Reevaluation of the Harboe assay as a standardized method of assessment for the hemolytic performance of ventricular assist devices.

The Harboe spectrophotometric assay is regarded as one of the safest and most reproducible methods for measuring plasma free hemoglobin (pfHb). However, there is still some ambiguity in the application of the assay when assessing the hemolytic performance of ventricular assist devices (VADs). The purpose of this study was to reexamine and compare values of pfHb obtained using different concentrations of plasma diluent (Na(2) CO(3) ) as cited by various studies such that a standard practice may be recommended for the application of the Harboe assay in the hemolytic evaluation of VADs, allowing reliable comparisons to be made between laboratories. As a means to examine the Harboe assay, a BioMedicus BPX-80 was tested using both whole blood and a washed suspension of red blood cells (RBCs). Results show that for whole blood, the pfHb may be underestimated by 13-23%, dependent upon the concentration of Na(2) CO(3) diluent solution. This trend was not observed for the washed suspension of RBCs. Furthermore, it is shown that the concentration of diluent influences the stability of a sample. The results of this study show that the problems associated with the incongruity of pfHb readings are a direct result of the precipitation of proteins from the plasma under alkaline conditions; as the molarity of the diluent controls pH, it becomes essential to use the appropriate concentration of Na(2) CO(3) diluent in order to avoid turbidity of the solution and the consequent misrepresentation of pfHb values. Such standardization is pertinent when measuring the very low levels of pfHb observed during the in vivo testing of modern ventricular assist devices.