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a Department of Cardiothoracic Surgery, Center for Heart and Lung Disease, Lund University Hospital, Lund, Sweden
b Department of Electrical Measurements, Faculty of Engineering, LTH, Lund University, Lund, Sweden
Accepted for publication December 26, 2007.
* Address correspondence to Dr Jönsson, Department of Cardiothoracic Surgery, Lund University Hospital, SE-221 85 Lund, Sweden (Email: henrik.jonsson{at}med.lu.se).
| Dr Petersson discloses that he has a financial relationship with ErySave AB.
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Abstract |
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Methods: Blood samples were collected from 24 patients undergoing routine cardiac surgery with cardiopulmonary bypass. Arterial and shed blood was analyzed using the Coulter counter technique to establish the number and size of particles. The composition of these lipid particles was compared with that of adipose tissue from the mediastinum using gas chromatography. Scanning electron microscopy was used to visualize the lipid particles in samples of shed blood.
Results: Lipid particles in the size range of 10 to 60 µm were characterized in shed mediastinal blood, and more than 300,000 particles per milliliter of blood were found. Triglyceride profiles in these lipid particles and in adipose tissue were similar, suggesting that their origin is the mediastinum. Scanning electron microscopy showed spherical formations corresponding in size to the particles counted using the Coulter counter.
Conclusions: During the past decade attention has focused on microembolism in cardiac surgery, and this study has helped define the problem. Different strategies, such as eliminating the use of shed mediastinal blood or purifying the blood by different techniques, may improve the results of cardiac surgery in the future.
In cardiac surgery with cardiopulmonary bypass (CPB), retransfusion of shed mediastinal blood is common practice to minimize the blood loss. It has been shown that this blood is contaminated with lipids, which may act as emboli [1]. Furthermore, these particles pass through the CPB circuit and find their way into various organs [2].
In the search for the pathogenesis of CPB-derived brain damage, Moody and colleagues [3–6] documented the presence of small capillary and arteriolar dilatations in autopsy specimens from patients who had undergone cardiac surgery with CPB, and it was later confirmed that these small capillary and arteriolar dilatations were traces of lipid deposition in the brain capillaries.
In a recent porcine study with radioactively tagged lipids, it was shown that these emboli not only find their way into the brain but also, in vast numbers, into the liver, the kidneys, and the heart [2]. Although it is understandable that the retransfusion of shed mediastinal blood containing lipid material will form emboli in several organs, little is known about the nature and physical properties of these lipid particles as they occur in shed blood.
Previously, authors have found lipid particles with light microscopy in CPB [7, 8]. Despite the lack of detailed knowledge about these particles, they continue to be the subject of lively debate. Several techniques have been proposed to avoid them. Some surgeons discard the shed mediastinal blood altogether to eliminate the potential embolic load. A number of new techniques are in use or are emerging. For example, closed-circuit CPB and cell-saving devices are already in use [9], and particle separation by ultrasound (PARSUS) and sedimentation-based separation are under development [10–12].
The aims of this study were to confirm the existence of these particles in human blood and to characterize them thoroughly using various techniques.
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Material and Methods |
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Coulter Counter Analysis
A Multisizer 3 Beckman Coulter counter with a 100-µm aperture probe (Beckman Coulter Inc, Fullerton, CA) was used for particle size determination [13]. The narrow aperture between the electrodes constitutes the sensing zone through which suspended particles pass. In the sensing zone each particle displaces its own volume of electrolyte. The volume displaced is recorded as a voltage pulse, the height of each pulse being proportional to the volume of the particle. The device was programmed to count and determine the size of all particles with diameters between 2 and 60 µm, at 0.2-µm intervals.
All blood samples were divided into two separate aliquots of 7 and 3 mL. The 7-mL aliquot was centrifuged at 4,200 rpm for 45 minutes to separate lipid material from blood cells. Twenty microliters of the supernatant was diluted with 100 mL of saline solution, and was used for analysis in the Coulter counter. In the protocol used, particles were counted for 100 seconds. From the 3-mL aliquot of noncentrifuged blood, 1 µL of blood was diluted with 100 mL of saline solution. A fixed volume of 10 mL was then analyzed in the Coulter counter.
Gas Chromatography
Gas chromatography was performed on mediastinal adipose tissue and the supernatant of shed blood from the first 9 patients. The samples were prepared in a three-step process including extraction, lipid fractioning, and transesterification [14, 15]. Neutral lipids and phospholipids were then analyzed separately using a Hewlett-Packard HP-5 column in a Hewlett-Packard 6890 gas chromatograph (both supplied by Agilent Technologies Sweden AB, Kista Sweden).
Scanning Electron Microscopy
Supernatant from shed blood was mixed with a 1% solution of OsO4 (Link Nordic, Stockholm, Sweden), which was dissolved in 0.1 mol/L Sörensen buffer at 7.2 pH. It was placed on a SuperFrost glass slide (Menzel-Gläser, Braunschweig, Germany) to allow for sedimentation. The samples were then dried and transferred to buttons for scanning electron microscopy. The samples were covered with a 15-nm layer of gold in a Polaron E5150 scanning electron microscopy coating unit (Quorum Technologies Ltd, East Sussex, UK). Scanning electron microscopy was performed with a Philips 515 scanning electron microscope (Philips, Amsterdam, The Netherlands).
Data Analysis
All data are presented as mean ± one standard deviation unless otherwise stated. To determine the lipid particle content in whole (noncentrifuged) blood, subtraction analysis was performed. First, normal particle or cell content of blood from the arterial line was determined for each patient using Coulter counter analysis. The same analysis was then performed on the shed mediastinal blood. The number of particles not normally occurring in arterial blood was determined by subtracting the background level (arterial blood) from that in shed blood. The final result was corrected for the difference in hematocrit in the samples. Particles were grouped in size ranges of 5 µm to determine the distribution of particle sizes in noncentrifuged blood. Particles smaller than 10 µm were excluded to avoid interference from any remaining blood cells.
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Comment |
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When using cardiotomy suction, these lipid particles are introduced into the CPB circuit and present an immediate embolic threat to patients undergoing open heart surgery. In a porcine model, it was shown that lipid particles entered the circulation and found their way into several organs [2]. Emboli in this size range have been found in the brains of dogs in an experimental model [1], and in patients who died several years after cardiac surgery [5, 16]. Even though this study did not investigate the route of the particles through the CPB circuit, it is likely that they would pass the circuit and end up as embolic material in different organs.
When lipids are added to a hydrophilic liquid such as blood, they do not dissolve but remain in suspension. For lipids to dissolve in water or blood, emulsifiers and energy must be added. We did not determine whether the particles were emulsified or not. However, the finding of separate particles in both Coulter counter analysis and electron microscopy indicates that the particles were in fact emulsified. A vast number of agents normally found in blood can serve as emulsifiers (eg, circulating albumin, and phospholipids derived from cell membranes and membranes from naturally occurring lipids). In addition, hemolysis in blood passing through the CPB circuit provides an abundant source of cell membranes (phospholipids) that can act as emulsifiers. Whether lipid particles are emulsified or not can play an important role in the interaction with the CPB circuit, as most of the surfaces are hydrophobic, eg, polyvinylchloride, silicone tubing, polyethylene, and microporous polypropylene, in the filtering system and oxygenator. Hydrophilic particles, such as blood cells and emulsified lipids, will thus not adhere to the surfaces in the CPB circuit, but pass freely into the circulation.
The Coulter counter analysis is a novel method of measuring particles in blood during cardiac surgery. The method is well established in the accurate determination of particles such as microspheres, blood cells, bacteria, and cells from cell cultures [13, 17]. In this study we were able to count tens of thousands of particles in each sample quickly and accurately. Moreover, the device also determines the volume of each particle.
When introducing new analysis techniques, sample preparation may alter the original composition, leading to erroneous or unrepresentative results. Sample centrifugation is a potential source of error, because strong forces are applied to biologic matter, which may alter its structure. However, the subtraction analysis of noncentrifuged blood revealed a similar pattern of size distribution for particles larger than 10 µm as compared with centrifuged shed blood (Figs 1, 2). It is therefore probable that the centrifugation process did not affect the size distribution of lipid particles in the suspension. Scanning electron microscopy clearly showed spherical particles in the same size range as found using the Coulter counter (Fig 4). Substances added to the samples before analysis could also lead to errors. In this study we only added saline solution at room temperature, which is unlikely to affect the composition of the particles. Based on our experience, the Coulter counter seems to be a reliable and robust method of determining the size and number of lipid particles in blood.
Gas chromatography was used to determine the lipid composition in the supernatant of shed blood and in adipose tissue from the mediastinum. Similar patterns were found. However, this analysis was not corrected for other lipids, such as endogenous lipids and the anesthetic agent propofol. Correction for these lipids could affect the results. Gas chromatography should therefore only be used to provide an indication of the potential source of these lipids.
We did not address the possible clinical effect of the emboli on the patients in this study. We have, however, learned that each milliliter of shed blood can contain hundreds of thousands of lipid particles. Other authors have shown that lipid material will end up as emboli in various organs throughout the body [1, 2, 5]. It is thus reasonable to assume that retransfusing shed mediastinal blood will cause organ damage. Two recent studies using cell-saving devices to wash mediastinal blood have shown conflicting results on neuropsychological outcome [18, 19]. One study showed a clear reduction on the incidence of cognitive decline, whereas the other reported none. With this combined knowledge, it is difficult to ignore these particles and the threat they may pose to the patient.
A novel technique has been used to characterize lipid particles in shed blood, which has opened new opportunities in embolism research. In addition, we have highlighted the need for further research into the potential threat of these particles to patients undergoing cardiac surgery.
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This article has been cited by other articles:
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  H. Jonsson, A. Eyjolfsson, S. Scicluna, P. Paulsson, and P. Johnsson Circulating particles during cardiac surgery Interactive CardioVascular and Thoracic Surgery, May 1, 2009; 8(5): 538 - 542. [Abstract] [Full Text] [PDF]
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