References Does Liver Biopsy Overestimate Liver Iron Concentration?

functionality of SP cells that stain low for Hoechst. This is a valid concern to which we respond below. Data on the toxicity of H33342 have been available for decades, as it is well known that H33342 is powerful inhibitor of DNA sythesis.4 From these early experiments, it was very evident that the effects on cell viability varied tremendously between different cell types and Hoechst dye concentration.4,5 Work by Van Zant and Fry5 found that most hematopoietic progenitors, with the exception of colony-forming unit– megakaryocyte (CFU-Meg) and CFU-erythroid (CFU-E), were functionally unaffected after H33342 staining. Interestingly, CFU-spleen (CFUS), a heterogeneous population consisting of both short and long-term HSCs, was the least sensitive to Hoechst exposure under conditions similar to the ones used for our SPstain. In addition, experiments that we reported in 1997, in which we compared the long-term repopulation activity of whole SP cells with that of SP cells additionally exposed to Hoechst in the presence of the efflux inhibitor verapamil, demonstrated no differences in terms of function, as assayed by competitive transplantation experiments.2 We have also compared the clonogenicity of highly purified HSCs that were either stained or not with Hoechst 33342, and our results indicate that at least in vitro, there are no significant detrimental effects associated with Hoechst exposure.3 Even though Hoechst staining does involve a great deal of viability loss in nonhematopoietic tissues,6 as pointed out, it does not seem to cause any functional impairment within the primitive hematopoietic compartments. Therefore, we consider that our results do represent an inherent difference in the self-renewal capacity of cells within the SP subset that is directly correlated with their ability to efflux dye.