Download Recommendations for Thromboelastography

Please review the draft manuscript and send comments to Meera Chitlur ([email protected]) with a
copy to Subcommittee Chairman on Factor VIII and IX, Flora Peyvandi ([email protected]).
Recommendations for Thromboelastography/Thromboelastometry in Patients with
Bleeding disorders
Meera Chitlur1, Benny Sorensen2, Georges E Rivard3, David Lillicrap4, Ken Mann5,
Midori Shima6, Guy Young7
1 Children’s Hospital of Michigan, Detroit, MI, USA; 2 Guy's & St Thomas' NHS Foundation Trust,
London, UK; 3 CHU Sainte-Justine, Montréal, QC, Canada, 4 Queens University, Kingston, ON, Canada;
5 University of Vermont, Colchester, VT, USA; 6 Nara Medical University, Kashihara city, Nara, Japan; 7
Children’s Hospital Los Angeles, Los Angeles, CA, USA.
Introduction:
Hemostasis is maintained through complex interactions between coagulant and anti-coagulant proteins,
cellular components and endothelium. Currently assessment of hemostasis involves plasma-based
assays of clotting proteins and studying platelets. There is now a growing interest in the use of so-called
global hemostasis assays like thrombelastography and thromboelastometry which measure the
viscoelastic changes occurring during clot formation, for the study of coagulation disorders. These assays
are performed using whole blood and provide the opportunity to assess the kinetics of clot formation in
the presence of the plasma and cellular blood components.
The ISTH-SSC WP on standardization of thromboelastography was established to determine a standard
methodology for performing thromboelastography in patients with hemophilia both at baseline and
following treatment with prohemostatic medications. In this manuscript the WP has put forth
recommendations for performing thromboelastography/ thromboelastometry in patients with hemophilia
and other coagulation factor deficiencies with the exclusion of von Willebrand factor deficiency, as the test
may require modification to increase its sensitivity for von Willebrand disease.
Differences between Thrombelastography/Thromboelastometry:
The two instruments currently available that utilize the principles of thromboelastography are the
TEG®5000 (Haemonetics Corp., Braintree, MA, USA) and the ROTEM® delta (Tem International GmbH,
Munich, Germany). The resultant analysis from the TEG®5000(TEG) is referred to as
Thrombelastography while that from the ROTEM® delta (ROTEM) is called Thromboelastometry. The
thromboelastograph consists of a heated cylindrical sample cup into which is suspended a pin. In the
TEG, the cup oscillates at ±4°45’ every 5 seconds and the pin is suspended freely into the cup by a
torsion wire. In the ROTEM, the cup is stationary while the pin transduction system oscillates at ±4°45’
every 6 seconds. With the initiation of coagulation, the forming clot results in a physical connection
(presumably by strands of fibrin) between the cup and the pin, transferring the torque of the cup to the
pin. The rate of clot formation and its elastic strength affect the magnitude of motion of the pin its range of
oscillation. In the TEG, a mechanical-electrical transducer is used to convert the rotation of the pin to an
electrical signal which is then recorded by a computer. In the ROTEM, an optical detection system is
used to generate the electrical signal which is recorded by a computer. With clot lysis, the transfer of
Please review the draft manuscript and send comments to Meera Chitlur ([email protected]) with a
copy to Subcommittee Chairman on Factor VIII and IX, Flora Peyvandi ([email protected]).
motion from the cup to the pin is interrupted. For both instruments, computer software produces both
quantitative parameters (see below) and a graphical representation (figure 1) which allows one to
evaluate the different phases of clotting and deduce the adequacy of the coagulation and fibrinolytic
pathways. The sample volume in the TEG is 360µL and 340 µL for the ROTEM. Coagulation may be
initiated purely by contact activation with the cup (called “native”), or with specific activators can either
target the contact pathway or the extrinsic pathway to reduce the time to clot formation and add a
measure of reliability to the assays. . For the TEG, the manufacturer provides a vial coated with kaolin for
activation of the intrinsic pathway while for the extrinsic pathway, the use of recombinant human tissue
factor (TF, mostly Innovin®, Dade Behring, Miami, FL) has been deployed by investigators in “homemade” reagents. For the ROTEM, the manufacturer provides reagents for both intrinsic activation -INTEM
(partial thromboplastin phospholipid made from rabbit brain), and EXTEM (thromboplastin/recombinant
tissue factor) for extrinsic factor activation. Mixing of the contents in the sample cup is done with the
oscillation of the cup over the pin in the TEG and by re-aspirating into the automated pipette in the
ROTEM.
The parameters measured in both systems are similar but have a slightly different nomenclature. The
measures commonly used in the evaluation of patients with bleeding disorders are:
1.
2.
3.
4.
5.
Coagulation time: R on TEG and CT on ROTEM
Clot Formation time: K on TEG and CFT on ROTEM
Max. Clot Firmness: MA on TEG and MCF on ROTEM
Shear Elastic Modulus Strength: G on both instruments.
α/Angle-Rate of polymerization of clot: α on both instruments.
Pre-analytical Variables affecting thromboelastographic results:
As with any coagulation assay, incorrect blood collection techniques and sampling result in activation of
the coagulation system and therefore erroneous results on thromboelastography. Paying attention to the
following details will minimize these variables:
1. Blood collection tubes: For Kaolin or INTEM, standard (buffered) sodium citrate (3.2%) blood
collection tubes should be used. If EXTEM or TF is considered, it is important that the collection
tube contain corn trypsin inhibitor (CTI) prior to the blood being placed in the tube as studies have
shown that clotting times are much shorter if there is any contact activation. The addition of the
CTI in the collection tube, prior to collection of the blood sample, to achieve a final concentration
of 0.1mg/ml decreases this activation (1). The ideal method would involve testing a native
sample, however this is impractical. Although studies have shown that there is a tendency
towards hypercoaguability with citrated samples due to inadequate inhibition of thrombin
generation (2), most studies with thromboelastography have utilized citrated blood. The addition
of CTI to the tube may therefore be beneficial to overcome this effect as well, thereby allowing for
the use of citrated samples with extrinsic pathway activation. Of note, the currently available CTI
agent is not sterile and therefore collection tubes should not be directly connected to the
individual undergoing phlebotomy.
1. Application of a tourniquet for venous sampling: Venous stasis during blood collection has been
known to result in increased variability in coagulation test results(3). In children it is sometimes
extremely difficult to obtain a peripheral venous blood draw, and while the preferred method
would be to obtain the blood without application of a tourniquet, it may become necessary to
apply a light tourniquet and release this once the vein is accessed.
2. Needle size: A 21G or larger needle is recommended to obtain the specimen for
thromboelastographic testing. Smaller needles have been demonstrated to result in platelet
activation (3).
3. Multiple sampling: Repeated sampling from the same tube has been known to result in activation
of platelets as well as coagulation factors and therefore should be avoided if possible(2).
4. Resting time: The sample may be allowed to sit for 30 minutes but not longer than 2 hours prior to
the running the test. If storage beyond 30 minutes is required, the tube should remain capped to
prevent the escape of CO2 and change in pH, and attempts should be made to keep the sample
at 37°C.
With the above in mind, the WP hereby makes the following recommendations for the use of
thromboelastography in patients with coagulation factor deficiency:
Recommendations for the Kaolin/INTEM Method: Blood should be collected as described above. For the
TEG, 1 mL should be transferred into the kaolin vial and 340μL then transferred into the cup into which
20μL of calcium chloride had been placed. For the ROTEM, the blood is transferred directly into the
device for automated pipetting. Since both reagents are supplied by the manufacturers, their potency is
standardized ensuring reliable results.
Recommendations for the Tissue Factor method: Most of the initial work in patients with hemophilia was
performed using TF as the activator since it was believed that this was a more physiologic representation
of the in vivo coagulation process. These studies have used dilutions of the commercially available TF,
Innovin®. A high TF dilution of 1:17,000 (approximately 0.35pM, final estimated concentration which may
vary slightly depending the patient hematocrit) and low TF dilution (1:42,000 dilution, approximately
0.15pM) have both been studied with some studies suggesting the higher concentration is effective while
others have shown the lower concentration to be more effective(4, 5) This issue remains controversial.
We suggest performing preliminary studies with both dilutions to determine the most appropriate one for
the specific study one is performing. The 1:17,000 dilution can be prepared as follows: 10 µL of Innovin®
is added to 160 µL of saline diluent (final concentration = 1:17). 100 µL of the 1:17 dilution of TF is then
added to 900 µL of diluent (final concentration = 1:170). Finally 990 µL of the 1:170 dilution of TF is added
to 1000 µL of diluent to obtain the desired final concentration of about 1:17,000 (assuming a final
hematocrit in the cup of about 30%). A similar approach can be followed for the 1:42,000 dilution. Once
prepared, 20µL of the dilute TF is placed in the specimen cup, followed by 20 µL of calcium chloride for
recalcification of the citrated blood sample. Since the maximum volume in the specimen cup cannot
exceed 360 µL, 320 µL of blood is added instead of 340 µL as with Kaolin in the TEG. The same
procedure may be applied to both the TEG and the ROTEM with manual pipetting. Otherwise, for the
ROTEM, the EXTEM reagent may be used as the extrinsic pathway activator, but it is important to be
aware that the results may differ from that obtained with the TF dilution described above as the
concentration of the activators are not identical.
Comparison of the TF and Kaolin methods: This remains a much debated and controversial question
since both activators have strengths and weaknesses. The advantage of kaolin or INTEM is that the
method is very simple and the reagents are standardized. The main disadvantage is the non-physiologic
nature of contact system activation. The advantage of TF is the physiologic activation of clotting while the
disadvantages include the lack of standardization of the potency of the TF and the requirement with the
TEG of making a “home-made” TF reagent using commercial TF intended for other uses. Although with
the ROTEM, a TF reagent is provided (EXTEM) by the manufacturer, there are concerns regarding the
potential variability in different lots. All of these issues with TF have led to significant variability between
studies using the same brand of TF. In a study comparing kaolin to TF, kaolin performed as well as a low
concentration of TF and better than a higher concentration(5). In a recent study, it was shown that with a
well characterized TF reagent (Baxter Healthcare, Hyland Division) at a concentration of 5pM combined
with CTI and relipidated in 75%PC/25%PS, improved the reproducibility of thromboelastographic
measurements significantly(6). Further research will be required to determine which method will be the
most reliable in predicting clinical outcomes.
Conclusion: This WP has concluded that thromboelastography can be performed with either of the
available instruments and that both the contact activation and tissue factor activation methods continue to
be used in clinical trials. Further research to determine a correlation between the laboratory results and
clinical outcomes are required before these assays can be recommended for clinical use.
Bibliography:
1.
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blood clot formation after ex vivo addition of plasma substitutes: improvements of the induced
coagulopathy with fibrinogen concentrate. Br J Anaesth2005 Mar;94(3):324-9.
2.
Zambruni A, Thalheimer U, Leandro G, Perry D, Burroughs AK. Thromboelastography with
citrated blood: comparability with native blood, stability of citrate storage and effect of repeated
sampling. Blood Coagul Fibrinolysis2004 Jan;15(1):103-7.
3.
Lippi G, Franchini M, Montagnana M, Salvagno GL, Poli G, Guidi GC. Quality and reliability of
routine coagulation testing: can we trust that sample? Blood Coagul Fibrinolysis2006 Oct;17(7):513-9.
4.
Sorensen B, Ingerslev J. Tailoring haemostatic treatment to patient requirements - an update on
monitoring haemostatic response using thrombelastography. Haemophilia2005 Nov;11 Suppl 1:1-6.
5.
Young G, Zhang R, Miller R, Yassin D, Nugent DJ. Comparison of kaolin and tissue factor activated
thromboelastography in haemophilia. Haemophilia May;16(3):518-24.
6.
Foley JH, Butenas S, Mann KG, Brummel-Ziedins KE. Measuring the mechanical properties of
blood clots formed via the tissue factor pathway of coagulation. Anal Biochem Mar 1;422(1):46-51.
Figure 1: Thromboelastograph: