Novel and rapid enumeration method of peripheral blood stem cells using automated hematology analyzer.

INTRODUCTION: The number of infused CD34(+) cells is crucial to the success of peripheral blood stem cell transplantation (PBSCT). Here, we present, for the first time, a new method of enumerating hematopoietic progenitor cells (HPCs) for PBSCT. METHOD: This novel method is based on hemolysis and chemical staining, followed by flow cytometry-based optical detection, conducted using an automated hematology analyzer (XN series, Sysmex). CD34(+) cells and HPCs were compared in 76 granulocyte colony-stimulating factor (G-CSF)-mobilized blood or apheresis samples taken from healthy donors (n = 18) or patients undergoing autologous PBSCT (n = 6). RESULTS: There was a strong correlation between the numbers of HPCs and CD34(+) cells (R(2)  = 0.958). The expected total number of HPCs in the final products, which was estimated from HPCs in pre-apheresis PB or mid-apheresis products, also correlated well with the total number of CD34(+) cells in the final products. The change in HPCs in PB closely resembled that of CD34(+) cells during mobilization. Experiments using immunomagnetic beads suggested that the majority of CD34(+) cells existed in HPCs, and vice versa. CONCLUSION: Hematopoietic progenitor cells may serve as surrogates for CD34(+) cells in PBSCT. However, further investigations are required to verify this.

Immature platelet fraction measurement: a future guide to platelet transfusion requirement after haematopoietic stem cell transplantation.

summary. The decision to prophylactically transfuse platelets is dependent on the platelet count, careful regular clinical assessment and agreed local protocol. The ability to predict when platelet recovery will occur should allow a more reasoned approach to platelet transfusion. An increase in reticulated platelets demonstrates impending platelet recovery. A new rapid automated method to assess reticulated platelets, the immature platelet fraction (IPF), is described, and its clinical utility assessed. The IPF is identified by flow cytometry with the use of a nucleic acid specific dye in the reticulocyte channel on the Sysmex XE-2100. Fifty healthy adult volunteers were used to establish the normal range. Patients where platelet marrow production or destruction might be abnormal were studied, and some patients followed serially during treatment. Thirty patients receiving cytotoxic chemotherapy were tested, and 13 of these patients followed serially. Fifteen patients post-autologous or allogeneic transplant were followed daily for platelet count and IPF percentage to monitor platelet recovery. The method demonstrates good reproducibility and stability. The recovery phase of thrombocytopenia in most chemotherapy and transplant patients was preceded by a rise in IPF percentage several days prior to platelet recovery. In particular, patients undergoing autologous transplantation (n = 8) using peripherally collected stem cells have a characteristic IPF percentage motif, with a rise one or two days prior to engraftment. The automated IPF is a useful parameter in the clinical evaluation of the thrombocytopenic patient and has the potential to allow optimal transfusion of platelet concentrates.

Distinct methylated profiles in Helicobacter pylori dependent and independent gastric MALT lymphomas.

BACKGROUND: Gastric mucosa associated lymphoid tissue (MALT) lymphomas are clinically subdivided into Helicobacter pylori dependent and independent, according to H. pylori infection and the therapeutic course. In previous reports it has been suggested that H. pylori independent cases develop from H. pylori dependent cases, and sometimes transform into high grade diffuse large B cell lymphomas (DLBCLs). METHODS: To better understand the pathogenesis of H. pylori dependent and independent MALT lymphomas, we analysed the methylation profiles of eight independent CpG islands, including p15, p16, p73, hMLH1, death associated protein kinase, MINT1, MINT2, and MINT31 in H. pylori dependent and independent MALT lymphomas, DLBCLs, and H. pylori associated chronic gastritis. RESULTS: We first confirmed that H. pylori independent cases had a high incidence of t(11;18)(q21;q21) (4/8 cases) and aberrant BCL10 expression (7/8 cases) compared with H. pylori dependent cases and gastric DLBCLs. In the methylation pattern study, all 13 H. pylori dependent MALT lymphomas had more than four methylated loci while H. pylori independent cases had less than two. According to the previous criterion, all H. pylori dependent MALT lymphomas (13/13, 100%) and five of 10 (50%) DLBCLs were classified as CpG island methylator phenotype positive (CIMP+). In contrast, all H. pylori independent MALT lymphomas were CIMP-. CONCLUSION: The distinct methylation pattern together with lack of chromosomal translocation in H. pylori dependent MALT lymphomas suggest that H. pylori dependent and independent MALT lymphomas have a different pathogenesis.

Mutations in c-kit gene exons 9 and 13 in gastrointestinal stromal tumors among Japanese.

Gain-of-function mutation in c-kit proto-oncogene exon 11 has been described in about 20 -- 50% of gastrointestinal stroma tumor (GIST). Recently, additional mutational hot-spots in exon 9 and exon 13 of the c-kit gene have been reported in GISTs without mutations of exon 11, but a subsequent report in a Western population indicated that only a small portion of GISTs (eight of 200 GISTs, 4%) showed mutations in these regions. In this study, we evaluated mutations in exon 9 and exon 13 of the c-kit gene by both polymerase chain reaction-single strand conformation polymorphism analysis and direct sequencing in 48 GISTs in a Japanese population, for which the clinicopathological and immunohistochemical features and mutations in exon 11 had previously been reported. C-kit gene mutation in exon 9, representing insertion of GCC TAT, was identified in only 4 of 48 GISTs (8%), and none of the GISTs had mutations in exon 13. All four GISTs with mutation in exon 9 were high-risk, and the patients died of multiple tumor metastasis. Mutations in exon 9 and exon 13 of the c-kit gene were also rare events in Japanese GISTs and were related to a poor prognosis. These results in Japanese are consistent with those in Western populations, although a preferential occurrence of GISTs with exon 9 mutation in the small intestine, which was suggested in a previous report, was not observed.

p53 mutation in carcinomas arising in ovarian cystic teratomas.

Carcinomas arising in mature cystic teratomas of the ovaries from nine women were examined for the presence of p53 mutations. The nine tumors comprised six squamous cell carcinomas, one squamous cell carcinoma in situ, one undifferentiated small cell carcinoma, and one mucoepidermoid carcinoma. Abnormal nuclear accumulation of the p53 protein was observed in four of the tumors. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue blocks and subjected to polymerase chain reaction (PCR) for specific amplification of the p53 gene exons 5-8, followed by direct chemiluminescence sequencing analysis. A frameshift mutation in exon 8 (codon 278, CCT > del T; stop at codon 344) was detected in one poorly differentiated squamous cell carcinoma. The samples were also evaluated for the possible association of 'benign' and 'malignant' types of human papillomavirus (HPV) by PCR using universal primer sets. None of the samples contained detectable HPV genome. These data suggest that p53 mutations are relatively uncommon in secondary carcinomas developing in ovarian dermoid cysts, although the number of samples studied was admittedly small.

Genetic constitution of germ cells in intervarietal and interspecific chimeras of Brassica induced by in-vitro grafting.

The characteristics of intervarietal and interspecific chimeras synthesized by the graft-culture method were determined by morphology, anthocyanin pigmentation pattern, and crossing. In an intervarietal chimera between 'YR-ranpou' (green cabbage) and 'Ruby ball' (red cabbage) in Brassica oleracea, a segregation phenomenon was noted in which seeds giving rise to purple and green plants were both produced in a single capsule in F1 progeny from crosses of chimeras with YR ranpou, the anthocyanin-free graft partner type. The degrees of segregation varied, reflecting the structure of the chimeras. YR ranpou-dominant chimeras produced capsules in which seeds gave rise to green plants at a high frequency, while Ruby ball-dominant chimeras produced capsules in which seeds in one capsule gave rise to purple plants at a high frequency. Mixed chimeras produced capsules with green plants or purple plants more regularly than did other chimeral types. Furthermore, a chimeral type in which seeds gave rise to green and purple plants was found in 3.2% of the total crosses. Segregation patterns in the progenies corresponded with the chimeral types. Chlorophyll-deficient variation (resulting in variegation or the production of albino plants) was found at a frequency of 2.6%. These results show that chimeric tissues are actually in a mixed state and that either the ovary develops from more than two cells or else that variation occurs in the germ-cell layer. In interspecific chimeras between Ruby ball and Komatsuna (B. campestris) various types of chimeras generally showed low pollen fertility, few capsules, and low seed-setting. Progenies from selves (geitonogamy), open crosses and crosses with the two parental species produce a predominantly homogeneous genotype showing either the Ruby ball or the Komatsuna type. Only two crosses produced four interspecific hybrids which expressed variations in their morphological and isozymic characters.