Automated Quantitation of Hemoglobin-Based Blood Substitutes in Whole Blood Samples

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Abstract
It is necessary to develop methods for accurate monitoring of cell-free hemoglobin in circulation. Routine monitoring of circulating cell-free hemoglobin will be useful for evaluating the efficacy of blood substitute administration and for determining the clearance rates of the blood substitute from circulation. In addition, discriminating between cell-free hemoglobin and cell-associated hemoglobin will enable accurate determination of RBC indices, mean cell hemoglobin and mean corpuscular hemoglobin concentration, in individuals receiving hemoglobin-based blood substitutes. As colorimetric methods used by hematology analyzers to quantitate the hemoglobin value of a blood sample cannot distinguish between cell-associated and cell-free hemoglobin, it is currently not feasible to quantitate the levels of hemoglobin substitutes in circulation. The advent of a technology that measures volume and hemoglobin concentration of individual RBCs provides an alternative strategy 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 hemoglobin substitutes in the circulation of recipients of these blood substitutes.