The presence of allo-antibodies in the serum of a recipient awaiting hematopoietic stem cell transplantation (HSCT) may have an impact on transfusion efficiency and/or donor choice, especially in the absence of an identical sibling donor. Prior to transplantation, donor specific anti-HLA (Human Leukocyte Antigen) antibodies (DSA) have a recognized effect on transplant outcome, correlated with the increasing MFI value and with the ability of such antibody to fix the complement fraction. Anti-platelet antibodies (anti-HLA class I and anti-HPA [Human Platelet Antigen]) are better involved in transfusion inefficiency and can be responsible for refractory status. ABO incompatibilities require a specific treatment of the graft in presence of high titer to avoid hemolytic adverse effects. Investigations of these antibodies should be carried out on a regular basis in order to establish appropriate transfusion recommendation, select an alternative donor when possible or adapt the source of cells. After transplantation, in case of delayed recovery or graft rejection, long term aplasia, persistent mixed chimerism or late release, and after elimination of the main clinical causes, a biological assessment targeted on the different type of antibodies will have to be performed in order to orient towards the cause or the appropriate therapy. Further studies should be carried out to determine the impact of anti-MICA antibodies and recipient specific anti-HLA antibodies, on the outcome of the transplantation.

BACKGROUND: Routine, high-resolution human leukocyte antigen (HLA) genotyping by next generation sequencing within clinical immunogenetics laboratories can now provide the full-length gene sequence characterization of fully phased HLA alleles. This powerful technique provides insights into HLA variation beyond the traditionally characterized antigen recognition domain, providing sequence annotation across the entire gene including untranslated and intronic regions and may be used to characterize novel alleles from massively parallel sequencing runs.
METHODS: We evaluated the utility of the Omixon Holotype HLA assay to generate credible, fully phased full-length gene consensus sequences for 50 individuals at major histocompatibility complex, class I, A (HLA-A), HLA-B, and HLA-C loci (300 genotyped alleles in total) to identify and characterize novel class I HLA alleles using our downstream analytical pipeline.
RESULTS: Our analysis revealed that 7.7% (23/300) of genotyped class I HLA alleles contain novel polymorphisms. Interestingly, all of the novel alleles identified by our analysis were found to harbor sequence variations within intronic regions of the respective locus. In total our analysis identified 17 unique novel class I HLA alleles from 23 of the 300 genotyped alleles and generated full-length gene sequence annotations for 9 previously incompletely annotated HLA class I allele sequences derived from 14 of the 300 genotyped alleles.
CONCLUSIONS: The demonstrated utility of the Omixon Holotype HLA assay in combination with our downstream analytical framework to generate fully phased, full-length gene consensus sequences for the identification and characterization of novel HLA alleles, facilitates the study of HLA polymorphism beyond the antigen recognition domain in human health and disease.

Immune responses to cross-conserved T cell epitopes in novel H1N1 influenza may explain reports of diminished influenza-like illnesses and confirmed infection among older adults, in the absence of cross-reactive humoral immunity, during the 2009 pandemic. These cross-conserved epitopes may prove useful for the development of a universal H1N1 influenza vaccine, therefore, we set out to identify and characterize cross-conserved H1N1 T cell epitopes. An immunoinformatic analysis was conducted using all available pandemic and pre-pandemic HA-H1 and NA-N1 sequences dating back to 1980. Using an approach that balances potential for immunogenicity with conservation, we derived 13 HA and four NA immunogenic consensus sequences (ICS) from a comprehensive analysis of 5,738 HA-H1 and 5,396 NA-N1 sequences. These epitopes were selected because their combined epitope content is representative of greater than 84% of pre-pandemic and pandemic H1N1 influenza strains, their predicted immunogenicity (EpiMatrix) scores were greater than or equal to the 95th percentile of all comparable epitopes, and they were also predicted to be presented by more than four HLA class II archetypal alleles. We confirmed the ability of these peptides to bind in HLA binding assays and to stimulate interferon-γ production in human peripheral blood mononuclear cell cultures. These studies support the selection of the ICS as components of potential group-common H1N1 vaccine candidates and the application of this universal influenza vaccine development approach to other influenza subtypes.

OBJECTIVE: To determine the function and phenotype of CD8(+) T-cells targeting consensus and autologous sequences of entire HIV-1 Nef protein.
METHODS: Multiparameter flow cytometry-based analysis was used to evaluate the responses of two treatment naïve HIV-infected individuals, during primary and the chronic phases of infection.
RESULTS: A greater breadth and magnitude of CD8 IFN-γ responses to autologous compared to clade-B consensus peptides was observed in both subjects. Cross recognition between autologous and consensus peptides decreased in both subjects during progression from primary to chronic infection. The frequencies of TEMRA and TEM CD8(+) T-cells targeting autologous peptides were higher than those targeting consensus peptides and were more polyfunctional (IFN-γ(+) Gr-B(+) CD107a(+)).
CONCLUSIONS: Our data indicate superior sensitivity and specificity of autologous peptides. The functional and maturational aspects of \"real\" versus \"cross-recognized\" responses were also found to differ, highlighting the importance of a sequence-specific approach towards understanding HIV immune response.

OBJECTIVE:   Transfusion-related acute lung injury (TRALI) is associated with the passive transfusion of leucocyte antibodies in blood products. Blood Transfusion Services have adopted a number of different strategies for reducing the incidence of TRALI, but, while these have been successful, TRALI has not been completely eliminated. Many Transfusion Services have introduced leucocyte antibody screening of donors to further reduce TRALI. This report describes the results of donor leucocyte antibody screening within NHS Blood and Transplant and the guidelines that have been developed for Transfusion Services within the United Kingdom (UK) to reduce the incidence of TRALI.
METHODS:   Blood samples from newly recruited female apheresis donors were tested for human leucocyte antigens (HLA) class I and class II antibodies and granulocyte-specific antibodies.
RESULTS:   A total of 1157 female donors were evaluated. Three hundred and fifteen (27·23%) donors had HLA class I or II antibodies and were returned to red cell component donation. Fifty-seven (6·77%) of the remaining 842 donors were found to have granulocyte-specific antibodies of which 11 (1·31%) had HNA-specific antibodies. A total of 818 donors (70·70%) were accepted for platelet apheresis, 336 donors (29·04%) were returned to red cell component donation, and three donors with HNA-3a antibodies (0·26%) were deferred from therapeutic donation.
CONCLUSIONS:   Female donors with leucocyte antibodies were identified in a stratified screening programme. Donors with antibodies were either directed to red cell donation or deferred. This process, combined with other measures that have already been introduced, is anticipated to further reduce the incidence of TRALI.

A key role in cell-mediated immunity is dedicated to the major histocompatibility complex (MHC) molecules that bind peptides for presentation on the cell surface. Several in silico methods capable of predicting peptide binding to MHC class I have been developed. The accuracy of these methods depends on the data available characterizing the binding specificity of the MHC molecules. It has, moreover, been demonstrated that consensus methods defined as combinations of two or more different methods led to improved prediction accuracy. This plethora of methods makes it very difficult for the non-expert user to choose the most suitable method for predicting binding to a given MHC molecule. In this study, we have therefore made an in-depth analysis of combinations of three state-of-the-art MHC-peptide binding prediction methods (NetMHC, NetMHCpan and PickPocket). We demonstrate that a simple combination of NetMHC and NetMHCpan gives the highest performance when the allele in question is included in the training and is characterized by at least 50 data points with at least ten binders. Otherwise, NetMHCpan is the best predictor. When an allele has not been characterized, the performance depends on the distance to the training data. NetMHCpan has the highest performance when close neighbours are present in the training set, while the combination of NetMHCpan and PickPocket outperforms either of the two methods for alleles with more remote neighbours. The final method, NetMHCcons, is publicly available at www.cbs.dtu.dk/services/NetMHCcons , and allows the user in an automatic manner to obtain the most accurate predictions for any given MHC molecule.

BACKGROUND: HLA antibodies might contribute to the pathogenesis of transfusion-related acute lung injury (TRALI). HLA antibody detection methods include ELISA, flow cytometry, and multiplex bead-based assays, as well as the older lymphocytotoxicity assay, and it is not obvious how to compare results across platforms.
METHODS: Five hundred twenty-five serum samples were selected from 7841 donors in the Leukocyte Antibody Prevalence Study (LAPS) repository based on risk for the development of HLA antibodies, using the number of pregnancies as the risk factor. Subjects included 81 males and females with 0 (n = 187), 1 (n = 67), or 2+ pregnancies (n = 190). Replicate frozen serum aliquots were sent blinded to four different HLA antibody assay manufacturers for detection using five different assays.
RESULTS: The flow cytometry and multiplex bead based-assays typically resulted in a larger proportion of HLA antibody positive samples compared with ELISA based assays. Latent variable analysis was used to derive a new set of consensus cutoffs, which yielded similar sensitivities across test platforms and increased concordance amongst assays. Assay agreement was higher in ever pregnant females than in males and never-pregnant females.
CONCLUSIONS: Different assays resulted in varied positivity rates when the manufacturer\'s suggested cutoffs were used, demonstrating that care needs to be taken when comparing clinical outcomes data generated using different HLA antibody assays and testing platforms. The method used here, involving latent variable analysis, presents one possible approach to calculating comparable cutoffs that result in broad agreement across assays with respect to positivity designation.

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Iron overload in humans is associated with a variety of genetic and acquired conditions. Of these, HFE hemochromatosis (HFE-HC) is by far the most frequent and most well-defined inherited cause when considering epidemiological aspects and risks for iron-related morbidity and mortality. The majority of patients with HFE-HC are homozygotes for the C282Y polymorphism [1]. Without therapeutic intervention, there is a risk that iron overload will occur, with the potential for tissue damage and disease. While a specific genetic test now allows for the diagnosis of HFE-HC, the uncertainty in defining cases and disease burden, as well as the low phenotypic penetrance of C282Y homozygosity poses a number of clinical problems in the management of patients with HC. This Clinical Practice Guideline will therefore, focus on HFE-HC, while rarer forms of genetic iron overload recently attributed to pathogenic mutations of transferrin receptor 2, (TFR2), hepcidin (HAMP), hemojuvelin (HJV), or to a sub-type of ferroportin (FPN) mutations, on which limited and sparse clinical and epidemiologic data are available, will not be discussed. We have developed recommendations for the screening, diagnosis, and management of HFE-HC.

OBJECTIVE: Lack of consensus in HLA antibody reporting in proficiency schemes has previously been attributed to a number of differing factors. This study was set up to eliminate the majority of these factors by reducing analysis to a pure data handling exercise.
METHODS: Anonymised raw data files for LABScreen Single Antigen class I and II and related patient information were provided to seven participating centres. The centres reported back the HLA antibody specificities according to their single antigen bead reporting policy. Details of the reporting policy of each centre were retrospectively requested by questionnaire.
RESULTS: The number of HLA antibody specificities reported by the different centres varied widely. Software analysis called more HLA antibody specificities than any of the centres. None of the centres matched consensus for reported HLA class I specificities on any of the datasets, and no two centres reported the exact same HLA class I antibody profile; consensus was reached by one centre for HLA class II antibody specificities reported from two of the datasets. Retrospective review found data handling practice between centres to vary widely.
CONCLUSIONS: Lack of agreement exists between UK centres in regard to HLA antibody specificity analysis. The fact that the required analysis was limited to interrogation of supplied data files makes the observation more concerning. The root cause of this variation is differences in data handling practice between the participating centres.