Etsuko Amakishi

Neonatal alloimmune thrombocytopenia caused by an antibody specific for a newly identified allele of human platelet antigen-7

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is a neonatal disorder characterized by maternal alloimmunization against fetal platelet (PLT) antigens inherited from the father. A healthy 30‐year‐old Japanese woman (Hit) gave birth to her second child after an uneventful pregnancy. Nine hours after birth, the infant presented with severe petechiae and a PLT count of 6 × 109/L.

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti-human platelet antibodies

Background: 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.

The first two cases of neonatal alloimmune thrombocytopenia associated with the low-frequency platelet antigen HPA-21bw (Nos) in Japan.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is a disorder characterized by maternal alloimmunization against paternal fetal platelet antigens. Two healthy, unrelated Japanese women each gave birth to a child with severe NAIT.

The IgE‑dependent pathway in allergic transfusion reactions: involvement of donor blood allergens other than plasma proteins

On transfusion, several plasma proteins can cause anaphylaxis in patients deficient in the corresponding plasma proteins. However, little is known about other allergens, which are encountered much more infrequently. Although it has been speculated that an allergen-independent pathway underlying allergic transfusion reactions (ATRs) is elicited by biological response modifiers accumulated in blood components during storage, the exact mechanisms remain unresolved. Furthermore, it is difficult even to determine whether ATRs are induced via allergen-dependent or allergen-independent pathways. To distinguish these two pathways in ATR cases, we established a basophil activation test, in which the basophil-activating ability of supernatants of residual transfused blood of ATR cases to whole blood basophils was assessed in the presence or absence of dasatinib, an inhibitor of IgE-mediated basophil activation. Three of 37 supernatants from the platelet concentrates with ATRs activated panel blood basophils in the absence, but not in the presence, of dasatinib. The basophil activation was inhibited by treatment of anti-fish collagen I MoAb in one case, suggesting that the involvement of fish allergens may have been present in donor plasma. We concluded that unknown non-plasma proteins, some of which had epitopes similar to fish antigens, in blood component may be involved in ATRs via an allergen/IgE-dependent pathway.

Detection of antibodies against human platelet antigens 15a and 15b by using a cell line panel.

The platelet surface membrane contains a variety of molecules, including ABO blood type antigens (Curtis et al, 2000), human leucocyte antigens (HLAs), human platelet antigens (HPAs) and Nak antigen. Antibodies to these molecules have been regarded as the principal causes of various reactions elicited by platelet transfusion, such as refractoriness and post-transfusion purpura. These antibodies are also thought to cause neonatal alloimmune thrombocytopenia and idiopathic thrombocytopenic purpura (Smith et al, 1995; Bordin et al, 1997). The HPA-15 (Gov) alloantigen system is localized on the CD109 protein, a glycosylphosphatidylinositol-linked glycoprotein of 175 kDa that is found on several tissues and also on a subset of haematopoietic stem cells, progenitor cells and activated platelets and T cells. HPA-15 alleles differ by an A/C single nucleotide polymorphism at position 2108 of the coding region of CD109 cDNA, resulting in a Tyr/Ser substitution at CD109 amino acid 682 (Schuh et al, 2002). Despite differences between races, the genotypic frequencies of HPA-15a and HPA-15b antigens are roughly equal (Cardone et al, 2004).

Detection of anti-human platelet antibodies against integrin α2β1 using cell lines

BACKGROUND Antibodies against human platelet antigens (HPA) are a cause of thrombocytopenia. Detection of rare anti-HPA antibodies using platelet preparations is difficult and would be improved by an alternative method that does not require platelets. In the present study, we describe the establishment of cell lines that stably express specific HPA associated with integrin α2β1 and the application of these cell lines for detecting anti-HPA-5a and anti-HPA-5b antibodies. MATERIALS AND METHODS Complementary DNA of the integrin α2 variants HPA-5b, -13b and -18b were individually transfected into K562 cells using retroviral vectors. Expression of integrin α2 was confirmed by flow cytometric analysis, immunoprecipitation and western blotting analysis. To verify whether the cell line panel was suitable for clinical diagnosis, we analysed its properties using monoclonal antibody-specific immobilisation of platelet antigens (MAIPA) and well-characterised serum samples. RESULTS Exogenous integrin α2 expression was observed in the transfected cells for over 6 months. The cell line panel specifically detected previously characterised anti-HPA-5a and anti-HPA-5b antisera. No reactivity was observed with control sera, including normal sera and HLA antisera. DISCUSSION We successfully established a cell line panel to facilitate the sensitive and reliable detection of anti-HPA-5a and anti-HPA-5b antibodies.

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies

Granulocyte immunofluorescence and granulocyte agglutination tests are standard methods for detecting human neutrophil antigen (HNA) antibodies (Abs); however, these require a typed panel of neutrophils, which can be time-consuming to develop, and it remains difficult to determine antibody specificity in some cases. We established and evaluated four detection systems for HNA-1a Abs based on an HNA-1a-expressing cell line (KY cells) and antigen capture. We additionally evaluated a commercial solid-phase system. Eleven HNA-1a antibody-positive samples, including the World Health Organization Reference Reagent, and 40 serum samples derived from male blood donors were used as positive and negative control samples, respectively. Although specificity was >0.90 in all systems evaluated, the sensitivity varied among the systems. The KY cell-based monoclonal antibody specific immobilisation of granulocyte antigens (KY-MAIGA) system using certain, but not all, monoclonal Abs, and the solid-phase system revealed higher sensitivity than other systems. In conclusion, the KY-MAIGA and commercial solid-phase systems were superior in terms of specific and sensitive detection of HNA-1a Abs.

A new transfectant panel cell line-based MoAb-independent antigen capture assay system for detection of CD36 antibody

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.

Re-evaluation of HNA-1 antibodies for their reactivity to different recombinant variants of FcγRIIIb

The Fcgamma receptor IIIb (FcγRIIIb) carries the human neutrophil antigen (HNA)‐1 system. The three FCGR3B alleles encode four antigens: one allele encodes HNA‐1a, and two alleles encode two antigens each, HNA‐1b and HNA‐1d and HNA‐1b and HNA‐1c, respectively. Several other non‐synonymous substitutions have also been reported. Little is known about the properties of HNA‐1d antibodies because HNA‐1d was recently discovered, and HNA‐1d antibodies have been reported only in a couple of cases. These findings raise the possibility that the polymorphisms in the HNA‐1 system and spectrum of the specificity of resulting antibodies are more complex than previously thought. We address this possibility in this work.