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Anesth Analg 1999;88:483
© 1999 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

The Effects of In Vitro Hemodilution with Gelatin, Hydroxyethyl Starch, and Lactated Ringer’s Solution on Markers of Coagulation: An Analysis Using SONOCLOTTM

Christoph Konrad, MD*, Timo Markl, CM*, Guido Schuepfer, MD*, Helmut Gerber, MD Prof.*, and Markus Tschopp, LT{dagger}

*Department of Anesthesiology and Intensive Care and {dagger}Hematology Laboratories, Kantonsspital, Lucerne, Switzerland

Address correspondence and reprint requests to Christoph Konrad, MD, Department of Anesthesiology and Intensive Care, Kantonsspital, CH-6000 Lucerne 16, Switzerland. Address e-mail to Fieber_Konrad{at}Compuserve.com


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Blood-saving strategies have recently been established to avoid allogeneic transfusion during surgery or after trauma. This includes an expanding use of crystalloids and colloids. These solutions interfere with coagulation systems, but quantitative measurements are still lacking. The SONOCLOTTM (Sienco Company, Morrison, CO) analysis (SCT), a viscoelastic test, measures clot formation and includes information on the cellular, as well as the plasmatic coagulation, system. To quantify hemodilutional effects on in vitro coagulation, we studied gelatin (G), hydroxyethyl starch 6% (HES; molecular weight 450,000), and lactated Ringer’s solution (RL) in 33% and 66% dilutions measuring routines laboratory and SCT variables. Hemodilution with RL tended to increase in vitro coagulability. Among the tested colloids, G had the least impact on markers of coagulation. G33% did not differ significantly from the undiluted control group. HES had the largest impact on markers of coagulation compared with G and RL. In conclusion, SCT provides a fast and easy to perform bedside test to quantify in vitro hemodilution.

Implications: The effects of progressive hemodilution on coagulation are difficult to measure. SONOCLOT analyses provide an easy to perform test with fast information on cellular and plasmatic coagulation properties. Among colloids, hydroxyethyl starch has the largest impact on markers of coagulation compared with gelatin or lactated Ringer’s solution.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
During elective or emergency surgery, blood-saving strategies include infusion of colloids or crystalloids to restore intravascular volume and to avoid allogeneic transfusions. To minimize the transmission of infectious diseases, these regimens are increasing, even in patients with coronary artery disease (1). Dilution itself and platelet dysfunction are known origins of postoperative bleeding disorders (2). Colloids (3), as well as lactated Ringer’s solution (RL) (4) significantly influence blood coagulation. Routine coagulation variables clinically available are unable to quantify these interferences. Therefore, fast and easy to use variables are desirable to assess the coagulation state.

The SONOCLOTTM (Sienco Company, Morrison, CO) analysis (SCT), a viscoelastic test, is a simple bedside diagnostic tool that includes analysis of platelet function, cellular and plasmatic coagulation for clot formation, maturation, and lysis. The SCT is superior to thromboelastography (TEG) in terms of practicality cost, and availability (5). No special solid platform for shock absorption is necessary for the SCT. The SCT can be performed very easily, and dynamic test results are available after a few minutes, and so may guide clinical therapy, such as perioperative bleeding problems, very effectively (6,7). Regarding sensitivity and specificity, the TEG and SCT are comparable, but taken together, SCT and TEG were able to increase diagnostic accuracy significantly (8). Using TEG and/or SCT, coagulation changes were found in parturients (9,10), patients with cancer (11,12), patients with sepsis (13), and in other patient populations (14). Therefore, clinically relevant decisions could be made using these viscoelastic tests. There are some limitations of the SCT. In contrast to the high accuracy of the SCT during the clot formation process, impaired clot lysis (e.g., caused by aspirin intake) is less accurate than the TEG, probably because of the method of clot activation (5,15–17).

Thus, we investigated the influence of moderate and profound in vitro hemodilution with gelatin (G), hydroxyethyl starch (HES), and RL on whole blood coagulation measuring SCT and standard laboratory variables.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
After institutional ethical approval, 10 healthy volunteers without any drug intake donated 50 mL of blood. The test solutions 4% G (Braun Co., Emmenbrücke, Switzerland), 6% HES (molecular weight 450,000; Fresenius Co., Bad Homburg, Germany) and RL (Braun Co.) were added to test tubes containing blood from the volunteers. Sample 1 (no dilution) was used for baseline values; Sample 2 was diluted with the test solutions to 33%; Sample 3 was diluted to 66%. In the same manner, the blood in the SCT cuvette was either undiluted (Sample 1) or diluted to 33% (Sample 2) or 66% (Sample 3). Thirty-three percent dilution corresponded to one part of the tested solution per two parts blood, and 66% dilution corresponded to two parts of tested solution per one part blood. Transferring the in vitro test design to an in vivo situation results in a calculated plasma expander concentration of approximately 25 mL/kg after 33% dilution and 50 mL/kg after 66% dilution.

The following laboratory tests were performed in the three samples: hematocrit, fibrinogen, red blood cell and platelet count, prothrombin time, activated partial thromboplastin time, and SCT (activated clotting time [ACT], clot rate, time to peak, clot signal at peak, and clot signal over time), (Fig. 1). All tests were performed immediately after obtaining the blood samples. For routine testing, all blood samples were taken at once, whereas samples for SCT were taken just before the test run because storage of blood samples for SCT analysis is impossible.



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  		Figure 1. Normal SONOCLOT signals and their interpretations. • = variable inflection point; start of retraction induced by platelets.

 
The principle of the SCT is the registration of the difference of viscoelastic properties of a forming blood clot (5). The SCT uses impedance difference over time caused by clotting in a fresh whole blood sample, which is oscillated by a vibrating transducer (200 Hz, distance 1 µm, temperature 37°C (17). The test cuvette contains 5 mg of celite activator. The following measurements were performed (5): ACT is the onset time of the beginning of fibrin formation, which is comparable to the conventional measured ACT; clot rate is the rate of fibrin formation from fibrinogen (primary slope R1). After primary fibrin formation as measured by the clot rate, there is a variable inflection point showing the start of fibrin retraction, which is activated by platelets. The second slope (R2), especially the peak value, represents completion of clot formation, including all necessary portions of plasmatic and cellular clot formation. Amplitude of the peak value represents fibrinogen concentration, and time to peak is a value for the speed and quality of fibrin formation. The decline (R3) after the peak value is caused by squeezing serum out of the clot matrix and further clot retraction, which is induced by platelets. Platelet dysfunction is seen by lack of an inflection point between R1 and R2, a prolongation of the time to peak, and a missing down slope after the maximal peak R3. Further decrease (after peak) is caused by clot lysis, which is hardly observed during standard SCT.

Study participants were male nonsmokers with no history of any chronic or acute diseases. Volunteers with acute or chronic medication intake (<3 wk) were excluded.

To calculate any differences between the coagulation results, nonparametric analysis of variance and adequate post hoc tests were used as appropriate using Statistica 5.1 (StatSoft Inc., Tulsa, OK). For multiple testing, the Bonferroni correction was applied. Power analyses were performed using GB-STAT 6.5 (Dynamic Microsystems Inc., Silver Spring, MD). The {alpha} error was set at 0.05 and the ß error at 0.1. Data are presented as median ± SD. For graphical presentation, box plots including the median were chosen, where the box represents 25%–75% of the entire data. Nonoutlier ranges, outliers, and extremes are also given. A P value <0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Ten volunteers took part in this investigation, (median age 32 ± 7 yr, body weight 74 ± 10 kg, body height 180 ± 10 cm, and body mass index 23 ± 2.7). As expected, standard coagulation variables decreased with dilution, and marked differences between the groups were observed only after 66% dilution. (Table 1)


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  		Table 1. Coagulation Variables
 
In contrast, SCT variables differed significantly between colloids and crystalloids earlier. The results of the SCT are presented in the order that they were generated by the SCT signature (Fig. 1). ACT (liquid phase) was significantly prolonged in the G66% and HES66% groups compared with baseline and RL66% (power 80%–90%). G66% and HES66% samples differed significantly from all other groups (Fig. 2). Clot rate during gel formation was significantly lower in all groups except RL33% and G33% compared with baseline (power >95%). HES66% and G66% differed significantly from all groups except HES33% (power >95%). RL showed the least impact on clot rate compared with the colloids (Fig. 3). Compared with baseline, the time to peak was significantly delayed with the HES66%, G66%, and RL66%. There were also significant differences between RL33% and G66% and HES66%, as well as between HES66% and HES33% and G33% regarding time to peak (power 80%–90%) (Fig. 4). Clot signal at peak was significantly lower in the G66% and HES66% groups compared with baseline (power >95%). The RL groups showed no significant difference compared with baseline (Fig. 5).



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  		Figure 2. SonoACT showed slight hypercoagulability in the 33% diluted groups. Within the 66% diluted groups; lactated Ringer’s solution (RL) had the least impact on SonoACT. HES = hydroxyethyl starch, G = gelatin.

 


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  		Figure 3. Hydroxyethyl starch (HES) had a large impact on the clot rate regardless of the degree of dilution, which was most visible in the 33% diluted samples.

 


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  		Figure 4. Time to peak was minimally affected by lactated Ringer’s solution (RL) even in the 66% diluted group.

 


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  		Figure 5. Maximal clot signal at peak was minimally affected by all lactated Ringer’s solution (RL) groups.

 
In the RL33% and G33% groups, there was a tendency for shortening of the ACT. There was also a slight activation of clot formation during the liquid phase and gel formation as measured by ACT and clot rate in the 33% diluted groups, except HES33%. Later during clot formation, this activation was no longer observed. Regarding this effect, HES showed the least impact on coagulation markers. Routine variables did not reflect this activation.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
SCT is a very fast and easy to use test that provides information on viscoelastic properties of the clot over nearly the entire in vitro clot formation and maturation process. SCT presents a consecutive series of defined clot signals, such as ACT, clot rate (fibrin formation), or time to peak (fibrin retraction). From these series, clot rate was the most rapid variable, revealing significant differences among the diluted solutions. During the liquid phase of clot formation, ACT showed only minimal changes, as did routines variables. Time to peak, which represents clot tightening activated by platelets, was significantly affected by HES in contrast to the clot amplitude.

Using SCT results regarding effects of hemodilution on coagulation markers were comparable to those obtained with the TEG (3). Progressive hemodilution resulted in significant changes of the clot signal, especially when HES was used. This can be explained by the profound impact of HES on factor VIII and subgroups, as well as inhibition of platelet aggregation and adhesion during clot formation (18). The effects of G on coagulation were less profound than those of HES, and can be explained by binding of G to fibronectin resulting in a reduction of free G (19) and by a more profound extravasation of G compared with HES in vivo (20).

RL or G at a moderate hemodilution of 33% resulted in a slightly shortening of the liquid phase (ACT) with an earlier start of gel formation of the clot, which could be interpreted as an activation of the initial coagulation cascade (18). This activation of coagulation was not observed during the later clot formation process. In our investigation, this effect was seen for G33% and RL33%, but not for HES, as other authors have also found with the TEG (21,22). This phenomenon that has been described for crystalloids as an early hypercoagulability effect seems to be nonspecific, due to modest hemodilution rather than a specific effect of hemodilution with saline (21). A recently published study found an enhancement of in vivo thrombin formation due to the procoagulant effect of decreased plasma concentrations of antithrombin III after the administration of 1000 mL of 0.9% saline or HES200/0.5. In contrast to saline, HES200 did not show this phenomenon (23). No procoagulant effect of the HES solution was seen, probably due to an antiplatelet effect.

SCT and TEG measure dynamic viscoelastic properties of clot formation and provide information on plasmatic, as well as cellular, coagulation. TEG relies on the production of a fibrin connection with the cup and torsion swing, whereas SCT responds as soon as fibrin formation starts (5,24). SCT is a fast, easy to use test that can be performed at the bedside. In contrast to the TEG, no special platform for the absorption of shock waves is necessary, and the machine can be easily transferred to the patient if necessary. The clot rate, which reflects the influences on coagulation by hemodilution, is available within 10–15 min. In contrast, TEG is more time-consuming and requires more logistics.

This study has some limitations: in vitro assays miss physiological reactions during progressive hemodilution, such as recruitment of additional resources of the coagulation system. Another disadvantage is the change of calcium concentration, as well as pH, by dilution with the tested solutions. However, surgical stress, tissue damage, and/or endothelial injury and their effects on coagulation are eliminated in an in vitro model (22). Nevertheless, moderate hemodilution in previously healthy volunteers in terms of influence on blood coagulation seems to be safe (25), especially when G or medium molecular weight HES is used (26).

In conclusion, different effects on coagulation variables between the tested solutions were seen with the SCT analysis. At the same degree of hemodilution, HES showed a significant impact on markers of coagulation as measured by the SCT. Clot rate was the earliest variable that showed significant differences on coagulation variables after progressive in vitro hemodilution.


   Footnotes
 
Presented at the annual meeting of the American Society of Anesthesiologists, Orlando, FL, October 17–21, 1998.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication November 25, 1998.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press