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Background: In previous studies, we demonstrated that the basophil activation test, which is performed using patient blood and the supernatants from transfused blood components, was able to elucidate not only the causative relationship between allergic transfusion reactions and the transfusion but also the mechanisms behind allergic transfusion reactions. However, for a large number of allergic transfusion reactions, patients are in a state of myelosuppression, and the basophil activation test cannot be performed for these patients because there are insufficient numbers of peripheral blood basophils., Study Design and Methods: To overcome this obstacle, we developed a passive immune basophil activation test, in which patient plasma and residually transfused blood are used as the patient's sources of immunoglobulin E and allergen, respectively, whereas healthy volunteer basophils serve as the responder cell source. The passive immune basophil activation test was performed for two patients who had severe allergic transfusion reactions, using supernatants of the residual platelet concentrates and the patients' own immunoglobulin E., Results: There were no differences in either surface immunoglobulin E or activation in response to allergens between untreated basophils and so-called quasi-basophils, in which immunoglobulin E was replaced by a third party's immunoglobulin E. In these patients, the supernatants of the residual platelet concentrates exclusively activated basophils in response to quasi-basophils onto which the patients' immunoglobulin E, but not a third party's immunoglobulin E, was bound., Conclusion: The passive immune basophil activation test may help clarify the causal relationship between allergic transfusion reactions and transfused blood, even when patients experience myelosuppression., (© 2017 AABB.)

Background: Platelet concentrates (PCs) are the most common blood components eliciting nonhemolytic transfusion reactions (NHTRs), such as allergic transfusion reactions and febrile reactions. However, the precise mechanisms of NHTRs in PC transfusion remain largely unknown. Previous studies reported that mitochondria-derived damage-associated molecular patterns (DAMPs) could be important mediators of innate cell inflammation. Platelets (PLTs) represent a major reservoir of mitochondria in the blood circulation. The aim of this study was to determine the possible involvement of mitochondrial DAMPs in NHTRs., Study Design and Methods: The amount of mitochondrial DAMPs was determined as an index of total copy numbers of mitochondrial DNA (mtDNA), including mtDNA itself and free mitochondria, using quantitative real-time polymerase chain reaction. To examine whether neutrophils, monocytes, and basophils were activated by mitochondrial DAMPs in vitro, an in vitro whole blood cell culture assay was performed., Results: In blood components associated with NHTRs, the mean total mtDNA concentration was highest in PCs followed in order by fresh-frozen plasma and red blood cells. The amount of mtDNA in NHTR PCs was higher than that in control PCs without NHTRs. The mitochondrial DAMPs present in NHTR PCs was high enough to activate neutrophils, monocytes, and basophils, when costimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or HLA antibodies., Conclusion: PLT-derived mitochondrial DAMPs are candidate risk factors for the onset of NHTRs., (© 2016 AABB.)

Background and Objectives CD36 antibody ( Ab) causes several disorders: neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and non-haemolytic transfusion reactions. However, there is no gold-standard test for CD36 Ab. Materials and Methods We developed a transfectant panel cell line-based Mo Ab-independent antigen capture assay system for detection of CD36 Ab and compared it with the monoclonal antibody-specific immobilization of platelet antigens ( MAIPA) system in terms of sensitivity and specificity. Results Our new system was characterized by (1) gene-transfected cell lines, but not panel platelets; (2) not being hampered by HLA Abs; and (3) no need to use CD36 Mo Abs to ensure the antigen specificity of this detection system. In addition, it showed a much better receiver operating characteristic curve than the MAIPA system. Conclusions The present results indicate that our new system permits highly sensitive and specific detection of CD36 Ab. [ABSTRACT FROM AUTHOR]

A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies. Wild-type β3, HPA-1b, -6b, -7b and -7 variant cDNA as well as wild-type αIIb and HPA-3b cDNA were individually co-transduced with wild-type αIIb and β3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test. Of the 12 sera containing HPA-1a ( n = 2), HPA-3a ( n = 6), HPA-6b ( n = 3) or HPA-7 variant ( n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA-3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay. [ABSTRACT FROM AUTHOR]

Although flow cytometric (FCM) analysis is one of the most widely used approaches to screen the presence of leucocyte antibodies, it has several drawbacks. First, neutrophils and, especially, monocytes exhibit high background reactivity. Second, to determine antibody specificity, it is often necessary to examine not only neutrophils and monocytes but also other lineage cells including T cells, B cells and platelets. Therefore, we attempted to establish an FCM analysis system in which four lineages of leucocytes and platelets are simultaneously tested with low background. FCM analysis was performed using ethylene diamine tetraacetic acid-anticoagulated whole blood as cell sample without any cell preparation. Discrimination of five cell lineages was carried out based on the differences in forward vs. side scatter distribution and in the expression of CD4, CD20 and CD14. When anti-HNA (human neutrophil antigen) 1b antiserum was applied to HNA 1b-positive blood samples, only neutrophils were unambiguously positive. When anti-Naka (anti-CD36) antiserum was applied, only platelets and monocytes were positive. The background reactivity of neutrophils and monocytes was low enough. When anti-human leucocyte antigen (HLA) class II antiserum was tested, only B-lymphocytes and monocytes were positive. When anti-HLA class I antiserum was tested, all the five-lineage cells were positive. [ABSTRACT FROM AUTHOR]