Wide Temperature Range Testing with ROTEM Coagulation Analyses

Abstract

Mild induced hypothermia is used for neuroprotection in patients successfully resuscitated after cardiac arrest. Temperature-dependent effects on rotational thromboelastometry (ROTEM^®) assays with EXTEM^®, FIBTEM^®, or APTEM^® in cardiac arrest patients have not previously been studied. Ten patients with out-of-hospital cardiac arrest who underwent induced hypothermia were studied during stable hypothermia at 33°C. ROTEM temperature effects on EXTEM, FIBTEM, and APTEM assays were studied at temperatures set between 30°C and 42°C. Citrated whole blood test tubes were incubated in temperature-adjusted heating blocks and then investigated at respective temperature in the temperature-adjusted ROTEM. The following variables were determined: clotting time (CT), clot formation time (CFT), α-angle, and maximum clot firmness (MCF). The results from hypo- and hyperthermia samples were compared with the samples incubated at 37°C using the Wilcoxon matched-pairs signed-rank test. A p-value of <0.05 was considered significant. CT-EXTEM^® and CT-APTEM^® were prolonged by hypothermia at 30°C (p<0.01 for both) and 33°C (p<0.05 for both). Hyperthermia at 42°C shortened CT-EXTEM (p<0.05) and CT-APTEM (p<0.01). CFT-EXTEM^® and CFT-APTEM^® were markedly prolonged by hypothermia at 30°C, 33°C, and 35°C (p<0.01 for all except CFT-EXTEM, 35°C [p<0.05]). The α-angle-EXTEM was markedly decreased at 30°C, 33°C, and 35°C (p<0.01) but increased at 40°C (p<0.05) and 42°C (p<0.01); α-angle-APTEM showed similar results. MCF was unchanged at different temperatures for all tests. ROTEM (EXTEM, FIBTEM, and APTEM assays) revealed a hypocoagulative response to in vitro-applied hypothermia in the blood of cardiac arrest patients reflected in the prolonged clot initiation and decreased clot propagation. Hyperthermia showed the opposite effects. Clot firmness was not affected by temperature.

Introduction

Hypothermia is frequently used as a therapeutic option to protect against neurological deficits in comatose survivors after cardiac arrest (Bernard et al., 2002; Holzer et al., 2002; Peberdy et al., 2010) and in cardiac surgery (Luehr et al., 2014). In trauma, the combination, hypothermia, acidosis, and coagulopathy, is commonly referred to as the lethal triad and affects mortality (Mikhail, 1999). Deep hypothermia has been shown to affect both coagulation and platelet function (Wolberg et al., 2004; Ortmann et al., 2012). Whether mild induced hypothermia, 33°C–36°C, has the same effect is still debated (Varon and Acosta, 2009; Polderman, 2012).

The effect of hypothermia on thromboelastography (TEG^®) (Heinius et al., 2002; Shimokawa et al., 2003; Martini et al., 2008; Ivan et al., 2011) and rotational thromboelastometry (ROTEM^®, Pentapharm GmbH, Munich, Germany), using INTEM^® or HEPTEM^® assays (Dirkmann et al., 2008; Rundgren and Engström, 2008; Spiel et al., 2009), has previously been investigated. Two of these studies were performed on cardiac arrest patients (Spiel et al., 2009; Ivan et al., 2011) and the others on healthy volunteers (Dirkmann et al., 2008; Rundgren and Engström, 2008) or animals (Heinius et al., 2002; Shimokawa et al., 2003; Martini et al., 2008). These studies show that during hypothermia (30°C–35°C), the time of clot initiation and the speed of clot propagation are increased, but maximal clot strength and firmness are essentially unchanged compared with normothermia. EXTEM^®, FIBTEM^®, and APTEM^® are the other ROTEM^® assays (Table 1) with different activators that have emerged and are extensively used in trauma patients (Schochl et al., 2010). To the best of our knowledge, the effect of different temperatures on these assays in cardiac arrest patients has not been investigated. The aim of the present study was to investigate the temperature-dependent effects on ROTEM (EXTEM, FIBTEM, and APTEM assays) using whole blood isolated from patients who suffered an out-of-hospital cardiac arrest (OHCA) and were treated with hypothermia.

Table 1.

Overview of ROTEM Assays

Assay	Activator	Additives	Information
EXTEM^®	Tissue factor		Global test. Plasmatic coagulation factors, fibrin polymerization, platelet function and count.
FIBTEM^®	Tissue factor	Platelet inhibition by cytochalasin D	Fibrin status. Identification of polymerization disorders or deficiency.
APTEM^®	Tissue factor	Fibrinolysis inhibition by aprotinin	Detection or exclusion of hyperfibrinolysis.
INTEM^®	Contact activator		Global test. Plasmatic coagulation factors, fibrin polymerization, platelet function and count.
HEPTEM^®	Contact activator	Heparin inactivation by heparinase	Screening test in the presence of heparinase.

Materials and Methods

This prospective observational study was approved by the regional ethical review board in Lund (registration numbers 411/2004, 223/2008, and 2013/284) and included comatose survivors of OHCA of all origins at the Department of Intensive and Perioperative Care, Skåne University Hospital, Lund, Sweden. Informed and written consent was obtained from all survivors. Patients were eligible if they had return of spontaneous circulation after nontraumatic OHCA of all origins, were comatose (GCS≤7) upon admission, and were >18 years old. Patients were treated according to the standard of care after cardiac arrest with body temperature at 33°C for 24 hours.

During stable hypothermia, blood was drawn through an arterial catheter using the Safedraw™ PMSET 1DT (Argon Critical Care Systems, Singapore, Singapore) and collected in tubes (BD Vacutainer Systems, Plymouth, United Kingdom). Blood samples were analyzed by conventional coagulation tests, prothrombin time (PT)/international normalized ratio (INR), activated partial thromboplastin time (aPTT), platelet count, fibrinogen, and C-reactive protein (CRP), along with ROTEM.

Conventional laboratory analyses were performed at the accredited hospital laboratory according to the standard procedure. The reference range for platelets is 165–387×10⁹/L for adult women and 145–348×10⁹/L for adult men. The reference range for PT/INR is 0.9–1.2, for aPTT is 22–44 seconds, and for fibrinogen is 2–4 g/L. Normal value for CRP is <3 mg/L.

ROTEM

Blood was collected in 4.5-mL tubes containing 0.109 M citrate (BD Vacutainer Systems). The tubes were incubated for 30–60 minutes at specified temperatures of 30°C, 33°C, 35°C, 37°C, 40°C, and 42°C in heating blocks. Temperatures were ensured with a temperature probe in reference sample tubes stored in the heating blocks. Analyses were performed with the ROTEM instrument set on the incubation temperatures. Tissue factor was used to trigger coagulation for all assays (EXTEM, FIBTEM, and APTEM). Thrombin-mediated platelet activation and fibrin polymerization are reflected on EXTEM, whereas fibrin polymerization is selectively shown on FIBTEM by inhibiting platelet–fibrin interactions using cytochalasin D. In APTEM, aprotinin is added to eliminate any fibrinolytic component in the sample. A summary of the various tests is shown in Table 1.

The following parameters were analyzed and digitally recorded:

Clotting time (CT): time from the start of measurement until the first signs of clotting— reflects initiation of clotting with thrombin and fibrin formation and then the initial start of clot polymerization (on ROTEM defined as the time until a 2 mm amplitude is detected on the thromboelastogram).

Clot formation time (CFT): time after CT until a clot firmness of 20 mm is detected on the thromboelastogram— reflects clot formation dynamics (clot propagation) and depends on fibrin polymerization, stabilization of the clot with platelets, and F XIII.

The α-angle: also reflects clot formation dynamics (clot propagation) and is defined by the angle between the center horizontal line and a tangent to the curve through the 2 mm amplitude point.

Maximum clot firmness (MCF): firmness of the clot— increasing stabilization of the clot by the polymerized fibrin, platelets, as well as F XIII.

Statistics

Variables were considered nonparametric (Gaussian distribution not assumed) and were summarized using the median with 25th and 75th percentiles Q2 (Q1 and Q3). Results for 30°C, 33°C, 35°C, 40°C, and 42°C samples were compared with results from the normothermic blood samples (37°C) using two-tailed, Wilcoxon matched-pairs signed-rank test. p-Values <0.05 were considered significant. All statistical analyses were performed using the GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA).

Results

Whole blood from 10 cardiac arrest patients was included. For patient demographics, see Table 2.

Table 2.

Patient Demographics and Laboratory Results (n=10)

Age (years)	63±9
Male (%)	5 (50)
SAPS3	68±18
EMR (%)	51±29
Bystander CPR (%)	8 (80)
Time to ROSC (min)	27 (5–45)
30-Day mortality (%)	5 (50)
PCI (%)	6 (60)
Dual platelet inhibition^a	6 (60)
Origin of cardiac arrest
Acute myocardial infarction (%)	7 (70)
Primary arrhythmia (%)	2 (20)
Hypoxia (%)	1 (10)
Laboratory results^b
Platelets, 10⁹/L	176±61
PT/INR	1.1±0.1
aPTT, seconds	36±9
Fibrinogen, g/L	3.6±1.1
CRP, mg/L	68±34

Mean±SD or medians with range (min–max).

Ticagrelor and aspirin.

At stable hypothermia, 33°C.

SAPS3, simplified acute physiology score 3; EMR, estimated mortality risk; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation; PCI, percutaneous coronary intervention; PT/INR, prothrombin time/international normalized ratio; aPTT, activated partial thromboplastin time; CRP, C-reactive protein.

Results from the conventional blood analyses are shown in Table 2. Results from ROTEM analyses are shown in Table 3 and Figures 1 and 2. The most important findings are presented below. All p-values represent a comparison with the results obtained at normothermia (37°C).

FIG. 1.

ROTEM^® (EXTEM^® and APTEM^® assays) on whole blood from cardiac arrest patients in stable hypothermia. The blood was incubated at specified temperatures of 30°C, 33°C, 35°C, 37°C, 40°C, and 42°C before analysis. Analyses were performed with the ROTEM instrument set on the incubation temperatures. Clotting time (CT). Clotting formation time (CFT). Box plots presented as the median with interquartile range and min–max whiskers. *p<0.05. **p<0.001.

FIG. 2.

ROTEM (EXTEM, FIBTEM, and APTEM assays) on whole blood from cardiac arrest patients in stable hypothermia. The blood was incubated at specified temperatures of 30°C, 33°C, 35°C, 37°C, 40°C, and 42°C before analysis. Analyses were performed with the ROTEM instrument set on the incubation temperatures. Maximum clot firmness (MCF). Box plots presented as the median with interquartile range and min–max whiskers. *p<0.05. **p<0.001.

Table 3.

ROTEM Analyses (n=10)

	30°C	33°C	35°C	37°C	40°C	42°C
CT, seconds
EXTEM	87 (79–113)^a	76 (69–89)^b	70 (66–76)	72 (63–84)	66 (53–79)	56 (46–68)^b
APTEM	98 (75–123)^a	76 (62–128)^b	74 (68–87)	74 (64–85)	64 (60–85)	58 (50–66)^a
CFT, seconds
EXTEM	152 118–192)^a	112 (92–148)^a	108 (80–121)^b	90 (63–100)	73 (58–82)^a	73 (52–78)^a
APTEM	162 (128–189)^a	119 (85–145)^a	106 (81–122)^a	92 (65–103)	74 (59–86)^a	66 (47–89)^a
α-Angle
EXTEM	66 (59–70)^a	70 (66–75)^a	72 (69–74)^a	76 (72–77)	77 (75–78)^b	79 (78–80)^a
APTEM	64 (75–68)^a	69 (65–73)^b	71 (67–74)^b	73 (70–77)	76 (74–78)^a	78 (75–80)^a
MCF, mm
EXTEM	58 (52–71)	60 (56–70)	62 (57–68)	64 (59–69)	64 (59–67)	61 (58–68)
FIBTEM	18 (13–22)	20 (13–24)	20 (15–25)	22 (16–24)	22 (13–24)	22 (14–25)
APTEM	58 (52–69)	62 (57–71)	60 (56–68)	62 (57–66)	62 (58–72)	59 (57–67)

p<0.01 and ^bp<0.05 indicates significance level when compared to 37°C.

CFT, clot formation time; CT, clotting time; MCF, maximum clot firmness.

CT-EXTEM^® and CT-APTEM^® were prolonged by hypothermia at 30°C (p<0.01 for both) and 33°C (p<0.05 for both). Hyperthermia at 42°C shortened CT-EXTEM (p<0.05) and CT-APTEM (p<0.01) (Table 3 and Fig 1).

CFT-EXTEM^® and CFT-APTEM^® were prolonged by hypothermia at 30°C, 33°C, and 35°C (p<0.01 for all except CFT-EXTEM, 35°C [p<0.05]) (Table 3 and Fig. 1).

The α-angle-EXTEM^® was decreased at 30°C, 33°C, and 35°C (p<0.01) but increased at 40°C (p<0.05) and 42°C (p<0.01). The α-angle-APTEM^® showed similar results (Table 3 and Fig. 2).

No temperature-dependent differences were observed for MCF-EXTEM^®, MCF-FIBTEM^®, and MCF-APTEM^® (Table 3 and Fig. 2).

Discussion

In this prospective observational study in OHCA patients treated with mild induced hypothermia, we have demonstrated that in vitro-applied hypothermia prolonged the clot initiation and decreased the clot propagation as measured with ROTEM (EXTEM and APTEM assays). Hyperthermia had the opposite effects. Clot strength (MCF) was not affected by temperature in this study. Our results are in agreement with previous studies, which were performed with TEG or other ROTEM assays with nontissue factor-activated agonists (Heinius et al., 2002; Shimokawa et al., 2003; Martini et al., 2008; Rundgren and Engström, 2008; Spiel et al., 2009; Ivan et al., 2011).

Viscoelastic instruments such as TEG and thromboelastometry (ROTEM) reflect both the initiation of the coagulation cascade and its propagation and the final clot structure, revealing dynamic interactions of fibrin polymerization, platelet function, and fibrinolysis at different temperatures, not detected by routine coagulation tests (Ganter and Hofer, 2008). However, in hypo- and hyperthermia, there are many factors affecting hemostasis that are not measurable with TEG or ROTEM. OHCA patients often develop postresuscitation stress responses after cardiac arrest, presumably due to the low-flow state during cardiac arrest, followed by a reperfusion injury, which causes the procoagulative systemic inflammatory response syndrome (Adrie et al., 2005; Koch et al., 2013). Furthermore, several studies have shown increased platelet activity in conjunction with mild induced hypothermia (Xavier et al., 2007; Högberg et al., 2009; Scharbert et al., 2010; Ortmann et al., 2012). These factors that strengthen hemostasis in OHCA patients during hypothermia are offset by delayed and slower clot formation demonstrated with thromboelastometry in this and previous studies (Heinius et al., 2002; Shimokawa et al., 2003; Martini et al., 2008; Rundgren and Engström, 2008; Spiel et al., 2009; Ivan et al., 2011). In patients who underwent percutaneous coronary intervention, the procoagulative effects described above are also offset by dual platelet inhibition medication. Gibbs (2009) confirmed that the effect of these powerful drugs is not detectable with thromboelastometry, which warrants the treatment of patients with and without dual platelet inhibition in one coherent group when evaluating ROTEM results, as was performed in the present study by us.

In this and other studies (Dirkmann et al., 2008; Rundgren and Engström, 2008; Meyer et al., 2013), in vitro-applied hyperthermia (39°C–42°C) strengthened coagulation as demonstrated by decreasing ROTEM CT, CFT, and an increasing α-angle (Table 3 and Figs. 1 and 2). This indicates that clot initiation and propagation is faster in the febrile patient, thus inducing a possible hypercoagulable state.

The current investigation was performed on OHCA patients to ascertain temperature-dependent effects on the ROTEM analysis. This population was chosen since healthy volunteers and animals in an experimental model probably do not have the same coagulation profile as OHCA patients. However, this may also be a limitation of this study since OHCA patients are a divergent cohort. We also acknowledge the inherent limitations of this investigation due to its in vitro design.

Measuring hemostasis is very complex. Multiple pathways for platelet activation coagulation exist, and some are flow and shear stress dependent (Schött and Johansson, 2013). Different in vitro tests reflect limited characteristics of hemostasis. Our study broadens the understanding of how temperature affects ROTEM, using EXTEM, FIBTEM, and APTEM assays, and the demonstrated delayed coagulation start may be part of the explanation for the bleeding diathesis seen in hypothermic patients (Schmied et al., 1996).

Conclusion

ROTEM (EXTEM, FIBTEM, and APTEM assays) revealed a hypocoagulative response to in vitro-applied hypothermia in the blood of cardiac arrest patients reflected in the prolonged clot initiation and decreased clot propagation. Hyperthermia showed the opposite effects. Clot firmness was not affected by temperature.

Footnotes

Author Disclosure Statement

Thomas Kander, Jens Brokopp, and Ulf Schött have no competing financial interests. Hans Friberg has received lecture fees from Bard Medical.

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