Reference ranges for rotational thromboelastometry in male Sprague Dawley rats

Viscoelastic hemostatic assays are useful point of care tools in guiding therapy in trauma and cardiac surgery patients, as well as patients with other perioperative bleeding disorders or risk factors for coagulopathy [1, 2]. Sprague Dawley rats are commonly used in experimental models, including those examining the coagulopathy resultant from trauma and hemorrhagic shock. Thromboelastometry and thromboelastography have been applied in various experimental protocols for evaluation of clot formation and fibrinolysis in rats [3‐6], and recognition of the utility of research involving these viscoelastic hemostatic assays has made their use more commonplace. Despite the widespread use of rodent models in coagulation studies, there is a lack of reference ranges for thromboelastometric parameters in the literature for Sprague Dawley rats. Lang et al. have described reference ranges in humans following a multi-centre approach employing blood donors, clinical personnel and other volunteers [7] and these ranges are used as the basis for treatment of coagulopathies in clinical care. Additionally, significant inter-species differences in the coagulation profiles between humans and various experimental animals have been previously reported [8, 9]. This established inter-species variation underscores the importance of defining reference intervals for individual species, and implies that reference ranges that define coagulopathies in one species do not do so in another. Additionally, often, when small animals are used, the use of the mini cup and pin system is required due to the obtainment of small volume aliquots at multiple time points. There are no established human reference ranges in the mini cup and pin system that are available for comparison, although it is established that differences exist between the two systems [10].

Clot formation in vivo is composed of two major pathways, extrinsic and intrinsic, which converge to regulate a complex biomolecular cascade leading to coagulation and fibrinolysis. The Extrinsic Tissue Factor pathway is tested by the EXTEM assay by ROTEM®. The results of this assay may be influenced by extrinsic coagulation factors, platelets and fibrinogen [7, 11]. The purpose of this study was to analyze citrated whole blood from a cohort of healthy rats to determine baseline thromboelastometry parameters as demonstrated by the EXTEM test in an effort to establish normal reference ranges that can be used to diagnose clinically significant coagulopathies in animal research.

Representative ROTEM® traces from individual animals are shown in Fig. 2. Descriptive statistics and histograms indicated mostly normal distribution of the ROTEM® results by D’Agostino and Pearson normality test (representative histograms are shown in Fig. 3). Mean and median values were calculated from individual animal values for each EXTEM parameter, and standard deviation was calculated to formulate reference ranges. Reference ranges are presented as the 2.5- and 97.5-percentiles in Table 1, as is standard for the reporting of human reference ranges [7].

Numerous studies have analyzed the clinical use of ROTEM and thromboelastography (TEG) in a variety of settings, including trauma, cardiac surgery and liver transplantation. The clinical applications of these viscoelastic assays in humans are well established for understanding individual coagulopathic and fibrinolytic profiles in patients, but their use in translational research related to hemostasis involving animal models is challenged by a scarceness of data indicating reference ranges. The importance of understanding clotting parameters in animals has been highlighted in recent publications exploring larger animal models for hemostatic alterations in cardiovascular surgery and haemorrhage [12, 13]. Rats can be useful models for hemostatic analysis in diverse pathologic states, including those commonly managed clinically with the aid of TEG or ROTEM.

No prior reference ranges for rotational thromboelastometry have been reported in Sprague Dawley rats. Considering the importance of accurate and translatable animal models for the study of coagulation and fibrinolysis physiology, particularly in investigating pathologic states, the authors wished to address this paucity in the literature to provide a guide for interpretation of rotational thromboelastometry data. The selection of this system and the EXTEM test, both widely established in the experimental and clinical settings, may allow for broader applicability of the results obtained from this study. As described by Siller-Matula et al., EXTEM results are more similar among species than NATEM or INTEM data, implying that the EXTEM test may be the ROTEM assay that may provide the most translational data [8].

Each parameter measured by ROTEM can assist in determining the need for transfusion and the choice of resuscitative fluid or therapeutic intervention. In humans, well-established reference ranges can indicate abnormalities in clot formation, strength, or breakdown. A prolonged clotting time parameter suggests the need to substitute clotting factors, such as fresh frozen plasma, factor concentrates, or anticoagulant inhibitors. A prolonged clot formation time suggests the need for fibrinogen or thrombocyte concentration, which may be administered as cryoprecipitate, fibrinogen concentrate, or fresh frozen plasma. Decreased maximum clot firmness, or low MCF, suggests potential bleeding, and may indicate the need for administration of fibrinogen, thrombocyte concentrate, and/or antifibrinolytics. Alternatively, a reduced alpha angle, shortened CFT, and high MCF indicate hypercoagulation. The lysis index parameters assist in decision making regarding the use of anti-fibrinolytic agents [1, 11, 14].

In previous literature, the differences between the mini-cup versus the conventional cup systems for ROTEM® have been reported as considerable, with mini-cup results demonstrating lower amplitudes and higher variability in the parameters reflecting the kinetics of clot formation in human blood [15]. Although the mini-cup model may be applicable beyond small rodent models in the pediatric patient population, reference ranges do not exist for this system in humans either. Despite the paucity of data from which to compare our work; the reference ranges that we provide are in agreement with the values reported by Griffin et al. They report, in a group of Sprague Dawley rats under isoflurane anesthesia (n = 6), a mean value of CT = 38, CFT = 38, α = 83, CFR = 84, maxV = 39, and ML = 8. All of these values are within the normal ranges that we have defined. Darlington et al. also report “baseline” ROTEM parameters for a group of Sprague Dawley rats (n = 7–11) that agree with our findings (MCF = 55, CT = 48, α = 73) [16].

A possible limitation to consider in this study is the potential for differences in results stemming from variability in the processing of samples and rotational thromboelastometry assays. In a prior multi-center comparative analysis of human blood tested for coagulation parameters, no significant differences in results were reported despite existing disparities in the pre-analytical factors, such as sampling technique or variations in the concentration of anticoagulant in aliquots [7]. Nonetheless, precautions to minimize machine or operator variability were followed. The device utilized was kept per manufacturer standards with weekly maintenance and calibration tests, as well as periodic professional servicing. EXTEM reagents were stored per manufacturer guidelines and utilized prior to their expiration date. The performance of collection and processing of samples was limited to two individuals. Another limitation in this study is the number of animals that was used in this experiment. Because the number of animals was defined by a power calculation for a different experiment for which the animals were used, 43 animals was not a specific number required for this study. Despite this limitation, the reference ranges that are presented here are much more robust than others that have been reported in cats (n = 25) [9], sheep (n = 6), pig (n = 6), and rabbit (n = 6) [8].

Following rotational thromboelastometry assays with mini-cup and pin system and EXTEM test in a cohort of male Sprague Dawley rats, we have formulated a standard and reproducible protocol and a series of species-specific reference ranges for commonly used parameters to assess coagulation and fibrinolysis in a rat animal model. The established reference indices may serve as guidelines in experimental design and analysis for future diagnostic and translational thromboelastometry assays in rodents.