Scottish, United Kingdom
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Boyle J.,UK National Institute for Biological Standards and Control | Thorpe S.J.,UK National Institute for Biological Standards and Control | Hawkins J.R.,UK National Institute for Biological Standards and Control | Lockie C.,UK National Institute for Biological Standards and Control | And 10 more authors.
Vox Sanguinis | Year: 2013

Background and Objectives The aim of the study was to evaluate, in an international collaboration, four lyophilised genomic DNA preparations, selected from genotyped and phenotyped donors by the study organisers, for their suitability to standardise and control blood group genotyping procedures for common ancestral Caucasian and Black African alleles. Materials and Methods Twenty-nine laboratories performed 'blind' testing of replicated ampoules of the candidate reference reagents, RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234), using a range of genotyping procedures, most commonly classical PCR using allele or sequence specific primers. Results The majority of laboratories reported blood group genotypes in accordance with those determined by the study organisers and the serological phenotypes. Despite an overall high level of accuracy in genotyping, the identified errors and inconsistencies, and the limited genotyping capabilities of many laboratories, confirmed the need for validated reference materials to control test procedures. Conclusions The establishment of RBC1, RBC4, RBC5 and RBC12 as World Health Organization Reference Reagents will facilitate international standardisation of blood group genotyping and ensure that such tests are sufficiently sensitive and specific.© 2012 International Society of Blood Transfusion.


Hall A.M.,University of Aberdeen | Zamzami O.M.,University of Aberdeen | Whibley N.,University of Aberdeen | Hampsey D.P.,University of Aberdeen | And 4 more authors.
Haematologica | Year: 2012

Background: Interleukin-17A is the signature cytokine of the Th17 subset and drives inflammatory pathology, but its relevance to autoantibody-mediated diseases is unclear. Th1 cells secreting interferon-γ have been implicated in autoimmune hemolytic anemia, so the aim was to determine which cytokine is more closely associated with disease severity. Design and Methods: Interferon-γ and interleukin-17A were measured in the sera of patients with autoimmune hemolytic anemia and healthy donors, and in peripheral blood mononuclear cell cultures stimulated with autologous red blood cells, or a panel of peptides spanning red blood cell autoantigen. Results: Serum interleukin-17A, but not interferon-γ, was significantly raised in patients with autoimmune hemolytic anemia (P<0.001), and correlated with the degree of anemia. Interleukin-17A was also more prominent in the responses of peripheral blood mononuclear cells from patients with autoimmune hemolytic anemia to red blood cells, and, again unlike interferon-γ, significantly associated with more severe anemia (P<0.005). There were no interleukin-17A responses to red blood cells by peripheral blood mononuclear cells from healthy donors. Specific autoantigenic peptides were identified that elicit patients' interleukin-17A responses. Conclusions: Interleukin-17A makes a previously unrecognized contribution to the autoimmune response in autoimmune hemolytic anemia, challenging the model that the disease is driven primarily by Th1 cells. This raises the possibility that Th17, rather than Th1, cells should be the target for therapy. © 2012 Ferrata Storti Foundation.


Hall L.S.,University of Aberdeen | Hall L.S.,Academic Transfusion Medicine Unit | Hall A.M.,University of Aberdeen | Pickford W.,University of Aberdeen | And 5 more authors.
Haematologica | Year: 2014

The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant Thelper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (P=0.008, Mann-Whitney rank sum test) or subcutaneously (P=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group. © 2014 Ferrata Storti Foundation.


Stephen J.,University of Aberdeen | Cairns L.S.,University of Aberdeen | Pickford W.J.,University of Aberdeen | Vickers M.A.,University of Aberdeen | And 3 more authors.
Blood | Year: 2012

The K blood group remains an important target in hemolytic disease of the newborn (HDN), with no immune prophylaxis available. The aim was to characterize the Th response to K as a key step in designing specific immunotherapy and understanding the immunogenicity of the Ag. PBMCs from K-negative women who had anti-K Abs after incompatible pregnancy, and PBMCs from unimmunized controls, were screened for proliferative responses to peptide panels spanning the K or k single amino acid polymorphism. A dominant K peptide with the polymorphism at the C terminus elicited proliferation in 90% of alloimmunized women, and it was confirmed that responding cells expressed helper CD3 +CD4 + and "memory" CD45RO + phenotypes, and were MHC class II restricted. A relatively high prevalence of background peptide responses independent of alloimmunization may contribute to K immunogenicity. First, cross-reactive environmental Ag(s) pre-prime Kell-reactive Th cells, and, second, the K substitution disrupts an N-glycosylation motif, allowing the exposed amino acid chain to stimulate a Th repertoire that is unconstrained by self-tolerance in K-negative individuals. The dominant K peptide was effective in inducing linked suppression in HLA-transgenic mice and can now be taken forward for immunotherapy to prevent HDN because of anti-K responses. © 2012 by The American Society of Hematology.


Whibley N.,University of Aberdeen | Whibley N.,Aberdeen Group | Whibley N.,University of Pittsburgh | Maccallum D.M.,Aberdeen Group | And 9 more authors.
European Journal of Immunology | Year: 2014

Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3+ regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3+ Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4+Foxp3+ population that correlated positively with fungal burden. Depletion from Foxp3hCD2 reporter mice in vivo confirmed that Foxp3+ cells exacerbated fungal burden and inflammatory renal disease. The CD4+Foxp3+ population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3- cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt, and secreting IL-17A. The expanded Foxp3+ T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3+ T cells promotes Th17-cell responses that drive pathology. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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