(522f) Transfusion Effects on WBC Adhesion in Sickle Cell Disease Patients

Sickle cell disease (SCD) is a devastating hemoglobin disorder where an inherited mutation causes the production of hemoglobin S (HbS) in a fraction of RBCs (%S, the fraction of RBCs with HbS). Unlike healthy hemoglobin that is free floating, HbS can spontaneously form crystalline domains that cause RBC shape change and rigidification. Rigid RBCs contribute to a number of complications such as increased clotting, increased risk for capillary occlusions, decreased O₂ delivery, systemic inflammation, organ damage, anemia, reduced WBC adhesion, and reduced quality and length of life. One of the most common treatments for SCD is a transfusing the patient with healthy RBCs. This effectively dilutes the %S by increasing total RBC count ( ~%Hct) making the blood less rigid on average and treating anemia. However, little is understood how the extent of transfusion affects WBC adhesion. This study seeks to describe how the interplay between %Hct, %S, and transfusion affect WBC adhesion by testing WBC adhesion in vitro on both a series of ex vivo transfusions of healthy RBCs to SCD patient blood samples in parallel with the patient receiving a transfusion. We show %S is directly related to blood rigidity via ektacytometry and the diversity of WBC adhesion changes as a function of transfusion. Finally, we propose a model to predict the most common extent of transfusion that results in maximized WBC adhesion using only clinically relevant and directly measurable variables.

Methods: Informed consent was obtained before blood collection from all subjects and parents of minors involved in the study. This study has been approved by the University of Michigan Internal Review Board (IRB-MED and IRB-HSBS). Healthy RBCs were separated from whole blood samples using centrifugation with the plasma and white blood cells stored separately. SCD patient blood was collected both pre and post transfusion. Both pre and post samples were sent to ektacytometry to assess rigidity. A confluent layer of HUVEC was allowed to grow on coverslips and inflamed 4 hours prior to use. Flow experiments were completed in a parallel flow plate chamber at 500 s-1 to mimic relevant shear conditions. WBC adhesion to the HUVEC was imaged using light microscopy during a flow experiment for all blood samples.

Result and Conclusions: This work quantifies WBC adhesion across a gradient of %Hct and compares them to both the pre and post transfusion values, Figure 1. We validate our ex vivo transfusion against a parallel real transfusion and find good agreeance. We find that %S and %Hct play a large role in WBC adhesion which aligns with previously published literature. Transfusions simultaneously reduce %S and increase %Hct making them inversely related. Using theses metrics together can be used to predict WBC adhesion maximum transfusion point in a clinically relevant matter. Future investigations will be made into quantifying SCD patient outcome changes based off of extent of transfusion.