A2 Donors Research Articles

Blood group antigen studies using CdTe quantum dots and flow cytometry.

New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs]) as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC) antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I) lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion, this methodology with high sensibility and specificity can be applied to study a variety of RBC antigens, and, as a quantitative tool, can help in achieving a better comprehension of the antigen expression patterns on RBC membranes.

Transplantation of ABO A2 Kidneys Into O Recipients: Do IgM Anti-A1 Titers Matter?

Purpose: Previous studies have shown successful kidney transplantation from ABO A2 donors into O or B recipients with low anti-A titers. To increase organ availability the new UNOS kidney allocation system (KAS) will incorporate A2 to B allocation. Criteria for A2 to O or B kidney transplants (txs) vary, from anti-A IgG titers (DTT treated serum) <1:8 (1) to anti-A IgM/G <1:16 or IgM/G > 1:8 with pre-tx plasmapharesis (2). Some report higher early graft failure with IgG titers >1:8, others found no correlation between IgG titers and acute rejection (AR) or allograft loss (1,2). The UNOS KAS does not provide anti-A titer criteria for A2 to B transplants. We report a high incidence of early AR or thrombotic microangiopathy (TMA) in A2 to O living donor (LD) transplant recipients with low IgG but high IgM/G titers. Methods: A retrospective review identified seven A2 to O LD kidney txs over 12 years at our institution. Both IgM/G and IgG (DTT treated) titers were reported. All patients (pts) received antibody induction and Tac/MMF/Pred maintenance. Results: Results are summarized in Table 1. All pts transplanted had pre-tx IgG anti-A titers <1:8. 4 pts had early allograft dysfunction with AR or TMA or both (mean 7 days post tx; range 2-16 d) with one graft loss POD 8 due to accelerated AR and cortical necrosis. Of these 4 pts, 3 had IgM/G titers >1:8 and the 4th had IgM/G=1:8. Of the three pts without rejection, one had IgM/G > 1:8.Table: No Caption available.Conclusions: Although good long term outcomes were achieved, our data show a high incidence of early AR and/or TMA and suggest caution in A2 to O kidney txs with high recipient anti-A IgM/G titers despite low IgG titers. Steps to lower anti-IgM/G pre-tx may reduce the risk of early AR or TMA. 1. Transpl 2005;80:75 2 Trans Proc 2001;33:3327 DISCLOSURES:Shaffer, D.: Grant/Research Support, Novartis, Astellas.

Flow Cytometric Detection of Anti-AB Antibody Titers in Blood Group O Recipients of Blood Group A2 Donor Kidneys

AimABO-incompatible kidney transplantation has been accepted for end-stage renal failure patients who have no ready opportunity for a deceased or living donor. Antibody titration for ABO-incompatible renal transplantation is not only difficult but also lacks conformity among laboratories. Herein we analyzed 20 living related renal transplant couples to detect recipient anti-A2 antibody using flow cytometric analysis. Materials and methodsPatients were admitted to our center for renal transplantation between January 1999 and December 2010. All but four of them had undergone a previous renal transplantation from an ABO-compatible donor but experienced graft failure. All donor blood groups were subtyped by our blood bank using a lectin-based dilution assay. To detect recipient anti-A2 antibody titers we used a tube hemagglutination method. A/B antibody titer analysis by flow cytometry incubated serially diluted serum samples with donor erythrocytes. Each analysis was repeated three times over a 2-week period using an older and the last sera simultaneously. ResultsThe 13 male and 7 female patients showed our overall mean age of 32 ± 12 years. All patients had panel-reactive antibody levels below 15%. The level of flow cytometric antibody titers did not vary upon repeated analysis (P = .01). When compared with the tube method there was a discrepancy of the level at which the antibody titer became negative. DiscussionFlow cytometric antibody titration is a practical and rapid technique to determine the amount of anti-A2 antibody in renal recipients.

Recommendations for transfusion in ABO‐incompatible hematopoietic stem cell transplantation

TransfusionVolume 52, Issue 2 p. 456-458 Recommendations for transfusion in ABO-incompatible hematopoietic stem cell transplantation Diarmaid O'Donghaile MB BCh, Diarmaid O'Donghaile MB BCh e-mail: [email protected] , Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MDSearch for more papers by this authorWalter Kelley DO, Walter Kelley DO e-mail: [email protected] , Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MDSearch for more papers by this authorHarvey G. Klein MD, Harvey G. Klein MD e-mail: [email protected] , Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MDSearch for more papers by this authorWilly A. Flegel MD, Willy A. Flegel MD e-mail: [email protected] , Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MDSearch for more papers by this author Diarmaid O'Donghaile MB BCh, Diarmaid O'Donghaile MB BCh e-mail: [email protected] , Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MDSearch for more papers by this authorWalter Kelley DO, Walter Kelley DO e-mail: [email protected] , Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MDSearch for more papers by this authorHarvey G. Klein MD, Harvey G. Klein MD e-mail: [email protected] , Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MDSearch for more papers by this authorWilly A. Flegel MD, Willy A. Flegel MD e-mail: [email protected] , Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MDSearch for more papers by this author First published: 12 January 2012 https://doi.org/10.1111/j.1537-2995.2011.03465.xCitations: 21Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume52, Issue2February 2012Pages 456-458 RelatedInformation

Plasmapheresis treatment of antibody‐mediated rejection in an A2 donor to O pediatric liver transplant recipient

It is safe to transplant kidneys from blood group A2 donors into O recipients if the latter have low titers of anti-A antibodies. However, in liver transplantation, O and B recipients of A2 donor livers are not routinely screened for anti-blood group antibodies because of the immuno-absorptive capacity of the liver and the low incidence of antibody-mediated rejection. Herein, we report a rare case of combined cell and antibody-mediated rejection in a pediatric blood group O recipient of an A2 donor liver, and rescue of the allograft using PP and IVIG.

Immunologically Targeting the Leukaemia Phosphoproteome

AbstractAbstract 1016Posttranslationally modified (PTM) antigens, particularly phosphorylated antigens, comprise a significant component of tumour antigens and are thought to be represented within anti-tumour immune responses. Moreover, PTM antigens may circumvent the barrier of central tolerance thus offer a new paradigm for immunologically targeting malignant disease.Our previous work has shown that altered signal transduction in neoplastic myeloid and lymphoid cells can generate novel phosphopeptides that are uniquely and differentially presented on malignant cells by class I MHC molecules.Here, we have examined the peripheral blood CD8+ and CD4+ T cell immune responses against phosphopeptide antigens, displayed on primary haematolymphoid malignancies, in healthy donors and patients with leukaemia. Using HLA class-I and class-II tetramers in addition to cytotoxicity assays, we have found that these T cell responses are highly cytotoxic in both in vitro and in vivo preclinical models of adoptive immunotherapy. Our data suggest that recall responses are present to a significant proportion of previously identified class-I restricted phosphorylated antigens in healthy donors. Furthermore, in ex-vivo stimulation renders T cell responses to more than 90% of phosphopeptide antigens tested. These T cells recognise and kill primary tumour cells in a phosphopeptide-specific manner. Ten out of 10 HLA A2 donors and 9 out of 10 HLA B7 donors each respond to up to 7 different phosphopeptides from our pools (50 to 100 different phosphopeptides from CLL and AML). Some of the most relevant cytotoxic CD4+ T cell responses generated are directed towards CLL-specific phosphoproteins such as phosphorylated CD19, CXCR4 and CD20 whereas some of the most relevant cytotoxic CD8+ T cell responses generated target CLL specific phosphopeptides derived from LSP1, MRCL3 and NRC1I3. All these antigens are either overexpressed or uniquely expressed on CLL primary tumour cells. High affinity, tetramer-binding T cells against phosphopeptides derived from BCL2, cMyc, GFI1, MLL transcription factor, LIM, RUNX1, SKI, GRK and MAP3K, have been successfully generated from healthy donors which elicit strong cytotoxic responses against primary tumours. No responses have been elicited to the nonphosphorylated counterparts. Furthermore, some of the phosphopeptides are uniquely displayed on primary AML tissue and derived from established leukaemogenic oncoproteins making them extremely attractive targets.These data support the hypothesis that T cells specific for PTM antigens are represented within the peripheral T cell repertoire and thus are not subject to central tolerance and can be exploited to recognise and destroy neoplastic cells.As haematolymphoid malignancies do not typically downregulate MHC molecules, T cell adoptive transfer can now be envisaged while phosphopeptide vaccination strategies are also a clear objective. Disclosures:No relevant conflicts of interest to declare.

Prevalence, serologic and genetic studies of high expressers of the blood group A antigen on platelets*

Objective/Aim: The aim of this study is to describe the distribution of the platelet blood group A antigenicity in Euro-Brazilians (EUBs) and Afro-Brazilians (AFBs).Background: A small but significant proportion of individuals express high levels of A or B antigen on their platelets corresponding to the erythrocyte ABO group. The mechanism of increased antigen expression has not been elucidated.Material/Methods: A cohort of 241 blood group A donors was analysed by flow cytometry. Although mean fluorescence intensity (MFI) is a typical continuous variable, platelets were screened and divided into two categories: low expressers (LEs) and high expressers (HEs). A three-generation family was investigated looking for an inheritance mechanism.Results: The prevalence of the HE platelet phenotype among group A1 donors was 2%. The mean of MFI on platelets of A1 subgroup of EUBs differs from that of AFBs (P = 0·0115), whereas the frequency of the HE phenotype was similar between them (P = 0·5251). A significant difference was found between sexes (P = 0·0039). Whereas the serum glycosyltransferase from HE family members converted significantly more H antigen on group O erythrocytes into A antigens compared with that in LE serum, their ABO, FUT1 and FUT2 genes were consensus. The theoretically favourable, transcriptionally four-repeat ABO enhancer was not observed.Conclusion: The occurrence of HE in several members suggests familial aggregation. Indeed, in repeated measures, stability of the MFI values is suggesting an inherited condition. Factors outside the ABO locus might be responsible for the HE phenotype. Whether the real mechanism of inheritance is either of a polygenic or of a discrete Mendelian nature remains to be elucidated.

Transplantation of A2 Kidneys into B and O Recipients Leads to Reduction in Waiting Time: USRDS Experience

Strategy of transplanting kidneys from A2 donors into patients with blood group B and O recipients has been used to alleviate the long waiting times. We used an inception cohort of US Renal Data System data base with patients older than 18 years who underwent renal transplantation between January 1995 and July 2006. The primary outcome variable was allograft loss (including death). Bivariate analysis of factors associated with receiving A2 or A2B kidneys was performed with chi-square testing for categorical variables (Fisher's exact test used for violations of Cochran's assumptions) and Student's t test for continuous variables (Mann-Whitney U test used for nonnormally distributed variables). There were 150,118 first kidney transplants of whom 113 received kidney transplant from A2 to O, and 125 patients received A2 to B kidney transplant. Compared with other recipients from the same blood group, recipients of A2 kidneys had significantly shorter wait times. O recipients had a median wait time of 1.63 years (range 0.00-17.21 years), whereas O recipients who received A2 kidneys had a median wait time of 0.70 years (range 0.02-1.47 years; P<0.001). B recipients had a median wait time of 1.90 years (range 0.00-17.52 years), whereas B recipients who received A2 kidneys had a median wait time of 0.74 years (range 0.10-5.21 years; P<0.001). There was no significant difference in graft loss or death between A2 to O and B versus all other recipients. The results showed that comparatively few patients received A2 to B or O kidney transplant.

Defining the Molecular Mechanisms Responsible for the ABO High Expresser Phenotype.

Abstract 2119Poster Board II-96ABO(H) blood group antigen expression on platelets varies widely among normal blood donors. An ABO ‚High Expresser' phenotype (HXP) that exhibits significantly increased A and/or B antigen expression on platelets has been identified in ∼7% of normal donors. HXP has been implicated in both platelet-refractoriness and neonatal alloimmune thrombocytopenic purpura, however, the underlying molecular and genetic elements that mediate this phenomenon are not well-defined.To investigate the mechanisms underlying HXP, blood samples were collected from 231 group A (180 A1and 51 A2) and 310 group O individual apheresis platelet donors. Quantitative expression of platelet A and H antigen were then assessed by flow cytometry of platelet-rich plasma. In total, 10 A1 donors (5.6%) exhibited HXP. Analysis of the platelet A antigen expression in these individuals identified 8 HXP donors who exhibited ‚type I' HXP (normal bimodal population of platelets, but with predominant A antigen expression) whereas 2 individuals exhibited ‚type II' HXP (a single uniform population of platelets, strongly positive for blood group A expression). Both types of HXP were found to be a stable donor characteristic.ABO(H) determinants have also been identified on the N-linked glycans of the plasma von Willebrand factor (VWF), and influence plasma VWF levels and susceptibility to proteolysis by ADAMTS13. To determine whether HXP was platelet-specific, blood group A antigen expression on plasma VWF from group A donors was determined. Interestingly, blood group A antigen expression on plasma VWF was concordantly increased in donors with type I and type II HXP, indicative of increased glycosyltransferase expression in HXP individuals.To ascertain whether increased glycosyltransferase expression contributes to HXP, ABO genotype was determined for all 231 group A donors by PCR-RFLP analysis. Genotype at the ABO locus on 9q34 exerts a dosage effect on glycosyltransferase expression. 80% HXP (all type I) donors were genotyped A1A1. suggesting increased A transferase activity contributes to type I HXP. Despite this, the majority of A1A1individuals (67%) did not exhibit HXP, and 2 HXP donors were found to possess the A2 allele, which expresses limited A transferase enzymatic activity. Collectively, this data clearly demonstrates the contribution of additional factors to ABO genotype that contribute to HXP.To identify additional HXP modifiers, potential enhancer repeat elements upstream of the ABO gene were examined in group A donors, including those with HXP. Typically, A1alleles contain a single 43-base pair repeat within a minisatellite positive regulatory region upstream of the ABO gene. In contrast, A2and O1alleles contain four 43bp repeats, which are associated with a 100-fold enhancement of transcriptional activity. Analysis of this enhancer region demonstrated two HXP donors with A1alleles containing four copies of the 43-base pair repeat. Consequently, this allele would be predicted to modulate A transferase expression via enhanced ABO gene transcription.In conclusion, we have demonstrated the multi-factorial nature of the regulatory elements mediating platelet type I and type II HXP. A1alleles containing novel upstream enhancer repeats identified in donor individuals may represent a novel genetic mediator of HXP, and contribute to the pathophysiology associated with this phenomenon independently of ABO genotype. Disclosures:No relevant conflicts of interest to declare.

Application of Flow Cytometry to Monitor Antibody Levels in ABO Incompatible Kidney Transplantation

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

Long-Term Survival of Kidneys Transplanted from Live A2 Donors to O and B Recipients

The long-term outcome of kidneys transplanted from blood group A(2) live donors into blood group O or B candidates is not known. From 1986 through 2006, we transplanted eight blood group O patients and one blood group B patient with kidneys from blood group A(2) live donors. Immunosuppression was no different for these patients than for ABO-compatible recipients. All patients received methylprednisolone, cyclosporine or tacrolimus and azathioprine or mycophenolate mofetil with or without antibody induction (monoclonal or polyclonal). Of the nine live-donor A(2) to O and B transplants performed, seven grafts remain functioning. One of those seven was lost to follow-up at 9.2 years with a functioning kidney. Of the remaining six patients, length of follow-up is 10.4, 6.5, 5.3, 4, 2.1 and 1 years. Of the two patients who lost their grafts, one died with a functioning graft (DWFG) at 8.8 years and one lost his graft at 13.2 years due to noncompliance with immunosuppression. These data show that good long-term graft survival can be expected in live-donor A(2) to O and B transplantation despite some of those patients experiencing the type of clinical problems seen with ABO-compatible transplants.

Apart from Blood Group A2, ABO Antigen Incompatible Platelet Transfusions Result in Significantly Lower Corrected Count Increments (CCI) in Children.

Background: In contrast to the transfusion of red blood cells there is no evidence for stringent rules on ABO-matching in platelet transfusion.Patients and methods: In a prospective study in pediatric patients we investigated the efficacy of randomly transfused ABO blood group identical and out-of-group (antigen incompatible or antibody [isoagglutinins] incompatible) leukocyte-depleted single donor apheresis platelets (SDP). Four hundred SDP (median, 5/child, range, 1–68) were transfused to 50 children (29 girls and 21 boys, age 0.2 y–18.5 y) with thrombocytopenia due to chemotherapy or aplastic anemia. In the majority, indications for platelet transfusion were not based on platelet counts <5 x 109/L, but were first of all prophylactical; e.g. before lumbar puncture, intramuscular injection, or minor surgery. Exclusion criteria were: splenomegaly, fever ≥38.5°C, major hemorrhage or transfusion reactions. Transfusion efficacy was measured by the 1-hour CCI. (CCI = increase in PLT count [109/L] x body surface area [m2]/number of PLT given [1011]). Expression of A antigen on platelets was assessed by flowcytometry. Accounting for the many patients receiving multiple transfusions, linear mixed effects (LME) analysis was used for univariate and multivariate analyses of the effect of ABO compatibility status on transfusion efficacy.Results: Comparison of data between ABO identical and out-of-group transfusions is summarized in the table. [Display omitted] In addition, by multivariate LME the following parameters were significantly and independently associated with CCI: time since apheresis, apheresis device, volume of SDP, irradiation of SDP, and age of the recipients.Discussion: CCI after ABO antigen incompatible transfusions was significantly lower compared to identical transfusions (p=0.018, multivariate analysis), particularly after antigen incompatible transfusion of A1 platelets (p=0.011, multivariate analysis). In patients with blood group O or B, transfused platelets expressing high levels of A antigen were rapidly cleared from the circulation as demonstrated by flowcytometry. Because donors with blood group A2 do not express A antigen on their platelets, after transfusion of A2 SDP there was no difference in CCI as compared to ABO identical transfusions (p=0.76, multivariate analysis). This led to a more striking (p=0.004, multivariate analysis) difference of CCI between ABO identical and antigen incompatible transfusions after excluding A2 SDP from the analysis.Conclusions: If ABO identical SDP are not available, our data strongly support an ABO antigen compatible transfusion strategy for obtaining the best possible CCI - with the only equally effective exception being SDP from A2 donors given to group O or B recipients. However, this would require an extended ABO blood group typing in platelet donors, since by routine typing blood groups A1 and A2 are not differentiated. Although we have not observed significant hemolytic transfusion reactions, ABO antibody incompatible SDP are known to have an inherent risk for it, but relating to CCI they seem to be comparable to ABO identical SDP.

Influence of Human Leukocyte Antigen Haplotypes on Acute Graft Versus Host Disease Incidence after Allogeneic Hematopoietic Stem Cell Transplantation.

There is increasing evidence that the Human Leukocyte Antigen (HLA) alleles may influence the incidence of acute graft versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (alloHSCT). Differential presentation of recipient antigens to donor-derived T-cells by the respective HLA molecules may be a responsible factor. We have reported a decreased rate of aGVHD in HLA DR15 positive patients (pt) undergoing alloHSCT for myeloid malignancies (Blood , 2006; 107:1970). We investigated the influence of different HLA alleles and the three most common HLA haplotypes in US Caucasians on grade 2–4 acute GVHD (graded using published criteria) in HLA matched alloHSCT. We conducted a retrospective review of 195 related and 70 unrelated consecutive first myeloablative alloHSCT pt treated from 01/1992 to 4/2005 at our center. HLA typing was determined by either molecular (n=176) or serologic (n=89) methods. Pt characteristics included: AML (n=84), CML (n=62), NHL (n=47), ALL (n=41), MDS (n=20), HD (n=7) and CLL (n=4); median age 39 years (range 6–66), < 40 years (n=130); Male (n=168), Female (n=97); Caucasian (>95%); Donor-Recipient Sex Mismatch (n=112); Total Body Irradiation (TBI) conditioning regimens (n=205); Etoposide/Cyclophosphamide(Cy)/TBI (n=122), Busulfan(Bu)/Cy (n=28), Bu/TBI (n=27), Thiotepa/TBI (n=23), Cy/TBI (n=22), Fludarabine/Melphalan (n=8), Thiotepa/Carboplatin (n=8) or other combinations (n=27) and peripheral blood stem cell source (n= 35). HLA Haplotypes analyzed for their effect on aGVHD were A1B8DR3 (n=25), A3B7DR15 (n=20) and A2B44DR4 (n=13). There was no significant difference in the pt characteristics (donor relation, conditioning regimen, donor-recipient sex match, diagnosis, age, gender, performance status, disease status at transplant, stem cell source and GVHD prophylaxis) by each of the three haplotypes. We analyzed prior published HLA alleles that were associated with an increased or decreased risk of aGVHD and pt characteristics for their effect on grade 2–4 aGVHD incidence. By univariate analysis, donor relation (unrelated vs. related, 52% vs. 38%, p=0.0002), HLA A2 (44% vs. 33%, p=0.05) and HLA B8 were associated with increased aGVHD (61% vs. 37%, p=0.005) whereas HLA B18 was associated with decreased aGVHD (19% vs. 43%, p=0.017) in contrast to prior reports using serological typing only. By multivariate analysis, HLA A2 (RR=1.5, 95%CI: 1.0 to 2.2, p=0.04), HLA B8 (RR=1.9, 95%CI: 1.1 to 3.0, p=0.01) and unrelated donor (RR= 1.9, 95%CI: 1.3 to 2.9, p=0.001) were associated with increased aGVHD. HLA haplotype multivariate analysis demonstrated increased risk of aGVHD with haplotypes A1B8DR3 and A2B44DR4 (A1B8DR3: 64% vs. 32%, RR 2.6, 95% CI: 1.1 to 5.9, p=0.012 and A2B44DR4: 61% vs. 39%, RR 3.5, 95% CI: 1.7 to 7.5, p=0.04) whereas haplotype A3B7DR15 had less aGVHD compared to others (18% vs. 42%, RR 0.31, 95%CI: 0.098 to 0.98, p=0.049). This is the first published report of the effect of HLA haplotypes on aGVHD incidence. Results of our haplotype analysis substantiate our findings of single allele effects of HLA A2, HLA B8 and HLA DR15 suggesting a differential effect on aGVHD incidence. The ability to predict aGVHD after alloHSCT using HLA haplotypes may improve outcomes by allowing for individualized GVHD prophylaxis regimens.

Implications of ABO Error Rates in Proficiency Testing for Solid Organ Transplantation

Implications of ABO Error Rates in Proficiency Testing for Solid Organ Transplantation

Development of CMRF-56 blood dendritic cell preparations for immunotherapy of multiple myeloma

17572 Background: Despite advances in therapy for multiple myeloma, such as the use of proteosome inhibitors or stem cell transplantation, it is usually incurable. One promising therapeutic approach is to eradicate residual disease after stem cell transplantation by immune therapy. Method: The CMRF-56 monoclonal antibody detects an antigen expressed on blood dendritic cells (BDC) that is expressed after a short in vitro culture period. With clinical trail applications in mind, we have developed a procedure for isolation of CMRF-56+ BDC using CliniMACS. Cell culture, antibody staining and immunoselection occur in a closed system, compatible with regulatory requirements for cell therapy. Simultaneously, our team has developed of a bioprocess for production of the murine biotinylated CMRF-56 antibody for clinical trials. The bioprocess incorporates components that remove contaminating DNA, host cell proteins, viral particles and endotoxin. Results: Positive immunoselection using this antibody produces a BDC population containing some contaminating CMRF-56 positive B cells and monocytes. CliniMACS selection of PBMC from an apheresis produces enough BDC for a vaccination program with an average recovery of 1%, purity of 38%, yield of 48% and viability of 70%. These CMRF-56 dendritic cell preparations have been used to generate strong T cell responses to influenza matrix peptide and the putative tumour antigen MART-1. In order to discover and validate additional antigens capable of inducing effective immune responses against multiple myeloma, we have tested numerous of potential target antigens. It has been possibe to generate moderate specific immune responses against HM1.24 and mucin 1 peptides in normal HLA A2 donors. Using the CMRF-56 dendritic cell preparation as antigen presenting cells, it is possible to generate strong cytotoxic T cell responses against a lysate of the myeloma cell line U266. Indicating the potential usefulness of a whole cell lysate in generating anti-myeloma T cells for immunotherapy. Conclusions: These studies have provided the necessary pre-clinical data to commence a Phase I Clinical Trial in 2006 using CMRF-56 BDC preparations loaded with myeloma and control antigens in myeloma patients with minimal residual disease. No significant financial relationships to disclose.

Adult ABO‐incompatible liver transplantation, using A2 and B donors

The longer waiting time for a liver graft in patients with blood group O makes it necessary to expand the donor pool for these patients. This applies in both urgent situations and for elective patients. We report on our experience with ABO-incompatible liver transplantation using A2 and B non-secretor donors here. Between 1996 and 2005, 12 adult blood group O recipients (seven male/five female) received ABO-incompatible cadaveric liver grafts (10 A2 donors, two B non-secretor donors). The indications were either rapid deterioration of liver function or hepatocellular cancer, in blood group O recipients, where an ABO-identical/compatible graft was not available. Mean recipient age was 54+/-8 (mean+/-SD) yr. All pre-operative CDC crossmatches were negative. The initial immunosuppression was induction therapy with antithymocyte globulin (n = 3), interleukin 2 receptor antagonists (n = 3) or anti-CD20 antibody (rituximab) (n = 1), followed by a tacrolimus-based protocol. Three patients underwent plasmapheresis post-transplantation. Baseline biopsies were taken before or immediately after reperfusion of the graft and after grafting when clinically indicated. No pre-operative plasmapheresis, immunoadsorption or splenectomies were performed. Patient and graft survival was 10/12 (83%) and 8/12 (67%), respectively, with a 6.5-month median follow-up (range 10 days to 109 months). Two patients (B non-secretor grafts) died of multiorgan failure probably because of a poor condition before transplantation. Three patients were retransplanted. Causes of graft loss were bacterial arteritis (n = 1), death with a functioning graft (n = 1) and portal vein thrombosis (n = 2). In one of the patients with portal vein thrombosis, an anti-A titer increase occurred concomitantly, and ABO incompatibility as the cause of the thrombosis cannot be excluded. Seven acute rejections occurred in five patients and all were reversed by steroids or increased tacrolimus dosage. The pre-transplant anti-A titers tested against A1 red blood cells were 1 to 128 (NaCl technique) and 4 to 1024 (indirect antiglobulin technique, IAT); the maximum postoperative titers were 16 to 2048 (NaCl) and 256 to 32,000 (IAT). The favorable outcome of A2 to O grafting, with a patient survival of 10/10 and a graft survival of 8/10, makes it possible to also consider this blood group combination in non-urgent situations. The use of non-secretor donor grafts is interesting but has to be further documented. There was no hyperacute rejection or increased rate of rejection. Anti-A/B titer changes seem not to play a significant role in the monitoring of ABO-incompatible liver transplantation.

The genetic and phenotypic basis of blood group A subtypes in Koreans

A serological and genetic study of Korean blood donors with phenotypic group A subtypes was performed. There were 176 donors with phenotypic A subtypes identified. Exons 6 and 7 from 57 representative donors were sequenced. The A(var) allele (784 G > A) was cloned and sequenced, and a family study demonstrating its inheritance and unusual serological characteristics was performed. The A102 allele was the most frequently identified allele in phenotypically A2 (58%, 11/19) and A2B (68%, 17/25) donors. Anti-A1 was rarely present amongst A2 and A2B donors. The family study revealed that the A(var) allele was expressed as phenotype A(weak)B in A(var)/B heterozygote members, but as phenotype O in A(var)/O heterozygotes. The most frequent allele in Korean donors with the A2 phenotype differs from its Caucasian counterpart, as does the frequency of anti-A1. The A(var) allele demonstrates allelic enhancement in A(var)/B heterozygotes.

Determinants of ABH expression on human blood platelets

AbstractPlatelets express ABH antigens, which can adversely effect platelet transfusion recovery and survival in ABH-incompatible recipients. To date, there has been no large, comprehensive study comparing specific donor factors with ABH expression on platelet membranes and glycoconjugates. We studied ABH expression in 166 group A apheresis platelet donors by flow cytometry, Western blotting, and thin layer chromatography relative to donor age, sex, A1/A2 subgroup, and Lewis phenotype. Overall, A antigen on platelet membranes, glycoproteins, and glycosphingolipids was linked to an A1 red blood cell (RBC) phenotype. Among A1 donors, platelet ABH varied significantly between donors (0%-87%). Intradonor variability, however, was minimal, suggesting that platelet ABH expression is a stable, donor-specific characteristic, with 5% of A1 donors typing as either ABH high- or low-expressers. Group A2 donors, in contrast, possessed a Bombay-like phenotype, lacking both A and H antigens. Unlike RBCs, ABH expression on platelets may be determined primarily by H-glycosyltransferase (FUT1) activity. Identification of A2 and A1 low expressers may increase the availability and selection of crossmatched and HLA-matched platelets. Platelets from group A2 may also be a superior product for patients undergoing A/O major mismatch allogeneic progenitor cell transplantation. (Blood. 2005;105:3356-3364)

Analysis of Memory T Lymphocytes Activity Following Stimulation with HLA-A24, A1 and Cw4 Restricted 9- and 10-mer Cytomegalovirus pp65 Overlapping Peptides.

In healthy subjects, primary infection with Cytomegalovirus (CMV) is usually mild or asymptomatic and is effectively controlled by the cell-mediated immune response. However, in immune compromised individuals, such as those with AIDS or after bone marrow transplantation, CMV reactivation is associated with significant morbidity until the individual's immune system is completely reconstituted. One means of preventing post-transplant CMV infection is adoptive immunotherapy using CMV-specific cytotoxic T cells (CTLs) from the transplant donor. Several 9- and 10-mer HLA class I restricted peptides derived from the immune dominant CMV 65 kd matrix phosphoprotein (pp65) have been shown to produce CMV-specific CTLs. Two overlapping HLA-A24 restricted peptides have been specifically described: pp65 341–349 and pp65 341–350. These are 9- and 10-mer peptides that overlap except for the last amino acid phenylalanine (F) at the C-terminus [QYDPVAALF(F)]. Despite their similarity, the ability of these peptides to induce a T cell response has been reported to differ. Although it has been generally accepted that a unique CMV peptide is bound and presented by each separate HLA class I molecule, recent studies suggest that certain peptides are more promiscuous and may be presented by more than one HLA Class I antigen. For example, the 9-mer pp65 341–349 has been shown to stimulate CTLs from both HLA-A24 and Cw4 donors, while the 10-mer pp65 341–350 has been shown to be reactive with both HLA-A24 and A1 donors. The current investigation sought to compare the potency of these two peptides and determine the optimum peptide size for effective CMV adoptive immune therapy. Both peptides were tested for their ability to stimulate CMV-specific CTLs in HLA-A24, HLA-A1, and HLA-Cw4 restriction. In addition, a pp65 16-mer that included the 9- and 10-mers was tested for its ability to reactivate either CD8+ or CD4+ memory T cells. IFN-γ mRNA transcript as well as protein production were measured by in vitro cell culture assays. Peptide stimulations were performed on isolated CD8 and CD4 T lymphocytes by inducing the cells for 3 hours after a 2-week in vitro sensitization. The goal of the investigation was to determine whether both the 9- and the 10-mer peptides maintained high levels of CTL stimulation over time for all HLA restrictions studied. Moreover, it was important to investigate whether stimulation with the 16-mer, followed by restimulation by the two smaller peptides embedded within the larger sequence, led to effective T cell memory immune response. The 9- and 10-mer peptides effectively stimulated CTLs from HLA-A24, HLA-A1, and HLA-Cw4 CMV seropositive donors. Although both 9- and 10-mer were able to maintain high levels of stimulation over time for all restrictions, the 9-mer induced highest responses in cells expressing HLA-A24 (S.I. 4.07–528) or HLA-Cw4 (S.I. 4.15–483) while the 10-mer induced highest responses in cells expressing HLA-A24 (S.I. 3.5–528) or HLA-A1 (S.I. 8.25–615). The 16-mer peptide was also able to stimulate T cells from all HLA-A24, A1 and Cw4 donors (S.I. 6.95, 4.96, 5.02) at levels that are well maintained over time. This data confirmed that both the 9- and the 10-mer peptides are promiscuous and not restricted to a single HLA antigen. These peptides that have the ability to produce CMV-specific CTLs in patients with several different HLA types present a practical advantage over peptides that are restricted only to a single HLA type, and thus are optimal for CMV adoptive immune therapy.

1848: Long-Term Outcome of Renal Transplantion Using Hla Mismatched A2 Donor Kidneys

You have accessJournal of UrologyDiscussed Poster, Wednesday, May 12, 2004, 8:00am - 12:00 pm1 Apr 20041848: Long-Term Outcome of Renal Transplantion Using Hla Mismatched A2 Donor Kidneys Patrick Luke, Vaishali Kamik, Mahms Mohammed, Andrew House, Norman Muirhead, David Hollomby, Vivian McAlister, and Anthony Jevnikar Patrick LukePatrick Luke More articles by this author , Vaishali KamikVaishali Kamik More articles by this author , Mahms MohammedMahms Mohammed More articles by this author , Andrew HouseAndrew House More articles by this author , Norman MuirheadNorman Muirhead More articles by this author , David HollombyDavid Hollomby More articles by this author , Vivian McAlisterVivian McAlister More articles by this author , and Anthony JevnikarAnthony Jevnikar More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)39040-2AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "1848: Long-Term Outcome of Renal Transplantion Using Hla Mismatched A2 Donor Kidneys." The Journal of Urology, 171(4S), p. 488 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 171Issue 4SApril 2004Page: 488 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Patrick Luke More articles by this author Vaishali Kamik More articles by this author Mahms Mohammed More articles by this author Andrew House More articles by this author Norman Muirhead More articles by this author David Hollomby More articles by this author Vivian McAlister More articles by this author Anthony Jevnikar More articles by this author Expand All Advertisement PDF downloadLoading ...