転移性がんの治療方針の策定や転移メカニズムの解明の為には，CTC を採血サンプル中から出来るだけ損傷を与えずに取りこぼし無く回収し，回収したCTC の生理学的性質や遺伝子変異を迅速に計測する技術が必要となる。 本稿では非浸襲的で高感度なCTC 分離精製法開発の取り組みについて概観した後，我々が開発を進めている画像認識型セルソーターシステムと超高速PCR のCTC 分離精製，遺伝子解析への応用可能性について論じた。

Current evidence supports “cancer stem cell hypothesis” that a small subpopulation composed of cancer stem cells (CSCs) in a tumor can have self-renewing ability and tumorigenesis, and generate tumor to recapitulate a heterogeneous population of cancer cells in hierarchical fashion. CSC hypothesis brings a paradigm shift for cancer biology and treatment strategies. In breast cancer, representative markers of CSCs have reported to be CD44+/CD24- and ALDH1, which enable us to isolate cancer cells with tumorigenesis and self-renewal. We have researched ribophorin II (RPN2) that is associated with drug resistance and stemness in breast cancer. RPN2 is one of oligosaccaryltransferase complex that is responsible for N-glycosylation of proteins. By controlling N-glycosylation of p-glycoprotein, RPN2 is causative of drug resistance in breast cancer patients receiving docetaxel. Furthermore, RPN2 is also controlling tumorigenesis and metastatic ability of breast CSCs with CD44+/CD24-. When RPN2 was suppressed in breast CSCs, CSCs lost both characteristics. We have also researched the mechanisms how to control breast CSCs by RPN2. Circulating tumor cells (CTCs) are crucial process to tumor metastasis that causes cancers to be incurable. In several cancers including breast cancer, CTCs were detected in patients with early and advanced diseases. Current researches revealed that CTC was an independent prognostic factor and a predictive marker in metastatic breast cancer. In addition, breast CTCs were reported to change HER2 status from primary breast cancer. Although their significance is not completely recognized, it is important to clarify molecular biology of CTCs and association between CTCs and CSCs. Here, we review the experimental and clinical evidence for the involvement of CSCs in breast cancer development, focusing on the common characteristics of CTCs.

血球形態分類および計数は，ロマノフスキー染色，網赤血球は核酸染色した塗抹標本の光学顕微鏡観察を参照法としているが，近年，参照法としてのフローサイトメトリーによる血球分類が複数の国際的な標準化組織において検討されている。その活動は，国際血液学標準化委員会（ICSH）と米国臨床検査標準化協議会（NCCLS）が中心を担ってきた。NCCLS は国際的標準化活動を行うことを明確にするために，臨床・検査標準協会（CLSI）と改名した。そして，ICSH 委員により発足した国際検査血液学会（ISLH）が設立された。2006 年にICSH はISLH の標準作業部会と改組した。その後，ICSH の活動アクティビティが高まり，CLSI との協調を強めていった。現在，血液検査分野の国際的標準化活動はICSH（ISLH）とCLSI で行われている。2010 年にはICSH とCLSI の共同会議が開催された。血球分類の参照法としてのフローサイトメトリーによる細胞表面抗原検査法は，ISLH とICSH により血小板計数の参照法（2001），CLSI とICSH により網赤血球計数ガイドライン（CLSI H44-A2：2004），CLSI により白血球５分類のガイドライン（CLSI H20-A2：2007）で提案がなされた。白血球５分類のガイドラインは，具体的な抗体パネル・測定法などが示されていなかった。2007 年，ICSH はCLSI H20-A2 の改定を目標として「Extended Differential Project」を発足し，マルチカラーフローサイトメトリーによる異常細胞を含む白血球分類の参照法の検討を開始した。これを受けて，日本検査血液学会（JSLH）においても検討を開始した。本講演では，フローサイトメトリーによる血球分類の動向として，①血球分類の国際的な標準化組織の動向，②標準化組織による血小板参照法と白血球参照法の現状および課題，③ICSH およびCLSI の今後の方向性について述べる。

ISO15189 is the specialized international standard developed by International Organization for Standardization in 2003 for medical laboratories. As of July 2011, 59 medical laboratories in Japan have been accredited. A wide range of overseas laboratories in Asia, Australia and European countries have also been accredited. Our laboratory was Japan’s first ever laboratory accredited under ISO15189 in September 2005. Against this background, we submit this report on initiatives undertaken to make flow cytometric testing at our laboratory meet ISO15189 requirements, the effects of the accreditation, as well as any anticipated future challenges. In order to meet ISO15189 requirements, a quality management system was established by producing and compiling documents and records, including a quality manual. Developing standard operating procedures for flow cytometric testing has allowed us to standardize education for testing personnel and to maintain the quality of the testing process. Although ISO15189 requires laboratories to participate in several kinds of proficiency testing, flow cytometric testing is not included at the moment in any of the major external quality assessment programs implemented in Japan. Our laboratory therefore only participates in the external quality assessment program organized by the supplier. An inter-laboratory comparison has therefore been undertaken in cooperation with laboratories at nearby hospitals, in order to see if this is feasible on a continuous basis. We believe that standardized testing will be necessary in the future in order to keep flow cytometric testing as an item for ISO15189 accreditation and that establishing an official external quality assessment program would be important to achieve that end.

Although multiple injections with IL-18 following burn injury remarkably restore IFN-γ production and thereby increase mouse survival after Eschericia coli (E. coli) infection, it has yet to be fully elucidated how the IL-18 therapy affects the functions of phagocytic cells. We investigated the effect of IL-18 therapy on the functions and interactions of Kupffer cells and NK cells in burn-injured mice. C57BL/6 mice received a 20% full-thickness burn injury, followed by multiple injections with IL-18. Although burn-injured mice decreased the expression of IL-18 receptors on the NK/NKT cells 5 days after injury, multiple IL-18 injections restored their expression. IL-18 treatment also augmented Kupffer cell phagocytosis. Although burn-injury decreased the number of CD68⁺ Kupffer cells with phagocytic activity, IL-18 treatment partially restored their proportion, and augmented phagocytosis-induced ROS production in CD68⁺ Kupffer cells after the injection of heat-killed E. coli. Consistently, IL-18 restored the impaired E. coli killing activity of Kupffer cells of burn-injured mice. Such Kupffer cell activation by IL-18 was abrogated by the deletion of NK cells or IFN-γ. We summarized that IL-18 therapy in burn-injured mice enhanced the function of CD68⁺ Kupffer cells via the activation of liver NK cells and augmentation of their IFN-γ production, thereby improving their survival after E. coli infection.

There are problems that tumor-like cells appear in transplantation therapy using iPS cells (iPSCs). Contamination and proliferation of undifferentiated cells that do not respond to differentiation induction cause tumorigenesis. The iPSCs grafts require completely differentiated cells and/or tissues in vitro unlike the mesenchymal stem cells. In this report, we survey recent topics in prevention of tumorigenesis risks of iPSCs with referring the knowledge from our experiments. Undifferentiated iPSCs extremely resemble to ES cells by transmission electron microscope analysis. The resistant undifferentiated cells after induction were detected by fluorescent microscope and flow cytometer. These cells were selectively eliminated by fluorescent labeling plus laser and radiation exposures. By irradiation to differentiated cells, GFP positive cells, those are undifferentiated iPS cells, were decreased without disorder in myocardial beats. Irradiated iPS cells were also injected into the testis of SCID mouse to examine teratoma formations. Although non-irradiated cells formed teratomas in all the mice examined, irradiated cells formed no teratoma and localized in the testis. Thus, it was suggested that irradiation before the transplantation selectively eliminates residual undifferentiated cells and inhibits post transplantation tumorigenesis in mouse iPSCs. Irradiation will be possible to cell / tissues before transplantation of human iPSCs. It is necessary to perform analysis of the cellular safety included the influence of gene functions in differentiated normal cells by irradiation.