Harumichi Matsukura

Minimum infectious dose of hepatitis B virus in chimpanzees and difference in the dynamics of viremia between genotype A and genotype C

BACKGROUND: In planning optimal hepatitis B virus (HBV) blood screening strategies, the minimum infectious dose and early dynamics of HBV need to be determined for defining the window period for HBV DNA as well as for hepatitis B surface antigen (HBsAg).

Titration of hepatitis B virus infectivity in the sera of pre‐acute and late acute phases of HBV infection: Transmission experiments to chimeric mice with human liver repopulated hepatocytes

Studies of hepatitis B virus (HBV) infection in non‐human primates such as chimpanzees are no longer possible due to ethical considerations and the endangered status of chimpanzees since April 2007 in Japan. A human hepatocyte transplanted chimeric mouse was used to characterize HBV infectivity in serial stages of acute infection. Chimeric mice were inoculated intravenously with serum samples obtained from an experimentally infected chimpanzee with HBV. Sera from the pre‐acute phases (i.e., rump‐up viremia prior to anti‐HBc) and late acute phases (i.e., declining phase of HBsAg and anti‐HBcAb positive) were collected from the chimpanzees 57 and 244 days after inoculation. These sera contained 2.6 × 106 and 2.8 × 106 copies/ml of HBV DNA, respectively. Three chimeric mice inoculated intravenously with 100 µl of pre‐acute serum (equivalent to 100 copy of HBV DNA) developed an HBV infection. The three chimeric mice that received 100 µl of pre‐acute serum (equivalent to 101 copies of HBV DNA), developed high levels of serum HBV DNA. None of the three chimeric mice inoculated with 100 µl of 1:104 dilution (equivalent to 101 copies of HBV DNA) of late‐acute serum was infected, while only one of three chimeric mice inoculated with 100 µl of 1:103 dilution (equivalent to 102 copies of HBV DNA) of late‐acute serum developed an HBV infection. Based on these results, chimeric mice can be used as animal models for the study of HBV infectivity, pathogenesis and control. The results show that pre‐acute phase HBV serum is about 100‐times more infectious than late acute phase serum. J. Med. Virol. 80:2064–2068, 2008.

Persistent infection by human parvovirus B19 in qualified blood donors

Persistent parvovirus B19 infection with a low viral load has been reported in immunocompromised and in immunocompetent individuals (reviewed in Parsyan and Candotti). Large cross-sectional studies using highly sensitive DNA amplification methods have also demonstrated persistent B19 infection. Recently, Lefere and colleagues conducted a longitudinal study of nonimmunodeficient patients who were multitransfused with red blood cells, demonstrating that asymptomatic chronic B19 infections may persist for a long period. To characterize the natural course of persistent B19 infections, we conducted the following longitudinal study using an in-house TaqMan polymerase chain reaction method for B19 DNA and enzyme immunoassays to detect B19 immunoglobulin M (IgM) and immunoglobulin G (IgG; Denka Seiken, Tokyo, Japan). This study was approved by the ethical Committee of the Japanese Red Cross Osaka Blood Center. In Japan, all donated blood is tested for B19 infection with an in-house receptor-mediated hemagglutination method that detects B19 antigen as a marker of a high viremic stage of infection (cutoff, approx. 2.5 ¥ 10 IU/mL B19 DNA; data not shown). Using this method, we identified 102 cases of B19 infection among 979,052 blood donors in Osaka between 1997 and 1999. We were able to test the plasma samples of 20 of these 102 donors (15 male, 5 female; mean age, 34.3 years) when they returned for subsequent blood donations for viral load and B19-specific IgG and IgM. We did not examine the donors at their first visit because B19 antigen-positive blood was automatically disqualified and disposed. The mean duration of follow-up was 838 days (range, 101-1749 days). The results of sequential viral load testing for all donors are shown in Fig. 1A. In the first 6 months, we observed a rapid decline in plasma B19 DNA, which decreased continuously, but never became undetectable. Median plasma B19 viral loads for samples tested within every 6 months are shown in Fig. 1B. We analyzed the B19 antibody for all donors during the study period (Fig. 2A). For 9 donors (Donors 1-9) with both IgG and IgM, IgM became undetectable, while for 9 others (Donors 10-18), only B19 IgG was detected, presumably because B19 IgM had decreased to an undetectable level before the second visit. The remaining 2 donors (Donors 19 and 20) had B19 IgM– detectable until the last visit (at 729 and 743 days). Although the initial profile for B19 antibodies showed different patterns, once established, B19-specific IgG persisted in all 20 donors. Summaries for 3 representative cases corresponding to each of these patterns for IgM, IgG, and viral load are presented in Fig. 2B. Consistent with previous studies that suggest that B19 DNA may persist for a long period in immunocompetent individuals, we observed persistent B19 infection in healthy blood donors in the present longitudinal study. During the follow-up period, none of the 20 infected blood donors reported symptoms of B19 infection, although they retained high levels of B19 IgG and low viral load. Our data suggest that in healthy individuals, the B19 plasma viral load declines to below 10 IU per mL in approximately 1 year and to 10 IU per mL in approximately 2 years after an acute (high viremia) infection. The patterns of plasma B19 viral load observed in our study may be useful for identifying more suitable blood donors in circumstances where B19 NAT is unavailable. We encourage further studies with a larger sample size to validate these preliminary findings. Harumichi Matsukura, BS Sachiko Shibata, MT Yoshihiko Tani, MD, PhD Hirotoshi Shibata, MD, PhD Rika A. Furuta, PhD e-mail: furuta@osaka.bc.jrc.or.jp Japanese Red Cross Osaka Blood Center Osaka, Japan B A

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.

Metagenomic profiling of the viromes of plasma collected from blood donors with elevated serum alanine aminotransferase levels

In Japanese Red Cross (JRC) blood centers, blood collected from donors with serum alanine aminotransferase (ALT) levels of more than 60 U/L are disqualified even if serologically negative for transfusion‐transmitted infections (TTIs). To assess potential risks of TTIs in plasma with elevated serum ALT levels in the current donor screening program of the JRC, we conducted a metagenomic analysis (MGA) of virome profiles in the plasma of blood donors with or without elevated serum ALT levels.

Defining the Jr(a–) phenotype in the Japanese population

The Jr(a–) phenotype is rare in European and North American populations but is not so rare in Japanese and other Asian populations. Recently, two groups have established the connection between the Jr(a–) phenotype and the ATP‐binding cassette, member G2 (ABCG2) gene and concluded that ABCG2‐null alleles encode the Jr(a–) phenotype. In Japanese Red Cross Blood Centers, the Jr(a–) phenotype is found with a prevalence of 0.05% among blood donors, and we applied DNA‐based genotyping to investigate the molecular basis of the Jr(a–) phenotype in Japan, in addition to serologic typing.

Reevaluation of confirmatory tests for human T‐cell leukemia virus Type 1 using a luciferase immunoprecipitation system in blood donors

Recently, Japanese Red Cross blood centers have changed the confirmatory test method from an indirect immunofluorescence (IF) technique to Western blotting (WB) for antibodies against human T‐cell leukemia virus Type 1 (HTLV‐1). In this study, these HTLV‐1 tests were assessed using another sensitive method, that is, a luciferase immunoprecipitation system (LIPS), to identify a better confirmatory test for HTLV‐1 infection.

A novel mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a blood donor with the AmB phenotype

The Am and Bm phenotypes are characterized by weak expression of the A or B antigens, respectively, by red blood cells with a normal expression by the saliva of secretors. Deletion of the regulatory element in the first intron of the ABO gene and disruption of the GATA motif in the element were found to be responsible. In this study, we identified a novel mutation within the GATA motif (G>C substitution at position c.28 + 5830) in the regulatory element of the A allele that might diminish transcription activity causing the generation of the AmB phenotype.

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.