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Background and Objectives: Platelet concentrates suspended in a platelet additive solution (PAS‐PC) are associated with a reduction in allergic response and are suitable for preparing pathogen‐inactivated PC. We aimed to develop an efficient platform for the dual preparation of PAS‐PC and platelet‐poor plasma. Materials and Methods: PAS‐PC was prepared in six steps by using a hollow‐fibre system based on cross‐flow filtration: priming, loading PC, loading PAS, collection of filtered liquid (flow‐through) and collection of platelets by washing with PAS followed by washing with air. In this study, the efficacy of platelet and plasma protein recovery and characteristics of recovered PAS‐PC and flow‐through plasma were analysed in detail. Results: Recoveries of platelet in PAS‐PC and plasma protein in the flow‐through were 95.4% ± 3.7% and 61.6% ± 5.0%, respectively. The residual plasma protein in PAS‐PC was 34.1% ± 2.8%. Although the expression level of CD62P, a platelet activation marker, in recovered platelets was approximately 1.2‐fold of that in original platelets, swirling patterns were well retained, and aggregation in PAS‐PC was not visible. Agonist‐induced aggregabilities, platelet morphology and hypotonic shock recovery were conserved. The patterns of plasma protein and lipoprotein in the flow‐through were comparable with those in the original PCs. The multimeric pattern analysis of VWF remained unaltered. Conclusion: We propose a highly efficient preparation system that enables the simultaneous production of PAS‐PC and platelet‐poor plasma. It also achieves a high recovery of functionally well‐retained platelets with very low activation. [ABSTRACT FROM AUTHOR]

The risk of sepsis through bacterial transmission is one of the most serious problems in platelet transfusion. In processing platelet concentrates (PCs), several methods have been put into practice to minimize the risk of bacterial transmission, such as stringent monitoring by cultivation assays and inactivation treatment by photoirradiation with or without chemical agents. As another potential option, we applied a light-emitting diode (LED) with a peak emission wavelength of 265 nm, which has been shown to be effective for water, to disinfect PCs. In a bench-scale UV-LED exposure setup, a 10-min irradiation, corresponding to an average fluence of 9.2 mJ/cm2, resulted in >2.0 log, 1.0 log, and 0.6 log inactivation (mean, n = 6) of Escherichia coli, Staphylococcus aureus, and Bacillus cereus, respectively, in non-diluted plasma PCs. After a 30-min exposure, platelet counts decreased slightly (18 ± 7%: mean ± SD, n = 7); however, platelet surface expressions of CD42b, CD61, CD62P, and PAC-1 binding did not change significantly (P>0.005), and agonist-induced aggregation and adhesion/aggregation under flow conditions were well maintained. Our findings indicated that the 265 nm UV-LED has high potential as a novel disinfection method to ensure the microbial safety of platelet transfusion. [ABSTRACT FROM AUTHOR]

Background and objectives: Although HLA‐eliminated platelets can facilitate transfusions to patients possessing HLA antibodies, no such products are currently available commercially perhaps because the platelet collection rate is not yet economically viable. We have improved this process' efficiency by employing a hollow‐fibre system at the last step of the production process after an acid and a reaction buffer have been washed out conventionally by centrifugation. Materials and methods: HLA‐eliminated platelets were prepared via four distinct steps: chilled on ice, treated with an acid solution, diluted and finally washed using the hollow‐fibre system. The efficiency of this platelet recovery process was determined. The resulting products' platelet characteristics, including a capacity for HLA expression, were evaluated in vitro and compared in detail to their corresponding originals. Results: The average efficiency of platelet recovery was 91%. Although the expression levels of CD62P, a molecular marker for platelet activation, were approximately threefold higher on new platelets than on the original platelets, their HLA expression levels were lower. The phagocytosis assay, with monoclonal antibodies and cognate HLA antibody‐containing sera, suggested that HLA‐ABC molecules on the cell surface were sufficiently removed. The platelet functions, including the agonist‐induced aggregability and adherence/aggregability of the collagen‐coated plates under certain conditions, were conserved and not significantly different from the original ones. Conclusion: We propose a novel preparation system for producing HLA‐eliminated platelets without centrifugation, which ensures a highly efficient, and therefore, much more economical method of platelet recovery that also retains their key functionality. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Antibodies against human platelet antigens (HPAs) cause thrombocytopenias. It is thus important to know the frequency of "b" allotypes in each HPA system for the diagnosis and treatment of anti‐HPA antibody‐mediated thrombocytopenia. Study Design and Methods: Genomic DNA was extracted from peripheral blood cells obtained from 2170 blood donors in Japan and was subjected to high‐resolution melt (HRM) analysis using polymerase chain reaction for each of the HPA genes, using 23 primer pairs. For genotyping, the resulting amplicons were classified based on their HRM curves. In some cases, direct sequence analysis was performed after HRM analysis to determine nucleotide substitutions. In cases where amino acid substitutions were predicted, protein expression levels were examined in a cell line using 293T cells. Results: The frequencies of each of the HPA‐b genotypes were as follows: HPA‐1b, 0.4%; HPA‐2b, 11.8%; HPA‐3b, 41.3%; HPA‐4b, 0.8%; HPA‐5b, 4.3%; HPA‐6b, 1.9%; HPA‐15b, 48.8%; HPA‐21b, 0.6%; and "b" allotype in the other HPA systems, 0.0%. Twenty‐eight variants were found; nine of them were predicted to cause amino acid substitution. However, expression analysis revealed that they did not affect protein expression levels on the cell surface. Conclusion: Nine HPA systems are of primary importance in Japan in potentially triggering thrombocytopenia via the HPA antibodies. Similar studies in other countries or races, together with ours, could provide basic information for clinicians in multiethnic societies. [ABSTRACT FROM AUTHOR]

Oxaliplatin-based chemotherapy is widely used to treat advanced colorectal cancer (CRC). Sinusoidal obstruction syndrome (SOS) due to oxaliplatin is a serious type of chemotherapy-associated liver injury (CALI) in CRC patients. SOS is thought to be caused by the sinusoidal endothelial cell damage, which results in the release of unusually-large von Willebrand factor multimers (UL-VWFMs) from endothelial cells. To investigate the pathophysiology of CALI after oxaliplatin-based chemotherapy, we analyzed plasma concentration of von Willebrand factor (VWF) and the distribution of VWFMs in CRC patients. Twenty-three patients with advanced CRC who received oxaliplatin-based chemotherapy with (n = 6) and without (n = 17) bevacizumab were analyzed. CALI (n = 6) and splenomegaly (n = 9) were found only in patients who did not treated with bevacizumab. Plasma VWF antigen (VWF:Ag) and serum aspartate aminotransferase (AST) levels increased after chemotherapy only in patients without bevacizumab. VWFM analysis in patients who did not receive bevacizumab showed the presence of UL-VWFMs and absence of high molecular weight VWFMs during chemotherapy, especially in those with CALI. In addition, plasma VWF:Ag and AST levels increased after chemotherapy in patients with splenomegaly (n = 9), but not in patients without splenomegaly (n = 14). Histological findings in the liver tissue of patients who did not receive bevacizumab included sinusoidal dilatation and microthrombi in the sinusoids. Many microthrombi were positive for both anti-IIb/IIIa and anti-VWF antibodies. Plasma UL-VWFM levels might be increased by damage to endothelial cells as a result of oxaliplatin-based chemotherapy. Bevacizumab could prevent CALI and splenomegaly through inhibition of VWF-rich platelet thrombus formation. [ABSTRACT FROM AUTHOR]

Thrombotic thrombocytopenic purpura ( TTP) has not yet been reported to be associated with mutations in the Wiskott- Aldrich syndrome ( WAS) gene. WAS is an X-linked recessive disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. A broad spectrum of mutations in the WAS protein ( WASP) gene have been identified as causing the disease. In this study, we report on a 2-month-old Japanese boy who presented with cytomegalovirus ( CMV) infection and TTP. The activity of von Willebrand factor cleaving metalloproteinase, ADAMTS13 was low and the antibody against ADAMTS13 was positive (3.6 Bethesda U/mL). Although TTP was improved by plasma exchange and steroid pulse therapy, thrombocytopenia persisted and regular transfusions of irradiated platelets were needed. Tiny platelets were found on a peripheral blood smear. CMV genome was positive in peripheral blood by polymerase chain reaction and the CMV viremia continued to persist despite intravenous gancyclovir therapy. Through direct sequencing of genomic DNA of the WASP gene in the patient, we identified a novel mutation of WASP gene: the seventh nucleotide in exon 11 ( G) had been deleted (1345del G). This mutation causes a frameshift and a stop codon at amino acid 470. Western blotting demonstrated a truncated WAS protein. To our knowledge, this is the first report describing TTP in WAS patients with novel mutation in the WASP gene. [ABSTRACT FROM AUTHOR]

Abstract: To examine the presence of CD34-positive cells and intranuclear factors in acute coronary thrombi, we compared thrombi in patients with type 2 diabetes mellitus (DM, n = 21) and without DM (n = 29). Immunohistochemical staining revealed the constitutive presence of platelets, fibrin, erythrocytes, neutrophils, extracellular high-mobility group box 1 (HMGB-1), and histone H3 in all thrombi. There were significantly more oval or flat CD34-positive cells and significantly larger HMGB-1-positive areas in the thrombi from patients with DM. The flat CD34-positive cells expressed ecto-nucleoside triphosphate diphosphohydrolase (a platelet aggregation inhibitor). The number of CD34-positive cells was negatively correlated with the serum levels of glucose and hemoglobin A1c, whereas the HMGB-1-positive area was positively correlated with the levels of serum glucose. The paucity of CD34-positive cells and the high levels of HMGB-1 expression in acute coronary thrombi from patients with type 2 DM could facilitate thrombus formation. [Copyright &y& Elsevier]

We encountered the case of a 4-year-old boy with thrombotic microangiopathy (TMA) of unknown etiology. Verotoxin-induced hemolytic uremic syndrome (HUS), Streptococcus-pneumoniae-related HUS, factor H deficiency, drug-induced thrombotic thrombocytopenic purpura (TTP), and ADAMTS13 (von Willebrand factor-cleaving protease; a disintegrin-like and metalloprotease with thrombospondin type 1 repeats)-related TTP were excluded. His condition was refractory to anticoagulants and plasma exchange, and his clinical course was catastrophic, with central nervous system symptoms and progressive renal failure. However, factual treatment of intravenous gamma globulin (IVIG) ended the hemolysis and resulted in a rise in platelet count. He fully recovered except for end-stage renal failure, but he underwent a successful renal transplant after peritoneal dialysis. He has not suffered a relapse of TMA or an allograft rejection for 4 years. IVIG might be an option for some patients with TMA of unknown etiology refractory to conventional treatment. [ABSTRACT FROM AUTHOR]

BACKGROUND: ADAMTS13 specifically cleaves unusually large von Willebrand factor (VWF) multimers, which induce platelet thrombi formation under high shear stress. ADAMTS13 activity is deficient in patients with thrombotic thrombocytopenic purpura (TTP). The determination of plasma levels of ADAMTS13 activity is a prerequisite for a differential diagnosis of thrombotic microangiopathies. Here, a unique and highly sensitive enzyme immunoassay (EIA) of ADAMTS13 activity is described. STUDY DESIGN AND METHODS: ADAMTS13 hydrolyzes the peptide bond between Y1605 and M1606 of VWF. In this assay, a recombinant fusion protein (GST-VWF73-His) is used as a substrate. A panel of mouse monoclonal antibodies (MoAbs) that specifically recognizes Y1605, which is the C-terminal edge residue of the VWF-A2 domain and is generated by the enzymatic cleavage, has been produced. These antibodies were prepared with a synthetic decapeptide, termed N-10 (1596-DREQAPNLVY-1605), as the immunogen. Twenty-six clones specific to N10 were obtained, and one anti-N10 MoAb was used in this study. RESULTS: With horseradish peroxidase–conjugated anti-N10 MoAb, a standard enzyme assay was established. This assay was highly sensitive, and the detection limit was 0.5 percent of the normal. Further, an inhibitor of ADAMTS13 was measured to a level of 0.1 Bethesda units per mL. ADAMTS13 activity was measured in 20 patients with Upshaw-Schulman syndrome, a congenital TTP, and 61 acquired TTP patients. The activity measured by this assay and by the classic VWF multimer assay showed high correlation. CONCLUSION: A convenient and highly sensitive EIA for ADAMTS13 activity has been established. This assay can be introduced for routine laboratory work in transfusion medicine. [ABSTRACT FROM AUTHOR]