The purpose of this study is to provide a reliable technique for performing thrombelastography in rodent coagulation research. The importance of developing a standard method of performing TEG is to improve reproducibility and facilitate comparison of results in the literature and between investigators. TEG is a versatile and comprehensive tool which measures specific components of coagulation, increasing its use in diverse clinical settings such as cardiac surgery (3
), liver transplant (2
), trauma (4
), sepsis (14
), and hemophilia (15
). Because TEG can function in multiple clinical arenas, and relies on proper performance by the operator, following a standard technique and choosing the appropriate type of TEG to perform are critical in achieving consistent results.
Previous studies have evaluated coagulation between species using TEG, using various activators (i.e. tissue factor, kaolin, and celite) (16
). Kaolin and celite activate the contact pathway via Factor XII, while tissue factor is used to activate thrombin through the TF:VIIa complex. Alternatively, native TEG employs whole blood without the use of an activator. Additionally, kaolin, celite, and native TEGs can all be performed using citrated whole blood. While citrated kaolin TEG has been used to assess feline coagulation (17
), the relative hypercoagulability of other laboratory animals, including rodents, renders kaolin activator unnecessary and non-citrated whole blood impractical (5
). Previous research supports the use of citrated whole blood as optimal for the performance of TEG in small laboratory animals (18
). Since there are multiple methods of performing TEG on specialized patient populations and in specific research settings, it can be plagued by variability leading to inconsistent, or worse, misleading results. In the following section, several methods for optimizing performance of the TEG are presented (summarized in ).
Thrombelastography for Rodents: Technical Considerations.
The first issue is optimal mixing of citrate. After collecting the citrated blood, invert 5 times to mix, and place the sample on its side. Storing the blood horizontally instead of vertically prevents the blood from layering and reduces the chance of premature coagulation during storage. When inverting the sample to mix, it is critical to do so gently. Shaking or vortexing blood will cause hemolysis and substantial platelet activation. Allow citrated whole blood to sit for 15-30 minutes before running TEG. This step is critical to limit variability, as previous clinical studies have shown that citrated blood requires time to equilibrate before running the TEG (19
The second issue in the preparation of the TEG sample is to discharge the 340 μl blood sample from the pipette gently into TEG cup. Calcium at the bottom of the cup will diffuse into the citrated blood. Pipetting to mix blood provokes contact activation.
Third, blood sample activity degrades over time. We have found optimal results when running samples within 2 hours. Furthermore, performing multiple TEGs from the same blood sample can substantially alter coagulation integrity (20
One of the strengths of the TEG is the ability to use the animal as its own control, comparing coagulation integrity before and after a stimulus. Therefore, collecting a baseline blood sample that closely mimics the animal’s coagulation status at rest is critical. In animal models in which a hypercoagulable state is induced (trauma, sepsis, cancer), placing the activated citrated blood on a rocker can prevent coagulation during the 30 minute sample equilibration period. It is not necessary to agitate citrated blood from a healthy animal. Refrigerating blood or placing blood on ice can affect platelet function and alter results. Additionally, oil from hands can induce fibrinolysis, so gloves should be worn when handling samples, cups, and pins.
Blood collection from the IVC requires laparotomy, which causes substantial tissue injury. Tail vein amputation is an invasive method of blood collection. The orbital vein has been used as a convenient means of blood collection; however, this technique is believed to cause contact activation. Cardiac puncture is an additional option, but it is technically demanding, especially when performing serial TEG measurements.
When comparing cardiac puncture to femoral arterial blood sampling, the results were erratic, as withdrawing blood through a needle for the cardiac puncture likely activates platelets. Because rats have a substantially higher platelet count than humans, this effect may have been more pronounced. For these reasons, we feel that collecting blood from the femoral artery using the animal’s blood pressure to receive the blood into a citrated tube is the most practical collection technique.
As in other coagulation assays, considerable variation between species and even strains of laboratory animal exists. Reference ranges in Sprague Dawley rats using TEG have been described in this paper. Using citrated native blood in the rodent allows the investigator familiar with TEG in the clinical arena to yield comparable numerical values compared to kaolin-activated citrated human blood. Laboratories using TEG for research purposes should establish their own reference ranges using the method in order to determine normal values for their target animal population.