Summary
The method of cytofluorometric measurement of the contents of Hb and nuclear DNA on a single erythroid cell (Fukuda et al., 1975, 1977 a) was used for the quantitative analysis of the erythropoiesis in normal human bone marrow.
The intracellular Hb in an erythroid cell was converted to fluorescent porphyrin after removing the Giemsa staining by irradiation with violet light in the presence of SH-donor (mercaptoethylamine hydrochloride, MEA) and its nuclear DNA was subsequently stained with pararosaniline Feulgen staining. With the two quantitative parameters, Hb content and DNA amount, the erythroid cells in normal human bone narrow were classified into 6 classes of different maturation stages (EI-EIV).
The morphological characteristics of the most primitive erythroblast (EI cells) were described. The “proerythroblasts” which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb, thereby indicating that they are rather aberrations from the normal steps of cell maturation. The DNA amounts of “orthochromatic erythroblasts” (EV cells) showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA from the erythroblast is not exclusively due to mechanical expulsion of a whole intact nucleus.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Awai, M., Okada, S., Takebayashi, J., Kubo, T., Inoue, M., Seno, S.: Studies on the mechanism of denucleation of the erythroblast. Acta haemat. 39, 193–202 (1968)
Böhm, N., Sprenger, E.: Fluorescence cytophotometry: A vauable method for quantitative determination of nuclear Feulgen-DNA. Histochemie 16, 100–118 (1968)
Böhm, N.: Fluorescence cytophotometric determination of DNA. In: Techniques of Biochemical and Biophysical Morphology, vol. I (ed. by D. Glick, R.M. Rosenbaum) p. 90–141. New York-London-Sydney-Toronto: John Wiley and Sons 1972
Flatmark, T.: A histochemical technique for the demonstration of cytochrom c. Nature 200, 1112–1113 (1963)
Förster, T.: Fluorescent organischer Verbindung. Göttingen: Vandenhoeck & Ruprecht 1951
Fujita, S., Yoshida, S., Fukuda, M.: Improvement of technique to minimize non-specific absorption in microspectrophotometric measurements of nuclear DNA. Acta histochem. cytochem. 4, 126–136 (1971)
Fujita, S., Ashiara, T., Fukuda, M., Hattori, T., Yoshida, S.: Combination of Feulgen cytofluorometry with 3 H-thymidine autoradiography. Acta histochem. cytochem. 6, 205–211 (1973)
Fujita, S.: DNA cytofluorometry on large and small nuclei stained with pararosaniline Feulgen. Histochemie 36, 193–199 (1973)
Fujita, S., Fukuda, M.: Irradioation of specimen by excitation light before and after staining with pararosaniline Feulgen: A new method to reduce non-specific fluorescence in cytofluorometry. Histochemistry 40, 59–67 (1974)
Fujita, S., Ashihara, T., Fukuda, M.: Simultaneous measurement of DNA content and grain count on an autoradiograph of Feulgen stained cells. Histochemistry 40, 155–163 (1974)
Fukuda, M., Isemura, T., Maruo, N., Nakanishi, K., Fujita, S.: Cytofluorometric measurement of contents of nuclear DNA and intracellular porphyrin converted from heme or hemoglobin on a single erythroid cell. Acta histochem. cytochem. 8, 331–341 (1975)
Fukuda, M., Nakanishi, K., Mukainaka, T., Shima, A., Fujita, S.: Combination of Feulgen nuclear reaction with immunofluorescent staining for photoproducts of DNA after UV-irradiation. Acta histochem. cytochem. 9, 180–192 (1976a)
Fukuda, M., Nakanishi, K., Mukainaka, T., Shima, A., Fujita, S.: Cytofluorometric determination of excision repair or UV-induced pyrimidine dimers in Ehrlich ascites cancer cells. Acta hictochem. cytochem. 9, 203–213 (1976b)
Fukuda, M., Isemura, T., Maruo, N., Nakanishi, K., Fujita, S.: Cytofluorometric quantitation of the hemoglobin and DNA contents of a single erythroid cell. Acta histochem. cytochem. (1977a) (in press)
Fukuda, M., Nakanishi, K., Sawamura, I., Fujita, S.: Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry. Histochemistry (1977b) In press
Granick, S., Levere, R.D.: The intracellular localization of heme by a fluorescence technique. J. Cell Biol. 26, 167–176 (1965)
Holtzer, H.: DNA dependent events during myogenesis and erythrogenesis. Seitai-no-Kagaku 25, 43–51 (1974)
Israel, O., Pappenheim, A.: Über die Entkernung der Säugetiererythroblasten. Virchows Arch. path. Anat. 143, 419–447 (1986)
Lewis, S.M.: The constituent of normal blood. In: Blood and its Disorders, vol. I (ed. by R.M. Hardisty, D.J. Weatherall) p. 3–67. London: Blackwell Scientific Publication 1974
Rind, H.: Kinetik der Erythroblastenentkernung mit Mikrofilmdemonstration (Phasenkontrast). Folia haemat. (Lpz.) 74, 262–266 (1963)
Rosse, C.: Small lymphocyte and transitional cell populations of the bone marrow; their role in the mediation of immune and hemopoietic progenitor cell functions. In: International Review of Cytology, vol. 45 (ed. by G.H. Bourne, J.F. Danielli) p. 156–290. New York-San Francisco-London Academic Press 1976
Thorell, B.: Studies on the formation of cellular substances during blood cell formation. Acta Med. Scand., suppl. 200 (1947)
Undritz, E.: Sandoz Atlas of Haematology. Basel: Sandoz 1952
Weintraub, H., Campbell, G.LE.M., Holtzer, H.: Primitive erythropoiesis in early chick embryogenesis. I. Cell cycle kinetics and control of cell division. J. Cell Biol. 50, 652–668 (1971)
Wintrobe, M.M.: Clinical Hematology, V. Philadelphia: Lea and Febiger 1961
Author information
Authors and Affiliations
Additional information
Partly supported by Alexander von Humboldt-Stiftung and Deutsche Forschungsgemeinschaft DFG-Grant Bo 395/3
Rights and permissions
About this article
Cite this article
Fukuda, M., Maruo, N., Isemura, T. et al. Cytofluorometric analysis of cell proliferation and differentiation of the human erythroblasts. Histochemistry 52, 317–327 (1977). https://doi.org/10.1007/BF00508404
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00508404