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J Thorac Cardiovasc Surg 2002;124:377-386
© 2002 The American Association for Thoracic Surgery
Cardiopulmonary Support and Physiology (CSP) |
From the Heart Center, Department of Surgery and Perioperative Science, Division of Cardiothoracic Surgery, Umeå University Hospital, Umeå, Sweden.
Supported by the Swedish Society for Medical Research and funds of the Medical Faculty, Umeå University Hospital; the Swedish Medical Research Council (12X-11204); the Swedish Heart and Lung Foundation, and the Heart Foundation of North Sweden.
Received for publication July 11, 2001. Revisions requested Aug 21, 2001; revisions received Nov 27, 2001. Accepted for publication Dec 12, 2001. Address for reprints: K. Gunnar Engström, MD, PhD, Heart Center, Department of Surgery and Perioperative Science, Cardiothoracic Division, Umeå University Hospital, S-901 85 Umeå, Sweden (E-mail: Gunnar.Engstrom.us{at}vll.se).
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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1,2 This brain damage has several possible causes. General effects of low blood pressure and cardiopulmonary bypass (CPB) with systemic inflammatory response are plausible, but in addition, embolic mechanisms are commonly suspected. 3 These embolic sources might either be air bubbles 4 or particulate matter. 5 They might also be described in terms of microembolism and macroembolism, which can be the result of detached particles from a calcified ascending aorta 6 or plastic or silicon material generated from the CPB circuit. 7,8 These particles enter the aorta through the arterial line. Recently, the pericardial suction blood (PSB) has been suggested as a source of fat-related deposits in the brain capillaries of animals 9 and human subjects 10 after CPB exposure. PSB is contaminated with cellular and liquid fat from the wound. 10 It is further possible that activated white blood cells and platelets add to this deterioration. 11,12 We aimed to describe the capillary-pore flow properties of PSB with the hypothesis that fat contamination can be monitored with this technique. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Collection system of PSB
An emptied 2-L saline bag (Fresenius-Kabi) was interpositioned between the cardiotomy roller pump and the cardiotomy reservoir. The vent line was not part of the PSB system. The PSB-containing bag was continuously weighed with an analog scale. The density of PSB was determined to be 1.055 kg/L, and PSB weight was converted to volume. After the release of the side-biting clamp, the collected PSB was gently mixed before blood sampling and reinfusion. Venous blood and PSB, each in 20-mL samples, were carefully aspirated from the venous reservoir inlet and from the PSB collection bag. Identical syringes were used, and the samples were sent to the laboratory for immediate analysis.
Media
A Krebs-Ringer-HEPES buffer (KRH) was used (NaCl, 135 mmol/L; KCl, 4.7 mmol/L; KH2PO4, 1.2 mmol/L; and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 20 mmol/L) supplemented with 0.02% wt/vol human serum albumin, 5.0 mmol/L glucose, and 2.56 CaCl2. The buffer was prefiltered through a 0.45-µm Millipore (Millipore Corp, Bedford, Mass) membrane and had an osmolality of 300 mosm and a pH of 7.4, which was calibrated by adding NaOH.
Blood sample processing for capillary-pore analyses and cellular counts
In the initial 20 patients, PSB and venous blood were processed to explore their different subcomponents. This process is shown in Figure 1. In 10 of these samples, the PSB was allowed to drain through a cardiotomy screen filter. A cardiotomy filter was taken from inside a standard reservoir (Monolyth VRF 40; Sorin, Saluggia, Italy) to accomplish this. This filter is a traditional nylon 30-µm screen mesh from which circular pieces (13 mm) were punched out and mounted in a standard filter holder (Nuclepore Corp, Pleasanton, Calif). This prefiltering was done without applying driving force to simulate the spontaneous emptying of PSB into the cardiotomy reservoir.
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). The 2 blood samples were resuspended in excess KRH to 50 mL and recentrifuged to wash out remaining plasma. This centrifugation and resuspension was repeated twice. In the sample from which the buffy coat had been aspirated, the remaining contaminating leukocyte cells were absorbed by using a leukocyte filter (Pall RC 400; Pall Biomedical Inc, Fajardo, NY). This was because buffy coat aspiration alone only removes about 50% of the leukocytes. This technique was a modified version of our previous method and is efficient in removing contaminating leukocytes and the great majority of platelets. 13
After washing, the blood cells were resuspended to achieve a 40% hematocrit level. A small sample was centrifuged (10,000g for 10 minutes) for microhematocrit measurement, which was read in a microscope. On the basis of this exact cell-volume fraction, the blood was diluted to precisely reach the 5% target hematocrit level, which is a recommended concentration for capillary-pore flow analysis. 14
Measurement of capillary-pore flow
The experiments were all carried out at room temperature. 14 The capillary-pore flow properties of the blood subcomponents were evaluated by using polycarbonate membranes with a 5-µm nominal pore diameter (pore density, 4.0 x 105; SN: 113613 Lot: 54M8B46; Nuclepore Corp, Pleasanton, Calif). The filters were mounted in the KGE flowmeter (ESTRAB, Umeå, Sweden; Figure 2), which has been well described elsewhere. 15 A hydrostatic constant driving pressure of 600 Pa was used. A digital balance (Mettler PM 480 Deltarange; Mettler instrumente AG, Greifensee, Switzerland) measurement was continuously acquired by using a standard personal computer. The onset and termination (3.15 seconds) of the flow was controlled by the computer through a pneumatic slide valve. The flow was derived from an accumulated volume at a 7.5-Hz sampling rate, assuming a 1.0 kg/L density of the low hematocrit resuspension (approximately 1 mg-1 µL). Each filter was calibrated with a blank run before each blood sample (see flow curve in Figure 3, upper panel). At flow onset, there is acceleration before a steady state is reached. The acceleration phase was omitted from the calculations. 15 All presented data refer to the 1- to 3-second window. At steady state, the flow declines with time as a result of a gradual occlusion of the pores by particles or rigid cells. The negative curve slope is an indicator for capillary-occluding property. The results are presented as relative flow (blood sample divided by blank medium; Figure 3, lower vs upper panel) to compensate for filter variations. From a linear regression of the relative curve, 1 to 3 seconds, the intercept at 1 second expresses the initial filtration rate (IFR). The decline in relative flow represents the clogging slope (CS). It is important to emphasize that as plasma was removed and the blood cells were resuspended in KRH, protein-induced cell aggregation did not occur in the experiments. The platelets are present in the plasma sample but are reduced in number to about 30% in the whole-blood resuspension or near completely eliminated in the erythrocyte resuspension because they are trapped in the leukocyte prefilter.
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A microscopy method was developed with the aim of demonstrating the mechanisms behind differences in plasma capillary-pore flow. The 5-µm Nuclepore membrane was mounted in a 13-mm Pop-Top filter holder (Nuclepore Corp), through which 1 mL of plasma was filtered. The membrane deposit was imprinted on a noncoated microscope glass slide. The imprint was air-dried for 30 seconds and fixed in 10% formaldehyde in phosphate buffer, followed by staining without dehydration. The staining had a hydrophilic-basophilic component (Hematoxylin-Mayer) and a hydrophobic fat stain (oil red in 50% isopropanol). After drying, a cover slip was mounted with gelatin. The occurrence and type of deposit was evaluated under the microscope. Because the great majority of deposits consisted of fat-positive material, a 10x objective (Carl Zeiss, Obekochen, Germany) was used to provide an optimal overview. The slides were coded, and the amounts of deposits were evaluated with a semiquantitative scale (minimum-maximum = 0-4). The observer was blinded to the slide identity.
Statistics
Analysis for differences between venous blood and PSB was done with the paired Student t test. Differences between groups of patients, with and without a cardiotomy filter, were determined by means of unpaired testing. The results demonstrated a close to normal distribution. The capillary-pore flow data were evaluated by using linear regressions with a least-sum square method. The microscopy data were evaluated by means of exact probability calculation. Mean ± SEM values are given.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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).
Blood cell characteristics, PSB versus venous blood
The PSB differed from venous blood (Table 2). The 30-µm cardiotomy filter did not affect the PSB cell counts, and in Table 2 the results were pooled (n = 20). The PSB and venous blood had similar hemoglobin concentrations, and there were no artifacts from hemodilution. However, the leukocyte counts showed differences between groups. The overall numbers were reduced in PSB, which solely reflects the neutrophilic cells. Platelets were found to be increased compared with levels seen in venous blood. For comparison, the blood cell counts before and after the operation are also shown (Table 2).
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, upper panel). The flow data are better expressed on their relative scale versus blank flow to compensate for filter variations (Figure 3, lower panel; Table 3). The addition of the 30-µm cardiotomy filter did not improve the PSB plasma-flow properties. The IFR at 1 second (Table 3) illustrates the relative effect of plasma in this model. The plasma-flow properties did not correlate with plasma platelet counts.
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). There were no detectable differences between the behavior of venous blood and PSB (Figure 4). In this setting the leukocytes have a great effect on the filter-occluding properties, and leukocyte counts showed a strong inverse association to flow. The IFR was sensitive to neutrophilic and macrophage counts (P < .05 and P < .01 for linear correlation, respectively). The CS was sensitive to lymphocyte and platelet counts (P < .05). The addition of the 30-µm cardiotomy filter appeared to decrease clogging, as indicated by the CS measurement (P < .05, Table 3).
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and 5, respectively). PSB did not differ from that of venous blood, and the 30-µm filter did not add detectable changes (Figure 5). However, when the 2 groups were pooled (n = 20), there was a close-to-significant (P < .06) steeper CS in PSB, possibly indicating an erythrocyte trauma.
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The PSB free hemoglobin demonstrated no correlation to PSB plasma viscosity. The plasma relative viscosity was not different between PSB and venous plasma (1.35 ± 0.07 vs 1.33 ± 0.06, respectively).
Microscopy results
The filter imprint revealed deposits of fat-positive staining in PSB. In the microscopic evaluation the PSB showed positive results in 5 of 5 experiments (P < .05). In terms of the semiquantitative scale (0-4 points), the venous plasma showed an average result of 0.6, and PSB plasma showed and average result of 3.2 (median, 1 vs 3). Typical deposits are demonstrated in Figure 6.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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16 Despite extensive research, this problem remains without a definitive solution. 1,2,17 A recent concern is the recycling of PSB. PSB might contribute to the systemic inflammatory response that occurs after CPB. 3 It is known today that PSB also contains fat from the wound. 10 Liquid fat is not a natural part of blood, which might be exemplified by fat-resembling microemboli (small capillary and arteriolar dilations) found in the small-diameter arteries of the brain after CPB. 9,10
We have previously studied the capillary-pore flow function of autotransfusion blood and fat contaminants after knee operations 18,19 and found markedly impaired results in terms of plasma flow. This finding was interpreted as being caused, in large part, by contaminating particles. After cardiac operations, mediastinal drain blood is autotransfused into the venous system, and clogging material might end up in the pulmonary circulation. However, during cardiac operations and CPB arterial reinfusion, such debris can be expelled into the cerebral circulation. Furthermore, in a previous study of ours 20 on CPB-induced blood changes, it was found that leukocytes triggered a marked reduction in 5-µm capillary-pore flow. This appeared both as leukocyte activation but also as a result of leukocytosis during CPB. Of great interest was the finding that the plasma improved in capillary-pore flow behavior because of the crystalloid prime at CPB onset but was then significantly reduced during CPB. This suggested a mechanism of gradual particle contamination from the continuously added PSB into the CPB circuit.
This study is a follow-up to our previous work 20 and was designed to describe the capillary-pore flow properties of PSB in an in vitro model. For this purpose, PSB was tested in a specially designed PSB collection system. From a clinical point of view, this system worked without apparent effects on CPB management or patient safety. The patients tolerated this retained volume. The PSB was poor in leukocytes, mainly neutrophilic cells. The hemoglobin concentration was equal to that of venous blood. The mechanisms behind the neutropenia in the PSB are unknown but can in theory reflect an activation and adhesion to the wound tissue.
We used a method to measure the flow of controlled suspensions of blood through well-defined narrow capillary pores, here of 5 µm in diameter and 11 µm in length. These pores and applied methods are well described in the literature 14 and have many clinical implications. 21 However, over the years, artifacts have been described, including by our group. 15 Although the experimental principle might appear simple, the biophysics are complex. However, the method used in this study provided results that might help in the understanding of how blood works in human capillaries during CPB and PSB reinfusion. Our initial focus was on PSB leukocytes, and the method again proved sensitive to leukocytes. However, the leukocyte contribution to the positive findings was negligible in comparison with PSB plasma characteristics. When PSB plasma was compared with that of venous plasma, an obvious deterioration was detected. The PSB plasma capillary flow indicated a markedly increased capillary clogging ability, and at 3 seconds, the flow was only about 30% of that for venous plasma and, in fact, was close to zero in many of the experiments. The leukocyte flow spectrum was studied by using resuspended whole-blood cells, but PSB did not differ from venous blood in this respect. The erythrocytes were tested after leukocyte absorption but were also unaffected. One reservation was a close-to-significant steeper CS slope in PSB erythrocytes, which suggests cellular trauma. PSB demonstrated major hemolysis, but the nonlysed cells functioned well. It is important to emphasize that the blood cells were washed and free from plasma, and therefore cell aggregation did not occur. The PSB hemolysis constituted a major portion of free hemoglobin detected in the circulation, as described by others. 22 Another concern that is often recognized is the effect of platelet activity in CPB systems. The whole-blood fraction of resuspended cells correlated with not only leukocytes (CS) but also with platelet counts (IFR). Platelets were found in the plasma fraction, but plasma-flow properties did not correlate with platelet counts. In general, platelets have a smaller diameter to that of the in vitro capillaries used in this model and therefore do not interfere with the results. The effect of a 30-µm cardiotomy filter was also tested, but there was no evidence of beneficial function in this respect.
The impaired PSB plasma flow can be explained by 2 possible mechanisms: either the viscosity is changed or the plasma contained contaminating particles. The PSB viscosity was tested but did not demonstrate differences versus the venous plasma. Contaminating particles were investigated by means of a newly developed microscopy method. Filter deposits were imprinted on to a glass slide for fixation and staining. Two stains were added to examine the possibility of either basophilic cellular presence, such as platelets and leukocytes, or hydrophobic material, such as fat. The presence of fat was clear (Figure 6).
In conclusion, these results support previous findings that suggested the presence of fat microemboli of PSB. 10 This method might help in the development of new means to monitor and avoid fat contamination from PSB during CPB. Although it remains difficult to extrapolate the in vitro findings to clinical consequences, these findings are of enough magnitude to strongly question the routine use of unprocessed PSB.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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