Taiko Seno

The RHD gene is highly detectable in RhD-negative Japanese donors.

Recent molecular studies on the Rh blood group system have shown that the Rh locus of each haploid RhD-positive chromosome is composed of two structural genes: RHD and RHCE, whereas the locus is made of a single gene (RHCE) on each haploid RhD-negative chromosome. We analyzed the presence or absence of the RHD gene in 130 Japanese RhD-negative donors using the PCR method. The RhD-negative phenotypes consisted of 34 ccEe, 27 ccee, 17 ccEE, 26 Ccee, 19 CcEe, 1 CcEE, and 6 CCee. Among them, 36 (27.7%) donors demonstrated the presence of the RHD gene. Others showed gross or partial deletions of the RHD gene. These results were confirmed by Southern blot analysis. Additionally, the RHD gene detected in the RhD-negative donors seemed to be intact through sequencing of the RhD polypeptide cDNA and the promoter region of RHD gene. The phenotypes of these donors with the RHD gene were CC or Cc, but not cc. It suggested that there is some relationship between the RHD gene and the RhC phenotypes in RhD-negative individuals. In Caucasian RhD-negative individuals, the RHD gene has not been found outside of the report of Hyland et al. (Hyland, C.A., L.C. Wolter, and A. Saul. 1994. Blood. 84:321-324). The discrepant data on the RHD gene in RhD-negative donors between Japanese and Caucasians appear to be derived from the difference of the frequency of RhD-negative and RhC-positive phenotypes. Careful attention is necessary for clinicians in applying RhD genotyping to clinical medicine.

Heterogeneity of the phenotype Jk(a-b-) found in Japanese.

Fourteen Jk(a‐b‐) persons were detected by testing 638,460 Osaka blood donors with an automated 2 M urea technique. Two of these 14 Jk(a‐b‐) samples were quite different from the hitherto reported Jk(a‐b‐) phenotype, and a family study showed that the mode of the inheritance was dominant. The red cell membranes of these Jk(a‐b‐) samples were studied by polyacrylamide gel electrophoresis and unusual protein bands (apparent mw, 67,000 d) were detected.

A Japanese family with two sisters apparently homozygous for Mk

Abstract. Two Japanese sisters with consanguineous parents have M‐N‐ En(a‐) Wr(a‐b‐) S‐s‐U‐ red cells and are therefore apparently homozygous for Mk; the third reported family with members of this genotype. The serum of the proposita (ORCMK) contained anti‐EnaTS, anti‐EnaFR and possibly anti‐Wrbwhereas the serum of her MkMksister contained no atypical antibodies. Total absence of sialoglycoproteins α and δ from red cell membranes of an Mkhomozygote was demonstrated by lactoperoxidase‐catalysed radioiodination of accessible tyrosine residues with subsequent SDS polyacrylamide gel electrophoresis and autoradiography, and by use of a monoclonal antibody directed at the cytoplasmic portion of α‐sialoglycoprotein.

SAT, a ‘new’ low frequency blood group antigen, which may be associated with two different MNS variants

Summary. A new private blood group antigen, SAT, was identified in an NFLD‐Japanese woman as a result of testing 10,480 blood donors with a serum containing anti‐NFLD and anti‐SAT. Three other sera were subsequently also shown to contain anti‐SAT. The donors family showed that SAT is inherited as a dominant character and may be associated with a weak M antigen. Serological and immunochemical analysis revealed no other aberrations in the MNS system.

Clinical Evaluation of Transfusion of Prestorage-Leukoreduced Apheresis Platelets

With increased use of platelet concentrate (PC) in recent years, adverse reactions to PC transfusion have received much clinical attention. Most of these reactions stem from white blood cells (WBC) contaminating the transfused PC. Several are thought to be preventable by removing WBC before PC storage. Methods: We routinely filtered apheresis PC either during collection or immediately afterwards and monitored various indicators of platelet quality during storage. After transfusion to patients, transfusion efficacy and the type, severity, and frequency of posttransfusion side effects were compared with those of a control group in which PC was filtered at the bedside. Results: Prestorage-filtered PC contained an average of 3.1±0.7 × 1011 platelets and 0.9±1.2 × 106 contaminating leukocytes. Measurement of platelet function and metabolic indicators revealed normal values throughout the storage period. CD62 measurement revealed no undue platelet activation after filtration or during the storage period. Cytokine, histamine, bradykinin, and complement levels showed no significant increase after filtration or during storage. Transfusion efficacy and overall side effect incidence rates were similar for the prestorage- and bedside-filtered groups, but reactions of the bedside-filtered group included serious reactions such as breathing difficulties and shock. No serious reactions were noted in the prestorage-filtered group. Conclusion: Filtering PC before storage has no adverse effect on PC quality and may reduce the severity of post transfusion side effects.

Detection of Rh23 in the partial D phenotype associated with the DVa category

The Rh blood group system, which is the most polymorphic of the blood group systems, is of major importance in transfusion medicine. The partial D phenotypes are classified into different categories according to the absence of one or more D epitopes. Most partial D phenotypes are generated from gene conversion events between the RHD and the RHCE genes. The low-frequency antigen, Rh23 (Dw), is characteristic of the DVa category.1 The DVa red cell (RBC) is also characterized as being negative when tested with monoclonal antibodies (MoAbs) to epitopes D1 and D5 in the 9epitope model.2 The molecular basis for the DVa category has been reported, in which either the entire RHD exon 5 (DVa Hus) or a portion of it (DVa Kou) was replaced by the RHCE equivalents.3 Recently, we reported the presence of a new RHD variant (DVa-like) that closely resembles the DVa category both structurally and serologically.4 In that study, we attempted to detect the expression of Rh23 on the DVa-like RBC membrane by using a polyclonal anti-Rh23 from a donor of blood group A. After the absorption test, the antibody reacted with a control DVa category RBC in an indirect antiglobin test, showing a higher titer, and was negative with normal RBCs. Rh23 was detected on the RBC membrane in three DVa and six DVa-like individuals. These results showed that the DVa-like sample was categorized as DVa category (Table 1). Various amino acid (aa) substitutions existed on the fourth loop of the Rh polypeptides in the DVa category. Detection of Rh23 on the RBC membrane is necessary for classification of the DVa category. The molecular structures of the mutated RHD genes from the DVa (Jpn) and the International Society of Blood Transfusion (ISBT) 49 categories were very similar.4,5 Both phenotypes exhibited four aa substitutions as compared to the aa sequence in the intact D polypeptide: Phe223Val, Glu233Gln, Val238Met, and Val245Leu. However, one aa difference (Ala226Pro) between these two phenotypes was found on the fourth external loop of the D polypeptide. It is known that Pro226 and Ala226 are the E/e polymorphism in the CE polypeptide. This result indicated that the hybrid RHD-CE-D genes of DVa ( Jpn) and ISBT 49 generated through a gene-conversion event were derived from the RHe and RHE genes, respectively. Avent et al.5 also described ISBT 49 as negative when tested with some epitope D8 MoAbs. We threorized that DVa (Jpn) was different from ISBT 49. A previous study3 proposed that the aa residing at position 233 was probably involved in the Rh23 epitope, because the difference in the extracellular region between the D polypeptides and those of the DVa category was only at aa 233 (Glu/Gln), while the other aa substitution at 223 (Phe/Val) was located in the intramembrane domain in all DVa RBCs. In our previous study,4 one aa substitution was detected at 233 on the fourth loop of the Rh polypeptide in S.M. and H. K. individuals. In this serologic study, the S.M. sample with the Glu233Gln substitution was serologically positive for Rh23, while the H.K. and M.I. samples with the Glu233Lys substitution were negative (Table 1). These results suggested that Rh23, which can be expressed on the RBC membrane, results from only one aa substitution (Glu233Gln) in the D polypeptide. We also described the reactivity of the partial D RBCs (in 9-epitope model) with MoAbs (Table 2). The DVa (Jpn) category was confirmed by the lack of epitopes D1 and D5 in this study. Then, the DVa (S.M.) RBCs were negative with MoAbs to epitope D1 and with one MoAb (P3X35 [Table 2]) to epitope D5. This result may indicate that the substitution Glu233Gln was associated not only with the expression of Rh23 but also with the loss of epitopes D1 and D5 in the DVa (S.M.) sample. We also showed that one substitution, Glu233Lys in new partial D phenotype, DHK, results in the lack of various RhD epitopes (Table 2). Negative reactions were observed with MoAbs to epitopes D1, D4, D5, D6/7 (a part), and D9 (a part). This information will allow further study of the epitopes of D.

Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh–green fluorescence protein fusions in K562 cells

Mutations detected in 161 weak D samples from Caucasians have been classified into 16 types. Because flow cytometry using monoclonal anti‐D antibodies (mAbs) has shown that weak D red cells display type‐specific antigen density, these mutations in transmembranous regions have been assigned weak D phenotypes. The present study attempts to confirm or refute this assignment.

Detection and quantitation of ABO RBC chimerism by a modified coil planet centrifuge method

BACKGROUND : Differential agglutination procedures and flow cytometric analysis have been used for detecting and quantitating mixed cell populations. For more than 20 years in our laboratory, a differential agglutination method using the coil planet centrifuge and polyclonal anti‐A or anti‐B has been used. However, it is now difficult to obtain polyclonal antisera, and it is unknown whether MoAbs can take the place of polyclonal antisera in the coil planet centrifuge method.

Isolation, characterization, and family study of DTI, a novel partial D phenotype affecting the fourth external loop of D polypeptides

BACKGROUND: The Rh system is the most polymorphic of the blood group systems and is of major importance in transfusion medicine. The partial D phenotypes lack one or more of the D epitopes. These variants appear to have arisen through hybrid RhD‐CE‐D or by spontaneous point mutations in RhD. The serologic findings and the molecular characterization of a novel partial D phenotype, termed DTI, are presented here.

A Japanese family with two Kp (a‐b‐c+) members, presumed genotype Kpc/Ko

While screening blood donors with anti‐Kpb, an unusual phenotype in the Kell blood group system was found. The proposita and her sister are Kp(a‐b‐c+). Unlike previously described individuals of this rare phenotype, the genotype of the two sisters is presumably Kpc/Ko: their parents are presumably KpbIKpc and Kpb/Ko