Hiromi Ihara

Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay

BACKGROUND : Studies were conducted using samples from early and late‐stage HBV‐infected persons to determine the pool size at which PCR had better sensitivity than a sensitive HBsAg chemoluminescence immunoassay (CLIA‐HBsAg).

Virus Trap in Human Serum Albumin Nanotube

Infectious hepatitis B virus (HBV), namely Dane particles (DPs), consists of a core nucleocapsid including genome DNA covered with an envelope of hepatitis B surface antigen (HBsAg). We report the synthesis, structure, and HBV-trapping capability of multilayered protein nanotubes having an anti-HBsAg antibody (HBsAb) layer as an internal wall. The nanotubes were prepared using an alternating layer-by-layer assembly of human serum albumin (HSA) and oppositely charged poly-L-arginine (PLA) into a nanoporous polycarbonate (PC) membrane (pore size, 400 nm), followed by depositions of poly-L-glutamic acid (PLG) and HBsAb. Subsequent dissolution of the PC template yielded (PLA/HSA)(2)PLA/PLG/HBsAb nanotubes (AbNTs). The SEM measurements revealed the formation of uniform hollow cylinders with a 414 ± 16 nm outer diameter and 59 ± 4 nm wall thickness. In an aqueous medium, the swelled nanotubes captured noninfectious spherical small particles of HBsAg (SPs); the binding constant was 3.5 × 10(7) M(-1). Surprisingly, the amount of genome DNA in the HBV solution (HBsAg-positive plasma or DP-rich solution) decreased dramatically after incubation with the AbNTs (-3.9 log order), which implies that the infectious DPs were completely entrapped into the one-dimensional pore space of the AbNTs.

Efficiency of Donor Screening for Human Parvovirus B19 by the Receptor‐Mediated Hemagglutination Assay Method

Background and Objectives: Mass screening for parvovirus B19 (B19) by receptor–mediated hemagglutination assay (RHA) may be inadequate to eliminate the virus from plasma pools. We characterized B19 carriers detected in blood donor screening by RHA to explain why some carriers were not detected by RHA. Materials and Methods: Donor plasma was screened for B19 by RHA, B19 DNA by nested polymerase chain reaction (PCR) assay, and anti–B19 by enzyme immunoassay. Results: B19 DNA–positive specimens (n = 73) screened from 456,665 donors were divided into group A (n = 41) with high DNA and high RHA titers, group B (n = 16) with low DNA and low RHA titers, and group C (n = 16) RHA–negative. Most (37/41) of the group A samples were without anti–B19, while most (15/16) of the group B samples were positive for anti–B19. Group C specimens were screened by PCR from 3,042 random RHA–negative specimens. Analysis of samples from early infections revealed that the viremic period, corresponding to group A, lasted only 8–10 days after infection. The RHA reactivity fell rapidly with the appearance of anti–B19 and disappeared 28–30 days after infection, thus corresponding to group B. The RHA reactions of group B specimens were often unstable, probably because of the formation of immune complexes. The B19 DNA–positive, RHA–negative state lasted for several months, which corresponded to group C. Conclusion: Only group A specimens are reliably eliminated in donor screening by RHA. Therefore, although donors with high B19 DNA could be screened out by testing for B19 by the RHA, most B19 carriers, with low B19 DNA and RHA–negative, will not be eliminated.

Nomenclature of Granulocyte Alloantigens

and the detection of new ones has made a new nomenclature for these antigens necessary. Neutrophil alloantibodies are known to cause neonatal alloimmune neutropenia, immune neutropenia after bone marrow transplantation, refractoriness to granulocyte transfusions, and severe transfusion-related acute lung injury. With respect to the increasing use of granulocyte transfusions this is of special importance, since alloimmunization to granulocyte antigens can decrease the recovery and half-life of transfused granulocytes, can prevent their migration to sites of infection and can cause severe pulmonary transfusion reactions. The Granulocyte Antigen Working Party of the ISBT agreed at their meeting in S’Agaró, Spain, in 1998, to establish a new nomenclature system for well-defined neutrophil alloantigens, which is based on the following rules: (1) Neutrophil antigens are to be called HNA for ‘human neutrophil antigen(s)’. (2) MemISBT Working Party

Interaction between desialylated hepatitis B virus and asialoglycoprotein receptor on hepatocytes may be indispensable for viral binding and entry.

Summary.  The cellular receptor for hepatitis B virus (HBV) infection has not yet been identified. The purpose of this study was to address the possibility of participation by desialylated HBV and the asialoglycoprotein receptor (ASGP‐R) exclusively expressed on liver parenchymal cells, in infection. Assays for viral binding and entry were performed by culturing a hepatoblastoma cell line, HepG2, and HBV particles derived from the HBV carrier in the presence or absence of neuraminidase (NA). Viral binding and entry were clearly enhanced in the presence of NA, and the enhancement of the binding could be blocked by asialo‐fetuin and ethylenediamine‐tetraacetic acid (EDTA). In addition, covalently closed circular (CCC)‐DNA, as a marker of infectivity, was detected in the presence of NA, but not in its absence. The optimal concentration of NA raised infectivity more than 1000 times. We concluded that this method makes it feasible to evaluate the infectivity of HBV in vitro and that ASGP‐R may be a specific HBV receptor once viral particles are desialylated.

Impact of chemiluminescent enzyme immunoassay screening for human parvovirus B19 antigen in Japanese blood donors

To reduce the risk of human parvovirus B19 (B19V) transmission through contaminated blood for transfusion and plasma‐derived products, the Japanese Red Cross (JRC) Blood Centers introduced B19V antigen screening by chemiluminescent enzyme immunoassay (CLEIA‐B19V) in 2008.

Efficacy of HBV NAT of pooled donor samples

We scientists, researchers, and practitioners in transfusion medicine frequently lament that we are not given respect and recognition by other scientific and medical disciplines. One explanation is that publications ascribed to us are easily criticized as not very scientific. Perhaps responsibility for quality should be shared by authors, reviewers, and editors involved in the publication of these reports. A case-in-point is the multicenter transfusion trial involving children with sickle cell anemia published recently in TRANSFUSION.1 This article is only one of several multicenter trials published over the past few years describing investigated and recommended transfusion practices for sickle cell anemia patients in various clinical situations (e.g., preoperative setting). These reports are characterized by large numbers of patients who are enrolled in transfusion study arms with either no concurrent control subjects assigned by randomization, no concurrent controls at all, or a partially controlled study design with some, but not all, centers enrolling control subjects if and as they wish, all of which lead to possible bias by “picking and choosing” control subjects for comparison with study subjects. These reports also give firm conclusions and recommendations for practices and guidelines that are to be applied nationally, despite acknowledging, somewhat after the fact, that their reported results are not definitive and require confirmation by future randomized clinical trials. The latter factor, of course, greatly hampers future clinical trials because data reported from flawed studies create clinical impressions and “standards of practice” that discourage participation by unbiased physicians and/or institutions. In a recent trial,1 the lack of concurrent controls assigned by randomization (i.e., patients who were given RBCs that were not antigen matched vs. patients in the study group who were given antigen-matched RBCs) is particularly distressing because a randomized study design easily could have been justified scientifically and ethically. In addition, the chosen experimental design was flawed because at least three experimental variables — compared by the authors to historical controls from other publications — were being tested at the same time rather than only one. These three variables were: 1) the transfusion of RBC units matched to avoid exposure and possible alloimmunization to key antigens (E, C, K); 2) exclusive transfusion of WBC-reduced RBC units; and 3) aspects of overall care that likely differed from historical controls (i.e., the papers cited, from which control data were obtained for comparison of alloimmunization rates with those in the current study, were published between 1978 and 1994). The reported endpoint of diminished alloimmunization to RBC antigens1 most likely is correctly explained by the transfusion of antigen-matched RBCs. However, the fact that 80 percent of the patients who developed significant RBC alloantibodies exhibited antibody specificity against the antigens intended to be avoided by matching seems inconsistent with the authors’ conclusion that “This study proves that an extended RBC-matching policy is both effective. . . .”1(p.1089) Moreover, the possible contribution of the transfusion of WBC-reduced RBC units to lowering the alloimmunization rates must be considered. Theoretically, WBC reduction should lower alloimmunization only to HLA class I antigens, not to RBC antigens. However, because theoretical factors that seem so logical sometimes are incorrect when actually tested scientifically, it is unfortunate that the historical control patients were not transfused with WBCreduced RBC units. Similarly, the lower rates of transfusion reactions reported by Vichinsky et al.1 might have been influenced by transfusing WBC-reduced RBC units. All of these confounding factors would have been addressed by random assignment of patients as either concurrent controls who received WBC-reduced RBC units not matched for C, E, and K or as study subjects given WBC-reduced and antigen-matched units. The authors state1 that many blood banks currently do not prophylactically select RBC units by antigen matching (i.e., there is no standard of practice prohibiting participation in a randomized trial) and that there is need for a randomized trial. Based on the authors’ statements, why was a properly designed study not done? As stated by the authors, the majority of patients with sickle cell anemia receive RBC transfusions, and the optimal selection of RBC units to be transfused is extremely important for many reasons (e.g., availability, cost, need for “minority” donor recruitment programs). Thus, properly designed and conducted clinical trials are mandatory to provide sound information for optimal transfusion recommendations. Patients with sickle cell anemia and their families deserve transfusion practices based on solid data. Hopefully, definitive clinical trials will be facilitated and conducted through funding of the Transfusion Medicine/Hemostasis Clinical Research Network by the National Heart, Lung, and Blood Institute. Ronald G. Strauss, MD University of Iowa DeGowin Blood Center Department of Pathology and Pediatrics University of Iowa College of Medicine 200 Hawkins Drive, C250 GH Iowa City, IA 55242 e-mail: Ronald.strauss@uiowa.edu

Evaluation of HBs Antigen Screening for Blood Donors Using a Fully Automated Chemiluminescent Immunoassay.

Hepatitis B-type virus (HBV) screening at Japanese Red Cross Blood Centers is carried out by agglutination method. The risk of post-transfusion hepatitis B (PTHB) has markedly decreased, especially since the hemagglutination inhibition test (HI) for anti-HBc and the passive hemagglutination test (PHA) for anti-HBs were introduced to screening in addition to the reversed passive hemagglution test (RPHA) for HBsAg in 1989. However, PTHB had not been completely eliminated even using the current system (RPHA, HI & PHA) which is rather complicated and time-consuming. We therefore evaluated the PRISM-HBsAg assay (Abbott), a fully automated assay based on chemiluminescence technology, in comparison with EIA-HBsAg (Auszyme overnight protocol, Dainabot) and our present HBV system.Sensitivity of PRISM-HBsAg (0.08ng/ml in ad panel and 0.06ng/ml in ay panel) was higher than that of EIA-HBsAg (0.23ng/ml in ad panel and 0.11ng/ml in ay panel). In 87 HBV-DNA-positive panels that were RPHA-negative (or weakly positive) and HI-positive, 59 and 50 were positive with PRISM-HBsAg and EIA-HBsAg, respectively.A total of 20, 041 donor specimens were tested simultaneously by PRISM-HBsAg, EIA-HBsAg and RPHA. The number of disqualified samples was 98, 91, 71 and their determination ratio (disqualified samples/initially positive samples×100) was 96.1%, 20.8% and 19.7%, respectively. These results show that the determination ratio of PRISM-HBsAg assay is clearly higher than that of EIA-HBsAg and RPHA. There were 19 RPHA-negative and PRISM-HBsAg-positive samples, 17 of which were HI-positive and disqualified according to the criteria of our present HBV system. Of 313 samples disqualified because of their high HI titer despite their negative PRISM-HBsAg and EIA-HBsAg, 10 were PCR-positive with DNA preparation concentrated from 10ml samples. These results indicate that the sensitivity and specificity of RPISM-HBsAg assay are significantly higher than those of EIA-HBsAg and RPHA. However, anti-HBc screening is still necessary, because some HBV-DNA-positive samples with high anti-HBc activity are PRISM-HBsAg-negative.

Hepatitis B virus DNA (HBV-DNA) in anti-HBc positive blood donors.

Hepatitis B virus DNA (HBV-DNA) in 391 anti-HBc positive blood samples were tested by nested polymerase chain reaction. DNA was prepared from pellets of plasma samples after ultra-centri fugation.Of 135 HBsAg weakly positive samples, 127 (93.3%) were HBV-DNA positive. Anti-HBc was the only HB maker for 156 samples, 57 of which (36.50) were HBV-DNA positive, whereas only 6 (3.8%) were positive if the samples were not concentrated. Of 100 anti-HBs positive donors, 6 were HBV-DNA positive. The positive rate of HBV-DNA tended to be high in the samples with high titre of anti-HBc, especially when the anti-HBc was the only HB marker or when HBsAg was positive only by EIA.Thus, it is reasonable to exclude blood units with high titre of anti-HBc even if HBsAg is undetectable in order to prevent post-transfusion hepatitis B.