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Accurate dosing of protamine reversal following on-pump cardiac surgical procedures is challenging, with both excessive and inadequate administration recognised to increase bleeding risk. We aimed to examine the relationship between three ratios for heparin reversal and markers of haemostasis.A retrospective analysis of a prospectively collected database was undertaken at a single tertiary cardiac unit, reviewing all cases of on-pump coronary artery bypass grafts and single valve replacements from 01/01/2011 to 31/12/2015. The ratio between total intra-operative heparin and protamine was stratified to three groups (low: ≤0.6 mg per 100 IU of heparin, moderate: 0.6-1.0 and high: >1.0) and related to the primary outcome of red blood cell (RBC) transfusion, with secondary outcomes being the number of units transfused, the haemoglobin differential and mediastinal drain output at 4 hours.Of the 803 patients identified, 338 received a blood transfusion, with 1035 units being used. Eighteen percent of individuals (145) received a low ratio, 50% (404) received a moderate ratio and 32% (254) a high ratio. Using the moderate group as a reference, the low dose group was 56.5% less likely to have received a RBC transfusion (OR 0.435; 95% CI 0.270:0.703 p=0.001) while the high dose group carried a 241% increased association with transfusion (OR 3.412; 95% CI 2.399:4.853 p<0.001). For those transfused, a lower protamine:heparin ratio was associated with a lower number of units transfused, lesser haemoglobin differential and less mediastinal drain output.Higher doses of intra-operative protamine relative to heparin are associated with greater risk of transfusion and post-operative bleeding.