Automated quantitation of hemoglobin-based blood substitutes in whole blood samples

It is necessary to develop methods for accurate monitoring of cell-free hem oglobin in circulation. Routine monitoring of circulating cell-free hemoglo bin will be useful for evaluating the efficacy of blood substitute administ ration and for determining the clearance rates of the blood substitute from circulation. In addition, discriminating between cell-fi-ee hemoglobin and cell-associated hemoglobin will enable accurate determination of RBC indic es, mean cell hemoglobin and mean corpuscular hemoglobin concentration, in individuals receiving hemoglobin-based blood substitutes. As colorimetric m ethods used by hematology analyzers to quantitate the hemoglobin value of a blood sample cannot distinguish between cell-associated and cell-free hemo globin, it is currently not feasible to quantitate the levels of hemoglobin substitutes in circulation. The advent of a technology that measures volum e and hemoglobin concentration of individual RBCs provides an alternative s trategy for quantitating the cell-associated hemoglobin in a blood sample. We document that the combined use of cell-based and colorimetric hemoglobin measurements provides accurate discrimination between cell-associated and cell-free hemoglobin over a wide range of hemoglobin levels. This strategy should enable rapid and accurate monitoring of the levels of cell-free hemo globin substitutes in the circulation of recipients of these blood substitu tes.