Neutralization of heparin using a protamine titration assay and the activated clotting time test

Abstract

Many of the clinical protocols currently used to calculate the protamine (Pr) dosage for heparin neutralization following cardiopulmonary bypass (CPB) utilize an empirical Pr to heparin ratio. Pr titration, though a proven method of dosage calculation, is inherently tedious and thus not routinely used in the operating room (OR). In the present study Pr titration was performed by preparation of diatomaceous earth-activated clotting time (ACT) test tubes containing known amounts of lyophilized Pr. The ACT determined in these test tubes was called the P-ACT. The Pr titration curve was constructed using a status (heparinized) ACT (S-ACT) and P-ACT at Pr concentrations of 10, 15, 20 and 30 mcg/ml. The ACT was performed using an automated technique (Hemochron). The intercept of the linear regression dose-response neutralization curve (ACT vs Pr concentration) with the baseline ACT was the calculated Pr concentration required for neutralization. Addition of calculated Pr to in vitro heparinized donor blood normalized the ACT and non-activated clotting time (NACT). A two-point dose-response curve (S-ACT and P-ACT, 15 mcg/ml) produced equivalent results to the linear regression dose-response calculation (r=.95). In 30 CPB patients infusion of the calculated Pr (based on the two-point assay and the patient's blood volume, adjusted for changes during CPB) normalized the ACT and produced clinical hemostasis. This assay provides a method of Pr quantification in the OR reducing the likelihood of over- or under-infusion of protamine and the adverse consequences associated with either.