Molecular mechanisms of the cellular internalization of human thioredoxin (TRX) have been largely unknown. Although TRX has no typical signal sequence, TRX is released from cells and enters into cells. To analyze the mechanisms of releasing or internalization of TRX, we prepared a mutant TRX (TRX-C35S), in which active site Cys35 is substituted by serine. Flowcytometric analysis revealed that Alexa488-labeled TRX-C35S binds more on the cell surface of the HTLV-I positive T cell lines comparing with the HTLV-I negative cell lines. The binding of TRX-C35S was inhibited by DTT or excess amount of wild type TRX. The binding and internalization of TRX-C35S was observed more on the cell surface of CD25 positive cells. Since CD25 is a marker of activated T cells, the binding and the internalization of TRX may be associated with the activation of T cells.

Flow cytometry is an indispensable methodology for immunological tests like immuno phenotyping of leukemic cells. Recently, it is becoming increasingly important to measure absolute count of particular cell populations, for example, enumerating CD34+ cells in hematopoietic stem cell transplantation and counting CD4+ T cells in HIV infection. Thus, quality control processes only focused on verifying optical alignment and instrument settings are not sufficient for quality assurance for clinical flow cytometry. Operators also need to pay close attention to both the precision and the accuracy of their measurements. Therefore, not only internal quality control but also external quality control is necessary for meaningful quality assurance for clinical flow cytometry. While the implementation of internal QC processes is possible by the use of appropriate control materials, there are no official external QC programs for clinical flow cytometry available in Japan. The Interlaboratory Quality Assurance Program (IQAP) is an external QC program provided by Beckman Coulter. In this program, the accuracy and the precision of measurements from each participant are simultaneously evaluated and compared to the pooled data from all participants. In this paper, I briefly illustrated the basic outlines of quality assurance for clinical flow cytometry as well as important points to consider when interpreting statistical data. I then summarized some IQAP results and showed the effectiveness of process automation for better precision, the importance of accurate lymphocyte gating for better accuracy, and the usefulness of CD45 for more appropriately gating lymphocytes.

Improvement of staining and measuring methods for DNA or cell cycle analyses by flow cytometry (FCM) has been continued by many researchers for years. Japan Cytometry Society (JCS) and the organizations which were past of JCS have borne the central role in Japan. Now, the application range of FCM is greatly expanded and to operate FCM machine became very easy. This paper has been written for the purpose of recognizing again the notes for improving coefficient variation(CV). It is necessary to understand that CV value and DNA histogram may be influenced on various factors. Furthermore, it is important to understand about a possibility of changing the DNA amount of G0/G1 cells or the affinity between double strands DNA and propidium iodide can change, when we analyze DNA histogram.

Clinical and research laboratories are getting to face an increasing number of requirements on the validation and standardization of their procedure and machinery reliance. Flowcytometric analysis is one of those.On the view of the flowcytometer, it should be managed to be stay reliable with some standardized procedure,however its method for calibration and validation is not widely accepted. We have evaluated several factors for the machinery calibration to settle reliability as follows; (1)utility of the CaliBRITE^TM beads with FACSComp^TM software on many Becton Dickinson(BD) cytometers as general practice; (2) utility of the DNA QC Particles tovalidate the linearity for the cell cylce analysis; (3) utility of the SPHERO^TM Rainbow QC Kit to be reliable of stability of the each detector on the light intensity and signal resolution. The result shows that ; (1)the CaliB-RITE^TM beads provide information on signal separation, compensation, the sensitibity of each detector and the flow stabilizastaion, and it settels appropriate instrument setting for peripheral blood specimens. And those information can be monitored by the Levy-Jennings charts as a time-being data. (2) The DNA QC Particles can be validate the linearlity of the FL2 channel detector and its parameters. It also can be separate the single cell from the doublets and others. Actual proof of the CV is possible as a result. (3) The QuantiBRITE^TM beads can be used for the quantification of the antigen-antibody reaction, and the SPHERO^TM Rainbow QC Kit is useful for the evaluation of the signal intensity and the lineality for the quantification.

A flow cytometer is used for rapid analyses of cells or chromosomes. For an optical system of flow cytometer, it is important to adjust the center of illuminating laser beam to a jet-stream. Microspheres or leukocytes are used for focus adjustment. Adjusted best position is the location where fluorescence of standard samples becomes the brightest. Manual adjustment of the optical-axis is not only complicated but also inaccurate. A slit-scan flow cytometer measures the morphological information along the objects being analyzed. The laser beam size of our instrument at the tightly focused position has dimensions of 0.95 in the flow direction by 85.0 across with a depth of focus being only 1. At unfocused position, width of the slit becomes wider and intensity of the peak beam becomes weaker. To guarantee best focusing, we examined a method of opticalaxis adjustment for the slit-scan flow cytometer by controlling position of the jet-stream based on fluorescence intensities of microspheres passed. The computer controlled optical adjustment system ensures stable and reliable measurements by the slit-scan flow cytometer.

トランスフェクションマイクロアレイ（TMA）は生細胞を用いた大規模網羅的な機能解析のための実験手法として有用である。これまでは、限られた種類の細胞にのみ適用可能であったためその有用性が必ずしも生かされてはいなかった。我々はECMタンパク質を最適に用いることによりトランスフェクション効率を改善することに成功した。 それによりTMAを神経系細胞に適応することを可能とした。本報告において、これまで困難であった、神経系細胞に対してsiRNAをもちいた特異的な機能阻害実験のマイクロアレイ化ができることを明らかとした。

泌尿器腫瘍内微小環境で微量に発現されている遺伝子転写物を検出する目的で，RT-PCR法を用いた遺伝子発現解析の基礎的検討を行った。 RNA検体はすべてACHN細胞より抽出した。負荷RNA量が62.5ngから500ngの範囲では，GAPDH転写物のRT-PCR産量は，負荷RNA量とほぼ直線的に関連し増加した（r=0.994, P=0.0039）。また，125ngのRNA量を負荷し，25から40サイクルの増幅回数においては，TGF-βI型およびII型リセプターの遺伝子転写物に対するRTPCR産量は，それぞれ理論値に近い指数関数的な増加を示した。TGF-β1遺伝子転写物は微量なため，各反応に1,000ngのRNA量を負荷し，併せて30サイクル以上の増幅回数が必要であった。 RT-PCR法は目標とする遺伝子転写物が微量で，また検体量が少量でも施行可能であった。

我々はこれまでに2倍体V79細胞をK-252aで高次に多倍体化し，クローニングした細胞から3倍体V79細胞の樹立化に成功している。しかし，3倍体V79細胞の多倍体化については不明である。本研究では，多くの培養細胞を多倍体化するデメコルチン（紡錘糸形成阻害剤）を用い，3倍体V79細胞の多倍体化を検討した。デメコルチンで処理された2倍体，3倍体V79細胞は増殖が停止し，1，2，3，4日の処理時間とともに多倍体化が顕著に表れた。しかし，デメコルチン処理後の細胞は高次倍数体の細胞としては増殖せず，2倍体細胞が4ならびに6倍体，3倍体細胞が3ならびに6倍体細胞として観察された。これらの結果から3倍体V79細胞は2倍体細胞と異なった倍数変化を示すことが示唆された。

We have previously reported that the expression of amino acid transporter, CD98 was high on the lineage marker negative precursor cells including hematopoietic stem cells in murine bone marrow. To clarify the expression of CD98 on the hematopoietic stem cells, we analyzed “side population” (SP) cells by modifying the BD LSR cytometer. We observed the typical SP pattern in Hoechst 33342-stained mouse bone marrow cells by a simple modification to the standard optical configuration of the BD LSR benchtop flow cytometer. In normal bone marrow cells, the expression of CD98 on the lineage marker negative SP cells was low ~ medium. To analyze CD98 expression on SP cells at the early bone marrow reconstitution, we administrated 5-fluorouracil (5-FU) into mice. The proportion of SP cells in the bone marrow was the order of the mice 4 days after 5-FU administration > the mice 7 days after 5-FU administration > normal mice, it seemed to increase the expression of CD98 on SP cells of lineage marker negative in the bone marrow after 4 days. The proportion of SP cells in the fetal liver cells was approximately 13%, in great numbers, and especially, the SP cells concentrated in the lineage^-CD49f^-CD98^hi cell fraction. Therefore, though CD98 expression in the hematopoietic stem cell was low ~ intermediate, the possibility of increasing the expression with cell division and differentiation to progenitor cells in which have pluripotency was shown.

グリオーマの顕微鏡下での手術において，腫瘍の境界を決定するのに困難な場合があり，どこまでを切除すべきかは大きな問題である。 我々はグリオーマの手術において，腫瘍を部位別に採取し，術中迅速標本（FS）で腫瘍細胞の浸潤を確認しているが，永久標本（PS）とレーザースキャニングサイトメーター（LSC）にてDNA-ploidyを測定し，腫瘍の境界部決定について検討した。 【症例】astrocytoma 20例中grade II 3例，grade III 7例，grade IV 10例を対象とした。 【方法】術中に中心部（C），辺縁部（E）（術者が腫瘍と判断した部まで，ニューロナビゲーターでは画像上造影さ れている部まで），周辺部（P）の組織を採取した。LSCでは小標本をガラススライドに塗沫標本を作成し，核染色後，DNA-ploidyを算出した。 【結果】（1）grade II：FSではP部は腫瘍細胞の浸潤はみられず，全例diploidパターンを示した。（2）grade III：FSではP部は4例に浸潤例をみたが，浸潤のない部まで摘出し得た。LSCではP部は全例diploidパ ターンであった。（3）grade IV：FSではP部は少量の細胞残存例として2例あった。LSCではP部に2例のaneuploidパターンがあった。（4）ナビゲーターで（P）に浸潤例があった。 【結論】悪性グリオーマでは顕微鏡下で全摘出しても，FS，LSCでは腫瘍細胞が残存していることが多く，FS, LSCは境界切除部位決定に有用と考えられる。また、FSの結果はPSとも相違ないことが証明された。

We investigated the chromosome aberrations of colorectal cancer with Genomic Array CGH, and analyzed those relations to lymph node or distant metastasis. There were significant associations between the loss of 8p and the depth of cancer, between the gain of 7q and lymph node metastasis, and between the gain of 8q and liver metastasis, respectively. When we analyzed the chromosome aberrations of both primary and liver metastatic tumor, almost identical changes were found in each case. Genomic Array CGH, which enabled to express the characteristics of each cancer, may contribute to practice ‘ the tailor maid treatment' (personal therapy).

Because the relative DNA content of tumor cells (DNA ploidy) is easily determined by flowcytometric analysis, DNA ploidy has been examined as a prognostic factor of solid carcinoma. However, it is still unknown why DNA aneuploid tumors exhibit a poor prognosis, and how we should clinically correspond to improve the prognosis. In this report, we reviewed the results of recent clinical trials considering the status of tumor DNA ploidy. The results suggest that more fundamental investigations and clinical studies would be needed to clarify the detailed mechanisms of the biological malignancy of DNA aneuploid carcinomas for clinical application in cancer treatment, because several clinical trials revealed that neither adjuvant chemotherapy nor irradiation therapy improved the prognosis of DNA aneuploid carcinomas.