The effects of cryopreservation on red blood cell microvesiculation, phosphatidylserine externalization, and CD47 expression.

BACKGROUND: Since the 1950s, cryopreservation has been used in transfusion medicine for long-term storage of phenotypically rare red blood cells (RBCs). Recent reports have identified phosphatidylserine (PS) exposure, loss of CD47 expression, and membrane microvesiculation as important indicators of RBC storage lesion and in vivo survival. The purpose of this study was to assess the effects of RBC cryopreservation and prefreeze storage length on these novel markers of membrane injury and to correlate them to traditional RBC quality indicators. STUDY DESIGN AND METHODS: Leukoreduced RBC units were collected in citrate-phosphate-dextrose (CPD)-saline-adenine-glucose-mannitol (SAGM), hypothermically stored (1-6 degrees C) for 2 to 3 days or 13 to 14 days after collection, and then cryopreserved in 40 percent (wt/vol) glycerol. In vitro RBC quality was assessed before freeze, after thaw, and 24 hours after thaw by evaluating RBC recovery, hemolysis, sterility, residual glycerol, adenosine triphosphate, extracellular potassium, RBC indices, and morphology. RBC membrane microvesiculation, PS externalization, and CD47 expression changes were evaluated using flow cytometry. RESULTS: Leukoreduced CPD-SAGM RBCs showed acceptable in vitro quality after deglycerolization, according to conventional assays. Cryopreservation alone did not induce significant changes in PS exposure, CD47 expression, and membrane microvesiculation. Prolonged prefreeze storage, however, resulted in a significant increase in RBC PS exposure and microvesiculation after 24 hours of postthaw hypothermic storage (1-6 degrees C). No significant changes in CD47 expression were detected. CONCLUSION: High-glycerol cryopreservation does not induce microvesiculation, PS exposure, and loss of CD47 expression in RBC membranes. Since prolonged prefreeze storage can result in RBC membrane injury during the postdeglycerolization storage period, more defined criteria for this variable should be adopted.

In human leukemia cells ephrin-B-induced invasive activity is supported by Lck and is associated with reassembling of lipid raft signaling complexes.

Proteins of the ephrin-B group operate in nonlymphoid cells through the control of their migration and attachment, and are crucial for the development of the vascular, lymphatic, and nervous systems. Ephrin-B activity is deregulated in various nonlymphoid malignancies; however, their precise role in cancer has only started to be addressed. We show here that ephrin-B1, a member of the ephrin-B group, is expressed in pediatric T-cell leukemias, including leukemia cell line Jurkat. Treatment of Jurkat cells with ephrin-B-stimulating EphB3 enhances ephrin-B1 phosphorylation and induces its relocalization into lipid rafts. These events are mediated by the T lineage-specific kinase, Lck, as ephrin-B1 phosphorylation and lipid raft association are blocked in the Lck-deficient clone of Jurkat, JCAM1.6. Ephrin-B1 also induces colocalization of the CrkL and Rac1 cytoskeleton regulators and initiates in leukemic cells a strong repulsive response. The absence of Lck blocks ephrin-B1-induced signaling and repulsion, confirming the essential role for Lck in ephrin-B1-mediated responses. This shows a new role for ephrin-B1 in the regulation of leukemic cells through the Lck-dependent Rac1 colocalization with its signaling partner, CrkL, in lipid rafts. In agreement with its repulsive action, ephrin-B1 seems to support metastatic properties of leukemic cells, as suppression of ephrin-B1 signaling inhibits their invasiveness. Because ephrin-B1-activating EphB proteins are ubiquitously expressed, our findings suggest that ephrin-B1 is likely to play an important role in the regulation of malignant T lymphocytes through the control of lipid-raft-associated signaling, adhesion, and invasive activity, and therefore may represent a novel target for cancer treatment.