DIRECT DETECTION OF RED BLOOD CELL FRAGMENTS

Abstract

Today, several types of blood pumps are available for a variety of clinical applications. Over the last decade these pumps have been continuously improved to reduce hemolysis. Today, the Normalized Index of Hemolysis (NIH) is the only strategy to evaluate pump-induced blood cell damage and to assess the hemolytic characteristics of prototype blood pumps. However, this spectrophotometric method has disadvantages since cell damage is indirectly calculated from free plasma hemoglobin levels. The aim of this study is to establish a new method that is able to detect red blood cell (RBC) fragments directly. Method: Whole blood flow cytometry was used to quantify circulating RBC fragments derived from a roller pump (Sarns Inc. 2M 6002) and a centrifugal pump (Baylor Gyro C1E3). The pumps were tested in a mock circuit for four hours (5L/min flow against 100 mm Hg pressure head) using fresh human anti-coagulated blood (1IU heparin /ml, ACT 250 sec.). RBC fragments were quantified by PE-labeled glycophorin A antibody specific for erythrocytes. RBC fragments were smaller than the intact RBC population and the fragments overlapped with the platelet population in size based on forward and side light scattering measurements. RBC fragments were differentiated from platelets based on their positive PE fluorescence. Results: Untreated control samples showed a concentration of 1,080±190 RBC fragments. For the roller pump, the values for RBC fragments increased rapidly by ∼10-fold after 15 min (9900/μl) and tremendously (185,000/μl) after 240 min. In contrast, for centrifugal pump, there is no increase of RBC fragments after 15 min (1070/μl) and only a modest increase of about 3-fold at 240 min. Conclusion: These results demonstrate, that the centrifugal pump has advantages in terms of hemolysis compared with roller pumps. More important, these studies demonstrate, that it is now feasible using flow cytometry to evaluate the extent of blood cell damage in artificial organs. By directly detecting the presence of red blood cell fragments, this sensitive method seems to have advantages over the NIH calculation.