In this prospective, exploratory study we demonstrated that plasma HBP increases due to heparin administration and the stimulation caused by CPB. Furthermore, the HBP concentration decreases immediately after CPB cessation as a result of protamine administration. By the first postoperative day, HBP reaches concentrations below those previously indicated as a threshold for the detection of infections with organ dysfunction.
Cardiac surgery using CPB induces a systemic inflammatory response including neutrophil activation, also leading to endotoxemia, ischemia and reperfusion injury (1, 3). Infections occur in 5–21% of cases after cardiac procedures and have negative effects on surgical outcomes including prolonged hospitalization, development of multiorgan dysfunction and increased hospital mortality (19). Therefore, early detection of post-operative infections is essential in patients undergoing major surgery. In this study, we confirmed that biomarkers commonly used to detect infections (e.g. CRP and WBC) are unspecifically elevated postoperatively, making it hard to separate systemic inflammation caused by surgical trauma from post-operative infections. Therefore, there is a need for a specific and reliable biomarker for early detection of postoperative infections.
To our knowledge, two studies have described HBP levels in cardiac surgery. Pesonen et al showed a 39-fold increase in HBP concentration during cardiac surgery with CPB and Pan et al demonstrated that elevated HBP levels after the release of cross-clamp predicted myocardial injury related cardiogenic-shock after cardiac surgery (14, 20). However, neither study did fully describe the intra-and postoperative dynamics of HBP or investigate whether the administration of heparin and protamine has any effect on HBP levels.
In our study, we showed that the administration of heparin caused a significant increase in HBP in both CABG and complex cardiac surgery patients. Whole blood from both healthy donors and CABG patients did not show a similar increase in HBP concentration when stimulated ex vivo, suggesting that the increase in HBP was not derived from direct stimulation of neutrophils or other blood cells. HBP is a highly positively charged molecule that has been shown to interact with negatively charged molecules and receptors, such as the glycocalyx of the endothelium. Therefore, we hypothesized that limited neutrophil activation occurs during the initial surgical trauma, which releases some HBP that binds to negatively charged molecules on the endothelial surface, such as proteoglycans (21). We used cultured endothelial cells to show that HBP adsorbs to the endothelial surface within 20 minutes and is then released within five minutes of adding heparin. We therefore suggest that the administration of high doses of negatively charged heparin competes with the binding of HBP to the negatively charged receptors on the endothelium. HBP is subsequently bound by heparin and released into the blood stream, rendering it detectable by ELISA. Our data indicate that the complex between heparin and HBP is not cleared from the circulation, as HBP levels decrease only after neutralization of heparin by protamine. These proposed mechanisms are summarized in Fig. 6.
Heparin itself has, in both in vitro and in vivo studies, been demonstrated to have beneficial effects in blocking HBP-induced vascular leakage and renal inflammation (21). Therefore, even if heparin induces an HBP elevation its effects are likely blocked by the presence high doses of heparin. This is supported by our finding that, although HBP levels during cardiac surgery with CPB exceed those seen in septic patients, the surgical patients are hemodynamically stable and do not bear any clinical resemblance to patients presenting with septic shock. Although we cannot exclude that HBP has an effect on capillary permeability in this setting, the clinical picture indicates that the HBP released after heparin administration and during CPB lacks its usual biological activity.
After the initial rise five minutes after heparin administration, HBP concentration dropped slightly five minutes after CPB started in CABG patients but not in those undergoing complex surgery. This drop was most likely due to hemodilution of the patients when starting CPB. The CPB circuit of patients undergoing complex surgery is primed with albumin, mannitol and occasionally red blood cells, while it is primed with crystalloids in CABG surgery patients. Crystalloids are ionic salt solutions similar to those commonly used to disrupt ionic charge-charge interactions between molecules, such as the interaction between HBP and heparin (22, 23). Therefore, it is likely that the administration of crystalloids induced a partial disruption of the circulating HBP-heparin complex, allowing some HBP to adsorb to the endothelium, resulting in reduced plasma HBP. Contact between blood and the CPB causes a systemic inflammatory response which leads to neutrophil activation and additional neutrophil release. This is the likely reason for the increase of HBP concentration during CPB (24), supported by the finding of a similar increase of MPO, which is released by activated neutrophils (25).
A significant drop in HBP was seen 5 minutes after protamine administration and the cessation of CPB. In the separate group of patients where protamine was delayed until 5 minutes after CPB end, HBP levels remained elevated until protamine was administered, suggesting that protamine, and not the lack of CPB stimulation, is the major cause of the drop in HBP concentration. Ex vivo stimulation of healthy blood with protamine did not block HBP release, suggesting that protamine does not affect the neutrophils themselves. Protamine is a highly positively charged molecule that binds strongly to heparin. We showed that, although protamine interfered slightly with the ELISA, it was not to such extent that this would explain the drop in HBP levels, and this interference was reversed in the presence of heparin. Therefore, we speculate that protamine displaces HBP from binding to heparin in the blood, and the free HBP is again able to bind to the negatively charged receptors of the endothelium and eventually to be cleared from the circulation, resulting in a rapid drop in HBP levels (Fig. 6).
At ICU arrival, HBP concentration still differed significantly between the two groups but was below the threshold for infection with organ dysfunction in CABG patients (13). In patients undergoing lung surgery, HBP levels at ICU arrival were significantly lower than in CABG patients indicating that the major mechanism of postoperatively elevated HBP is CPB rather than surgical trauma and that surgical trauma has a limited effect on postoperative HBP values. However, on postoperative day one, HBP concentrations were below 30 ng/mL in all study subjects, except for the one patient who later died after nine days on ECMO.
In a recent study on patients undergoing cardiac surgery with CPB, Pan et al showed that HBP predicted postoperative cardiogenic shock (20). Interestingly, in the study by Pan et al, mean HBP concentration was 91 ng/mL after 24 hours and 84 ng/mL after 48 hours also in cases not complicated by cardiogenic shock. This contradicts our findings with all but one patient having HBP concentrations below 30 ng/mL on the first postoperative day. Pan et al did not measure pre-operative HBP levels and therefore we cannot put the postoperative HBP levels in relation to preoperative concentrations. The group used a different HBP ELISA kit and without the pre-operative samples we do not know whether the ELISA kit measures higher baseline HBP or is more effected by hemolysis or any of the medications given, which could all affect detected HBP levels. Furthermore, their finding is surprising, considering that patients in the group without cardiogenic shock did not have significant rates of other postoperative complications that might activate neutrophils and considering a previous observation by our research group that the half-life of HBP in the blood is only two minutes (unpublished data).
In current clinical routine, CRP and WBC are used as primary biomarkers for the detection of postoperative infections. However, WBC is unspecifically elevated after surgery, as is CRP, reaching its peak value on the third postoperative day. In this study we could demonstrate that HBP levels significantly decreased already at arrival to the ICU whereas CRP, WBC, IL-6 or IL-8 all peaked at a later time point. MPO had similar dynamics as HBP but was slightly more elevated on post-operative day one and has the downside of not being available for analysis in clinical routine. Therefore, our data suggest that HBP, which is currently available for rapid analysis at our institution and is advancing toward routine clinical use, indeed has the potential of serving as a screening tool for infections after surgery, even after the use of CPB.
We acknowledge that this study has several limitations. First, although this is an exploratory study, the study samples size was small with consequent risk of type II errors. Postoperative samples were not collected consecutively or at completely uniform time points due to logistic considerations. We are aware that the optimal control group would have consisted of patients undergoing off-pump cardiac surgery and we included one such patient in our study. However, at our institution, we do not perform off-pump surgery by routine and therefore we used patients undergoing lung surgery via antero-lateral thoracotomy as a reference group. Although the groups are not perfectly comparable, we believe that using a reference group of patients undergoing lung surgery with significant surgical trauma but the absence of CPB was an acceptable substitute. Furthermore, our intention was to always centrifuge the blood samples immediately on collection, but in some cases, centrifugation was delayed up to 20 minutes. We cannot exclude the possibility that this had an effect on the ratio of components in the blood samples. Lastly, we did not test the binding strength for HBP to heparin in the presence or absence of protamine to confirm our suggested mechanism of HBP increase and decrease following heparin and protamine administration as this was beyond of the scope of the current study.