Trends in the total numbers of HBV and HCV carriers in Japan from 2000 to 2011.

We estimated the total number of undiagnosed HBV and HCV carriers and patients with hepatitis virus-related disease in Japan according to 6 different groups classified by their natural histories during 2011. In 2011, the total number of carriers and patients infected with HBV or HCV was estimated according to 6 groups using government reports and reports from the hepatitis epidemiology research group of The Ministry of Health, Labor and Welfare in Japan. In 2011, the total number of hepatitis virus carriers was estimated to be 2 090 128-2 840 128 in which the estimated number of undiagnosed HCV and HBV carriers was 776 826 (HBV: 481 470; HCV: 295 356). The total number of treated patients, as either inpatients or outpatients, was estimated to be 811588 (HBV: 303 366; HCV: 520 600) in 2011. It is presumed that many carriers shirk consultation for many reasons, such as patients' misunderstanding, lack of awareness and forgetfulness of their positive status. The numbers of infected patients who did not seek treatment increased gradually to 501 714-1 251 714 (HBV: 333 791-483 791; HCV: 167 923-767 923) in 2011. Compared to 2000, the number of undiagnosed carriers was significantly reduced in 2011 probably because of the well-organized, effective national hepatitis virus screening system that has been launched by the Japanese government since 2002. Moreover, the increase in the number of untreated persons who are aware of their positive status shows that more effort should be invested in improving the referral system from screening centres to core hospitals.

Investigation of antibody to severe fever with thrombocytopenia syndrome virus (SFTSV) in blood samples donated in a SFTS-endemic area in Japan.

The risk of transfusion-transmitted infection (TTI) for severe fever with thrombocytopenia syndrome virus (SFTSV) is a concern because person-to-person transmission resulting from contact with SFTSV-contaminated blood has been reported. To obtain information regarding the risk of TTI-SFTSV, antibody testing was performed for blood samples donated in an severe fever with thrombocytopenia syndrome-endemic area in Japan. No antibody-positive samples were detected among 3990 samples. This finding suggested that there were few cases of SFTSV infection among donors and that the risk of TTI-SFTSV was also estimated low in Japan.

Silent KEL alleles identified from Japanese individuals with the Ko phenotype.

BACKGROUND AND OBJECTIVE: The rare Ko phenotype lacks all 36 antigens in the Kell blood system. The molecular basis of the Ko phenotype has been investigated, and more than 40 silent KEL alleles are reported by many investigators. The majority of silent alleles are the KEL*02 background. Here, we report molecular genetic analysis of the KEL gene in Japanese individuals with the Ko phenotype. MATERIALS AND METHODS: The Ko phenotype was screened from Japanese blood donors for several years using monoclonal anti-Ku or anti-K14 by an automated blood grouping system PK7300. Kell-related antigens were typed by standard tube tests. Genomic DNA was extracted from the blood samples, and KEL gene was analysed by polymerase chain reaction (PCR) and Sanger sequencing. RESULTS: We collected 35 Ko blood samples with K-k-, Kp(a-b-), Js(a-b-) and K14-. PCR and sequence analysis revealed that 11 individuals were homozygous for a mutant KEL allele with a c.299G>C (p.Cys100Ser) mutation (rs. 200268316). Three individuals were homozygous for the KEL*02N.24 allele that is c.715G>T (p.Glu239*), and one individual was homozygous for the KEL*02N.40 allele that is c.1474C>T (p.Arg492*). Five individuals were homozygous for novel KEL alleles with single-nucleotide mutations, four individuals had a c.2175delC (p.Pro725 fs*43), and one individual had a c.328delA (p.Arg110 fs*79). The remaining 15 individuals were compound heterozygous, and eight new alleles were identified from them. CONCLUSIONS: We identified three known and ten new silent KEL alleles from Japanese individuals with the Ko phenotype. The KEL allele with the c.299G>C (p.Cys100Ser) mutation was the most frequent.

The B allele with a 5·8 kb deletion in intron 1 of the ABO gene is the major allele in Japanese individuals with Bm and A1 Bm phenotypes.

Bm and A1 Bm phenotypes are the most frequent ABO variants in the Japanese population. The B antigen on Bm red blood cells is only detectable by adsorption and elution tests, and plasma B-transferase activity is usually detected at half or less levels compared with that of common B. Recently, a B allele lacking an erythroid cell-specific transcription enhancer in intron 1 of the ABO gene was identified from individuals with Bm and A1 Bm phenotypes, which could explain the unique serologic properties of Bm . In the Japanese Red Cross Society, eight Blood Centers tested blood samples from donors throughout Japan and collected blood samples from 888 Bm and 415 A1 Bm individuals. DNA analysis revealed that 1300 of 1303 (99·77%) individuals had the B allele with a 5·8 kb deletion (c.28 + 5110_10889del), which included the enhancer element.

Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains.

BACKGROUND AND OBJECTIVES: Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the 'repository'), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. MATERIALS AND METHODS: Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10-25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. RESULTS: Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. CONCLUSION: The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

Prevalence of RHD alleles in Japanese individuals with weak D phenotype: Identification of 20 new RHD alleles.

We identified 46 different RHD alleles from 226 Japanese individuals with weak D phenotype, 26 of which had been previously described and 20 that were novel. Among these weak D individuals, the alleles with c.960G>A, c.845G>A (RHD*15) or c.1013T>C (RHD*01W.24) mutations were most prevalent with relative occurrences of 36·7%, 15·9% and 9·7%, respectively. These findings demonstrate that the prevalence of common weak D alleles in the Japanese population significantly differs from that of Caucasian populations.

Weak D alleles in Japanese: a c.960G>A silent mutation in exon 7 of the RHD gene that affects D expression.

BACKGROUND AND OBJECTIVES: The molecular basis of the weak D phenotype has been investigated for many years, and more than 80 different alleles producing weak D phenotypes have been identified. Most alleles producing weak D phenotypes have a single missense mutation in exons corresponding to a transmembrane domain of the RhD polypeptide. We report here RHD alleles with single nucleotide mutations in Japanese accounting for weak expression of D antigen. METHODS: Seventy-five blood samples with a weak D phenotype were detected from 763 408 blood donors by standard serological methods. Forty-five of the 75 blood donors were available for RHD gene analysis by PCR and sequencing using genomic DNA and reticulocyte mRNA. Real-time PCR was performed to estimate the relative amounts of the RHD transcripts. RESULTS: We detected 16 different RHD alleles in the 45 individuals with weak D by nucleotide sequencing; 12 were newly identified. Thirty-two of the 45 individuals had an RHD allele with a single missense mutation, while the other 13 individuals had RHD with a c.960G>A silent mutation in exon 7. Red blood cells of these 13 individuals showed direct agglutination with anti-D at a strength of 3+ or less. Semi-quantitative analysis of the RHD transcripts by real-time PCR revealed that the cDNA samples with the c.960G>A mutation showed a significant increment of exon 7 skipping compared with the common RHD. CONCLUSION: Reduced expression of D antigen is caused not only by missense mutation of the RHD gene, but also by silent mutation that may affect splicing.

Estimation of the infectious viral load required for transfusion-transmitted human T-lymphotropic virus type 1 infection (TT-HTLV-1) and of the effectiveness of leukocyte reduction in preventing TT-HTLV-1.

BACKGROUND AND OBJECTIVES: The risk of transfusion-transmitted human T-lymphotropic virus type 1 infection (TT-HTLV-1) after prestorage leucocyte reduction (LR) remains unknown, as the proviral load in the blood component that would cause TT-HTLV-1 is undetermined. On the basis of the distribution of HTLV-1 proviral load among HTLV-1-sero-positive blood donors, we attempted to estimate the proviral load for transfusion-related infectivity. We also discuss the effectiveness of LR in preventing TT-HTLV-1. MATERIALS AND METHODS: The HTLV-1 proviral load in 300 HTLV-1-sero-positive blood donors was determined by real-time polymerase chain reaction analysis. The proviral load required for transfusion-related infectivity was estimated using historical TT-HTLV-1 frequency data from a retrospective study on patients who had received blood from HTLV-1-sero-positive blood donors and the distribution pattern of HTLV-1 proviral load among blood donors. RESULTS: HTLV-1 proviral loads ranged between < 0.01 and 25.0 copies per 100 leucocytes. Historical data showed TT-HTLV-1 frequency to be 80%. Assuming that 80% of the 300 sero-positive samples are infectious, it is estimated that the transfer of ≥ 9 × 10(4) cells containing the HTLV-1 provirus is required to establish TT-HTLV-1. CONCLUSION: The residual number of HTLV-1-infected cells after LR is substantially lower than the viral load necessary for TT-HTLV-1. LR therefore appears to be effective in minimizing the incidence of TT-HTLV-1.

Molecular basis for D- Japanese: identification of novel DEL and D- alleles.

BACKGROUND AND OBJECTIVES: The occurrence of D- is approximately 0.5% in Japanese, but DEL in apparently D- individuals is relatively common compared with that in Caucasian populations. On the basis of molecular genetics, we examined D- Japanese blood donors. METHODS: A standard serological technique was used for RhD typing, and we selected 3526 D- blood samples. Genomic DNA obtained from whole blood was used for RHD analysis by polymerase chain reaction (PCR) and sequencing. Multiplex PCR to detect all of the RHD exons and use of PCR-sequence-specific primer (PCR-SSP) to detect RHD deletion (RHD*01N.01) and c.1227G>A mutation (for RHD*01EL.01) were performed. RESULTS: Multiplex PCR and PCR-SSP revealed that 3091 of 3526 D- individuals (87.7%) were homozygous for RHD*01N.01, and 318 individuals (9.0%) had the RHD*01EL.01/RHD*01N.01 or RHD*01EL.01/RHD*01EL.01 genotype. The other 103 in the 3526 individuals (2.9%) had the known D-CE-D hybrid allele, RHD*01N.04, and the association of RHCE*Ce with RHD*01EL.01 as well as RHD*01N.04 was observed. The remaining 14 individuals had RHD*01N.01 hemizygous with one of the following alleles: RHD*01N.06 (3), RHD*01N.07 (1), RHD*04N.01 (1), RHD*DEL8 (1), RHD with c.761C>G (p.Ser254Ter) (2), RHD with c.1252T>A (p.Ter418Lysex26) (2) and apparently common RHD (4). Adsorption and elution tests with anti-D revealed that the individuals with c.761C>G mutation were D- while the individuals with c.1252T>A mutation were DEL. CONCLUSIONS: The RHD genotype of more than 96% of D- Japanese could be determined by conventional PCR-SSP. In addition, we identified a novel DEL allele having c.1252T>A mutation and a novel RHD silencing allele having c.761C>G nonsense mutation.

Efficacy of the Mirasol pathogen reduction technology system against severe fever with thrombocytopenia syndrome virus (SFTSV).

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tickborne virus in the Bunyaviridae family. This virus has recently been found in China, Japan and Korea. The risk of transfusion-transmitted SFTSV infection (TTI-SFTSV) is a concern because person-to-person transmission resulting from contact with SFTSV-contaminated blood has been reported. Therefore, we investigated the efficacy of the Mirasol pathogen reduction technology (PRT) system for inactivating SFTSV in vitro. The Mirasol PRT system achieved a > 4.11 log10 reduction value (LRV) for SFTSV. In conclusion, we showed that the Mirasol PRT system could potentially be used to reduce the risk of TTI-SFTSV.

A novel DO null allele with a c.268C>T (p.Gln90Stop) mutation in Japanese.

The Dombrock blood group system consists of two antithetical antigens, Do(a) (DO1) and Do(b) (DO2), and seven high-prevalence antigens, Gy(a) (DO3), Hy (DO4), Jo(a) (DO5), DOYA (DO6), DOMR (DO7), DOLG (DO8) and DOLC (DO9). Do(a) /Do(b) polymorphism is associated with c.793A>G (p.Asn265Asp) in exon 2 of the DO (ART4) gene, and the corresponding alleles are named DO*01 and DO*02. The rare Donull or Gy(a-) phenotype lacks all Dombrock antigens, and the DO null alleles vary with both DO*01 and DO*02 backgrounds. We report a novel DO null allele, which has a c.268C>T (p.Gln90Stop) nonsense mutation with a DO*02 background identified from four unrelated Gy(a-) Japanese individuals.

Anticancer effects of gemcitabine are enhanced by co-administered iRGD peptide in murine pancreatic cancer models that overexpressed neuropilin-1.

BACKGROUND: Impaired drug transport is an important factor that reduces the efficacy of anticancer agents against pancreatic cancer. Here, we report a novel combination chemotherapy using gemcitabine (GEM) and internalised-RGD (iRGD) peptide, which enhances tumour-specific drug penetration by binding neuropilin-1 (NRP1) receptor. METHODS: A total of five pancreatic cancer murine models (two cell line-based xenografts (CXs) and three tumour grafts (TGs)) were treated with either GEM (100 mg kg(-1), q3d × 4) alone or GEM plus iRGD peptide (8 µmol kg(-1)). Evaluation of NRP1 expression in xenografts and 48 clinical cancer specimens was performed by immunohistochemistry (IHC). RESULTS: We identified a subset of pancreatic cancer models that showed NRP1 overexpression sensitive to iRGD co-administration. Treatment with GEM plus iRGD peptide resulted in a significant tumour reduction compared with GEM monotherapy in CXs, but not remarkable in TGs. Potential targets of iRGD were characterised as cases showing NRP1 overexpression (IHC-2+/3+), and these accounted for 45.8% of the clinical specimens. CONCLUSIONS: Internalised RGD peptide enhances the effects of co-administered drugs in pancreatic cancer models, its efficacy is however only appreciable in those employing cell lines. Therefore, the clinical application needs to be given careful consideration.

Right portal vein embolization with absolute ethanol in major hepatic resection for hepatobiliary malignancy.

BACKGROUND: This study aimed to evaluate the safety and efficacy of preoperative right portal vein embolization (PVE) with absolute ethanol in patients with hepatobiliary malignancies. METHODS: PVE was performed via a percutaneous transhepatic ipsilateral approach, and the right portal branch was embolized with absolute ethanol. Technical success and complications following PVE, and changes in liver enzyme levels were evaluated. Changes in future liver remnant (FLR) and FLR/total functional liver volume ratio were calculated. Complications following hepatic resection were assessed. RESULTS: A total of 83 patients with hepatobiliary malignancies (53 men, 30 women; mean age 68 years) underwent right PVE. Tumour types were hilar cholangiocarcinoma (37), liver metastases (14), gallbladder cancer (13), intrahepatic cholangiocellular carcinoma (10) and hepatocellular carcinoma (HCC) (9). PVE was performed successfully in all patients. Four patients (5 per cent) developed complications following PVE (liver abscess 2, left portal vein thrombosis 1, pseudoaneurysm 1), but this did not preclude hepatic resection. Liver enzyme levels rose transiently after PVE. The mean FLR and FLR/total functional liver volume increased after PVE (from 366 to 513 cm(3) and from 31 to 43 per cent respectively; both P < 0·001). Changes in the FLR and FLR/total functional liver volume ratio were comparable between patients with HCC and those with other malignancies (42 and 44 per cent, and 12 and 12 per cent, respectively). Sixty-nine of 83 patients underwent hepatic resection at a median of 25 days after PVE, with no postoperative mortality. CONCLUSION: Preoperative right PVE with absolute ethanol is safe and effective for induction of selective hepatic hypertrophy in patients with hepatobiliary malignancy.

JK null alleles identified from Japanese individuals with Jk(a−b−) phenotype.

The Kidd blood group system consists of three common phenotypes: Jk(a+b−), Jk(a−b+) and Jk(a+b+), and one rare phenotype, Jk(a−b−). Jka/Jkb polymorphism is associated with c.838G>A (p.Asp280Asn) in exon 9 of the JK (SLC14A1) gene, and the corresponding alleles are named JK*01 and JK*02. The rare phenotype Jk(a−b−) was first found in a Filipina of Spanish and Chinese ancestry, and to date, several JK null alleles responsible for the Jk(a−b−) phenotype have been reported. We report seven novel JK null alleles, 4 with a JK*01 background and 3 with a JK*02 background, identified from Jk(a−b−) Japanese.

Two novel null HLA-A alleles with identical exon 4 nonsense mutations: HLA-A*24:183N and A*02:356N.

HLA-A*24:183N, A*02:356N, and A*02:15N share the same stop codon at the same position in exon 4.

Parallel comparison of apheresis-collected platelet concentrates stored in four different additive solutions.

BACKGROUND AND OBJECTIVES: Partially replacing plasma with additive solutions in platelet (PLT) concentrates (PCs) may help to reduce transfusion reactions. Constituents of PLT additive solutions (PASs) have been revealed to affect the quality of PCs. Previous studies involved pairwise comparison of identical PLTs with two different PASs or multicomparison using random PLTs with three or more PASs. In this study, we performed parallel comparison using PCs from identical donors with four PASs. In addition to traditional parameters, the release of bioactive substances and plasma proteins was assessed. MATERIALS AND METHODS: Platelets collected four times by apheresis from three donors were suspended in Intersol, SSP+, Composol or M-sol with 35% autologous plasma. The PC parameters, including PLT activation markers, glucose consumption, chemokines and plasma proteins, were assessed during 5-day storage. RESULTS: Mean PLT volumes were decreased in SSP+, Composol and M-sol after 5-day storage, with significant differences, whereas the hypertonic shock response (HSR) was decreased only in Intersol. Glucose consumption was faster in Intersol and M-sol than in SSP+ or Composol. PLT activation, determined as CD62P, sCD62P, sCD40L and RANTES, was significantly higher in Intersol than the other three PASs. No marked change was observed in fibrinopeptide A and C3a in any PASs. CONCLUSIONS: M-sol, SSP+ and Composol effectively preserved the quality of PCs. PLT activation was significantly enhanced in Intersol compared with the other three PASs. These effects seem to depend on magnesium and potassium as a constituent. Parallel comparison further verified that the PC quality largely depended on PASs but not donors.

Analysis of 66 patients definitive with transfusion-associated graft-versus-host disease and the effect of universal irradiation of blood.

BACKGROUND: Transfusion-associated graft-versus-host disease (TA-GVHD) is a potentially fatal adverse reaction to blood transfusion. Although TA-GVHD was formerly considered to be rare and to occur only in immunocompromised patients, it was confirmed to occur even in immunocompetent patients in Japan, based on a definitive diagnostic test for TA-GVHD using highly polymorphic microsatellite repeat sequences. We clarify the clinical picture of TA-GVHD via definitive diagnosed cases and argue the validity of blood irradiation for TA-GVHD prevention. PATIENTS AND METHODS: Two-hundred and ninety patients who were suspected of having TA-GVHD and referred to us for diagnostic testing from October 1992 to August 1999 were analysed for the associated clinical characteristics and risk factors. Effects of universal irradiation were followed up until 2010. RESULTS: Sixty-six of the 290 study patients were diagnosed as having definite TA-GVHD by microsatellite DNA analysis. Regarding the symptoms of patients with definite TA-GVHD, a fever of over 38 °C, erythema and leucocytopenia were found in virtually all of these patients. Among patients in whom human leucocyte antigen (HLA) typing was carried out, TA-GVHD almost always developed in HLA heterozygous patients following the transfusion of HLA homozygous blood. TA-GVHD was reported significantly more frequently in older patients. In this study, TA-GVHD was caused by the transfusion of HLA one-way match blood stored for 14 days. CONCLUSION: No cases of TA-GVHD development have been confirmed since 2000, when the supply of irradiated blood products became widespread. No major problems have been encountered since the start of universal irradiation, more than 10 years ago.

Natural killer-mediated lysis of normal and malignant target cells, and its regulation by monocytes.

After depletion of monocytes, natural killer (NK) cells were partially purified from peripheral blood by Percoll density gradient sedimentation. The NK cells were then cultured for 1 d and assayed for their cytotoxicity against various types of normal and malignant target cells. All types of target cells tested were found to be susceptible to NK cells. The susceptible targets were autologous T and B lymphocytes, mitogen-induced T and B blasts, monocytes, large granular lymphocytes, autologous or allogeneic lymphoma and leukemia cells isolated from patients, and cultured cell lines, including those resistant to interferon-activated lymphocytes. Such a broad spectrum of cytotoxicity was demonstrated in 1 d of culture, and freshly prepared NK cells were not cytotoxic, or, if anything, were less cytotoxic. Monocytes and their supernatants, added throughout the course of culture, markedly inhibited the development of their cytotoxicity. These results may suggest that, although NK cells having ability to lyse autologous normal and malignant target cells are present in vivo, their lytic activity is regulated by coexisting monocytes.