Tracking Syngeneic Leukocytes after Transfusion of Non-leukoreduced Blood in a Murine Model

Introduction

Red Blood Cell (RBC) transfusions are used worldwide as a lifesaving procedure in many health care settings. Several Western European countries and Canada have introduced white blood cell (WBC) reduction as a universal standard to prevent potential adverse effects of allogeneic blood transfusions. There is an ongoing controversy regarding its appropriateness in other countries such as North America .WBC reduction has been shown to reduce febrile, non-hemolytic transfusion reactions,alloimmunization to leukocyte antigens and transmission of cytomegalovirus.Potential, but not established, benefits of leukoreduction include reduction of transfusion associated immunomodulation, bacterial overgrowth, reperfusion injury following cardiopulmonary bypass and RBC storage lesion. Additionally, the potential to reduce infection by pathogens that are primarily associated with WBCs may extend beyond cytomegalovirus. The U.S. Food and Drug Administration (FDA) supports the use of leukocytes reduced blood ,but leukoreduction has not been fully implemented by all blood establishments. This in vivo study examines where syngeneic leukocytes can be found in the recipient after transfusion of nonleukoreduced blood in a time-dependent murine model in order to provide further insights into potential mechanisms of WBC-associated adverse events.

Materials and methods:Animals:All animal experiments received institutional approval by the Regierungspräsidium Darmstadt, Germany. 12–14 weeks old, male enhanced green fluorescent protein (EGFP) transgenic C57BL/6- Tg(CAG-EGFP)1 Osb/J mice (kindly provided by Dr. Johannes Holfeld and Prof. Michael Grimm, Innsbruck, Department of cardiothoracic surgery, University Hospital Innsbruck, Austria), which express EGFP in all blood cells with the exception of erythrocytes, were used as blood donors. 10–12 weeks old C57BL/6 wildtype mice (Janvier Laboratory, St Berthevin Cedex, France) were used as blood recipients.

Blood collection and transfusion:Animal handling and surgery were performed according to the European guidelines. Blood donor mice were anesthetized via intraperitoneal application of ketamine/xylazine (100/10 mg/kg) and anesthesia was maintained with isoflurane per inhalationem. Blood was retrieved via an intra-arterial catheter, anticoagulated with 14% citrate-phosphatedextrose-adenine (CPDA)-1 (Sigma-Aldrich, Munich, Germany) and pooled. It was then centrifuged for 15 min at 600 x g, adjusted to a hematocrit of 70% to 75% by removing plasma, and stored in Eppendorf tubes at 4°C until transfusion the next day. C57BL/6 wildtype mice were randomized into a control group (n=3) and a transfusion group (n=6). Each mouse of the latter group was transfused with 110 μl of EGFP-labeled blood via a tail vein.

Control mice received the same volume of 0.9% sodium chloride balanced electrolyte solution. Blood and organs of three transfused mice and one control mouse were harvested on day 3 and day 5 after transfusion, respectively. For the extraction of bone marrow, the femur was explanted, the epiphyses were cut off and the medullary cavity flushed with PBS.