- Email: [email protected]
Reappearance of Circulating Heparin in Whole Blood Heparin Concentration-Based Management Does Not Correlate With Postoperative Bleeding After Cardiac Surgery
Reappearance of Circulating Heparin in Whole Blood Heparin Concentration-Based Management Does Not Correlate With Postoperative Bleeding After Cardiac Surgery Junko Ichikawa, MD,* Mitsuharu Kodaka, MD,* Keiko Nishiyama, MD,* Yuji Hirasaki, MD,† Makoto Ozaki, MD,† and Makiko Komori, MD* Objective: The Hepcon Heparin Management System (HMS) facilitates administration of higher heparin and lower protamine doses, which may affect bleeding potential due to heparin rebound. The present study evaluated heparin rebound in patients for whom the Hepcon HMS was used to determine whether point-of-care tests detect residual heparin and residual heparin is associated with postoperative blood loss. Design: Prospective study. Setting: Tertiary care center affiliated with a university hospital. Participants: Adults undergoing elective cardiac surgery requiring cardiopulmonary bypass. Interventions: In blood samples obtained at baseline, at 2 minutes, and at 1, 2, 4, 6, and 24 hours after heparin neutralization, heparin concentrations were measured using an automated chromogenic assay. Activated coagulation time (ACT), activated partial thromboplastin time (APTT), and thromboelastometry 2 hours after heparin neutralization also were examined in the last 22 study patients enrolled.
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OAGULOPATHY AFTER CARDIOPULMONARY BYPASS (CPB) is caused by multiple factors, such as disturbed hemostatic function due to hemodilution, coagulation factor depletion, platelet dysfunction, and activation of the fibrinolytic system.1 Heparin rebound after adequate heparin neutralization2 also is thought to contribute to microvascular coagulopathy. The incidence of heparin rebound varies from 4% to almost 100% of patients,3–5 based on variations in the heparin and protamine dosing strategy and the methods used to assess anticoagulant activity, and the definition of heparin rebound. Although heparin management based on activated coagulation time (ACT) is still common, ACT is not sensitive enough to detect low heparin concentrations that might occur in the presence of protamine reversal, hemodilution, low platelet numbers, or even excess protamine.6,7 On the other hand, the Hepcon Heparin Management System (HMS) Plus (Medtronic, Minneapolis, MN) provides whole blood heparin concentration measurements using a heparin dose-response (HDR) assay and automated protamine titration on an individual basis,7,8 because heparin sensitivity and the heparin clearance rate, as well as the amount of protamine required for heparin reversal, vary considerably from patient to patient.9 Whether the higher heparin levels used during CPB and the reduced dose of protamine necessary for heparin neutralization provided by the Hepcon HMS are related to heparin rebound, however, remains unclear. The aim of the present study was to evaluate heparin rebound using heparin concentration-based heparin monitoring and to determine whether laboratory and point-of-care tests accurately reflect the presence of circulating heparin and whether residual circulating heparin is associated with postoperative bleeding.
Measurements and Main Results: All 31 patients had measurable heparin levels 2 hours after protamine administration; 22 patients exhibited a primary failure to reverse heparin after protamine administration, and 9 patients had measureable heparin levels 2 hours after complete heparin reversal (ie, heparin rebound). The thromboelastometric variable, INTEM-CT:HEPTEM-CT ratio, correlated with heparin concentration (r ¼ 0.72), but ACT (r ¼ –0.12), APTT (r ¼ 0.36), and whole blood heparin concentration, determined using the Hepcon HMS, did not. Peak heparin concentration (0.18 ⫾ 0.07 U/mL) at 4 hours was not correlated with mediastinal blood loss. Conclusion: Circulating heparin detected by the chromogenic assay was too low to be clinically significant based on postoperative bleeding, although all 31 patients had residual heparin or heparin rebound at 2 hours after protamine administration with use of the Hepcon HMS. & 2014 Elsevier Inc. All rights reserved. KEY WORDS: heparin rebound, hepcon/HMS system, cardiopulmonary bypass, thromboelastometry, bleeding
METHODS After obtaining institutional review board approval and written informed consent from each patient, 32 adult patients scheduled for elective cardiac surgery requiring CPB were studied prospectively. Exclusion criteria included a history of any known coagulopathies, liver dysfunction, reoperations, preoperative abnormal coagulation profiles (international normalized ratio Z1.3, APTT 433 sec), and exposure to heparin, warfarin, clopidogrel, or direct thrombin inhibitors in the preceding 14 days. No attempt was made to standardize the anesthesia, as the standard practice varies widely among professionals. The Hepcon HMS was used for all patients, and heparin (LEO Pharma, Ballerup, Denmark) was administered based on the results. A kaolin ACT of 480 seconds was determined to be the target value for the HDR on the Hepcon HMS. Because the calculation of the volume of a patient based on the body surface area is approximate, however, a 3,000 to 5,000 IU loading dose of heparin was added to the CPB pump prime. All patients underwent normothermic CPB using a membrane oxygenator and biocompatible circuits (Capiox, RX-15 or 25; Terumo Corporation, Tokyo, Japan). Depending on the size of the patient, the extracorporeal circuit was primed with 550 mL of sodium bicarbonate, 30 g of mannitol, and 5 mg of betamethasone per kilogram of body
From the *Department of Anesthesiology, Tokyo Women’s Medical University Medical Center East; and †Department of Anesthesiology, Tokyo Women’s Medical University Hospital, Tokyo, Japan. Address reprint requests to Junko Ichikawa, MD, Department of Anesthesiology, Tokyo Women’s Medical University Medical Center East, 2-1-10 Nishiogu, Arakawa-ku, Tokyo 116-8567, Japan. E-mail: [email protected] © 2014 Elsevier Inc. All rights reserved. 1053-0770/2601-0001$36.00/0 http://dx.doi.org/10.1053/j.jvca.2013.10.010
Journal of Cardiothoracic and Vascular Anesthesia, Vol ], No ] (Month), 2014: pp ]]]–]]]
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weight when the body surface area was o2.0 m2 or 800 mL of bicarbonate solution when the body surface area was Z2.0 m2. After CPB termination, heparin was reversed using protamine sulfate based on the Hepcon HMS results. The heparin was considered neutralized once the ACT returned to the baseline level, and additional doses of protamine were not required based on the Hepcon HMS. A cell-saver collection device (Cell Saver; Haemonetics Corporation, Braintree, MA) was used in all patients. Red blood cells were administered to patients with a hematocrit level of less than 20% during CPB or 30% thereafter. Allogeneic blood products were transfused based on visual assessment of empirical microvascular bleeding. Blood samples (4 mL) were drawn via an indwelling arterial catheter after discarding approximately 6 deadspace volumes of the catheter and were collected immediately into 3.2% sodium citrate tubes (Venoject II, Terumo Corporation, Tokyo, Japan). Samples were obtained at the following times: (1) at baseline after the induction of anesthesia; (2) at 2 minutes after heparin neutralization using protamine sulfate; and at (3) 1 hour, (4) 2 hours, (5) 4 hours, (6) 6 hours, and (7) 24 hours after the end of the protamine infusion. Blood was centrifuged at 2,000 g for 20 minutes to obtain platelet-poor plasma for measurement of the heparin concentration. The plasma heparin levels were measured in the laboratory using an automated chromogenic assay. In this assay, heparin is analyzed as a heparin-antithrombin complex after the addition of purified human antithrombin to the plasma sample. An excess of factor Xa then was added to the sample and neutralized by the heparin-antithrombin complex. The residual Xa level was quantified using a synthetic chromogenic substrate (lower limit of detection by chromogenic assay, 0.04 U/mL). The authors defined heparin rebound when the plasma heparin concentration was more than 0.04 U/mL after complete heparin neutralization. In the last 22 patients enrolled in the study, blood samples obtained at 2 hours after the end of protamine infusion also were used for analysis of the whole blood heparin concentration, ACT (hemochron 401; International Technidyne Corporation, Edison, NJ), and APTT, and to perform thromboelastometry (ROTEMs; Tem Innovations GmbH, Munich, Germany). ROTEM was performed using citrated whole blood and intrinsically activated tests (INTEM test: 20 μL of 0.2-M CaCl2, 20 μL of thromboplastin-phospholipid and ellagic acid, 300 μL of blood; and the HEPTEM test: Addition of 10 μL heparinase). As the coagulation time (CT) values mainly depend on the concentrations of coagulation factors and their inhibitors, the CT values were measured using the INTEM and HEPTEM tests. Additionally, the following demographic and surgical data were collected: Sex; body weight (kg); height (cm); age (years); CPB duration (min); aortic cross-clamp time (min); use of tranexamic acid (No. of patients); chest tube drainage (mL) at 1, 2, 4, 6, and 18 hours after intensive care unit admission (a measure of postoperative blood loss); and the use of blood products (RBCs, FFP, platelet concentrate) during the intraoperative or 18-hour postoperative period. Serial heparin concentrations were compared with the values at baseline and at heparin neutralization using a repeated measures analysis of variance followed by a paired t-test with Holm’s correction. Pearson’s correlation coefficients were determined between heparin concentration at 4h and blood loss at 1, 2, 4, or 6 hours, or total blood loss, and heparin concentration at 2 hours and various laboratory tests (ACT, APTT, ROTEM) at 2 hours after protamine neutralization. A pilot study of the chromogenic assay for plasma heparin revealed that the standard deviation of heparin concentrations was approximately 0.1 U/L. In this case, the required number of samples was estimated to be greater than 13, when alpha was set at 0.05/5 (five times comparison) and beta was set at 0.8 in a one-sided comparison. An unpaired t test was used to compare the patient data, perfusion time, heparin and protamine dose, and blood loss at 2 hours between incomplete reversed heparin and heparin rebound.
ICHIKAWA ET AL
Table 1. Characteristics of Patients Enrolled in This Study No. of patients
31
Sex Age (yr) Weight (kg) Height (cm) CPB duration ACC time (min) Baseline ACT (sec) Mean CPB ACT (sec) After CPB ACT (sec) Total heparin dose (U102) Total heparin (U/kg) Total protamine dose (mg) Total protamine (mg/kg) Protamine : heparin ratio Tranexamic acid (No. of patients) 2 h chest drainage (mL) 4 h chest drainage (mL) 6 h chest drainage (mL) 18 h chest drainage (mL)
Male (22)/Female (9) 71.1 ⫾ 6.9 62.1 ⫾ 13.4 161.5 ⫾ 11.0 122.4 ⫾ 32.1 94.2 ⫾ 25.7 146.7 ⫾ 16.2 492.3 ⫾ 52.0 125.3 ⫾ 15.0 236.7 ⫾ 54.7 381.0 ⫾ 78.2 116.8 ⫾ 30.7 1.88 ⫾ 0.47 0.56 ⫾ 0.49 15 143.8 ⫾ 115.8 275.2 ⫾ 240.5 340.6 ⫾ 272.3 489.4 ⫾ 331.2
NOTE. Data are shown as the mean ⫾ SD. Abbreviations: ACC, aortic cross clamp; ACT, activated coagulation time; CPB, cardiopulmonary bypass.
Results are presented as the mean ⫾ SD. The criterion for rejection of the null hypothesis was p o 0.05. All the statistical analyses, except statistical power analyses determined using G*Power 3.1, were performed using SPSS software (version 11.0; Chicago, IL).
RESULTS
One patient was excluded from the statistical analysis because of excessive bleeding requiring an additional dose of protamine; the remaining 31 patients were included in the analysis. None of the patients required surgical re-exploration postoperatively. The study patient characteristics are shown in Table 1. The surgical procedures performed in the 31 patients were as follows: 21 cases of valve replacement, 8 cases of both valve replacement and coronary artery bypass grafting, and 2 cases of aortic replacement. The mean dose of heparin, as estimated using the Hepcon HMS system, was 23,667 ⫾ 5,466 U. None of the patients required additional heparin administration before instituting CPB due to failure to achieve their respective target ACT. This resulted in an ACT of 492 ⫾ 52 seconds. The reversal dose of protamine sulfate was 122 ⫾ 31 mg. The postoperative ACT (125 ⫾ 15 sec) was significantly different from the baseline time of 147 ⫾ 16 seconds (p o 0.01). The mean and standard deviation of heparin concentrations measured at 2 minutes after heparin neutralization using protamine sulfate and at 1, 2, 4, and 6 hours, after the end of the protamine infusion using the automated chromogenic assay were 0.09 ⫾ 0.05 U/mL, 0.11 ⫾ 0.07 U/mL, 0.14 ⫾ 0.07 U/mL, 0.18 ⫾ 0.07 U/mL, and 0.14 ⫾ 0.05 U/mL, respectively (Fig 1). Although heparin reversal based on the Hepcon HMS results was considered neutralization, 22 of the 31 patients exhibited residual heparin (0.09 ⫾ 0.05 U/mL) after the initial protamine administration, representing unreversed
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HEPARIN MANAGEMENT SYSTEM AND CIRCULATING HEPARIN
Table 2. Characteristics and Perioperative Data of Patients with Incomplete Reversed Heparin and Heparin Rebound Heparin Incomplete Reversed Heparin n ¼ 22
Age Sex (male/female) Weight (kg) CPB duration (min) ACC time (min) Heparin Initial bolus (U/kg) Total dose (U/kg) Protamine dose (mg/kg) Heparin-protamine ratio 2 h chest drainage
Fig 1. Plasma heparin concentration from the induction of anesthesia (baseline) until 24 hours after ending the protamine infusion. Values are shown as mean ⫾ SD; *p o 0.01 v baseline. y p o 0.05 vs. values immediately after protamine infusion.
heparin, rather than heparin rebound. The plasma heparin concentrations at 1, 2, 4, and 6 hours after protamine administration among the patients with incomplete heparin reversal were not significantly higher than those after the initial protamine neutralization. Although only 9 (29%) of the 31 patients had no measureable heparin after the initial protamine administration, the heparin concentrations in all patients included in the analysis were elevated at 2 hours after protamine neutralization, peaking at 0.18 ⫾ 0.07 U/mL at 4 hours, and were similar to the preoperative level at 24 hours after protamine neutralization (Fig 1). Thus, heparin rebound, as characterized by the chromogenic assay detection of circulating heparin after complete heparin reversal, was observed in 29% of the patients at 2 hours after heparin neutralization. Nevertheless, all the data obtained using the Hepcon HMS showed the absence of circulating heparin at that time. The patient data, perfusion time, heparin and protamine dose, and blood loss at 2 hours did not differ significantly between the unreversed heparin group and the true heparin rebound group (Table 2). The heparin concentration at 2 hours after neutralization was correlated poorly with laboratory anticoagulant tests, such as the ACT (r ¼ –0.12, p ¼ 0.638) and APTT (r ¼ 0.36, p ¼ 0.306). On the other hand, the INTEM-CT:HEPTEM-CT ratio (a ROTEM variable) correlated with the heparin concentration (r ¼ 0.72, p o 0.001, Fig 2). The heparin concentration at 4 hours after protamine administration was not correlated significantly with the mediastinal blood loss at 1 hour (r ¼ –0.35, p ¼ 0.101), 2 hours (r ¼ –0.35, p ¼ 0.101), 4 hours (r ¼ –0.32, p ¼ 0.127), or 6 hours (r ¼ –0.37, p ¼ 0.092), or total blood loss (r ¼ –0.35, p ¼ 0.106).
Rebound n¼9
p Value
71.3 ⫾ 6.1 (16/6) 59.2 ⫾ 9.4 125.2 ⫾ 35.8 94.7 ⫾ 28.1
62.3 ⫾ 14.1 (6/3) 66.1 ⫾ 16.9 123.0 ⫾ 23.6 97.3 ⫾ 21.3
0.10
286.5 ⫾ 60.8 388.7 ⫾ 76.4 1.92 ⫾ 0.51 0.57 ⫾ 0.2 168.9 ⫾ 108.0
268.6 ⫾ 80.7 355.7 ⫾ 85.2 1.75 ⫾ 0.29 0.53 ⫾ 0.2 127.1 ⫾ 118.7
0.53 0.34 0.38 0.52 0.42
0.27 0.84 0.81
NOTE. Data are shown as the mean ⫾ SD. Abbreviations: ACC, aortic cross-clamp; CPB, cardiopulmonary bypass.
heparin results when assayed within a few minutes after the administration of protamine, indicating incompletely reversed heparin, rather than heparin rebound. The frequency of heparin activity at 6 hours after protamine administration was higher than that described in previous reports.3–5 Several factors may account for the discrepancy between these findings and those of others. First, the Hepcon HMS was used to administer heparin and protamine, rather than a conventional fixed dose based on weight. This system allows for a higher heparin concentration to be maintained during CPB and significantly reduces the amount of protamine required, compared with ACT-based monitoring.10 In the present study, the mean protamine-heparin ratio of 0.56 was smaller than that of a fixed-dose regimen and was monitored based on the ACT. Gravlee et al11 reported that larger doses of heparin increased the incidence of postoperative heparin rebound. Second, the Hepcon HMS provides less precise heparin concentration measurements. Hardy et al12 reported that the
DISCUSSION
The results of an anti-factor Xa chromogenic substrate assay indicated that all the patients had detectable circulating heparin levels at 2 hours after protamine administration, based on the Hepcon HMS results. Of the 31 patients, 22 exhibited positive
Fig 2. Box plot showing the relationship between the plasma heparin concentration and the INTEM-CT:HEPTEM-CT ratio for thromboelastometry. (correlation coefficient, r ¼ 0.72; p o 0.001).
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heparin concentrations determined using the Hepcon HMS do not agree with laboratory determinations of heparin concentration. One reason for this is that the majority of Hepcon HMS cartridges allow for the detection of heparin concentrations by discrete increments, rather than a continuous value, and cannot differentiate intermediate values or particularly low levels of heparin. In addition, the method used to determine the heparin level differs between the Hepcon HMS and the laboratory method. In the laboratory method, exogenous antithrombin III is added in excess to bind all the heparin molecules;13 only endogenous antithrombin III is activated in the Hepcon HMS. Third, the indicator of measurable circulating heparin differs depending on the report. If the circulating heparin level was measured based on the ACT,6,7 APTT,11 or whole blood heparin concentration as measured using Hepcon HMS, the incidence of measurable circulating heparin in this study would have been zero. Nine (29%) of the patients exhibited true heparin rebound, characterized by the reappearance of heparin in the blood following initial adequate neutralization. Heparin rebound occurs because not all the heparin is bound to and cleared by the protamine.14 Rather, a proportion remains bound nonspecifically to plasma proteins and vascular cells, providing a reservoir of heparin.15 Another factor is the difference in the pharmacokinetic half-life between protamine and heparin.16 The latter mechanism is a minor contributor to heparin rebound, because the heparin concentration was elevated at 2 hours after protamine neutralization in this study. No statistically significant differences in patient data, perfusion time, heparin and protamine doses, and blood loss at 2 hours were observed between those with incomplete reversed heparin and those with heparin rebound. Considering that the mechanism of heparin rebound is comparable to incomplete reversal of heparin as a result of inadequate clearance by protamine, these results could suggest that the onset of detectable heparin varies based on the proportion of free heparin to bound heparin. Monitoring heparin concentrations using an anti-factor Xa chromogenic substrate assay causes an unavoidable delay between the blood sampling and completion of the laboratory report. Point-of-care testing should enable immediate and tighter control of anticoagulation. In this study, the ROTEMderived CT based on intrinsically activated tests with (HEPTEM) or without (INTEM) heparinase were more sensitive to changes in heparin activity than the APTT and ACT values. Postoperative ACT values were shortened when compared with those at baseline levels, when measurable heparin was detected. The finding that APTT and the ACT were not correlated with the presence of heparin in the plasma was consistent with previous findings.6,7,17 Mittermayr et al18,19 reported that a ROTEM analysis could detect heparin up to a concentration of 1.0 U/mL, regardless of whether the blood sample had been diluted. In the present study, the correlation coefficient between the laboratory anti-Xa determination of the heparin concentration and the CT-INTEM:CT-HEPTEM ratio was 0.72. The correlation coefficient determines whether the two methods are related however, and not whether their findings agree.20 Thus, the CT-INTEM:CT-HEPTEM ratio cannot be used
ICHIKAWA ET AL
interchangeably in clinical settings, but can be used as a predictor of heparin rebound. Despite the detection of circulating heparin in all patients at 2 hours after heparin neutralization, residual heparin was not correlated with postoperative bleeding. This result may be associated with the lower perioperative blood loss in patients in whom the Hepcon HMS was used.10,21 Heparin dosing does not influence postoperative bleeding when a consistent protamine neutralization technique is used.11 Moreover, Koster et al22 reported that a higher heparin concentration decreases thrombin activation, fibrinolysis, and neutrophil activation, compared with ACT-based heparin management. The reduced doses of protamine used for heparin neutralization after CPB can result in a lower potential for protamine toxicity, such as protamine-induced platelet inhibition.23 There were several limitations to the present study. First, the decision to transfuse blood products was left to the attending anesthesiologist and physician in the intensive care unit. None of the patients required platelet transfusion. Eleven of the 31 patients who required blood transfusion received preoperative autologous blood products. Second, approximately two-thirds of patients were retransfused with cell saver blood intraoperatively. Although cell savers adequately remove 95% heparin from retransfused blood, a few studies have demonstrated that small amounts of heparin could remain in the prepared retransfused blood during intraoperative autotransfusion. Therefore, residual heparin in autologous retransfused blood may affect heparin levels 2 hours after protamine administration. Third, no attempt was made to standardize administration of tranexamic acid regarding use, timing and dose, which confounds measures of blood loss. Finally, the sample selection did not include a large number of high-risk patients with prolonged extracorporeal circulation times. CONCLUSIONS
In conclusion, circulating heparin detected by the chromogenic assay was too low to be clinically significant based on postoperative bleeding, although all the patients had measurable heparin levels at 2 hours after protamine administration based on the Hepcon HMS results. Of the 31 patients, 22 (71%) exhibited incomplete heparin reversal, and 9 (29%) had true heparin rebound. A ROTEM variable, the INTEM-CT:HEPTEM-CT ratio, provided more sensitive and specific information regarding residual heparin, compared with the ACT, APTT, and whole blood heparin level as determined using the Hepcon HMS. Further studies are needed to determine whether ROTEM analysis is suitable for detecting heparin in clinical settings. ACKNOWLEDGMENTS The authors would like to thank Dr. Satoshi Hagihira (Department of Anesthesiology and Intensive Care Medicine, Osaka University), Kouichi Tsuzaki (Department of Anesthesiology, Keio University School of Medicine), and Satoru Shimizu (Medical Research Institute, Tokyo Women's Medical University) for their advice concerning the present study.
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REFERENCES 1. Karkouti K, McCluskey SA, Syed S, et al: The influence of perioperative coagulation status on postoperative blood loss in complex cardiac surgery: A prospective observational study. Anesth Analg 110: 1533-1540, 2010 2. Hyun BH, Pence RE, Davila JC, et al: Heparin rebound phenomenon in extracorporeal circulation. Surg Gynecol Obstet 115: 191-198, 1962 3. Teoh KH, Young E, Blackall MH, et al: Can extra protamine eliminate heparin rebound following cardiopulmonary bypass surgery? J Thorac Cardiovasc Surg 128:211-219, 2004 4. Gundry SR, Drongowski RA, Klein MD, et al: Postoperative bleeding in cardiovascular surgery. Does heparin rebound really exist? Am Surg 55:162-165, 1989 5. Pifarré R, Babka R, Sullivan HJ, et al: Management of postoperative heparin rebound following cardiopulmonary bypass. J Thorac Cardiovasc Surg 81:378-381, 1981 6. Gravlee GP, Case LD, Angert KC, et al: Variability of the activated coagulation time. Anesth Analg 67:469-472, 1988 7. Ottesen S, Stormorken H, Hatteland K: The value of activated coagulation time in monitoring heparin therapy during extracorporeal circulation. Scand J Thorac Cardiovasc Surg 18:123-128, 1984 8. Jobes DR, Schwartz AJ, Ellison N, et al: Monitoring heparin anticoagulation and its neutralization. Ann Thorac Surg 31: 161-166, 1981 9. Dietrich W, Braun S, Spannagl M, et al: Low preoperative antithrombin activity causes reduced response to heparin in adult but not in infant cardiac-surgical patients. Anesth Analg 92:66-71, 2001 10. Despotis GJ, Joist JH, Hogue CW Jr, et al: The impact of heparin concentration and activated clotting time monitoring on blood conservation. A prospective, randomized evaluation in patients undergoing cardiac operation. J Thorac Cardiovasc Surg 110:46-54, 1995 11. Gravlee GP, Rogers AT, Dudas LM, et al: Heparin management protocol for cardiopulmonary bypass influences postoperative heparin rebound but not bleeding. Anesthesiology 76:393-401, 1992 12. Hardy JF, Bélisle S, Robitaille D, et al: Measurement of heparin concentration in whole blood with the Hepcon/HMS device does not agree with laboratory determination of plasma heparin concentration
using a chromogenic substrate for activated factor X. J Thorac Cardiovasc Surg 112:154-161, 1996 13. Teien AN, Abildgaard U, Höök M, et al: Anticoagulant activity of heparin: Assay of bovine, human and porcine preparations by amidolytic and clotting methods. Thromb Res 11:107-117, 1977 14. Ellison N, Beatty CP, Blake DR, et al: Heparin rebound. Studies in patients and volunteers. J Thorac Cardiovasc Surg 67:723-729, 1974 15. Teoh KH, Young E, Bradley CA, et al: Heparin binding proteins. Contribution to heparin rebound after cardiopulmonary bypass. Circulation 88: II420-425, 1993 16. Kolf WJ, Effler DB, Groves LK, et al: Disposable membrane oxygenator (heart-lung machine) and its use in experimental surgery. Cleve Clin Q 23:69-97, 1956 17. Taneja R, Marwaha G, Sinha P, et al: Elevated activated partial thromboplastin time does not correlate with heparin rebound following cardiac surgery. Can J Anaesth 56:489-496, 2009 18. Mittermayr M, Margreiter J, Velik-Salchner C, et al: Effects of protamine and heparin can be detected and easily differentiated by modified thrombelastography (Rotem): an in vitro study. Br J Anaesth 95:310-316, 2005 19. Mittermayr M, Velik-Salchner C, Stalzer B, et al: Detection of protamine and heparin after termination of cardiopulmonary bypass by thrombelastometry (ROTEM): Results of a pilot study. Anesth Analg 108:743-750, 2009 20. Bland JM, Altman DG: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1: 307-310, 1986 21. Jobes DR, Aitken GL, Shaffer GW: Increased accuracy and precision of heparin and protamine dosing reduces blood loss and transfusion in patients undergoing primary cardiac operations. J Thorac Cardiovasc Surg 110:36-45, 1995 22. Koster A, Fischer T, Praus M, et al: Hemostatic activation and inflammatory response during cardiopulmonary bypass: impact of heparin management. Anesthesiology 97:837-841, 2002 23. Shigeta O, Kojima H, Hiramatsu Y, et al: Low-dose protamine based on heparin-protamine titration method reduces platelet dysfunction after cardiopulmonary bypass. J Thorac Cardiovasc Surg 118:354-360, 1999
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OAGULOPATHY AFTER CARDIOPULMONARY BYPASS (CPB) is caused by multiple factors, such as disturbed hemostatic function due to hemodilution, coagulation factor depletion, platelet dysfunction, and activation of the fibrinolytic system.1 Heparin rebound after adequate heparin neutralization2 also is thought to contribute to microvascular coagulopathy. The incidence of heparin rebound varies from 4% to almost 100% of patients,3–5 based on variations in the heparin and protamine dosing strategy and the methods used to assess anticoagulant activity, and the definition of heparin rebound. Although heparin management based on activated coagulation time (ACT) is still common, ACT is not sensitive enough to detect low heparin concentrations that might occur in the presence of protamine reversal, hemodilution, low platelet numbers, or even excess protamine.6,7 On the other hand, the Hepcon Heparin Management System (HMS) Plus (Medtronic, Minneapolis, MN) provides whole blood heparin concentration measurements using a heparin dose-response (HDR) assay and automated protamine titration on an individual basis,7,8 because heparin sensitivity and the heparin clearance rate, as well as the amount of protamine required for heparin reversal, vary considerably from patient to patient.9 Whether the higher heparin levels used during CPB and the reduced dose of protamine necessary for heparin neutralization provided by the Hepcon HMS are related to heparin rebound, however, remains unclear. The aim of the present study was to evaluate heparin rebound using heparin concentration-based heparin monitoring and to determine whether laboratory and point-of-care tests accurately reflect the presence of circulating heparin and whether residual circulating heparin is associated with postoperative bleeding.
Measurements and Main Results: All 31 patients had measurable heparin levels 2 hours after protamine administration; 22 patients exhibited a primary failure to reverse heparin after protamine administration, and 9 patients had measureable heparin levels 2 hours after complete heparin reversal (ie, heparin rebound). The thromboelastometric variable, INTEM-CT:HEPTEM-CT ratio, correlated with heparin concentration (r ¼ 0.72), but ACT (r ¼ –0.12), APTT (r ¼ 0.36), and whole blood heparin concentration, determined using the Hepcon HMS, did not. Peak heparin concentration (0.18 ⫾ 0.07 U/mL) at 4 hours was not correlated with mediastinal blood loss. Conclusion: Circulating heparin detected by the chromogenic assay was too low to be clinically significant based on postoperative bleeding, although all 31 patients had residual heparin or heparin rebound at 2 hours after protamine administration with use of the Hepcon HMS. & 2014 Elsevier Inc. All rights reserved. KEY WORDS: heparin rebound, hepcon/HMS system, cardiopulmonary bypass, thromboelastometry, bleeding
METHODS After obtaining institutional review board approval and written informed consent from each patient, 32 adult patients scheduled for elective cardiac surgery requiring CPB were studied prospectively. Exclusion criteria included a history of any known coagulopathies, liver dysfunction, reoperations, preoperative abnormal coagulation profiles (international normalized ratio Z1.3, APTT 433 sec), and exposure to heparin, warfarin, clopidogrel, or direct thrombin inhibitors in the preceding 14 days. No attempt was made to standardize the anesthesia, as the standard practice varies widely among professionals. The Hepcon HMS was used for all patients, and heparin (LEO Pharma, Ballerup, Denmark) was administered based on the results. A kaolin ACT of 480 seconds was determined to be the target value for the HDR on the Hepcon HMS. Because the calculation of the volume of a patient based on the body surface area is approximate, however, a 3,000 to 5,000 IU loading dose of heparin was added to the CPB pump prime. All patients underwent normothermic CPB using a membrane oxygenator and biocompatible circuits (Capiox, RX-15 or 25; Terumo Corporation, Tokyo, Japan). Depending on the size of the patient, the extracorporeal circuit was primed with 550 mL of sodium bicarbonate, 30 g of mannitol, and 5 mg of betamethasone per kilogram of body
From the *Department of Anesthesiology, Tokyo Women’s Medical University Medical Center East; and †Department of Anesthesiology, Tokyo Women’s Medical University Hospital, Tokyo, Japan. Address reprint requests to Junko Ichikawa, MD, Department of Anesthesiology, Tokyo Women’s Medical University Medical Center East, 2-1-10 Nishiogu, Arakawa-ku, Tokyo 116-8567, Japan. E-mail: [email protected] © 2014 Elsevier Inc. All rights reserved. 1053-0770/2601-0001$36.00/0 http://dx.doi.org/10.1053/j.jvca.2013.10.010
Journal of Cardiothoracic and Vascular Anesthesia, Vol ], No ] (Month), 2014: pp ]]]–]]]
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2
weight when the body surface area was o2.0 m2 or 800 mL of bicarbonate solution when the body surface area was Z2.0 m2. After CPB termination, heparin was reversed using protamine sulfate based on the Hepcon HMS results. The heparin was considered neutralized once the ACT returned to the baseline level, and additional doses of protamine were not required based on the Hepcon HMS. A cell-saver collection device (Cell Saver; Haemonetics Corporation, Braintree, MA) was used in all patients. Red blood cells were administered to patients with a hematocrit level of less than 20% during CPB or 30% thereafter. Allogeneic blood products were transfused based on visual assessment of empirical microvascular bleeding. Blood samples (4 mL) were drawn via an indwelling arterial catheter after discarding approximately 6 deadspace volumes of the catheter and were collected immediately into 3.2% sodium citrate tubes (Venoject II, Terumo Corporation, Tokyo, Japan). Samples were obtained at the following times: (1) at baseline after the induction of anesthesia; (2) at 2 minutes after heparin neutralization using protamine sulfate; and at (3) 1 hour, (4) 2 hours, (5) 4 hours, (6) 6 hours, and (7) 24 hours after the end of the protamine infusion. Blood was centrifuged at 2,000 g for 20 minutes to obtain platelet-poor plasma for measurement of the heparin concentration. The plasma heparin levels were measured in the laboratory using an automated chromogenic assay. In this assay, heparin is analyzed as a heparin-antithrombin complex after the addition of purified human antithrombin to the plasma sample. An excess of factor Xa then was added to the sample and neutralized by the heparin-antithrombin complex. The residual Xa level was quantified using a synthetic chromogenic substrate (lower limit of detection by chromogenic assay, 0.04 U/mL). The authors defined heparin rebound when the plasma heparin concentration was more than 0.04 U/mL after complete heparin neutralization. In the last 22 patients enrolled in the study, blood samples obtained at 2 hours after the end of protamine infusion also were used for analysis of the whole blood heparin concentration, ACT (hemochron 401; International Technidyne Corporation, Edison, NJ), and APTT, and to perform thromboelastometry (ROTEMs; Tem Innovations GmbH, Munich, Germany). ROTEM was performed using citrated whole blood and intrinsically activated tests (INTEM test: 20 μL of 0.2-M CaCl2, 20 μL of thromboplastin-phospholipid and ellagic acid, 300 μL of blood; and the HEPTEM test: Addition of 10 μL heparinase). As the coagulation time (CT) values mainly depend on the concentrations of coagulation factors and their inhibitors, the CT values were measured using the INTEM and HEPTEM tests. Additionally, the following demographic and surgical data were collected: Sex; body weight (kg); height (cm); age (years); CPB duration (min); aortic cross-clamp time (min); use of tranexamic acid (No. of patients); chest tube drainage (mL) at 1, 2, 4, 6, and 18 hours after intensive care unit admission (a measure of postoperative blood loss); and the use of blood products (RBCs, FFP, platelet concentrate) during the intraoperative or 18-hour postoperative period. Serial heparin concentrations were compared with the values at baseline and at heparin neutralization using a repeated measures analysis of variance followed by a paired t-test with Holm’s correction. Pearson’s correlation coefficients were determined between heparin concentration at 4h and blood loss at 1, 2, 4, or 6 hours, or total blood loss, and heparin concentration at 2 hours and various laboratory tests (ACT, APTT, ROTEM) at 2 hours after protamine neutralization. A pilot study of the chromogenic assay for plasma heparin revealed that the standard deviation of heparin concentrations was approximately 0.1 U/L. In this case, the required number of samples was estimated to be greater than 13, when alpha was set at 0.05/5 (five times comparison) and beta was set at 0.8 in a one-sided comparison. An unpaired t test was used to compare the patient data, perfusion time, heparin and protamine dose, and blood loss at 2 hours between incomplete reversed heparin and heparin rebound.
ICHIKAWA ET AL
Table 1. Characteristics of Patients Enrolled in This Study No. of patients
31
Sex Age (yr) Weight (kg) Height (cm) CPB duration ACC time (min) Baseline ACT (sec) Mean CPB ACT (sec) After CPB ACT (sec) Total heparin dose (U102) Total heparin (U/kg) Total protamine dose (mg) Total protamine (mg/kg) Protamine : heparin ratio Tranexamic acid (No. of patients) 2 h chest drainage (mL) 4 h chest drainage (mL) 6 h chest drainage (mL) 18 h chest drainage (mL)
Male (22)/Female (9) 71.1 ⫾ 6.9 62.1 ⫾ 13.4 161.5 ⫾ 11.0 122.4 ⫾ 32.1 94.2 ⫾ 25.7 146.7 ⫾ 16.2 492.3 ⫾ 52.0 125.3 ⫾ 15.0 236.7 ⫾ 54.7 381.0 ⫾ 78.2 116.8 ⫾ 30.7 1.88 ⫾ 0.47 0.56 ⫾ 0.49 15 143.8 ⫾ 115.8 275.2 ⫾ 240.5 340.6 ⫾ 272.3 489.4 ⫾ 331.2
NOTE. Data are shown as the mean ⫾ SD. Abbreviations: ACC, aortic cross clamp; ACT, activated coagulation time; CPB, cardiopulmonary bypass.
Results are presented as the mean ⫾ SD. The criterion for rejection of the null hypothesis was p o 0.05. All the statistical analyses, except statistical power analyses determined using G*Power 3.1, were performed using SPSS software (version 11.0; Chicago, IL).
RESULTS
One patient was excluded from the statistical analysis because of excessive bleeding requiring an additional dose of protamine; the remaining 31 patients were included in the analysis. None of the patients required surgical re-exploration postoperatively. The study patient characteristics are shown in Table 1. The surgical procedures performed in the 31 patients were as follows: 21 cases of valve replacement, 8 cases of both valve replacement and coronary artery bypass grafting, and 2 cases of aortic replacement. The mean dose of heparin, as estimated using the Hepcon HMS system, was 23,667 ⫾ 5,466 U. None of the patients required additional heparin administration before instituting CPB due to failure to achieve their respective target ACT. This resulted in an ACT of 492 ⫾ 52 seconds. The reversal dose of protamine sulfate was 122 ⫾ 31 mg. The postoperative ACT (125 ⫾ 15 sec) was significantly different from the baseline time of 147 ⫾ 16 seconds (p o 0.01). The mean and standard deviation of heparin concentrations measured at 2 minutes after heparin neutralization using protamine sulfate and at 1, 2, 4, and 6 hours, after the end of the protamine infusion using the automated chromogenic assay were 0.09 ⫾ 0.05 U/mL, 0.11 ⫾ 0.07 U/mL, 0.14 ⫾ 0.07 U/mL, 0.18 ⫾ 0.07 U/mL, and 0.14 ⫾ 0.05 U/mL, respectively (Fig 1). Although heparin reversal based on the Hepcon HMS results was considered neutralization, 22 of the 31 patients exhibited residual heparin (0.09 ⫾ 0.05 U/mL) after the initial protamine administration, representing unreversed
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HEPARIN MANAGEMENT SYSTEM AND CIRCULATING HEPARIN
Table 2. Characteristics and Perioperative Data of Patients with Incomplete Reversed Heparin and Heparin Rebound Heparin Incomplete Reversed Heparin n ¼ 22
Age Sex (male/female) Weight (kg) CPB duration (min) ACC time (min) Heparin Initial bolus (U/kg) Total dose (U/kg) Protamine dose (mg/kg) Heparin-protamine ratio 2 h chest drainage
Fig 1. Plasma heparin concentration from the induction of anesthesia (baseline) until 24 hours after ending the protamine infusion. Values are shown as mean ⫾ SD; *p o 0.01 v baseline. y p o 0.05 vs. values immediately after protamine infusion.
heparin, rather than heparin rebound. The plasma heparin concentrations at 1, 2, 4, and 6 hours after protamine administration among the patients with incomplete heparin reversal were not significantly higher than those after the initial protamine neutralization. Although only 9 (29%) of the 31 patients had no measureable heparin after the initial protamine administration, the heparin concentrations in all patients included in the analysis were elevated at 2 hours after protamine neutralization, peaking at 0.18 ⫾ 0.07 U/mL at 4 hours, and were similar to the preoperative level at 24 hours after protamine neutralization (Fig 1). Thus, heparin rebound, as characterized by the chromogenic assay detection of circulating heparin after complete heparin reversal, was observed in 29% of the patients at 2 hours after heparin neutralization. Nevertheless, all the data obtained using the Hepcon HMS showed the absence of circulating heparin at that time. The patient data, perfusion time, heparin and protamine dose, and blood loss at 2 hours did not differ significantly between the unreversed heparin group and the true heparin rebound group (Table 2). The heparin concentration at 2 hours after neutralization was correlated poorly with laboratory anticoagulant tests, such as the ACT (r ¼ –0.12, p ¼ 0.638) and APTT (r ¼ 0.36, p ¼ 0.306). On the other hand, the INTEM-CT:HEPTEM-CT ratio (a ROTEM variable) correlated with the heparin concentration (r ¼ 0.72, p o 0.001, Fig 2). The heparin concentration at 4 hours after protamine administration was not correlated significantly with the mediastinal blood loss at 1 hour (r ¼ –0.35, p ¼ 0.101), 2 hours (r ¼ –0.35, p ¼ 0.101), 4 hours (r ¼ –0.32, p ¼ 0.127), or 6 hours (r ¼ –0.37, p ¼ 0.092), or total blood loss (r ¼ –0.35, p ¼ 0.106).
Rebound n¼9
p Value
71.3 ⫾ 6.1 (16/6) 59.2 ⫾ 9.4 125.2 ⫾ 35.8 94.7 ⫾ 28.1
62.3 ⫾ 14.1 (6/3) 66.1 ⫾ 16.9 123.0 ⫾ 23.6 97.3 ⫾ 21.3
0.10
286.5 ⫾ 60.8 388.7 ⫾ 76.4 1.92 ⫾ 0.51 0.57 ⫾ 0.2 168.9 ⫾ 108.0
268.6 ⫾ 80.7 355.7 ⫾ 85.2 1.75 ⫾ 0.29 0.53 ⫾ 0.2 127.1 ⫾ 118.7
0.53 0.34 0.38 0.52 0.42
0.27 0.84 0.81
NOTE. Data are shown as the mean ⫾ SD. Abbreviations: ACC, aortic cross-clamp; CPB, cardiopulmonary bypass.
heparin results when assayed within a few minutes after the administration of protamine, indicating incompletely reversed heparin, rather than heparin rebound. The frequency of heparin activity at 6 hours after protamine administration was higher than that described in previous reports.3–5 Several factors may account for the discrepancy between these findings and those of others. First, the Hepcon HMS was used to administer heparin and protamine, rather than a conventional fixed dose based on weight. This system allows for a higher heparin concentration to be maintained during CPB and significantly reduces the amount of protamine required, compared with ACT-based monitoring.10 In the present study, the mean protamine-heparin ratio of 0.56 was smaller than that of a fixed-dose regimen and was monitored based on the ACT. Gravlee et al11 reported that larger doses of heparin increased the incidence of postoperative heparin rebound. Second, the Hepcon HMS provides less precise heparin concentration measurements. Hardy et al12 reported that the
DISCUSSION
The results of an anti-factor Xa chromogenic substrate assay indicated that all the patients had detectable circulating heparin levels at 2 hours after protamine administration, based on the Hepcon HMS results. Of the 31 patients, 22 exhibited positive
Fig 2. Box plot showing the relationship between the plasma heparin concentration and the INTEM-CT:HEPTEM-CT ratio for thromboelastometry. (correlation coefficient, r ¼ 0.72; p o 0.001).
4
heparin concentrations determined using the Hepcon HMS do not agree with laboratory determinations of heparin concentration. One reason for this is that the majority of Hepcon HMS cartridges allow for the detection of heparin concentrations by discrete increments, rather than a continuous value, and cannot differentiate intermediate values or particularly low levels of heparin. In addition, the method used to determine the heparin level differs between the Hepcon HMS and the laboratory method. In the laboratory method, exogenous antithrombin III is added in excess to bind all the heparin molecules;13 only endogenous antithrombin III is activated in the Hepcon HMS. Third, the indicator of measurable circulating heparin differs depending on the report. If the circulating heparin level was measured based on the ACT,6,7 APTT,11 or whole blood heparin concentration as measured using Hepcon HMS, the incidence of measurable circulating heparin in this study would have been zero. Nine (29%) of the patients exhibited true heparin rebound, characterized by the reappearance of heparin in the blood following initial adequate neutralization. Heparin rebound occurs because not all the heparin is bound to and cleared by the protamine.14 Rather, a proportion remains bound nonspecifically to plasma proteins and vascular cells, providing a reservoir of heparin.15 Another factor is the difference in the pharmacokinetic half-life between protamine and heparin.16 The latter mechanism is a minor contributor to heparin rebound, because the heparin concentration was elevated at 2 hours after protamine neutralization in this study. No statistically significant differences in patient data, perfusion time, heparin and protamine doses, and blood loss at 2 hours were observed between those with incomplete reversed heparin and those with heparin rebound. Considering that the mechanism of heparin rebound is comparable to incomplete reversal of heparin as a result of inadequate clearance by protamine, these results could suggest that the onset of detectable heparin varies based on the proportion of free heparin to bound heparin. Monitoring heparin concentrations using an anti-factor Xa chromogenic substrate assay causes an unavoidable delay between the blood sampling and completion of the laboratory report. Point-of-care testing should enable immediate and tighter control of anticoagulation. In this study, the ROTEMderived CT based on intrinsically activated tests with (HEPTEM) or without (INTEM) heparinase were more sensitive to changes in heparin activity than the APTT and ACT values. Postoperative ACT values were shortened when compared with those at baseline levels, when measurable heparin was detected. The finding that APTT and the ACT were not correlated with the presence of heparin in the plasma was consistent with previous findings.6,7,17 Mittermayr et al18,19 reported that a ROTEM analysis could detect heparin up to a concentration of 1.0 U/mL, regardless of whether the blood sample had been diluted. In the present study, the correlation coefficient between the laboratory anti-Xa determination of the heparin concentration and the CT-INTEM:CT-HEPTEM ratio was 0.72. The correlation coefficient determines whether the two methods are related however, and not whether their findings agree.20 Thus, the CT-INTEM:CT-HEPTEM ratio cannot be used
ICHIKAWA ET AL
interchangeably in clinical settings, but can be used as a predictor of heparin rebound. Despite the detection of circulating heparin in all patients at 2 hours after heparin neutralization, residual heparin was not correlated with postoperative bleeding. This result may be associated with the lower perioperative blood loss in patients in whom the Hepcon HMS was used.10,21 Heparin dosing does not influence postoperative bleeding when a consistent protamine neutralization technique is used.11 Moreover, Koster et al22 reported that a higher heparin concentration decreases thrombin activation, fibrinolysis, and neutrophil activation, compared with ACT-based heparin management. The reduced doses of protamine used for heparin neutralization after CPB can result in a lower potential for protamine toxicity, such as protamine-induced platelet inhibition.23 There were several limitations to the present study. First, the decision to transfuse blood products was left to the attending anesthesiologist and physician in the intensive care unit. None of the patients required platelet transfusion. Eleven of the 31 patients who required blood transfusion received preoperative autologous blood products. Second, approximately two-thirds of patients were retransfused with cell saver blood intraoperatively. Although cell savers adequately remove 95% heparin from retransfused blood, a few studies have demonstrated that small amounts of heparin could remain in the prepared retransfused blood during intraoperative autotransfusion. Therefore, residual heparin in autologous retransfused blood may affect heparin levels 2 hours after protamine administration. Third, no attempt was made to standardize administration of tranexamic acid regarding use, timing and dose, which confounds measures of blood loss. Finally, the sample selection did not include a large number of high-risk patients with prolonged extracorporeal circulation times. CONCLUSIONS
In conclusion, circulating heparin detected by the chromogenic assay was too low to be clinically significant based on postoperative bleeding, although all the patients had measurable heparin levels at 2 hours after protamine administration based on the Hepcon HMS results. Of the 31 patients, 22 (71%) exhibited incomplete heparin reversal, and 9 (29%) had true heparin rebound. A ROTEM variable, the INTEM-CT:HEPTEM-CT ratio, provided more sensitive and specific information regarding residual heparin, compared with the ACT, APTT, and whole blood heparin level as determined using the Hepcon HMS. Further studies are needed to determine whether ROTEM analysis is suitable for detecting heparin in clinical settings. ACKNOWLEDGMENTS The authors would like to thank Dr. Satoshi Hagihira (Department of Anesthesiology and Intensive Care Medicine, Osaka University), Kouichi Tsuzaki (Department of Anesthesiology, Keio University School of Medicine), and Satoru Shimizu (Medical Research Institute, Tokyo Women's Medical University) for their advice concerning the present study.
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using a chromogenic substrate for activated factor X. J Thorac Cardiovasc Surg 112:154-161, 1996 13. Teien AN, Abildgaard U, Höök M, et al: Anticoagulant activity of heparin: Assay of bovine, human and porcine preparations by amidolytic and clotting methods. Thromb Res 11:107-117, 1977 14. Ellison N, Beatty CP, Blake DR, et al: Heparin rebound. Studies in patients and volunteers. J Thorac Cardiovasc Surg 67:723-729, 1974 15. Teoh KH, Young E, Bradley CA, et al: Heparin binding proteins. Contribution to heparin rebound after cardiopulmonary bypass. Circulation 88: II420-425, 1993 16. Kolf WJ, Effler DB, Groves LK, et al: Disposable membrane oxygenator (heart-lung machine) and its use in experimental surgery. Cleve Clin Q 23:69-97, 1956 17. Taneja R, Marwaha G, Sinha P, et al: Elevated activated partial thromboplastin time does not correlate with heparin rebound following cardiac surgery. Can J Anaesth 56:489-496, 2009 18. Mittermayr M, Margreiter J, Velik-Salchner C, et al: Effects of protamine and heparin can be detected and easily differentiated by modified thrombelastography (Rotem): an in vitro study. Br J Anaesth 95:310-316, 2005 19. Mittermayr M, Velik-Salchner C, Stalzer B, et al: Detection of protamine and heparin after termination of cardiopulmonary bypass by thrombelastometry (ROTEM): Results of a pilot study. Anesth Analg 108:743-750, 2009 20. Bland JM, Altman DG: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1: 307-310, 1986 21. Jobes DR, Aitken GL, Shaffer GW: Increased accuracy and precision of heparin and protamine dosing reduces blood loss and transfusion in patients undergoing primary cardiac operations. J Thorac Cardiovasc Surg 110:36-45, 1995 22. Koster A, Fischer T, Praus M, et al: Hemostatic activation and inflammatory response during cardiopulmonary bypass: impact of heparin management. Anesthesiology 97:837-841, 2002 23. Shigeta O, Kojima H, Hiramatsu Y, et al: Low-dose protamine based on heparin-protamine titration method reduces platelet dysfunction after cardiopulmonary bypass. J Thorac Cardiovasc Surg 118:354-360, 1999