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We studied the biological features of eosinophils in a patient with chronic eosinophilic leukemia and a unique t(2;5)(p23;q35) translocation. Microscopic and cytochemical studies revealed no particular abnormalities, although more than 90% of the peripheral eosinophils had a density lighter than 1.080 g/ml. Clonogenic assay disclosed that myeloid progenitor cells possessed the translocation, although in vitro eosinophilopoiesis seemed normal, and there were also hematopoietic cells with a normal karyotype. In a surface marker study, EGI was positive on 34.0% of the eosinophils, while EG2 positivity was only 0.5%. Eosinophilopoietic growth factors and adhesion molecules were virtually absent with the exception of GM-CSF and CD11b. Functional studies showed that chemotaxis for C5a was normal, although that fix IL-2 or FMLP was attenuated. In addition, leukotriene C4 production was decreased while O2- production was intact. These findings indicated that our patient's eosinophils were not in an activated state despite their extreme hypodensity, and suggested that the leukemic eosinophils had slight defects of cellular function. These characteristics may have saved the patient from the multiple organ damage which occurs in typical hypereosinophilic syndrome.

No relevant conflicts of interest to declare.

Abstract 1097

With the exception of CD5, the clinical implications of aberrant T-cell antigen expression on B cells in diffuse large B cell lymphoma (DLBCL) have not been well studied. The present retrospective study aimed to determine associations between T cell antigen expression and patients′ characteristics and prognosisPatients, Material and Methods: 249 cases with B-cell lymphoma (BCL) newly-diagnosed in our Institute between January 2002 and August 2009 were biopsied and also analyzed by flow cytometry. Biopsy specimens were fixed in formalin, stained with Hematoxylin-Eosin, and also immunostained. Histological subtypes were defined according to the World Health Organization Classification Version 3. Flow cytometric (F/C) analysis was performed following standard methods.The aberrant expression of one or more T cell surface antigens on B cells (CD2, 3, 4, 7, 8) was assessed. Statistical analysis was conducted to seek associations between aberrant positive and negative cases and patients′ background laboratory data, and prognosis.This study was approved by the Ethics Committee of Kitasato University School of Medicine.150 DLBCL, 68 Follicular lymphoma (FL), 17 Marginal zone lymphoma (MZL), 6 MCL and 4 CLL patients were tested. Of the DLBCL cases, 12 (8%) showed aberrant T cell antigen expression with 4, 1, 5, 1 and 1 patients′ B cells positive for CD2, CD4, CD7, CD8 and CD7 + 8 respectively. Aberrant expression among the FL, MCL, MZL and CLL cases was seen in 0, 1, 1 and 1 patient, respectively.Among the DLBCL patients, T antigen-negative cases tended to have higher WBC counts and greater CD20 expression by F/C analysis. No statistically significant associations with gender, age, IPI, clinical stage, laboratory data, expression of CD5 and other markers, ABC/GCB, or karyotype were observed between the positive and negative groups.96 of the 150 DLBCL patients received rituximab-chemotherapy. Again, there were no statistically significant differences between the two groups in overall survival and response to treatment.1) CD2 and CD7 are relatively common among aberrant T cell antigen- positive DLBCL. 2) No statistically significant differences were observed between the two groups in terms of background and prognosis. 3) Both CD8-positive DLBCL patients experienced a very aggressive clinical course and rapidly succumbed to their disease. CD8-positive DLBCL cases may in general have poorer survival. 4) Further analysis will be necessary to confirm these results.No relevant conflicts of interest to declare.

Abstract 5088

Abstract 5000

The anti-CD20 antibody Rituximab has improved the disease-free survival and overall survival of patients with several types of B-cell lymphoma, in which CD20 is expressed on the cell surface and in the cytoplasm. Flow cytometry indicated that CD20 expression varies from null, weak to strong. Despite its clinical importance, the influence of CD20 expression levels on lymphoma therapy has not been well elucidated. Patients, Material and214 cases were analyzed, all newly diagnosed with B-cell lymphoma at our institute from January in 2002 to April in 2009. All were biopsied and analyzed by flow cytometry. The biopsy specimens were fixed in formalin and stained with Hematoxylin-Eosin; they were also immunostained. Histological subtypes were defined according to the World Health Organization Classification Ver. 3. The mean florescence intensities of CD20 and CD19 were determined by flow cytometry, and cytoplasmic expression of CD20 was determined by immunohistochemistry. 1) The cases were categorized as follows: A: CD20-negative, B: CD20/19 less than 20%, C: CD20/19 20-50% and D: CD20/19 more than 50%. Patients' backgrounds, pathological diagnoses, primary lesions, etc. were also analyzed. 2) Among DLBCL cases, 76 treated with R-chemo were selected and analyzed with respect to treatment response and overall survival.1) Diagnoses: DLBCL 128, Follicular lymphoma 58, MALT 7, CLL 4 and so on. Among the DLBCL cases, immunohistochemistry indicated six (4%) were CD20-negative and three were CD20-positive; the ages in the CD20-negative DLBCL group tended to be high (74.28 vs 64.36, p=0.06, t-test). Weak CD20 expression was observed in 15 cases (B: 4, C:11). No statistically significant differences were seen with respect to gender, IPI, clinical stage, biopsy lesion, CD5 expression or Karyotype. No FL cases were CD20-negative, but two ( 3.4%) showed low CD20 expression. 2) Kaplan-Meier analysis of 76 cases of DLBCL treated with R-Chemo showed a significantly lower overall survival for Group A, (CD20-negative) while there was no significant difference in overall survival of Groups B+C(CD20-weak) and D(CD20-normal). Our results indicate that even patients with lower levels of CD20 expression in B-cell lymphoma may respond favorably to anti-CD20 therapy. Further studies will be necessary to explore this possibility.No relevant conflicts of interest to declare.

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder, and eradication of Helicobacter pylori (HP) has been demonstrated to be an effective and tolerable firstline treatment for the patients infected with HP in Japan. However the mechanism has still remained to be uncovered. Recently CD4+CD25high+Foxp3+ regulatory T cells (Tregs), which regulate autoreactive T cells, have been identified, and suggested to play an important role in pathogenesis of ITP, as well as other autoimmune disorders. It has been shown that Tregs are reduced and suppressed in the ITP patients. And a recent report has demonstrated that the defective Tregs are restored upon the rituximab treatment. However, the effects of HP eradication therapy on Tregs have not been determined. The aim of this study is to investigate the circulating Tregs in ITP patients treated with HP eradication. And we also attempted to elucidate the mechanisms of the treatment. The peripheral blood CD4+CD25high+ Tregs were measured by flow cytometry before and after the treatment in 21 Japanese adults with HP-positive chronic ITP. We also confirmed the expression of Foxp3 in this cellular population with the permeabilized mononuclear cells. Among 21 patients, the platelet counts increased in 13 cases (responders), but not in 9 cases (non-responders). In responders the numbers of Tregs have been restored, but not in non-responders after the treatment. It is interesting here that the amounts of Tregs were still transiently elevated in an early phase (2–3weeks) after the treatment in some non-responders without recovery of the platelet counts. Furthermore, in three cases, who failed in pylorus elimination, Tregs were also transiently increased in number associated with brief recovery of the platelet counts, and reduced to the initial level in about two months. After the successful re-eradication, the numbers of Tregs and platelets have been restored. From these results it is shown that HP eradication can modulate Tregs to increase the platelet counts for ITP patients. We also demonstrate that the increase of Tregs by HP eradication was more rapid than that by rituximab, which required about three months. Further, it might be suggested that there are two phases of the therapy with HP eradication for ITP. In an initial stage, the therapy itself could have an effect modulating the immune systems to potentiate Tregs. Some drugs, such as macrolide antibiotics including clarithromycin have been demonstrated to be a potent immunomodulator. However, this phase is not sufficient for the successful treatment, as shown in the cases of failure in HP elimination. In a retentive stage, extermination of HP is also necessary for sustainment of restored Tregs.

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder, and eradication of Helicobacter pylori (HP) has been demonstrated to be an effective and tolerable firstline treatment for the patients infected with HP in Japan. However the mechanism has still remained to be uncovered. Recently CD4+CD25high+Foxp3+ regulatory T cells (Tregs), which regulate autoreactive T cells, have been identified, and suggested to play an important role in pathogenesis of ITP, as well as other autoimmune disorders. It has been shown that Tregs are reduced and suppressed in the ITP patients. And a recent report has demonstrated that the defective Tregs are restored upon the rituximab treatment. However, the effects of HP eradication therapy on Tregs have not been determined. The aim of this study is to investigate the circulating Tregs in ITP patients treated with HP eradication. And we also attempted to elucidate the mechanisms of the treatment. The peripheral blood CD4+CD25high+ Tregs were measured by flow cytometry before and after the treatment in 21 Japanese adults with HP-positive chronic ITP. We also confirmed the expression of Foxp3 in this cellular population with the permeabilized mononuclear cells. Among 21 patients, the platelet counts increased in 13 cases (responders), but not in 9 cases (non-responders). In responders the numbers of Tregs have been restored, but not in non-responders after the treatment. It is interesting here that the amounts of Tregs were still transiently elevated in an early phase (2–3weeks) after the treatment in some non-responders without recovery of the platelet counts. Furthermore, in three cases, who failed in pylorus elimination, Tregs were also transiently increased in number associated with brief recovery of the platelet counts, and reduced to the initial level in about two months. After the successful re-eradication, the numbers of Tregs and platelets have been restored. From these results it is shown that HP eradication can modulate Tregs to increase the platelet counts for ITP patients. We also demonstrate that the increase of Tregs by HP eradication was more rapid than that by rituximab, which required about three months. Further, it might be suggested that there are two phases of the therapy with HP eradication for ITP. In an initial stage, the therapy itself could have an effect modulating the immune systems to potentiate Tregs. Some drugs, such as macrolide antibiotics including clarithromycin have been demonstrated to be a potent immunomodulator. However, this phase is not sufficient for the successful treatment, as shown in the cases of failure in HP elimination. In a retentive stage, extermination of HP is also necessary for sustainment of restored Tregs.

It is sometimes confusing to distinguish idiopathic thrombocytopenic purpura (ITP) from thrombocytopenia due to dysmegakaryopoiesis, as seen in myelodysplastic syndrome (MDS) patients, especially MDS with isolated thrombocytopenia. In this study, we investigated the useful parameters for the different diagnosis of thrombocytopenia. The number of reticulated platelets reflects the rate of thrombopoiesis, and this clinical utility has been established in the laboratory diagnosis of thrombocytopenia due to increased peripheral platelet destruction, such as autoimmune thrombocytopenic purpura (AITP). However, the number of reticulated platelets has not been well investigated in the patients with myelodysplatsic syndrome (MDS), while some of them are misdiagnosed as ITP. The aim of this study is to evaluate the diagnostic utility of the measurements of reticulated platelets as well as other parameters of platelets, such as MPV (mean platelet volume), P-LCR (platelet larger cell ratio) and PDW (platelet distribution width). The reticulated platelets, expressed as the immature platelet fraction (IPF) were determined in 108 ITP and 57 MDS patients using the Sysmex XE-2100 blood cell counter with upgraded software (Sysmex, Kobe, Japan). This system enabled rapid, inexpensive, automated, stable measurements of reticulated platelets compared with the flow cytometry system, of which consensus method has not yet been identified to provide acceptable intra- and inter-laboratory results. The platelet counts in ITP and MDS patients were equivalent (ITP, 7.99 ± 0.40 × 104/μL; MDS, 8.05 ± 0.57× 104/μL). The IPF values in ITP patients (10.4 ± 0.61%) were significantly higher than those in MDS (5.82 ± 0.63%), and the inverse correlation between the IPF and the platelet counts was observed among the ITP patients, but not among the MDS. Both MPV and PDW in MDS (10.6 +/− 0.15 fL and 12.2+/−0.41 fL, respectively) were significantly higher than in ITP (7.7 +/− 0.38 fL and 9.4 +/− 0.48 fL, respectively), while P-LCR in MDS (28.7 +/− 1.2%) and ITP (23.6 +/− 1.3%) were not significantly different. Although MPV was correlated with IPF among either group, the correlation between IPF and either PDW or P-LCR was weak among MDS (IPF × PDW, r=0.673; IPF × P-LCR, r=0.660) compared with ITP (IPF × PDW, r=0.779; IPF × P-LCR, r=0.803). Next we precisely investigated the clinical features of the minor population of MDS with higher IPF. Most of these patients revealed the significantly higher values of PAIgG (Platelet-associated IgG) and/or poor response to the blood transfusion, suggesting the possibility of associated autoimmune mechanisms. The patients of MDS in overt leukemic stage also recorded higher IPF even if they had no or few blood transfusion. The IPF would be a useful parameter to distinguish ITP from MDS with isolated thrombocytopenia, which has been shown to have a favorable prognosis.

It is sometimes confusing to distinguish idiopathic thrombocytopenic purpura (ITP) from thrombocytopenia due to dysmegakaryopoiesis, as seen in myelodysplastic syndrome (MDS) patients, especially MDS with isolated thrombocytopenia. In this study, we investigated the useful parameters for the different diagnosis of thrombocytopenia. The number of reticulated platelets reflects the rate of thrombopoiesis, and this clinical utility has been established in the laboratory diagnosis of thrombocytopenia due to increased peripheral platelet destruction, such as autoimmune thrombocytopenic purpura (AITP). However, the number of reticulated platelets has not been well investigated in the patients with myelodysplatsic syndrome (MDS), while some of them are misdiagnosed as ITP. The aim of this study is to evaluate the diagnostic utility of the measurements of reticulated platelets as well as other parameters of platelets, such as MPV (mean platelet volume), P-LCR (platelet larger cell ratio) and PDW (platelet distribution width). The reticulated platelets, expressed as the immature platelet fraction (IPF) were determined in 108 ITP and 57 MDS patients using the Sysmex XE-2100 blood cell counter with upgraded software (Sysmex, Kobe, Japan). This system enabled rapid, inexpensive, automated, stable measurements of reticulated platelets compared with the flow cytometry system, of which consensus method has not yet been identified to provide acceptable intra- and inter-laboratory results.