A novel haemodilution chip mounted on the total thrombus-formation analysis system, a flow chamber system, enables stable analysis of platelet products under low platelet concentration/high shear conditions.

BACKGROUND AND OBJECTIVES: The total thrombus-formation analysis system (T-TAS) can quantitatively analyse the contribution of platelets to haemostasis using reconstituted blood samples. However, it is unsuitable in cases with low platelet counts. We introduced a haemodilution (HD) chip with a shallow chamber depth, adapted to low platelet counts and high shear conditions (1500 s-1). MATERIALS AND METHODS: Blood samples were prepared by mixing red blood cell products, standard human plasma and platelet products; the final platelet count was 50 × 103/µL. Aggregation tests were performed by using the aggregation inducers collagen, adenosine diphosphate (ADP) and ristocetin. Samples with 2-, 4- and 9-day-old platelet products (N = 10) were evaluated. RESULTS: The HD chip enabled the stable analysis of the haemostatic function of all samples at a platelet count of 50 × 103/µL. Haemostatic function was correlated with ADP aggregation (time to 10 kPa [T10]: r = -0.53; area under the curve for 30 min: r = 0.40) and storage period (T10: r = 0.44). CONCLUSION: The HD chip-mounted T-TAS can stably analyse haemostatic function under low platelet counts and high shear conditions; this approach is expected to serve as a bridge to in vivo haemostatic tests with experimental animals.

X-irradiated umbilical cord blood cells retain their regenerative effect in experimental stroke.

Although regenerative therapy with stem cells is believed to be affected by their proliferation and differentiation potential, there is insufficient evidence regarding the molecular and cellular mechanisms underlying this regenerative effect. We recently found that gap junction-mediated cell-cell transfer of small metabolites occurred very rapidly after stem cell treatment in a mouse model of experimental stroke. This study aimed to investigate whether the tissue repair ability of umbilical cord blood cells is affected by X-irradiation at 15 Gy or more, which suppresses their proliferative ability. In this study, X-irradiated mononuclear (XR) cells were prepared from umbilical cord blood. Even though hematopoietic stem/progenitor cell activity was diminished in the XR cells, the regenerative activity was surprisingly conserved and promoted recovery from experimental stroke in mice. Thus, our study provides evidence regarding the possible therapeutic mechanism by which damaged cerebrovascular endothelial cells or perivascular astrocytes may be rescued by low-molecular-weight metabolites supplied by injected XR cells in 10 min as energy sources, resulting in improved blood flow and neurogenesis in the infarction area. Thus, XR cells may exert their tissue repair capabilities by triggering neo-neuro-angiogenesis, rather than via cell-autonomous effects.

Development of a recombinant hepatitis B immunoglobulin derived from B cells collected from healthy individuals administered with hepatitis B virus vaccines: A feasibility study.

BACKGROUND: In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg). STUDY DESIGN AND METHODS: B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein-Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG1 expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry. RESULTS: Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells. DISCUSSION: We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful.

A novel quantitative method to evaluate the contribution of platelet products to white thrombus formation in reconstituted blood under flow conditions.

BACKGROUND AND OBJECTIVES: Currently, the quality of platelet (PLT) products is evaluated using a series of in vitro tests, which only analyse PLTs as an inspection material. However, it would be ideal to assess the physiological functions of PLTs under conditions similar to the sequential blood haemostatic process. In this study, we attempted to establish an in vitro system where the thrombogenicity of PLT products was evaluated in the presence of red blood cells (RBCs) and plasma using a microchamber under constant shear stress (600/s). MATERIALS AND METHODS: Blood samples were reconstituted by mixing PLT products, standard human plasma (SHP) and standard RBCs. Each component was serially diluted keeping the other two components fixed. The samples were applied onto a flow chamber system (Total Thrombus-formation Analysis System [T-TAS]), and white thrombus formation (WTF) was assessed under large arterial shear conditions. RESULTS: We observed a good correlation between the PLT numbers in the test samples and WTF. The WTF of samples containing ≦10% SHP was significantly lower than those containing ≧40% SHP, and no difference was observed in WTF among samples containing 40%-100% SHP. WTF significantly declined in the absence of RBCs, whereas no change in WTF was observed in the presence of RBCs, over haematocrit range of 12.5%-50%. CONCLUSION: The WTF assessed on the T-TAS using reconstituted blood may serve as a new physiological blood thrombus test to quantitatively determine the quality of PLT products.

Gap Junction-Mediated Transport of Metabolites Between Stem Cells and Vascular Endothelial Cells.

We have previously demonstrated that small molecular transfer, such as glucose, between hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) and vascular endothelial cells via gap junctions constitutes an important mechanism of stem cell therapy. Cell metabolites are high-potential small-molecule candidates that can be transferred to small molecules between stem cells and vascular endothelial cells. Here, we investigated the differences in metabolite levels between stem cells (HSCs and MSCs), vascular endothelial cells, and the levels of circulating non-hematopoietic white blood cells (WBCs). The results showed remarkable differences in metabolite concentrations between cells. Significantly higher concentrations of adenosine triphosphate (ATP), guanosine triphosphate (GTP), total adenylate or guanylate levels, glycolytic intermediates, and amino acids were found in HSCs compared with vascular endothelial cells. In contrast, there was no significant difference in the metabolism of MSCs and vascular endothelial cells. From the results of this study, it became clear that HSCs and MSCs differ in their metabolites. That is, metabolites that transfer between stem cells and vascular endothelial cells differ between HSCs and MSCs. HSCs may donate various metabolites, several glycolytic and tricarboxylic acid cycle metabolites, and amino acids to damaged vascular endothelial cells as energy sources and activate the energy metabolism of vascular endothelial cells. In contrast, MSCs and vascular endothelial cells regulate each other under normal conditions. As the existing MSCs cannot ameliorate the dysregulation during insult, exogenous MSCs administered by cell therapy may help restore normal metabolic function in the vascular endothelial cells by taking up excess energy sources from the lumens of blood vessels. Results of this study suggested that the appropriate timing of cell therapy is different between HSCs and MSCs.

Comparison of the programmed freezer method and deep freezer method in the manufacturing of frozen red blood cell products.

BACKGROUND AND OBJECTIVES: Frozen-thawed red blood cells (FTRCs) are useful blood components to patients with rare blood phenotypes. However, frozen red blood cells (FRCs) sometimes cause significant haemolysis after thawing due to the freeze/thaw process. In this study, we aimed to focus on the former process and reduce process-related haemolysis. MATERIALS AND METHODS: Five-day-old red blood cells (RBCs) (5D) or 9-week-old RBCs (9 W) were glycerolized, pooled and split into two aliquots. RBCs were frozen using either the programmed freezer (PF) method or the deep freezer (DF) method. After 4-8 weeks, the FRCs were thawed and washed. In vitro characteristics were compared between the PF and DF methods. Nine week were used as a starting material for FTRCs with the assumption that they can mimic disqualified FTRCs with respect to Hb recovery. RESULTS: The PF method resulted in a significantly higher Hb recovery rate than the DF method (5D: 85.9 ± 2.1 vs. 81.1% ± 3.5%, p < 0.001) (9 W: 56.8 ± 4.0 vs. 52.4% ± 3.5%, p < 0.001). Both 5D and 9W-derived FTRCs immediately after preparation prepared by the PF method were more resistible to haemolysis than those prepared by the DF method. On the other hand, there were no significant differences between PF and DF methods in Adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). CONCLUSION: The PF method was more suitable for RBC freezing than the DF method in terms of Hb recovery in FTRCs. Although it was only 4%-5%, the improvement in the Hb recovery rate will contribute to a more stable supply.

Passive immune basophil activation test for the identification of allergic episodes from various adverse events elicited by haematopoietic cell transplantation: A pilot study.

BACKGROUND AND OBJECTIVES: Haematopoietic cell transplantation (HCT) therapy tends to be associated with various complications including engraftment failure, regimen-related toxicities, and infectious diseases. In addition, HC infusion itself occasionally elicits adverse events (AEs), one of the most common AEs is an allergic reaction. As appropriate laboratory tests have not yet been established to distinguish allergy-mediated AEs from other complications, clinical responses for HCT-related AEs can only be nonspecific. In this pilot study, using passive immune basophil activation test (pi-BAT), we attempted to distinguish an HC infusion-induced allergic reaction from various HCT-related AEs. MATERIALS AND METHODS: Using pi-BAT, we examined 34 patients who underwent HCT, that is, 11 with AEs and 23 without AEs as controls. RESULTS: Two of the eleven AE cases were pi-BAT positive and, the rest of nine AE cases were negative, while all non-AE cases were negative. Both of the two positive cases showed erythema, tachycardia, plus cough. Because erythema is one of the representative symptom of allergy, those cases could be classified as allergic reaction cases or anaphylaxis cases if tachycardia and cough were concomitant symptoms of erythema. Among the nine AEs with pi-BAT negative result, four cases showed urticaria, four showed vomiting plus diarrhoea, and one showed cough. Urticaria case was strongly suspected of allergy, however, the AE cases were pi-BAT negative. CONCLUSION: The pi-BAT may be useful as an auxiliary diagnostic tool to confirm the possible involvement of HC infusion in HCT-related AEs and identify an immunologic mechanism for HCT-related hypersensitivity reactions.

Sensitivity and specificity of passive immune-basophil activation test to detect allergic transfusion reactions.

BACKGROUND: The basophil activation test (BAT), performed with patient blood samples and supernatants from transfused blood, was developed to elucidate the mechanistic relationship between transfusion and the resultant allergic transfusion reactions (ATRs). This test cannot be performed on myelosuppressed patients and neonates because of the absence of basophils. Therefore, we devised the passive immune basophil activation test (pi-BAT) using patients' plasma and residual transfused blood as sources of immunoglobulin E and allergen, respectively, and the basophils of healthy volunteers served as a source of the responder cells. The sensitivity and specificity of the pi-BAT, however, remained largely unknown. STUDY DESIGN AND METHODS: In this study, the pi-BAT was performed on 31 patients with nonhemolytic transfusion reactions including nine non-ATR and 22 ATR (12 mild and 10 moderate-to-severe) cases to examine its sensitivity and specificity. RESULTS: Nine of the 10 cases with moderate-to-severe ATR tested positive, whereas all the non-ATR cases negative, strongly indicating immunoglobulin E and allergens are involved in the pathogenesis underlying the blood transfusion-triggered adverse effects. CONCLUSION: Thus, we propose that pi-BAT can be used to detect moderate-to-severe ATRs and their underlying mechanisms.

Immunoglobulin (Ig)G antibodies against IgE identified by basophil activation test as the putative causative agent of a serious allergic transfusion reaction: potential utility of the test as a new safety measure for allergic transfusion reactions.

BACKGROUND: In most cases of allergic transfusion reactions (ATRs), the causative agents have not been identified and the mechanisms are largely unknown, with a few exceptions. The basophil activation test (BAT) was recently introduced in the field of transfusion to investigate the causal relationships between ATRs and transfusion, as well as the mechanisms behind them. STUDY DESIGN AND METHODS: The BAT was used to screen the residual supernatants (SNs) of 43 blood components associated with serious ATRs for those that can activate basophils of many healthy volunteers. The SNs were then fractionated by centrifugal ultrafiltration and protein G column chromatography and each separated fraction was reexamined by the BAT. RESULTS: Of the 43 such blood components, one activated basophils from 19 of 21 healthy volunteers. In the blood component, the IgG antibody against IgE was identified as a putative causative agent. CONCLUSION: Blood donors who possessed the IgG antibody against IgE may be dangerous to transfusion recipients. The BAT would be useful in identifying such high-risk blood donors, when it is used to screen the blood components associated with serious ATRs for residual SNs that can activate the basophils of many healthy volunteers.

Possible Utility of the Basophil Activation Test for the Analysis of Mechanisms Involved in Allergic Transfusion Reactions.

Allergic transfusion reactions (ATRs) are the most common adverse reactions occurring during transfusion of blood components. Although most reactions are mild and involve cutaneous manifestations, severe ATRs including life-threatening anaphylaxis may also occur. The mechanisms of ATRs are largely unknown because they have not been well studied. One of the reasons for this may be the absence of a standard assay system for investigating these processes. Basophils and/or mast cells are key effector cells in immediate-type allergic reactions. They possess the unique ability to degranulate upon cross-linking of specific IgE bound on the membrane-bound, high-affinity IgE receptor or upon direct stimulation by exposure to allergens. Basophils are present in peripheral blood, unlike mast cells which are located in tissues. Therefore, basophils are valuable for the clinical testing of allergy. Consequently, the basophil activation test (BAT) was developed as a simple blood test for the diagnosis of allergic reactions to substances such as foods, inhalants, medicines and venom. In the last decade, the BAT has also been applied to transfusion medicine; 5 pilot studies revealed that the supernatants of the responsible blood products activated basophils in the BAT in 13 ATR cases, suggesting a causal relationship between ATRs and transfusion. In this review, we describe those cases and explore the potential utility of the BAT as a test performed in reference laboratories for the analysis of ATRs. We also describe the weaknesses, pitfalls, and unanswered issues of this assay.

Clinical utility of a passive immune basophil activation test for the analysis of allergic transfusion reactions.

BACKGROUND: In previous studies, we demonstrated that the basophil activation test, which is performed using patient blood and the supernatants from transfused blood components, was able to elucidate not only the causative relationship between allergic transfusion reactions and the transfusion but also the mechanisms behind allergic transfusion reactions. However, for a large number of allergic transfusion reactions, patients are in a state of myelosuppression, and the basophil activation test cannot be performed for these patients because there are insufficient numbers of peripheral blood basophils. STUDY DESIGN AND METHODS: To overcome this obstacle, we developed a passive immune basophil activation test, in which patient plasma and residually transfused blood are used as the patient's sources of immunoglobulin E and allergen, respectively, whereas healthy volunteer basophils serve as the responder cell source. The passive immune basophil activation test was performed for two patients who had severe allergic transfusion reactions, using supernatants of the residual platelet concentrates and the patients' own immunoglobulin E. RESULTS: There were no differences in either surface immunoglobulin E or activation in response to allergens between untreated basophils and so-called quasi-basophils, in which immunoglobulin E was replaced by a third party's immunoglobulin E. In these patients, the supernatants of the residual platelet concentrates exclusively activated basophils in response to quasi-basophils onto which the patients' immunoglobulin E, but not a third party's immunoglobulin E, was bound. CONCLUSION: The passive immune basophil activation test may help clarify the causal relationship between allergic transfusion reactions and transfused blood, even when patients experience myelosuppression.

Mitochondrial damage-associated molecular patterns as potential proinflammatory mediators in post-platelet transfusion adverse effects.

BACKGROUND: Platelet concentrates (PCs) are the most common blood components eliciting nonhemolytic transfusion reactions (NHTRs), such as allergic transfusion reactions and febrile reactions. However, the precise mechanisms of NHTRs in PC transfusion remain largely unknown. Previous studies reported that mitochondria-derived damage-associated molecular patterns (DAMPs) could be important mediators of innate cell inflammation. Platelets (PLTs) represent a major reservoir of mitochondria in the blood circulation. The aim of this study was to determine the possible involvement of mitochondrial DAMPs in NHTRs. STUDY DESIGN AND METHODS: The amount of mitochondrial DAMPs was determined as an index of total copy numbers of mitochondrial DNA (mtDNA), including mtDNA itself and free mitochondria, using quantitative real-time polymerase chain reaction. To examine whether neutrophils, monocytes, and basophils were activated by mitochondrial DAMPs in vitro, an in vitro whole blood cell culture assay was performed. RESULTS: In blood components associated with NHTRs, the mean total mtDNA concentration was highest in PCs followed in order by fresh-frozen plasma and red blood cells. The amount of mtDNA in NHTR PCs was higher than that in control PCs without NHTRs. The mitochondrial DAMPs present in NHTR PCs was high enough to activate neutrophils, monocytes, and basophils, when costimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or HLA antibodies. CONCLUSION: PLT-derived mitochondrial DAMPs are candidate risk factors for the onset of NHTRs.

High-resolution melting analysis as an alternative method for human neutrophil antigen genotyping.

Human neutrophil antigen (HNA)-typed granulocyte panels are widely used to screen for the presence of HNA antibodies and to determine antibody specificity. Many laboratories screen donors for HNA genotypes using low-throughput methods such as allele-specific polymerase chain reaction (PCR), PCR-restriction fragment-length polymorphism, and multiplex PCR. In the present study, we used a high-resolution melting (HRM) analysis to determine HNA genotypes. For the HRM analysis, purified genomic DNA samples were amplified via PCR with HNA-specific primers. Nucleotide substitutions in genes encoding HNAs were differentiated on the basis of the HRM curves, and the results of HRM and DNA sequencing analyses were determined to be in complete agreement. The gene frequency of HNA-1a, -1b, -1c, -3a, -3b, -4a, -4b, -5a, and -5b in the Japanese population was consistent with the previous reports. Our results suggest that HRM analysis can be used for genotyping HNA antigens determined by single nucleotide substitutions.

The IgE-dependent pathway in allergic transfusion reactions: involvement of donor blood allergens other than plasma proteins.

On transfusion, several plasma proteins can cause anaphylaxis in patients deficient in the corresponding plasma proteins. However, little is known about other allergens, which are encountered much more infrequently. Although it has been speculated that an allergen-independent pathway underlying allergic transfusion reactions (ATRs) is elicited by biological response modifiers accumulated in blood components during storage, the exact mechanisms remain unresolved. Furthermore, it is difficult even to determine whether ATRs are induced via allergen-dependent or allergen-independent pathways. To distinguish these two pathways in ATR cases, we established a basophil activation test, in which the basophil-activating ability of supernatants of residual transfused blood of ATR cases to whole blood basophils was assessed in the presence or absence of dasatinib, an inhibitor of IgE-mediated basophil activation. Three of 37 supernatants from the platelet concentrates with ATRs activated panel blood basophils in the absence, but not in the presence, of dasatinib. The basophil activation was inhibited by treatment of anti-fish collagen I MoAb in one case, suggesting that the involvement of fish allergens may have been present in donor plasma. We concluded that unknown non-plasma proteins, some of which had epitopes similar to fish antigens, in blood component may be involved in ATRs via an allergen/IgE-dependent pathway.

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies.

Granulocyte immunofluorescence and granulocyte agglutination tests are standard methods for detecting human neutrophil antigen (HNA) antibodies (Abs); however, these require a typed panel of neutrophils, which can be time-consuming to develop, and it remains difficult to determine antibody specificity in some cases. We established and evaluated four detection systems for HNA-1a Abs based on an HNA-1a-expressing cell line (KY cells) and antigen capture. We additionally evaluated a commercial solid-phase system. Eleven HNA-1a antibody-positive samples, including the World Health Organization Reference Reagent, and 40 serum samples derived from male blood donors were used as positive and negative control samples, respectively. Although specificity was >0.90 in all systems evaluated, the sensitivity varied among the systems. The KY cell-based monoclonal antibody specific immobilisation of granulocyte antigens (KY-MAIGA) system using certain, but not all, monoclonal Abs, and the solid-phase system revealed higher sensitivity than other systems. In conclusion, the KY-MAIGA and commercial solid-phase systems were superior in terms of specific and sensitive detection of HNA-1a Abs.

Detection of anti-human platelet antibodies against integrin α2ß1 using cell lines.

BACKGROUND: Antibodies against human platelet antigens (HPA) are a cause of thrombocytopenia. Detection of rare anti-HPA antibodies using platelet preparations is difficult and would be improved by an alternative method that does not require platelets. In the present study, we describe the establishment of cell lines that stably express specific HPA associated with integrin α2ß1 and the application of these cell lines for detecting anti-HPA-5a and anti-HPA-5b antibodies. MATERIALS AND METHODS: Complementary DNA of the integrin α2 variants HPA-5b, -13b and -18b were individually transfected into K562 cells using retroviral vectors. Expression of integrin α2 was confirmed by flow cytometric analysis, immunoprecipitation and western blotting analysis. To verify whether the cell line panel was suitable for clinical diagnosis, we analysed its properties using monoclonal antibody-specific immobilisation of platelet antigens (MAIPA) and well-characterised serum samples. RESULTS: Exogenous integrin α2 expression was observed in the transfected cells for over 6 months. The cell line panel specifically detected previously characterised anti-HPA-5a and anti-HPA-5b antisera. No reactivity was observed with control sera, including normal sera and HLA antisera. DISCUSSION: We successfully established a cell line panel to facilitate the sensitive and reliable detection of anti-HPA-5a and anti-HPA-5b antibodies.

Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.