Flow cytometric detection of human red cells labeled with a fluorescent membrane label: potential application to in vivo survival studies

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Abstract
In vivo survival studies of human red cells (RBCs) are commonly carried out by using chromium-51 (51Cr), a gamma-emitting radionuclide, as the cell label. The effects of labeling human RBCs with PKH-2, a nonradioactive lipophilic fluorescent dye that binds to the cell membrane, and the feasibility of detecting the labeled cells by flow cytometric analysis were investigated. Optimal labeling, defined as maximum mean fluorescence intensity with minimal cell-to-cell variability in fluorescence intensity and minimal cell loss, was achieved with the use of 15.0 x 10(-6) M (15.0 x 10(-6) mol/L) PKH-2 and a cell concentration of 4.0 x 10(9) RBCs per mL. Both freshly drawn and stored RBCs could be labeled, but RBCs stored for more than 20 days did not take up the label as uniformly as fresher cells. Although labeling with PKH-2 did not interfere with the detection of ABO, Rh(D), or common minor RBC antigens by routine serologic methods, it resulted in a morphologic appearance resembling echinocytosis and an increased resistance to osmotic lysis by hypotonic saline. RBCs labeled by this method could be quantitated accurately in blood samples in which their proportion was 0.01 percent, or 1 labeled cell in 10,000 cells. This method holds promise as a simple, reliable, and sensitive method for the detection of labeled human RBCs, but the in vivo significance of the label's effects on cell morphology and osmotic fragility is not known. Further studies directly comparing PKH-2-labeled and 51Cr-labeled RBCs will be necessary to establish the accuracy of the former method in determining the in vivo survival of human RBCs.