibility test yielded positive results with T and B lymphocytes. After completing the therapy, the crossmatch test showed negative results in 4 cases with T lymphocytes and positive results with B lymphocytes. Plasmapheresis sessions effectively reduced circulating antibodies. However, the combination of rituximab and plasmapheresis alone did not achieve a negative crossmatch test required for kidney transplant. It is crucial to assess the reduction of donor-specific antibody quantity, considering both the percentage and the mean fluorescence intensity. To avoid false-positive results in crossmatch analysis, drug half-life must be considered. Laboratories should have various crossmatch techniques, such as flow cytometry and single-antigen microsphere bead assay technology, available for research and urgent cases that require crossmatch analysis.

Phenotypic Frequency of Clinically Significant Blood Group Antigens (Rh, Kell, Kidd, and Duffy) in Blood Donors of the Blood Center at a Tertiary Care Referral Teaching Hospital in Tirupati, Andhra Pradesh, South India

ABSTRACT Background and Objectives: The knowledge of various blood group antigen and phenotype frequencies in a population is important in selecting compatible blood in alloimmunized patients. Such information in Indian population is very limited. Hence this study was carried out to determine the frequency of clinically significant antigens. Such information in Indian population is very limited. Methods: This study was carried out to determine the frequencies of the D, C, c, E, e, K, k, Fya, Fyb, Jka, and Jkb antigens in 1024 blood donors at our department. Samples were collected for extended antigen typing during March 2016–February 2017 for D, C, c, E, e, K, k, Fya, Fyb, Jka, and Jkb antigens using monoclonal antisera by column agglutination technology. Antigenic and phenotypic frequencies were expressed as percentages. Results: Among the Rh system, “e” antigen was found in 98.6% of donors, followed by D (92%), C (89%), c (56%), and E (17%) with DCe/DCe (R1 R1, 41.4%) as the most common phenotype. “k” was found to be positive in 97% of donors, and K+k− phenotype was found in 0.1% of donors. For Kidd and Duffy blood group system, Jk(a+b+) and Fy(a+b+) were the most common phenotypes with frequency of 56.2% and 47.5%, respectively. MNS antigens were not included on account of financial constraints. Conclusion: Database for antigen frequency of various blood group systems in native donors helps provide antigen-negative compatible blood units to patients with multiple antibodies in order to formulate in-house red cells for antibody detection and identification and for preparing donor registry for rare blood groups.

Second and Third Kidney Transplants.

Renal transplant is the best procedure for patients with end-stage renal disease. Although an ideal kidney transplant should survive for the lifetime of each recipient, there may be a need for a second, third, or even a fourth retransplant. The outcomes of these kidney allografts, surgical approaches, immunology issues, and drug therapies warrant greater focus. Pediatric kidney retransplant is even more important because these patients are more immunologically responsive to donor antigens and because they need longer allograft survival. Although kidney retransplant provides a survival advantage for patients who would otherwise remain on the wait list and/or hemodialysis, careful patient selection is crucial for second, third, and fourth renal transplants. Despite the shortage of donor organs, outcomes, manageable complications, and economic considerations support earlier kidney retransplants rather than delayed retransplants. Preoperative vascular imaging, appropriate induction therapy, regular monitoring of renal function, and regular surveillance for malignancy and infection are more important in the retransplanted kidneys than in cases of first kidney transplants. The lack of robust data on optimal clinical management of these retransplant recipients has contributed to substantial variations in clinical practice among different centers. In this review, we discuss medical and surgical approaches in the cases of second and third kidney transplants.

Human Leukocyte Antigen Typing in Organ Transplantation: Methods, Clinical Relevance, and Practical Applications

ABSTRACT Transplantation of tissues and organs is one of the greatest achievements of this century. Antigens which vary between members of same species are known as allo-antigens. Difference in the allo-antigens between the donor and the recipient represents the antigenic stimulus, which can cause rejections. Adaptive immunity identifies self from non-self. The main objective of the immune response is to identify the cell surface molecules (major histocompatibility complex /MHC) expressed on the donor cells. It is imperative that human leukocyte antigens (HLA) antigens are identified to gauge the mismatches. Presence of pre formed HLA antibodies or formation of de-novo HLA antibodies against these mismatched antigens can lead to antibody mediated rejections and decreased allograft survival. Identification and monitoring of these antibodies pre transplant and post-transplant by performing a virtual cross-match with mismatched donor antigens help in planning and adjusting immunosuppression. A precise and adequate HLA typing of the donor and recipient is required for virtual cross-match. HLA typing technologies have advanced from serological typing to molecular technologies, which can now help identify the donor tissue to allelic level. Methods of HLA typing and their applications with cases have been described in this article.

Evaluation of Methods to Obtain Peripheral Blood Mononuclear Cells From Deceased Donors for Tolerance-Induction Protocols.

Regulatory cell therapies have shown promise in tolerance-induction protocols in living donor organ transplantation. These protocols should be pursued in deceased donor transplantation. Donor peripheral mononuclear cells (PBMCs) are an optimal source of donor antigens for the induction of donor-specific regulatory cells. During the development of a regulatory cell tolerance-induction protocol with organs from deceased donors, we compared 3 methods of obtaining PBMCs from deceased donors focusing on cell yield, viability, and contamination of unwanted cell types. PBMC procurement methods: 1. During organ procurement at the time of cold perfusion, blood was collected from the vena cava and placed into a 10-liter blood collection bag, and thereafter transported to Karolinska University Hospital, where leukapheresis was performed (BCL). 2. Blood was collected via the vena cava into blood donation bags before cold perfusion. The bags underwent buffy coat separation and thereafter automated leukocyte isolation system (BCS). 3. To collect PBMCs, leukapheresis was performed via a central dialysis catheter on deceased donors in the intensive care unit (ICU) prior to the organ procurement procedure (LEU).All 3 methods to obtain PBMC from deceased donors were safe and did not affect the procurement of organs. BCL contained around 50% of NK cells in lymphocytes population. LEU had a highest yield of donor PBMC among 3 groups. LEU had the lower amount of granulocyte contamination, compared to BCS and BCL. Based on these results, we choose LEU as the preferred method to obtain donor PBMC in the development of our tolerance-induction protocol.

A comprehensive study of rare Rhesus phenotype case

Introduction. The RH system includes major antigens D, C/c and E/e encoded by two closely related RHD and RHCE genes. Correct identifi cation of Rh antigens in both donors and recipients is the key to proper transfusion practice. However, there are cases when Rh antigens cannot be detected by standard serological typing. For example, –D– phenotype has no expression of C, c, E, and e antigens on the surface of erythrocytes due to various genetic rearrangements in the RHCE gene.Aim: to present a study of a family where two siblings have a defi cient -D-phenotype with a normal rhesus phenotype in the parentsMaterials and methods. A comprehensive study of family N., including parents and two sons was conducted. Initially, an unusual phenotype -D- was identifi ed in the siblings, who are currently donors. All family members identifi ed themselves as Tatars. Serology tests were performed using gel cards. Genomic DNA of family members, as well as cDNA of siblings, was examined by allele-specifi c PCR, exon-scanning assay, and Sanger sequencing. In addition, copy number analysis was performed to identify rearrangements in the RHD and RHCE genes.Results. During serological typing of siblings, only the D antigen was revealed, while the C/c and E/e antigens were absent. Molecular genetic analysis suggested that the cause of the phenotype –D– in the brothers was a hybrid allele RHCE-D(3-8)- CE in homozygous status, forming a haplotype inherited from each parent with the normal RHD allele. The sequence of the fi rst two exons in the hybrid allele corresponded to RHCE*C allele. The parents were heterozygous for the identifi ed allele, so the expression of C/c and E/e antigens was not altered.Conclusion. Two donors with the –D– phenotype were assessed by comprehensive study. Identifi cation of the genetic causes of such variants in recipients is necessary to ensure safety during transfusion of erythrocyte-containing blood components. Genotyping of donors with Rh-defi cient phenotypes is also highly recommended in order to predict the molecular structure of Rh antigens.

T-cell receptor sequencing reveals selected donor-reactive CD8+ T cell clones resist antithymocyte globulin depletion after kidney transplantation

High frequencies of donor-reactive memory T cells in the periphery of transplant candidates prior to transplantation are linked to the development of posttransplant acute rejection episodes and reduced allograft function. Rabbit antithymocyte globulin (rATG) effectively depletes naïve CD4+ and CD8+ T cells for >6 months posttransplant, but rATG’s effects on human donor-reactive T cells have not been carefully determined. To address this, we performed T cell receptor β-chain sequencing on peripheral blood mononuclear cells aliquots collected pretransplant and serially posttransplant in 7 kidney transplant recipients who received rATG as induction therapy. We tracked the evolution of the donor-reactive CD4+ and CD8+ T cell repertoires and identified stimulated pretransplant, CTV-(surface dye)-labeled, peripheral blood mononuclear cells from each patient with donor cells or third-party cells. Our analyses showed that while rATG depleted CD4+ T cells in all tested subjects, a subset of donor-reactive CD8+ T cells that were present at high frequencies pretransplant, consistent with expanded memory cells, resisted rATG depletion, underwent posttransplant expansion and were functional. Together, our data support the conclusion that a subset of human memory CD8+ T cells specifically reactive to donor antigens expand in vivo despite induction therapy with rATG and thus have the potential to mediate allograft damage.

Islet transplantation outcomes in type 1 diabetes and transplantation of HLA-DQ8/DR4: results of a single-centre retrospective cohort in Canada

In solid organ transplantation, HLA matching between donor and recipient is associated with superior outcomes. In islet transplantation, an intervention for Type 1 diabetes, HLA matching between donor and recipient is not performed as part of allocation. Susceptibility to Type 1 diabetes is associated with the presence of certain HLA types. This study was conducted to determine the impact of these susceptibility antigens on isletallograft survival. This is a single-centre retrospective cohort study. This cohort of transplant recipients (n=268) received islets from 661 donor pancreases between March 11th, 1999 and August 29th, 2018at the University of Alberta Hospital (Edmonton, AB, Canada). The frequency of the Type 1 diabetes susceptibility HLA antigens (HLA-A24, -B39, -DQ8, -DQ2 and-DQ2-DQA1∗05) in recipients and donors were determined. Recipient and donor HLA antigens were examined in relation to time to first C-peptide negative status/graft failure or last observation point. Taking into account multiple transplants per patient, we fitted a Gaussian frailty survival analysis model with baseline hazard function stratified by transplant number, adjusted for cumulative islet dose and other confounders. Across all transplants recipients of donors positive for HLA-DQ8 had significantly better graft survival (adjusted HRs 0.33 95% CI 0.17-0.66; p=0.002). At first transplant only, donors positive for HLA-DQ2-DQA1∗05 had inferior graft survival (adjusted HR 1.96 95% CI 1.10-3.46); p=0.02), although this was not significant in the frailty analysis taking multiple transplants into account (adjusted HR 1.46 95% CI 0.77-2.78; p=0.25). Other HLA antigens were not associated with graft survival after adjustment for confounders. Our findings suggest islet transplantation from HLA-DQ8 donors is associated with superior graft outcomes. A donor positive for HLA-DQ2-DQA1∗05at first transplant was associated with inferior graft survival but not when taking into account multiple transplants per recipient. The relevance of HLA-antigens on organ allocation needs further evaluation and inclusion in islet transplant registries and additional observational and interventional studies to evaluate the role of HLA-DQ8 in islet graft survival are required. None.