1 Clinic for Anaesthesia and Intensive Care Medicine, 2 Division of Theoretical Surgery and 3 Department of Pediatrics, Innsbruck Medical University, Innsbruck, Austria
* Corresponding author: Clinic for Anaesthesia and Intensive Care Medicine, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail: markus.mittermayr{at}uibk.ac.at
Accepted for publication June 16, 2005.
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Abstract |
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Methods. Undiluted and diluted blood samples from 26 healthy volunteers were spiked with increasing concentrations of heparin (0.1, 0.2, 0.4, 0.8 and 1 U ml1). In addition, undiluted blood was spiked with protamine hydrochloride (0.1, 0.2, 0.4, 0.8 and 1.6 U ml1), and we tested the effect of protamine on the reversal of heparin 0.4 U ml1. Heparin-containing samples were analysed using the heparin-sensitive INTEM test and the heparinase-containing HEPTEM test; protamine series were also analysed with the EXTEM test (tissue factor activation).
Results. CT by the INTEM test [CT-INTEM; median (min/max)] increased significantly and dose-dependently with increasing concentrations of heparin [control, 175 s (146/226); heparin, 1.0 U ml1 1320 s (559/2100); P<0.001] and protamine [control, 172 s (150/255); protamine, 1.6 U ml1 527 s (300/1345); P<0.0001]. Up to heparin concentrations of 0.4 U ml1, results were similar in undiluted and diluted blood samples. As expected, CT-HEPTEM remained within the normal range for all tested heparin concentrations (median 180183 s), but increased similarly to CT-INTEM for increasing protamine concentrations.
Conclusion. CT measurement using the Rotem® technique appears to be a valuable tool for heparinprotamine management. For detection of heparin alone, protamine alone and the two combined, the ratio of CT-INTEM:CT-HEPTEM can be used to distinguish the effects of heparin excess (CT-INTEM:CT-HEPTEM>1) from those of protamine excess (CT-INTEM:CT-HEPTEM=1).
Keywords: blood, anticoagulants, heparin ; measurement techniques, thrombelastography, Rotem® ; protamine
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Introduction |
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Coagulopathy following CPB originates from haemodilution, coagulation factor depletion, platelet dysfunction, and activation of the fibrinolytic system. In addition, residual heparin and protamine excess impair the coagulation process and platelet function. The high heparin concentrations needed during CPB are commonly monitored by measuring activated clotting time (ACT). However, Murray and colleagues2 showed that ACT is not very sensitive to the low heparin concentrations that might be present after protamine reversal. Moreover, a fixed dose of protamine is usually calculated from the total heparin dose. This is followed by ACT measurement to confirm adequacy of protamine administration. Thus, protamine is frequently administered in repeated doses, although elevated ACT after protamine can be caused by residual heparin, protamine excess or other factors influencing coagulation.3
Thrombelastographic techniques can be used to determine the dynamics and quality of clot formation in whole blood, and to detect coagulation inhibitors such as heparin. Intrinsic activation plus heparinase neutralization (HEPTEM test) can be used to identify the presence of heparin and its effects on coagulation by comparison with the intrinsically activated measurement containing no heparinase (INTEM test).4 5
In patients after CBP, use of the conventional Thrombelastograph® (TEG®) and a modification of it (Rotem®) have been shown to decrease transfusion requirement and facilitate the differentiation of coagulopathic from surgical bleeding.6 7
However, in vitro studies using non-activated TEG® and human blood spiked with heparin showed that concentrations as low as 0.2 U ml1 virtually prevented clot formation, indicating that higher concentrations of heparin may not be differentiated from lower concentrations of heparin.8 In addition, few data are available on signs of protamine excess in TEG® analysis. The goal of our study was to clarify whether in vitro heparin concentrations greater than 0.2 U ml1 can be measured and differentiated using the Rotem®-derived measurement of coagulation time (CT). In addition, we wanted to determine whether protamine effects are also detectable, and whether they can be differentiated from heparin effects. Since haemodilution, frequently present in surgical patients, might influence the accuracy of heparin detection, measurements were performed in undiluted and diluted blood spiked with heparin.
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Methods |
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Blood was drawn without stasis from an antecubital vein through a 19 G butterfly needle and collected in four 10 ml citrate-containing tubes (S-Monovette; Sarstedt, Nümbrecht, Germany) for assessment in four thrombelastograph analysis series. In addition, we performed standard coagulation tests and blood cell counts (prothrombin time; partial thromboplastin time, aPTT; concentrations of antithrombin, fibrinogen; platelet count, and red blood cell count).
Test samples were analysed using modified thrombelastography (Rotem®; Pentapharm, Munich, Germany), which is based on the TEG® system, after Hartert, reviewed by Mallet and Cox.9 Technical details of the Rotem® analyser have been described elsewhere.10 Activated tests accelerate the measurement process and enhance reproducibility compared with conventional TEG® analysis of native blood.11 In addition, activation of test samples also decreases the high sensitivity of native TEG® against inhibitors of coagulation, such as heparin. Thus, the probability of discriminating low and moderate heparin concentrations might be improved with the Rotem® technique. A typical Rotem® tracing and its interpretation is given in Figure 1.
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To investigate the effects of heparin, protamine, both combined and the effects of heparin in 40% diluted blood with modified 3% gelatin solution (Gelofusin®; Braun, Melsungen, Germany) four test series were performed for each volunteer. Gelatin was used since colloids are known to exert a greater effect on coagulation than do crystalloids and gelatin solution is routinely used in our cardiac patients. Spiked and control aliquots were stored at room temperature for 20 min without agitation and subsequently analysed. To avoid the bias of time effects three Rotem® devices were used, each allowing assessment of four measurements and thus parallel assessment of all samples from one series. The Rotem® devices used were tested daily for proper function using quality control serum (Pentapharm). To avoid channel bias, the sequence of channels was rotated after each complete measurement series. In addition, the time sequence of measurement series (heparin, protamine, heparin plus protamine, heparin in diluted blood samples) was varied.
All measurements were run for at least 45 min. According to previously published thrombelastographic results, it is possible to have undetectable clot formation with increasing heparin concentrations. Since minimal clot formation after 35 min is of no clinical relevance, we decided to discontinue measurement when initiation of coagulation, CT time (normal range 100240 s) was prolonged to about 10-fold the upper normal value (more than 35 min), and the observation was assigned a CT of 2100 s, an angle of 0° and a maximum clot firmness (MCF) of 0 mm.
The five heparin and protamine dilutions tested were prepared fresh daily in sterile containers. The heparin solutions were prepared using 0.9% sodium chloride from unfractionated porcine heparin (1000 IU ml1; Ebewe Pharma, Unterach, Austria) and the protamine dilution from Protamin ICN® (protamine hydrochloride 1000 U ml1; ICN Pharmaceuticals, Frankfurt, Germany). According to the manufacturer's instructions, 1 IU protamine hydrochloride antagonizes 1 IU heparin (1 IU protamine hydrochloride is comparable to 0.01 mg protamine sulphate).
Dilutions were calculated to achieve the final concentrations in the various 1 ml aliquots of citrated blood by adding 20 µl of heparin or protamine dilution. For the series measuring combined heparin and protamine, one control aliquot of 1 ml citrated blood was left untreated; heparin was then added to the remaining 9 ml citrated blood at a final concentration of 0.4 U ml1 and 1 ml aliquots were subsequently spiked with protamine.
In addition to control measurements, the following final concentrations of heparin, protamine or both were investigated using the Rotem® analyser: (i) heparin series (heparin 0.1, 0.2, 0.4, 0.8 and 1 U ml1); (ii) heparin in 40% diluted blood (heparin 0.1, 0.2, 0.4, 0.8 and 1 U ml1); and (iii) protamine series (protamine 0.1, 0.2, 0.4, 0.8 and 1.6 U ml1); (iv) combined heparin 0.4 U ml1 and protamine series (protamine 0, 0.2, 0.4, 0.8 and 1.6 U ml1).
Statistical methods
A non-parametric Friedman analysis of variance was used for the analysis of the influences of heparin and protamine on thrombelastographic variables. For comparison of Rotem® results with baseline measurements and between the different investigated concentrations of heparin and protamine, the Wilcoxon test was used. All statistical tests were two-sided, and P<0.05 was considered significant.
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Results |
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Heparin series
Compared with control samples, addition of heparin (0.11.0 U ml1) resulted in a significant dose-dependent increase in CT-INTEM above the upper normal range (P<0.001), and all CT values differed significantly from each other (Fig. 2A).
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Up to a heparin concentration of 0.4 U ml1 a clot was detectable in all samples analysed using the INTEM test. At heparin 0.8 U ml1 in 2/26 samples and at heparin 1.0 U ml1 in 8/26 samples no clot was detectable (CT2100 s).
Similar results for CT-INTEM and CT-HEPTEM were observed when heparin concentrations were measured in 40% diluted blood using a modified gelatin solution (Fig. 2B). Compared with undiluted blood, CT-INTEM and CT-HEPTEM results were comparable up to heparin concentrations of 0.4 U ml1. At heparin concentrations of 0.8 and 1.0 U ml1, no clot was detected more frequently in diluted samples (12/26 and 18/26 measurements respectively) than in undiluted samples.
In order to test the ability of Rotem® to detect even small concentrations of heparin, we compared the CT-INTEM and HEPTEM values of control samples with those spiked with 0.1 U ml1 heparin. Assuming that CT-INTEM is at least 10% higher than CT-HEPTEM when heparin is present, the CT-INTEM:CT-HEPTEM ratio was seen to increase in all samples in the heparin series, meaning that 26 of 26 heparin-containing samples (heparin 0.1 U ml1) were correctly identified as containing heparin, while 25 of 26 control samples were correctly identified as containing no heparin and one control sample showed a false-positive result of containing heparin.
Protamine series
Compared with control samples, addition of protamine (0.11.6 U ml1) produced a significant dose-dependent increase in CT-INTEM (P<0.05), while all CT values differed significantly from each other (Fig. 2C). CT-EXTEM increased inconsistently from 56 s (86/45) in control samples to 64 s (88/16), 67 s (109/49), 64 s (98/48), 68 s (110/39) and 82 s (118/58) at protamine 0.1, 0.2, 0.4, 0.8 and 1.6 U ml1 respectively. In contrast to the results for the heparin series, CT-HEPTEM values also increased with increasing protamine concentrations, because heparinase cannot neutralize protamine effects (Fig. 2C).
Heparinprotamine series
As described in Methods, our experiments used protamine hydrochloride; 1 U protamine hydrochloride antagonizes 1 U heparin. As long as heparin concentrations were greater than protamine concentrations (protamine 0 and 0.2 U ml1), CT-INTEM values were higher than CT-HEPTEM values, while protamine excess caused a similar increase in CT-INTEM and CT-HEPTEM (Fig. 2D).
Other Rotem® variables
Increasing concentrations of heparin resulted in a significant dose-dependent decrease in MCF in the INTEM test (P<0.001), mostly to values outside the normal range, while variable changes in MCF were observed in the HEPTEM test and in the INTEM test and EXTEM test in the protamine series. These changes were not dose-related and remained mainly within the normal range. In diluted blood samples, MCF (INTEM and HEPTEM) was already below the low normal range at baseline and changed inconsistently with increasing heparin concentrations (Table 1).
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Discussion |
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Nielsen13 also demonstrated the ability of native non-activated TEG® in detecting heparin in blood samples from rabbits receiving heparin stepwise from 10 U kg1 up to a cumulative dose of 30 U kg1. In this experiment, TEG® variables were more sensitive to changes in heparin activity than were aPTT and ACT values. However, the data show that the ability of TEG® to discern heparin increases of more than 20 U kg1 is poor, because no discernible clot can be observed.
In the study by Zmuda and colleagues,8 the in vitro addition of heparin 0.2 U ml1 already abolished clot formation, as measured by non-activated TEG®. In contrast, in our study at heparin concentrations of 0.8 U ml1 no clot formation occurred in only 2/26 samples, whereas this was true for 8/26 samples at the heparin concentration of 1 U ml1. However, it has been shown that thrombelastographic results derived from activated tests demonstrate less variability than those obtained from non-activated native blood samples.9 14 In addition, in non-activated TEG®, thrombin formation is triggered by the relatively weak contact activation occurring between blood and the plastic surfaces of the pin and cup of the TEG®/Rotem® analyser. In activated Rotem® tests, coagulation is triggered by ellagic acid, an activator of the contact pathway (activation by the factors XII, XI, IX, VIII, X and V). This principle also applies to other activators, such as kaolin and Celite. However, these substances have a much greater tendency to sedimentation and were therefore replaced with ellagic acid in Rotem® analysis (communication with Pentapharm). Due to the stronger activation, initiation of coagulation is less sensitive to the presence of inhibitors in activated TEG® compared with non-activated TEG®, a fact already described by Caprini and colleagues in 1976.15
Protamine binds ionically to the numerous negative charges of heparin. In addition, protamine causes anticoagulant effects that prolong ACT, meaning that ACT measurements can be misleading when deciding whether additional protamine should be used for complete heparin reversal. It is not clear whether this anticoagulant effect of protamine is clinically important and what concentrations of protamine are critical for inhibiting coagulation. However, several studies have confirmed the impairment of platelet function in the presence of heparin/protamine complexes or protamine alone.1619 Griffin and colleagues12 also showed that the platelet inhibitory effects of protamine are more pronounced under conditions of high shear stress, and they also observed a prolonged prothrombin time and aPTT with increasing protamine concentrations. The authors showed that the therapeutic window for protamine is narrower than previously documented, since protamine excess at heparin:protamine ratios greater than 1:1.5 already contributed directly to platelet dysfunction. In our experiments we also observed that protamine excess prolonged CT values and decreased MCF in the INTEM test, which shows good correlation with aPTT measurements20 but changed inconsistently in the tissue-factor-activated EXTEM test.
We can only speculate that our observation might be the result of an unspecific, charge-dependent impairment of the amplification step of coagulation by protamine, interfering with activation of factors FVIII and FIX on the surface of activated platelets, which is of minor importance in tests using TF for activation.
To our best knowledge, only one study using TEG® to monitor the effects of heparin also investigated the effects of protamine by analysing recalcified blood samples without activation.8 In this study, protamine at 8.3 µg ml1 showed a similar, albeit incomplete, reversal of heparin-induced prolongation (heparin 0.2 or 0.5 U ml1) of reaction time (with regard to CT) as did the threefold concentration of 25 µg ml1. However, the protamine concentrations tested in this study were both well above a heparin:protamine ratio of 1.0, meaning that in this study it was mainly the effects of protamine excess that were measured. The present study shows that CT-INTEM increases with heparin and protamine. By calculating the CT-INTEM:CT-HEPTEM ratio, which increases with heparin excess but remains at 1 with protamine excess, we can distinguish impairment of coagulation caused by heparin or protamine. CT measurements depend also on concentrations of coagulation factors that might be critically reduced in a surgical patient. Therefore, it seems necessary to measure CT-HEPTEM before and about 15 min after protamine administration. If CT-HEPTEM is already increased before protamine administration, the influence of coagulation factor depletion on CT measurements needs to be considered. If CT-HEPTEM is prolonged only after protamine administration, the result of CT-HEPTEM and the relation to CT-INTEM reflects the effect of excess protamine, because it is unlikely that a patient exhibits coagulation factor depletion during this short period, except in the rare cases of excessive blood loss.
The limitation on our study is the fact that we investigated the effects of protamine and heparin on Rotem® variables only in vitro. However, it is unlikely that the unspecific binding of heparin to proteins, endothelial cells and other cells or the in vivo pharmacokinetics of heparin and protamine alter the accuracy of this measurement technique in detecting heparin and protamine.
In conclusion, we demonstrate here that Rotem® analysis can detect heparin with high sensitivity, regardless of whether blood is diluted, and can detect it up to a concentration of 1.0 U ml1. We show for the first time that the effect of even low concentrations of excess protamine on coagulation can be measured with this technique. Further, the effects of heparin and protamine on coagulation can be clearly distinguished from each other.
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References |
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