Flow Cytometric Analysis of Red Cell Surface Markers To Detect Changes Related to Red Cell Storage
JJ Taylor, D Bernard, C LeVeque, CC Chang. The Methodist Hospital and The Methodist Hospital Research Institute, Houston, TX
Background: Evidence has accumulated over 15 years that critically ill and cardiac surgery patients have poorer outcomes when transfused with stored older red cells (storage more than 14 days) compared with newer blood. The goal of the current study is to use flow cytometry assays to provide a more quantitative and accurate determination of changes of surface markers/antigens that occur with RBC storage. This may eventually lead to clinical assays to avoid the transfusion of the “old” blood unit to critically ill patients and to maximize the efficacy of usage of stored blood simultaneously.
Design: Samples were obtained from each donor and the related packed (P) RBC unit: 3 ml of fresh blood obtained at the time of donation, and 3 ml of blood from the PRBC unit at day of preparation (day 0), and at day 7, day 10, day 12, day 14, day 16, day 20, day 24 and day 35 after collection. Expression of phosphatidyl serine (PS), CD44, CD47, CD324, CD35, CD55, CD71, and CD59 was evaluated by flow cytometry. The ratio of mean fluorescence intensity (MFI) of each marker to MFI of isotype controls in RBC of each sample were obtained.
Results: RBC in storage showed a gradually decreased expression of CD47, CD55, and CD59 as well as gradual increased expression of PS over the period of storage. CD47, CD55, and CD59 showed statistically significant decreases from the time of collection at day 16, 35, 12 after storage respectively. CD55 showed decreasing expression between day 12 and day 14 but statistical significance was not reached. PS expression showed an increase at day 35 although not statistically significant. The remaining markers tested did not show significant changes over storage.
Conclusions: Reduced expression of CD55 and CD59 suggest that transfused aged RBC may be more susceptible to complement-mediated RBC lysis. The progressive decrease in CD47 antigen expression in stored RBC implies that loss of CD47 during storage may target transfused RBC for clearance from circulation. Thus, transfused aged RBC could lead to shorter life span of RBC than expected. In addition, the lysed RBC by complement mediated process and/or activation of macrophages could impact the immune system and induce a cytokine storm after transfusion of aged RBC. These findings suggest that the stored RBC may lose some of the surface markers during storage and that changes of surface marker expression on RBC determined by flow cytometry may serve as a sensitive surrogate marker to determine the aging process of stored packed RBC.
Wednesday, March 24, 2010 1:00 PM
Poster Session VI # 207, Wednesday Afternoon