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Keywords:

  • Image analysis;
  • fluorescence microscopy;
  • lead disease

Abstract

The quantitation of the non-heme porphyrin content of circulating erythrocytes is an important tool in the screening, diagnosis, and management of lead disease, iron deficiency anemia, and certain porphyrias. Useful information about these pathological conditions may be obtained from the distribution of the non-heme porphyrin content of individual red blood cells. An image-based cytometry system was developed and used to determine the cellular distributions of zinc protoporphyrin (ZPP) in the erythrocytes of normal and lead-exposed human subjects. The fluorescence cytometry system described here lends itself to a wide range of statistical studies of cell populations, in which extrinsic fluorescent probes or antibodies may be used instead of porphyrin. Image-based cytometry appears to offer a number of important advantages over the more conventional flow cytometry systems, including greater sensitivity, applicability to a wider range of cellular parameters, and the ability to retain images of each cell used in the survey for subsequent examination. Pairs of absorption and fluorescence images of many fields of dispersed immobilized red cells were acquired by means of a program which controls the microscope stage, focussing, shutters, filter wheels, and a cooled, slow-scan CCD camera. The observed marked differences in the ZPP distributions of erythrocytes from donors with chronic and acute lead exposure are discussed in terms of the metabolism of internalized lead.