INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In critically ill patients, the major indication for red blood cell (RBC) transfusion is the correction or the improvement of oxygen delivery to sustain tissue oxygen demand (1). However, this practice is not supported by firm clinical or experimental data. On the contrary, blood transfusion may affect the different determinants of the oxygen delivery-oxygen uptake relationship. First, blood transfusion is not always associated with an increase in oxygen delivery (2). The rise in oxygen-carrying capacity may indeed be counterbalanced by a decrease in blood flow, secondary to a higher blood viscosity. Second, even in the presence of an increased oxygen delivery, blood transfusion is not always associated with an increased oxygen uptake. This has been attributed to either the absence of oxygen deficit or to the inability of blood transfusion to correct a debt in tissue oxygenation (3). Evaluation of blood transfusion effects on oxygen uptake therefore requires situations in which oxygen uptake-oxygen delivery dependency can be identified. Even if blood transfusion would prove to be effective in restoring oxygen uptake in these conditions, its relative efficacy has to be compared with other standard forms of treatment such as increasing blood flow. Indeed, from a physiological point of view, increases in blood flow could theoretically augment the perfused capillary area by increases in filling pressures or microvascular vasodilation, resulting in increased oxygen uptake. The effects of blood transfusion on oxygen uptake may be less predictable than the effect of blood flow as the rise in hematocrit will increase blood viscosity, which may alter regional microvascular blood flow (4).

The present study addressed this issue in a model of oxygen uptake-oxygen supply dependency during cardiopulmonary bypass in anesthetized dogs. Providing a similar increase in oxygen delivery, the effects of "fresh" red blood cell transfusion on oxygen uptake were compared with those of an increase in blood flow.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Anesthesia and Surgical Preparation

All experimental procedures used in this investigation were performed according to the NIH guidelines and were approved by the Erasme Institutional Animal Investigation Committee. The study included 12 mongrel dogs (27.6 ± 3.7 kg). After 12 h of fasting, animals were anesthetized with an intravenous administration of 5 mg/kg of ketamine. Endotracheal intubation was performed, and mechanical ventilation was started (Elema 900 B; Siemens, Solna, Sweden) with air (fraction of inspired oxygen [FI_O2] = 0.21). The respiratory rate was set at 12/min and the tidal volume was adapted to obtain a Pa_CO2 between 35 and 40 mm Hg. Anesthesia was maintained with a continuous infusion of ketamine at 0.2 mg/kg/min and pancuronium bromide at 0.1 mg/kg followed by repeated boluses of 1 to 2 mg/h. A femoral artery catheter was advanced into the abdominal aorta through the right femoral artery for arterial pressure monitoring and blood gas analysis. In each animal, hemoglobin concentration was lowered to 10 g/dl by normovolemic hemodilution. The blood withdrawn was replaced by the same volume of 4% modified fluid gelatin (Gelofusin; Braun, Melsungen, Germany). In these acute conditions, modified fluid gelatin has been found as efficient as low molecular starch in maintaining intravascular volume (5).

A median sternotomy was performed. After administration of 5 mg/ kg of heparin, cannulas were inserted in the aortic root and the right atrial appendage. The blood circulation was maintained by a roller pump (Stöckert Instrumente GmbH, München, Germany) through a combined pediatric heat-exchanger-oxygenator (Midiflo D705; Sorin Dideco, Mirandola, Italy). The CPB circuit was primed with 4% modified fluid gelatin (Gelofusin; Braun). The prime volume was adjusted to obtain a hemoglobin concentration of 6 g/dl on cardiopumonary bypass. Sodium bicarbonate was added to obtain a pH about 7.35. After controlling the anticoagulation (activated coagulation time [ACT] greater than 450 s), the bypass was started at a flow of 120 ml/kg/min. The aorta was then clamped and the heart stopped using 200 ml of cardioplegic solution (Ringer lactate with 30 mEq/L potassium chloride). A left ventricular venting sump was then inserted. Cardiopulmonary bypass was performed at 38° C. Gas flow and FI_O2 were adapted throughout the experiment to maintain a Pa_CO2 between 30 and 35 mm Hg and a Pa_O2 above 100 mm Hg. Mixed venous O₂ saturation (Sv_O2) was continuously monitored (Oxystat SW0200 monitor; Baxter-Bentley, Irvine, CA). To compensate for insensible water losses during the experimental procedure, each dog received a saline infusion given at a rate of 10 ml/kg during the first hour and 1 ml/kg/h thereafter. Throughout the experimental procedure, additional volumes of gelatin were administered into the venous reservoir to maintain the hemoglobin level of 6 g/dl. ACT was maintained above 450 s throughout the entire procedure by additional doses of heparin (1-2 mg/kg).

After aortic cross-clamping, a period of 15 min was allowed to achieve steady state, defined by a stable mean arterial pressure and Sv_O2. Baseline measurements of arterial pressure and pump flow were then recorded. Immediately thereafter, arterial and venous blood samples were drawn from the arterial and venous lines for the measurement of blood gas tensions (ABL 2; Radiometer, Copenhagen, Denmark). Hemoglobin concentration and oxygen saturation were measured by cooximetry, with the instrument calibrated for canine blood (OSM3; Radiometer). Each sample was analyzed at least twice, with a variability between measurements less than 5%. Serum lactate concentration was assessed enzymatically by an automated analyzer (Kontron, Basel, Switzerland).

Total vascular resistance (TVR), arterial oxygen content (Ca_O2), mixed venous oxygen content (Cv_O2), DOO₂, O₂, oxygen extraction ratio (O₂ER), and venoarterial gradient for PCO₂ (VAP_CO2) were calculated using the following formulas:

TVR (d/s/cm⁵) = (mean arterial pressure [MAP]/pump flow) × 79.9

Ca_O2 (m/dl) = (Hb × 1.39 × Sa_O2) + (0.0031 × Pa_O2)

Cv_O2 (m/dl) = (Hb × 1.39 × Sv_O2) + (0.0031 × Pv_O2)

DO₂ (ml/kg/min) = (pump flow × Ca_O2 × 10) /weight

O₂(ml/kg.min) = (pump flow × [Ca_O2  Cv_O2] × 10)/weight

O₂ER (%) = (Ca_O2  Cv_O2)/Ca_O2

VAP_CO2 (mm Hg) = Pv_CO2  Pa_CO2

Experimental Protocol

After baseline measurements, oxygen delivery was reduced by a progressive decrease in pump flow by steps of 10 ml/kg/min. At each step, a 10-min period was allowed to achieve a new steady state before another set of measurements was performed. At the same time, O₂ and DO₂ were calculated. Oxygen supply dependency was considered to be present when two successive experimental steps showed a decrease in calculated O₂ value. This particular point, corresponding to a O₂ value of 15-20% below baseline, was defined as control 1.

The experimental protocol is illustrated in Figure 1. A randomized two-period crossover design with baseline measurements was used in this study. All animals were subjected to two consecutive different interventions, to obtain an increase in systemic oxygen supply of at least 40%. These interventions consisted of either an augmentation in pump flow or an increase in hemoglobin concentration obtained by the transfusion of "fresh" dog red blood cells (stored in citrate phosphate adenine for less than 3 d). Dogs were randomized according to the order of administration of the two consecutive interventions. After the first intervention, either pump flow or hemoglobin level was returned to the values obtained in the control 1 conditions (control 2) before the second was administered. After the second intervention, pump flow or the hemoglobin level was again returned to the control 1 condition values (control 3). Hemoglobin level was decreased by normovolemic hemodilution using dog's plasma and 4% modified fluid gelatin. At this point, oxygen delivery was further decreased by a progressive reduction in pump flow to approximately 20 ml/kg/min to define the complete O₂-DO₂ relationship. Serum lactate was measured at each time point, except during the two intervention periods.

View larger version (24K): [in this window] [in a new window]   Figure 1.   Illustration of the experimental protocol.

In each animal, the O₂-DO₂ relationship was analyzed from all experimental points. Critical DO₂, defined as the DO₂ value below which O₂ becomes DO₂ dependent, was determined using the mathematical analysis developed by Samsel and Schumacker (6). Briefly, paired sets of linear regressions were calculated, after sorting the DO₂ and O₂ paired values with increasing DO₂ for all possible combinations of points separated into low (supply-dependent) and high (supply-independent) DO₂ groups. Points were constrained to fall on either regression line, but never on both. The pair of regressions with the lowest sum of the standard errors of estimate was taken as the set that best fit the data. The values of DO₂ and O₂ at the intersection point were than calculated using the two regression equations. The extraction ratio at the critical point was calculated by dividing O₂crit by DO₂crit. The critical value of DO₂ was also determined in each animal from a plot of serum lactate versus DO₂ using the same computing method described above.

Statistical Analysis

The statistical analysis of the effects of the two interventions consisted in an analysis of variance for a 2 × 2 crossover design with baseline measurements (7). First, carryover effects (first order and second order) were tested. As carryover effects were not significant, data after treatment from both periods were combined and compared using an analysis of variance for repeated measures. If the F value reached the level of significance, pairwise comparisons were made, using a Tukey's Honestly Significant Difference test. For all tests, a p value < 0.05 was considered significant. All values are expressed as mean ± SD.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Table 1 shows data obtained at the beginning (baseline) at the critical point and at the end of the experiments and data at the critical point. In our study conditions, critical DO₂ was 6.6 ± 1.8 ml/kg/min. Critical DO₂ obtained from lactate concentrations was similar to the one identified from O₂ measurements (6.4 ± 2.0 ml/kg/min).

Data obtained before and after each intervention are presented in Table 2. Two different interventions were applied to animals in oxygen supply-dependent conditions to obtain an increase in DO₂ of at least 40%. The first intervention consisted of an increase in pump flow from 1.64 ± 0.44 to 2.73 ± 0.67 L/min (p < 0.001), while arterial oxygen content was maintained constant (Table 2, column T2). This intervention resulted in a significant increase in MAP, while TVR did not change. The second intervention consisted of an increase in hemoglobin concentration from 6.4 ± 0.9 to 11.1 ± 1.3 g/dl (p < 0.001), while pump flow and arterial oxygen saturation were maintained constant (Table 2, column T4). This intervention resulted in a significant increase in MAP and in TVR. TVR was also significantly higher after the hemoglobin increase than after the pump flow increase. Both interventions were associated with a similar increase in DO₂ resulting in a similar increase in O₂, back to the supply-independent part of the O₂-DO₂ relationship (Table 2 and Figure 2). The venoarterial gradient for PCO₂ decreased in a similar manner with the two interventions.

View larger version (12K): [in this window] [in a new window]   Figure 2.   Effects of RBC transfusion or increased pump flow on oxygen uptake. The relationship between O2 delivery (DO2) and O2 uptake ( O2) has been generated from data obtained in all animals at baseline, at the critical point, and at the end of the experiment. (Asterisks) Treatment by "fresh" RBC transfusion; (circles) treatment by increased pump flow. *p = 0.02 versus before treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In conditions of oxygen supply dependency, a similar increase in oxygen delivery, either by transfusion of "fresh" red blood cells or increment in pump flow, resulted in a similar increase in oxygen uptake.

Numerous studies have addressed the effects of red blood cell transfusion on tissue oxygenation in critically ill patients, with conflicting results (3, 8). Differences in experimental design were obviously confounding factors. However, the absence of a clear demonstration of a O₂-DO₂-dependent status was a major weakness in these studies. Only when transfusion is carried out in this condition is red blood cell administration expected to increase tissue oxygenation.

Two recent experimental studies specifically address this issue. In rats placed into supply dependency conditions by hemodilution, Fitzgerald and coworkers (12) and Sielenkämper and coworkers (13) demonstrated that "fresh" (stored for less than 6 d) red blood cell transfusion improved tissue oxygenation. The present study shows similar results in dogs placed into supply-dependency conditions during cardiopulmonary bypass. Moreover, our data demonstrate that "fresh" red blood cell transfusion was as efficacious as an increase in blood flow not only to improve oxygen delivery, but also to restore oxygen uptake. The effects of hematocrit on oxygen uptake may be less predictable than the effect of blood flow. Indeed, the rise in hematocrit will be associated with an increase in blood viscosity, which in turn may alter blood flow distribution at the capillary level. Using intravital microscopy, Vicaut and coworkers (14) showed that hemoconcentration to a hematocrit of 55% did not modify capillary density but was associated with an increase in the number of capillaries containing stationary red blood cells. These results illustrate that acute hemoconcentration increases the heterogeneity of capillary perfusion by excluding capillaries with high hematocrit from perfusion (4). However, at all shear rates, the relationship between hematocrit and whole blood viscosity is exponential. This might explain why increasing the hemoglobin level within the normal physiological range to improve oxygen delivery resulted in a similar increase in oxygen uptake than an increase in blood flow.

Our results are in contrast to those reported by Lorente and coworkers (15). In patients presenting a supply-dependent oxygen uptake, only increase in blood flow by dobutamine was able to improve tissue oxygenation, in opposition to transfusion of allogeneic units of red blood cells. However, improvement in oxygen delivery resulting from these two treatments was not equivalent. Furthermore, by its direct calorigenic effects, dobutamine could also increase oxygen uptake (16). Finally, the authors did not specify the age of the red blood cells they used to increase oxygen supply. Importantly, it has been shown that only "fresh" red blood cell transfusion and not old (> 28 d storage) improved tissue oxygenation in oxygen supply-dependent conditions (12, 13). The apparent inefficacy of old red blood cell transfusion to increase oxygen uptake has been attributed to a 2-3 diphosphoglycerate depletion and to the decreased deformability of the red blood cells (17- 19). These two "storage" effects would result in a relative inability of old red blood cells to increase oxygen delivery at the cellular level. Our results may have clinical implications regarding the therapeutic approach when acute improvements in tissue oxygenation are required. Future studies have to determine whether it is preferable to use fresh red blood cells in this setting and to evaluate treatments that either protect or restore red blood cell properties before transfusion.

To evaluate the effects of "fresh" red blood cell transfusion on oxygen uptake, we need an experimental model in which the different determinants of oxygen delivery could be accurately controlled. We therefore used a cardiopulmonary bypass model. Critical oxygen delivery observed in this model is somewhat lower than that obtained with other models, such as hemorrhagic shock, anemia, or cardiac tamponade (5, 20). Our model is distinguished from these because it excludes the heart and lungs from the systemic circulation and because it uses nonpulsatile blood flow. As for other models, critical oxygen delivery obtained from lactate measurements was comparable to the one estimated from oxygen uptake measurements (5, 23, 26, 27).

Several points deserve discussion. Anesthesia was maintained with ketamine. Anesthetic agents have been shown to alter the O₂-DO₂ relationship in a dose-dependent manner (23). Among these agents, ketamine appeared to have the least effects on this relationship. This is probably because of its stimulant properties on the central nervous system (28).

Oxygen supply dependency was defined when two successive experimental points showed a decrease in calculated oxygen uptake. Oxygen delivery and oxygen uptake at the different control points (Table 2) were consistently 15-20% below critical values (Table 1). This indicated that interventions were administered when animals were in supply-dependent conditions.

Interventions aimed at an increase in systemic oxygen delivery by at least 40%. This end point was chosen to ensure improvement in oxygen delivery far above the calculated critical point. With such an increase, possible confounding factors related to a mathematical coupling between oxygen delivery and oxygen uptake determination were minimized. Moreover, to reduce possible errors as a consequence of mathematical coupling, we used the same model of tubing in the pump circuit for all experiments. Pump flow was measured electronically and was validated by a stroboscopic device. Direct measurement of oxygen consumption using a metabolic cart would have been a means of confirming the effects of DO₂ changes in oxygen uptake. However, membrane oxygenators presented a significant air leak that precluded any reliable measurement of oxygen consumption.

In conclusion, in oxygen supply-dependent conditions, "fresh" RBC transfusion and increased blood flow are equally effective in restoring tissue oxygenation.