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Recommendations for Thromboelastography/Thromboelastometry in Patients with Hemophilia

Please review the draft manuscript and send comments to Meera Chitlur (MCHITLUR@dmc.org)  with a copy to Subcommittee Chairman on Factor VIII and IX, Flora Peyvandi (info@florapeyvandi.eu).

 

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, Montr�al, 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 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 360�L and 340 �L 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 “home-made” 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. Coagulation time: R on TEG and CT on ROTEM
  2. Clot Formation time: K on TEG and CFT on ROTEM
  3. Max. Clot Firmness: MA on TEG and MCF on ROTEM
  4. Shear Elastic Modulus Strength: G on both instruments.
  5. 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.
  2. 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.
  3. 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).
  4. 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).
  5. 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 �L of Innovin� is added to 160 �L of saline diluent (final concentration = 1:17). 100 �L of the 1:17 dilution of TF is then added to 900 �L of diluent (final concentration = 1:170). Finally 990 �L of the 1:170 dilution of TF is added to 1000 �L 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, 20�L of the dilute TF is placed in the specimen cup, followed by 20 �L of calcium chloride for recalcification of the citrated blood sample. Since the maximum volume in the specimen cup cannot exceed 360 �L, 320 �L of blood is added instead of 340 �L 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. Fenger-Eriksen C, Anker-Moller E, Heslop J, Ingerslev J, Sorensen B. Thrombelastographic whole 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:


 

Please review the draft manuscript and send comments to Meera Chitlur (MCHITLUR@dmc.org)  with a copy to Subcommittee Chairman on Factor VIII and IX, Flora Peyvandi (info@florapeyvandi.eu).

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