Increased Free Heme Levels and Red Blood Cell Transfusions Are Associated with Thrombosis in Neonates and Infants Undergoing Cardiac Surgery for Congenital Heart Disease
Authors: Stolla Moritz , Henrichs Kelly , Cholette Jill M. , Pietropaoli Anthony , Phipps Richard P. , Spinelli Sherry , Blumberg Neil 
Published: November 2019

Abstract
Background: Elevated free heme levels have been shown to be associated with thrombosis in a variety of diseases as well being experimentally causative in vitro and in vivo. Stored red blood cells hemolyze both in the bag and in the recipient, raising the question of whether red blood transfusion is prothrombotic. Transfusions have been associated with an increased risk of thrombosis in several patient cohorts, including one randomized trial. While neonates and infants are generally considered to be in a slightly hypocoaguable state, certain complex congenital heart diseases and their surgical repair can put them at increased risk for thrombosis. Red blood cell transfusions may further increase this risk for thrombosis. It is unclear if transfusions result in increased heme levels and if this increase is associated with thrombotic events in this setting.
Method: We retrospectively analyzed samples from 39 children who underwent cardiac surgery with cardiopulmonary bypass from 2009-2010, including 15 children who developed a post-operative thrombosis and 24 age matched controls from the same randomized trial of washed blood transfusions. The infants/children were between 0 days to 60 months of age. All patients received leukoreduced, irradiated and ABO identical red blood cell products. Seven patients in the thrombosis group and 13 patients in the non-thrombosis group received washed products (47% and 54%, respectively, no significant difference). Free heme was quantitated by colorimetric analysis of previously frozen citrated plasma samples pre-surgery, immediately post-operatively and 6 and 12 hours post-operatively.
Results: Patients that developed thrombosis post-operatively had significantly greater increased average heme levels over time (pre-surgery to 12 hours post-surgery) compared to patients that did not develop thrombosis (39.8μM vs. 29.3 μM respectively, p<0.001). The analysis of different time points revealed significantly higher heme levels immediately pre-surgery (30.7μM vs. 17.4 μM p=0.032), and 6 hours post surgery (38μM vs. 26.5μM, p=0.028), with a trend for higher levels after 12 hours of surgery (38.7μM vs. 28.3μM, p=0.17) for patients who developed thrombosis during their hospitalization. Interestingly, other hemolysis parameters, such as hemoglobin, haptoglobin, and iron did not differ significantly between the two groups. The patients in the thrombosis group received significantly more blood transfusions than the group without thrombosis (8 vs. 2 respectively, p<0.001) with a significantly higher dose of transfusions (910ml vs. 561ml, p<0.01). The average number of transfusions was 4 before the thrombotic event occurred and the average time from the last transfusion to the clinical detection of thrombosis was 12h.
Conclusion: Increased cell-free heme levels are associated with a higher risk of thrombosis in a transfused pediatric cardiac surgical cohort. Higher heme levels are also associated with the number and volume of transfusions. This is a potentially important finding since it highlights for the risk for transfusion-associated thrombosis in another vulnerable patient cohort. Further studies are warranted to examine the possible mechanistic role of heme in contributing to thrombosis in transfused patients.

Disclosures
No relevant conflicts of interest to declare.