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Keywords:

  • cat;
  • dog;
  • equine;
  • thromboelastography;
  • thromboelastometry;
  • TEG;
  • ROTEM

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Databases and Search Terms Used
  5. Discussion
  6. Footnotes
  7. References

Objective

To systematically examine the evidence on activating agents and test protocols for the thrombelastography (TEG) and rotational thrombelastometry (ROTEM) viscoelastic point-of-care instruments and to identify knowledge gaps.

Design

Ten questions were considered, the primary question addressed the use of activating agents and secondary questions addressed assay temperature, length of analysis, pipetting, sample volume, reproducibility, and quality controls. Standardized, systematic evaluation of the literature was performed. Relevant articles were categorized according to level of evidence (LOE). Consensus was developed regarding conclusions for application of concepts to clinical practice.

Setting

Academic and referral veterinary medical centers.

Results

PubMed and CAB abstracts were searched. Twenty papers were initially identified concerning the primary question; 16 were in support of the questions (LOE 2 Good, LOE 3 Good, LOE 5 Good, LOE 6 Good, LOE 5 Fair, LOE 6 Fair); and 4 were neutral (LOE 3 Good, LOE 6 Good, LOE Fair, LOE 5 Fair). Additional papers were evaluated post hoc during manuscript preparation.

Conclusions

Overall, there is a body of evidence from veterinary and human medicine that strongly suggests that TEG or ROTEM assays using citrated samples that employ an activator have significantly lower inherent variability than those that use recalcification alone. There is also strong evidence in dogs, cats, and humans that the results obtained using different activators are not directly comparable. There is no evidence to suggest that any one activating agent is superior to another for all patient populations, or drug monitoring indications. As such, use of more than one assay for complete thromboelastographic evaluation of a patient's coagulation system may be warranted. Standardization of the concentrations of activators would be beneficial.


Abbreviations
CV

coefficient of variation

rhTF

recombinant human tissue factor

LOE

level of evidence

PICO

Population-Intervention-Comparison-Outcome

R

reaction time (TEG variable)

ROTEM

rotational thrombelastometry

TEG

thrombelastography

TF

tissue factor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Databases and Search Terms Used
  5. Discussion
  6. Footnotes
  7. References

The incorporation of activators in viscoelastic testing protocols significantly impacts assay results. Activators standardize initiation of clot formation, speed-up clot kinetics, and stabilize clot formation. Thus, in theory, activators make assays less sensitive to preanalytical variation and also minimize analytical variation. The effect of activators on analytical variation has been investigated in dogs, cats, and horses.[1-8]

The most commonly used activators in veterinary medicine are recombinant human tissue factor (rhTF) and kaolin. Tissue factor (TF) plays a pivotal role in initiation and propagation of coagulation in vivo and is identified as the main activator of coagulation in inflammatory states.[9] Its use as an activator of coagulation in equine thrombelastography (TEG) assays has been validated employing high concentrations (1:100; 1:3,600) of TF, and in cats and dogs at concentrations of 1:50,000.[1, 2, 4, 5] Kaolin is a negatively charged compound that activates coagulation via the intrinsic pathway.[10] In humans, kaolin-activated TEG has been shown to be more robust than nonactivated assays, and less sensitive to preanalytical variation such as the variation seen due to storage of samples for prolonged periods of time.[11]

Variation of an assay can be divided into analytical, intra- and interindividual variation and the contribution of each of these components to the overall assay variation can be obtained by a nested analysis of results obtained from repetitive sampling of healthy individuals.[12] Direct comparison of these components of variation across various TEG methodologies can reveal the effect of activators on the analytical variation and the impact of pre-analytical sources of variation (such as sampling technique, handling, and storage) on the results.[13, 14] Characterization of assay variation is critical to interpretation and application of assay results to clinical decision making. Unfortunately few studies on biological variation have been carried out in either human or veterinary medicine.[14-16] The objectives of this domain were to systematically examine the evidence on activating agents and test protocols for the TEG and rotational thromboelastometry (ROTEM) viscoelastic point-of-care instruments and to identify knowledge gaps.

Databases and Search Terms Used

  1. Top of page
  2. Abstract
  3. Introduction
  4. Databases and Search Terms Used
  5. Discussion
  6. Footnotes
  7. References

PubMed1 and CAB Abstracts2 were searched using these terms as “topic” (CAB Abstracts) or “all fields” (PubMed): ((dog OR canine) OR (cat OR feline) OR (horse OR equine)) AND ((TEG OR thromboelastograph*) OR (ROTEM OR thromboelastom*)). PubMed and CAB abstracts were searched. Twenty papers were initially identified concerning the primary question; 16 were in support of the questions (level of evidence (LOE) 2 Good, LOE 3 Good, LOE 5 Good, LOE 6 Good, LOE 5 Fair, LOE 6 Fair); and 4 were neutral (LOE 3 Good, LOE 6 Good, LOE Fair, LOE 5 Fair). Additional papers were evaluated post hoc during manuscript preparation.

Evidence summary

Population-intervention-comparison-outcome (PICO) question

In companion animals with hemostatic abnormalities (P), does routine use of activators for citrated samples (I) compared with recalcification only (C), alter TEG/ROTEM testing results (O)?

Conclusion

The routine use of activators for TEG/ROTEM assays in companion animals is recommended.

Evidence summary

Evidence from multiple studies in companion animals (LOE 2–3, Good-Fair) (Table 1) suggests that standard parameters from TEG or ROTEM assays using citrated whole blood samples that employ an activator have lower variability compared with those using recalcification alone.[2-4, 7, 17-19] One study in cats (LOE 3, Good) suggested that variability is comparable between assay types.[6] It should also be noted that one study (LOE 3, Good) identified significant interoperator variation even with use of a TF-activated TEG assay.[2] It has been argued that potent activators may mask mild changes in coagulation capacity and that such a decrease in analytical sensitivity could potentially be of clinical importance.[2, 11] However, the use of a mild activator, resulting in reduced assay susceptibility to preanalytical factors will likely increase sensitivity for intraindividual changes, due to reduced effect of preanalytical variation. Overall the available evidence argues for use of an activating agent or agents for veterinary TEG/ROTEM assays.

Table 1. Summary of agonist usage in veterinary TEG/ROTEM assays published to date
ARTICLEAGONIST USEDAGONIST CONCENTRATIONSYSTEMSPECIES
Alwood et al, 2007 [50]Recalcification only20 μL 0.2M CaCl2TEGFELINE
Andreasen et al, 2012[30]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE
Ao et al, 2001[51]Recalcification onlyNot specifiedTEGCANINE
Banerjee et al, 2011[6]Recalcification only20 μL CaCl2TEGFELINE
 Recalcification + KaolinProprietary  
 Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)  
Bauer et al, 2011[52]Recalcification + KaolinProprietaryTEGCANINE
Bauer 2012[53]Recalcification + KaolinProprietaryTEGCANINE
Bauer et al, 2010[54]Recalcification + KaolinProprietaryTEGCANINE
Bauer et al, 2009[17]Recalcification + KaolinProprietaryTEGCANINE
Bjornvad et al, 2012[31]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGFELINE
Brainard et al, 2011[55]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Brooks et al, 2009[25]Recalcification only20 μL 0.28mM CaCl2TEGCANINE
 Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)  
Callan et al, 2006[56]Recalcification + rhTF (Innovin)4 pmol/LROTEMCANINE
Donahue et al, 2011[57]Recalcification onlyNot specifiedTEGCANINE
Dunkel et al, 2010[58]Recalcification only20 μL 0.2M CaCl2TEGEQUINE
Epstein et al, 2011[18]Recalcification only20 μL 0.2M CaCl2TEGEQUINE
 Recalcification + rhTF (Innovin)1:3,600  
Epstein et al, 2009[2]Recalcification only20 μL 0.2M CaCl2TEGEQUINE
 Recalcification + rhTF (Innovin)1:3,600  
Eralp et al, 2011[59]Recalcification + KaolinProprietaryTEGCANINE
Fenty et al, 2011[60]Recalcification + KaolinProprietaryTEGCANINE
Fiakpui et al, 2012[61]Recalcification only20 μL 0.2M CaCl2TEGFELINE
Flint et al, 2011[62]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Flint et al, 2012[3]Recalcification only20 μL 0.2M CaCl2TEGCANINE
 Recalcification + KaolinProprietary  
Foley et al, 2012[63]Recalcification + rhTF (Innovin)1:15,000TEGCANINE
Garcia-Pereira et al, 2012[45]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Giordano et al, 2010[64]Recalcification + InTEM (Ellagic acid)ProprietaryROTEMEQUINE
 Recalcification + ExTEM (rhTF)Proprietary  
Goggs et al, 2012[65]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Gonzales et al, 2011[66]Recalcification + KaolinProprietaryTEGCANINE
Goodwin et al, 2011[67]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Hall et al, 2012[42]Recalcification + KaolinProprietaryTEGFELINE
Jessen et al, 2008[24]Recalcification + KaolinProprietaryTEGCANINE
 Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE
Karpf et al, 2011[68]Kaolin onlyProprietaryTEGCANINE
Klose et al, 2011[69]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Knudsen et al, 2011[70]Kaolin onlyProprietaryTEGCANINE
Koenigshof et al, 2012[46]Recalcification + KaolinProprietaryTEGCANINE
Kristensen et al, 2008[32]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE
Leclere et al, 2009[5]Recalcification only20 μL 0.2M CaCl2TEGEQUINE
 Recalcification + rhTF (Innovin)1:3,600  
Macieira et al, 2007[71]Recalcification + Kaolin25 mg/mLTEGEQUINE
 Recalcification + rhTF (Innovin)1:18,000  
Margaritis et al, 2009[72]rhTF (Innovin) only1:200,000TEGCANINE
Marschner et al, 2010[4]Recalcification only20 μL 0.2M CaCl2TEGFELINE
 Recalcification + KaolinProprietary  
 Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)  
McMichael et al, 2011[22]Recalcification + InTEM (Ellagic acid)ProprietaryROTEMEQUINE
 Recalcification + ExTEM (rhTF)Proprietary  
Mendez-Angulo et al, 2011[41]Recalcification only20 μL 0.2M CaCl2TEGEQUINE
Mendez-Angulo et al, 2010[73]Recalcification only20 μL 0.2M CaCl2TEGEQUINE
Nielsen et al, 2011[33]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE
O'Marra et al, 2012[74]Recalcification + KaolinProprietaryTEGCANINE
Oberg et al, 2010[75]Recalcification + KaolinProprietaryTEGFELINE
Othman et al, 2009[76]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Othman et al, 2010[23]Recalcification only20 μL 0.2M CaCl2TEGCANINE
 Recalcification + rhTF (Innovin)1:30,000TEGCANINE
Otto et al, 2000[77]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Paltrinieri et al, 2008[19]Recalcification only20 μL 0.2M CaCl2ROTEMEQUINE
 Recalcification + InTEM (Ellagic acid)Proprietary  
 Recalcification + ExTEM (rhTF)Proprietary  
Pittman et al, 2010[78]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Poston et al, 2004[79]??TEGCANINE
Prasad et al, 2008[80]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Rioja et al, 2012[34]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE
Rose et al, 2011[81]Recalcification + rhTF (Innovin)1:3,600TEGCANINE
Saavedra et al, 2011[82]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Sinnott and Otto, 2009[83]Recalcification onlyNot specifiedTEGCANINE
Smith et al, 2010[7]Recalcification only20 μL 0.2M CaCl2ROTEMCANINE
 Recalcification + InTEM (Ellagic acid)Proprietary  
 Recalcification + ExTEM (rhTF)Proprietary  
 Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)  
Taggart et al, 2012[47]Recalcification + KaolinProprietaryTEGCANINE
Vilar-Saavedra et al, 2011[84]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Vilar-Saavedra et al, 2011[85]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Vilar-Saavedra et al, 2008[86]Recalcification only20 μL 0.2M CaCl2TEGCANINE
Wagg et al, 2009[87]Recalcification + rhTF (Innovin)1:3,600TEGCANINE
Wiinberg et al, 2008[36]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE
Wiinberg et al, 2009[35]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE
Wiinberg et al, 2007[16]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE
Wiinberg et al, 2005[1]Recalcification + rhTF (Innovin)1:50,000 (0.119 pmol/L)TEGCANINE

PICO questions

(i) In companion animals with hemostatic abnormalities (P), does the use of one activator for citrated samples (I) compared with another activator (C) alter TEG/ROTEM testing results (O)?

(ii) In companion animals with hemostatic abnormalities (P), does the use of an activator at a given concentration (I) compared with another concentration (C) alter TEG/ROTEM testing results (O)?

Conclusions

Standardization of activators for TEG/ROTEM assays in companion animals is recommended. We suggest that 2 or more TEG/ROTEM assays be used to completely evaluate a patient's coagulation system. For contact-pathway evaluation in companion animals using TEG, we recommend the use of proprietary kaolin tubes. For evaluation of the TF pathway in horses using TEG, we suggest using diluted Innovin 1:3,600. For evaluation of the TF pathway in small animals we suggest using diluted Innovin at both 1:3,600 and 1:50,000. We recommend use of proprietary In-TEM and Ex-TEM reagents with the ROTEM system.

Evidence summary

Evidence from 3 companion animals studies (LOE 2–3, Good),[3, 4, 6] and 3 human studies (LOE 6, Good-Fair),[11, 20, 21] suggests in dogs and cats that the results obtained using different activators are not directly comparable. Although the evidence for this in horses is less compelling (LOE 3, Good-Fair),[8, 19, 22] the evidence overall strongly suggests that results from different assays cannot be used interchangeably and argues for standardization between centers. However, correlation of activator selection to clinical state remains unknown and further studies are needed to determine criteria for clinical decisions. Currently, there is no evidence to suggest that any one activating agent is superior to another in any patient population for the detection of bleeding, thrombosis, or for drug monitoring indications. As such, use of more than one assay for complete evaluation of a patient's coagulation system may be warranted and specific assays may be preferable for certain patient populations, therapeutic monitoring scenarios or for evaluating different components of the hemostatic system, such as platelets, thrombin, and fibrinolytic potential.[23-27]

The availability of proprietary reagents (standardized at source) for contact pathway evaluation using TEG and for both contact and TF pathway evaluation using ROTEM makes comparability of these assays more straightforward. Standardization of the concentrations and preparations of dilute rhTF (Innovin) for dogs, cats, and horses would be beneficial. However, the affinity between human TF and its ligand FVII in the different animal species varies such that the same concentration of TF may have very different effects in the individual species. Evidence from 3 human studies (LOE 6, Good-Fair), shows that different agonist concentrations produce dissimilar results, arguing for standardization of agonist concentrations.[11, 28, 29] A 1:50,000 dilution of rhTF has been validated and used in one ROTEM and multiple TEG publications in small animals.[1, 4, 6, 7, 16, 24, 25, 30-36] Experimental work using ROTEM suggests that ex vivo contact activation has a greater effect on ROTEM parameters when coagulation is initiated using this low TF concentration compared with a higher concentration.[7] As such, use of the higher TF concentration (1:3,600) is likely to provide a robust assay that is less sensitive to preanalytical variation. A more sensitive assay may be obtained using lower dilutions. A robust assay may be more appropriate for routine clinical use, while use of more than one concentration may provide a more complete picture and will enable future metaanalysis to be conducted.

PICO question

In companion animals with hemostatic abnormalities (P), does the use of quality controls (I) compared with no quality control (C) alter TEG/ROTEM testing results (O)?

Conclusion

We recommend manufacturer's guidelines regarding routine use of quality controls be followed.

Evidence summary

No studies were available which specifically addressed the question of quality controls for TEG-ROTEM assays. Several studies have performed external quality assurance assessments on TEG/ROTEM systems.[37, 38]

PICO question

In companion animals with hemostatic abnormalities (P), does use of manual pipetting (I) compared to automatic pipetting (C) alter TEG/ROTEM testing results (O)?

Conclusion

There is insufficient evidence to conclude that one pipetting technique is superior to the other. We suggest careful manual pipetting be used for TEG assays. We suggest automatic pipetting be used for ROTEM assays.

Evidence summary

The pipetting technique used for the 2 assays is different. The TEG system typically involves a manual technique whereas ROTEM uses an automated technique. Pipetting for the TEG system is straightforward and single portion reagents have been developed for the ROTEM system to reduce the pipetting complexity for ROTEM assays.[39] Only one study (LOE 6, Good) evaluated the impact of standardizing the pipetting between the 2 systems by employing solely the manual method in both TEG and ROTEM recalcification only assays.[40] This study also included a consistent mixing step. Results from the 2 machines were positively correlated, but significant differences in all 4 standard variables were identified between the 2 machines. This study suggests that differences exist between the 2 platforms that cannot be accounted for by pipetting alone.

PICO question

In companion, animals with hemostatic abnormalities (P), does timing of reagent and sample mixing (I)(C) alter TEG/ROTEM testing results (O)?

Conclusion

There is insufficient evidence to make recommendations regarding mixing of samples and reagents for TEG. For both TEG and ROTEM, we suggest that blood samples be added to reagents (eg, calcium chloride [CaCl2], rhTF) in prewarmed cups immediately prior to introduction of the pin per the manufacturers’ instructions. For ROTEM, we suggest that reagents should be mixed using the automatic pipette per the manufacturers’ instructions.

Evidence summary

There is no direct published evidence, regarding when reagents and sample should ideally be mixed prior to introduction of the pin, in order to get a result that most precisely resembles in vivo coagulation potential of the patient. The time from mixing until the start of the assay will likely affect the result. Some reagents take time to interact and for those incubation could be beneficial, while other reagents quickly lose effect and the assay using them should be started without delay. Ultimately the effect of timing should be established and mixing and incubation should be standardised for each assay.

PICO question

In companion animals with hemostatic abnormalities (P), does fixed analysis time (I) compared to variable analysis time (C) alter TEG/ROTEM testing results (O)?

Conclusions

For routine clinical purposes, we suggest that all TEG/ROTEM assays be run until all values pertinent to individual patients are available. For research purposes, we suggest that all TEG/ROTEM assays be run until values for fibrinolysis are available such that they may be included in related publications.

Evidence summary

There is no relevant evidence.

PICO question

In companion animals with hemostatic abnormalities (P), does duplicate analysis (I) compared to single analysis (C) alter TEG/ROTEM testing results (O)?

Conclusion

We suggest that duplicate assays are not required when activators are used. We suggest that if recalcification only assays are used that duplicate samples be performed. We recommend repeated sampling from 1 tube is avoided when duplicate assays are performed (ie, 2 tubes will be required). We suggest individual laboratories determine if and or when repeat analyses of erroneous samples is required.

Evidence summary

Evidence from 4 studies in horses (LOE 2–3, Good-Fair), 3 studies in dogs (LOE-Good), and 2 studies in cats (LOE 3, Good) provides information related to evaluation of analytical variation through the use of duplicate samples.[1, 3-5, 8, 16, 19, 41, 42] Results of these studies consistently document significantly greater intraindividual variation in TEG parameters in nonactivated samples compared to kaolin-activated and rhTF-activated samples. In a study of 15 cats using native, kaolin-activated, and rhTF-activated samples, native samples were found to have the highest coefficient of variation (CV) while kaolin-activated samples had the lowest CV in all TEG parameters measured.[4] Laursen et al (2013) documented similar increased intraindividual variation in nonactivated compared with TF-activated and kaolin-activated samples in healthy horses suggesting greater sensitivity to preanalytical sources of variation.[8] In those studies (using dogs, cats, and horses) using 1 activator for the evaluation of hemostasis with duplicate samples performed, a low analytical variance was observed in both TF-activated and kaolin-activated samples suggesting high reproducibility between tests.[1, 5, 16, 41] These studies suggest that duplicate assays are not necessary when activators are used for TEG analysis. Duplicate samples may be helpful when recalcified nonactivated samples are used.

PICO question

In companion animals with haemostatic abnormalities (P), does use of fixed sample volume (I) compared to variable sample volume (C) alter TEG/ROTEM testing results (O)?

Conclusions

There is insufficient evidence to recommend specific sample volumes for TEG/ROTEM assays. We suggest that manufacturer's guidelines regarding sample volumes be followed for routine assays.

Evidence summary

Studies addressing this question are limited. Mini-cups with reduced total volumes are available for the ROTEM system. Sample volumes used in standard TEG/ROTEM assays are different. TEG assays typically require 360 μL total volume compared to 340 μL total for ROTEM assays. One study used 320 μL for both assays (LOE 6, Poor) although the rationale for this was not directly discussed.[43] However, no mention was made to the effect that it was problematic for TEG analyses. One crossover study of TEG/ROTEM for evaluation of fibrin-based clotting in whole blood samples ensured consistent blood/reagent ratios in every assay (LOE 6, Poor).[44] This study found differences in the resulting measurements between the 2 platforms. These studies suggest that similar reaction volumes can be used in both systems without problem, but that results from the 2 platforms are not directly comparable.

PICO question

In companion animals with hemostatic abnormalities (P), does analysis at 37°C (I) compared to analysis at the patient's body temperature (C) alter TEG/ROTEM testing results (O)?

Conclusions

There is insufficient evidence to recommend use of specific temperatures for each species. We suggest all routine TEG/ROTEM assays are performed at 37°C. In patients with marked hypothermia or hyperthermia, we suggest clinicians consider evaluating TEG/ROTEM tracings performed at 37°C and at the patient's core temperature.

Evidence summary

The majority of TEG/ROTEM assays reported in veterinary medicine have been analyzed at 37°C (LOE 2–3, Good-Poor). Two studies (LOE 3, Good) have reported TEG analyses performed at 39°C.[45, 46] One study using kaolin-activated TEG (LOE 3, Good) compared incubated and tested canine whole blood samples at a range of clinically relevant temperatures (27–42°C).[47] Each sample was analyzed in parallel with a control sample from the same dog rested at room temperature and analyzed at 37°C. Simulated hypothermia led to significant increases in K times and significant reductions in α values. Temperature variations between 27°C and 42°C did not significantly alter R times or maximum amplitude values. Altering the analysis temperature affects estimates of clot formation kinetics rather than clot initiation or maximum clot strength. The evidence suggests that analysis temperature should be standardized in order to enable comparison of data within and between centers. In addition, evidence from 1 study in ROTEM (LOE 3, Good) suggests that an in-cup incubation period of at least 5 minutes is required before blood held at room temperature is fully rewarmed to 37°C.[7] Although no published work has investigated the time required for samples to warm from room temperature to 37°C in the TEG machine, unpublished observations suggest that rewarming is complete within 150 seconds.3

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Databases and Search Terms Used
  5. Discussion
  6. Footnotes
  7. References

Overall, evidence from veterinary and human medicine strongly suggests that TEG or ROTEM assays using citrated samples that employ an activator have significantly lower inherent variability than those that use recalcification alone.[1, 4, 8, 16, 17]

Numerous sources of preanalytical variation can affect the results of an assay. Some of the most important include sample collection, handling, transport, and storage, which are all difficult to control. Nonactivated TEG has demonstrated a higher sensitivity to preanalytical sources of variation than assays using an activator, suggesting that assays with activators should preferably be used. However, other aspects such as the possible effect of activator concentration on assay precision and diagnostic sensitivity must be considered. Standardization of the concentrations of dilute rhTF (Innovin) for dogs, cats, and horses would be beneficial and might be based on the prevalence of each method in the available literature. The availability of proprietary reagents (standardized at source) for contact pathway evaluation using TEG and for both contact and TF pathway evaluation using ROTEM makes comparability of these assays more straightforward.

There is also strong evidence in dogs, cats, and humans that the results obtained using different activators are not directly comparable.[1, 4, 16, 17] The evidence for this in horses is less compelling.[1, 4, 8, 16, 17] However, there is no evidence to suggest that any one activating agent is superior to another for all patient populations, or drug monitoring indications. As such use of more than one assay for complete thromboelastographic evaluation of a patient's coagulation system may be warranted.

For particular patient populations, eg, Scott syndrome or hemophilia A, particular assays may be preferable. For certain therapeutic monitoring scenarios such as following low molecular weight heparin (LMWH) administration, other assays may be preferable. As such, the available evidence argues for the use of an activating agent or agents for veterinary TEG/ROTEM assays and in addition, that some standardization of activating agent or agents in use is required to improve comparability of results between centers.

In order to trust the generated results completely, it is essential that proper sample handling be used, that, the analysis has been carried out according to protocol, and that test system is functioning normally. In order to ensure the latter, we recommend following the manufacturer's guidelines regarding routine use of quality controls be followed and that the results are tracked. This includes both assay and system controls and we suggest each laboratory set up additional QC protocols according to local needs and guidelines.

The 2 systems use different pipetting techniques. For a trained operator, there should be minimal difference between using manual and automated pipetting; however, for an untrained operator, it will likely be of benefit to use the automated system. However, automated pipetting is only possible as long as proprietary assays are used and therefore it will likely be impossible to avoid all manual pipetting when running assays on animals. Therefore, we suggest careful manual pipetting be used for TEG assays and automatic pipetting be used for ROTEM assays, when possible.

There is insufficient evidence to make general recommendations regarding the optimal way to mix samples and reagents for TEG or ROTEM. Unless it has been demonstrated that there is no effect of different protocols, it is always recommended to adhere to 1 protocol in order to limit interassay variability. Assays where a pharmacological agent is spiked into the blood sample to predict effect of treatment are expected to be particular sensitive to differences in protocol, due to variation in contact time between the blood and the agent.

The length of the analysis is relevant in relation to which component of the coagulation system is being evaluated. For general routine clinical screening, we suggest that all TEG/ROTEM assays be run until all values pertinent to individual patients are available. Once a specific defect has been identified it may be relevant only to run the assay as long as it takes to evaluate that specific component. For research purposes, we suggest that all TEG/ROTEM assays be run until values for fibrinolysis are available such that they may be included in related publications. In general, it is evident from this literature review that there are still many knowledge gaps regarding TEG/ROTEM analyses and therefore it is important to report as much data as possible in future publications, so that these gaps can be filled.

Several studies have shown that there is lower analytical variation when using activators and in general this variation is below 20%, suggesting that it is not necessary to run these assays in duplicate. On the other hand assays if assays are only recalcified and run without an activator, data shows that the analytic variation for some parameters is very high, which particularly poses a challenge in relation to critical decision levels and when following the same patient with several measurements.[1, 4, 16, 17] Consequently, we suggest that duplicate assays are not required when activators are used, whereas with recalcification only assays duplicate samples should be performed. We recommend repeated sampling from one tube is avoided when duplicate assays are performed in order to avoid preactivating the sample.[49, 50] We suggest that manufacturer's guidelines regarding sample volumes be followed for routine assays. The blood volume recommended by the manufacturers ensures an optimal contact between cup/pin and blood, relative to the traction produced by the machines. Less blood may be required to produce a measurable clot, but to what extent it reflects the endogenous clotting potential of the patient is currently unknown. One study has used 320 μL for both assays, although the rationale for this was not directly discussed,[43] and no mention was made to the effect that it was problematic for TEG analyses. Another crossover study of TEG/ROTEM for evaluation of fibrin-based clotting in whole blood samples ensured consistent blood/reagent ratios in every assay between the two platforms.[44] These studies suggest that similar reaction volumes can be used in both systems without problem, but found differences in the resulting measurements, suggesting that results from the 2 platforms are not directly comparable.

Although 37°C is lower than the normal body temperature in most animals, coagulation assays in veterinary medicine have traditionally been carried out at this temperature and in order to standardize, we suggest all routine TEG/ROTEM assays are performed at 37°C, as are all other coagulation assays. However, in patients with marked hypothermia or hyperthermia, we suggest clinicians consider evaluating TEG/ROTEM tracings both at 37°C and at the patient's core temperature, in order to evaluate the effect of body temperature on coagulation potential.

  1. 1
  2. 2
  3. 3

    Brainard BM. Personal communication. 2013.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Databases and Search Terms Used
  5. Discussion
  6. Footnotes
  7. References