Anesth Analg 2003;96:1209-1213
© 2003 International Anesthesia Research Society
GENERAL ARTICLES
The Influence of Induced Hypothermia for Hemostatic Function on Temperature-Adjusted Measurements in Rabbits
Mitsuru Shimokawa, MD,
Katsuyasu Kitaguchi, MD,
Masahiko Kawaguchi, MD,
Takanori Sakamoto, MD,
Meiko Kakimoto, MD, and
Hitoshi Furuya, MD
Department of Anesthesiology, Nara Medical University, Japan
Address correspondence and reprint requests to Mitsuru Shimokawa, MD, Department of Anesthesiology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. Address e-mail to mshimoka{at}naramed-u.ac.jp
 |
Abstract
|
|---|
In hypothermic patients, a tendency to bleed may be observed even when hemostatic tests seem to be normal. Coagulation and platelet function tests are usually performed at 37°C. We investigated the influence of induced hypothermia on temperature-adjusted hemostasis function testing using Sonoclot Analyzer® (Sonoclot®) and Thromboelastography® (TEG®). Anesthesia was induced and maintained with IV ketamine and fentanyl on 15 male New-Zealand White rabbits. A water blanket was used to induce hypothermia to 30°C and to rewarm to 37°C. Blood samples were obtained at four points: before hypothermia, at 34°C, at 30°C, and after rewarming. Standard coagulation tests were performed at 37°C (C method), and simultaneously, real temperature hemostasis function tests (R method) were run. In Sonoclot®, activated clotting time and time to peak increased and clot rate decreased significantly at 30°C in the R method compared with those in the C method. In TEG®, reaction time and clot formation time were prolonged and clot formation rate was diminished at 30°C in the R method compared with those in the C method. Induced hypothermia delayed the coagulation cascade and reduced platelet function. During hypothermia, hemostatic measurements should be performed at real temperature to avoid overestimating patient hemostatic function based on results measured at the standard 37°C.
IMPLICATIONS: We investigated the influence of induced hypothermia on temperature-adjusted hemostasis function tests in rabbits using Sonoclot Analyzer® and Thromboelastography®. Induced hypothermia delayed the coagulation cascade and reduced platelet function. The conventional coagulation tests performed at 37°C failed to detect these hypothermia-induced degradations in hemostasis performance.
|
Introduction
|
|---|
Mild to moderate hypothermia (30°C–35°C) is used as an effective neuroprotective treatment in clinical and laboratory studies. However, hypothermia has various problems and side effects that may be avoidable (1). One of these complications is hemostatic dysfunction caused by hypothermia (2). Conventionally, coagulation and platelet function tests are performed at 37°C, even when patient body temperature is low, and it is thought that the hemostatic function measured at 37°C provides an accurate measure of hemostatic function during hypothermia. However, hemostatic function measured at 37°C may not show the patient’s actual condition, and measurement of hemostatic function at the patient’s real temperature may be very important during hypothermia treatment in clinical settings. In a previous report (3), we suggested that the conventional measurement at 37°C overestimated the actual hemostatic functions in neurosurgical patients during induced mild hypothermia. However, there was a possibility that anesthetics or aggressive operative insults influenced hemostasis performance in these patients. Therefore, to isolate the effect of hypothermia on hemostatic function, we conducted this study on healthy rabbits not undergoing any additional surgical procedures.
|
Methods
|
|---|
The Animal Experiment Committee of Nara Medical University approved the present study. Fifteen male New Zealand White rabbits weighing 2.0–2.5 kg (mean, 2.1 kg) were used.
Each rabbit was given 30 mg/kg IM of ketamine, and a 24-gauge catheter was placed in the marginal ear vein. Then, a continuous infusion of 25 mg · kg-1 · h-1 of ketamine and 25 µg · kg-1 · h-1 of fentanyl was started. The trachea was intubated via tracheostomy, and pancuronium (0.4 mg/kg) was administered. The lungs were ventilated mechanically with 50% nitrous oxide and 50% oxygen. The femoral artery was exposed and cannulated to monitor arterial blood pressure and to collect blood samples. Esophageal temperature was continuously monitored using a thermometer (Mon-a-Therm, Mallinckrodt, St Louis, MO). The whole body was wrapped with a water blanket to control body temperature.
Blood samples of 1.9 mL were obtained when the esophageal temperature reached the following four conditions: 37°C–39°C before hypothermia (Control; Pre), 34°C and 30°C after hypothermia had begun, and 37°C after rewarming (Post). Body temperature control was a target of 30°C for cooling and then a target of 37°C for rewarming via water blanket, and we took samples along the way.
Lactated Ringer’s solution was infused at the rate of 3–6 mL · kg-1 · h-1 during the experiment. Mechanical ventilation was controlled to maintain end-tidal carbon dioxide to be 30–40 mm Hg (STPD). Sodium bicarbonate was administered to keep the pH value at 7.3–7.5. Infusion rates and anesthetics were controlled to maintain changes of <20% from control values for mean arterial blood pressure, heart rate, and hemoglobin. When shivering and body movement were recognized, pancuronium (0.2 mg/kg) was IV administered.
Blood coagulation and platelet function at 37°C (conventional manner; C method) and those at real temperature (temperature-adjusted; R method) were simultaneously measured using two Sonoclot Analyzers® (Sonoclot®; Sienco Inc, Wheat Ridge, CO). The temperature was adjusted to real temperature for one Sonoclot®, whereas the other Sonoclot® remained at the standard 37°C. Sonoclot® temperature settings were rotated for each animal to reduce any instrument variance.
The Sonoclot® measures viscosity of fluids and gels and reports this measurement in a normalized scale called clot signal units. The time varying clot signal measurements for a clotting test is called a sonoclot signature (Fig. 1). Analytic results calculated from the signature include the activated clotting time (ACT), clot rate (CR), time to peak (TP), and peak angle (PA). In this study, Sonoclot® data were collected using glass bead contact activation.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 1. Sonoclot® signature and variables. Activated clotting time (ACT) is a measure of the performance of clotting factors. Clot rate (CR) is the slope of the sonoclot signature during initial fibrin formation and characterizes the rate of fibrin formation. Time to peak (TP) is a measure of how fast platelets activate and retract the clot. Peak angle (PA) is a new sonoclot signature indicator (4) that is thought to quantify the strength or quality of clot retraction.
|
|
Prothrombin time, activated partial thromboplastin time, and fibrinogen were measured using Drihemato® (CGO1, A&T, Yokohama, Japan). Platelet and hemoglobin were calculated using automatic blood cell counter (LC-630, Horiba Ltd, Kyoto, Japan).
In 12 rabbits, computerized Thromboelastography® (TEG®) was performed using the TEG5000® (Haemoscope Corp, Skokie, IL) having two chambers with variable temperature controls. Data of reaction time (r), clot formation time (k), coagulation time (r + k), clot formation rate (angle), and maximum amplitude (MA) were obtained from thromboelastograms (Fig. 2) of C and R methods with celite activation. Hemostatic analysis was conducted using whole blood within 2 min of blood collection.
View larger version (11K):
[in this window]
[in a new window]
|
Figure 2. Thromboelastogram® and variables. Reaction time (r) is the time when the first fibrin clotting is detected. Clot formation time (k) is the time it takes for the graph to widen from 1 mm to 20 mm (variable of the clot firmness). Coagulation time (r + k) is the total time of r and k. Clot formation rate (angle) is the rate of clot growth. Maximum amplitude (MA) indicates the maximum strength of the clot.
|
|
All the values obtained are presented as mean ± SD. Sonoclot® data were analyzed using two-way analysis of variance with repeated measures. Data of Drihemato® and platelet count were analyzed using one-way analysis of variance with repeated measures. Post hoc comparisons were conducted using Turkey-Kramer test. TEG® data were analyzed using the Wilcoxon’s signed rank test. P values of <0.05 were regarded as statistically significant.
|
Results
|
|---|
The esophageal temperature took an average of 2.5 h to reach 30°C from Pre and an average of 2 h to reach Post from 30°C. No significant changes in the variables of arterial blood pressure, heart rate, arterial blood gas, and hemoglobin were observed at each measurement time.
Sonoclot® Experiment
In the R method, ACT results at 34°C, 30°C, and Post were significantly prolonged from that at Pre. In the C method, the ACT result at Post was significantly prolonged compared with Pre. At 30°C, a significant increase in ACT was observed between the R method and C method. The CR at 30°C decreased significantly from that at Pre in the R method. During inter-method, the CR at 30°C in the R method reduced significantly compared with that in the C method. The TP at 30°C increased significantly from that at Pre in the R method. No significant difference in PA was observed in either intra- or inter-method data during the whole procedure (Fig. 3).
TEG® Experiment
Results for r, k, and r + k in the R method at 30°C were significantly increased from those in C method at 30°C and from those at Pre in the R method. The angle at 30°C in the R method was decreased significantly compared with that at Pre and that in the C method. However, no differences in MA were observed during the procedure in either intra- or inter-method data (Table 1).
Activated partial thromboplastin time at 30°C was significantly higher than that at Pre. Platelet count substantially decreased from 168 x 103/µL to approximately 125 x 103/µL between Pre and Post, and the P value for the change was significant at 0.0197. There were no significant differences in prothrombin time and fibrinogen among the measurement points (Fig. 4).
View larger version (25K):
[in this window]
[in a new window]
|
Figure 4. Changes in prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and platelet in response to the body temperature. Data presented as mean ± SD. *P < 0.05 versus Pre.
|
|
|
Discussion
|
|---|
Hemostatic dysfunction during hypothermia is believed to occur by enzyme inhibition, platelet alteration, fibrinolytic increasing, or combinations thereof (5). However, coagulation and platelet function tests are generally performed at 37°C, even when the samples are obtained from hypothermia patients. There have been few reports in which measurements were conducted both at body temperature and in a conventional manner at 37°C, and the data were compared using the same sample. Results by Douning et al. (6), Kettner et al. (7), and Fukuda et al. (8) suggest that coagulation measurement at 37°C may not reflect the real hemostatic function in hypothermic patients. However, these findings reflect the influence of factors other than hypothermia.
Because the influences of surgery and anesthesia were not considered in our previous report (3), healthy animal subjects were used in this study to reduce those influences. Inhaled anesthetics believed to have an effect on hemostatic function such as halothane (9,10) and propofol (9,11) were avoided. Instead, ketamine and fentanyl were used as IV anesthetics because we expected them to minimally influence hemostatic function (9,12,13) .
Measurement was performed using two Sonoclots® and one dual-chamber TEG5000®, all with variable temperature controls. Our TEG® results agreed with previously reported results (6,7) in some respects but differed in others.
Douning et al. (6) demonstrate in liver transplantation cases that differences caused by hyperthermia are hardly observed between conventional and real temperature methods. The contradiction between our result and Douning et al. (6) probably can be attributed to existing preoperative coagulopathy caused by serious liver dysfunction. However, Kettner et al. (7) report in extra corporeal circulation cases that r and k at the lowest temperature (33.3°C) increase approximately 200% compared with baseline measurements (36.2°C). In the present study, although the measurement temperature was less than 33.3°C (30°C), r and k increased only 170% compared with Pre measurements. The difference between our results and theirs may be because of platelet and coagulation factor stress and hemostatic alterations related to extra corporeal circulation and surgery. However, Kettner et al. agree that hemostatic function tends to reduce as the body temperature decreases, even in the measurement at 37°C, and that coagulation function is significantly reduced in the measurement at real temperature. These results support our Sonoclot® ACT and CR results. Moreover, TEG® clot formation rate (angle) results at real temperature decreased with hypothermia, but there was no difference of MA in either previous or present studies.
In TEG®, angle shows the speed of blood clot formation, whereas MA shows completed clot strength. In Sonoclot®, TP reflects the time for clot retraction to develop, whereas PA is thought to reflect both fibrinogen and clot retraction quality. Results of this study indicated that the time to clot retraction, including the coagulation time, increased because of hypothermia. However, once clot retraction began, hypothermia had no impact on clot strength. These results clarify the reduction of platelet function during hypothermia observed in the temperature-adjusted measurements.
It is assumed that platelets are taken into the liver or spleen temporarily during hypothermia, and the number of circulating platelets decreases (14). However, the number of blood platelets continued decreasing and did not recover after the rewarming in this study. The cause is not hemodilution for blood collecting because a change of hemoglobin hardly decreases. Mahajan et al. (15) report that a large quantity of tissue thromboplastin returns from the impaired organs because of rapid rewarming and makes it easy to develop disseminated intravascular coagulation (DIC). However, it was unclear whether the platelet reductions seen in this study were caused by a delay in recovery process from hypothermia or DIC induced during the rewarming.
We found it interesting that clot retraction on Sonoclot® showed a hemostatic recovery tendency, although the number of platelets continued to decrease. This suggests that the number of platelets and the platelet function do not simultaneously recover after rewarming. In clinical practice, close attention should be paid to platelet reduction, the delay of hemostatic functional recovery, and the development of DIC at rewarming.
In conclusion, coagulation and platelet function in rabbits under hypothermic anesthesia showed greater reduction when measured at 30°C than when measured at 37°C. Results measured at real temperature (30°C) show that the actual hemostatic function during hypothermia undergoes more deterioration than shown when measured at 37°C. The conventional measurement conducted at 37°C failed to detect hypothermic-induced degradations in hemostatic performance.
|
Acknowledgments
|
|---|
The authors thank IMI CO, LTD, and Rico Trading CO, LTD, for their support and Jon Henderson for proofreading the manuscript.
|
References
|
|---|
- Clifton GL, Miller ER, Choi SC, et al. Lack of effect of induction of hypothermia after acute brain injury. N Engl J Med 2001; 344: 556–63.[Abstract/Free Full Text]
- Bock M, Muller J, Bach A, et al. Effects of preinduction and intraoperative warming during major laparotomy. Br J Anaesth 1998; 80: 159–63.[Abstract/Free Full Text]
- Shimokawa M, Kitaguchi K, Furuya H. Influence of induced mild hypothermia on temperature adjusted sonoclot analysis in neurosurgical patients. Jpn J Reanimatology 2002; 21: 18–21.
- Miyaguchi O, Yaku H, Izuta S, et al. Perioperative analysis of coagulation/platelet function on Sonoclot. J Jpn Soc Intensive Care Med 1999; 6: S150.
- Watts DD, Trask A, Soeken K, et al. Hypothermic coagulopathy in trauma: effect of varying levels of hypothermia on enzyme speed, platelet function, and fibrinolytic activity. J Trauma 1998; 44: 846–54.[ISI][Medline]
- Douning LK, Ramsay MA, Swygert TH, et al. Temperature corrected thrombelastography in hypothermia patients. Anesth Analg 1995; 81: 608–11.[Abstract]
- Kettner SC, Kozek SA, Groetzner JP, et al. Effects of hypothermia on thrombelastography in patients undergoing cardiopulmonary bypass. Br J Anaesth 1998; 80: 313–7.[Abstract/Free Full Text]
- Fukuda A, Ishida H, Kubota M, et al. Clinical data obtained through coagulation testing suggests that hypothermia exerts influence on a patient’s blood coagulation reaction. Rinsho Byori 2000; 48: 1102–8.[Medline]
- Gibbs NM. The effect of anaesthetic agents on platelet function. Anaesth Intensive Care 1991; 19: 495–505.[ISI][Medline]
- Dogan IV, Ovali E, Eti Z, et al. The in vitro effects of isoflurane, sevoflurane, and propofol on platelet aggregation. Anesth Analg 1999; 88: 432–6.[Abstract/Free Full Text]
- De La Cruz JP, Carmona JA, Paez MV, et al. Propofol inhibits in vitro platelet aggregation in human whole blood. Anesth Analg 1997; 84: 919–21.[Abstract]
- Gaszynski W, Jezewska D, Kanski A. Studies of blood clotting system during dissociative anaesthesia with ketamine. Anaesth Resusc Intensive Ther 1976; 4: 47–51.[Medline]
- Referowska M. Changes in some blood clotting and fibrinolysis factors during anaesthesia with neuroleptanalgesic agents. Anaesth Resusc Intensive Ther 1975; 3: 141–6.[Medline]
- Hessel EA, Schmer G, Dillard DH. Platelet kinetics during deep hypothermia. J Surg Res 1980; 28: 23–34.[ISI][Medline]
- Mahajan SL, Myers TJ, Baldini MG. Disseminated intravascular coagulation during rewarming following hypothermia. JAMA 1981; 245: 2517–8.[ISI][Medline]
Accepted for publication November 21, 2002.
This article has been cited by other articles:
|
|
|
|
 
M. Rundgren and M. Engstrom
A Thromboelastometric Evaluation of the Effects of Hypothermia on the Coagulation System
Anesth. Analg.,
November 1, 2008;
107(5):
1465 - 1468.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Iwata, J. W. Newburger, D. Zurakowski, and R. A. Jonas
Postoperative Hypothermia and Blood Loss After the Neonatal Arterial Switch Procedure
Ann. Thorac. Surg.,
November 1, 2007;
84(5):
1627 - 1632.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Scharbert, M. Kalb, C. Marschalek, and S. A. Kozek-Langenecker
The effects of test temperature and storage temperature on platelet aggregation: a whole blood in vitro study.
Anesth. Analg.,
April 1, 2006;
102(4):
1280 - 1284.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
, C method (measured at 37°C); 
, R method (measured at adjusted temperature). *P < 0.05 intra-group; 
P < 0.05 versus C method.
