Tomoya Hayashi

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.

Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens, antibody detection strategies, and genotyping

Alloimmunisation to platelets leads to the production of antibodies against platelet antigens and consequently to thrombocytopenia. Numerous molecules located on the platelet surface are antigenic and induce immune-mediated platelet destruction with symptoms that can be serious. Human platelet antigens (HPA) cause thrombocytopenias, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. Thirty-four HPA are classified into 28 systems. Assays to identify HPA and anti-HPA antibodies are critically important for preventing and treating thrombocytopenia caused by anti-HPA antibodies. Significant progress in furthering our understanding of HPA has been made in the last decade: new HPA have been discovered, antibody-detection methods have improved, and new genotyping methods have been developed. We review these advances and discuss issues that remain to be resolved as well as future prospects for preventing and treating immune thrombocytopenia.

Establishment of a novel method for detecting Naka antibodies by using a panel cell line

common in prolonged pregnancies. When associating meconium with fetal distress, it is important to note that finding meconium by itself does not necessarily imply that fetal stress is currently present. It might occur as a rather common phenomenon in postterm pregnancies. These pregnancies, however, tend to have a greater incidence of placental insufficiency and thus require more often an operative delivery (vaginal or abdominal). Furthermore a prolonged firstand/or second-stage labor does not necessarily lead to obvious fetal distress if delivery is initiated in time. The analysis of our own data shows no correlation between the duration of labor and TNC count, with or without meconium. The in utero UCB collection has been shown to be more efficacious compared to the ex utero technique. In this letter there were significant differences in collection techniques among the groups. Interestingly, although there were more ex utero UCB collections in the meconium group, the TNC yield was higher. Here, we would suggest a multivariate statistical analysis to exclude any confounding variables that influence the TNC count. This would strengthen the issue whether meconium staining itself is an independent factor for TNC yield. Such an analysis would also help to identify the impact of the gestational age and birth weight for the TNC count since in Solves’s data these two variables are significantly higher in the meconium group and might bias the results. In conclusion, all of the present studies suggest that fetal distress during labor as indicated by low umbilical arterial pH or operative delivery (vaginal or abdominal) leads to a mobilization of hematopoietic progenitor cells into the UCB. The physiology of the mobilization of stem cells into the UCB during labor and delivery is not well understood and further studies are needed to better understand which cytokines mediate stem cell mobilization and which stem cell populations then can be collected depending on fetal distress. Gwendolin Manegold, MD e-mail: gmanegold@uhbs.ch Wolfgang Holzgreve, MD Carolyn Troeger, MD Department of Obstetrics and Gynecology University Women’s Hospital Basel Basel, Switzerland

Application of the bactericidal activity of ε-poly-L-lysine to the storage of human platelet concentrates.

BACKGROUND: ε‐Poly‐l‐lysine (ε‐PLL) is a polypeptide comprising approximately 30 l‐lysine subunits generated by bond formation between α‐carboxy and ε‐amino groups. It is an approved antimicrobial food preservative in Japan. However, the efficacy of ε‐PLL as an antibacterial additive for storage of human platelet concentrates (PCs) is not known.

A Leu55 to Pro substitution in the integrin αIIb is responsible for a case of Glanzmann's thrombasthenia

Summary. Glanzmanns thrombasthenia (GT) is a hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin αIIbβ3. A new mutation, a T to C substitution at base 258 in the αIIb gene, leading to the replacement of Leu55 with Pro, was found by sequence analysis of a patients αIIb cDNA. In transfection experiments using COS7 cells, the cells co‐transfected with the mutated αIIb cDNA containing C258 and wild‐type β3 cDNA scarcely expressed the αIIbβ3 complex. The Leu55 to Pro substitution in the αIIb gene was found to be responsible for this case of Glanzmanns thrombasthenia.

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.

High-resolution melting method for genotyping human platelet antigens on ITGB3 Exon 11.

According to the Immuno Polymorphism Database (IPD: http://www.ebi.ac.uk/ipd/hpa/), 21 types of human platelet antigens (HPAs) can be identified by genomic incompatibility; these HPAs are located on four types of platelet (PLT) molecules. The genotype frequencies of HPAs vary among races and HPA types. ITGB3 gene mutations responsible for the antigenicity of approximately half of HPAs, particularly Exon 3 and Exon 11, are hot spots. Three rare human PLT antigens are located on Exon 11 of ITGB3, that is, HPA-8bw, -11bw, and -21bw. The point mutations in HPA-11bw and HPA-8bw are located 16 and 24 bp downstream from that in HPA-21bw, respectively. HPA genotyping is important for the diagnosis and prevention of thrombocytopenia to determine PLT transfusion refractoriness and neonatal alloimmune thrombocytopenic purpura (NAIT). Several methods have been developed for HPA genotyping. High-resolution melting method (HRM) is based on the principles that melting temperature depends on the difference in DNA sequences, for example, minimum value of point mutations. Since HRM was developed as a simple and costeffective alternative to other closed-tube genotyping methods, we applied HRM using one primer set without fluorescent probe for efficient screening of HPA-8bw, HPA-11bw, and HPA-21bw genotypes. Blood donors were randomly selected after informed consent was obtained. All genomic samples were extracted from whole blood samples using the DNA whole blood kit (QuickGene, Fujifilm, Tokyo, Japan). We used 2155 genomic samples in the HRM analysis. Polymerase chain reaction (PCR) and melting analyses were performed using a real-time PCR cycler (Rotor-Gene Q, Qiagen GmbH, Hilden, Germany) using an HRM PCR kit (Type-it, Qiagen). All procedures were performed according to the manufacturer’s instructions using the following primer set: 5′-TCCTTCAGAGAATGTGTGGAGTGT-3′ and 5′-TAAGCTCTTTCACTGACTCAATCTCG-3′. Thermal cycling was performed as follows: 10 seconds at 95°C, 30 seconds at 52°C, and 10 seconds at 72°C for 40 cycles. Melting curve was acquired with a ramp rate of 0.1°C/ second to 95°C to produce a continuous fluorescence curve. Melting data were analyzed using the Rotor-Gene 1.7.94 program and exported as a computer spreadsheet (Excel, Microsoft Corp., Redmond, WA). To normalize the data, Regions 1 and 2 were set at 76 to 78 and 82 to 84°C, respectively. The total analysis time for PCR and melting was approximately 2 hours under these conditions. During the genotyping of genomic samples, artificial DNA fragments were used as controls that were analyzed in parallel with the samples. Four control DNA fragments (ITGB3 genomic DNA 29990-30210), wild-type, HPA-8bw, HPA-11bw, and HPA-21bw, were synthesized by Operon Biotechnology (Tokyo, Japan). Heterogeneous artificial DNA samples were prepared as a mixture of equal amounts of wild-type and each rare HPA DNA. HRM analysis of control DNA showed that rare HPAs, that is, wild-type/HPA-8bw, HPA-8bw/bw, wild-type/HPA-11bw, HPA-11bw/bw, wild-type/HPA-21bw, and HPA-21bw/bw, produced visibly different curves from the wild-type DNA (Fig. 1). However, it may be difficult to distinguish HPA8bw/bw and wild-type/HPA-11bw from HPA-11bw/bw and wild-type/HPA-21bw, respectively. Therefore, we aimed to retrieve the rare HPAs. Next, the retrieved genomic DNAs were analyzed to determine their HPAs by DNA sequence analysis. The results acquired from 2155 genomic samples are shown in Table 1. HRM analysis showed that 2140 samples had wild-type HPAs whereas 15 samples had rare HPAs. Representative data are shown in Fig. 1. All samples identified as rare antigens using HRM were confirmed as wildtype/HPA-21bw by DNA sequence analysis. To validate the results, all samples were analyzed by conventional PCR with sequence-specific priming (SSP) using specific primers for wild-type and HPA-21bw. No discrepancy was observed between the HRM results for rare HPAs and conventional PCR-SSP for wild-type/HPA-21bw. In this study, the frequency of HPA-21bw was approximately 0.7% in the Japanese population. Because NAIT was recently associated with HPA-21 incompatibility in Japan and the United States, HPA-21bw should receive more attention in the future. We now plan to determine the HPA-21bw frequency in other races. These results suggest that applying the HRM method to Exon 11 of ITGB3 might at least discriminate DNA containing rare HPA from wild-type DNA. HRM analysis should be used to distinguish the wild-type from other rare antigens. It was difficult to assess HPA-8a/b and HPA-11a/b genomic DNA because of their low global frequencies. Thus, further