Neutralization of Heparin Using a Protamine Titration Assay and the Activated Clotting Time Test

_____________ _ (J. Extra-Corpor. Techno/. 19 [3] p. 358-364 Fall1987, 16 ref.) 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 1 0, 15, 20 and 30 meg/mi. 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 doseresponse 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 twopoint assay and the patient's blood volume, adjusted for changes during CPB) normalized the ACT and Direct communications to: Dr. Frank LaDuca, Research Division, International Technidyne Corporation, 23 Nevsky St. Edison, NJ 08820 358 produced clinical hemostasis. This assay provides a method of Pr quantification in the OR reducing the likelihood of overor under-infusion of protamine and the adverse consequences associated with either. Introduction ______________ _ Protamine sulfate is used to neutralize the heparin infused during cardiovascular surgery procedures requiring cardiopulmonary bypass (CPB). The amount of heparin neutralized by protamine is in direct proportion to the amount of protamine present in the blood. Thus, titration methods can be accurately used to quantify the amount of protamine required to neutralize a given quantity of circulating heparin. Clinically useful protamine titration methods were originally described by Allen et a!. 1 and later modified by Perkins et aJ.2 and Hurt et a!. 3 These protocols involved in vitro addition of varying amounts of liquid protamine to heparinized blood and determination of the amount of protamine required to normalize the whole blood clotting time. Recently Dutton et a!. 4 described a similar protamine titration methodology, using the Activated Clotting Time (ACT) test as the means to determine the amount of protamine necessary to neutralize heparin and normalize the ACT. The ACT is the preferential method of heparin monitoring during CPBY Yet the clinical use of a protamine titration assay based on ACT technology has been limited due to the lack of a convenient and accurate assay. Many surgical teams continue to use one of several modifications of the Heparin Dose-Response Curve described by Bull et aJ.7 In this procedure, the patient's heparin concentration at the end of cardiopulmonary bypass is calculated based on an ACT determination. By converting this ACT into a heparin concentration, the protamine dose required to neutralize this heparin load is calculated using an empirical protamine to heparin ratio. In many clinical settings the ACT value is not used in determining protamine dose. Rather, protamine dosage is calculated based on a ratio of protamine to total heparin infused during the procedure. 8 10 Another method of dose calculation used is an automated heparin-protamine titration assay which is independent of the ACT methodology and incorporates an empirical protamine to heparin ratio. 6 Because of the pharmacology of protamine, however, it is important to quantify protamine dose and avoid the possible morbidity associated with underor over-infusion of protamine. 10 14 For these reasons, and since more accurate quantification of protamine dose can be achieved by using titration assays than by using empirical data, protamine titration is a preferred method of dose calculation. This paper describes a convenient and accurate method for protamine titration using the principle of dose-response heparin neutralization as monitored by the automated ACT. Materials and Methods ________ _ Protamine Titration Assay. Stock solution of protamine sulfate was prepared in 0.9% NaCl to a final concentration of 1 mg/ml. Protamine was added in varying amounts (10-160 mel) to Hemochrona brand test tubes ( # H106) and lyophilized in a vacuum dryer. Diatomaceous earth, 12 mg (celiteb), was added to each tube, the tubes were evacuated (to draw 2.5 mls), capped, and stored at room temperature. Test tubes containing protamine and diatomaceous earth were designated P-ACT test tubes. The titration assay was performed by addition of exactly two mls of blood to each tube resulting in final protamine concentrations of 5 to 80 meg/mi. Titration assays using liquid protamine were performed by addition of varying amounts of protamine (10-160 mel) to plastic tubes. Blood was added in an appropriate volume producing final protamine concentrations of 5-80 mel/mi. After mixing, 2.0 ml was transferred to a Hemochron ACT test tube (#CA510) and the ACT determined using standard Hemochron procedure. In some studies the non-activated clotting time was performed using #5412 test tubes. For in vitro studies, blood was obtained by venipuncture from a normal donor. One aliquot was immediately transferred to a #CA510 test tube for determination of the baseline ACT, and a second alia International Technidyne Corp., Edison, NJ 08820 b Johns Manville Corp., Denver, CO 80201 quot immediately added to a plastic tube containing heparin (derived from either porcine intestinec or beef lungct) in final concentration of 1-5 units/mi. Following mixing, 2.0 ml was transferred to a #CA510 test tube, for determination of the status ACT (S-ACT), and to each P-ACT test tube previously prepared. The ACT and P-ACT values were determined using Hemochron Models 400 or 800. These values were graphed on standard graph paper (y-axis) versus the corresponding protamine concentration (x-axis). The dose-response neutralization curve was constructed from the S-ACT to the baseline ACT by plotting the linear regression line of best fit among the P-ACT values falling within this range. The protamine value at the point of intercept of the neutralization curve with the baseline ACT represented the unit protamine concentration (UPC) required to normalize the ACT. Protamine Index Factor: The heparin neutralizing capacity of the protamines used clinically were quantified against the protamine used in the titration assay. This was done by simultaneous determination of the dose-response neutralization curve of a heparinized blood specimen using both protamine preparations. For each protamine the UPC value was determined and the Index Factor (IPs) calculated by the formula: IPs= UPC (clinical protamine) UPC (reference protamine) Clinical Studies: Patients ranged in age from 7 months to 74 years. The surgical procedures requiring CPB included: coronary artery bypass graft (CABG), heart valve replacement, combination CABG and valve replacement, repair of congenital septal defect and heart transplant. Following determination of the baseline ACT, the patient was given a bolus heparin dose of 3 mg/kg (body weight). Heparin used was either porcine intestinal or beef lung. The ACT value was determined five minutes following the heparin infusion and the Heparin Dose-Response Curve constructed. The patient's blood volume (BV) was estimated from the height and weight. 16 The adjusted blood volume (ABV) was calculated with the formula: ABV = BV + pump prime volume + cardioplegic volume. The Final ABV was calculated at the end of the procedure by the formula: Final ABV = ABV residual volume left in pump. c Eli Lilly and Co., Indianapolis, IN 46285 d The Upjohn Co., Kalamazoo, MI 49003