Yumiko Tanaka

The Kinetics of Immune Reconstitution after Cord Blood Transplantation and Selected cd34+ Stem Cell Transplantation in Children: Comparison with Bone Marrow Transplantation

The present study compares immune reconstitution after allogeneic cord blood transplantation (CBT) and CD34+ stem cell transplantation (CD34-SCT) with that after bone marrow transplantation (BMT). Eighty-eight children who underwent CBT (20 patients), BMT (58), and CD34-SCT (10) were enrolled, and lymphocytes and T-, B-, and natural killer—lymphocyte subsets were monitored for more than 5 years after transplantation. CBT recipients showed significant increases in (1) total lymphocyte counts (P < .001), (2) CD4+/CD8+ cell ratios (P < .01), (3) CD4+ and CD4+CD45RA+ cells (P < .001), (4) CD8+CD11b+ cells (P < .001), and (5) CD19+ and CD19+CD5+ cells (P < .0001) and marked decreases in the frequencies of CD8+ and CD8+CD11b- cells (P < .0001). CD34-SCT recipients showed lower lymphocyte counts in the first 6 months and an emergence of lymphocyte and CD4+CD45RA+ cells at approximately 9 months and 1 year. Both CBT and CD34-SCT recipients showed increased frequencies of CD56+ cells at 1 month (CD34-SCT versus BMT, P < .001) but decreased frequencies after 6 months (CBT versus BMT, P < .001). Lymphoproliferative responses to exogenous interleukin 2 were constantly lower in CBT and CD34-SCT recipients than in BMT recipients.These results suggest that the delay in immune reconstitution after CBT in the early phase was mainly qualitative and related to the immaturity of cells, whereas the delay in CD34-SCT was mainly quantitative in the first several months. Int J Hematol. 2003;77:399-407.

Establishment of a xenograft model of human myelodysplastic syndromes.

Background To understand how myelodysplastic syndrome cells evolve from normal stem cells and gain competitive advantages over normal hematopoiesis, we established a murine xenograft model harboring bone marrow cells from patients with myelodysplastic syndromes or acute myeloid leukemia with myelodysplasia-related changes. Design and Methods Bone marrow CD34+ cells obtained from patients were injected, with or without human mesenchymal stem cells, into the bone marrow of non-obese diabetic/severe combined immunodeficient/IL2Rγnull hosts. Engraftment and differentiation of cells derived from the patients were investigated by flow cytometry and immunohistochemical analysis. Results Co-injection of patients’ cells and human mesenchymal stem cells led to successful engraftment of patient-derived cells that maintained the immunophenotypes and genomic abnormalities of the original patients. Myelodysplastic syndrome-originated clones differentiated into mature neutrophils, megakaryocytes, and erythroblasts. Two of the samples derived from patients with acute myeloid leukemia with myelodysplasia-related changes were able to sustain neoplastic growth into the next generation while these cells had limited differentiation ability in the murine host. The hematopoiesis of mice engrafted with patients’ cells was significantly suppressed even when human cells accounted for less than 1% of total marrow mononuclear cells. Histological studies revealed invasion of the endosteal surface by patient-derived CD34+ cells and disruption of extracellular matrix architecture, which probably caused inhibition of murine hematopoiesis. Conclusions We established murine models of human myelodysplastic syndromes using cells obtained from patients: the presence of neoplastic cells was associated with the suppression of normal host hematopoiesis. The efficiency of engraftment was related to the presence of an abnormality in chromosome 7.

Performance evaluation of platelet counting by novel fluorescent dye staining in the XN-series automated hematology analyzers.

Conventional automated hematology analyzers have limitations in platelet measurements such as poor accuracy and precision in the low count range and interference by nonplatelet particles. In order to improve it, the newly developed XN‐Series automated hematology analyzers (Sysmex Corporation, Kobe, Japan) have been installed with a new dedicated channel for platelet analysis (PLT‐F), which is based on a fluorescence flow cytometry method with uses of a novel fluorescent dye specifically staining platelets. We evaluated the basic performance of this new PLT‐F channel.

Altered expression of CD45 isoforms in differentiation of acute myeloid leukemia

Specific expression of different CD45 isoforms can be seen in various stages of differentiation of normal nucleated hematopoietic cells. Association of membrane expression of CD45 isoforms and differential levels of leukemia cells was studied in 91 cases with de novo acute myeloid leukemia (AML). Membrane expression of CD45RA and CD45RO was analyzed by flow cytometry and their expression patterns were compared with AML subtypes classified according to the French–American–British (FAB) classification. CD45RA was essentially expressed in all of the FAB myelocytic subtypes (M0–M3). Its expression in percentage was lower in the most differentiated subtype of AML (M3) when compared with other myelocytic subtypes. CD45RO expression was rarely observed in cases with myelocytic subtypes (1/56 cases of M0, M1, M2, and M3) except for the minimally differentiated myelocytic subtype (M0) or those with potential for differentiation to T‐cell lineage where three of 12 cases showed CD45RO expression. When leukemia cells of an M3 case were differentiated to mature granulocytes by treatment of all‐trans‐retinoic acid, they showed increasing expression of CD45RO. In subtypes with a monocytic component (M4 and M5), both of CD45RA and CD45RO expression were observed and mutually exclusive. When 10 cases of M5 were subdivided by the differential level into undifferentiated (M5a) and differentiated monocytic leukemia (M5b), expression of CD45RA and CD45RO was strictly restricted to cases with M5a and M5b, respectively. These results suggest that CD45 isoform expression in AML characterizes differential levels both in myelocytic and monocytic lineages and specifically disturbed in each subtype. The assessment of CD45 isoform expression appears to provide an insight on biological characteristics and a useful supplementary test for differential diagnosis of AML subtypes. Am. J. Hematol. 62:159–164, 1999.

Clinical and pathological features of B-cell non-Hodgkin lymphomas lacking the surface expression of immunoglobulin light chains.

Abstract Background: The flow cytometric analysis of surface immunoglobulin light chains (sIgL) is used as a simple method for evaluating monoclonal B-cell proliferation. However, the sIgL expression, κ or λ, is occasionally undetectable in cases with B-cell non-Hodgkin lymphomas (B-NHL). The purpose of this study was to investigate the clinical and pathological characteristics of these B-NHL cases. Methods: We retrospectively analyzed 50 cases with previously untreated sIgL-negative B-NHL. All of these cases had been diagnosed at Tokai University Hospital between January 2001 and February 2011. Their medical charts were reviewed. Results: These cases had several clinical features: diffuse large B-cell lymphoma (DLBCL) (72%), a high serum lactate dehydrogenase level (66%), clinical stage III and IV (68%), and complex karyotypes (58%). Seven out of eight evaluated patients (87%) did not express cytoplasmic IgL, and the DNA rearrangement pattern of IgL showed diversity in 10 analyzed patients. The 5-year event-free survival of all the sIgL-negative B-NHL cases was significantly better with rituximab-containing chemotherapies in comparison to the regimens without it (57.9% vs. 17.9%, p=0.0207), although there was no statistical significance when the DLBCL cases were analyzed (56.6% vs. 22.2%, p=0.1530). Conclusions: These findings suggest that sIgL-negative B-NHL cases predominantly developed DLBCL in advanced disease, but were heterogeneous at the molecular level.

A Case of Quadruple Malaria Infection Imported from Mozambique to Japan

A 35-year-old Japanese man had an intermittent fever and mild headache for eight weeks after he returned to Japan from working in Mozambique. He had taken antimalarial prophylaxis (doxycycline) for 25 weeks, and stopped taking this drug two weeks after his return. Microscopic examination of a peripheral blood smear showed a mixed infection with Plasmodium vivax, P. falciparum, and P. ovale. In addition, a nested polymerase chain reaction and subsequent sequencing detected specific DNA sequences of four species of Plasmodium, including P. malariae. The patient was successfully treated with artemether-lumefantrine and primaquine phosphate. The present case is a rare instance of a mixed infection with four species of Plasmodium. Nonimmune persons in malaria-endemic areas may have a risk of mixed infection. All four species must be identified by using sensitive and specific tests, such as a nested polymerase chain reaction, in addition to conventional morphologic identification.

A simple screening method for the diagnosis of chronic myeloid leukemia using the parameters of a complete blood count and differentials

BACKGROUND This study aimed to develop a simple and inexpensive method using the complete blood count (CBC) and differentials to screen for chronic myeloid leukemia (CML). METHODS The receiver operating characteristic (ROC) curves of each CBC parameter, differential and the neutrophil alkaline phosphatase (NAP) score using CML and non-CML cases were generated to determine effective cut-off values. They were applied to the review of randomly-selected 45,608 samples for validation. RESULTS The leukocyte count showed the highest area under the ROC curve (AUC) value (0.909) among the CBC parameters. In the absolute counts of differentials, the AUC was the highest in basophils (0.982), followed by immature granulocytes (IGs) (0.975), which had cut-off values of 0.43 × 109/L and 0.46 × 109/L, respectively. The AUC of the NAP score was 0.963 at a cut-off value 122. In the validation, the absolute basophil counts were elevated in 280 samples from 96 cases, including 22 CML cases. In contrast, the absolute IG counts were elevated in 1310 samples from 516 cases, including only 17 CML cases. Three newly-diagnosed CML cases whose data were analyzed sequentially at the CML onset consistently met the basophil criteria before the IG criteria. CONCLUSIONS The absolute basophil count is effective for screening for CML.

Triage of lymphoid malignancies in the peripheral blood using the Extended Immunofluorescent Application of the CELL-DYN Sapphire automated hematology analyzer.

Automated hematology analyzers are useful for differentiation of white blood cells. However, they often fail to detect infiltration of lymphoid malignancies in the peripheral blood, particularly when the tumor cells appear infrequently or are morphologically similar to normal lymphocytes. Microscopic observations by experts may lead to the identification of the suspicious cells, but such examinations often cannot confirm whether they are neoplastic. Following the identification of suspicious cells, an immunophenotypic analysis of these cells is needed to determine whether there is monoclonal proliferation, but this requires staff that are proficient in the techniques and time for sample preparation and analysis. The development of a simple and rapid discriminating ‘‘triage’’ assay system for lymphoid lineage cells is therefore needed in hospital laboratories. There have been several automated hematology analyzers that have provided additional functions for the evaluation of abnormal leukocyte fractions detected by common light and laser technology. For example, the Advia Leukocyte Hematology Analyzer (Siemens) has a peroxidase channel to evaluate blasts and atypical lymphocytes as large unstained cells (LUCs) (1). XE-2100 and 5000 (Sysmex) instruments can also detect blasts through the immature information (IMI) channel, which were reported to correspond to CD34 and CD34 hematopoietic stem cells and progenitors (2, 3). However, these technologies are dependent on the biochemical