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 CaliBRITETM beads with FACSCompTM 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 SPHEROTM 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-RITETM 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 QuantiBRITETM beads can be used for the quantification of the antigen-antibody reaction, and the SPHEROTM 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.
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-CD98hi 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.
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.