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Ann Thorac Surg 2005;80:6-14
© 2005 The Society of Thoracic Surgeons

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Hawley H. Seiler resident award paper

Using a Miniaturized Circuit and an Asanguineous Prime to Reduce Neutrophil-Mediated Organ Dysfunction Following Infant Cardiopulmonary Bypass

^a Division of Pediatric Cardiac Surgery and Pediatric Perfusion Services, Doernbecher Children’s Hospital, Oregon Health & Science University, Portland, Oregon
^b Bonfils Blood Center, University of Colorado Health Sciences Center, Denver, Colorado
^c Division of Cardiovascular Surgery, Hospital for Sick Children, Toronto, Ontario, Canada

Accepted for publication February 1, 2005.

^_* Address reprint requests to Dr Ungerleider, Oregon Health & Science University, Dept. of Cardiothoracic Surgery, Mail Code DC8S, 3181 SW Sam Jackson Park Road, Portland, OR97201 (Email: ungerlei{at}ohsu.edu).

Presented at the Fifty-first Annual Meeting of the Southern Thoracic Surgical Association, Cancun, Mexico, Nov 2–4, 2004.

	Abstract

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 
BACKGROUND: Contemporary infant cardiopulmonary bypass circuits require a blood prime. Blood, especially when stored, generates an inflammatory response, and may contribute to organ dysfunction following cardiopulmonary bypass. We determined whether using a miniaturized circuit and an asanguineous prime attenuated the post-bypass inflammatory response, and improved right ventricular and pulmonary function.

METHODS: Sixteen infant piglets were placed into 3 groups based on prime components: group I (fresh blood), group II (stored blood), and group III (miniaturized circuit and asanguineous prime). Piglets were placed on cardiopulmonary bypass (100 mL·kg^–1·min^–1), cooled to 18°C, and underwent continuous perfusion (50 mL·kg^–1·min^–1) for 30 minutes. They were rewarmed and separated from bypass. Serum tumor necrosis factor-, right ventricular function, and pulmonary function were measured before and 30 minutes after bypass. Neutrophil priming activity in fresh and stored donor blood was also assessed.

RESULTS: Animals in group III had significantly improved cardiopulmonary function than the groups receiving blood (right ventricular cardiac index [mL·kg^–1·min^–1]: group I [18.8 ± 4.8], group II [21.5 ± 6.2], and group III [81.2 ± 11.4], p < 0.001; and pulmonary vascular resistance index [dynes·mL^–1·kg^–1]: group I [1169 ± 409], group II [1610 ± 486], and group III [214 ± 63], p = 0.03). Tumor necrosis factor- (pg·mL^–1) was lower in group III (1465 ± 39) than in the groups receiving blood (3940 ± 777), p = 0.002. Neutrophil priming activity (nmol·min^–1) was also higher in stored blood (3.7 ± 6) than in fresh blood (1.9 ± 0.2), p = 0.02.

CONCLUSIONS: We have devised a unique miniaturized circuit that allows an asanguineous prime without hemodilution in an infant swine model. The employment of this circuit attenuates the post-bypass inflammatory response and has salutary effects on cardiopulmonary function.

	Introduction

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 
The Hawley H. Seiler Resident Award is presented annually to the resident with the oral presentation and manuscript deemed the best of those submitted for the competition. This Award was inaugurated in 1997 to honor Dr Seiler for his contributions and dedicated service to the Southern Thoracic Surgical Association.

Conventional cardiopulmonary bypass (CPB) circuits in infants are large relative to the patient size, and therefore require a blood prime. There is increasing evidence that current extracorporeal circuits, which utilize large priming volumes, produce deleterious hemodynamic effects and postoperative cardiopulmonary dysfunction [1, 2]. In addition, recent literature has demonstrated that use of blood products is an important independent predictor for multiple organ failure in susceptible patients, including those exposed to CPB, through the initiation of endogenous immunomodulatory cascades [3–7]. Blood products, especially when stored, amplify the inflammatory response by delaying neutrophil (PMN) apoptosis and enhancing PMN priming [3–7].

Cardiopulmonary bypass, through the exposure of blood to foreign surfaces as well as ischemia-reperfusion events that occur in specific tissue beds, results in an activation of the systemic inflammatory response [8–11]. The hyperinflammatory state is an important contributor to post-CPB organ dysfunction in neonates [11–14]. Accordingly, the use of steroids, ultrafiltration, and other immunomodulators have indicated salutary effects in both animal models as well as in infants following CPB [15–19]. Despite these strategies, organ dysfunction remains a significant problem in infants particularly after the use of hypothermic low flow perfusion (HLF) [9, 14, 18, 19].

The employment of circuit miniaturization that avoids the use of a blood prime is one novel idea that has the potential to improve outcomes following infant CPB. We have therefore developed a miniaturized circuit that allows an asanguineous prime without hemodilution to test the hypothesis that using this circuit attenuates the inflammatory response and improves cardiopulmonary and coagulation function following HLF. This study will also determine whether the use of stored blood products, through enhanced neutrophil priming, augments the deleterious effects of HLF.

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 
Animal Groups and Surgical Procedures
Sixteen neonatal Yorkshire piglets (3 to 5 kg) were placed into three groups based on the components of the priming solution. Group 1 (n = 5) underwent HLF using a conventional circuit primed with fresh donor blood; group II underwent HLF using a conventional circuit primed with blood (n = 5) harvested 6 days prior to use; and group III (n = 6) underwent HLF using a mini-circuit (109 mL) to achieve an asanguineous prime. Animals were studied with the approval of our institution’s Animal Care and Use Committee in compliance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1985).

Donor swine, weighing 40 to 50 kg, were used to supply blood for the extracorporeal circuit in groups I and II. The donors were fully anesthetized and systemically heparinized. The blood was harvested sterilely through cannulation of the carotid artery and stored at –4°C in citrate phosphate dextrose solution until use (CPD Blood Pack Unit; Baxter Healthcare, Deerfield, IL). An aliquot of blood (10 mL) was collected and centrifuged at 3000 rpm for 20 minutes to collect plasma for neutrophil-priming assays on the day of collection (Day 0), and at Day 6.

The piglets were weighed to the nearest gram, and then premedicated with Telozole (8 mg/kg) (Baxter Healthcare, Deerfield, IL). Peripheral venous access was obtained and used to administer a bolus of fentanyl citrate (10 µg/kg). After intubation through a surgical tracheostomy, anesthesia was maintained with isoflurane (0.5%–1.0%) and a fentanyl citrate infusion (100 µg/H). An infant pressure cycled ventilator (Sechrist Industries, Anaheim, CA) was used for ventilation, with peak inspiratory pressures of 25 mm Hg, and positive end-expiratory pressures of 3 mm Hg. Arterial PO₂ of 200 to 250 mm Hg was achieved by adjusting the partial pressure of oxygen in either the ventilator or CPB oxygenator. An arterial PCO₂ of 35 to 45 mm Hg was maintained by adjusting minute ventilation or the rate of the sweep gas of the CPB circuit. Rectal and esophageal temperature probes were placed to ensure accurate measurements of core temperatures during cooling and rewarming intervals. Catheters (V-8; Bolab, Lake Havasu, AZ) were inserted into the right femoral artery and vein for blood gas monitoring and measurements of mean aortic and central venous pressure, respectively. Under sterile conditions, the heart was exposed through a median sternotomy. A pulmonary artery ultrasonic flow probe (Transonic Systems, Ithaca, NY) was placed around the main pulmonary artery for measurement of cardiac output. A V-5 catheter (Bolab, Lake Havasu, AZ) was inserted into the left atrium (LA), through the appendage for left atrial pressure measurements, and a V-5 catheter was also inserted into the pulmonary artery (PA), distal to the ultrasonic flow probe, to measure pulmonary artery pressures. Systemic heparinization (500 IU/kg) was given intravenously to maintain the kaolin activated clotting time greater than 480 seconds. After instrumentation and baseline data acquisition, the aortic root (10 Fr) and right atrial appendage (22 Fr) were cannulated through purse string sutures. Complete instrumentation and cannulation are depicted in Figure 1. Normothermic CPB was established at a rate of 100 mL·kg^–1·min^–1 and mean systemic arterial pressure 40 to 50 mm Hg. The animal was then perfusion cooled using pH-stat to 18°C over 20 minutes. The heart was packed in ice and the myocardial temperature maintained at 15 to 22°C. At the end of the cooling period, continuous low flow (CLF) bypass was instituted for 30 minutes at 50 mL·kg^–1·min^–1. At the conclusion of this interval the animal was rewarmed to normothermia over a minimum of 30 minutes at flow rates of 100 mL·kg^–1·min^–1, and weaned from CPB. After 30 minutes following the discontinuation of CPB, the animal was euthanized with Beuthanasia-D (1 mL/10 kg) (Baxter Healthcare, Deerfield, IL) and weighed to the nearest 10 g.

View larger version (114K): [in this window] [in a new window]   Fig 1. Photograph of acute neonatal piglet preparation, including instrumentation and cannulation.

Data Acquisition and Sample Preparation
Data of pulmonary and cardiac function were obtained at baseline prior to CPB and again 30 minutes after the discontinuation of CPB using a Gould TA-6000 chart recorder (Gould, Valley View, OH) and PowerLab 4.2 software (AD Instruments, Castle Hill, Australia). All animals were allowed to stabilize for 10 minutes prior to each data acquisition point. During data acquisition, the animal was held at end expiration. The measurements included arterial blood gas (Instrumentation Laboratories Synthesis 10, Lexington, MA), arterial blood pressure, heart rate, left atrial pressure (LAP), pulmonary artery pressure (PAP), pulmonary artery flow, dynamic pulmonary compliance (C_dyn), and ventilator settings. Pressure and flow data were sampled for 10 seconds and digitized at 500 Hz. Two to three sets of data are obtained at each time point under a steady-state condition and over a physiologic range of arterial pressure. All data were stored as a computer file for later analysis. The following equations were used to determine cardiopulmonary function:

Venous blood samples are drawn at baseline and at 30 minutes after CPB. These were placed in vacutainer tubes and sent for hematocrit, platelet count, serum osmolarity, and thromboelastogram analysis performed by the OHSU core laboratory. In addition, plasma was collected by centrifugation for cytokine assays, and for neutrophil priming activity as described by Biffl and colleagues [3].

Coagulation Function
One 5-mL aliquot of venous blood was collected at baseline and again 30 minutes after cessation of CPB for thromboelastographic analysis (Haemoscope, Skokie, IL). The samples were sent in citrated tubes, and processed by the Oregon Health & Science (OHSU) laboratory according to established protocols with heparinase to neutralize the effects of heparin sodium [20, 21]. Using proprietary software, an overall coagulation index is generated. Blood samples with a coagulation index of more than 3.0 are considered to be hypercoagulable, whereas samples with an index of less than –3.0 are considered hypocoagulable [20, 21].

Neutrophil O₂^– Production (Priming) and TNF- Assay
Superoxide production was measured, as described by Biffl and coworkers [3], using the donor samples at Day 0, 2 days after storage, and 6 days after storage. Briefly, superoxide production was measured by the O₂^– dismutase-inhibitable reduction of cytochrome c. Isolated PMNs (3.75x10⁵/well) are stimulated with formyl-methionyl-leucyl-phenylalanine (1 µmol/L) and immediately placed in a microplate reader (THERMOmax with Softmax software; Molecular Devices, Menlo Park, CA) for kinetic measurement of O₂^– production. Absorbance at 550 to 450 nm are measured every 20 seconds for 5 minutes. The maximal rate of O₂^– production (V_max) is determined by the absorbance curve over 5 points. An extinction coefficient of 8.4x10^–3 L/mol/min is used as determined for the 150-µL reaction volume and the 550-nm filter in the microplate reader. Data are recorder as V_max, nmol O₂^–/3.75x10⁵ PMNs/min. An enzyme-linked immunosorbent assay (ELISA) porcine cytokine kit (R&D Systems Inc, Minneapolis, MN) was used to assay the serum concentration of TNF- at 3 time points. The sensitivity of the ELISA kit was 2.8 to 5.0 pg/mL, and the range for the standards was from 0 to 1500 pg/mL. Serum samples with concentrations greater than 1500 pg/mL were diluted in deionized water at 1 to 2, and 1 to 5 dilutions, to measure serum concentrations.

Percent Lung Water and Total Body Water Gain
Peripheral lung specimens, weighing 1 to 2 g were taken from the anterior right middle lobe of each animal at the end of the experiment. To calculate the lung water content, the lung tissue was weighed fresh, and then dessicated in a warming oven at 40°C for 24 hours, prior to being reweighed dry.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA). All data are expressed as a mean ± standard error of the mean (SEM). Paired t tests were used to compare pre-CPB and post-CPB measurements for each animal. Analysis of variance (ANOVA) was used to compare the baseline and post-CPB measurements for control and both experimental groups using the Tukey’s posttest to correct for multiple comparisons. A p value of less than 0.05 was considered significant.

	Results

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 
Arterial Blood Gas and Hemodynamic Data
Baseline hemodynamic data, presented in Table 1, were not different between experimental groups. Arterial PO₂ was slightly higher before CPB in group I, but otherwise baseline arterial blood gases, osmolarity, and hematocrit were similar between all groups (Table 2). Following CPB, mean aortic pressure was slightly higher in group I (45 ± 9) and mean pulmonary artery pressure was slightly lower (18 ± 4) than the groups primed with blood (aortic pressure 27 ± 5 [group I] and 34 ± 12 [group II]; mean pulmonary artery pressure 22 ± 2 [group I] and 23 ± 1 [group II]), although statistical significance was not reached (Table 3). Metabolic acidosis was significantly improved in group III (–1.3 ± 1.0) compared with those animals in group II (–8.2 ± 2.9; p = 0.037). The miniaturized circuit effectively prevented hemodilution because the post-CPB hematocrits were not significantly different among groups (Table 4).

Cardiopulmonary Function Following CPB
Right ventricular cardiac index was significantly better in group III than in groups I and II (p < 0.001) following CPB (Fig 2). Pulmonary vascular resistance index was less (p = 0.033 overall; p = 0.019 between groups III and II) in group III than in the groups primed with blood, which is illustrated in Figure 3. The percent decrease in dynamic pulmonary compliance (C_dyn) was also less in group III (18.0 ± 5.7) than in group I (38.1 ± 4.3) and group II (42.2 ± 4.9; p = 0.022 overall; p = 0.007 between groups II and III. Derived post-CPB hemodynamic data are additionally depicted within Table 5.

View larger version (35K): [in this window] [in a new window]   Fig 2. Right ventricular cardiac index (RVCI) was significantly better in group III than in groups I and II (**p < 0.001) following cardiopulmonary bypass (CPB).

View larger version (25K): [in this window] [in a new window]   Fig 3. Pulmonary vascular resistance index (PVRI) measurements before cardiopulmonary bypass (CPB) and 30 minutes following separation from CPB. In group III, the animals with the miniaturized circuit, the index was significantly improved compared to the other groups primed with blood, p = 0.03 (**p < 0.05).

TNF- and PMN Priming

View larger version (16K): [in this window] [in a new window]   Fig 4. Tumor necrosis factor- (TNF-) production was significantly lower (**p < 0.002) in the animals primed with crystalloid than in those animals primed with blood.

View larger version (17K): [in this window] [in a new window]   Fig 5. Storage of blood resulted in enhanced neutrophil priming activity, as measured by superoxide production, compared with fresh blood (**p = 0.02), although activity was still present even in the fresh blood samples.

	Comment

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 
Conventional CPB is especially problematic in neonates because the large foreign surface area compared to body weight required for circuit priming results in significantly more hemodilution than their adult counterparts [1, 2, 22]. For this reason, children under 4 months old receive the largest number of blood components during CPB compared with any other age population [22]. Up to 50% of these infants will experience significant cardiac or pulmonary dysfunction following CPB and many others exhibit dysregulated coagulation [10, 23].

Recent evidence suggests that blood product transfusion enhances the inflammatory response to CPB and increases myocardial and pulmonary dysfunction [1, 2, 24–30]. Blood transfusion has also been shown to be a major risk factor for postinjury multiorgan system failure, including transfusion related acute lung injury (TRALI) [3–7]. Infants exposed to CPB have been found to be at increased risk following blood usage because the circuit systematically primes host PMNs and activates endothelial tissue beds [6, 26, 29].

The neutrophil has been implicated as a primary effector cell in the pathogenesis of postinjury hyperinflammation leading to TRALI and multiorgan system failure [3–7, 27]. Parry and colleagues [31] also demonstrated a putative effect of neutrophil deganulation on placental and cardiac dysfunction following CPB in a fetal sheep model. The plasma from stored red blood cells directly primes PMNs for cytotoxicity, prompting the release of lytic enzymes (sPLA2, superoxide [O₂^–], and elastase) [3, 4]. In addition, recent studies have documented delayed apoptosis of neutrophils in patients receiving blood transfusions [3, 28]. In support of these findings, stored blood products had a significantly higher concentration of primed neutrophils in our study.

Several strategies have been developed in an attempt to alleviate the adverse effects of blood product usage in infant cardiac surgery, including ultrafiltration, leukoreduction, red blood cell washing, using "fresh" blood as opposed to stored blood, and the use of lipid-free, cytokine-free blood substitutes [18, 19]. Pharmacologic agents such as steroids and aprotinin have also demonstrated efficacy in attenuating inflammation following infant heart surgery, although their use may produce other untoward effects [15–17]. However, controversy exists since no studies conclusively demonstrate whether PMN-mediated inflammation is altered by these strategies, and the expense of universal leukocyte reduction for all blood products is prohibitive because many units become outdated and must be discarded without ever being used [3, 4].

The reduction of prime volume by minimizing circuit length may alleviate the post-CPB hyperdynamic response and improve coagulation function [1, 2, 22, 24–27, 29]. Additionally, small feasibility studies have shown that a low-volume, asanguineous prime can be achieved safely in neonates and in the conduct of fetal cardiac bypass [22, 31]. Wabeke and coworkers [2] reported vacuum-assisted venous drainage in a rabbit model of CPB indicated that the use of a smaller prime (90 vs 330 mL) normalized resistance in the peripheral microcirculation. Jansen and associates [1] similarly found that colloid osmotic pressure was maintained in patients primed with a low-volume circuit, which improved postoperative fluid balance and led to superior cardiopulmonary function.

Our data agree with these findings. Neonatal piglets, in which the miniaturized circuit was utilized, had markedly improved cardiopulmonary function following CPB and significantly less extracellular/interstitial fluid accumulation. The superior right ventricular performance likely reflects improved pulmonary function as the lungs are at increased risk of both inflammation-mediated cytotoxicity and ischemia-reperfusion following CPB. The pulmonary circuit therefore would benefit greatly from methods to abrogate these effects. The use of the miniaturized circuit and avoidance of blood provided a significant reduction in the TNF- burden following HLF, and removed the primed PMNs that are the main substrate responsible for the activation of these cytotoxic cascades. This translated into improved pulmonary compliance, decreased pulmonary vascular resistance, decreased total lung water, and improved gas exchange within the susceptible pulmonary tissue bed.

No clear consensus exists regarding the role of temperature on the systemic inflammatory response following CPB, as recent reports in the adult population have indicated equivocal results over a wide temperature spectrum [32, 33]. Similar cytokine profiles and effector cells are evident following normothermic bypass, suggesting common underlying mechanisms that are independent of temperature [32, 33]. Thus, although this study focused on HLF, inflammation related to normothermic bypass may also be reduced with a strategy of circuit miniaturization.

We were unable to find any change in the thromboelastogram trace in our study. Although there is evidence that inflammation could play an important role in the pathogenesis of bleeding diatheses, this model may not be an ideal one for its evaluation [20, 21, 23]. Blood use, though enhancing the inflammatory response, also provides platelets and clotting factors that may negate any adverse effects. The inability to detect differences could also be due to a lack of statistical power owing to our small sample size.

Other limitations in this study include:

1 The lack of a direct comparison between a conventional circuit without blood and a miniaturized circuit to distinguish whether the beneficial effects we have shown are secondary to reduced circuit size or the avoidance of blood per se. In this infant model we could not use such a circuit without hemodilution, which has its own deleterious sequelae

2 Absence of direct histologic or genetic evidence of a reduced inflammatory state, such as decreased mRNA or protein levels of TNF-, necrotizing factor- B, interleukin-6, or interleukin-1, particularly within the pulmonary tissue bed

3 Our data included only acute changes occurring within 30 minutes following the bypass interval. The relatively short duration of follow-up was mandated because the uniformly poor cardiopulmonary function in those animals receiving a blood prime precluded the use of a chronic preparation

Despite these limitations, this report documents the extensive, adverse end-organ effects of conventional circuit size and blood use in a neonatal swine model of CPB. Accordingly, we have devised a unique miniaturized circuit that allows an asanguineous prime in infants without hemodilution. In addition, we have demonstrated that using this circuit effectively decreases the inflammatory response and has salutary effects on cardiopulmonary function following CPB.

	Discussion

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 
DR IRVING L. KRON (Charlottesville, VA): I want to congratulate you, Tara, on a well presented study and this is the usual sophisticated work from Ungerleider’s lab. They have clearly demonstrated superiority of this miniature circuit without a blood prime. The key issue seems to be inflammation resulting in increased pulmonary water and thus RV dysfunction. I have three questions for you.

Is there a downside to using this technology clinically? Did you consider using blood in the miniaturized circuit? In other words, is the issue the circuit, which is much smaller, or the blood that you put in it?

And, finally, are there issues of the preparation? We have a lot of familiarity with this infant swine prep with lung transplantation. They tend to have increased pulmonary vascular resistance if you fool with them in any way, so maybe you can project how this might work out in a neonate. Thank you.

DR KARAMLOU: Thank you, Dr Kron, for your questions. With regard to your question relating to using the circuit rather than the volume, I think that these two are inextricably related in that an asanguineous prime in an infant necessitates a miniaturized circuit and therefore we could not separate the two without producing hemodilution, which, as you know, has its own deleterious effects.

In addition, another part of this study, which I didn’t present today in the interest of time, was that we were interested in looking at coagulation function via thromboelastography (TEG), which would be influenced by drastic hematocrit differences. Hemodilution was therefore an even more important issue in that platelets and clotting factors would also be affected if we used a larger circuit. So, therefore we did not look specifically at whether the reduction in circuit surface area or priming volume per se accounted for our findings, and I cannot provide a specific answer to your question.

With regard to your other question relating to the applicability of a porcine model versus a human model; there have been several homologous results from other animal species, indicating the beneficial effects of circuit miniaturization. There was a notable report from Jansen et al using rabbits, which showed similar findings in that peripheral resistance was also normalized when a clear prime was utilized.

And I think more importantly, in the human theater, if you look at the New England Journal study which was recently published, or other studies in the trauma literature looking at both pediatric and adult patients, there is no question that any blood transfusion, but particularly stored blood product usage, has significant deleterious effects, mediated by a two-hit model (ie, if there is an initial insult, which in this preparation is likely the instrumentation followed by cardiopulmonary bypass, that these patients are extremely susceptible to transfusion-related enhanced neutrophil cytotoxicity such as present in this model.)

And could you please repeat your first question?

DR KRON: What is the downside to this sort of preparation if used clinically?

DR KARAMLOU: Thank you. I think the most evident downside is that we could not use an arterial filter in this preparation because that would necessitate a further increase in the prime. Certainly we do not advocate removing the arterial filter, although I think that there is some data that may question its utility. But I think in our preparation that is certainly one of the most obvious downsides.

The other downside is that the efficiency of the oxygenator and the degree that you can actually increase the pump flow rates are limited certainly by the circuit size. And finally, using vacuum-assisted venous drainage, although we have had good results clinically in Oregon, also may have some unfavorable effects, specifically with regards to lysis of the cellular elements within blood.

DR ANDREW LODGE (Durham, NC): I too would like to congratulate you for a well designed and well presented study. I have two questions since Dr Kron already asked the third. One relates to the experimental preparation. You mentioned that you obtained the blood from the donor animals by exsanguination, which is obviously not the usual protocol for human donors, and you might speculate that as the blood is obtained from the donor animal as dying from exsanguination, it might contain various cytokines and inflammatory mediators that could impact your results. I wonder if you took that into account and separated the blood into the initially drained blood and used that preferentially?

The second question relates to the use of the stored blood. Usually institutions that have access to whole blood use it well before the age of 6 days or else resort to component blood products, and I wonder if you could expand on the differences in results between groups I and II, particularly the tumor necrosis factor (TNF) levels, whether they were different between groups I and II, and what impact you think the age of the stored blood may have had on the differences in those groups?

DR KARAMLOU: Thank you, Dr Lodge, for your questions. With regard to the exsanguination of the donor animals, that is a very good point. Actually because the prime was not excessive, even in the animals receiving the blood prime, we harvested the blood initially and placed it into one CPD pack, which was a 300 CPD or close to a 300 CPD pack. Because we were using adult donor swine, the animals experienced almost no hemodynamic impairment at that point, and we terminated blood collection after that time point so that if the animal did develop hemodynamic compromise and the blood became acidotic or other untoward things may have happened, we elected to not use those blood products. So hopefully that effect was minimized, although not completely eradicated, as you suggest.

Small sample size necessitated combining the "blood groups" into one single group for the statistical analysis, and therefore we could not directly determine whether or not there was a significant difference between old and stored blood with respect to tumor necrosis factor-alpha production.

However, I think that the crux of this study is that when you separate the blood based on the duration that it was stored, and clearly, the fresh and stored blood were separated in the neutrophil priming assay, that there is a significant difference, and this is in agreement, as I mentioned, with all of the studies done very elegantly by Chris Silliman’s group in Denver looking at transfusion-related lung injury in trauma patients that have shown exactly the same findings.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 
This work was supported in part by a generous grant from the Children’s Heart Foundation and the Medical Research Foundation of Oregon.

	Footnotes

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 
^_* Recipient of the 2004 Hawley H. Seiler Resident Award.

	References

Top Abstract Introduction Material and Methods Results Comment Discussion Footnotes Acknowledgments References

 

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