Heparin-protamine balance after neonatal cardiopulmonary bypass surgery

Abstract

Essentials Heparin-protamine balance (HPB) modulates bleeding after neonatal cardiopulmonary bypass (CPB). HPB was examined in 44 neonates undergoing CPB. Post-operative bleeding occurred in 36% and heparin rebound in 73%. Thrombin-initiated fibrin clot kinetic assay and partial thromboplastin time best assessed HPB.

Summary: Background Neonates undergoing cardiopulmonary bypass (CPB) are at risk of excessive bleeding. Blood is anticoagulated with heparin during CPB. Heparin activity is reversed with protamine at the end of CPB. Paradoxically, protamine also inhibits blood coagulation when it is dosed in excess of heparin. Objectives To evaluate heparin-protamine balance in neonates undergoing CPB by using research and clinical assays, and to determine its association with postoperative bleeding. Patients/Methods Neonates undergoing CPB in the first 30 days of life were studied. Blood samples were obtained during and after surgery. Heparin-protamine balance was assessed with calibrated automated thrombography, thrombin-initiated fibrin clot kinetic assay (TFCK), activated partial thromboplastin time (APTT), anti-FXa activity, and thromboelastometry. Excessive postoperative bleeding was determined by measurement of chest tube output or the development of cardiac tamponade. Results and Conclusions Of 44 neonates enrolled, 16 (36%) had excessive postoperative bleeding. The TFCK value was increased. By heparin in neonatal blood samples, but was only minimally altered by excess protamine. Therefore, it reliably measured heparin in samples containing a wide range of heparin and protamine concentrations. The APTT most closely correlated with TFCK results, whereas anti-FXa and thromboelastometry assays were less correlative. The TFCK and APTT assay also consistently detected postoperative heparin rebound, providing an important continued role for these long-established coagulation tests in the management of postoperative bleeding in neonates requiring cardiac surgical repair. None of the coagulation tests predicted the neonates who experienced postoperative bleeding, reflecting the multifactorial causes of bleeding in this population.

Keywords: cardiopulmonary; hemostasis; heparin; neonate; protamine.

Fig. 1.. Protamine restores heparin inhibition of thrombin generation when not in excess of heparin.

Adult pooled plasma was incubated with the indicated amounts of protamine (mg/mL) and thrombin generation was detected with CAT. All lines are averages of duplicate runs. (A) Assays performed in the absence of heparin. (B) Assays performed in the presence of 0.167 U/mL heparin. Ratios of heparin (U/mL) to protamine (mg/mL) ranged from 1 to 10 and are indicated in parentheses. When the heparin:protamine ratio was <1, no thrombin was generated.

Fig. 2.. Unbalanced heparin and protamine do not affect the TFCK assay.

(A) Thrombin was added to plasma containing (red line) or lacking (black line) protamine (0.025 mg/mL) and the indicated amount of heparin. (B) The TFCK ratio increases with increasing amounts of heparin at heparin(U/mL) to protamine (mg/mL) ratios ranging from 0.04 to 0.4. (C) Thrombin was added to plasma lacking (red bars) or containing (blue bars) heparin (0.05 U/mL) in the presence of the indicated amount of protamine The last two bars show the rate of fibrin formation without addition of protamine in the absence (hatched) or presence of heparin.

Fig. 3.. The TFCK ratio correlates to CAT assay results performed with neonatal samples.

Neonatal plasma TFCK ratio was correlated to thrombin generation parameters obtained from TF-initiated CAT assays using neonatal PRP. In all panels, red dots represent bleeding neonates and black dots represent non-bleeding neonates. (A) Correlation of TFCK ratio to CAT thrombin generation rate (TGR) at Post-CPB, r=−0.47, p=0.0015; (B) Correlation of TFCK ratio to CAT TGR at Post-Op, r=−0.66, p<0.0001; (C) Correlation of TFCK ratio to CAT lagtime at Post-CPB, r=0.72, p<0.0001; (D) Correlation of TFCK ratio to CAT lagtime at Post-Op, r = 0.78, p<0.0001); (E) Correlation of TFCK ratio to CAT peak thrombin at Post-CPB, r =−0.67, p<0.0001; (F) Correlation of TFCK ratio to CAT peak thrombin at Post-Op, r =−0.77, p<0.0001.

Fig. 4.. The TFCK ratio correlates most strongly to aPTT in Post-Op neonatal samples.

Neonatal plasma TFCK ratio was compared to other clinical assays performed with neonatal plasma. In all panels, red dots represent bleeding neonates and black dots represent non-bleeding neonates. (A) Correlation of TFCK ratio to aPTT at Post-CPB, r=0.39, p=0.011; (B) Correlation of TFCK ratio to aPTT at Post-Op, r=0.70, p<0.0001; (C) Correlation of TT ratio to anti-FXa activity at Post-CPB, r= 0.49, p= 0.0008; (D) Correlation of TT ratio to anti-FXa activity at Post-Op, r=0.54, p=0.0002; (E) Correlation of TFCK ratio to thromboelastography (intem-heptem) at Post-CPB, r=0.27, p=0.075; (F) Correlation of TFCK ratio to thromboelastography (intem-heptem) at Post-Op, r=0.43, p=0.005.

Fig. 5.. Post-operative heparin rebound is detected with TFCK ratio.

(A) TFCK ratio from Post-CPB (circles) and Post-Op (squares) in non-bleeder (black) and bleeder (red) groups. Neonates ranked from lowest to highest TFCK ratio in Post-Op samples are presented on the x-axis. (B) Receiver-operator curve (ROC) analysis comparing post-operative heparin rebound detected by TFCK ratio to that detected by aPTT, CAT TGR, thromboelastography (TEG) or anti-FXa activity. The aPTT (Black, AUC=0.89, 95% CI: 0.78–0.99) correlated best with TFCK ratio for detection of heparin rebound, followed by CAT TGR (Blue, AUC=0.73, 95% CI: 0.57–0.90), thromboelastography (Green, AUC=0.69, 95% CI: 0.46–0.92) and anti-FXa (Red, AUC=0.60. 95% CI: 0.37–0.82).