Multiple trauma is associated with accidental hypothermia (<35°C) in 12–66% of all cases (Hildebrand et al., 2009; Mommsen et al., 2013). In numerous studies, the injury severity score (ISS) was higher in hypothermic compared to normothermic patients (Martin et al., 2005; Shafi et al., 2005; Bukur et al., 2012; Mommsen et al., 2013; Winkelmann et al., 2019). For instance, in one study the ISS was 35.0 in the patients diagnosed with hypothermia at hospital admission, while it was 29.2 in normothermic patients (Trentzsch et al., 2012). Furthermore, a lower body temperature is independently associated with increased odds of death (odds ratio, 3.03; 95% CI, 2.62–3.51) (Wang et al., 2005).

Hypothermia impairs clotting factor activity and platelet function. In multiple trauma patients these undesirable effects on the coagulation system are intensified by hemorrhage, inflammation, acidosis, but also by therapeutic interventions like volume resuscitation and the administration of drugs, which act directly or indirectly on the coagulation system (Moore et al., 2021). Besides detrimental effects on the coagulation system, hypothermia increases morbidity and mortality in trauma patients (Jurkovich et al., 1987; Wolberg et al., 2004; Gerecht, 2014; Vardon et al., 2016; Ise et al., 2020; van Veelen and Brodmann Maeder, 2021). In mild hypothermia (35°–32°C), coagulopathy results primarily from inhibited platelet adhesion. Wolberg et al. showed that platelet aggregation and adhesion were significantly reduced at 33.0°C compared with 37.0°C (p < 0.05) and below 33.0°C, enzyme activity and platelet function were significantly reduced (Wolberg et al., 2004).

The diagnosis of hypothermia-associated coagulopathy is still challenging. The negative effects of hypothermia on plasma coagulation and cellular hemostasis cannot be identified with standard laboratory techniques such as platelet count, prothrombin time (PT) and activated partial thromboplastin time (aPTT), because these parameters are usually measured at the default of 37°C (Martini et al., 2008). Rotational thromboelastometry (ROTEM) is increasingly used as point-of-care analysis of trauma-associated coagulopathy and can better identify trauma associated coagulopathy, already at the bed-side (Johansson et al., 2009; Park et al., 2009; Schöchl et al., 2010; Ise et al., 2020). The ROTEM gamma and delta devices further provide the possibility to measure blood samples at variable temperatures according to the present temperature of the patient. In addition, multiple-electrode aggregometry (MULTIPLATE) has been used recently to assess platelet function and is more frequently being used to assess hypothermia associated impairment in platelets (Kander et al., 2014; Würtz et al., 2014). However, direct comparisons of coagulation alterations with different blood temperatures by ROTEM and MULTIPLATE are not available. In a previous study our research group has evaluated the overall impact of hypothermia on coagulation and have introduced Real Time Live Confocal Microscopy as a new tool to visualize hypothermia-induced impairment of thrombin (Wallner et al., 2020). This in vitro follow-up study was designed to focus on the clinical aspects of hypothermia-associated coagulopathy and on the important role of platelets which is evaluated by comparing measurements of ROTEM and MULTIPLATE with low blood temperatures that clinically conform to mild to severe hypothermia.

This study provides evidence that mild to moderate hypothermia have detrimental effects on coagulation as shown by ROTEM and MULTIPLATE. Severe hypothermia inhibits the function of platelets as shown by the linear decrease in platelet function in MULTIPLATE, but ROTEM does not reflect these changes. Our study shows that viscoelastic tests like ROTEM were unable to reflect the adverse effects of severe hypothermia on platelets, as ROTEM may overestimate clot stability and clot firmness by increased blood viscosity at very low temperatures.

This study is a follow-up study of a previously published analysis, and was designed to systematically analyze clinically important effects of several stages of hypothermia on various components of the coagulation system (Wallner et al., 2020). The preceding study focused on real time live confocal microscopy which enabled to visualize various hypothermia associated changes in the coagulation system as a result of the impairment of thrombin and platelets. In contrast to the previous study, this study specifically focuses on the detection of coagulopathy with point-of-care methods like ROTEM and MULTIPLATE at the different stages of hypothermia. This study was conducted as an in-vitro study analyzing whole blood from healthy volunteers. The blood samples were cooled to predefined temperatures and coagulation tests including ROTEM and MULTIPLATE were performed starting from the lowest temperature (13.7°C) to normothermia (37.0°C). ROTEM and MULTIPLATE have been used for the analysis of hypothermia-associated effects on coagulation in previous studies (Kettner et al., 2003; Rundgren and Engström, 2008; Ruzicka et al., 2012). We did not find any significant difference between female and male participants. However, a difference in the coagulation system is described in the literature, with a trend towards higher levels of both fibrinogen and Factor VII in women as well as a higher platelet count and higher platelet reactivity compared to men (Ossei-Gerning et al., 1998; Ranucci et al., 2019).

Patients out of hospital and in the hospital are at risk of hypothermia (Brown et al., 2012; Rauch et al., 2021). Hypothermia may be life threatening in bleeding and traumatized patients. For example, a systematic review on the effects of mild hypothermia on blood loss reported that a drop in core temperature by 1°C is associated with 10–15% increase of intraoperative bleeding in major surgery and 12% increase of packed red blood cells transfusion (Rajagopalan et al., 2008). In a state-wide study performed in Pennsylvania, mortality in multiple trauma patients increased exponentially from <5% at 37°C to almost 50% at <32°C (Wang et al., 2005). Frequently, in trauma patients only standard coagulation test (blood count, PT, aPTT and Fibrinogen-count) are performed.

Those parameters fail to accurately represent the coagulative state of the patient for two reasons. The first reason is that testing of routine parameter is not performed at the patients’ temperature, but samples are warmed by the analyzer by default to 37.0°C. The second reason is that PT or aPTT fail to detect relevant coagulation abnormalities caused by hypothermia. A study showed that ROTEM is better than PT and aPTT in detecting clinically relevant clotting abnormalities and pointed out that there were no significant changes in PT or aPTT at 32.0°C (Martini et al., 2008). Another recent study confirmed that standard coagulation parameters (PT and aPTT) do neither correspond with ROTEM results nor with patients’ blood loss. However, a significant correlation (p = 0.001) was seen between fibrinogen levels and alpha angles (Sharma et al., 2018). Thus, we assume that our study may be a valuable contribution to accurately assess hypothermia-associated coagulopathy.

In the present study, the proposed temperature range for mild hypothermia (clinical stage 1 in the Swiss staging system) is <35–32°C (Pasquier et al., 2019). Using the criteria of hypothermia, we observed that all ROTEM parameters (EXTEM CT, A5, CFT, MCF and alpha-angle) were significantly impaired in mild hypothermia below baseline (37°C). A drop in blood temperature to 32°C resulted in a significant reduction of CT, CFT, A5 and the alpha-angle.

An in vitro study that compared coagulation at 36°C with coagulation at 32°C using ROTEM confirmed that coagulation was impaired, as expressed by an increased clotting time by 50% at 32°C (Kettner et al., 2003). The same study stated that clot formation was delayed (alpha-angle of 52.9° at 32.0°C vs. 61.8° at 36.0°C) in temperature-adjusted measurements, which indicates that hypothermia reduces plasmatic coagulation and platelet reactivity, but it showed no change if the test temperature was performed at 37°C (Kettner et al., 2003). Mild induced (therapeutic) hypothermia is used for neuroprotection in patients successfully resuscitated after cardiac arrest. In these patients, a body temperature between 36.0 and 33.0 ± 1°C is targeted after the cardiac arrest (Hypothermia after Cardiac Arrest Study Group, 2002; Holzer et al., 2005; Rout et al., 2020; Dankiewicz et al., 2021; Nolan et al., 2021). Furthermore, mild hypothermia is currently undergoing studies in several other indications, e.g., after neonatal asphyxia, myocardial infarction, and traumatic brain injury (Shankaran, 2009; Crossley et al., 2014; Datta, 2017).

The impact of induced hypothermia on coagulation within the range used after cardiac arrest produced minimal but significant prolongation of CT (at 34.0°C) in the study by Rundgren et al. A randomized controlled trial assessing blood loss and transfusion requirements in patients undergoing total hip arthroplasty have shown that intra- and postoperative blood loss was significantly higher in the patients with lower core temperatures: 2.2 versus 1.7 L, (p < 0.001) when temperature was decreased from 36.4 to 35.0°C (Schmied et al., 1996; Rundgren and Engström, 2008). However, a study by Wolberg et al. noted that thrombin generation is not affected when temperature is decreased from 37.0 to 33.0°C (Wolberg et al., 2004).

In severe hypothermia (18 and 13.7°C) this study confirmed increasingly impaired ROTEM parameters. The CT was almost doubled compared to baseline values at 18°C. MULTIPLATE results were also significantly impaired and increasing malfunction was evident with decreasing temperature (Supplementary Table S1), resulting in an almost 100% reduction of the AUC at 13.7°C for all applied tests parameters (ADP, ASPI and TRAP). Impairment of coagulation factor enzymatic activity at low temperature is known in the literature, but these studies do not report the degree of impairment in vivo (Reed et al., 1990; Reed et al., 1992; Meng et al., 2003). As shown by the present data, hypothermia has detrimental effects on platelet but also on coagulation factors. Hypothermia has a significant negative impact on thrombin generation since it primarily inhibits the initiation phase of thrombin generation (Martini, 2009). The physiological effect of thrombin as a promotor of coagulation factors V, VIII and XI is inhibited. A study performed on survivors after out-of-hospital cardiac arrest treated with targeted temperature management (TTM) at 33 ± 1°C confirmed that TTM impaired thrombin generation. These studies support the findings of our study. Even though we did not measure thrombin directly, we demonstrated the detrimental effects of hypothermia on thrombin generation indirectly through a prolonged CT, a change in alpha-angle and significantly reduced platelet function as demonstrated in MULTIPLATE. A previous study described dynamic changes in the metabolism of fibrinogen during hypothermia. Hypothermia at 32°C primarily inhibits the initiation phase and decreases fibrinogen synthesis as compared to normothermia (Martini, 2007; Martini, 2009; Martini, 2010).

Severe hypothermia, besides having a significant impact on humoral coagulation, is further impairing platelet count and platelet function, adding to the negative consequence on all parts of coagulation. In our study, MULTIPLATE was used to analyze the temperature-associated effect on platelets. We found a significant correlation between low temperatures and platelet dysfunction. At normothermia platelet activity was normal but started to decrease with lower temperature, with almost no detectable platelet activity at and below 18.0°C.

Controversy continues concerning platelet function at mild and moderate hypothermia. A study performed by Scharbert et al. stated that in clinical stage 1 hypothermia (34°C), overall platelet aggregation was increased compared to normothermia (37°C) (Scharbert et al., 2010). However, in severe hypothermia the negative impact on platelet mobility and aggregation is unquestioned (Patt et al., 1988; Van Poucke et al., 2014; Jeppesen et al., 2018). Wolberg et al. stated that mainly platelets and tissue factor-bearing cells can potentially be affected by temperature (Wolberg et al., 2004). Also, platelet aggregation and adhesion were significantly reduced at 33.0°C by comparison with 37.0°C (platelet aggregation size 46.5 at 37.0°C vs. 28.1 at 33.0°C). Below 33°C, however, both enzyme activity and platelet function were reduced (Wolberg et al., 2004). The detrimental effects of hypothermia on platelet function are of utmost importance in multiple trauma patients. Tissue damage and shock-related hypoperfusion in multiple trauma render platelets hyporesponsive (Neal, 2020; Vulliamy et al., 2021). Traumatic brain injury creates a hypocoagulable state by depleting clotting factors systemically and provoking platelet inhibitors (Maegele et al., 2017). The effect of hypothermia and multiple trauma are synergistic.

The present study showed inconsistent effects of hypothermia on platelets by ROTEM and MULTIPLATE. MCF in EXTEM was unchanged even at lowest temperature of 13.7°C but significant reductions were shown in all the MULTIPLATE measurements. MCF represents the strength and firmness of the clot, to which platelets play a substantial role. The unchanged MCF in this study might lead to the false conclusion that the hypothermic patient still has intact clot formation and platelets. The study by Rundgren et al. found that MCF was the variable least impaired at low temperatures (25°C) and concluded that the hypothermia-associated effect is more likely to be related to humoral coagulation factors (Rundgren and Engström, 2008). The authors further argued that this finding suggests that a general impairment of procoagulant enzyme systems occurs that affect hemostatic and platelet activity.

Another study by Kettner et al. found no change in clot strength, which is the variable equivalent to MCF in ROTEM (Kettner et al., 2003). Also, Solomon et al. pointed out that MCF was inappropriate as a parameter because clot amplitude and clot elasticity were not properly correlated (Solomon et al., 2015). Their study indicated that platelet contribution to clot strength was underestimated by using MCF and that MCF also remains unchanged across a range of platelet counts (from 10,000 to 100,000/μL). This was in accordance with previous studies and can be explained by the fact that ROTEM measures the viscosity of the blood and the clot. Cooled blood demonstrates a higher viscosity and ROTEM, as a viscometer, measures false normal values especially in MCF (Wallner et al., 2020).

ROTEM is more frequently used as point-of-care testing and has already been integrated into treatment algorithms for massive hemorrhage. Considering only CT and MCF as clinically important parameters, clinically relevant hypothermia induced impairment of platelets will be missed.

In 2020 a case report of a 2-year old boy surviving neurologically intact an unintentional drop to a core temperature of 11.8°C was reported (Mroczek et al., 2020). However, the clinical relevance between the 13.7°C as lowest temperature used in our study and the 11.8°C is minor because only two individuals unintentionally reached and survived these temperatures. We are not aware of any differences in the function of coagulation at these ultra-deep temperatures and only very few patients will ever reach these temperatures. In order to cool the (normothermic) blood samples to the set temperature we had to incubate all the samples of our healthy volunteers for 30 min in a water-bath at the desired temperature before we commenced the analysis. We acknowledge the fact that the situation in hypothermic trauma patients is different because the blood samples in hypothermic patients already represent the exact temperature of the patient and do not need to be cooled further. The blood samples were systematically cooled to specifically predefined temperatures and coagulation tests were performed starting from 13.7 to 37.0°C. By starting from the lowest temperature, we wanted to demonstrate that the adverse effect on coagulation are first and foremost caused by the low temperature and not by other impairing factors, such as storing of the blood samples. Using a single blood sample at every temperature stage would have been better. We further acknowledge that in multiple trauma patients several factors impair the coagulation system such as hemorrhage, inflammation, acidosis, but also therapeutic interventions like volume resuscitation and the administration of drugs which act directly or indirectly on the coagulation system. We acknowledge that low temperatures in-vitro do not directly resemble hypothermia in-vivo. The clinical picture of hypothermia constitutes of numerous pathophysiological changes like inflammation, release of acute phase proteins and cytokines, and activation of the complement system. In the present study we aimed to isolate the distinct effects of low temperatures on platelets and the hemostatic system. The difference between MCF in ExTEM and FibTEM to subtract the fibrinogen component and thereby estimate the contribution of platelets to the clot has not been calculated as a surrogate marker.

Hypothermia impairs coagulation by prolonging coagulation clotting time and by decreasing the velocity of clot formation in ROTEM measurements. MULTIPLATE testing confirms a linear decrease in platelet function with decreasing temperatures, but ROTEM fails to adequately detect hypothermia induced impairment of platelets.