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© 2000 International Anesthesia Research Society
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Abstract |
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Isovolemic hemodilution is used to decrease the incidence of blood transfusions. However, the effects of the degree of hemodilution and the fluid used on hemostasis are controversial. We tested the hypothesis that hemodilution and the fluid administered would adversely alter Thrombelastographic® (Haemoscope, Skokie, IL) variables (reaction time,
angle and maximal amplitude). Conscious rabbits had blood sampled from ear arteries and diluted 0% or 75% in vitro with one of four solutions: 6% hetastarch in 0.9% NaCl, 5% human albumin in 0.9% NaCl, or balanced electrolyte solutions containing either 6% pentastarch or 6% hetastarch. Isoflurane-anesthetized rabbits were randomly assigned to groups (n = 9 per group) that underwent in vivo isovolemic hemodilution (75% of estimated blood volume removed), with blood replaced with one of the four solutions mentioned previously. In vitro hemodilution resulted in a significant (P < 0.05) decrease in hemostatic function (increase in reaction time, decrease in angle and maximal amplitude) that was largest after hemodilution with albumin. However, although in vivo hemodilution significantly (P < 0.05) decreased reaction time, increased the angle, and decreased maximal amplitude, there were no significant fluid-dependent effects. Implications: The effects of hemodilution and the fluid used on Thrombelastographic® (Haemoscope, Skokie, IL) variables are markedly different between in vitro and in vivo hemodilution studies.
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Introduction |
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Isovolemic hemodilution can reduce the need for allogeneic blood administration intraoperatively. Mathematical (1) and clinical (2) investigations have demonstrated that isovolemic hemodilution to a hemoglobin concentration as small as 5 g/dL is well tolerated by healthy, conscious volunteers and could potentially obviate the need for allogeneic transfusion with surgical blood losses between 5 and 10 L. Similarly, extreme isovolemic hemodilution (75% of red cell mass removed) in an anesthetized rabbit model demonstrated neither hepatic ischemia nor histologic injury (3). These investigations support the concept that low hematocrit values previously avoided may provide acceptable organ perfusion in the perioperative period.
Although organ perfusion may be maintained, the impact of isovolemic hemodilution on hemostatic function has been a controversial topic. Issues include the effects of the specific fluid and degree of hemodilution on hemostatic function in vitro (4–11) and in vivo (12). With regard to the administration of colloid solutions during isovolemic hemodilution and the preservation of hemostasis, the role played by the specific macromolecule, the solution that contains the macromolecule, and the degree of hemodilution in vitro and in vivo remain to be elucidated. The purpose of the current study was to determine whether hemostatic function, assessed by using a Thrombelastograph® (Haemoscope, Skokie, IL) in rabbits, would be adversely affected to the same extent in vitro and in vivo by the degree of hemodilution and the colloidal solution administered.
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Methods |
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The study was approved by our animal review committee. Conscious rabbits (n = 15) were restrained (<2 min) once a week and had 2 mL of blood aseptically drawn from central ear arteries. The blood samples were immediately diluted 0% or 75% with one of the following fluids: 5% human albumin in 0.9% NaCl (n = 9), 6% generic hetastarch in 0.9% NaCl (n = 9), Hextend® (n = 9), or PentaLyte® (n = 9). Hextend® and PentaLyte® are, respectively, 6% hetastarch and 6% pentastarch solutions containing balanced electrolytes (Na+ = 143 mmol/L, Cl- = 124 mmol/L, Ca+2 = 2.5 mmol/L, Mg+2 = 0.45 mmol/L, K+ = 3 mmol/L), glucose (5 mmol/L) and lactate buffer (28 mmol/L). All samples were mixed by twice inverting the 1.5 mL plastic sample tube containing the blood sample. Within 1 min after mixing, 360 µL of blood was placed in a disposable cup and inserted into a Thrombelastograph® that was computer controlled. The proper functioning of the Thrombelastograph® was confirmed daily with quality control standards purchased from Haemoscope. The following Thrombelastographic® variables were measured for each sample over a 1 h period at 37°C: reaction time (R, min, the time from when the blood sample is placed into the Thrombelastograph® cuvette until initial fibrin formation occurs as noted by a signal of 2 mm amplitude); coagulation time (K, min, the speed at which a clot forms with a certain viscoelastic strength; defined as the time from R until the amplitude of the Thrombelastographic® signal; 20 mm in amplitude); angle (
, degrees, the angle formed from R to the inflection point of the Thrombelastographic® signal as clot strength stabilizes; it is a measure of the speed of clot formation); and maximal amplitude (MA, mm, the largest amplitude of the Thrombelastographic® signal and a measure of clot strength). A detailed description of the methodology of the Thrombelastograph® has been presented in great detail elsewhere (6–12).
After this first series of experiments, a second series of rabbits (n = 6) had blood samples diluted 75% with 5% human albumin solution or with 5% human albumin solution containing 1.5 mmol/L Ca+2 (CaCl2 used to add Ca+2; normal rabbit whole blood Ca+2 ranged between 1.2 and 1.7 mmol/L in the current study). Blood samples had R, K, and MA determined as mentioned previously. Concurrent blood Ca+2 was determined with a blood gas analyzer. The second in vitro series of experiments was designed to determine whether hypocalcemia was the mechanism responsible for the albumin-associated hypocoagulable state noted in the first in vitro series.
In the conduct of in vivo experimentation, rabbits were anesthetized with 10 mg/kg IV ketamine via a marginal ear vein and subsequently administered inhaled 1% isoflurane carried in 99% oxygen. Isoflurane administration (inspired concentration) was monitored with an anesthetic specific monitor (BCI International, Model 8100, Waukesha, WI). After tracheotomy and placement of a 3.5 mm outer diameter endotracheal tube, mechanical ventilation with a Harvard Apparatus (Holliston, MA) ventilator was performed with PaCO2 maintained at 32–45 mm Hg. Pancuronium was administered 0.3 mg · kg-1 · hr-1 IV to facilitate mechanical ventilation. An 18-gauge catheter was placed in the right femoral artery for blood sampling and pressure recording on a polygraph (Model 7D, Grass Instruments, Quincy, MA). All rabbits received a maintenance infusion of lactated Ringer’s solution at 4 mg · kg-1 · hr-1. A 30 min equilibration period followed the completion of the surgical preparation and device insertion. After 30 min of equilibration, rabbits were randomly assigned to groups (n = 9 per group) administered 5% human albumin, 6% generic heta-starch, Hextend® or PentaLyte®. All fluids were warmed to 39°C in a water bath. Isovolemic hemodilution was performed by an exchange transfusion of 75% of the estimated blood volume (EBV), which was calculated as follows: ([weight in kg x 0.07 L/kg] x 0.75) x 1000 mL = 75% of the EBV in mL. A quantity of one of the test fluids equal to 75% of the EBV was infused IV while simultaneously, an equal volume of blood was removed from the femoral arterial catheter over a 10 min period. The pHa, PaCO2, PaO2, and Ca+2 of arterial blood were determined at 37°C after 30 min of equilibration and 1 h after isovolemic hemodilution. Hematocrit was determined after 30 min of equilibration and 1 h after isovolemic hemodilution by centrifuging blood samples contained in a microhematocrit tube for 3 min in a microcapillary centrifuge and then, manually determining the hematocrit on a microcapillary reader. Finally, blood samples were obtained after 30 min of equilibration and 1 h after isovolemic hemodilution and then, were immediately placed in a Thrombelastograph® for 1 h to determine R, K, , and MA, as previously mentioned.
All variables are expressed as mean ± STD. With regard to the Thrombelastograph®, it was decided a priori that blood samples that did not clot were to be assigned an R value of 60 min, a K value of 60 min, an value of 0°, and an MA of 0 mm. Analyses of the effects of hemodilution and the fluid administered on all variables in the first in vitro and the in vivo study were conducted by two-way analysis of variance. Post hoc analysis was conducted with the Tukey test. Analyses of the effects of hemodilution with 5% human albumin on whole blood Ca+2 and the effects of supplemental Ca+2 on Thrombelastographic® variables after hemodilution with 5% human albumin in vitro were conducted with the Student’s t-test. An error of = 0.05 was considered significant.
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Results |
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Hemodilution and the fluid used, significantly affected R, K,
, and MA values of whole blood (Table 1). The R values after 75% dilution in the hetastarch and albumin groups were significantly larger than the R values observed after 0% dilution. Further, the albumin group had a significantly larger R value after 75% dilution than any other group. The K values of all groups observed after 75% dilution were significantly larger than that observed after 0% dilution. Additionally, the hetastarch and albumin groups had significantly larger K values than the Hextend® and PentaLyte® groups after 75% dilution. However, all groups had significantly smaller values after 75% dilution compared with values observed after 0% dilution. The values of the albumin group after 75% were significantly less than those of any other group. Compared with values observed after 0% dilution, MA significantly decreased in all groups after 75% hemodilution. The albumin group had MA values significantly less than those of all other groups after 75% dilution. In the albumin group, six blood samples had no discernible clotting activity after 75% dilution.
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In the second series of experiments designed to determine whether dilution with albumin impaired the coagulation cascade by binding Ca+2, whole blood Ca+2 was significantly decreased from 1.56 ± 0.13 mmol/L observed at 0% dilution to 0.30 ± 0.01 mmol/L after 75% dilution with albumin. Blood samples diluted 75% with albumin supplemented with Ca+2 had Thrombelastographic® variables (R = 16.0 ± 5.4 min, K = 6.4 ± 2.3 min,
= 37.5 ± 11.3°, MA = 33.6 ± 6.9 mm) that were significantly different from blood diluted with albumin alone (R = 52.4 ± 18.6 min, K = 50.5 ± 23.4 min, = 9.0 ± 22.0°, MA = 8.2 ± 20.0 mm). Only one of the six samples diluted 75% with albumin had discernible clotting.
In contrast to the findings of the in vitro studies, R, , and MA values were significantly affected only by hemodilution after isovolemic hemodilution in vivo as displayed in Table 2. Isovolemic hemodilution significantly decreased R values, with no fluid-dependent differences in R values noted. K values were not significantly changed by hemodilution or by the fluid used. Isovolemic hemodilution significantly increased ; however, there were no fluid-dependent changes in values. Isovolemic hemodilution significantly decreased MA; however, the fluid used did not affect MA. Finally, with regard to the hemodynamic, arterial blood gas, hematocrit, and Ca+2 data displayed in Table 3, hemodilution significantly decreased only mean arterial pressure and hematocrit. When the entire cohort of rabbits was analyzed, the animals were on average diluted 74 ± 4% based on changes in hematocrit. The fluid used for hemodilution did not affect any of the variables (Table 3).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Many of our findings are in agreement with previous in vitro studies involving dilution of human blood. First, Thrombelastographic® values of blood obtained from either conscious or anesthetized rabbits before hemodilution are similar to that obtained from healthy adult humans (6,7,12,13). Rabbits appear to have R values 1–8 min longer than humans, K and
values well within the range of human values, and MA values 5–10 mm greater than human values (6,7,12,13). Human blood diluted 30% to 60% with a hydroxyethyl starch (molecular weight [MW] = 200,000 d) or 5% human albumin, demonstrated no change in R, an increase in K, and a decrease in both and MA compared with undiluted values (8). A similar study that hemodiluted samples up to 75%, demonstrated a dose-dependent increase in R and a decrease in both and MA (10). The largest decrease in hemostatic function was noted when 5% human albumin was used as the diluent (10). Ionized calcium concentrations <0.6 mmol/L severely impairs clotting kinetics in humans (14). Consequently, the present findings that the mechanism responsible for the albumin-mediated impairment of hemostasis after 75% hemodilution in vitro in the rabbit is likely secondary to a severe reduction of whole blood Ca+2 is consistent with human studies (10,14). Considered as a whole, Thrombelastographic® data generated in vitro with human or rabbit models appear comparable.
Our findings support the concept that the effects of hemodilution and the fluid used on hemostasis in vitro poorly predict changes in Thrombelastographic® data after comparable isovolemic hemodilution in vivo. After an approximately 75% hemodilution, R values were decreased in vivo, whereas R values were prolonged in vitro. K values were not changed after 75% hemodilution in vivo; however, in vitro K values were prolonged. Further, values were increased after 75% hemodilution in vivo, although decreased in vitro. Finally, MA decreased after 75% hemodilution both in vivo and in vitro, yet the values observed at 75% dilution in vivo were larger than those observed in vitro. Although in vivo hemodilution accelerated clot formation (albeit a clot with decreased viscoelastic strength), neither the macromolecule nor the vehicle composition of the fluid administered significantly affected any Thrombelastographic® variable.
Some investigators have attempted to simulate quasi in vivo Thrombelastographic® data after in vitro hemodilution by adding buffers and Ca+2 to the diluent of interest (8,9,11). Although maintaining pH and Ca+2 concentrations within physiologic ranges may attenuate fluid-specific pharmacodynamic effects on hemostasis, only an intact animal model can provide insight into pharmacokinetic effects of the specific fluid administered. Specifically, most hydroxyethyl starch solutions contain macromolecules that are polydispersoids (e.g., MW range of 30,000 to 2.2 million d; human albumin solution is a monodispersion with a MW of 66,000 d). For example, low MW hydroxyethyl starch species adversely affect coagulation variables in vitro (15,16); therefore, renal clearance of these starch species in vivo may result in Thrombelastographic® data significantly different from in vitro investigations. The comparison of in vitro and in vivo Thrombelastographic® investigation could be thought of as a continuum, wherein in vitro data represent the initial effects of dilution on hemostasis, and in vivo data represent the effects of compensatory homeostatic mechanisms on clotting.
Although the current study of isovolemic hemodilution in anesthetized rabbits did not demonstrate any fluid-specific effects on Thrombelastographic® variables, a recent clinical investigation by Gan et al. (17) did demonstrate fluid-specific differences in R and K values in patients undergoing surgeries associated with significant blood loss. Patients that were administered more than 20 mL/kg of Hextend® had significantly lower R and K values compared with patients receiving equivalent quantities of generic 6% hetastarch in 0.9% NaCl (17). Further, patients requiring red blood cell transfusions had a significantly lower blood loss during Hextend® administration as compared with patients administered 6% hetastarch. It is also important to note that most of the patients in this study were ASA physical status II and III undergoing abdominal surgery. Because laparotomy may cause occult hepatic hypoperfusion (18,19), concurrent exposure to citrated blood products could have resulted in poor clotting kinetics secondary to hypocalcemia in the 6% hetastarch group (17). We used a degree of isovolemic hemodilution (75% of EBV) that is not associated with either systemic or hepatic ischemia in rabbits (3).
In conclusion, we have demonstrated that extreme isovolemic hemodilution accelerates clotting kinetics with a concurrent decrease in clot strength in vivo in the anesthetized rabbit. The effect of isovolemic hemodilution on Thrombelastographic® variables was not influenced by any of the colloids administered in vivo. Only in vitro hemodilution demonstrated fluid-specific effects on Thrombelastographic® variables.
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Acknowledgments |
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Supported in part by a grant from BioTime, Inc., Berkeley, CA. and the Department of Anesthesiology, The University of Alabama at Birmingham, AL.
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References |
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[Abstract/Free Full Text] - Nielsen VG, Baird MS, Brix AE, Matalon S. Extreme, progressive isovolemic hemodilution with 5% human albumin, PentaLyte, or Hextend does not cause hepatic ischemia or histologic injury in rabbits. Anesthesiology 1999;90:1428–35.[Medline]
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[Abstract/Free Full Text] - Mortier E, Ongenae M, De Baerdemaeker L, et al. In vitro evaluation of the effect of profound haemodilution with hydroxyethyl starch 6%, modified fluid gelatin 4% and dextran 40 10% on coagulation profile measured by thrombelastography. Anaesthesia 1997;52:1061–64.[Web of Science][Medline]
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