Schlimp, Christoph J. MD*; Solomon, Cristina MD*†; Hochleitner, Gerald†; Zipperle, Johannes MSc*; Redl, Heinz PhD*; Schöchl, Herbert MD*‡
Coagulation monitoring using viscoelastic tests (thromboelastometry [ROTEM®, Tem Innovations, Munich, Germany] or thrombelastography [TEG®, Haemonetics Corp., Braintree, MA]) is increasingly used for rapid detection of coagulopathy and goal-directed hemostatic intervention.1,2 Rapid availability of results means that treatment can be tailored to the patient’s specific needs, and that the effect of treatment can be measured quickly after administration. This constitutes a “theragnostic” approach to managing the patient.3 One of the most important decision-making variables is fibrin polymerization,4 usually assessed by clot amplitude in the FIBTEM assay of ROTEM or the Functional Fibrinogen assay of TEG® (FFTEG). For both of these assays, coagulation is activated extrinsically, and platelet function is inhibited. However, interpretation of published clot amplitude values, such as the amplitude after 10 minutes of clot formation (A10), the maximum clot firmness (MCF), or the maximum amplitude, is complicated by the use of different functional fibrinogen polymerization assays (FFPAs),5 and the use of whole blood or plasma (platelet-rich, platelet-poor, or platelet-free [PFP]).6–9
Currently, there are no reports on whether the components of FFPAs interfere with fibrin polymerization or alter clot amplitude values, aside from the effects of platelet inhibition. In PFP, it should be possible to investigate the exclusive effect of FFPAs on fibrin polymerization.
Using various PFP preparations, we planned to use a ROTEM device to compare the extrinsically activated thromboelastometric assay EXTEM with 3 manufactured FFPAs: the commercially available FIBTEM assay (TEM Innovations, Munich, Germany); the experimental FIBTEM PLUS assay (TEM Innovations); and the Functional Fibrinogen Test® (FFTEG; Haemonetics Corp., Braintree, MA). According to TEM, the FIBTEM PLUS assay allows reliable measurement of the fibrin contribution to clot firmness even in the presence of 1,500,000 platelets/µL (personal communication, TEM Innovations, 2012).
The study hypothesis was that different FFPAs do not alter MCF as compared with EXTEM in ROTEM. Based on the initial study results, further experiments with cytochalasin D, abciximab, and dimethyl sulfoxide (DMSO; a solvent of cytochalasin D), as well as a stepwise dilution of the tissue factor (TF)-containing activation reagent, were performed to investigate the influence of assay constituents on the MCF measurements.
The study protocol for testing several FFPAs with blood/plasma samples from healthy volunteers was approved by the AUVA Ethics Committee (EK-AUVA 13/2011) in Vienna, which has jurisdiction over all trauma hospitals/institutions in Austria. Besides industrial plasma preparations, blood from a healthy volunteer (1 of the study authors) was used for preparation of PFP. We chose an experimental model with PFP preparations to exclude any platelet contribution to clot strength, and to be certain that this was the case in assays designed to block platelet activity.
For this study, 4 different preparations of PFP were used:
1. Standard Human Plasma (Siemens Healthcare Diagnostics, Marburg, Germany), which is a citrated mixed human plasma from healthy donors, stabilized with buffer, lyophilized, and prepared for use with H2O.
2. Calibration Plasma (IL Coagulation Systems, Instrumentation Laboratory, Munich, Germany), consisting of lyophilized human plasma, buffer, stabilizers, and preservatives, and prepared for use with H2O.
3. Octaplas® LG (Octapharma, Langenfeld, Germany), consisting of pooled fresh-frozen plasma containing sodium citrate dihydrate, sodium dihydrogen phosphate dihydrate, and glycine, was thawed at 37°C.
4. Fresh human PFP from a healthy volunteer (study author) was prepared in the following way. Using minimal stasis, antecubital venous blood was drawn through a 21-gauge butterfly needle. The first 2 mL were discarded. Blood was collected in 3-mL tubes containing 0.3 mL buffered 3.2% trisodium citrate, giving a blood:trisodium citrate volume ratio of 1:10 (Vacuette; Greiner Bio-One, Linz, Austria). Blood samples were centrifuged at 2200g for 15 minutes to obtain homologous platelet-poor plasma. The upper two-thirds of the plasma supernatant was centrifuged at 2800g for 15 minutes. After this step, the upper two-thirds of this plasma solution was frozen at −80°C. Before testing, the plasma was thawed at 37°C for approximately 30 minutes and then filtered through a 0.22-µm filter to inhibit any residual platelet activity, resulting in PFP suitable for ROTEM analysis.
Each plasma preparation was tested 8 times with 4 different assays using a ROTEM device, so a total of 32 runs were performed on each plasma preparation.
ROTEM Analysis of Plasma with EXTEM Assay
The cup, pin, tips, and reagents were prepared, and the channel was programmed for EXTEM. Using the automated ROTEM pipette, 20 µL of star-tem® reagent and 20 µL of ex-tem® reagent were added to the cup, followed by 300 µL of the tested plasma preparation. The test was initiated automatically via the pipette signal. The reaction mixture (total volume of 340 µL) was aspirated and then dispensed back into the cup, and the cup was set onto the pin. The ex-tem reagent contains recombinant TF and heparin inhibitor (hexadimethrine). The star-tem reagent contains calcium chloride (0.2 mol/L).
ROTEM Analysis of Plasma with FIBTEM and FIBTEM PLUSAssays
The cup, pin, tips, and reagents were prepared, and the channel was programmed for FIBTEM. Using the automated ROTEM pipette, 20 µL of ex-tem reagent and then 20 µL of fib-tem® reagent were added to the cup, followed by 300µL of the tested plasma preparation. The test was initiated automatically via the pipette signal. The reaction mixture (total volume of 340 µL) was aspirated and then dispensed back into the cup, and the cup was set onto the pin. The fib-tem reagent contains cytochalasin D and 0.2 mol/L calcium chloride. The fib-tem plus reagent contains cytochalasin D, the glycoprotein-IIb/IIIa receptor inhibitor tirofiban and 0.2mol/L calcium chloride.
ROTEM Analysis of Plasma with FFTEG Assay
The cup, pin, and tips were prepared, and the channel was programmed for NATEM. Citrated plasma (500 µL) was pipetted into the FFTEG vial containing lyophilized TF with a platelet inhibitor that binds to glycoprotein-IIb/IIIa receptors; the vial was swirled and inverted 5 times. To keep the plasma:reagent ratios consistent with TEG®, the following pipetting scheme was followed: 19 µL of star-tem and 20-µL physiologic saline were pipetted into the cup. Next, 301 µL of the mixture was pipetted from the vial into the cup. The test was initiated automatically via the pipette signal. The reaction mixture (total volume of 340 µL) was aspirated and then dispensed back into the cup, and the cup was set onto the pin.
For this study, further comparisons with EXTEM were performed using different batches of FIBTEM and FIBTEM PLUS from those used in Study 1, and other pipetted assays containing: abciximab (Assay 1); cytochalasin D (Assay 2); a combination of cytochalasin D and abciximab (Assay 3); or DMSO (the solvent of cytochalasin D; Assay 4). The aim of these experiments was to investigate the influence of assay constituents on the results of Study 1.
Preparation of Assays
Assay 1 (20 µL): Containing 5 µL (10 µg) abciximab (ReoPro®, Centocor B.V., Leiden, the Netherlands) + 15 µL of H2O.
Assay 2 (20 µL): Stock solution of 5 mg/mL cytochalasin D (Sigma Aldrich, Vienna, Austria), already dissolved in DMSO, was diluted 1:100 with H2O. The resulting cytochalasin D concentration of 50 µg/mL was chosen based on concentration experiments showing inhibition of the platelet contribution to clot elasticity with the fib-tem reagent and cytochalasin D (Fig. 1).
Assay 3 (25 µL): Containing 5 µL (10 µg) of abciximab + 20 µL of Assay 2 solution (cytochalasin D 50 µg/mL).
Assay 4 (20 µL): DMSO (Fluka, Sigma Aldrich, Buchs, Switzerland) was diluted 1:100 with H2O to achieve the same concentration as used in Assay 2.
For Study 2, fresh human PFP was tested 8 times with each assay; no other PFP preparations were investigated. Fresh human PFP was prepared in the same manner as described earlier for Study 1. For each set of experiments, a new PFP sample was prepared.
Experiment 1 aimed to reproduce the results of Study 1 by using what we assumed to be the same pharmacodynamic constituents: EXTEM, abciximab (Assay 1), cytochalasin D (Assay 2), and the combination of cytochalasin D and abciximab (Assay 3). The assays were run in parallel.
Experiment 2 aimed to reassess the differences between EXTEM, FIBTEM, FIBTEM PLUS, and cytochalasin D (Assay 2) by performing multiple parallel runs. Experiment 3 aimed to evaluate whether DMSO (Assay 4) exerts an influence on EXTEM or cytochalasin D (Assay 2) by performing multiple parallel runs.
ROTEM analysis of Study 2 was performed using the same principles as in Study 1. However, for all other assays besides EXTEM the channel was programmed for FIBTEM. Approximately 1 minute before the start of the procedure, 20 or 25 µL of assay solution or 20 µL of H2O (the latter in case of EXTEM to achieve the same volume of reaction mixture) was dispensed into the cup. Using the automated ROTEM pipette, 20 µL of star-tem and 20 µL of ex-tem were added to the cup, followed by 300 µL of PFP. Unlike in Study 1, the reaction mixture had a total volume of 360 or 365 µL.
Freshly prepared PFP was tested using ROTEM assays with the following constituents: 20 µL of the recalcifying reagent star-tem plus another 20 µL of either a stepwise dilution of the ex-tem reagent (1:1, 1:2, 1:4, 1:8, 1:20, 1:40, 1:100) or H2O (resulting in a nonactivated NATEM). ROTEM analysis was run on the same principles as in Studies 1 and 2, with a total reaction mixture volume of 340 µL. Also as in the previous studies, every test was performed 8 times.
Factor XIII deficient/inactivated plasma (Technoclone, Vienna, Austria) was tested using ROTEM, assays with the following constituents: 20 µL of the recalcifying reagent star-tem plus 20 µL of ex-tem reagent, fib-tem reagent, or fib-tem plus reagent. ROTEM analysis was performed using the same principles as in Studies 1 to 3, with a total reaction mixture volume of 340 µL. Every test was performed 6 times.
Data Collection and Statistics
ROTEM data were collected over a 30-minute period and analyzed for MCF (the main study parameter) and clot amplitude at 5, 10, 15, 20, 25, and 30 minutes. Clotting time (CT), which may be influenced by the TF concentration of the assay, was also recorded.
Based on preliminary experiments, 8 measurements for each FFPA were calculated as being sufficient to detect a 2-mm difference in mean MCF (±1 mm SD), providing a power of 80% and a 2-sided α of 5%. A Kruskal–Wallis 1-way analysis of variance was used to test for differences among all groups. The Kolmogorov–Smirnov test was used to ascertain whether the data were normally distributed and, based on this evaluation, the Mann–Whitney U test was used to compare the groups in this study. A 2-tailed P-value of <0.05 was considered significant. Data are expressed as medians with interquartile ranges. Calculations were performed using commercially available statistical software (GraphPad Prism 5, GraphPad Software, La Jolla, CA).
Median MCF values for each PFP preparation are shown in Table 1. Median values (interquartile ranges) for all plasma preparations tested were: 20.5 mm (17.25–22.0 mm) in EXTEM, 23.0 mm (18.5–24.0 mm) in FIBTEM, 23.0 mm (18.25–24.75 mm) in FIBTEM PLUS, and 18.0 mm (16.0–19.0 mm) in FFTEG. Kruskal–Wallis 1-way analysis of variance was significant for all groups. FIBTEM and FIBTEM PLUS gave significantly increased MCF compared with EXTEM (P< 0.01). FFTEG gave significantly decreased MCF compared with EXTEM (P < 0.001; Fig. 2).
Analysis of clot amplitudes at different time points revealed that the significant differences in MCF between EXTEM and FIBTEM/FIBTEM PLUS or FFTEG were already evident after 5 minutes of clot formation (A5, data not shown). Analysis of CT revealed that clotting takes a significantly longer time to begin in FFTEG (Fig. 3).
Mean MCF values of Assays 1 to 3 were not significantly different from EXTEM, showing that the platelet-inhibiting agents within the commercial assays are not responsible for the differences observed in Study 1 (Fig. 4).
Mean MCF values of FIBTEM and FIBTEM PLUS assays (different batches from Study 1) were not significantly different from each other, but they were significantly different from EXTEM (P < 0.05 and P < 0.01, respectively) and Assay 2 (P < 0.05 and P < 0.01, respectively; Fig. 5).
Mean MCF values of FIBTEM were significantly increased compared with EXTEM (P < 0.001), Assay 2 (P < 0.001), and Assay 4 (P < 0.05). Assay 4 was significantly higher than EXTEM (P < 0.05) but not Assay 2 (Fig. 6).
A reduction of the extrinsic activation in EXTEM through stepwise dilution of the ex-tem reagent revealed a highly significant prolongation of CT as compared with the undiluted ex-tem reagent (P < 0.001; Table 2). A significant reduction of MCF was found at and below a 1:8 dilution of the ex-tem reagent (Table 2).
Assessments of Factor XIII-deficient plasma revealed a median FIBTEM MCF of 12 mm (12–12 mm) and a median FIBTEM PLUS MCF also of 12 mm (11–12 mm). The median EXTEM MCF was 11 mm (10.75–11.00 mm), significantly lower than the result with either of the other 2 assays (P = 0.03 for FIBTEM; P = 0.04 for FIBTEM PLUS).
This is the first comparison of the FFTEG, FIBTEM, FIBTEM PLUS, and EXTEM viscoelastic assays in different PFP preparations. All FFPAs showed significantly different MCF values compared with EXTEM. In further experiments, we showed that the increase of MCF in FIBTEM and FIBTEM PLUS is not due to the presence of cytochalasin D, and that the reduction of MCF in FFTEG is not caused by the glycoprotein-IIb/IIIa inhibitor abciximab. We also found that activating PFP with decreasing concentrations of ex-tem reagent resulted in a significant reduction of MCF.
Our experimental model revealed unexpected and significant differences in viscoelastic clot strength that would not have been detected in whole blood or plasma with residual platelet activity. The results are surprising because they indicate that FFPAs either interfere with fibrin polymerization or exert different kinds of extrinsic activation. The study hypothesis that different FFPAs do not alter MCF as compared with the EXTEM assay in ROTEM was rejected. Based on this initial study result, further experiments with cytochalasin D, abciximab, and DMSO (a solvent of cytochalasin D) were performed. DMSO exerted only minor effects on MCF; therefore, we speculate that other unknown industrial stabilizing solvents in the FIBTEM assays are responsible for the increased clot firmness. Based on the experiments with Factor XIII-deficient plasma, we can also assume that fibrinogen is primarily involved, and not Factor XIII-dependent activation/cross-linking. Because FFPA components (e.g., TF, phospholipids [PLs]) have specific activities, manufacturers usually adjust the quantities to achieve desired outcomes. It is generally understood that activator levels affect the speed of clot initiation/propagation,10,11 and this study provides data to support that relationship. The present results also suggest that differences between assays in their concentrations of TF and PL may affect observed values for MCF/maximum amplitude. The basis for this inference is as follows: differences between assays despite the absence of platelets; relationship between TF concentration and MCF; lack of impact of cytochalasin D or abciximab on MCF. Lack of impact of cytochalasin D and abciximab on maximum clot strength has been shown previously.6,12 However, previous studies have indicated that neither TF nor PL affect MCF in viscoelastic coagulation assays performed in the absence of platelets.10,11 Further studies would be needed to shed light on this apparent contradiction (i.e., to understand precisely which assay components are responsible for the differences in MCF and the mechanism[s] of action). It is interesting that MCF with the FFTEG assay was lower than with FIBTEM in the present study, because in the presence of platelets the difference between these assays was reversed (higher MCF with FFTEG).5 This indicates that the difference between the assays relating to platelet inhibition is larger than it first appears. A further consideration is the use of ROTEM versus TEG®. The TEG® device has been shown to produce higher MCF values than ROTEM,5 indicating that the difference between FIBTEM and FFTEG in PFP is larger than the data from this study suggest.
What are the implications of the present results for clinical practice? Clot amplitude values from FFPAs may be used to influence coagulation management and to guide goal-directed hemostatic therapy; for example, by distinguishing between platelet deficiency and fibrinogen deficiency.1,2,4 Differences between FFPAs in clot amplitude values could affect therapeutic decisions in trauma-induced coagulopathy3,13 and cardiac surgery,14–16 where MCF is used to determine the need for fibrinogen supplementation. As shown in a recent study, a difference of 2 mm in FIBTEM MCF corresponds to a change in plasma fibrinogen concentration of approximately 0.2 g/L.15 In a bleeding patient, such a difference could have a significant effect on clinical decisions regarding fibrinogen supplementation. Dosing decisions may also be affected. Based on calculations of previous cardiac surgery studies, a difference of 2 mm in FIBTEM MCF could correspond to a difference in fibrinogen dose of 1 g in an 80-kg patient.14,16,17 Future experimental studies are needed to confirm whether the 2-mm difference we observed between FIBTEM and EXTEM with PFP also occurs with whole blood. However, the platelet-free whole blood needed for such investigations is not easy to obtain. In addition, the presence of additional influencing factors such as red blood cells would complicate the analysis. For instance, hematocrit was recently shown to influence MCF in ROTEM, a higher hematocrit dilutes the remaining absolute quantity of fibrinogen.18,19 FIBTEM MCF incorporates the effect of hematocrit on whole blood clot firmness whereas this is not reflected by plasma fibrinogen concentration.20 Despite the questions raised by our study, FFPAs remain essential tools for rapid, point-of-care evaluation of patients’ fibrin-based clotting and, therefore, their need for fibrinogen supplementation.
Our study has some limitations. Investigations using plasma generate information valuable for quality control of viscoelastic assays, but they do not reflect point-of-care theragnostic concepts with whole blood. Further studies are needed in this regard. Standardized comparison of extrinsically activated clotting with and without platelet inhibition is possible with the ROTEM device, by comparing EXTEM with FIBTEM (both assays are activated in the same way). Unfortunately, with TEG® there is no equivalent to the EXTEM assay, meaning that standardized comparison of FFTEG is not possible. Our only option was therefore to compare FFTEG with ROTEM assays, and this is another limitation of the study.
In conclusion, in several human PFP preparations (either fresh, pooled, or industrially processed), viscoelastic functional fibrinogen assays gave significantly different MCF values when compared with the extrinsically activated EXTEM assay. We speculate that unknown industrial stabilizing solvents are responsible for the enhanced clot firmness in the cytochalasin D-based ROTEM assays. The platelet inhibitors present in the FFPAs we investigated (i.e., cytochalasin D and abciximab) did not alter clot firmness in PFP. The lower MCF observed with the FFTEG could potentially be an effect of a lower concentration of TF in this assay. Reference ranges and cutoffs determined with one reagent cannot be applied to another reagent. The current experimental study would need to be validated in large patient samples to provide the clinical relevance of this finding. Nevertheless, FFPAs remain invaluable for rapid determination of the need for fibrinogen supplementation, and the use of FIBTEM cutoff values and dosing formulae for guiding hemostatic therapy should be continued.
Name: Christoph J. Schlimp, MD.
Contribution: Christoph Schlimp helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Christoph Schlimp has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Conflicts of Interest: The author has no conflicts of interest to declare.
Name: Cristina Solomon, MD.
Contribution: Cristina Solomon helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Cristina Solomon has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Conflicts of Interest: Cristina Solomon is an employee of CSL Behring, has acted as a consultant for CSL Behring, and received speaker honoraria and research support from Tem International and CSL Behring and travel support from Haemoscope Ltd (former manufacturer of TEG®).
Name: Gerald Hochleitner.
Contribution: Gerald Hochleitner helped design the study, analyze the data, and write the manuscript.
Attestation: Gerald Hochleitner approved the final manuscript.
Conflicts of Interest: Gerald Hochleitner is an employee of CSL Behring.
Name: Johannes Zipperle, MSc.
Contribution: Johannes Zipperle helped conduct the study and analyze the data.
Attestation: Johannes Zipperle approved the final manuscript.
Conflicts of Interest: The author has no conflicts of interest to declare.
Name: Heinz Redl, PhD.
Contribution: Heinz Redl helped design the study and analyze the data.
Attestation: Heinz Redl approved the final manuscript.
Conflicts of Interest: The author has no conflicts of interest to declare.
Name: Herbert Schöchl, MD.
Contribution: Herbert Schöchl helped design the study, analyze the data, and write the manuscript.
Attestation: Herbert Schöchl approved the final manuscript.
Conflicts of Interest: Herbert Schöchl has received speaker honoraria and research support from CSL Behring and Tem International.
This manuscript was handled by: Jerrold H. Levy, MD, FAHA.
We thank Anna Khadem from the Ludwig Boltzmann Institute for Experimental and Clinical Traumatology and AUVA Research Centre for her excellent technical and laboratory support. The FIBTEM and FIBTEM PLUS assays were provided by TEM Innovations GmbH. Final-stage editorial support was provided by Meridian HealthComms, with funding from CSL Behring. Neither TEM Innovations nor CSL Behring had any influence on study procedures or manuscript preparation. This was also true for other companies with which the authors had financial involvement.
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