Establishment and characterization of immortalized erythroid progenitor cell lines derived from a common cell source.

Immortalized erythroid progenitor cell lines, which exhibit potential for enucleated red blood cell (RBC) production, are expected to serve as an in vitro source of RBCs. These erythroid progenitor cell lines have previously been established from a variety of sources; however, large numbers of cell lines have not been established, characterized, and compared from a common cell source. In the present study, 37 cell lines were established from human bone marrow cells from a single donor. The time required for the establishment of each cell line varied greatly from 46 to 246 days. Of these lines, five were selected and their characteristics were analyzed. The cell lines established at the earliest time point showed better results in terms of both karyotype and differentiation potential than those established the latest. Moreover, obvious differences were noted even when cell lines were established at the earliest time point from the same source. These results suggest that it is important to select the best cell lines from ones established at the earliest time point for generating cell lines with low genomic abnormality and high differentiation ability. We have successfully generated an adult type of cell line with 50% cells carrying a normal karyotype and with 25% enucleation efficiency. These findings could be valuable in the development of an optimal method for establishing cell lines.

Application of immortalized human erythroid progenitor cell line in serologic tests to detect red blood cell alloantibodies.

BACKGROUND: Antibody screening in pretransfusion tests is necessary to avoid critical complications of blood transfusion. Although red blood cells (RBCs) expressing relevant alloantigen(s) have been used for serologic antibody screening, little attention has been given to the use of cell lines, in which blood group antigen gene(s) are transduced, as reagent RBCs for antibody screening. STUDY DESIGN AND METHODS: The use of an erythroid progenitor cell line for serologic tests was studied. The expression of blood group antigens of erythroid progenitor cells was analyzed by genotyping and flow cytometry. Serologic analysis including hemagglutination was performed using erythroid progenitor cells to evaluate their sensitivity for antibody detection. Overexpression of exogenous erythroid antigen by lentiviral transduction was carried out and investigated for antibody detection sensitivity. RESULTS: Erythroid progenitor cells contained a substantial amount of hemoglobin and expressed sufficient levels of blood group antigens to detect corresponding monoclonal antibodies. Furthermore, the cell line could acquire an exogenous RBC antigen after lentiviral transduction and detected corresponding monoclonal and alloantibodies with equal sensitivity to antigen-positive RBCs. CONCLUSION: Application of erythroid progenitor cell lines for screening for unexpected antibodies could be helpful in solving issues such as reagent availability associated with the conventional RBC-based assay. The genetic expandability of erythroid progenitor cell lines by gene modification techniques could lead to the development of more convenient reagent RBCs.

The HIV care cascade: Japanese perspectives.

Japan has been known as a low HIV-prevalence country with a concentrated epidemic among high-risk groups. However, it has not been determined whether Japan meets the 90-90-90 goals set by the Joint United Nations Programme on HIV/AIDS (UNAIDS)/World Health Organization (WHO). Moreover, to date, the HIV care cascade has not been examined. We estimated the total number of diagnosed people living with HIV/AIDS (PLWHA) (n = 22,840) based on legal reports to the Ministry of Health, Labour and Welfare by subtracting the number of foreigners who left Japan (n = 2,273) and deaths (n = 2,321) from the cumulative diagnosis report (n = 27,434). The number of total undiagnosed PLWHA was estimated by age and sex specific HIV-positive rates observed among first-time blood donors between 2011-2015 in Japan. Our estimates show that 14.4% (n = 3,830) of all PLWHA (n = 26,670) were undiagnosed in Japan at the end of 2015. The number of patients retained in care (n = 20,615: 77.3% of PLWHA), the percentage of those on antiretroviral therapy (n = 18,921: 70.9% of PLWHA) and those with suppressed viral loads (<200 copies/mL; n = 18,756: 70.3% of PLWHA) were obtained through a questionnaire survey conducted in the AIDS Core Hospitals throughout the country. According to these estimates, Japan failed to achieve the first two of the three UNAIDS/WHO targets (22,840/26,670 = 85.6% of HIV-positive cases were diagnosed; 18,921/22,840 = 82.8% of those diagnosed were treated; 18,756/18,921 = 99.1% of those treated experienced viral suppression). Although the antiretroviral treatment uptake and success after retention in medical care appears to be excellent in Japan, there are unmet needs, mainly at the surveillance level before patients are retained in care. The promotion of HIV testing and treatment programs among the key affected populations (especially men who have sex with men) may contribute to further decreasing the HIV epidemic and achieving the UNAIDS/WHO targets in Japan.

Analysis of HTLV-1 proviral load (PVL) and antibody detected with various kinds of tests in Japanese blood donors to understand the relationship between PVL and antibody level and to gain insights toward better antibody testing.

Adult T-cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T-cell leukemia virus type 1 (HTLV-1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti-HTLV-1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real-time PCR and other tests in 600 HTLV-1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV-1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV-1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV-1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus-positive samples, probably suggesting that antibody-based screening tests should incorporate multiple HTLV-1 antigens, such as Gag and Env antigens.

Unique clinical courses of transfusion-transmitted hepatitis E in patients with immunosuppression.

BACKGROUND: The high prevalence of specific immunoglobulin G for hepatitis E virus (HEV) in Japanese people raises the possibility of a high incidence of HEV-viremic blood donors and therefore frequent transfusion-transmitted HEV (TT-HEV). STUDY DESIGN AND METHODS: TT-HEV cases established in Japan through hemovigilance and those published in the literature were collected. Infectivity of HEV-contaminated blood components and disease severity in relation to immunosuppression were investigated. RESULTS: Twenty established TT-HEV cases were recorded over the past 17 years. A lookback study verified that five of 10 patients transfused with known HEV-contaminated blood components acquired HEV infection. The minimal infectious dose of HEV through transfusion was 3.6 × 104 IU. Nine of the 19 TT-HEV cases analyzed had hematologic diseases. Only two cases showed the maximal alanine aminotransferase level of more than 1000 U/L. Two patients with hematologic malignancy and two liver transplant recipients had chronic liver injury of moderate severity. CONCLUSION: The infectivity of HEV-contaminated components was 50%. Immunosuppression likely causes the moderate illness of TT-HEV, but it may lead to the establishment of chronic sequelae. Transfusion recipients, a population that is variably immunosuppressed, are more vulnerable to chronic liver injury as a result of TT-HEV than the general population is as a result of food-borne infection.

Comparison between in vitro properties of washed platelet concentrates suspended in M-sol and those in BRS-A, both of which were prepared with an automated cell processor.

BACKGROUND: Washed platelet concentrate (WPC) is prepared manually in general, but automated preparation is desirable to minimize variation in the WPC quality and enhance WPC production. Recently, the software was improved for an automated cell processor (ACP) to control all processes of WPC preparation. M-sol and BRS-A, which are mixtures of medical solutions, are widely used for WPC preparation with a manual method in Japan. In this study, we prepared WPC suspended in M-sol (WPC-M) or BRS-A (WPC-B) with the ACP, and compared their in vitro properties during 7-day storage. STUDY DESIGN AND METHODS: PC was divided into two equal aliquots for WPC-M and WPC-B. A divided PC, medical solutions and disposable materials were set in the ACP, and it was started to prepare WPC-M or WPC-B on Day 0. Prepared WPC was stored on a flatbed shaker until Day 7. RESULTS: The pH of WPC-M and WPC-B was maintained above 6.8 during the 7-day storage. The differences in aggregation (%), HSR (%), P-selectin expression, GPIbα expression, and phosphatidylserine expression between WPC-M and WPC-B were minimal until Day 3. CONCLUSION: The in vitro properties of WPC-B are not markedly different from those of WPC-M until Day 3.

Pulsed xenon flash treatment inactivates bacteria in apheresis platelet concentrates while preserving in vitro quality and functionality.

BACKGROUND: Pulsed xenon (Xe) flash without any photoreactive compounds has been shown to inactivate a type of bacteria spiked into platelet (PLT) suspension in plasma, but enhanced the PLT storage lesion (PSL). Predicting reduction of PSL with increasing bactericidal ability, pulsed Xe flash was filtered through a band-stop filter, which excluded ultraviolet (UV)A, UVB, and visible light. STUDY DESIGN AND METHODS: Apheresis PLT concentrates (PCs) inoculated with bacteria were irradiated with filtered Xe flash (fXe treatment). For in vitro functional quality assessment, PLT aggregation and thrombin generation together with other assays that monitor the PSL were investigated. RESULTS: Staphylococcus aureus and Streptococcus dysgalactiae could be inactivated without regrowth during 6 days of storage. PC variables, such as PLT count, concentrations of soluble CD40 ligand, and ratio of aggregated PLTs, were not significantly different between fXe-treated and untreated PCs after 6 days of storage, while PAC-1 binding increased in the fXe-treated PLTs. Responsiveness of fXe-treated PLTs to ADP was maintained over a 6-day storage period as shown by the up regulation of P-selectin expression and induction of both integrin αIIbß3 conformational change and PLT aggregation. The fXe-treated PLTs showed a sustained aggregation curve in response to ADP, whereas untreated PLTs transiently aggregated and then subsequently dissociated. Thrombin-generating kinetics of fXe-treated PLTs via PLT membrane surface were equivalent to those of untreated PLTs. CONCLUSIONS: The fXe treatment inactivated bacteria in apheresis PCs in plasma without additional chemical compounds. The fXe-treated PCs retained acceptable in vitro properties of PC quality and PLT functionality.

Genetic Analysis of HIV-1 in Japan: a Comprehensive Analysis of Donated Blood.

In Japan, the number of human immunodeficiency virus (HIV)-1 infections remains relatively low; nevertheless, the annual incidence of HIV-1 infection has not decreased. New infections remain a great concern, and an improved understanding of epidemiological trends is critical for public health. The env C2V3 and pol sequences of HIV-1 RNA from 240 early (1996-2001) and 223 more recent (2010-2012) blood donations were used to compare the distribution of virus subtypes and to generate phylogenetic trees. Subtype B was clearly predominant in both early and more recent donations (both were 88.3%), and CRF01_AE was the second most common subtype. Phylogenetic analysis revealed a peculiar epidemiological transition. Compared to early subtype B isolates from 2 major endemic areas (Tokyo and Osaka), the more recent subtype B isolates formed fewer tight clusters in phylogenetic trees (from 8 to 2 clusters in Tokyo and 5 to zero clusters in Osaka). Furthermore, mixing of HIV-1 infections between these 2 endemic areas appear to increase. Analysis of phylogenetic trees suggested that local outbreaks have become smaller in Japan; however, intermixing of viral types between these 2 areas was more evident in the more recent samples.

Evaluation of the protective ability of plasma from Japanese individuals against mosquito-borne viral infections.

Background: Most of the Japanese population is seropositive for anti-Japanese encephalitis virus (JEV) antibodies because of previous JEV vaccination or natural infection. Because the virological characteristics of JEV are similar to those of West Nile virus (WNV) and dengue virus (DENV), we hypothesized that anti-JEV antibodies can cross-react with WNV and DENV antigens, leading to protection against infection by these viruses. Methods: Using isolated intravenous immunoglobulin (IVIG) from plasma collected in Japan, neutralizing activities against WNV and DENV and antibody-dependent enhancement (ADE) of these viral infections were evaluated using an in vitro assay to determine the potency of immunity against these viruses. Results: The prepared IVIG showed considerable neutralizing activity of 2.57 log10 reduction factor against WNV infection but showed little effect against DENV infection. A strong correlation was observed between the neutralizing activity of individual plasma samples against JEV and WNV (ρ=0.768). Moreover, IVIG showed no significant ADE of WNV infection. Conclusions: Based on these results, we presume that the Japanese population is generally protected from WNV infection. Furthermore, IVIG prepared from plasma donations from Japanese individuals is expected to be an effective therapeutic agent based on its neutralizing activity against JEV and WNV.

First report of the isolation of Lactococcus garvieae from a platelet concentrate in Japan.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) remains a serious problem in transfusion. We have been conducting sterility tests on all PCs rejected by blood centers or hospitals due to abnormal appearances. We recently experienced a case in which discrepant results were obtained between the methods used to identify a bacterial species isolated from a PC, requiring further analyses. STUDY DESIGN AND METHODS: Bacteria were isolated from a PC using the BacT/ALERT system and plate culture. The species was identified using biochemical tests and molecular analysis. Phylogenetic trees were constructed using sequences of the 16S ribosomal RNA (rRNA) and superoxide dismutase (sodA) genes from the bacterial isolate and related species. In addition, the isolate was cultured at temperatures of 10°C and below to determine its growth activity at low temperatures. RESULTS: Biochemical tests determined that the isolate was Streptococcus alactolyticus, whereas molecular analysis determined that it was Lactococcus garvieae. These two species belonged to different clusters on the phylogenetic tree. Similar to L. garvieae, the isolate could grow at 10°C. CONCLUSIONS: We conclude that the isolate was L. garvieae according to molecular identification and its growth characteristic at 10°C. Molecular analysis enabled the identification of this species, which was difficult to classify by biochemical tests. Blood facilities need to be prepared with multiple techniques, including genetic analysis techniques, for identifying contaminating bacterial species. L. garvieae can grow at 10°C and can contaminate both red blood cell concentrates and PCs; thus, this species should be listed as a cryophilic bacterium that could threaten blood safety.

Reduction of bacteria and human immunodeficiency virus Type 1 infectivity of platelet suspension in plasma using xenon flash-pulse light in a bench-scale trial.

BACKGROUND: Current pathogen reduction systems for platelet concentrates (PCs) require addition of chemical compounds and/or reduction of plasma content in PCs. We have investigated a new method using xenon (Xe) flash-pulse light without additional compounds or plasma replacement. STUDY DESIGN AND METHODS: An aliquot of apheresis platelets (PLTs) in plasma inoculated with bacteria or human immunodeficiency virus Type 1 (HIV-1) was irradiated with Xe flash-pulse light (Xe flash phototreatment). Bacterial growth was monitored up to 6 days of storage, whereas HIV-1 infectivity was assayed just after treatment. Pairs of Xe flash-phototreated and untreated PCs were examined for PLT lesion during the storage period. RESULTS: Under the current conditions, a low titer (1.8 colony-forming units [CFUs]/mL) of Staphylococcus aureus did not proliferate during the 6-day storage period, but grew in some cases at high-titer (24.0 CFUs/mL) inoculation. HIV-1 infectivity was reduced by 1.8 log. PLT recovery of the treated PCs was lower than untreated ones. An increase of mean PLT volume and glucose consumption, together with a decrease of hypotonic shock response and pH, were enhanced by the treatment. CD62P- and PAC-1-positive PLTs increased after the treatment, indicating the induction of PLT activation. Among biologic response modifiers, soluble CD40 ligand was significantly increased in the treated PCs on Day 6. CONCLUSIONS: Xe flash phototreatment could prevent bacterial proliferation and reduce HIV-1 infectivity in 100% plasma PCs without any additional compounds, but enhanced PLT storage lesions. Further improvement is required to increase the potency of pathogen inactivation with reducing PLT damage.

Sequence analysis of two variable cytomegalovirus genes for distinction between transfusion- and breast milk-transmitted infections in a very-low-birthweight infant.

BACKGROUND: Cytomegalovirus (CMV) infections in very-low-birthweight infants can lead to serious clinical consequences. When CMV-related symptoms occur after transfusion, CMV transmission is often attributed to the transfusion products rather than to breast milk. However, it is sometimes difficult to distinguish between transfusion-transmitted and breast milk-transmitted CMV infections. PATIENT AND METHODS: A patient was born at 27 gestational weeks with a weight of 689 g. He was transfused with leukoreduced red blood cells (LR-RBCs), which were later found to be CMV seropositive and CMV DNA positive. He was also fed with CMV DNA-positive breast milk. Thereafter, he developed CMV disease with thrombocytopenia and jaundice. To determine the route of transmission, we analyzed the sequences of two variable CMV genes, UL139 and UL146, by direct sequence analysis. We also performed deep sequence analysis to determine whether there were polyclonal CMV strains in the LR-RBCs transfused. RESULTS: CMV DNA sequence-matching rates for the LR-RBCs and the patient's blood were 64.6% for the UL139 gene and 68.6% for the UL146 gene. In contrast, the sequences of these genes in the patient's blood were 100% matched with those in the breast milk. Furthermore, by deep sequence analysis, the CMV strain found in the patient's blood was not detected in the LR-RBCs transfused. CONCLUSION: The results indicate that the pathogenic CMV strain was transmitted through breast milk, which is consistent with the claims that transfusion-transmitted CMV infection due to leukoreduced blood products is uncommon.

Real-time measurement of platelet shape change by light scattering under riboflavin and ultraviolet light treatment.

BACKGROUND: The adoption of pathogen reduction technologies (PRTs) is considered for the implementation of safer platelet (PLT) transfusion. However, the effects of PRT treatment including irradiation with ultraviolet (UV) light on PLT shape have not yet been fully clarified. STUDY DESIGN AND METHODS: Leukoreduced PLT concentrates (PCs) were treated with riboflavin and UV light (Mirasol PRT, TerumoBCT). PLT shape and adenosine diphosphate (ADP)-induced shape change were evaluated by a light scattering method where the amplitude of the scattered signal intensity was measured as the indicator of the proportion of discoid PLTs. Using a modified fluorometer, the real-time effects of different wavelengths of UV light on PLT shape were examined over the range of 300 to 360 nm at the same dose. RESULTS: The proportion of discoid PLTs in the Mirasol PRT-treated PCs decreased immediately after treatment. The difference in the proportion between PRT-treated and untreated PLTs became larger with storage. Although this modification correlated significantly with the pH decrease and P-selectin expression, the Mirasol PRT-treated PLTs retained sufficient ability to undergo an ADP-induced shape change. In the study using the modified fluorometer, the proportion of discoid PLTs significantly decreased with the wavelength (< 320 nm) of irradiated UV light. CONCLUSION: Mirasol PRT treatment of PCs decreases the proportion of discoid PLTs, which seemed to be caused by the irradiation with UV light of short wavelengths (< 320 nm), not that of long wavelengths (≥ 320 nm) in the Mirasol PRT system. Modification of UV light wavelength may improve the quality of PRT-treated PCs.

Autoantibody Profiles in Collagen Disease Patients with Interstitial Lung Disease (ILD): Antibodies to Major Histocompatibility Complex Class I-Related Chain A (MICA) as Markers of ILD.

Interstitial lung disease (ILD) is frequently associated with collagen disease. It is then designated as collagen vascular disease-associated ILD (CVD-ILD), and influences patients' prognosis. The prognosis of acute-onset diffuse ILD (AoDILD) occurring in patients with collagen disease is quite poor. Here, we report our investigation of auto-antibody (Ab) profiles to determine whether they may be useful in diagnosing CVD-ILD or AoDILD in collagen disease. Auto-Ab profiles were analyzed using the Lambda Array Beads Multi-Analyte System, granulocyte immunofluorescence test, Proto-Array Human Protein Microarray, AlphaScreen assay, and glutathione S-transferase capture enzyme-linked immunosorbent assay in 34 patients with rheumatoid arthritis (RA) with or without CVD-ILD and in 15 patients with collagen disease with AoDILD. The average anti-major histocompatibility complex class I-related chain A (MICA) Ab levels were higher in RA patients with CVD-ILD than in those without (P = 0.0013). The ratio of the average anti-MICA Ab level to the average anti-human leukocyte antigen class I Ab level (ie, MICA/Class I) was significantly higher in RA patients with CVD-ILD compared with those without (P = 4.47 × 10(-5)). To the best of our knowledge, this is the first report of auto-Ab profiles in CVD-ILD. The MICA/Class I ratio could be a better marker for diagnosing CVD-ILD than KL-6 (Krebs von den lungen-6).

A new ABCG2 null allele with a 27-kb deletion including the promoter region causing the Jr(a-) phenotype.

BACKGROUND: The high-prevalence antigen Jr(a) is carried on the ATP-binding cassette transporter ABCG2. The ABCG2 gene consists of 16 exons and its translation start codon is located on the second exon. Although the occurrence of the Jr(a-) phenotype is rare, several ABCG2 null alleles have been reported. We report a new ABCG2 null allele having a large deletion in this study. STUDY DESIGN AND METHODS: The Jr(a) status was determined by standard serologic tests and genomic DNA was isolated from whole blood. Exons 1 to 16 and the 5'-untranslated region of the ABCG2 gene were analyzed by polymerase chain reaction and sequencing. Expression of the ABCG2 protein on red blood cells was examined by immunoblotting. RESULTS: A Jr(a-) blood donor had a novel allele having a 27-kb deletion including noncoding Exon 1 and the promoter region of ABCG2, and the donor was apparently homozygous for the allele. In addition, we found three more individuals having heterozygosity for the same allele, with ABCG2*01N.01 having c.376C>T (p.Q126X), but did not find the allele having the 27-kb deletion in 3000 Jr(a+) individuals. Immunoblotting revealed that the ABCG2 protein was not found to be expressed in the individual with homozygosity for the ABCG2 27-kb deleted and in two individuals with an ABCG2 27-kb deleted/ABCG2*01N.01 genotype, which indirectly allows to conclude that the 27-kb deletion is responsible for a null ABCG2 allele. CONCLUSION: We first identified an ABCG2 null allele (provisional ISBT allele number ABCG2*01N.23) having a large deletion including the promoter region.

Novel swine model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. Antibodies against human leukocyte antigens in donors' plasma are the major causes of TRALI. Several animal models of TRALI have been developed, and the mechanism underlying TRALI development has been extensively investigated using rodent models. Although sheep models of nonimmune TRALI have been developed, large-animal models of antibody-mediated TRALI are not yet available. STUDY DESIGN AND METHODS: To develop a swine model of TRALI, male Clawn strain miniature pigs were used. A monoclonal antibody (MoAb) against swine leukocyte antigens (SLAs) Class I (4G8, 0.3 or 1.0 mg/kg body weight [BW]) and a control antibody (1.0 mg/kg BW) were injected into the peripheral vein after priming with or without 1 µg/kg BW lipopolysaccharide (LPS; n = 3 each). Lung injury was assessed using PaO2 /FiO2 (P/F) ratio and by chest X-ray imaging. Histopathologic analysis was also conducted. RESULTS: Lung injury could be induced by injecting 4G8 at an amount of 1.0 mg/kg BW, after LPS. The P/F ratio 90 minutes after the administration of 4G8 significantly decreased (p < 0.05). Bilateral infiltration was shown in chest X-ray imaging. Lung injury was confirmed by histopathologic analysis. CONCLUSION: Lung injury in pigs was successfully induced by anti-SLA MoAb. Priming with LPS is a prerequisite for inducing lung injury and the amount of the antibody is a critical condition.

Establishment of culture systems for Genotypes 3 and 4 hepatitis E virus (HEV) obtained from human blood and application of HEV inactivation using a pathogen reduction technology system.

BACKGROUND: It has been demonstrated that the hepatitis E virus (HEV) can be transmitted via blood transfusion, and the risk of HEV transmission via transfusion has become a major global concern. An HEV culture system for blood-derived HEV has been sought to obtain valuable knowledge of the virus and the risk of HEV infection through blood products. STUDY DESIGN AND METHODS: We endeavored to establish an HEV culture system using RNA-positive blood specimens for Genotypes (G) 3 and 4 and applied this system to evaluate tissue culture infectious dose (TCID). We applied this method to investigate the potential of the Mirasol pathogen reduction technology (PRT) system (Terumo BCT) to inactivate live HEV in contaminated platelet samples (PLTs). PLTs were spiked with cultured HEV G3 or G4 and then treated with the Mirasol PRT system. PLTs were examined before and after the treatment for HEV load using TCID titration. RESULTS: We successfully established two strains for HEV production: the JRC-HE3 strain for G3 and the UA1 strain for G4. The Mirasol PRT system expressed more than 3 log inactivation for JRC-HE3 and more than 2 log inactivation for UA1. CONCLUSION: The Mirasol PRT system inactivated greater than 2 to 3 logs of live HEV in PLTs and can potentially be used to lower the possibility of blood-borne HEV transmission. The G3 and G4 HEV inocula identified in this study and the hepatoma cell culture system provide a new means to assess HEV infectious titer and to evaluate other pathogen reduction strategies.