Detection of Mutated Erythrocytes in Man

  • William L. Bigbee
  • Elbert W. Branscomb
  • Ronald H. Jensen
Part of the Environmental Science Research book series (ESRH, volume 30)


Two assay systems are being developed to measure the level of in vivo somatic mutations in human cells. Both are based on immunologic recognition and fluorescence-activated cell sorter enumeration of cells carrying variant proteins. The first assay is based on the detection of erythrocytes containing the single amino acid-substituted hemoglobins S or C. Frequencies of anti-hemoglobin S- and C-labeled red cells in the blood of normal hemoglobin A individuals were determined. In five samples, the S-frequencies ranged from 1.1 x 10-8 to 1.1 x 10-7. C-frequencies from 6.7 x 10-8 to 2.6 x 10-7 were observed in three samples. Methods to test the genetic validity of these results and to extend the measurements to additional point- and frameshift-mutant hemoglobins through the use of monoclonal antibodies are discussed. The second assay seeks to detect variant red cells arising as a result of “gene expression loss” and point mutations in the genes for the membrane-associated protein glycophorin A. Monoclonal antibodies, specific for the M and N allelic forms of the protein, have been produced and can be used to screen blood samples from MN heterozygotes for variant red cells which fail to present one of the glycophorin A forms. These antibodies may also be capable of detecting red cells in homozygotes expressing single amino acid-substituted glycophorin A.


Variant Cell Globin Gene Single Amino Acid Substitution Flow Sorter Germinal Mutation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    G. H. Strauss and R. J. Albertini, Enumeration of 6-Thioguan- ine-Resistant Peripheral Blood Lymphocytes in Man as a Potential Test for Somatic Cell Mutations Arising in vivo, Mutation Res., 61:353–379 (1979).CrossRefGoogle Scholar
  2. 2.
    T. H. J. Huismari and J. H. P. Jonxis, The Hemoglobinopathies: Techniques of Identification, Marcel Dekker, New York (1977).Google Scholar
  3. 3.
    Th. Papayannopoulou, T. C. McGuire, G. Lim, E. Garzel, P. E. Nute, and G. Stamatoyannopoulos, Identification of Haemoglobin S in Red Cells and Normoblasts, Using Fluorescent Anti-Hb S Antibodies, Brit. J. Haemat., 34:25–31 (1976).CrossRefGoogle Scholar
  4. 4.
    Th. Papanannouploulou, G. Lim, T. C. McGuire, V. Ahern, P. E. Nute, and G. Stamatoyannopoulos, Use of Specific Fluorescent Antibodies for the Identification of Hemoglobin C in Erytho- cytes, Amer. J. Hemat., 2:105–112 (1977).CrossRefGoogle Scholar
  5. 5.
    G. Stamatoyannopoulos, Possibilities for Demonstrating Point Mutations in Somatic Cells, as Illustrated by Studies of Mutant Hemoglobins, in: Genetic Damage in Man Caused by Environmental Agents (K. Berg, ed.), pp. 49–62, Academic Press, New York (1979)Google Scholar
  6. 6.
    K. Wang and F. M. Richards, Reaction of Dimethyl-3,3-dithio- bispropionimidate with Intact Human Erythrocytes, J. Biol. Chem., 250:6622–6626 (1975).Google Scholar
  7. 7.
    W. L. Bigbee, E. W. Branscomb, H. B. Weintraub, Th. Papayannopoulou, and G. Stamatoyannopoulous, Cell Sorter Immunofluorescence Detection of Human Erythrocytes Labeled in Suspension with Antibodies Specific for Hemoglobins S and C, J. Immunol. Meth., 45:117–127 (1981).CrossRefGoogle Scholar
  8. 8.
    J. J. Aragon, J. E. Feliu, R. A. Frenkel, and A. Sols, Perme- abilization of Animal Cells for Kinetic Studies of Intracellular Enzymes: In situ Behavior of the Glycolytic Enzymes of Erythrocytes, Proc. Natl. Acad. Sci. (U.S.A.), 77:6324–6328 (1980)ADSCrossRefGoogle Scholar
  9. 9.
    H. W. Goedde, H.-G. Benkmann, and L. Hirth, Ultrathin-Layer Isoelectric-focusing for Rapid Diagnosis of Protein Variants, Hum. Genet., 57:434–436 (1981).CrossRefGoogle Scholar
  10. 10.
    S. I. 0. Anyaibe and V. E. Headings, Identification of Inherited Protein Variants in Individual Erythrocytes, Biochem. Genet., 18:455–463 (1980).CrossRefGoogle Scholar
  11. 11.
    F. A. Garver, M. B. Baker, C. S. Jones, M. Gravely, G. Altay, and T. H. J. Huisman, Radioimmunoassay for Abnormal Hemoglobins, Science, 196:1334–1336 (1977).ADSCrossRefGoogle Scholar
  12. 12.
    J. A. Berzofsky, G. Hicks, J. Fedorko, and J. Minna, Properties of Monoclonal Antibodies Specific for Determinants of a Protein Antigen, Myoglobin, J. Biol. Chem., 255:11188–11191 (1980).Google Scholar
  13. 13.
    G. Stamatoyannopoulos, D. Lindsley, Th. Papayannopoulos, M. Farquhar, M. Brice, P. E. Nute, G. R. Serjeant, and H. Lehmann, Mapping of Antigenic Sites on Human Haemoglobin by Means of Monoclonal Antibodies and Haemoglobin Variants, Lancet ii, 952–954 (1981).CrossRefGoogle Scholar
  14. 14.
    H. F. Bunn, G. J. Schmidt, D. N. Haney, and R. G. Dluhy, Hemoglobin Cranston, an Unstable Variant Having an Elongated 3 Chain due to Nonhomologous Crossover between Two Normal 3 Chain Genes, Proc. Natl. Acad. Sci. (U.S.A.), 72:3609–3613 (1975).ADSCrossRefGoogle Scholar
  15. 15.
    G. Flatz, J. L. Kinderlerer, J. V. Kilmartin and H. Lehmann, Haemoglobin Tak: A Variant with Additional Residues at the End of the 3 Chains, Lancet i, 732–733 (1971).CrossRefGoogle Scholar
  16. 16.
    J. R. Shaeffer, G. J. Schmidt, R. E. Kingston, and H. F. Bunn, Synthesis of Hemoglobin Cranston, an Elongated 3 Chain Variant, J. Mol. Biol., 140:377–389 (1980).CrossRefGoogle Scholar
  17. 17.
    K. Imai and H. Lehman, The Oxygen Affinity of Haemoglobin Tak, a Variant with an Elongated 3 Chain, Biochim. Biophys. Acta, 412:288–294 (1975).Google Scholar
  18. 18.
    G. Stamatoyannopoulos, P. E. Nute, Th. Papayannopoulou, T. McGuire, G. Lim, H. F. Bunn, and D. Rucknagel, Development of a Somatic Mutation Screening System Using Hb Mutants, IV. Successful Detection of Red Cells Containing the Human Frameshift Mutants Hb Wayne and Hb Cranston Using Monospecific Fluorescent Antibodies, Am. J. Hum. Genet., 32:482–496 (1980).Google Scholar
  19. 19.
    H. Furthmayer, Structural Analysis of a Membrane Glycoprotein: Glycophorin A, J. Supramol. Struct., 7:121–134 (1977).CrossRefGoogle Scholar
  20. 20.
    C. G. Gahmberg, M. Jokinen, and L. C. Andersson, Expression of the Major Red Cell Sialoglycoprotein, Glycophorin A, in the Human Leukemic Cell Line K562, J. Biol. Chem., 254:7442–7448 (1979).Google Scholar
  21. 21.
    H. Furthmayer, Structural Comparison of Glycophorins and Im-munochemical Analysis of Genetic Variants, Nature, 271:519–524 (1978).ADSCrossRefGoogle Scholar
  22. 22.
    D. Pious and C. Soderland, HLA Variants of Cultured Human Lymphoid Cells: Evidence for Mutational Origin and Estimation of Mutation Rate, Science, 197:769–771 (1977).ADSCrossRefGoogle Scholar
  23. 23.
    P. Kavathas, F. H. Bach, and R. DeMars, Gamma Ray-Induced Loss of Expression of HLA and Glyoxalase I Alleles in Lymphoblastoid Cells, Proc. Natl. Acad. Sci. (U.S.A.), 77:4251–4255 (1980).ADSCrossRefGoogle Scholar
  24. 24.
    P. A. W. Edwards, Monoclonal Antibodies that Bind to the Human Erythrocyte-Membrane Glycoproteins Glycophorin A and Band 3, Biochem. Soc. Trans., 8:334–335 (1980).Google Scholar
  25. 25.
    P. N. Dean and D. Pinkel, High Resolution Dual Laser Flow Cytometry, J. Histochem. Cytochem., 26:622–627 (1978).CrossRefGoogle Scholar
  26. 26.
    M. J. A. Tanner and D. J. Anstee, The Membrane Change in En(a-) Human Erythrocytes, Biochem. J., 153:271–277 (1976).Google Scholar
  27. 27.
    H. Furthmayer, M. N. Metaxas and M. Metaxas-Bühler, Mg and Mc: Mutations within the Amino-Terminal Region of Glycophorin A, Proc. Natl. Acad. Sci. (U.S.A.), 78:631–635 (1981).ADSCrossRefGoogle Scholar
  28. 28.
    W. Dahr, M. Kordowicz, K. Beyreuther, and J. Krüger, The Amino- Acid Sequence of the Mc-Specific Major Red Cell Membrane Sialo- glyoprotein - An Intermediate of the Blood Group M- and N-Active Molecules, Hoppe-Seyler’s Z. Physiol. Chem., 362:363–366 (1981).CrossRefGoogle Scholar
  29. 29.
    R. M. Lawn, A. Efstratiadis, C. O’fConnell, and T. Maniatis, The Nucleotide Sequence of the Human β-Globin Gene, Cell, 21: 647–651 (1980).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1984

Authors and Affiliations

  • William L. Bigbee
    • 1
  • Elbert W. Branscomb
    • 1
  • Ronald H. Jensen
    • 1
  1. 1.Biomedical Sciences DivisionUniversity of California Lawrence Livermore National LaboratoryLivermoreUSA

Personalised recommendations