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2001 Fiscal Year Final Research Report Summary

A novel application of flow cytometry - Quantification of cell surface molecule density and its clinical application

Research Project

Project/Area Number 10470514
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Laboratory medicine
Research InstitutionUniversity of Tokyo

Principal Investigator

NAKAHARA Kazuhiko  University of Tokyo Hospital, Professor, 医学部・附属病院, 教授 (70101095)

Co-Investigator(Kenkyū-buntansha) WATANABE Takashi  Kyorin University, Faculty of Medicine, Associate Professor, 医学部, 教授 (00191768)
HIGASHI Katsumi  University of Tokyo Hospital, Assistant Professor, 医学部・附属病院, 助手 (50159109)
YONEYAMA Akiko  University of Tokyo Hospital, Lecturer, 医学部・附属病院, 講師 (50175684)
IKEDA Tadako  Kyorin University, Faculty of Medicine, Lecturer, 医学部, 講師 (90099242)
Project Period (FY) 1998 – 2000
Keywordsflow cytometry / cell surface antigen / antigen molecule density / cell size / FF index / quantification of cell surface molecule / cellular activation / clinical application
Research Abstract

Until now, cytometric immunophenotyping using flow cytometry (FCM) has focused mainly on the quantification of the proportion of cells bearing certain combinations of molecules. The purpose of this study is to explore an effective application of FCM to clinical medicine by using the information of individual cells.
For the qualification of cell surface molecule density, the forward light scatter (FSC) and fluorescence intensity (FI), obtained by FSM on an individual cell basis were plotted in a scattergram where x-and y-axis show FSC and FI, respectively. Where a proportional relationship between FI and FSC can been seen, the slope of the line of regression (or its absolute value) may be used as an indicator of the molecule density, which is referred to as the FF index. In this way, the FI vs FSC scattergram enables the evaluation of the relationship between the size of the cell and the amount of antigen expressed. In the case of CD4 positive lymphocytes, CD4-FI was actually found to be … More positively proportional to FSC, or their cell size. This indicates that the amount of CD4 molecules per size (or FSC) unit is invariable regardless of cell size. Accordingly, the slope of the resultant regression line, or FF index, could be an indicator of the molecule density. In practice, flow cytometric measurements on five parameters-FI (FI1, FI2 and FI3), FSC and SSC (side scatter) for individual cells taken from the FACScan flow cytometer were processed on a personal computer using newly developed software.
The applicability of the FF index method to clinical flow cytometry was tested on a few model systems. When CD4 positive T lymphocytes were divided into two subpopulations on the basis of the expression of CD45 isoforms and the density of CD4 molecule was compared between these two subpopulations, the FF index method clearly demonstrated that CD4 density on CD45-RO positive memory cells was significantly higher than that on CD45-RA naive cells. This may indicate that not only the total amount of CD4 molecules per cell, but also the density of the molecule on the surface of cell is under strict regulation, possibly depending on the functional nature of the cells.
Since the FF index is the indicator of molecule density, it is conceivable that this technique could be of great value in the study of, for instance, cellular activation, where changes in cell size are often associated with the alteration in the expression of cell molecules. To verify this possibility, the expression of cell surface molecules, CD4 and CD25 on CD4 positive T lymphocytes were analyzed before and after stimulation by ConA. Although both molecules showed a remarkable increase in their expression upon ConA stimulation, a significant difference in the extent of the increase in density could be seen between CD4 and CD25 molecules by using this technique. Thus, the FF index method could be useful in quantitative analysis of cell surface molecules, particularly where changes in cell size are involved.
The FF index method is a simple and convenient means for quantitative analysis of the expression of cell surface molecules in clinical flow cytometry, thereby enabling the evaluation of how alterations in the quantity of functionally important cell surface molecules underlie various physiological and pathological conditions. Less

  • Research Products

    (16 results)

All Other

All Publications (16 results)

  • [Publications] Y.Koike, K.Nakahara et al.: "Evaluation of thrombopoieses in thrombocytopenic disorders by simultaneous measurement of reticulated piatelets of whole blood and serum thrombopoietin concentrations"Thromb Haemost. 79. 1106-1110 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Yan, K.Nakahara et al.: "The increase of memory T cell subsets in children with idiopathic nephrotic syndrome"Nephron. 79. 274-278 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 中原一彦: "フローサイトメトリーによる白血病診断の有用性とFCM利用の新しい試み"Cytometry Research. 9sup. 31-32 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 渡邊卓, 中原一彦: "リンパ球細胞表面抗原密度定量の標準化に関する検討"Cytometry Research. 9sup. 94 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Watanabe, K.Nakahara: "Quantification of cell surface molecule density by flow cytometry"The Sixth Asian Conference of Clinical Pathology. 589 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 渡邊 卓: "細胞抗原定量化のための新指標"臨床病理. Vol.48補冊. 82 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 中原一彦: "応用サイトメトリー"Flow cytometry (FCM)造血器腫瘍細胞・成人. 64-68 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 渡邊 卓: "応用サイトメトリー"基礎知識-抗体. 7-10 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y. Koike, K. Nakahara et al.: "Evaluation of thrombopoesis in thrombocytopnic disorders by simultaneous measurement of reticulated platelets of whole blood and serum thrombopoietin concerntrations"Thromb Haemost. 79. 274-278 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Yan, K. Nakahara et al.: "The increase of memory T cell subsets in children with idopathic nephrotic syndrome"Nephron. 273(41). 274-278 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Nakahara: "Usefulness for a diagnosis of leukemia using flow cytometry and new trial of FCM utilization"Cytometry Research. 9sup. 31-32 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Watanabe, K. Nakahara: "Standardization of quantity measurement of lymphocyte surface antigen density"Cytometry Research. 9sup. 93 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Watanabe, K. Nakahara: "Qualification of cell surface molecule density by flow cytometry"The Sixth Asian Conference of Clinical Pathology. S89 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Watanabe: "A novel index for quantification of cell surface antigens"The Japanese Journal of Clinical Pathology. 48sup. 82 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K. Nakahara: "Flow cytometry (FCM), Adult hematological disorders"Applied Cytometry. 64-68 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Watanabe: "Basic knowledge antibodies"Applied Cytometry. 7-10 (2000)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2003-09-17  

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