Counting of RBC Variants Using Rapid Flow Techniques

Abstract

Presently there are important clinical, occupational and environmental needs for assays to estimate somatic mutation rates in human individuals. These methods are based on the enumeration of rare cells which present a variant phenotype as a result of somatic mutations having occurred in the DNA of single structural genes in precursor cells. To identify and quantitate such cells, an efficient and specific selection method must be employed; either clonogenic assay of mutant cells or immunologic detection of mutant gene products. Also, there must be an independent means for validating that the identified cells are, in fact, structural gene mutants, e.g., maintenance of the mutant phenotype under non-restrictive conditions or direct biochemical demonstration of the presence of a variant protein. In this paper, I will describe two assay systems we are developing based on high speed sorter detection of immunologically identified mutant human erythrocytes. Both take advantage of the ability of the flow sorter to rapidly screen large numbers of cells to enumerate and sort rare, presumptively mutant, cells labeled with fluorescent antibodies.

Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48 with the financial support of the National Institute of Environmental Health Sciences Interagency Agreement No. 222401-ES-00060 and the Environmental Protection Agency, Grant No. R808642-01.