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Autologous Blood Sequestration Using a Double Venous Reservoir Bypass Circuit and Polymerized Hemoglobin Prime

Neragi-Miandoab, Siyamek; Guerrero, J. Luis; Vlahakes, Gus J.

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After the appearance of human immunodeficiency virus (HIV) disease in the early 1980s, as well as increased awareness of the complications of hepatitis, attention in cardiac surgery has been directed toward blood conservation. Despite efforts at blood conservation, this surgical specialty still requires use of homologous blood products, particularly in an increasingly complex patient population. 1

Hemoglobin based oxygen carrying solutions (HBOCs) have been proposed for use as alternatives to traditional blood transfusions. The concept of using hemoglobin extracted from erythrocytes as a “blood substitute” dates to the 19th century. However, repeated demonstrations of toxicity led to a temporary suspension of research in 1978, after an important but unsuccessful clinical trial. 2 Stimulated in the 1980s by concern about the safety of the national blood supply, interest was renewed in this area. Modern purification technologies were applied to this effort, and previously observed toxicities were diminished. Numerous efficacy studies demonstrated that contemporary HBOC solutions could be used for near complete blood replacement without significant toxicity. 3

However, over the past decade, certain limitations to the efficacy of HBOCs have been noted, including rapid clearance from the circulation and oxidation to methemoglobin, 4 resulting in effective contribution to oxygen transport for only approximately 24 hours. Thus, as noted in recent clinical trials in cardiac surgery, 5,6 when HBOCs were used in a manner similar to conventional allogeneic red cell infusions, limited efficacy was obtained. In these clinical trials, less than one unit of net homologous blood conservation was noted.

To date, most clinical efforts to use HBOC solutions in cardiac surgery have used them as an alternative to traditional homologous blood infused after surgery. However, as demonstrated in these clinical trials, rapid clearance from the circulation, as well as autooxidation results in low level and short lived efficacy. If these materials are to achieve significant degrees of blood conservation, then alternative strategies must be designed to use their short-term contribution to oxygen carrying capacity.

Isovolemic hemodilution has been demonstrated to decrease homologous blood usage in fields such as vascular surgery, but it has not been widely applied to cardiac surgery. Studies have suggested that HBOCs might be used for acute blood donation before surgery 7; the concept involved is the acute replacement of donated autologous blood with HBOC, permitting acute autologous donation down to levels of hematocrit normally not accepted or tolerated by usual clinical practice. Blood loss during surgery, therefore, would contain less of the autologous red cell mass and clotting factors, and at the end of surgical blood loss, donated autologous blood can be returned to the patient with net conservation of blood. The present study extends this concept to cardiac surgery using a novel double venous reservoir bypass circuit.

One of the major causes of hemostatic disorders and bleeding after cardiac surgery is the dilution of clotting factors and the diminution of platelet number and function. 8,9 The hemostatic effect of fresh whole blood after cardiopulmonary bypass (CPB) has been shown in early studies. 10–12 Potentially, acute isovolemic hemodilution and sequestration of patients’ own blood during CPB could function as fresh whole blood and exert a hemostatic benefit after surgery.

In the present study, we developed a double venous reservoir cardiopulmonary bypass circuit that permits acute sequestration and protection from CPB of a significant portion of a patient’s autologous blood volume; this system uses an HBOC as a temporary oxygen carrier during CPB.

Materials and Methods

The following protocol was reviewed and approved by the Subcommittee on Research Animal Care, Massachusetts General Hospital. It complies with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (DHEW Publication No. (NIH) 86-23, revised in 1985, Office of Science and Health Reports, DRR.NIH, Bethesda, MA).

Fourteen mongrel dogs were studied (weight range, 18–22 kg). After anesthesia with isoflurane (1.5–2.5% in oxygen) and intubation, arterial and venous catheters were inserted for hemodynamic measurements and vascular access. By means of a left thoracotomy, the pericardium was opened and suspended to form a cradle to support the heart. A micromanometer tipped catheter was inserted into the left ventricle for pressure measurement. After systemic heparinization (3 mg/kg), the right atrium was cannulated for CPB drainage. The pulmonary artery and the left carotid artery were cannulated for control of left heart preload and afterload, respectively. The sinoatrial (SA) node was inactivated by crushing, and the right atrium was paced to achieve constant heart rate. Aortic pressure was independently controlled and kept constant by pumping blood into and out of the left carotid artery.

A bovine derived hemoglobin based oxygen carrying solution, HBOC-201, was used in this study. This material is a glutaraldehyde polymerized, ultrapurified bovine hemoglobin preparation that is currently in clinical trials in several surgical specialties. The properties of HBOC-201 are summarized in Table 1.

Table 1
Table 1:
Characteristics of Bovine Hemoglobin Based Oxygen Carrying Solution

As shown in Figure 1, a novel, experimental cardiopulmonary bypass circuit was created using two venous reservoirs. Two groups were studied (n = 7 each). In one group (control), a CPB circuit was used with a single reservoir and crystalloid priming solution (2 L of lactated Ringer’s solution). In a second group (HBOC), a double venous reservoir bypass circuit was used. One reservoir was initially empty (sequestration reservoir), and the second reservoir contained a prime (2000 + [50 × body weight {kg}] milliliters) that consisted of lactated Ringer’s solution and bovine HBOC in a 3:1 ratio. Before bypass and during subsequent stages of this study, arterial blood samples were taken for measurement of the following parameters: pO2, pCO2, pH, base excess, hematocrit, total hemoglobin, and platelet count.

Figure 1
Figure 1:
Schematic diagram of the preparation and experimental bypass circuit used. Note the use of a double venous reservoir system that permits perfusion from either reservoir. HBOC, hemoglobin-based oxygen carrying solution.

In the control group, bypass was instituted and conducted at 37°C. In the HBOC group, when bypass was instituted, perfusate was drawn from the primed reservoir. Initial systemic venous drainage was directed to the empty sequestration reservoir until approximately 900 to 1,000 ml had drained; venous drainage was then directed to the prime containing reservoir and bypass was conducted at 37°C.

In both groups, after a 30 minute period of stability on normothermic total bypass (100 ml/min/kg), preischemic left ventricle (LV) function curves were generated by pumping blood into the pulmonary artery, increasing by 500 ml steps, from 500 to 3,000 ml/min. Systemic pressure was maintained constant by adjustment of flow into or out of the carotid cannula, as needed. At each increment, peak LV and LV end-diastolic pressures and peak LV dP/dt were measured and preischemic function curves were generated. Blood samples were again taken as described.

After preischemic measurements were made, total bypass was restored at a flow of 75 ml/min per kg with systemic cooling to 23°C; the left ventricle was vented. The aorta was clamped and cold crystalloid potassium cardioplegia was infused in the aortic root (20 ml/kg); cardioplegia was repeated 35 minutes later (10 ml/kg). Perfusion at 23°C was maintained at a flow rate of 75 ml/min per kg. Subsequently, rewarming was begun, systemic flow was increased to 100 ml/min per kg, and the aortic clamp was removed after a total ischemic time of 60 minutes. The heart was cardioverted if spontaneous rhythm did not occur.

After 30 minutes of reperfusion, blood samples were again taken as described, after which the bypass circuit in the HBOC group was changed to incorporate the sequestration reservoir. In both groups, ventricular function studies were repeated as described and final blood samples were obtained.

All data are expressed as mean ± standard deviation. Statistical comparisons between control and HBOC groups were made by Student’s t-test; statistical significance was defined as p < 0.05.


All 14 experimental preparations were brought to completion without loss. As noted in Figure 2, the bypass protocol used in this study resulted in a significant degree of hemodilution, but as anticipated, addition of polymerized hemoglobin in the HBOC group resulted in maintenance of the total hemoglobin level, despite the low native canine hematocrit. Of particular note is the net conservation of platelets. As noted in the bottom panel of Figure 2, at the end of the protocol there was a significantly greater platelet concentration in the HBOC group. As seen in Table 2, this protocol resulted in base deficit in both groups, although the degree was significantly less in the HBOC group; in addition, the presence of HBOC did not alter oxygen or carbon dioxide exchange.

Figure 2
Figure 2:
Hematologic parameters measured at the four time points of this study. Note the degree of hemodilution used in this study and the additive effect on total hemoglobin of adding hemoglobin based oxygen carrying solution (HBOC) to the prime solution in the HBOC group. As shown in the bottom panel, there is net conservation of platelets in the HBOC group, with a greater platelet count at the end of the study. Controlversus HBOC, *p < 0.05.
Table 2
Table 2:
Gas Exchange and Metabolic Parameters*


In this experimental study, an HBOC solution was used as a temporary oxygen carrier to permit cardiopulmonary bypass using an extracorporeal circuit designed to sequester and protect a significant portion of the autologous blood volume from hypothermic bypass. Despite the extreme degree of hemodilution used, oxygen transport and delivery was adequate in the HBOC group. Furthermore, the autologous blood sequestration protocol resulted in net conservation of platelets as evidenced by a greater platelet count after bypass in the HBOC group. As shown in Figure 3, after reperfusion the control group, with diminished total hemoglobin, did not have the same degree of return of ventricular function as the HBOC group. There was no evidence in this study that addition of HBOC exacerbated reperfusion injury after global ischemia.

Figure 3
Figure 3:
Left ventricle (LV) end-diastolic pressure and contractility measured with cardiac preload regulated by right heart bypass. In particular, note the difference between the control and hemoglobin-based oxygen carrying solution (HBOC) preparations after global ischemia. Controlversus HBOC: *p < 0.05.

Experimental studies 7 have suggested that acute autologous donation of a substantial portion of the red cell mass can be achieved acutely with HBOCs used for replacement and short-term oxygen carrying capacity. The present study represents an extension of this principle. In the case of noncardiac surgery, the concept of extended, acute autologous donation uses the principle that blood lost during surgery will contain a smaller proportion of the patient’s red cell mass, leaving the acutely donated blood to be reinfused after surgical blood loss occurs.

In the case of cardiac surgery, the issue of blood loss is further complicated by the coagulation system abnormalities produced, particularly platelet number and function. The decrease in platelet count during cardiopulmonary bypass has been reported previously, 13,14 as well as an acquired, transient defect in platelet function. 15 The proposed protocol is intended to obviate some of these deleterious effects by protecting a significant portion of the autologous platelet mass from exposure to hypothermic bypass.

The present study incorporates the reinfusion of sequestered autologous blood; the relatively short time frame of this protocol and the lack of refrigeration needed for longer term storage should lead to preservation of platelet function. Although in this study we did not make direct measurements of platelet function, previously published studies examining the hemostatic effect of fresh whole blood 8,16 suggest that the strategy used in this study should produce a significant benefit in surgical hemostasis. Furthermore, administration of freshly drawn platelets to thrombocytopenic patients has been shown to be more effective than standard or previously frozen platelet concentrates. 17,18

The proposed protocol may result in platelet preservation superior to platelets stored by standard blood banking methods, because platelet harvest and storage techniques may decrease platelet viability. 19–21 Another potential explanation is that the preservation conditions for platelets may be better in whole blood than in platelet concentrates. These potential issues support the short-term sequestration concept used in this study.

As noted in the literature, 4 the presently unavoidable oxidation of HBOCs severely limit the duration of their efficacy. Furthermore, additional studies have shown that exposure to a blood-gas interface, such as during cardiopulmonary bypass, can substantially accelerate the rate of HBOC oxidation. 22 Although methemoglobin levels were not directly measured in this study, the duration of bypass relative to the known oxidation rate during bypass, as well as evidence that oxygen delivery to the periphery was adequate, strongly suggest that physiologically significant methemoglobin formation did not occur.

Reperfusion injury was not observed. Previous work from this laboratory 23 has suggested that some HBOC solutions that contain low molecular weight hemoglobin entities, such as hemoglobin dimers, may exacerbate reperfusion injury. In the present study, the HBOC solution contained a minimal amount of dimeric hemoglobin, and, as shown by the ventricular function curves generated, there was no significant decrement in LV function in the HBOC group. The only significant decrement in function observed was in the severely hemodiluted control animals.

Critique and Limitations of this Study

This study was conducted in an animal model without cardiac pathology. Although use in the setting of coronary artery disease might raise the question of possible impaired oxygen delivery with extreme hemodilution, one study suggested that by adding oxygen carrying capacity while decreasing viscosity, hemodilution with HBOCs might actually improve myocardial O2 delivery. 24 In the present study, an extraordinary degree of hemodilution was used in an experimental model where an adult size oxygenator was used for a pediatric size model. This extraordinary degree of hemodilution was used as proof of concept that an HBOC solution could supply systemic oxygen demands during bypass where much of the autologous red cell mass had been sequestered from the circulation.

The premise of this study is that the protocol being used results in improved platelet function by avoidance of cardiopulmonary bypass. However, this study only examined platelet number; there was no direct measurement of platelet function. The benefit of this acute sequestration technique in platelet conservation may be underestimated by this study protocol, because cardiopulmonary bypass was used after reinfusion of set-aside blood to complete ventricular function studies. Furthermore, in this acute study, longer term potential toxicities could not be examined.


This study demonstrates HBOCs can be used as a priming solution in cardiopulmonary bypass in a circuit that protects a portion of the autologous blood volume from the damaging effects of bypass. This acute autologous blood sequestration strategy may improve postoperative hemostasis by protection of a portion of the autologous platelet mass from exposure to cardiopulmonary bypass.


The hemoglobin based oxygen carrying solution used in this study (HBOC-201) was kindly supplied by the Biopure Corporation, Cambridge, MA. This work was supported by a Grant-in-Aid from the American Heart Association


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