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. 1989 Apr;134(4):733–739.

Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis.

R L Garcia 1, M D Coltrera 1, A M Gown 1
PMCID: PMC1879787  PMID: 2565087

Abstract

Cell kinetic information is an important adjunct to histologically-based tumor classifications. Presently, cell kinetic data can be obtained from slide-based material only with monoclonal antibodies such as Ki-67, which require the use of frozen sections and cannot be applied to archival, paraffin-embedded material. Monoclonal antibodies have recently been generated to PCNA/cyclin, a 36 kd, S-phase-associated nuclear protein. The authors investigated whether monoclonal antibody 19A2 could be used to identify proliferating cells within fixed, embedded tissue sections. Deparaffinized sections of 41 methacarn-fixed human tumors were immunostained with 19A2 using a streptavidin biotin immunoperoxidase system. A semiquantitative scoring system was used to evaluate the fraction of cells that were PCNA/cyclin-positive, and this score was compared with cell kinetic data obtained from parallel flow cytometric S-phase analysis that had been performed on fresh samples of the same tumors. While there was general agreement between the slide-based, antibody-derived and the flow cytometrically-derived cell kinetic information, some discrepancies were observed. Some of the latter represented cases in which the anti-PCNA/cyclin antibody preparations demonstrated significant heterogeneity in the numbers of proliferating cells in different regions of the tumor. In other cases, a significant fraction of the positive cells corresponded to nontumor stromal and/or inflammatory cells. In these cases, the slide-based method provided more information about the tumor cell population than did the flow cytometry data. It is concluded that semiquantitative immunocytochemical analysis with anti-PCNA/cyclin antibodies may represent a simple, reproducible, yet powerful technique for the routine analysis of cell kinetic data in alcohol-fixed, paraffin-embedded tissue by the surgical pathologist.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bacus S., Flowers J. L., Press M. F., Bacus J. W., McCarty K. S., Jr The evaluation of estrogen receptor in primary breast carcinoma by computer-assisted image analysis. Am J Clin Pathol. 1988 Sep;90(3):233–239. doi: 10.1093/ajcp/90.3.233. [DOI] [PubMed] [Google Scholar]
  2. Bravo R., Celis J. E. A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells. J Cell Biol. 1980 Mar;84(3):795–802. doi: 10.1083/jcb.84.3.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bravo R., Frank R., Blundell P. A., Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987 Apr 2;326(6112):515–517. doi: 10.1038/326515a0. [DOI] [PubMed] [Google Scholar]
  4. Celis J. E., Bravo R., Larsen P. M., Fey S. J. Cyclin: a nuclear protein whose level correlates directly with the proliferative state of normal as well as transformed cells. Leuk Res. 1984;8(2):143–157. doi: 10.1016/0145-2126(84)90135-8. [DOI] [PubMed] [Google Scholar]
  5. Celis J. E., Celis A. Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc Natl Acad Sci U S A. 1985 May;82(10):3262–3266. doi: 10.1073/pnas.82.10.3262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dean P. N., Jett J. H. Mathematical analysis of DNA distributions derived from flow microfluorometry. J Cell Biol. 1974 Feb;60(2):523–527. doi: 10.1083/jcb.60.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dubey D. P., Staunton D. E., Parekh A. C., Schwarting G. A., Antoniou D., Lazarus H., Yunis E. J. Unique proliferation-associated marker expressed on activated and transformed human cells defined by monoclonal antibody. J Natl Cancer Inst. 1987 Feb;78(2):203–212. [PubMed] [Google Scholar]
  8. Engström Y. Monoclonal antibodies against mammalian ribonucleotide reductase. Acta Chem Scand B. 1982;36(5):343–344. doi: 10.3891/acta.chem.scand.36b-0343. [DOI] [PubMed] [Google Scholar]
  9. Gerdes J., Lemke H., Baisch H., Wacker H. H., Schwab U., Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 1984 Oct;133(4):1710–1715. [PubMed] [Google Scholar]
  10. Gown A. M., Vogel A. M. Monoclonal antibodies to human intermediate filament proteins. II. Distribution of filament proteins in normal human tissues. Am J Pathol. 1984 Feb;114(2):309–321. [PMC free article] [PubMed] [Google Scholar]
  11. Hsu S. M., Soban E. Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J Histochem Cytochem. 1982 Oct;30(10):1079–1082. doi: 10.1177/30.10.6182185. [DOI] [PubMed] [Google Scholar]
  12. Jaskulski D., deRiel J. K., Mercer W. E., Calabretta B., Baserga R. Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin. Science. 1988 Jun 10;240(4858):1544–1546. doi: 10.1126/science.2897717. [DOI] [PubMed] [Google Scholar]
  13. Kurki P., Ogata K., Tan E. M. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry. J Immunol Methods. 1988 Apr 22;109(1):49–59. doi: 10.1016/0022-1759(88)90441-3. [DOI] [PubMed] [Google Scholar]
  14. Lloyd R. V., Wilson B. S., Varani J., Gaur P. K., Moline S., Makari J. G. Immunocytochemical characterization of a monoclonal antibody that recognizes mitosing cells. Am J Pathol. 1985 Nov;121(2):275–283. [PMC free article] [PubMed] [Google Scholar]
  15. Mitchell D., Ibrahim S., Gusterson B. A. Improved immunohistochemical localization of tissue antigens using modified methacarn fixation. J Histochem Cytochem. 1985 May;33(5):491–495. doi: 10.1177/33.5.3921605. [DOI] [PubMed] [Google Scholar]
  16. Miyachi K., Fritzler M. J., Tan E. M. Autoantibody to a nuclear antigen in proliferating cells. J Immunol. 1978 Dec;121(6):2228–2234. [PubMed] [Google Scholar]
  17. Ogata K., Kurki P., Celis J. E., Nakamura R. M., Tan E. M. Monoclonal antibodies to a nuclear protein (PCNA/cyclin) associated with DNA replication. Exp Cell Res. 1987 Feb;168(2):475–486. doi: 10.1016/0014-4827(87)90020-6. [DOI] [PubMed] [Google Scholar]
  18. Robbins B. A., de la Vega D., Ogata K., Tan E. M., Nakamura R. M. Immunohistochemical detection of proliferating cell nuclear antigen in solid human malignancies. Arch Pathol Lab Med. 1987 Sep;111(9):841–845. [PubMed] [Google Scholar]
  19. Takasaki Y., Deng J. S., Tan E. M. A nuclear antigen associated with cell proliferation and blast transformation. J Exp Med. 1981 Dec 1;154(6):1899–1909. doi: 10.1084/jem.154.6.1899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tanaka S., Hu S. Z., Wang T. S., Korn D. Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase alpha. J Biol Chem. 1982 Jul 25;257(14):8386–8390. [PubMed] [Google Scholar]
  21. Taylor I. W. A rapid single step staining technique for DNA analysis by flow microfluorimetry. J Histochem Cytochem. 1980 Sep;28(9):1021–1024. doi: 10.1177/28.9.6157714. [DOI] [PubMed] [Google Scholar]
  22. Thornthwaite J. T., Sugarbaker E. V., Temple W. J. Preparation of tissues for DNA flow cytometric analysis. Cytometry. 1980 Nov;1(3):229–237. doi: 10.1002/cyto.990010309. [DOI] [PubMed] [Google Scholar]

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