Kinya Uchihashi

Foxp3 expression on normal and leukemic CD4+CD25+ T cells implicated in human T-cell leukemia virus type-1 is inconsistent with Treg cells

Foxp3 is a master gene of Treg cells, a novel subset of CD4+ T cells primarily expressing CD25. We describe here different features in Foxp3 expression profile between normal and leukemic CD4+CD25+ T cells, using peripheral blood samples from healthy controls (HCs), human T‐cell leukemia virus type‐1 (HTLV‐1)‐infected asymptomatic carriers (ACs), patients with adult T‐cell leukemia (ATL), and various hematopoietic cell lines. The majority of CD4+CD25+ T cells in HCs were positive for Foxp3, but not all CD4+CD25+ T cells in ACs were positive, indicating that Foxp3 expression is not always linked to CD25 expression in normal T cells. Leukemic (ATL) T cells constitutively expressing CD25 were characteristic of heterogeneous Foxp3 expression, such as intra‐ and inter‐case heterogeneity in intensity, inconsistency with CD25 expression, and a discrepancy in the mRNA and its protein expression. Surprisingly, a discernible amount of Foxp3 mRNA was detectable even in most cell lines without CD25 expression, a small fraction of which was positive for the Foxp3 proteins. The subcellular localization of Foxp3 in HTLV‐1‐infected cell lines was mainly cytoplasmic, different from that of primary ATL cells. These findings indicate that Foxp3 has two facets: essential Treg identity and molecular mimicry secondary to tumorigenesis. Conclusively, Foxp3 in normal T cells, but not mRNA, is basically potent at discriminating a subset of Treg cells from CD25+ T‐cell populations, whereas the modulation of Foxp3 expression in leukemic T cells could be implicated in oncogenesis and has a potentially useful clinical role.

Clinical significance of detection of immature platelets: comparison between percentage of reticulated platelets as detected by flow cytometry and immature platelet fraction as detected by automated measurement

To the Editor: It is important to evaluate the megakaryocyte plateletproducing activity, particularly in patients with thrombocytopenia. Although flow cytometric analysis of the percentage of reticulated platelets (RP%) was introduced for this purpose and found to be useful as a non-invasive method (1), many issues have been raised, mostly related to its manual testing and the resultant difficulty in standardization. Thus, automatic measurement of young platelets with an elevated RNA was awaited. In this context, the usefulness of immature platelet fraction (IPF), a novel indicator determined using an automated hematology analyzer (Sysmex XE-2100; Sysmex Corp., Kobe, Japan), has been recently reported (2–6). In this study, we performed automated measurement of the IPF simultaneously with measurement of RP% by flow cytometry and compared the two parameters. Our aim was, in addition to evaluating the significance of IPF, to determine the feasibility of its measurement in routine laboratory practice.

An automated haematology analyzer XN-30 distinguishes developmental stages of falciparum malaria parasite cultured in vitro

BackgroundThe automated haematology analyzer XN-30 (Sysmex, Kobe, Japan) easily and rapidly detects malarial parasites in clinical blood samples using flow cytometry. The XN-30 analyzer is able to distinguish each developmental stage by measuring DNA content and cell size. Thus, it was expected to be capable of quantifying the developmental stages of cultured falciparum parasite. To achieve this requirement, a modified algorithm was tested for its validity and reliability using in vitro cultured falciparum parasite.ResultsThe XN-30 analyzer automatically measured each developmental stage as well as total parasitaemia. Comparison of the parasitaemia obtained using the XN-30 analyzer equipped with the modified algorithm with that obtained using microscopy examination of Giemsa-stained smears revealed the greater sensitivity and reproducibility of the former. The XN-30 analyzer also detected free merozoites and purified gametocytes.ConclusionsThe XN-30 analyzer allows the precise recognition and enumeration of total and each developmental stages of cultured falciparum parasites, and permits the sensitive and reproducible calculation of parasitaemia. The results indicate the potential of the XN-30 analyzer for basic research on malarial biology, anti-malarial drug discovery, and evaluation of drug efficacy.

Improved detection of minimal acute myeloid leukemia cells by the use of the combined parameters of XE-2100 hematology analyzer

For the diagnosis and therapy of acute leukemia, it is important to detect a small number of leukemic cells precisely. Although several automated hematology analyzers that carry blast‐detecting programs have been developed, they do not exert sufficient detection sensitivity to exceed the sensitivity of manual eye counting method.

Evaluation of cell count and classification capabilities in body fluids using a fully automated Sysmex XN equipped with high-sensitive Analysis (hsA) mode and DI-60 hematology analyzer system

The XN series automated hematology analyzer has been equipped with a body fluid (BF) mode to count and differentiate leukocytes in BF samples including cerebrospinal fluid (CSF). However, its diagnostic accuracy is not reliable for CSF samples with low cell concentration at the border between normal and pathologic level. To overcome this limitation, a new flow cytometry-based technology, termed “high sensitive analysis (hsA) mode,” has been developed. In addition, the XN series analyzer has been equipped with the automated digital cell imaging analyzer DI-60 to classify cell morphology including normal leukocytes differential and abnormal malignant cells detection. Using various BF samples, we evaluated the performance of the XN-hsA mode and DI-60 compared to manual microscopic examination. The reproducibility of the XN-hsA mode showed good results in samples with low cell densities (coefficient of variation; % CV: 7.8% for 6 cells/μL). The linearity of the XN-hsA mode was established up to 938 cells/μL. The cell number obtained using the XN-hsA mode correlated highly with the corresponding microscopic examination. Good correlation was also observed between the DI-60 analyses and manual microscopic classification for all leukocyte types, except monocytes. In conclusion, the combined use of cell counting with the XN-hsA mode and automated morphological analyses using the DI-60 mode is potentially useful for the automated analysis of BF cells.

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode “XN-BF gating algorithm” to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples.