Me- of Monitoring Heparin Therapy for

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1 Polybrene Neutralization as a Me- of Monitoring Heparin Therapy for Extracorporeal Circulation Eric D. Foster, M.D., Jesse I. Spector, M.D., Lilia Talarico, M:.D., Joel Umlas, M.D., C. Robert Valeri, M.D., Dusan B. Dobnick, M.D., and Robert L. Berger, M.D. ABSTRACT Dose-r'esponse effects of heparin and protamine in 34 adult patients undergoing cardiac operations were monitored by an in vitro analysis utilizing hexadimetharine bromide (Polybrene) neutralization. Heparin administered prior to cannulation for cardiopulmonary bypass in a dose of 3. mg (3 units) per kilogram of body weight, and 1.5 mg (15 units) per kilogram for each subsequent hour of bypass, routinely produced circulating heparin concentrations greater than 1. units per milliliter of plasma. A protaminci dose equal to 8% of the total number of milligrams of heparin given resulted in no detectable plasma h'eparin in 23 of the 34 patients one-half hour after administration. No patient required protamine in an amount greater than the total number of milligrams in the heparin dose to achieve heparin neutralization. Modest postoperalive chest tube drainage (mean, 784 ml in 48 hours) in these patients provides clinical support for low-dos,e protamine administration for heparin neutralization at the conclusion of cardiopulmonary bypass. Despite the prominent role heparin plays in cardiovascular surgery, the administration of this anticoagulant for extracorporeal circulation and its neutralization by protamine lack stan- From the Department of Cardiothoracic Surgery and Hematology, Boston Un iversity Medical Center, Boston, and the United States Naval BloodResearch Laboratory, Chelsea, MA. Supported in part by (;rant NO. RR , Division of Research Resources,-N<ational Institutes of Health, and the Naval Medical Research and Development Command, Research No. MI" , US Navy. The opinions and assertions herein are those of the authors and are not to be construed as official or as reflecting the views of the Navy Department or Naval Service at large. Accepted for publicaticm Dec 13, Address reprint requests to Dr. Foster, Division of Cardiothoracic Surgery, Suite ME 622, Albany Medical College, Albany, NY dardization. In a recent survey of cardiovascular surgeons across the United States, Bull and associates [21 received 3 different protocols for heparin-protamine therapy. The multiplicity of advocated regimens suggests an absence of precise understanding among clinicians regarding dose-response effects of heparin and protamine. The impression is that many of the current heparin-protamine protocols used by cardiac surgeons have been formulated on a trial-anderror basis. Among the numerous laboratory tests and their modifications suggested for monitoring heparin therapy, the most widely used have been the whole blood clotting time, partial thromboplastin time, thrombin time, and plasma clotting time [2-5, 6, 9-12, 141. The whole blood clotting time is time-consuming, poorly reproducible, and lacks adequate correlation with heparin doses [5, 141. The partial thromboplastin time lacks sensitivity in discriminating response at the extremely high and low dose ranges and does not correlate well with heparin doses [6,12,14]. The thrombin time has recently been simplified to allow determination of heparin units by a single assay [81. Interest in determining dose-response effects with the heparin-protamine protocol used for cardiac surgery at Boston University Medical Center stimulated our search for a simple, definitive analysis method. Of particular concern was an assessment of our practice of neutralizing heparin at the conclusion of c-diopulmo- nary bypass with smaller doses of protamine than are generally advocated* Our concept Of protarnine administration was generated by an earlier clinical study that demonstrated reduced postoperative bleeding in patients receiving protarnine doses [11' The purpose of this report is to present our 514

2 515 Foster et al: Polybrene Neutralization for Heparin Monitoring experience with an in vitro technique utilizing hexadimethrine bromide (Polybrene*) for monitoring dose-response effects of heparin and protamine in patients undergoing cardiac operations. Materials and Methods Thirty-four adult patients undergoing openheart operations for a variety of acquired cardiac lesions were studied (Table 1). Prior to cannulation for cardiopulmonary bypass, each patient received heparin,t 3. mg (3 units) per kilogram of body weight, intravenously. One hour after the first heparin dose, and at the start of each subsequent hour of cardiopulmonary bypass, additional heparin was administered in the amount of 1.5 mg (15 units) per kilogram. Anticoagulant neutralization was achieved by giving intravenous protamine, initially in an amount equal to 8% of the total number of milligrams in the dose of heparin. In some cases additional small quantities of protamine (25 to 5 mg) were given in the operating room if the surgeon thought total neutralization had not been achieved as evidenced by continued bleeding from cut surfaces and failure of visible clot formation. This additional protamine was administered on subjective grounds only, without the knowledge of laboratory test results indicating a need for protamine. In the operating room, after placement of vascular pressure monitoring catheters prior to the start of the operation, all patients had samples taken from the central venous line for analysis. *Abbott Laboratories, North Chicago, IL. theparin derived from beef lung, The Upjohn Co, Kalamazoo. MI. Subsequent samples were taken 3 minutes after each heparin or protamine dose in all patients. Additional samples were obtained in 26 patients 6 hours postoperatively and in 19 patients on the first day following operation to assess residual (rebound) heparin levels. The in vitro Polybrene neutralization technique as described by Grann and associates [31 for determining circulating heparin levels was used in this study. Reagents Bovine thrombin" (1, NIH unitslml) was reconstituted with.9'/ sodium chloride to a final concentration of 16.7 units per milliliter. Twomilliliter aliquots were stored at -2 C and retained activity for more than six months. At the time of use a tube of the thrombin solution was thawed and kept at room temperature. A stock solution of Polybrene was made containing 1 mg per milliliter of.9% sodium chloride. From this stock Polybrene solution, dilutions of.1 mg,.4 mg, and.8 mg per milliliter of.9% saline were made. These working solutions can be stored at 4 C for up to six months. Blood Collections To ensure catheter flushing and to obtain a representative specimen, blood was drawn from the central venous line by a two-syringe technique with the sample for analysis taken from the second plastic syringe. The blood was transferred to plastic tubes containing 3.8% sodium citrate (1 : 9 VN, 1 volume of anticoagulant to 9 volumes of blood). The specimens were cen- 'Parke-Davis, Detroit, MI. Table 1. Cardiac Procedure and Postoperative Chest Tube Drainage for 34 Patients Studied Cardiac Procedure Valve replacement Coronary artery bypass Ventricular aneurysm resection Total No. of Patients Mean 48 Hr Postop Chest Tube Drainage (ml) 862 (35-3,56) 75 (18-2,) 65 (43-87) 784 (18-3,56)

3 516 The Annals of Thoracic Surgery Vol 23 No 6 June 1977 trifuged at 2,4 g for 5 minutes at room temperature. Plastic tubes were used, and either the heparin assay was performed immediately or the plasma was stored at -2 C for future use. Procedure For each specimen, four 1 X 75 mm glass tubes were placed in a 37 C water bath and.5 ml of plasma was pipetted into each tube. The plasma was incubated for,at least 1 minute to allow it to reach 37 C. STEP 1. Into the first tube.5 ml of thrombin solution was pipetted.* The tube was mixed and allowed to incubate at 37 C for 1 minute. The presence of a firm clot indicated no heparin activity and the test was completed. STEP 2. If no clclt formed in Step 1,.5 ml of the.1 mg per milliliter Polybrene solution was added to the second tube, followed by.5 ml of thrombin addition as with the first tube. Formation of a solid clot after l minute of incubation at 37 C indicated a heparin level less than.2 unit per milliliter of plasma. STEP 3. If no clot formed in Step 2,.5 ml of the.4 mg per milliliter Polybrene was added to the third tube and the thrombin test repeated. Development of a firm clot indicated a plasma heparin level of.2 to.6 unit per milliliter plasma. STEP 4. If no clot formed in Step 3, the test was repeated in the fourth tube using the.8 mg per milliliter Polybrene solution. A solid clot indicated a heparin level of.6 to 1. unit per milliliter of plasma. Failure of clot formation meant that the heparin level was in excess of 1. unit per milliliter of plasma. The reproducibility of the assay was tested weekly by determining heparin-thrombin times on normal plasma to which known quantities of bovine heparin had been added. The reagents, which have a usable lifespan of six months, were nevertheless prepared fresh monthly. Results on normal plasma were invariably accurate to.1 unit of heparin per milliliter of plasma. Each patient underwent a preoperative and postoperative coagulation survey that included *5-Lambda precision micropipette, MLA Inc, Mount Vernon, NY. measurements of prothrombin time, partial thromboplastin time, fibrinogen levels, and direct platelet counts. A record was made of the chest tube drainage in all patients as an assessment of blood loss for the first 48 hours postoperatively. During the study period no alterations were made in the heparin-protamine protocol as outlined. Results of the Polybrene analysis indicating circulating heparin concentrations in each of the 34 patients were not made known by the investigators to members of the cardiac surgical team until the project was completed. Results The circulating heparin concentrations determined by the Polybrene neutralization technique at the sampling times for the 34 patients studied are recorded in Table 2. Five patients, including 1 who had more than 1. unit of heparin per milliliter of plasma, had detectable heparin concentrations preoperatively before the loading dose of anticoagulant was given prior to cardiopulmonary bypass. Following administration of the initial heparin dose, all 34 patients had plasma concentrations greater than 1. unit per milliliter. Due to the length of the cardiac procedure, 24 patients received a second heparin dose; in 3 cases a third dose was given. In each instance, one-half hour after the heparin was given, the patient s plasma concentration exceeded 1. unit per milliliter. Following the initial protamine administration, 24 patients received a second protamine dose; in 6 of these cases a third dose was given. One-half hour after the first dose of protamine, 23 patients had no circulating heparin detected. Nevertheless, on the basis of subjective criteria, the cardiac surgeon administered a second small protamine dose (25 to 5 mg) to 18 of these patients and a third protamine dose to 4 of them. Of the 11 patients who had detectable heparin levels after the first protamine dose, only 2 had greater than.2 unit per milliliter. On a subjective basis, the cardiac surgeon involved gave 6 of these 11 patients an additional amount of protamine, including the 2 individuals with circulating heparin concentrations of greater than

4 517 Foster et al: Polybrene Neutralization for Heparin Monitoring Table 2. Circulating Heparin Concentrations as Measured by Polybrene Analysis in 34 Patients Studied Sample Time Preoperative '/z hr after 1st heparin dose '/z hr after 2nd heparin dose '/z hr after 3rd heparin dose '/z hr after 1st protamine dose '/z hr after 2nd protamine dose hr after 3rd protamine dose 6 hr postoperative 24 hr postoperative Heparin Concentration (unitlml plasma) < > unit per milliliter. Five patients with heparin levels less than.2 unit per milliliter did not receive further protamine. The preoperative coagulation survey was normal for all the patients. Postoperatively, minor prolongations of the prothrombin and partial thromboplastin times were noted for the first 48 hours following operation. Platelet counts were markedly reduced in all patients postoperatively; however, none were lower than 5, per microliter. The fibrinogen levels were only slightly reduced, and no evidence of disseminated intravascular coagulation was detected postoperatively. The mean chest tube drainage for these 34 adult patients during the first 48 hours postoperatively was 784 ml (see Table 1). One individual in this group had excessive chest tube drainage meriting reexploration during the first 24 hours after an aortic valve replacement. In this case the Polybrene study indicated complete heparin neutralization after the first protamine administration. This patient did receive two subsequent protamine doses (25 mg each). Polybrene analysis showed no circulating heparin after the additional protamine, and no heparin was detected 6 hours postoperatively. At the time of reexploration no major bleeding source was found. The total 48-hour chest tube drainage for this patient was 3,56 ml. Among the 11 patients who had detectable circulating heparin levels after the first protamine dose, the postoperative chest tube drainage for the 6 who received additional protamine was 93 ml; for the 5 patients who did not get more protamine, the mean 48- hour chest tube drainage was 624 ml. Of the 26 patients studied who had Polybrene analysis 6 hours postoperatively, only 1 patient had a detectable circulating heparin level, with.2 to.6 unit per milliliter recorded. No heparin was found in this patienton the24-houranalysis. Chest tube drainage over 48 hours postoperatively was 95 ml. In 19 patients Polybrene analysis was performed 24 hours following operation; only 1 patient was found to have a detectable heparin level, at.2 to.6 unit per milliliter. No further studies were done on this patient, whose 48- hour chest tube drainage was 815 ml. Comment Prior to cardiopulmonary bypass 5 patients in our series had detectable circulating heparin levels. At this stage in the operative procedure the only source of exogenous heparin in these individuals was the heparinized solutions used to flush arterial pressure monitoring lines. The circulating heparin found in the 4 patients with less than.6 unit of heparin per milliliter of plasma is probably a result of excessive flushing of these vascular catheters. In retrospect, a technical measurement error is believed responsible for the reading of more than 1. unit of heparin per milliliter of plasma found in 1 patient before cardiac bypass. Correlations of circulating heparin concentrations and whole blood clotting times indicate that patients with heparin levels of.6 to 1. unit per milliliter of plasma have clotting times in

5 518 The Annals of Thxacic Surgery Vol 23 No 6 June 1977 excess of 1 hour, or indeterminate [3]. In this study, heparin administered in a dose of 3. mg (3 units) per kilogram for cardiac bypass produced circulating heparin concentrations greater than 1. unit of heparin per milliliter of plasma at 3 minutes in all patients. Although these results suggest excessive heparin levels, Bull and associates [2] have emphasized that there is great variation among patients in the sensitivity of their coagulation mechanism to heparin; and the heparin half-life varies among patients. This patient variability has prompted most clinicians to adapt the safe practice of giving too much rather than too little heparin. Although heparin concentrations greater than 1. unit per milliliter of plasma may be unnecessary during extracorporeal circulation, this project was not designed to produce data on which an objective statement could be made concerning optimal heparin levels for cardiac operations. What can be stated is that in this patient group there was no evidence of inadequate anticoagulation. This study confirmed that heparin neutralization at the completion of cardiopulmonary bypass is accomplished with small doses of protamine. Protamine administered in an amount equal to 8 / of the total number of milligrams of heparin given resulted in no detectable plasma heparin in 23 of 34 patients. In 9 of the 11 individuals who did have circulating heparin after the first protamine dose, the heparin concentration was less than.2 unit per milliliter of plasma, which corresponds to a whole blood clotting time of 12 to 25 minutes [3]. On subjective grounds, the cardiac surgeon s assessment that additional protamine was required was inaccurate in 18 cases. Furthermore, in the 11 patients with residual circulating heparin after the first protamine dose, the operating team s subjective appraisal that more protamine was needed was correct in only 6 cases. In none of the 34 patients did the total amount of protamine exceed the total number of milligrams of the heparin dose. In addition to establishing the principle of adequate heparin neutralization with smaller amounts of protamine, these findings reveal the inadequacy of the cardiac surgeon s subjective criteria for determining protamine requirements. Reliable laboratory techniques adaptable to the operating room setting provide more accurate measurements of heparin-protamine dose-response effects. The Polybrene assay will supply the surgeon with information concerning residual heparin activity so that protamine sulfate can be given on a more rational basis. Mean chest tube drainage 48 hours postoperatively in this series was modest and was similar to that noted in our earlier study [l]; this fact adds further support for reduced protamine doses to neutralize heparin following openheart procedures. Of the patients evaluated 6 and 24 hours after operation, only 2 had detectable heparin concentrations, with.2 to.6 unit per milliliter of plasma recorded. Following protamine administration in the operating room, no detectable heparin had been found in either case. Although heparin rebound may have occurred in these 2 individuals, it is possible that the circulating heparin concentrations represent excessive flushing of pressuremonitoring catheters with heparinized solutions. The 48-hour chest tube drainage was 815 and 95 ml, respectively, for these 2 patients. Based on experience from this study, we believe the Polybrene neutralization heparin assay is adaptable to the cardiac surgery setting, in both the operating room and the intensive care-recovery room. The equipment required is small and easily portable. The analysis technique is simple and can be performed after minimal training. End-points are clear-cut, with clot or no clot results. The procedure can be reduced to a one- or two-tube test in many cases by altering the sequence of steps so that in samples in which no heparin activity is suspected, only Step 1 is carried out; if excessive heparinization is suspected, only Step 4 is performed. Since development of clot in Step 1 or failure of clot formation in Step 4 are end-points, this eliminates the need to test other dilutions of Polybrene with the sample plasma. This heparin assay technique is based on thrombin clotting processes. It is sensitive to plasma heparin levels, and, theoretically, it is influenced by plasma concentrations of fibrinogen and fibrin split products. Hypofibrinogenemia and high levels of fibrin split products could inhibit the thrombin clotting se-

6 519 Foster et al: Polybrene Neutralization for Heparin Monitoring quence. However, clotting with this Polybrene neutralization assay in the absence of heparin occurs even with fibrinogen levels as low as 8 mg per 1 ml (measured by the Parfentjov method). Fibrin split products must be present in very high concentrations before they will exert a meaningful effect on this analysis technique [7]. These plasma levels of fibrinogen and fibrin split products are rarely, if ever, reached in the course of cardiac surgery [151. While equal units of bovine and porcine heparin produce comparable anticoagulation effects, variations in protamine neutralization of heparin derived from different animal sources are recognized. More protamine is required to neutralize an equal number of units of bovine heparin than porcine heparin [13]. Heparin derived from beef lung was used exclusively in the present study. The Polybrene neutralization heparin assay is equally applicable for analyzing porcine or bovine heparin levels, as long as variations in neutralization resulting from different animal heparin sources are considered. Conclusions We have reached the following conclusions based on our experience in this study. 1. Heparin administration prior to initiation of extracorporeal perfusion in a dose of 3. mg (3 units) per kilogram of body weight, with subsequent doses of 1.5 mg (15 units) per kilogram each hour of bypass, results in circulating heparin concentrations greater than 1. unit per milliliter of plasma. 2. Postoperative heparin neutralization with protamine is accomplished uniformly with a heparin-protamine dose ratio (in milligrams) of 1 : 1; in the majority of cases no circulating heparin is detected after protamine is administered in the heparin-protamine ratio of 1.: Modest chest tube drainage 48 hours postoperatively supports our practice of using reduced protamine doses to neutralize heparin. Plasma determinations at 6 and 24 hours after operation rarely reveal detectable heparin levels when small doses of protamine are employed at the conclusion of cardiac bypass. 4. Subjective criteria employed by the cardiac surgeon to assess the need for additional pro- tamine in the operating room are unreliable. Only objective data obtained from analysis of heparin-protamine dose-response effects should be employed. 5. Polybrene neutralization proved to be an effective method for assessing heparinprotamine dose response effects in patients undergoing cardiac operations. References 1. Berger RL, Krishnaswamy R, Ryan TJ: Reduced protamine dosage for heparin neutralization in open-heart operations. Circulation 37,38:Suppl 2:154, Bull BS, Korpman RA, Huse WM, et al: Heparin therapy during extracorporeal circulation: I. Problems inherent in existing heparin protocols. J Thorac Cardiovasc Surg 69:674, Grann VR, Homewood K, Golden W: Polybrene neutralization as a rapid means of monitoring blood heparin levels. Am J Clin Pathol58:26, Hattersley PG: Activated coagulation time of whole blood. JAMA 196:436, Hougie C: The Lee and White test for coagulation time. Am J Clin Pathol 36:537, Lee RI, White P: A clinical study of the coagulation time of blood. Am J Med Sci 145:495, Natelson EA, Dooley DF: Rapid determination of fibrinogen by thrombokinetics. Am J Clin Pathol 61:828, Penner JD: Experience with a thrombin clotting time assay for measuring heparin activity. Am J Clin Pathol 61:645, Rapaport SI, Ames SB: Clotting factor assay on plasma from patients receiving intramuscular or subcutaneous heparin. Am J Med Sci 234:678, Ray PK, Harper TA: Comparison of activated recalcification and partial thromboplastin tests as controls of heparin therapy. J Lab Clin Med 77:91, Reno JW, Rotman M, Grumline FC, et al: Evaluation of the BART test (a modification of the whole-blood activated recalcification time test) as a means of monitoring heparin therapy. 12. Schatz IJ, Hathaway JC: Heparin therapy and thrombin time. JAMA 186:74, Silverglade A: Biological equivalence of beef lung and hog mucosal heparins. Curr Ther Res 18:91, Spector I, Corn M: Control of heparin therapy with activated partial thromboplastin times. JAMA 21: 157, Umlas J: Fibrinolysis and disseminated intravascular coagulation in open-heart surgery. Transfusion 16:46, 1976