Endoscopic Diagnosis of Epithelial Subtypes of Superficial Non-Ampullary Duodenal Epithelial Tumors using Magnifying Narrow-Band Imaging.

INTRODUCTION: Superficial non-ampullary duodenal epithelial tumors (SNADETs) include low-grade adenoma (LGA) and high-grade adenoma or carcinoma (HGA/Ca) and are classified into two different epithelial subtypes, gastric-type (G-type) and intestinal-type (I-type). We attempted to distinguish them by endoscopic characteristics including magnifying endoscopy with narrow-band imaging (M-NBI). METHODS: Various endoscopic and M-NBI findings of 286 SNADETs were retrospectively reviewed and compared between G- and I-types and histological grades. M-NBI findings were divided into four patterns based on the following vascular patterns; absent, network, intrastructural vascular (ISV), and unclassified. Lesions displaying a single pattern were classified as mono-pattern and those displaying multiple patterns as mixed-pattern. Lesions showing CDX2 positivity were categorized as I-types and those showing MUC5AC or MUC6 positivity were categorized as G-types based on immunohistochemistry. RESULTS: Among 286 lesions, 23 (8%) were G-type and 243 (85%) were I-type. More G-type lesions were located oral to papilla (91.3 vs. 45.6%, p < 0.001), and had protruding morphology compared to those of I-types (65.2 vs. 14.4%, p < 0.001). The major M-NBI pattern was ISV in G-type (78.2 vs. 26.3%, p < 0.001), and absent for I-type (0 vs. 34.5%, p = 0.003). Three endoscopic characteristics; location oral to papilla, protruding morphology, and major M-NBI pattern (ISV) were independent predictors for G-type. Mixed-pattern was more common in HGA/Ca than LGA for I-type (77.0 vs. 58.8%, p = 0.01); however, there was no difference for those in G-type. CONCLUSION: Endoscopic findings including M-NBI are useful to differentiate epithelial subtypes.

Risk factors for skin, mucosal, and organ bleeding in adults with primary ITP: a nationwide study in Japan.

Bleeding manifestations in primary immune thrombocytopenia (ITP) range from skin petechiae to life-threatening intracranial hemorrhage (ICH). However, the relation between these various bleeding manifestations and the platelet count in ITP remains poorly characterized. Using a nationwide database of patients with ITP during the years 2005 to 2014 (10 years) in Japan, we analyzed 19 415 adult patients newly diagnosed with ITP, including 222 with ICH. The frequency of skin purpura was 64.8%, and this increased linearly with thrombocytopenia without a specific platelet count threshold. In contrast, mucosal bleeding (epistaxis and gingival bleeding) and organ bleeding (melena, hematuria, and ICH) increased exponentially with thrombocytopenia at a platelet count threshold of 10 to 15 × 109/L. Age showed a much weaker correlation than platelet count with skin and mucosal bleeding. However, the incidence of organ bleeding increased exponentially above 60 years of age. Multivariate analysis showed that the presence of mucosal bleeding was a risk factor for occurrence of melena and hematuria but not for ICH. The frequency of ICH was 1.1% and risk factors for ICH were age ≥60 years (odds ratio [OR], 3.09; 95% confidence interval [CI], 2.13-4.47; P < .001), platelet count <10 × 109/L (OR, 2.96; 95% CI, 2.11-4.15; P < .001), and the presence of hematuria (OR, 1.56; 95% CI, 1.04-2.35; P = .033). The relation between ICH and platelet count varied with age. This large-scale analysis of risk factors for bleeding in ITP has revealed distinct characteristics of skin, mucosal, and organ bleeding in adult patients with newly diagnosed ITP, thus indicating those who are at a high risk of severe organ bleeding.

Incidence of transfusion-related adverse reactions per patient reflects the potential risk of transfusion therapy in Japan.

OBJECTIVES: To describe the frequency of adverse reactions (ARs) after transfusion on both per transfused patient and per transfused unit bases. METHODS: We performed a retrospective analysis of data available from records of 6 hospitals on the total number of transfusions and documented ARs between January 2008 and December 2009 for RBCs, fresh-frozen plasma (FFP), and platelet concentrates (PCs). RESULTS: The incidence of ARs to RBCs, FFP, and PCs per transfused unit was 0.6%, 1.3%, and 3.8%, respectively. The incidence of ARs to RBCs, FFP, and PCs per patient was 2.6%, 4.3%, and 13.2%, respectively-almost 3-fold higher. Most RBC-ARs were febrile nonhemolytic transfusion reactions and allergic reactions, whereas most FFP-ARs and PC-ARs were allergic reactions. CONCLUSIONS: The incidence of ARs per transfused patient may reflect better the potential risk of transfusion with blood components, taking into account the characteristics of the transfused patient.

[Effects of anticoagulants and storage temperature on immature platelet fraction % (IPF%) values in stored samples measured by the automated hematology analyzer, XE-5000--utility of CTAD-anticoagulation and room temperature storage].

Measurement of reticulated platelet percentage (RP%) is thought to be a useful marker for differential diagnosis and analysis of platelet kinetics in patients with thrombocytopenic disorders. Two methods are used to detect RP; flow cytometric method and immature platelet fraction (IPF) method using automated hematology analyzers. Although IPF% measured by the automated hematology analyzers is simple and convenient, we already reported that IPF% values were highly fluctuated in stored whole blood sample with EDTA-2K at 4 degrees C day by day. In this study we investigated the stability of IPF% in blood samples obtained from 11 patients with chronic immune thrombocytopenic purpura (ITP) and 19 healthy volunteers using the automated hematology analyzer, XE-5000 (Sysmex) under various storage conditions. EDTA-2K, 3.13% sodium citrate, acid-citrate dextrose solution (ACD), citrate-theophylline-adenosine-dipyridamole solution (CTAD), or sodium fluoride was used as an anticoagulant. When blood samples obtained from healthy subjects were stored at 4 degrees C, IPF% values markedly increased in a time-dependent manner by any anticoagulant examined. On the other hand, there was no significant or only slight difference in IPF% values at room temperature (RT) storage except sodium fluoride. However, in patients with ITP the elevated IPF% values fluctuated widely in EDTA-2K, sodium citrate and ACD-anticoagulated samples even at RT storage. In contrast, IPF% values in CTAD samples stored at RT were highly stable in all patients with ITP up to 4 day storage. These results suggest that the measurement of IPF% by XE-5000 provides quite stable data up to 4 day-storage in ITP patients as well as healthy subjects under CTAD-anticoagulation and RT storage conditions.

Epidemiology of primary immune thrombocytopenia in children and adults in Japan: a population-based study and literature review.

The epidemiology of primary immune thrombocytopenia (ITP) is not well-characterized in the general population. Most published studies, which have included relatively small numbers of ITP patients, have been conducted in England or Scandinavian countries. No epidemiologic data from Asian countries have been published. This study describes the epidemiology of ITP in a Japanese population. We analyzed the database registry of the Ministry of Health, Labour, and Welfare of Japan, and extracted newly diagnosed acute and chronic ITP patients with a platelet count of <100 × 10(9)/L. From 2004 to 2007, 7,774 cases of ITP were reported, giving an overall incidence of 2.16/100,000/year. The incidence differed greatly between males and females, being 1.72 and 2.58, respectively. The median age of the total affected population was 56 years old. In male patients, there was a striking preponderance of boys below 4 years and a very high peak among those aged 75-89 years. In female patients, the number of ITP patients appeared to show a trimodal distribution by age, with the first peak representing patients below 4 years, the second peak those aged 20-34 years, and the third peak those aged 50-89 years. In conclusion, the incidence of ITP in Japan is not markedly different from that of European countries studied to date. This population-based study reveals that, contrary to previously published studies, the maximum age-specific incidence is in the eighth decade.

Effects of bisphosphonate administration on the bone mass in immune thrombocytopenic purpura patients under treatment with steroids.

Immune thrombocytopenic purpura (ITP) is an acquired hemorrhage condition involving accelerated platelet consumption caused by antiplatelet autoantibodies. Although various therapeutic strategies are used to treat patients with ITP, the standard treatment method is steroid therapy. The most important problem with steroid administration may be a prolonged use tendency in many cases, because there are many refractory chronic patients. To elucidate the effects of glucocorticoid on bone mineral density (BMD) in patients with ITP, we retrospectively evaluated the relationship between BMD and the total dose of glucocorticoid or the mean daily dose given. We observed decreased BMD in 66.7% of the patients with ITP to whom glucocorticoid was given, although normal bone BMD was observed in 28.6% of patients with ITP treated without steroids. The mean level of BMD was markedly decreased in steroid-treated patients compared with nonsteroid-treated patients (P < .01). The relationship between BMD and the total dose of glucocorticoid (P = .023) or the mean daily dose revealed a negative correlation (P = .022). Administration of bisphosphonate revealed a significant increase in bone mass in patients at 6 and 12 months after the start of bisphosphonate treatment, despite the aggravation of thrombocytopenia. In conclusion, glucocorticoid-induced osteoporosis was observed in patients with ITP, similar to situation seen in patients with other diseases. Bisphosphonate may be an effective agent for the prevention and treatment of glucocorticoid-induced osteoporosis in patients with ITP scheduled to receive long-term steroid treatment.

[Comparison between two methods for the measurement reticulated platelet and their clinical significance--flow cytometry (FCM) method and IPF method Using automated hematology analyzer (XE-2000)].

Reticulated platelet (RP) is thought to be a useful marker for differential diagnosis and analysis of platelet kinetics in patients with thrombocytopenia. In this study, we compared two methods for the measurement of RP: flow cytometric (FCM) method and immature platelet fraction (IPF) method using automated hematology analyzer (XE-2000). There was a relatively good correlation between RP% measured by FCM method and IPF% measured by IPF method in patients with idiopathic thrombocytopenic purpura (ITP) (Y = 0.806X-0.050, r = 0.634, p < 0.001). We then measured RP% and IPF% in 61 patients with ITP and 27 patients with aplastic anemia (AA). For the differential diagnosis for ITP, the sensitivity (82%) and specificity (93%) of FCM method were better than those of IPF method (sensitivity 67% and specificity 63%). Our data demonstrate the significant difference between two methods by analyzing clinical samples in parallel.

A phase II, open-label, sequential-cohort, dose-escalation study of romiplostim in Japanese patients with chronic immune thrombocytopenic purpura.

This phase II, multicenter, open-label, sequential-cohort, dose-escalation study was designed to evaluate the safety and efficacy of romiplostim, a novel peptibody that increases platelet production, in Japanese patients with chronic immune thrombocytopenic purpura (ITP). Sequential cohorts of four patients each received romiplostim (1, 3, or 6 microg/kg) subcutaneously on days 1 and 8 of the dose-escalation phase. Patients who achieved platelet responses (doubling of baseline platelet counts to > or =50 x 10(9)/L) continued romiplostim weekly during the treatment-continuation phase. Romiplostim produced dose-dependent increases in mean and peak platelet counts. Five patients received romiplostim during the treatment-continuation phase, with platelet counts > or =50 x 10(9)/L maintained in approximately half of the weekly assessments. Romiplostim was well tolerated. No severe, serious, or life-threatening adverse events were reported. No binding antibodies to romiplostim or thrombopoietin were detected. Romiplostim is safe and well tolerated in Japanese patients with chronic ITP and is effective in producing platelet count increases, consistent with the results from studies in non-Japanese patients. On the basis of these findings, a starting dose of 3 microg/kg was recommended for phase III evaluation of romiplostim in Japanese patients with chronic ITP.

[Four cases of pseudothrombocytopenia due to platelet cold agglutinins].

We report 4 cases of pseudothrombocytopenia due to platelet cold agglutinins. Case 1 was a 57 y.o. female whose platelet count was 97 x 10(3)/microl. Case 2 was a 37 y.o. male with a platelet count of 96 x 10(3)/microl. Case 3 was a 74 y.o. male with a platelet count of 28 x 10(3)/microl. Case 4 was a 62 y.o. female whose platelet count was 34 x 10(3)/microl. The platelet counts in these 4 cases were decreased and blood smears showed platelet clumping in blood drawn in a tube without anticoagulant just after withdrawal, as well as in blood drawn in a tube with anticoagulant. The platelets from these patients agglutinated at a temperature below 10 degrees C (case 1 and 4) and 24 degrees C (case 2). The immunoglobulin class of the platelet cold agglutinins in cases 1, 2 and 4 was IgM. Agglutinated platelets showed no activation marker, such as CD62P, CD63 or CD40L, on the surface of the platelets. The target antigen of cold agglutinins was GPIIb-IIIa in cases 1 and 2. We considered that the detection of platelet agglutination in blood without anticoagulant is important to diagnose pseudothrombocytopenia due to platelet cold agglutinins. Although this disease is considered to be very rare, we suspect that this disease may be misdiagnosed as pseudothrombocytopenia due to the presence of an anticoagulant, and overlooked.

Adiponectin acts as an endogenous antithrombotic factor.

OBJECTIVE: Obesity is a common risk factor in insulin resistance and cardiovascular diseases. Although hypoadiponectinemia is associated with obesity-related metabolic and vascular diseases, the role of adiponectin in thrombosis remains elusive. METHODS AND RESULTS: We investigated platelet thrombus formation in adiponectin knockout (APN-KO) male mice (8 to 12 weeks old) fed on a normal diet. There was no significant difference in platelet counts or coagulation parameters between wild-type (WT) and APN-KO mice. However, APN-KO mice showed an accelerated thrombus formation on carotid arterial injury with a He-Ne laser (total thrombus volume: 13.36+/-4.25 x 10(7) arbitrary units for APN-KO and 6.74+/-2.87x10(7) arbitrary units for WT; n=10; P<0.01). Adenovirus-mediated supplementation of adiponectin attenuated the enhanced thrombus formation. In vitro thrombus formation on a type I collagen at a shear rate of 250 s(-1), as well as platelet aggregation induced by low concentrations of agonists, was enhanced in APN-KO mice, and recombinant adiponectin inhibited the enhanced platelet aggregation. In WT mice, adenovirus-mediated overexpression of adiponectin additionally attenuated thrombus formation. CONCLUSIONS: Adiponectin deficiency leads to enhanced thrombus formation and platelet aggregation. The present study reveals a new role of adiponectin as an endogenous antithrombotic factor.

[Fragmented red cells are the major cause of spuriously high platelet counts in patients with fragmented red cells when counts are obtained by automated blood cell counter].

Platelet counts measured by automated blood cell counter often show spuriously high values when measuring samples contain particles of equal size to platelets. The major cause of spuriously high platelet counts in samples with fragmented red cells (FRC) is thought to be the FRC themselves. We studied the correlation between FRC and spuriously high platelet counts in 40 patients demonstrating FRC on blood smears. FRC were measured by manual hemocytometry and by flow cytometry using a monoclonal antibody against glycophorin A (GPA method). There was a significant correlation between spuriously high platelet counts and FRC by manual hemocytometry (r=0.60, p<0.001) or FRC by the GPA method (r=0.45, p<0.005). These data suggest that FRC are the major cause of spuriously high platelet counts in samples with FRC.

[A case of pseudothrombocytopenia due to platelet cold agglutinins].

We report a case of pseudothrombocytopenia due to platelet cold agglutinins. Platelet counts were decreased in blood drawn in a tube without anti-coagulant just after withdrawal as well as in blood drawn in a tube with anti-coagulant, such as EDTA-2K, MgSO4, citrate or heparin. In our case, platelet aggregates were noted on blood-smear made from blood samples obtained with and without anti-coagulant. RBC and WBC counts were within the normal range. Platelet aggregates mainly consisted of 2-5 platelets. Patient plasma agglutinated normal platelets at a temperature below 10 degrees C. Immunoglobulin class was determined as IgM by flow cytometry.

Negative regulation of platelet function by a secreted cell repulsive protein, semaphorin 3A.

Semaphorin 3A (Sema3A) is a secreted disulfide-bound homodimeric molecule that induces growth cone collapse and repulsion of axon growth in the nervous system. Recently, it has been demonstrated that Sema3A is produced by endothelial cells and inhibits integrin function in an autocrine fashion. In this study, we investigated the effects of Sema3A on platelet function by using 2 distinct human Sema3A chimera proteins. We detected expression of functional Sema3A receptors in platelets and dose-dependent and saturable binding of Sema3A to platelets. Sema3A dose-dependently inhibited activation of integrin alphaIIbbeta3 by all agonists examined including adenosine diphosphate (ADP), thrombin, convulxin, phorbol 12-myristate 13-acetate, and A23187. Sema3A inhibited not only platelet aggregation induced by thrombin or collagen but also platelet adhesion and spreading on immobilized fibrinogen. Moreover, Sema3A impaired alphaIIbbeta3-independent spreading on glass coverslips and aggregation-independent granular secretion. Sema3A inhibited agonist-induced elevation of filamentous action (F-actin) contents, phosphorylation of cofilin, and Rac1 activation. In contrast, Sema3A did not affect the levels of cyclic nucleotides or agonist-induced increase of intracellular Ca2+ concentrations. Thus, the extensive inhibition of platelet function by Sema3A appears to be mediated, at least in part, through impairment of agonist-induced Rac1-dependent actin rearrangement.

SHPS-1 negatively regulates integrin alphaIIbbeta3 function through CD47 without disturbing FAK phosphorylation.

CD47 (integrin-associated protein) serves as a receptor for thrombospondin-1 (TSP-1) and Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), and the TSP-1/CD47 interaction has been believed to augment integrin-mediated platelet function. Here, employing SHPS-1-immunoglobulin (Ig) as a ligand, we have newly demonstrated that CD47 acts as an inhibitory receptor for platelet function. The binding of SHPS-1-Ig was solely mediated by CD47, because CD47-deficient platelets failed to bind murine SHPS-1-Ig. The human SHPS-1/CD47 interaction inhibited the platelet aggregation induced by several kinds of agonists at a low concentration. Moreover, human SHPS-1 expressed on the cell surface as well as soluble SHPS-1-Ig markedly inhibited the platelet spreading on, but not initial adhesion to, immobilized fibrinogen. Again, neither murine SHPS-1 expressed on the cell surface nor murine SHPS-1-Ig inhibited the spreading of CD47-deficient platelets. We further investigated the tyrosine phosphorylation of signaling proteins during platelet spreading on immobilized fibrinogen. Unexpectedly, SHPS-1 inhibited alpha(IIb)beta(3)-mediated platelet spreading without disturbing focal adhesion kinase (FAK) tyrosine phosphorylation. Further examination revealed that SHPS-1 inhibited the tyrosine phosphorylation of alpha-actinin, a downstream effector of FAK, but not of cortactin. Thus, it is likely that the SHPS-1/CD47 interaction inhibits alpha(IIb)beta(3)-mediated outside-in signaling by interfering with the downstream pathway of FAK. Taken together, our data suggest that SHPS-1 negatively regulates platelet function via CD47, especially alpha(IIb)beta(3)-mediated outside-in signaling.

Is eradication therapy useful as the first line of treatment in Helicobacter pylori-positive idiopathic thrombocytopenic purpura? Analysis of 207 eradicated chronic ITP cases in Japan.

A retrospective study was performed to determine the prevalence of Helicobacter pylori (H pylori) infection, the effect of H pylori eradication on platelet counts, and the characteristic clinical features of chronic immune or idiopathic thrombocytopenic purpura (ITP) with H pylori infection. H pylori infection was found in 300 patients, a group that was significantly older (P < .005) and had more cases of hyperplastic megakaryocytes in the bone marrow (P = .01) than patients without H pylori infection. H pylori eradication therapy was performed in 207 H pylori-positive ITP cases, and the platelet count response was observed in 63% of the successful eradication group and in 33% of the unsuccessful eradication group (P < .005). In the successful group, the complete remission and partial remission rates were 23% and 42%, respectively, 12 months after eradication. In the majority of responders, the platelet count response occurred 1 month after eradication therapy, and the increased platelet count continued without ITP treatment for more than 12 months. H pylori eradication therapy was effective even in refractory cases, which were unresponsive to splenectomy. In conclusion, H pylori infection was involved in most ITP patients older than 40 years in Japan, and eradication therapy should be the first line of treatment in H pylori-positive ITP patients.

Amino acid mutagenesis within ligand-binding loops in alpha v confers loss-of-function or gain-of-function phenotype on integrin alpha v beta 3.

The crystal structure of alpha(v)beta(3) in complex with a cyclic RGD-containing ligand has recently been demonstrated. However, the functional significance of each residue within ligand binding loops has not been fully elucidated. Here, by employing alanine-scanning mutagenesis, we have examined the functional role of ligand contact residues in alpha(v). Tyr178 --> Ala substitution (Tyr178Ala) and Asp218Ala abolished a monovalent ligand, WOW-1 Fab binding as well as soluble fibrinogen binding, which is in perfect agreement with the crystallography. However, Asp150Ala showed no or only a modest inhibition of ligand binding. In contrast, Tyr substitution at Ala215 (Ala215Tyr) increased WOW-1 Fab binding, suggesting that the substitution increased the integrin affinity. The adhesion assay to immobilized fibrinogen showed essentially the same data as obtained using soluble ligands. Our present data indicate that Tyr178 and Asp218, but not Asp150 in alpha(v) is critically involved in ligand-binding and that Ala215 could regulate the affinity of alpha(v)beta(3).