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The influence of extracorporeal circulation on erythrocytes and flow properties of blood
J
THORAC CARDIOVASC SURG
1990;100:538-45
The influence of extracorporeal circulation on erythrocytes and flow properties of blood The influence of extracorporeal circulation on red blood cells and flow properties of blood was studied in 10 patients undergoing aorta-coronary bypass grafting. Blood samples were drawn on admission, under general anesthesia before the operation, during extracorporeal circulation, immediately after extracorporeal circulation, and 24 hours after extracorporeal circulation. Echinocytes were found during and shortly after extracorporeal circulation, but disappeared within 24 hours. Washing the cells in buffer restored the normal discocytic shape, which indicated that a plasma factor was responsible. Red ceU membrane lipids were not affected. Analysis of the membrane proteins revealed a decrease of ankyrin after extracorporeal circulation, which was prevented by protease inhibitors during prepara-. tion. This suggests an increased proteolytic activity of the plasma after extracorporeal circulation. Red cell deformability was not altered. Plasma viscosity and hematocrit were markedly reduced by hemodilution with the priming solution. Their low levels resulted in a low blood viscosity during extracorporeal circulation, which was even lower at 26° C than before or after the operation at 37° C. We conclude that the red cell is affected by extracorporeal circulation. The flow properties of blood, however, are not impaired, but are improved by hemodilution.
Walter H. Reinhart, MD, Peter E. Ballmer, MD, Franziska Rohner, MD, Peter Ott, PhD,' and P. Werner Straub, MD, Bern. Switzerland
Etracorporeal circulation (ECC) is a prerequisite for cardiac operations. Over the years the technique has been refined and now has a low mortality rate. I The post-ECC morbidity, however, is still substantial and warrants intensive postoperative care. Several factors have been implicated in this morbidity. A major factor is the generation of oxygen free radicals, which may lead to lipid peroxidation in cell membranes.e" A large number of abnormalities have been described, such as an activation of the complement system through the alternative pathway,S,6 a deposition of complement complexes on erythrocytes,? hemolysis.I-" activation of platelets? and blood clotting, I0 sequestration of leukocytes in the lungs, II. and leukopenia.f Recently a profound change of the red blood
cell (RBC) shape during ECC has been observed.F The RBC shape affects the flow properties of blood in the macrocirculation and microcirculation.P One may ask, therefore, what effect ECC has on blood rheology and if an impairment of the flow properties of blood could be responsible for intraoperative and postoperative disorders such as the decreased cerebral blood flow after ECc. 14 Many of these changes were observed with bubble oxygenators, which are known to be more traumatizing for blood than the newer types of membrane oxygenators.P In the present study we have investigated the influence of ECC with membrane oxygenators on the RBC shape, biochemical properties of the RBC membrane, and rheologic properties of blood. Patients
From the Department of Internal Medicine, Inselspital, and Institute of Biochemistry and Molecular Biology,' University of Bern, Bern, Switzerland. Received for publication July 26,1989. Accepted for publication Dec. I, 1989. Address for reprints: Dr. Walter Reinhart, Department of Internal Medicine, University of Bern, Inselspital, CH - 3010 Bern, Swit-
zerland,
12/1/19699
538
Ten patients (onewomanand nine men,aged 50to 67 years) with coronaryheart disease (four patientswith three-vessel disease,six patients with two-vessel disease),but withoutvalvular heart disease, werestudied.They were treated beforethe operation with nitrates (nine patients), i1-blocking agents (eight patients),and calcium-channel blockers (six patients).The following procedures weredone:18aorta-coronary bypasses, eight grafts with the internal mammary artery, and one ventricular aneurysmectomy. Halothane, fentanyl, and midazolam were usedfor anesthesia. During ECC 10gm mannitoland 4 ~I hep-
Volume 100 Number 4 October 1990
arin (200 U) per kilogram of body weight were administered. A membrane oxygenator (Maxima, Medtronic Inc., Minneapolis, Minn., or CML2, Cobe Laboratories, Lakewood, Colo.) was used. The system was primed with 2.5 L of a I: I mixture of 0.9% saline and Ringer's lactate with sodium bicarbonate 48 mmol/ L, potassium 10 mmol/L, magnesium 4 mmol/L, and calcium gluconolactobionate 3 gm. An arterial oxygen pressure of about 200 mm Hg was provided by an oxygen blender (Cobe Laboratories). Hypothermia was maintained at 26° C during ECC. The flow rate of the roller pump (Shiley-Stockert, Miinchen, Federal Republic of Germany) was 2.4 L/ min/m 2 at normothermia and 1.2 L/min/m 2 at hypothermia. An arterial filter (Bentley AF 1040 C, 40 ~m, Baxter Healthcare Corporation, Irvine, Calif.) was intercalated between the pump and the patient. The mean ECC time was 92 ± 40 minutes and the aortic clamping time, 60 ± 20 minutes. Informed consent was obtained from the patients before the operation. Blood anticoagulated with ethylenediaminetetraacetic acid was withdrawn on five different occasions: (1) on admission to the hospital before the operation, (2) during general anesthesia before the operation, (3) during ECC (60 minutes after the beginning of ECC), (4) immediately after changing from ECC to the patient's own circulation, and (5) 1 day after the operation. No blood transfusions were given during ECC.
Methods Drops of blood were fixed overnight in cacodylate-buffered 1% glutaraldehyde and viewed with a light microscope. The shape of the RBCs was classified according to the nomenclature of Bessis." Some specimens were processed for scanning microscopy (PSEM 500, Philips, Eindhoven, The Netherlands) by dehydration in increasing ethanol series, air-drying on a coverslip, and coating with gold palladium. The RBC membrane lipids were extracted from specimens obtained on admission, before ECC, and after ECC with the method of Rose and Oklander.!? The membrane phospholipids were separated by two-dimensional thin-layer chromatography according to Broekhuyse'" and quantitated as inorganic phosphorus with the method described by Rouser, Fleischer, and Yamamoto. 19 The fatty acid composition of the total membrane phospholipids was analyzed by gas-liquid chromatography. Membrane proteins were studied in RBC ghosts, which were obtained by lysing washed RBCs obtained before and after ECC in a tenfold volume of hypotonic (10 mOsm/kg water) phosphate buffer with four or fiveconsecutive washes. In a later series of experiments with 10 other patients the influence of proteases was eliminated. Heparinized blood was run over a column of acelluloseand microcellulose (I: I mixture) to remove white cells and platelets and was then lysed with the above-mentioned buffer to which the protease inhibitors pepstatin A (2 ~g/rnl) and phenylmethylsulfonyl fluoride (20 ~g/ml) were added. Electrophoresis was performed with 3.5% SDS polyacrylamide gels. Approximately 20 ~g protein with and without the reducing agent dithiothreitol was used. The gels were stained with Coomassie brillant blue and scanned with a photometer at a wavelength of 560 nm. The areas under the curve of the different membrane protein bands were determined. A closed loop of tubing in an ECC pump (Polystan, Copenhagen, Denmark) was used to test the influence of the roller pump alone on the RBCs. Heparinized blood was diluted with
RBCs during ECC 5 3 9
saline to a hematocrit value of 25% as during ECC and pumped at 140 rpm and a flow rate of 2.4 L/min (100 pump pas,sages per minute) for 0, 20, 40, and 80 minutes. Although the blood temperature rose gradually from 22 ° C at the beginning to 38° C after 80 minutes of pumping, hemolysis was only minimal ( 1.5%after 80 minutes). RBC morphology and membrane proteins were analyzed as described earlier. RBC deformability was assessed with a filtration test described previously'? with some slight modifications.U The RBCs were washed three times in phosphate-buffered saline (PBS) containing glucose 0.2 gm/dl and human serum albumin 0.25 gm/dl, The cells were resuspended in PBS with a hematocrit value of 10%. Some drops were fixed in glutaraldehyde for morphologic examination. The RBC suspension was then pumped at a flow rate of 0.5 ml/rnin through a polycarbonate filter with a mean pore diameter of 2.84 ± 0.17 ~m (Lot No. 62BIC58, Nuclepore Corp., Pleasanton, Calif.). The filtration pressure was measured on the upstream side of the filter. The relative filtration resistance of an RBC (fJ = filtration resistance in a pore containing an RBC in transit compared with a pore with suspending medium alone) was calculated as: fJ = 1 + ([Pi/Po] - 1) V/h, where Pi is the initial pressure rise, Po the filtration pressure for suspending medium alone measured before RBC filtration, V the fraction of the pore volume occupied by the RBC, and h the fractional volume of RBCs in suspension.P The flow properties of blood were assessed according to Chien 22 by measuring (I) plasma viscosity, (2) whole blood viscosity, which is influenced by the actual hematocrit, (3) viscosity of an RBC suspension in plasma with a normalized hematocrit value of 45%, and (4) viscosity of a suspension of washed RBCs in PBS with a hematocrit value of 45%, which reflects primarily RBC deformability, Viscosity was measured with a Couette-type viscometer (LS-30, Contraves, Ziirich, Switzerland). Whole blood and RBC suspensions were measured at four different shear rates, namely, 0.1, 0.87, 10.2, and 87.0 sec-I. The plasma viscositywas measured at 10.2,34.5, and 87.0 sec- 1 and the arithmetic mean taken into account. Viscosity measurements were performed at 37° C. Whole blood collected during ECC was also measured at 26° C, that is, the actual body temperature during ECC. Statistical analysis of the data was performed with a one-way analysis of variance. For membrane proteins, which were examined only twice for each patient, a paired t test was used.
Results RBC morphology was affected by ECC, which is illustrated in Fig. 1. The normal RBCs seen preoperatively (Fig. 1, A) underwent an echinocytic shape transformation during the ECC (Fig. 1, B), which was reversible when the RBCs were washed and resuspended in PBS with albumin 0.25 gmjdl(Fig.l, C). One day after ECC, the echinocytic shape had disappeared (Fig. 1, D). Normal RBCs of a healthy donor (Fig. 1, E) incubated in patients' ECC plasma underwent a strong echinocytic transformation (Fig. 1, F). The quantitative assessment of the RBC morphology is summarized in Table I. The mean RBC volume remained constant. Echinocytes increased in frequency
5 4 0 Reinhart et al.
The Journal of Thoracic and Cardiovascular Surgery
Fig. 1. Scanningelectronmicrographs showing the RBC morphology of a patientduringECC. A, RBCsin plasma preoperatively; B, RBCs in plasma during ECC; C, RBCs during ECC as in B, but washedthree timesin PBS; D, RBCs in plasma 24 hours after the operation; E, normal RBCs in preoperative plasma;F, normal RBCs in plasma from ECC. The bar represents 5 /-Lm. during ECC and after ECC, whereas stomatocytes declined. The frequency ofdiscocytes was decreased 1day after ECC. Note that sometimes large standard deviations were present, which indicates that the shape changes were more pronounced in certain patients than in others. No correlation was found between the degree of shape transformation and other parameters, such as severity of the underlying coronary heart disease, duration of the ECC, or preoperative and intraoperative medication. Analysis of the membrane phospholipids and their fatty acid composition revealed no difference before and after ECC (Table II). No evidence for peroxidation of fatty acids was observed, because the amount of polyunsaturated fatty acids (e.g., 20:4, 22:4, 22:5, or 22:6) was unaffected, and the ratio saturated/unsaturated fatty acids remained constant.
The analysis of the RBC membrane skeletal proteins after ECC revealed no difference in electrophoretic mobility of protein bands. High molecular weight complexes were not detected with either reduced or unreduced ghost preparations. The area under the curve of individual protein bands was measured and related to the area under the curve of the integral membrane protein band 3, which is an indicator of the amount of RBC membrane loaded Onthe gel. 23 The ratio ankyrin/band 3 was found to be 0.164 ± 0.026 and 0.118 ± 0.021 before and after ECC, respectively (p < 0.001), which suggests that about One fourth of ankyrin had been lost. Other important membrane proteins such as spectrin, actin, and band 4.1 remained constant in relation to protein band 3. In preparations with complete removal of white cells and platelets and the addition of protease inhibitors, however, ankyrin remained unaffected by the ECC (ratio ankyrin/
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Table I. Red cell morphology during ECC (mean ± standard deviation, n = 10) Intraoperative Preoperative Mean cell volume (fl) 91.1 Morphology (%) Discocytes In plasma 67.6 In buffer 68.6 Echinocytes In plasma 5.5 In buffer 5.8 Stomatocytes In plasma 26.8 In buffer 25.6 Morphology of normal RBCs (%) in patient plasma (n = 4) Discocytes 56.5 Echinocytes 36.4 Stornatocytes 7.1
Pre-ECC
ECC
Post-ECC
Postoperative
± 4.1
90.8 ± 4.4
91.1 ± 4.4
90.8 ± 3.8
91.7 ± 3.1
± 9.3 ± 7.2
67.3 ± 6.6 68.0 ± 8.5
60.3 ± 16.9 65.4 ± 10.4
63.8 ± 10.1 66.4 ± 5.5
50.6 ± 11.1t 67.9 ± 5.2
± 1.9 ± 3.7
6.3 ± 4.1 7.3 ± 4.4
25.5 ± 21.7* 7.5 ± 6.8
19.5 ± 13.5* 9.1 ± 5.9
5.2 ± 2.6 11.2 ± 5.0*
± 8.7 ± 7.8
26.5 ± 7.9 24.8 ± 10.0
14.1 ± 6.3t 27.1 ± 11.9
16.7 ± 7.5* 24.4 ± 6.8
44.1 ± 12.3§ 20.8 ± 4.4
± 35.5
54.6 ± 31.2 38.3 ± 37.6 7.1 ± 7.9
11.3 ± 22.3* 85.5 ± 26.7* 2.2 ± 4.4
17.9 ± 27.4 80.4 ± 30.8 1.7 ± 3.4
65.1 ± 5.2 5.3 ± 2.3 29.6 ± 5.6*
± 41.3
± 7.6
'p < 0.05 compared with preoperative values with a one-way analysis of variance.
tp < 0.0 I compared with preoperative values with a one-way analysis of variance. :j:p < 0.00 I compared with preoperative values with a one-way analysis of variance. §p < 0.0001 compared with preoperative values with a one-way analysis of variance.
band 3: 0.180 ± 0.025 and 0.182 ± 0.017, respectively). The mechanical shearing of the ECC pump in vitro for up to 80 minutes did not cause a significant loss of ankyrin and did not cause echinocytosis (not shown). The deformability of the RBCs, as assessed by the resistance to filtration of washed RBCs through narrow pores, was not affected (Table III). Whole blood viscosity changed drastically during ECC (Fig. 2). During general anesthesia, the whole blood viscosity was slightly lower than on admission and became markedly reduced during ECC, especially at low shear rates. Even at 26° C the whole blood viscosity was lower than pre-ECC and post-ECC values at 37° C. Postoperative values were similar to pre-ECC values with the exception of a high viscosity at the lowest shear rate. The change in whole blood viscosity during ECC was due to the hemodilution by the priming solution, which was reflected by the lower hematocrit value and plasma viscosity during and after ECC (Table III). When the hematocrit value was adjusted to 45%, the viscosity still remained lower during the operation, which was attributable to the lower plasma viscosity. Washed RBCs in buffer had an unchanged viscosity, indicating that the deformability of the RBCs was not affected by the ECC, which is in agreement with the filtration data. Discussion RBCs with a reduced deformability are an important cause of an impaired microcirculation and can decrease the supply of oxygen and nutrients to tissues.I" These
phenomena can eventually explain part of the morbidity after ECC. A decreased RBC deformability has been described after cardiac operations." For that reason we studied the influence of ECC on red blood cells and flow properties of blood. We found an echinocytic shape transformation of the RBCs during ECC. The normal biconcave discocyte is an equilibrium shape between stomatocytosis and echinocytosis." Our observation of a simultaneous decrease of stomatocytes with the increase of echinocytes is, therefore, better described as a shift of the RBC shape toward echinocytosis. Recently, Kamada and associates'? drew attention to a marked echinocytosis during ECC with bubble oxygenators. Because they did not examine fixed RBCs, artifacts such as the "glass effect,,27could not be excluded. Their study has therefore been questioned by Simpson.P' who did not observe any change in the RBCs during ECC. Our data on fixed RBCs and with the less traumatizing membrane oxygenator now confirm that indeed echinocytosis occurs during ECC. The echinocytosis was reversible within 24 hours or by removing the RBCs from the plasma and resuspending them in buffer, which is evidence that a plasma factor was responsible for the shape change. This hypothesis was corroborated by the echinocytogenic effect of ECC plasma on normal RBCs (Fig. 1, F). The responsible plasma factor was not yet present during anesthesia but was still active shortly after ECC, which excludes anesthetics or foreign surfaces as the cause of echinocytosis. Kamada and co-workers I 2 have postulated that an increased con-
The Journal of Thoracic and Cardiovascular Surgery
5 4 2 Reinhart et al.
Table II. Phospholipids of RBC membranes and their fatty acid composition (mean ± standard deviation, n = 6) Intraoperative Pre-ECC
Preoperative Phosphatidylcholine (%) Phosphatidylserine (%) Phosphatidylethanolamine (%) Sphingomyelin (%) Fatty acids (%) 16:0 18:0 18:1 18:2 20:3 20:4 22:4 22:5 22:6 24:0 24:1 Ratio saturated/unsaturated fatty acids
31.2 15,3 26.2 27,3 21.1 16.5 14.1 8.7 1.5 15.7 2.8 2.9 5.0 5.9 5.9 0.81
Post-ECC
± 0.8 ± 1.6
31.9 13.8 24.5 29.7
± 2,3 ± 2,3 ± 3.8 ± 3.2
31.0 14.9 25.8 28.4
± 1.2 ± 1.3 ± 1.2 ± 1.8 ± 0.4 ± 1.1 ± 0.7 ± 0.5 ± 0.6 ± 0.4 ± 0.6 ± 0.10
21.7 15.7 14.6 8.7 1.6 15.1 2.7 2.9 4.9 6.0 6.2 0.80
± 1.7 ± 1.4
21.6 ± 16,3 ± 14.7 ± 8.5 ± 1.4 ± 15.2 ± 2.8. ± 2.8 ± 4.8 ± 5.9 ± 6.1 ± 0.82 ±
± 1.6 ± 0.6
± l.l ± 2.0 ± OJ ± 0.6 ± 0.7 ± 0.4 ± 0.6 ± OJ ± 0.8 ± 0.07
± 1.6 ± 1.4 ± 1.4 ± 1.7
1.0 0.9 l.l 1.8 0,3 0.7 0.7 0.5 0.7 0.5 0.6 0.10
Table III. Blood rheology data during ECC (mean ± standard deviation, n = 10) Intraoperative
Relative RBC filtration resistance (fJ) Hematocrit (%) Plasma viscosity (cp) Blood viscosity (cp) at 87 sec- 1 Whole blood Hematocrit 45% in plasma Hematocrit 45% in buffer Blood viscosity (cp) at 0.1 sec"! Whole blood Hematocrit 45% in plasma Hematocrit 45% in buffer
Preoperative
Pre-ECC
ECC
Post-ECC
Postoperative
79.6 ± 18.5 44.7 ± 2.0 1.36 ± 0.05
74.0 ± 16.5 39.5 ± 4.4t 1.29 ± O.07t
65.7 ± 20.7 25.4 ± 2.2§ 1.00 ± 0.05§
6903 ± 21.2 25.9 ± 3.2§ 1.02 ± 0.04§
81.2 ± 19.5 38.7 ± 4.8:j: 1.31 ± 0.07*
4.86 ± 0,36 5.03 ± 0.22 3.63 ± 0.17
4.00 ± 0.57+ 4.72 ± o.ist 3.59 ± 0.13
2.26 ± 0.28§ 4.20 ± 0,34§ 3.59 ± 0.14
2.36 ± 0,37§ 4.16 ± O.l5§ 3.63 ± 0.13
3.92' ± 0.70§ 4.88 ± 0.16 3.46 ± O.13t
26.41 ± 3.54 26.60 ± 2.74 6.62 ± 0.58
16.97 ± 5.61§ 27.18 ± 2.94 6,37 ± 0.58
4.75 ± 2.09§ 29.28 ± 3.55 6.33 ± 0.48
4.63 ± 1.95§ 22.47 ± 3,38 6.17 ± 0.55
25.44 ± 6.18 33.38 ± 6.38* 6.35 ± 0.72
*p < 0.05 compared with preoperative values with a one-way analysis of variance. tp < 0.01 compared with preoperative values with a one-way analysis of variance. :j:p < 0.001 compared with preoperative values with a one-way analysis of variance. §p < 0.0001 compared with preoperative values with a one-way analysis of variance.
centration in free fatty acids, which was preventable by the addition of albumin to the priming solution, was the cause.Echinocytogenic substancessuchas lysophosphatidylcholine accumulate in plasma during storage.i?: 30 which may explainthe relativeprevalence of echinocytes among normal RBCs incubatedin thawed frozenplasma of patients (Tablel). The decreaseddiscocyte count I day after ECC may, among other causes, be attributed to postoperative blood transfusions given to all patients. RBC membrane phospholipids were not affected by ECC, nor wasa change in the fatty acid pattern observed. A peroxidation of the membrane lipids by oxygen free
radicals had to be considered because the RBCs had a most intense contact with the ECC apparatus and, in particular, with the membrane oxygenator.' This possibility, however, could be ruled out on the basis of the observation that the polyunsaturatedfatty acidsof RBC membrane phospholipids were not affected (Table II). Obviously the RBCs possess a sufficiently activeself-protectionagainstoxidative lipiddamage byenzymes suchas superoxide dismutase and catalase or the glutathione systemY·32 The mechanical stability and elasticity of the RBC membrane is provided by the membrane skeleton. It con-
Volume 100 Number 4
RBCs during ECC
October 1990
543
30,........-------------------......,
20 Q.
U
'-'
> in
~
o(J
(/)
> c
o 9 co
10
W
...J
oJ: ;:
O ..........- -------'----------'----------L...I 0.1
1
10
SHEAR RATE
100
(sec· 1)
Fig. 2. Plot of whole blood viscosityversus shear rate of 10 patients preoperatively, intraoperatively but before ECC, during ECC, and 24 hours postoperatively. The samples during ECC were measured at 37° C and at 26° C, that is, the average body temperature during this phase of the operation. The values are means ± standard deviation.
sists of a meshwork of proteins, primarily spectrin, actin, and protein band 4.1, which underlies the lipid bilayer. It is attached to the transmembranous protein band 3 by the anchoring protein band 2.1, called ankyrin. Oxidative damage causes the precipitation of hemoglobin and membrane proteins and the appearance of high molecular weight proteins detectable on gel electrophoresis.P We did not observe such high molecular weight proteins, which is another argument for the strong self-protection of the RBCs against oxidative damage. Ankyrin was reduced after ECC. This effect was not seen when protease inhibitors were added during ghost preparation. This indicates that the ECC increased the proteolytic activity of the plasma, possibly through an activation of leukocytes.II The fact that we were not able to induce a lossof ankyrin with an ECC pump in vitro suggests that the increased proteolytic activity is not simply due to the mechanical stress of the pump. The question arises as to whether the observed
echinocytosis had an influence on the flow properties of blood as suggested by other in vitro studies. 13, 34 The rheologic in vitro data of our study, namely, viscosity and RBC filtration, indicate that the RBC abnormalities seen during ECC are not relevant for blood flow. The marked reduction of the plasma viscosity by the dilution with priming solution and the simultaneous reduction of the hematocrit value both reduce whole blood viscosity in an additive manner and preponderate over any possible change induced by the abnormal RBC shape. A major determinant of viscosity is temperature. Hypothermia during ECC therefore increases blood viscosity. The reduction of plasma viscosity and hematocrit, however, were sufficient to keep the blood viscosity during ECC at 26° C below the preoperative values at 37° C. These data indicate that hemodilution during ECC is essential for blood rheology and that the use of blood for priming, as done in the early phases of ECC, could be deleterious. Tissue oxygenation is determined by RBC
The Journal of Thoracic and Cardiovascular Surgery
5 4 4 Reinhart et al.
transport efficiency, which can be estimated by the ratio hematocrit/viscosity.P At the highest shear rate (87 sec"), which is probably representative for the human circulation, the RBC transport efficiency fell from 9.1 preoperatively to 7.3 during ECC at 26° C; at all other shear rates it increased. We conclude that ECC leads to a reversible echinocytic shape transformation of RBCs. Blood viscosity is not increased by this RBC change but rather is decreased because of the marked hemodilution during ECC. It is therefore not likely that rheologic abnormalities are a contributing factor in post-ECC morbidity. We would like to thank Dr. Miihlemann and A. Wiesmann for their kind cooperation, Miss S. Kampfer for her excellent technical assistance, and Miss B. Urfer for typing the manuscript. REFERENCES I. Kirklin JW, Kirklin JK, Lell WA. Cardiopulmonary bypass for cardiac surgery. In: Sabiston DC Jr, Spencer FC, eds. Surgery of the chest. 4th ed. Philadelphia: WB Saunders, 1983:909-25. 2. Cavarocchi NC, England MD, O'Brien JF, et al. Superoxide generation during cardiopulmonary bypass: Is there a role for vitamin E? J Surg Res 1986;40:519-27. 3. Miki M, Tarnai H, Mino M, Yamamoto Y, Niki E. Freeradical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by a-tocopherol. Arch Biochem Biophys 1987;258:373-80. 4. Bolli R, Patel BS, Jeroudi MO, Lai EK, McCay PB. Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap a phenyl N-tert-butyl nitrone. J Clin Invest 1988;82:476-85. 5. Chenoweth DE, Cooper SW, Hugli TE, Stewart RW, Blackstone EH, Kirklin JW. Complement activation during cardiopulmonary bypass. N EnglJ Med 1981;304:497503. 6. Hammerschmidt DE, Stroncek DF, Bowers TK, et al. Complement activation and neutropenia occurring during cardiopulmonary bypass. J THORAC CARDIOVASC SURG 1981;81:370-7. . 7. Salama A, Hugo F, Heinrich D, et al. Deposition ofterminal C5b-9 complement complexes on erythrocytes and leukocytes during cardiopulmonary bypasses. N Engl J Med 1988;318:408-14. 8. Gans H, Krivit W. Problems in hemostasis during openheart surgery. IV. On the changes in the blood clotting mechanism during cardiopulmonary bypass procedures. Ann Surg 1962;155:353-9. 9. Gomes MR, McGoon DC. Bleeding patterns after openheart surgery. J THoRAc CARDIOVASC SURG 1970;60:8797. 10. "Davies GC, Sobel M, Salzman EW. Elevated plasma
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RBCs during ECC 545
oxia by tracheal insufflation of erythrocytes: role of red cell glutathione. Science 1985;227:756-9. 32. Agar NS, Sadrzadeh SMH, Hallaway PE, Eaton J~. Erythrocyte catalase: a somatic oxidant defense. J Clin Invest 1986;77:319-21. 33. Synder LM, Leb L, Piotrowski J, Sauberman N, Liu SC, Fortier NL. Irreversible spectrin-haemoglobin crosslinking in vivo: a marker for red cell senescence. Br J Haematol 1983;53:379-84. 34. Reinhart WH, Chien S. Red cell rheology in stomatocyteechinocyte transformation: roles of cell geometry and cell shape. Blood 1986;67:1110-8.
Bound volumes available to subscribers Bound volumesof THEJOURNAL OFTHORACIC AND CARDIOVASCULAR SURGERY are available to subscribers (only) for the 1990issues from the Publisher, at a cost of $58.00 ($75.00 international) for Vol. 99 (January-June) and Vol. 100(July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 60 days after publication of the last issue of the volume. The binding is durable buckram with the JOURNAL name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact Mosby-Year Book, Inc., Circulation Department, 11830 Westline Industrial Drive, St. Louis, Missouri 63146-3318, USA; phone (800) 325-4177, ext. 7351. Subscriptions must be in force to qualify. Bound volumes are not available hi place of a regular JOURNAL subscription.
THORAC CARDIOVASC SURG
1990;100:538-45
The influence of extracorporeal circulation on erythrocytes and flow properties of blood The influence of extracorporeal circulation on red blood cells and flow properties of blood was studied in 10 patients undergoing aorta-coronary bypass grafting. Blood samples were drawn on admission, under general anesthesia before the operation, during extracorporeal circulation, immediately after extracorporeal circulation, and 24 hours after extracorporeal circulation. Echinocytes were found during and shortly after extracorporeal circulation, but disappeared within 24 hours. Washing the cells in buffer restored the normal discocytic shape, which indicated that a plasma factor was responsible. Red ceU membrane lipids were not affected. Analysis of the membrane proteins revealed a decrease of ankyrin after extracorporeal circulation, which was prevented by protease inhibitors during prepara-. tion. This suggests an increased proteolytic activity of the plasma after extracorporeal circulation. Red cell deformability was not altered. Plasma viscosity and hematocrit were markedly reduced by hemodilution with the priming solution. Their low levels resulted in a low blood viscosity during extracorporeal circulation, which was even lower at 26° C than before or after the operation at 37° C. We conclude that the red cell is affected by extracorporeal circulation. The flow properties of blood, however, are not impaired, but are improved by hemodilution.
Walter H. Reinhart, MD, Peter E. Ballmer, MD, Franziska Rohner, MD, Peter Ott, PhD,' and P. Werner Straub, MD, Bern. Switzerland
Etracorporeal circulation (ECC) is a prerequisite for cardiac operations. Over the years the technique has been refined and now has a low mortality rate. I The post-ECC morbidity, however, is still substantial and warrants intensive postoperative care. Several factors have been implicated in this morbidity. A major factor is the generation of oxygen free radicals, which may lead to lipid peroxidation in cell membranes.e" A large number of abnormalities have been described, such as an activation of the complement system through the alternative pathway,S,6 a deposition of complement complexes on erythrocytes,? hemolysis.I-" activation of platelets? and blood clotting, I0 sequestration of leukocytes in the lungs, II. and leukopenia.f Recently a profound change of the red blood
cell (RBC) shape during ECC has been observed.F The RBC shape affects the flow properties of blood in the macrocirculation and microcirculation.P One may ask, therefore, what effect ECC has on blood rheology and if an impairment of the flow properties of blood could be responsible for intraoperative and postoperative disorders such as the decreased cerebral blood flow after ECc. 14 Many of these changes were observed with bubble oxygenators, which are known to be more traumatizing for blood than the newer types of membrane oxygenators.P In the present study we have investigated the influence of ECC with membrane oxygenators on the RBC shape, biochemical properties of the RBC membrane, and rheologic properties of blood. Patients
From the Department of Internal Medicine, Inselspital, and Institute of Biochemistry and Molecular Biology,' University of Bern, Bern, Switzerland. Received for publication July 26,1989. Accepted for publication Dec. I, 1989. Address for reprints: Dr. Walter Reinhart, Department of Internal Medicine, University of Bern, Inselspital, CH - 3010 Bern, Swit-
zerland,
12/1/19699
538
Ten patients (onewomanand nine men,aged 50to 67 years) with coronaryheart disease (four patientswith three-vessel disease,six patients with two-vessel disease),but withoutvalvular heart disease, werestudied.They were treated beforethe operation with nitrates (nine patients), i1-blocking agents (eight patients),and calcium-channel blockers (six patients).The following procedures weredone:18aorta-coronary bypasses, eight grafts with the internal mammary artery, and one ventricular aneurysmectomy. Halothane, fentanyl, and midazolam were usedfor anesthesia. During ECC 10gm mannitoland 4 ~I hep-
Volume 100 Number 4 October 1990
arin (200 U) per kilogram of body weight were administered. A membrane oxygenator (Maxima, Medtronic Inc., Minneapolis, Minn., or CML2, Cobe Laboratories, Lakewood, Colo.) was used. The system was primed with 2.5 L of a I: I mixture of 0.9% saline and Ringer's lactate with sodium bicarbonate 48 mmol/ L, potassium 10 mmol/L, magnesium 4 mmol/L, and calcium gluconolactobionate 3 gm. An arterial oxygen pressure of about 200 mm Hg was provided by an oxygen blender (Cobe Laboratories). Hypothermia was maintained at 26° C during ECC. The flow rate of the roller pump (Shiley-Stockert, Miinchen, Federal Republic of Germany) was 2.4 L/ min/m 2 at normothermia and 1.2 L/min/m 2 at hypothermia. An arterial filter (Bentley AF 1040 C, 40 ~m, Baxter Healthcare Corporation, Irvine, Calif.) was intercalated between the pump and the patient. The mean ECC time was 92 ± 40 minutes and the aortic clamping time, 60 ± 20 minutes. Informed consent was obtained from the patients before the operation. Blood anticoagulated with ethylenediaminetetraacetic acid was withdrawn on five different occasions: (1) on admission to the hospital before the operation, (2) during general anesthesia before the operation, (3) during ECC (60 minutes after the beginning of ECC), (4) immediately after changing from ECC to the patient's own circulation, and (5) 1 day after the operation. No blood transfusions were given during ECC.
Methods Drops of blood were fixed overnight in cacodylate-buffered 1% glutaraldehyde and viewed with a light microscope. The shape of the RBCs was classified according to the nomenclature of Bessis." Some specimens were processed for scanning microscopy (PSEM 500, Philips, Eindhoven, The Netherlands) by dehydration in increasing ethanol series, air-drying on a coverslip, and coating with gold palladium. The RBC membrane lipids were extracted from specimens obtained on admission, before ECC, and after ECC with the method of Rose and Oklander.!? The membrane phospholipids were separated by two-dimensional thin-layer chromatography according to Broekhuyse'" and quantitated as inorganic phosphorus with the method described by Rouser, Fleischer, and Yamamoto. 19 The fatty acid composition of the total membrane phospholipids was analyzed by gas-liquid chromatography. Membrane proteins were studied in RBC ghosts, which were obtained by lysing washed RBCs obtained before and after ECC in a tenfold volume of hypotonic (10 mOsm/kg water) phosphate buffer with four or fiveconsecutive washes. In a later series of experiments with 10 other patients the influence of proteases was eliminated. Heparinized blood was run over a column of acelluloseand microcellulose (I: I mixture) to remove white cells and platelets and was then lysed with the above-mentioned buffer to which the protease inhibitors pepstatin A (2 ~g/rnl) and phenylmethylsulfonyl fluoride (20 ~g/ml) were added. Electrophoresis was performed with 3.5% SDS polyacrylamide gels. Approximately 20 ~g protein with and without the reducing agent dithiothreitol was used. The gels were stained with Coomassie brillant blue and scanned with a photometer at a wavelength of 560 nm. The areas under the curve of the different membrane protein bands were determined. A closed loop of tubing in an ECC pump (Polystan, Copenhagen, Denmark) was used to test the influence of the roller pump alone on the RBCs. Heparinized blood was diluted with
RBCs during ECC 5 3 9
saline to a hematocrit value of 25% as during ECC and pumped at 140 rpm and a flow rate of 2.4 L/min (100 pump pas,sages per minute) for 0, 20, 40, and 80 minutes. Although the blood temperature rose gradually from 22 ° C at the beginning to 38° C after 80 minutes of pumping, hemolysis was only minimal ( 1.5%after 80 minutes). RBC morphology and membrane proteins were analyzed as described earlier. RBC deformability was assessed with a filtration test described previously'? with some slight modifications.U The RBCs were washed three times in phosphate-buffered saline (PBS) containing glucose 0.2 gm/dl and human serum albumin 0.25 gm/dl, The cells were resuspended in PBS with a hematocrit value of 10%. Some drops were fixed in glutaraldehyde for morphologic examination. The RBC suspension was then pumped at a flow rate of 0.5 ml/rnin through a polycarbonate filter with a mean pore diameter of 2.84 ± 0.17 ~m (Lot No. 62BIC58, Nuclepore Corp., Pleasanton, Calif.). The filtration pressure was measured on the upstream side of the filter. The relative filtration resistance of an RBC (fJ = filtration resistance in a pore containing an RBC in transit compared with a pore with suspending medium alone) was calculated as: fJ = 1 + ([Pi/Po] - 1) V/h, where Pi is the initial pressure rise, Po the filtration pressure for suspending medium alone measured before RBC filtration, V the fraction of the pore volume occupied by the RBC, and h the fractional volume of RBCs in suspension.P The flow properties of blood were assessed according to Chien 22 by measuring (I) plasma viscosity, (2) whole blood viscosity, which is influenced by the actual hematocrit, (3) viscosity of an RBC suspension in plasma with a normalized hematocrit value of 45%, and (4) viscosity of a suspension of washed RBCs in PBS with a hematocrit value of 45%, which reflects primarily RBC deformability, Viscosity was measured with a Couette-type viscometer (LS-30, Contraves, Ziirich, Switzerland). Whole blood and RBC suspensions were measured at four different shear rates, namely, 0.1, 0.87, 10.2, and 87.0 sec-I. The plasma viscositywas measured at 10.2,34.5, and 87.0 sec- 1 and the arithmetic mean taken into account. Viscosity measurements were performed at 37° C. Whole blood collected during ECC was also measured at 26° C, that is, the actual body temperature during ECC. Statistical analysis of the data was performed with a one-way analysis of variance. For membrane proteins, which were examined only twice for each patient, a paired t test was used.
Results RBC morphology was affected by ECC, which is illustrated in Fig. 1. The normal RBCs seen preoperatively (Fig. 1, A) underwent an echinocytic shape transformation during the ECC (Fig. 1, B), which was reversible when the RBCs were washed and resuspended in PBS with albumin 0.25 gmjdl(Fig.l, C). One day after ECC, the echinocytic shape had disappeared (Fig. 1, D). Normal RBCs of a healthy donor (Fig. 1, E) incubated in patients' ECC plasma underwent a strong echinocytic transformation (Fig. 1, F). The quantitative assessment of the RBC morphology is summarized in Table I. The mean RBC volume remained constant. Echinocytes increased in frequency
5 4 0 Reinhart et al.
The Journal of Thoracic and Cardiovascular Surgery
Fig. 1. Scanningelectronmicrographs showing the RBC morphology of a patientduringECC. A, RBCsin plasma preoperatively; B, RBCs in plasma during ECC; C, RBCs during ECC as in B, but washedthree timesin PBS; D, RBCs in plasma 24 hours after the operation; E, normal RBCs in preoperative plasma;F, normal RBCs in plasma from ECC. The bar represents 5 /-Lm. during ECC and after ECC, whereas stomatocytes declined. The frequency ofdiscocytes was decreased 1day after ECC. Note that sometimes large standard deviations were present, which indicates that the shape changes were more pronounced in certain patients than in others. No correlation was found between the degree of shape transformation and other parameters, such as severity of the underlying coronary heart disease, duration of the ECC, or preoperative and intraoperative medication. Analysis of the membrane phospholipids and their fatty acid composition revealed no difference before and after ECC (Table II). No evidence for peroxidation of fatty acids was observed, because the amount of polyunsaturated fatty acids (e.g., 20:4, 22:4, 22:5, or 22:6) was unaffected, and the ratio saturated/unsaturated fatty acids remained constant.
The analysis of the RBC membrane skeletal proteins after ECC revealed no difference in electrophoretic mobility of protein bands. High molecular weight complexes were not detected with either reduced or unreduced ghost preparations. The area under the curve of individual protein bands was measured and related to the area under the curve of the integral membrane protein band 3, which is an indicator of the amount of RBC membrane loaded Onthe gel. 23 The ratio ankyrin/band 3 was found to be 0.164 ± 0.026 and 0.118 ± 0.021 before and after ECC, respectively (p < 0.001), which suggests that about One fourth of ankyrin had been lost. Other important membrane proteins such as spectrin, actin, and band 4.1 remained constant in relation to protein band 3. In preparations with complete removal of white cells and platelets and the addition of protease inhibitors, however, ankyrin remained unaffected by the ECC (ratio ankyrin/
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Table I. Red cell morphology during ECC (mean ± standard deviation, n = 10) Intraoperative Preoperative Mean cell volume (fl) 91.1 Morphology (%) Discocytes In plasma 67.6 In buffer 68.6 Echinocytes In plasma 5.5 In buffer 5.8 Stomatocytes In plasma 26.8 In buffer 25.6 Morphology of normal RBCs (%) in patient plasma (n = 4) Discocytes 56.5 Echinocytes 36.4 Stornatocytes 7.1
Pre-ECC
ECC
Post-ECC
Postoperative
± 4.1
90.8 ± 4.4
91.1 ± 4.4
90.8 ± 3.8
91.7 ± 3.1
± 9.3 ± 7.2
67.3 ± 6.6 68.0 ± 8.5
60.3 ± 16.9 65.4 ± 10.4
63.8 ± 10.1 66.4 ± 5.5
50.6 ± 11.1t 67.9 ± 5.2
± 1.9 ± 3.7
6.3 ± 4.1 7.3 ± 4.4
25.5 ± 21.7* 7.5 ± 6.8
19.5 ± 13.5* 9.1 ± 5.9
5.2 ± 2.6 11.2 ± 5.0*
± 8.7 ± 7.8
26.5 ± 7.9 24.8 ± 10.0
14.1 ± 6.3t 27.1 ± 11.9
16.7 ± 7.5* 24.4 ± 6.8
44.1 ± 12.3§ 20.8 ± 4.4
± 35.5
54.6 ± 31.2 38.3 ± 37.6 7.1 ± 7.9
11.3 ± 22.3* 85.5 ± 26.7* 2.2 ± 4.4
17.9 ± 27.4 80.4 ± 30.8 1.7 ± 3.4
65.1 ± 5.2 5.3 ± 2.3 29.6 ± 5.6*
± 41.3
± 7.6
'p < 0.05 compared with preoperative values with a one-way analysis of variance.
tp < 0.0 I compared with preoperative values with a one-way analysis of variance. :j:p < 0.00 I compared with preoperative values with a one-way analysis of variance. §p < 0.0001 compared with preoperative values with a one-way analysis of variance.
band 3: 0.180 ± 0.025 and 0.182 ± 0.017, respectively). The mechanical shearing of the ECC pump in vitro for up to 80 minutes did not cause a significant loss of ankyrin and did not cause echinocytosis (not shown). The deformability of the RBCs, as assessed by the resistance to filtration of washed RBCs through narrow pores, was not affected (Table III). Whole blood viscosity changed drastically during ECC (Fig. 2). During general anesthesia, the whole blood viscosity was slightly lower than on admission and became markedly reduced during ECC, especially at low shear rates. Even at 26° C the whole blood viscosity was lower than pre-ECC and post-ECC values at 37° C. Postoperative values were similar to pre-ECC values with the exception of a high viscosity at the lowest shear rate. The change in whole blood viscosity during ECC was due to the hemodilution by the priming solution, which was reflected by the lower hematocrit value and plasma viscosity during and after ECC (Table III). When the hematocrit value was adjusted to 45%, the viscosity still remained lower during the operation, which was attributable to the lower plasma viscosity. Washed RBCs in buffer had an unchanged viscosity, indicating that the deformability of the RBCs was not affected by the ECC, which is in agreement with the filtration data. Discussion RBCs with a reduced deformability are an important cause of an impaired microcirculation and can decrease the supply of oxygen and nutrients to tissues.I" These
phenomena can eventually explain part of the morbidity after ECC. A decreased RBC deformability has been described after cardiac operations." For that reason we studied the influence of ECC on red blood cells and flow properties of blood. We found an echinocytic shape transformation of the RBCs during ECC. The normal biconcave discocyte is an equilibrium shape between stomatocytosis and echinocytosis." Our observation of a simultaneous decrease of stomatocytes with the increase of echinocytes is, therefore, better described as a shift of the RBC shape toward echinocytosis. Recently, Kamada and associates'? drew attention to a marked echinocytosis during ECC with bubble oxygenators. Because they did not examine fixed RBCs, artifacts such as the "glass effect,,27could not be excluded. Their study has therefore been questioned by Simpson.P' who did not observe any change in the RBCs during ECC. Our data on fixed RBCs and with the less traumatizing membrane oxygenator now confirm that indeed echinocytosis occurs during ECC. The echinocytosis was reversible within 24 hours or by removing the RBCs from the plasma and resuspending them in buffer, which is evidence that a plasma factor was responsible for the shape change. This hypothesis was corroborated by the echinocytogenic effect of ECC plasma on normal RBCs (Fig. 1, F). The responsible plasma factor was not yet present during anesthesia but was still active shortly after ECC, which excludes anesthetics or foreign surfaces as the cause of echinocytosis. Kamada and co-workers I 2 have postulated that an increased con-
The Journal of Thoracic and Cardiovascular Surgery
5 4 2 Reinhart et al.
Table II. Phospholipids of RBC membranes and their fatty acid composition (mean ± standard deviation, n = 6) Intraoperative Pre-ECC
Preoperative Phosphatidylcholine (%) Phosphatidylserine (%) Phosphatidylethanolamine (%) Sphingomyelin (%) Fatty acids (%) 16:0 18:0 18:1 18:2 20:3 20:4 22:4 22:5 22:6 24:0 24:1 Ratio saturated/unsaturated fatty acids
31.2 15,3 26.2 27,3 21.1 16.5 14.1 8.7 1.5 15.7 2.8 2.9 5.0 5.9 5.9 0.81
Post-ECC
± 0.8 ± 1.6
31.9 13.8 24.5 29.7
± 2,3 ± 2,3 ± 3.8 ± 3.2
31.0 14.9 25.8 28.4
± 1.2 ± 1.3 ± 1.2 ± 1.8 ± 0.4 ± 1.1 ± 0.7 ± 0.5 ± 0.6 ± 0.4 ± 0.6 ± 0.10
21.7 15.7 14.6 8.7 1.6 15.1 2.7 2.9 4.9 6.0 6.2 0.80
± 1.7 ± 1.4
21.6 ± 16,3 ± 14.7 ± 8.5 ± 1.4 ± 15.2 ± 2.8. ± 2.8 ± 4.8 ± 5.9 ± 6.1 ± 0.82 ±
± 1.6 ± 0.6
± l.l ± 2.0 ± OJ ± 0.6 ± 0.7 ± 0.4 ± 0.6 ± OJ ± 0.8 ± 0.07
± 1.6 ± 1.4 ± 1.4 ± 1.7
1.0 0.9 l.l 1.8 0,3 0.7 0.7 0.5 0.7 0.5 0.6 0.10
Table III. Blood rheology data during ECC (mean ± standard deviation, n = 10) Intraoperative
Relative RBC filtration resistance (fJ) Hematocrit (%) Plasma viscosity (cp) Blood viscosity (cp) at 87 sec- 1 Whole blood Hematocrit 45% in plasma Hematocrit 45% in buffer Blood viscosity (cp) at 0.1 sec"! Whole blood Hematocrit 45% in plasma Hematocrit 45% in buffer
Preoperative
Pre-ECC
ECC
Post-ECC
Postoperative
79.6 ± 18.5 44.7 ± 2.0 1.36 ± 0.05
74.0 ± 16.5 39.5 ± 4.4t 1.29 ± O.07t
65.7 ± 20.7 25.4 ± 2.2§ 1.00 ± 0.05§
6903 ± 21.2 25.9 ± 3.2§ 1.02 ± 0.04§
81.2 ± 19.5 38.7 ± 4.8:j: 1.31 ± 0.07*
4.86 ± 0,36 5.03 ± 0.22 3.63 ± 0.17
4.00 ± 0.57+ 4.72 ± o.ist 3.59 ± 0.13
2.26 ± 0.28§ 4.20 ± 0,34§ 3.59 ± 0.14
2.36 ± 0,37§ 4.16 ± O.l5§ 3.63 ± 0.13
3.92' ± 0.70§ 4.88 ± 0.16 3.46 ± O.13t
26.41 ± 3.54 26.60 ± 2.74 6.62 ± 0.58
16.97 ± 5.61§ 27.18 ± 2.94 6,37 ± 0.58
4.75 ± 2.09§ 29.28 ± 3.55 6.33 ± 0.48
4.63 ± 1.95§ 22.47 ± 3,38 6.17 ± 0.55
25.44 ± 6.18 33.38 ± 6.38* 6.35 ± 0.72
*p < 0.05 compared with preoperative values with a one-way analysis of variance. tp < 0.01 compared with preoperative values with a one-way analysis of variance. :j:p < 0.001 compared with preoperative values with a one-way analysis of variance. §p < 0.0001 compared with preoperative values with a one-way analysis of variance.
centration in free fatty acids, which was preventable by the addition of albumin to the priming solution, was the cause.Echinocytogenic substancessuchas lysophosphatidylcholine accumulate in plasma during storage.i?: 30 which may explainthe relativeprevalence of echinocytes among normal RBCs incubatedin thawed frozenplasma of patients (Tablel). The decreaseddiscocyte count I day after ECC may, among other causes, be attributed to postoperative blood transfusions given to all patients. RBC membrane phospholipids were not affected by ECC, nor wasa change in the fatty acid pattern observed. A peroxidation of the membrane lipids by oxygen free
radicals had to be considered because the RBCs had a most intense contact with the ECC apparatus and, in particular, with the membrane oxygenator.' This possibility, however, could be ruled out on the basis of the observation that the polyunsaturatedfatty acidsof RBC membrane phospholipids were not affected (Table II). Obviously the RBCs possess a sufficiently activeself-protectionagainstoxidative lipiddamage byenzymes suchas superoxide dismutase and catalase or the glutathione systemY·32 The mechanical stability and elasticity of the RBC membrane is provided by the membrane skeleton. It con-
Volume 100 Number 4
RBCs during ECC
October 1990
543
30,........-------------------......,
20 Q.
U
'-'
> in
~
o(J
(/)
> c
o 9 co
10
W
...J
oJ: ;:
O ..........- -------'----------'----------L...I 0.1
1
10
SHEAR RATE
100
(sec· 1)
Fig. 2. Plot of whole blood viscosityversus shear rate of 10 patients preoperatively, intraoperatively but before ECC, during ECC, and 24 hours postoperatively. The samples during ECC were measured at 37° C and at 26° C, that is, the average body temperature during this phase of the operation. The values are means ± standard deviation.
sists of a meshwork of proteins, primarily spectrin, actin, and protein band 4.1, which underlies the lipid bilayer. It is attached to the transmembranous protein band 3 by the anchoring protein band 2.1, called ankyrin. Oxidative damage causes the precipitation of hemoglobin and membrane proteins and the appearance of high molecular weight proteins detectable on gel electrophoresis.P We did not observe such high molecular weight proteins, which is another argument for the strong self-protection of the RBCs against oxidative damage. Ankyrin was reduced after ECC. This effect was not seen when protease inhibitors were added during ghost preparation. This indicates that the ECC increased the proteolytic activity of the plasma, possibly through an activation of leukocytes.II The fact that we were not able to induce a lossof ankyrin with an ECC pump in vitro suggests that the increased proteolytic activity is not simply due to the mechanical stress of the pump. The question arises as to whether the observed
echinocytosis had an influence on the flow properties of blood as suggested by other in vitro studies. 13, 34 The rheologic in vitro data of our study, namely, viscosity and RBC filtration, indicate that the RBC abnormalities seen during ECC are not relevant for blood flow. The marked reduction of the plasma viscosity by the dilution with priming solution and the simultaneous reduction of the hematocrit value both reduce whole blood viscosity in an additive manner and preponderate over any possible change induced by the abnormal RBC shape. A major determinant of viscosity is temperature. Hypothermia during ECC therefore increases blood viscosity. The reduction of plasma viscosity and hematocrit, however, were sufficient to keep the blood viscosity during ECC at 26° C below the preoperative values at 37° C. These data indicate that hemodilution during ECC is essential for blood rheology and that the use of blood for priming, as done in the early phases of ECC, could be deleterious. Tissue oxygenation is determined by RBC
The Journal of Thoracic and Cardiovascular Surgery
5 4 4 Reinhart et al.
transport efficiency, which can be estimated by the ratio hematocrit/viscosity.P At the highest shear rate (87 sec"), which is probably representative for the human circulation, the RBC transport efficiency fell from 9.1 preoperatively to 7.3 during ECC at 26° C; at all other shear rates it increased. We conclude that ECC leads to a reversible echinocytic shape transformation of RBCs. Blood viscosity is not increased by this RBC change but rather is decreased because of the marked hemodilution during ECC. It is therefore not likely that rheologic abnormalities are a contributing factor in post-ECC morbidity. We would like to thank Dr. Miihlemann and A. Wiesmann for their kind cooperation, Miss S. Kampfer for her excellent technical assistance, and Miss B. Urfer for typing the manuscript. REFERENCES I. Kirklin JW, Kirklin JK, Lell WA. Cardiopulmonary bypass for cardiac surgery. In: Sabiston DC Jr, Spencer FC, eds. Surgery of the chest. 4th ed. Philadelphia: WB Saunders, 1983:909-25. 2. Cavarocchi NC, England MD, O'Brien JF, et al. Superoxide generation during cardiopulmonary bypass: Is there a role for vitamin E? J Surg Res 1986;40:519-27. 3. Miki M, Tarnai H, Mino M, Yamamoto Y, Niki E. Freeradical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by a-tocopherol. Arch Biochem Biophys 1987;258:373-80. 4. Bolli R, Patel BS, Jeroudi MO, Lai EK, McCay PB. Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap a phenyl N-tert-butyl nitrone. J Clin Invest 1988;82:476-85. 5. Chenoweth DE, Cooper SW, Hugli TE, Stewart RW, Blackstone EH, Kirklin JW. Complement activation during cardiopulmonary bypass. N EnglJ Med 1981;304:497503. 6. Hammerschmidt DE, Stroncek DF, Bowers TK, et al. Complement activation and neutropenia occurring during cardiopulmonary bypass. J THORAC CARDIOVASC SURG 1981;81:370-7. . 7. Salama A, Hugo F, Heinrich D, et al. Deposition ofterminal C5b-9 complement complexes on erythrocytes and leukocytes during cardiopulmonary bypasses. N Engl J Med 1988;318:408-14. 8. Gans H, Krivit W. Problems in hemostasis during openheart surgery. IV. On the changes in the blood clotting mechanism during cardiopulmonary bypass procedures. Ann Surg 1962;155:353-9. 9. Gomes MR, McGoon DC. Bleeding patterns after openheart surgery. J THoRAc CARDIOVASC SURG 1970;60:8797. 10. "Davies GC, Sobel M, Salzman EW. Elevated plasma
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fibrinopeptide A and thromboxane B2 levels during cardiopulmonary bypass. Circulation 1980;61:808-14. Wilson JW. Pulmonary morphologic changes due to extracorporeal circulation: a model for "the shock lung" at cellular levelin humans. In: Forscher BK, Lillehei RC, Stubbs SS, eds. Shock in low- and high-flow states. Proceedings of a Symposium at Brook Lodge, Augusta, Michigan. Amsterdam: Exerpta Medica, 1972:160-71. Kamada T, McMillan DE, Sternlieb JJ, Bjork VO, Otsuji S. Erythrocyte crenation induced by free fatty acids in patients undergoing extracorporeal circulation. Lancet 1987;2:818-21. Meiselman HJ. Rheology of shape-transformed human red cells. Biorheology 1978;15:225-37. Henriksen L. Evidence suggestive of diffuse brain damage following cardiac operations. Lancet 1984;1:816-20. Van Oeveren W, Kazatchkine MD, Descamps-Latscha B, et al. Deleterious effects of cardiopulmonary bypass: a prospective study of bubble versus membrane oxygenation. J THORAC CARDIOVASC SURG 1985;89:888-99. Bessis M. Red cell shapes: an illustrated classification and its rationale. Nouv Rev Fr Hematol 1972;12:721-46. Rose HG, Oklander M. Improved procedure for the extraction of lipids from human erythrocytes. J Lipid Res 1965;6:428-31. Broekhuyse RM. Quantitative two-dimensional thin layer chromatography of blood phospholipids. Clin Chim Acta 1969;23:457-61. Rouser G, Fleischer S, Yamamoto A. Two-dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 1970;5:494-6. Reinhart WH, Usami S, Schmalzer EA, Lee MML, Chien S. Evaluation of red blood cell filterability test: influencesof pore size, hematocrit level, and flow rate. J Lab Clin Med 1984;104:501-16. Reinhart WH, Straub PW. Detection of less deformable erythrocytes in suspensions: evaluation of different rheological tests. Clin Hemorheol 1988;8:861-76.. Chien S. Biophysicalbehavior of red cellsin suspensions.In: MacN Surgenor D, ed. The red blood cell. 2nd ed. New York: Academic Press, 1975;2:1031-133. Coetzer TL, Lawler J, Liu SC, et al. Partial ankyrin and spectrin deficiency in severe atypical hereditary spherocytosis. N Engl J Moo 1988;318:230-4. Simchon S, Jan KM, Chien S. Influence of reduced red cell deformabilityon regional blood flow. Am J Physiol1987; 253:H893-903. Ekestrom S, Koul BL, Sonnenfeld T. Decreased red cell deformability following open-heart surgery. Scand J Thorac Cardiovasc Surg 1983;17:41-4. Deuticke B. Transformation and restoration of biconcave shape of human erythrocytes induced by amphiphilic agents and ionic environment. Biochim Biophys Acta 1968;163:494-500. Metha NG. Role of membrane integral proteins in the
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