Hirotoshi Shibata

Gene frequencies of human platelet antigens on glycoprotein IIIa in Japanese

Background: Polymorphism of glycoprotein IIIa on human platelets is one of the factors in alloimmunization that causes neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusion.

Prospective study on the risk of hepatocellular carcinoma among hepatitis C virus-positive blood donors focusing on demographic factors, alanine aminotransferase level at donation and interaction with hepatitis B virus

The risk for hepatocellular carcinoma (HCC) among asymptomatic hepatitis C virus (HCV) carriers is not well understood. A community‐based prospective study was conducted for over 8 years by record linkage to the Osaka Cancer Registry. The subjects were 1,927 individuals who were positive for anti‐HCV through screening for second‐generation HCV antibody (passive hemagglutination assay: ≥ 212) in voluntary blood donation. The risk factors for HCC and interaction between HCV and hepatitis B virus (HBV) infection were evaluated by including additional blood donors: 2,519 individuals positive for hepatitis B virus surface antigen (HBsAg) alone, 25 positive for both anti‐HCV and HBsAg, 150,379 negative for both anti‐HCV and HBsAg. The incidence of HCC (/105 person‐years) among the HCV‐positive individuals increased with age in both genders, ranging from 68 to 1,306 among those aged 45–74 years. In the HCV‐positive individuals, the cumulative risk of developing HCC between the ages of 40 and 74 year was 21.6% among males and 8.7% among females. A stepwise increase in risk was noted as the serum alanine aminotransferase level increased or serum cholesterol level at baseline decreased in multivariate Cox proportional hazard analysis. The 9‐year cumulative incidence of HCC among individuals positive for HCV alone, those positive for HBsAg alone and those positive for both was 3.0%, 2.0% and 12.0%, respectively. The age‐and‐sex‐adjusted rate ratio was 126, 102 and 572, respectively, when those negative for both were used as a reference. The results demonstrate an increased risk for HCC among asymptomatic HCV‐positive individuals in Japan. Coinfection with HBV and HCV carried a superadditive risk for HCC.

Genotyping of the ABO blood group system: analysis of nucleotide position 802 by PCR-RFLP and the distribution of ABO genotypes in a German population

Genotypes of the ABO blood group system were studied by PCR-RFLP analysis of the eight polymorphic nucleotide positions (ups) 261, 467, 526, 646, 703, 796, 802 and 803 of the cDNA from A transferase. In 169 unrelated German individuals, 17 genotypes were found and the calculated allele frequencies of A(Pro), A(Leu), B, O(T), O(A) and O2 were 0.2130, 0.0770, 0.0473, 0.4260, 0.2160 and 0.0207, respectively. These frequency data may provide useful additional information for disputed paternity and stain testing. A variant O allele, O2, was fout at a polymorphic frequency. As the nucleotide (np 261) of the O2 allele is the same as that of A and B alleles, the analysis of at least three nucleotide positions, i.e. ups 261, 526 and 802, is necessary to avoid mistyping of the ABO genotype.

Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers

Del (D‐elute) in the Rh blood group system is a variant with very weak D antigen and no agglutination is found by the indirect antiglobulin test. This variant is characterized by the presence of anti‐D eluate obtained after an adsorption‐elution test using anti‐D antibodies. We studied here the molecular genetic status of Del by using polymerase chain reaction with sequence‐specific primers (PCR‐SSP).

Simultaneous DNA Typing of Human Platelet Antigens 2, 3 and 4 by an Allele‐Specific PCR Method

We developed an allele‐specific polymerase chain reaction (ASPCR) method using originally designed primers to determine the genotype of the human platelet antigens (HPAs) 2, 3 and 4 in parallel. The results were compared with those obtained by PCR restriction fragment length polymorphism and the mixed passive hemagglutination test. Seventy‐three individuals were tested and the ASPCR results were in good agreement with those determined by the other two methods. This method enables the genotyping of HPA‐2, ‐3 and ‐4 in parallel without the use of platelets, platelet‐specific alloantibodies or restriction enzymes.

Genotyping of ABO blood groups by PCR and RFLP analysis of 5 nucleotide positions

The genotyping of ABO blood groups was performed using the polymerase chain reaction (PCR) method. The 4 DNA fragments containing the nucleotide position 261, 526, 703 and 796 of cDNA from A-transferase were amplified by PCR, and the amplified DNA subjected to restriction fragment length polymorphism (RFLP) analysis. The different nucleotide at position 803 was clearly distinguished by electrophoresis of the PCR products amplified with allele-specific primers. By analyzing the electrophoresis patterns, ABO genotyping was conclusively accomplished. The frequencies of ABO genotypes found in Japanese blood donors with A and B phenotypes were as follows: in the phenotype A group, AA =19.8 % and AO = 80.2%; and in the phenotype B group, BB =12.8% and BO=87.2%.ZusammenfassungDie Bestimmung der ABO-Blutgruppen wurde mittels Polymerase-Kettenreaktion (PCR) durchgeführt. Die vier DNA-Fragmente, die Nukleotidpositionen 261, 526, 703 und 796 von der cDNA der A-Transferase enthielten, wurden mittels PCR amplifiziert. Die amplifizierte DNA wurde einer Restriktionsfragmentl ängen-Analyse (RFLP) unterzogen. Nach Amplifikation mit allelspezifischen Primern konnte der Nukleotidunterschied an Position 803 durch Elektrophorese der PCR-Produkte klar getrennt werden. Die Bestimmung der ABO-Genotypen war durch die Analyse der eletrophoretischen Muster eindeutig durchführbar. Folgende Frequenzen der ABO-Genotypen von japanischen Blutspendern wurden für die Phänotypen A und B gefunden: In der Phänotypgruppe A, AA =19,8% und AO=80,2%, Phänotypgruppe B, BB=12,8% und BO= 87,2%.

Establishment of cell lines stably expressing HNA-1a, -1b, and -2a antigen with low background reactivity in flow cytometric analysis.

BACKGROUND: Antibodies to neutrophil antigens have been implicated in neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion‐related acute lung injury. Most often, neutrophil‐specific antibodies are directed toward human neutrophil antigen (HNA)‐1 (Fcγ receptor 3b) and HNA‐2a (CD177) in these disorders.

Distribution of abo genotypes and allele frequencies in a korean population

SummaryThe genotypes of the ABO blood group system were investigated in Korean living in Kangwon-Do area by PCR-RFLP analysis of the seven polymorphic nucleotide positions 261, 467, 526, 646, 703, 796 and 803 of the cDNA from A1 transferase. In 253 unrelated Korean individuals, 15 genotypes were found and the allele frequencies of A(Pro), A(Leu), B, O(T) and O(A) were 0.022, 0.209, 0.209, 0.360 and 0.200, respectively, with no deviation from Hardy-Weinberg expectations (Χ 2=2.145, d.f.=6, 0.90<p<0.95). As for the distribution of allele frequencies, a significant difference was noticed between the Korean and a Japanese (Χ 2=30.87, d.f.=4, p<0.001) and a German (Χ 2=127.76, d.f.=4, p<0.001) populations.

Double-blind test of human urinary macrophage colony-stimulating factor for allogeneic and syngeneic bone marrow transplantation: effectiveness of treatment and 2-year follow-up for relapse of leukaemia

Summary A randomized, double‐blind placebo‐controlled phase III clinical trial was performed to study the effects of human urinary macrophage colony‐stimulating factor (hM‐CSF) after allogeneic and syngeneic bone marrow transplantation (BMT) in 60 hM‐CSP treated and 59 placebo control patients. HM‐CSF was administered at a daily dose of 2 ± 105 units/kg from day 1 to day 14 after RMT. Significant differences between hM‐CSF and control patients were found in the recovery time to greater than 0. 5 ± 109 granulocytes/1 and the survival rate during the initial 120 d without retransplantation. There was no difference in the incidence or grade of graft‐versus‐host disease (GVHD). There was no difference in the rate of leukaemic relapse at 24–36 months after BMT in patients with acute lymphocytic. acute non‐lymphocytic, or monocytic leukaemia. The results of this trial show that human M‐CSF improves the outcome of BMT without any influence on the occurrence of leukaemic relapse or GVHD.

Genotype frequencies of the human platelet antigen, Ca/Tu, in Japanese, determined by a PCR-RFLP method

Recently, the polymorphism of a new human platelet antigen, Ca/Tu, was shown to be derived from a G‐A nucleotide substitution at base 1564 of GPIIIa cDNA, which leads to a single amino acid difference, Arg/Gln at amino acid 489 of GPIIIa. We developed a PCR‐RFLP method to determine the genotypes of Ca/Tu and their frequencies in a Japanese population. Fifteen Ca/Tua donors comprising 1 Ca/Tu(a/a) homozygous donor and 14 Ca/Tu(a/b) heterozygous donors were found among the 314 random donors analyzed. The frequencies of Ca/Tu genes were 0.025 (Ca/Tua) and 0.975 (Ca/Tub). The present study showed that the frequency of Ca/Tua individuals in the Japanese (15/314) was approximately 7‐fold higher than in the Finnish population (1/150) previously reported by Kekomäki et al. Therefore, attention must be given to the involvement of the Ca/Tu alloantigen in neonatal alloimmune thrombocytopenia and the refractoriness of platelet transfusion.