Keywords
blood coagulation tests - critical care - fibrinolysis - sepsis - thromboelastography
Introduction
Impairment of the fibrinolytic system is increasingly considered a key contributor
in sepsis-induced coagulopathy (SIC), which is a frequent and serious complication
to sepsis.[1] SIC is characterised by global activation of the coagulation system with microvessel
thrombosis and simultaneous consumption of coagulation factors leading to increased
bleeding risk. Its presentation can range from subclinical coagulation derangements
to disseminated intravascular coagulation (DIC) in the most severe cases.[2] The pathophysiology behind SIC is complex and not yet fully understood. However,
it is increasingly apparent that dysregulated fibrinolysis may play a pivotal role
in the development of SIC.[3]
[4]
[5] Thus, impaired fibrinolysis in sepsis may contribute to adverse outcomes such as
microvascular thrombosis, organ dysfunction, and mortality.[6]
[7]
[8]
[9]
[10]
Additionally, abnormalities in the fibrinolytic system have been described in other
critically ill patient populations including trauma patients,[11] postoperative patients,[12] and individuals with liver disease.[13] Currently, we have few, if any, options for assessing fibrinolysis in the routine
diagnostic laboratory.[14] Circulating plasma concentrations of pro- and antifibrinolytic proteins such as
plasminogen activator inhibitor 1 and thrombin-activatable fibrinolysis inhibitor
can be measured[14] but do not provide dynamic information about fibrinolytic capacity. Plasma-based
clot formation and lysis assays are available for research use and can provide more
detailed information on fibrinolytic capacity[9]
[15]
[16]; but due to lack of automation and standardisation, these assays are currently not
suitable for routine clinical use, especially not in critically ill patients where
short turnaround times are needed.
In contrast, viscoelastic haemostatic tests, e.g., rotational thromboelastometry (ROTEM®),
are implemented in routine laboratories or point-of-care settings worldwide and are
well suited for the acute setting as they have short runtimes and are semi-automated.
Furthermore, as the viscoelastic tests utilise whole blood, they enable a global evaluation
of fibrinolysis that considers the influence of cellular components on the fibrinolytic
process. However, the most widely used viscoelastic protocols are designed for use
in bleeding patients and are not sensitive toward hypofibrinolysis as they include
0% lysis in their reference interval. Thus, the evaluation and quantification of impaired
fibrinolysis in the clinical setting remains a challenge.[4]
In recent years, viscoelastic assays modified with tissue plasminogen activator (tPA)
or other plasminogen activators have been developed.[17]
[18]
[19]
[20] Exogenous tPA is added to stimulate fibrinolysis, which means that in contrast to
standard viscoelastic tests, full lysis is obtained within the clinically relevant
runtime of an hour. This allows the detection of hypofibrinolysis in the patient sample.
These assays have been used in sepsis patients with promising results.[21]
[22]
[23] However, such studies have lacked critically ill nonsepsis control groups and only
one study investigated associations between fibrinolytic status and clinical outcomes,
such as organ failure and mortality.[21]
Our research group has recently set up a modified ROTEM® with tPA based on standard
EXTEM reagents.[24] The primary objective of the present study was to investigate fibrinolytic capacity
using our novel ROTEM®-tPA assay in sepsis patients admitted to the intensive care
unit (ICU) compared with nonsepsis ICU patients and healthy individuals. Moreover,
we aimed to explore whether fibrinolytic impairment on the first day of admission
was associated with organ failure, venous thromboembolism (VTE), or 30-day mortality
in ICU patients both with and without sepsis.
Materials and Methods
Design and Study Population
The present study was designed as a prospective cohort study. Patients admitted to
the ICU at the Aarhus University Hospital, Denmark from September 2022 to April 2023
were screened for eligibility. Adult patients (≥18 years old) with an expected length
of ICU stay exceeding 12 hours were included. Patients with prior ICU admission within
the preceding 3 months or who had received antifibrinolytic or fibrinolytic treatment
within 24 hours before blood sampling were excluded. Patients were stratified based
on the presence of sepsis at the time of blood sampling according to the Sepsis-3
guidelines.[25] In cases of uncertainty regarding the sepsis diagnosis, the authors determined the
categorisation by assessing whether changes in the Sequential Organ Failure Assessment
(SOFA) score were most likely attributable to the patient's infection or to other
clinical conditions. A blood sample was collected within the first 24 hours of ICU
admission, specifically on the routine morning rounds on the day following admission.
Patients were exclusively included on weekdays for logistic reasons. Written informed
consent was obtained following the blood sampling procedure from the patient or next
of kin and from an independent ICU physician. Data on ROTEM®-tPA in healthy individuals
were obtained from 38 blood donors enrolled from the Department of Clinical Immunology,
Aarhus University Hospital.[24] The study was approved by the Central Denmark Region Committees on Health Research
Ethics (file no. 1-10-72-162-20).
Clinical Data
Clinical data were collected from the patients' electronic medical journals and ICU
observation charts and managed using the REDCap electronic data capture tools hosted
at the Aarhus University, Denmark.[26]
[27] At the time of study enrollment, information regarding age, sex, body mass index
(BMI), smoking status, and comorbidities was recorded. Presence of septic shock was
assessed in accordance with the Sepsis-3 guidelines.[25] Moreover, details on treatment prior to blood sampling were documented, including
treatment with extracorporeal membrane oxygenation, renal replacement therapy, major
surgeries, and the use of anticoagulant medication. Simplified Acute Physiology Score
(SAPS) III was assessed by the attending ICU physician at the time of admission.[28] DIC scores were calculated according to the International Society for Thrombosis
and Haemostasis (ISTH).[29] SIC scores[30] and the highest SOFA scores[25] on the day of blood sampling were also evaluated.
During the 30 days following ICU admission, data were prospectively collected regarding
length of ICU stay, administration of vasopressor medications, utilisation of renal
replacement therapy or mechanical ventilation during admission, 30-day all-cause mortality,
and VTE during the 30 days, verified by relevant imaging (e.g., ultrasound, computed
tomography scan). Imaging was obtained at the discretion of the attending physician.
Additionally, data regarding bleeding (World Health Organization [WHO] Bleeding Score
grade ≥ 1)[31] during the first 7 days of ICU admission were obtained.
Laboratory Methods
Rotational Thromboelastometry Modified with Tissue Plasminogen Activator
Blood was drawn from intra-arterial catheters or, if such a catheter was not in place,
by venipuncture with minimal stasis, and collected in citrated tubes (1.8 mL BD Vacutainer
3.2% sodium citrate). The collected samples were gently inverted and allowed to rest
for 30 minutes. The ROTEM®-tPA assay was performed as previously described.[24] Briefly, undiluted EXTEM reagent was used as the tissue factor (TF) source, and
STARTEM reagent as the calcium source, similar to the standard EXTEM protocol. Human
recombinant tPA (Calbiochem, Sigma-Aldrich, Merck, Darmstadt, Germany) was added to
the STARTEM reagent immediately before analysis to achieve a final concentration of
125 ng/mL tPA in the cup. The ROTEM® analysis was performed at 37°C with a runtime
of 60 minutes. All analyses were run in duplicate and mean parameter values were used.
For each channel, the following standard ROTEM® parameters were registered: clotting
time (CT, seconds), maximum clot firmness (MCF, mm), maximum velocity (MaxV, mm/min),
lysis index 45 (LI45, %), maximum lysis (ML, %), lysis onset time (LOT, seconds),
and lysis time (LT, seconds). ROTEM® parameters are depicted in [Fig. 1]. Patients who did not achieve an LOT or an LT during the 60-minute runtime due to
hypofibrinolysis were assigned an LOT and/or an LT of 3,600 seconds.
Fig. 1 ROTEM® coagulation and lysis parameters. CT, clotting time (seconds, time until an
amplitude of 2 mm is reached); FS, fibrinolysis speed (mm/min, clot breakdown speed
in mm/min between LOT and LT; LI45, lysis index 45 (% of clot amplitude of MCF at
45 minutes after CT); LOT, lysis onset time (seconds, time from CT to 15% decrease
in amplitude of MCF); LT, lysis time (seconds, time from CT until clot firmness has
decreased to 10% of MCF); MaxV, maximum velocity (mm/min, maximum clot formation speed);
MCF, maximum clot firmness (mm, the maximum amplitude reached); ML, maximum lysis
(%, maximum lysis detected during the runtime); t-AUCi, time to attain maximal clot
amplitude after reaching maximal clot formation velocity (minutes, time from MAXV-t
to MCF-t).
Time to attain maximal clot amplitude after reaching maximal clot formation velocity
(t-AUCi, min) was computed according to the methodology by Scarlatescu et al using
the formula: t-AUCi = (MCF-t + CT) − MaxV-t.[23]
Fibrinolysis speed (FS, mm/min), the clot breakdown speed between LOT and LT, was
calculated according to the approach for FSc outlined by Kuiper et al[22] using the formula: FS = Δamplitude (LT − LOT)/Δtime (LT − LOT). In cases where no
LT was available due to hypofibrinolysis, FS was calculated using the amplitude at
60 minutes: 
. If an LOT was unavailable due to hypofibrinolysis, the patient was assigned an FS
of 0 mm/min.
Routine Laboratory Analyses
Platelet count, activated partial thromboplastin time (aPTT), international normalised
ratio (INR), antithrombin, fibrinogen, arterial lactate, and routine laboratory markers
of inflammation and organ dysfunction were analysed at the automated routine laboratory
at the Department of Clinical Biochemistry according to ISO15189:2021 accredited protocols.
Leukocyte count and platelet count were analysed on Sysmex XN-9000 (Sysmex, Kobe,
Japan). INR (Medirox Owren's PT reagent), aPTT (Siemens Dade Actin FS reagent), fibrinogen
(functional, Clauss, Siemens Dade thrombin reagent), antithrombin (functional, Siemens
INNOVANCE reagent) were analysed on Sysmex C5100 (Sysmex, Kobe, Japan). Arterial lactate
was analysed on automated blood gas analysers (ABL800 and ABL90, Radiometer, Brønshøj,
Denmark) and markers of inflammation and organ dysfunction were analysed on Siemens
Atellica chemistry and immunochemistry analysers (Siemens Healthineers, Duisburg,
Germany).
Statistical Analysis
The primary endpoint was the difference in LT between sepsis and nonsepsis patients.
Our protocol was developed to yield LTs of 40 minutes in healthy individuals with
an assumed standard deviation (SD) of 10% (4 minutes).[24] We expected SD to be larger in an ICU population. With a study power (1 − β) of
0.9, a significance level (2α) of 0.05, a minimal relevant difference of 25%, and
an estimated SD in ICU patients of double that of healthy individuals (i.e., 8 minutes),
23 sepsis patients and 23 nonsepsis patients had to be included.
Since we found impaired fibrinolysis not only in sepsis patients but also in nonsepsis
ICU patients, we then defined a secondary endpoint: to investigate the association
between LT and mortality in the overall ICU cohort.
Normal distribution was assessed visually with quantile–quantile plots. Continuous
data were described using median and interquartile range for uniformity, since the
majority of data did not follow a Gaussian distribution. Categorical data were presented
as numbers and percentages. Differences in continuous variables between groups were
tested with the Mann–Whitney test, and Fisher's exact test was used for categorical
data. Association between whole-blood fibrinolysis parameters and mortality was analysed
using uni- and multivariate logistic regression and with the Kaplan–Meier method and
the log-rank test.
All statistical analyses and graphs were generated using GraphPad Prism version 9.5.0.730
for Windows (GraphPad Software, San Diego, California, United States).
Results
Patient Characteristics
A total of 310 patients were screened for eligibility. Of these, 62 were excluded
due to logistical reasons, 61 were excluded due to ICU admission within the preceding
3 months, 12 were excluded due to treatment with pro- or antifibrinolytic agents,
10 had admissions of less than 12 hours, and 6 did not consent to participate. Ultimately,
159 patients were enrolled, 30 sepsis patients and 129 nonsepsis patients.
Baseline clinical characteristics and data regarding bleeding, VTE, and mortality
in sepsis versus nonsepsis patients are reported in [Table 1].
Table 1
Clinical characteristics and bleeding, venous thromboembolism, and mortality data
of sepsis and nonsepsis patients
|
Characteristics
|
Sepsis patients (n = 30)
|
Nonsepsis patients (n = 129)
|
|
SAPS III score at admission
|
63 (48–79)
|
60 (46–73)
|
|
SOFA score day 1
|
9 (7–11)
|
9 (5–11)
|
|
Arterial lactate at admission (mmol/L)
|
1.7 (1.0–2.8)
|
1.6 (1.1–3.5)
|
|
DIC score
|
3 (2–4)
|
2 (2–3)
|
|
DIC score ≥ 5
|
6 (20%)
|
5 (4%)
|
|
SIC score
|
3 (2–4)
|
–
|
|
Septic shock
|
16 (53%)
|
–
|
|
Occurrence of VTE (within 30 d)
|
6 (20%)
|
7 (5%)
|
|
Any bleeding, WHO bleeding score ≥1 (within 7 d)
|
21 (70%)
|
89 (69%)
|
|
Mortality (within 30 d)
|
12 (40%)
|
25 (19%)
|
Abbreviations: DIC, disseminated intravascular coagulation; ICU, intensive care unit;
SAPS, Simplified Acute Physiology Score; SIC, sepsis-induced coagulopathy; SOFA, Sequential
Organ Failure Assessment; VTE, venous thromboembolism; WHO, World Health Organization.
Notes: Continuous variables are reported as medians (interquartile range), whereas
categorical variables are presented as numbers and percentages. The following parameters
have missing data: arterial lactate at admission (n = 2).
Sepsis patients and nonsepsis patients had comparable SAPS III scores, SOFA scores,
and arterial lactate at ICU admission, but sepsis patients demonstrated higher DIC
scores (3 [2–4] vs. 2 [2–3] than nonsepsis patients. Among the sepsis patients, 16
(53%) had septic shock.
Sepsis patients experienced significantly worse outcomes in terms of 30-day mortality
(40 vs. 19%) and VTE development (20 vs. 5%) compared with nonsepsis patients. However,
both groups showed a similar incidence of bleeding episodes during the first 7 days
of admission (70 vs. 69%).
ROTEM®-tPA in Sepsis and Nonsepsis Patients
Results of the ROTEM®-tPA analysis are shown in [Figs. 2] and [3]. Sepsis patients differed from nonsepsis patients across all ROTEM® parameters and
were characterised by a markedly impaired fibrinolytic capacity compared with both
nonsepsis patients and healthy individuals. Median LT was significantly prolonged
among sepsis patients compared with nonsepsis patients (3,600 [3,352–3,600] vs. 3,374 seconds
[2,175–3,600], p < 0.01). A larger proportion of sepsis patients displayed LTs above the 97.5 percentile
of the 38 healthy individuals (3,000 seconds[24] compared with nonsepsis patients (n = 24, 80% vs. n = 73, 57%). Overall, only very few patients demonstrated increased fibrinolysis,
as no sepsis patients and two nonsepsis patients had an LT below the 2.5 percentile
of the 38 healthy individuals (1,080 seconds).[24] ML was significantly lower in sepsis patients than in nonsepsis patients (23 [8–90]
vs. 94 [14–100] %, p = 0.02), as was FS (0.41 [0.0–1.4] vs. 1.6 [0.1–2.7] mm/min, p = 0.01). t-AUCi was higher in sepsis patients than nonsepsis patients, although the
difference was not statistically significant (17 [12–25] vs. 15 [11–21] minutes, p = 0.21). Furthermore, the sepsis population showed prolonged LOT as well as higher
LI45, all indicative of hypofibrinolysis.
Fig. 2 Coagulation and fibrinolysis parameters measured by ROTEM®-tPA in sepsis patients,
nonsepsis patients, and healthy individuals. Boxes represent medians with interquartile
ranges, and whiskers represent the 2.5 and the 97.5 percentiles. p-values were calculated with the Mann–Whitney test. t-AUCi, time to attain maximal
clot amplitude after reaching maximal clot formation velocity.
Fig. 3 Illustrations of ROTEM®-tPA tracings for sepsis patients, nonsepsis patients, and
healthy individuals. Median ROTEM® traces are depicted as solid lines and dotted lines
represent the interquartile range. Figure created with Biorender.com.
Within the nonsepsis group, fibrinolytic capacity ranged from normal to severely impaired.
Altogether, nonsepsis patients had substantially prolonged LOT, LT, and t-AUCi as
well as decreased ML and FS compared with healthy individuals. Thus, the nonsepsis
group were characterised by impaired fibrinolysis when compared with healthy individuals,
although not as pronounced as the sepsis group.
Regarding coagulation parameters, the sepsis group exhibited elevated MCF as well
as a higher MaxV compared with nonsepsis patients, indicating increased clot strength.
However, sepsis patients also demonstrated a slightly longer median CT compared with
controls as shown in [Fig. 2].
Association between Clinical Outcomes and Impaired Fibrinolysis
All 159 patients, including both sepsis and nonsepsis cases, were stratified according
to the presence of hypofibrinolysis defined as LT above the 97.5 percentile of 38
healthy individuals as previously calculated (50 minutes).[24] Most patients (61%) displayed LTs above the 97.5 percentile, indicating an impaired
fibrinolytic capacity, whereas the remaining patients (39%) had LTs below the 97.5
percentile, indicating normal fibrinolysis. Patients with LT below the 2.5 percentile
(n = 2) was included in the normal LT group. Clinical and outcome data for patients
with normal and impaired fibrinolysis are presented in [Table 2].
Table 2
Demographics, clinical characteristics, and bleeding, venous thromboembolism, and
mortality data of intensive care unit patients with normal and impaired fibrinolysis
|
Characteristics
|
Impaired fibrinolysis (n = 97)
|
Normal fibrinolysis (n = 62)
|
p
|
|
Female sex
|
61 (37%)
|
41 (34%)
|
–
|
|
Age (y)
|
67 (54–74)
|
63 (56–71)
|
–
|
|
BMI (kg/m2)
|
27 (24–31)
|
25 (23–30)
|
–
|
|
Current smoker
|
29 (29%)
|
16 (32%)
|
–
|
|
Days in hospital before ICU admission
|
0 (0–2)
|
0 (0–1)
|
–
|
|
Sepsis on day 1
|
24 (25%)
|
6 (10%)
|
–
|
|
SAPS III score at admission
|
63 (49–76)
|
59 (44–71)
|
0.15
|
|
SOFA score day 1
|
10 (7–11)
|
7 (4–10)
|
<0.01
|
|
Arterial lactate at admission (mmol/L)
|
1.9 (1.1–3.9)
|
1.4 (1.0–2.3)
|
<0.01
|
|
DIC score day 1
|
2 (2–3)
|
2 (2–3)
|
0.42
|
|
DIC score ≥ 5
|
9 (9%)
|
2 (3%)
|
0.20
|
|
Interventions before blood sampling
|
|
|
|
|
Renal replacement therapy (24 h)
|
5 (5%)
|
0 (0%)
|
–
|
|
ECMO (24 h)
|
9 (9%)
|
1 (2%)
|
–
|
|
Major surgery (7 d)
|
32 (33%)
|
7 (12%)
|
–
|
|
Platelet inhibitors (14 d)
|
32 (33%)
|
25 (40%)
|
–
|
|
Vitamin K antagonists (14 d)
|
7 (7%)
|
1 (2%)
|
–
|
|
Direct oral anticoagulants (3 d)
|
8 (8%)
|
13 (21%)
|
–
|
|
Heparins (24 h)
|
|
|
|
|
Low molecular weight, prophylactic dose
|
22 (23%)
|
12 (19%)
|
–
|
|
Low molecular weight, therapeutic dose
|
15 (15%)
|
5 (8%)
|
–
|
|
Unfractionated
|
15 (15%)
|
5 (8%)
|
–
|
|
No heparin
|
45 (46%)
|
40 (65%)
|
–
|
|
Comorbidities
|
|
|
|
|
Hypertension
|
40 (41%)
|
29 (43%)
|
–
|
|
Diabetes
|
21 (22%)
|
16 (26%)
|
–
|
|
Ischaemic heart disease
|
18 (19%)
|
19 (31%)
|
–
|
|
Solid cancer
|
12 (12%)
|
4 (6%)
|
–
|
|
Haematologic cancer
|
7 (7%)
|
3 (5%)
|
–
|
|
SARS-Cov-2
|
1 (1%)
|
1 (2%)
|
–
|
|
Occurrence of VTE (within 30 d)
|
11 (11%)
|
2 (3%)
|
0.08
|
|
Length of ICU stay (d)
|
4 (2–8)
|
2 (1–5)
|
0.03
|
|
Interventions during ICU admission
|
|
|
|
|
Mechanical ventilation
|
79 (81%)
|
33 (53%)
|
<0.001
|
|
Renal replacement therapy
|
15 (16%)
|
3 (5%)
|
0.04
|
|
Vasopressor treatment
|
90 (93%)
|
39 (63%)
|
<0.0001
|
|
Any bleeding, WHO bleeding score ≥1 (within 7 d)
|
75 (77%)
|
35 (57%)
|
<0.01
|
|
Mortality (within 30 d)
|
30 (31%)
|
7 (11%)
|
<0.01
|
Abbreviations: BMI, body mass index; DIC, disseminated intravascular coagulation;
ECMO, extracorporeal membrane oxygenation; ICU, intensive care unit; SAPS, Simplified
Acute Physiology Score; SARS-Cov-2, severe acquired respiratory syndrome coronavirus-2;
SIC, sepsis-induced coagulopathy; SOFA, Sequential Organ Failure Assessment; VTE,
venous thromboembolism; WHO, World Health Organization.
Notes: Continuous variables are reported as medians (interquartile range) while categorical
variables are presented as numbers and percentages. The following parameters have
missing data: BMI (n = 8), arterial lactate at admission (n = 2) and length of ICU stay (n = 2). Patients who died during ICU admission (n = 29) were excluded from the length of stay calculation. Further, there are missing
SOFA score component values on two patients and a score of 0 was imputed for missing
variables.
Table 3
Laboratory parameters of intensive care unit patients on day 1 of intensive care unit
admission
|
Parameter
|
Impaired fibrinolysis (n = 97)
|
Normal fibrinolysis (n = 62)
|
|
Platelet count (109/L)
|
221 (160–277)
|
209 (165–273)
|
|
D-Dimer (mg/L FEU)
|
3.0 (1.1–7.3)
|
3.9 (1.1–7.6)
|
|
International normalised ratio, INR
|
1.2 (1.1–1.3)
|
1.2 (1.1–1.3)
|
|
Activated partial thromboplastin time, aPTT (s)
|
27 (24–33)
|
26 (23–30)
|
|
Antithrombin (µmol/L)
|
0.80 (0.66–0.92)
|
0.87 (0.76–1.0)
|
|
Fibrinogen (µmol/L)
|
12.0 (8.7–15.9)
|
11.6 (8.4–14.7)
|
|
C-reactive protein, CRP (mg/L)
|
62 (40–194)
|
56 (16–139)
|
|
Leukocyte count (109/L)
|
13.1 (10.4–17.2)
|
10.0 (7.8–14.1)
|
Abbreviation: FEU, fibrinogen equivalent unit.
Notes: Reported as medians (interquartile range). The following parameters have missing
data: C-reactive protein (n = 1), leukocyte count (n = 1).
Patients with normal fibrinolysis and impaired fibrinolysis had comparable ages, BMI,
sex distribution, DIC scores, and SAPS III scores, but patients with impaired fibrinolysis
had higher SOFA scores and higher arterial lactate than patients with normal fibrinolysis.
A larger proportion in the normal fibrinolysis group had ischaemic heart disease than
in the impaired fibrinolysis group and cancer was slightly more prevalent among those
with impaired fibrinolysis than those with normal fibrinolysis. Apart from that, the
groups were comparable with regards to comorbidity prevalence.
Regarding biochemical parameters obtained on the first morning of admission ([Table 3]), patients with impaired fibrinolysis demonstrated higher C-reactive protein and
leukocyte count than those with normal fibrinolysis. The groups were, however, comparable
with regards to platelet count, D-Dimer, INR, aPTT, antithrombin, and fibrinogen.
All-cause 30-day mortality was 23%. In univariate analysis, age, SAPS-III score, SOFA
score, DIC score, and day 1 ROTEM®-tPA parameters LT, LOT, ML, FS, LI45, and t-AUCi
fibrinolysis were associated with 30-day mortality ([Table 4]). Impaired fibrinolysis on day 1, defined as LT > 50 minutes, was strongly associated
with 30-day mortality, odds ratio [OR] = 3.52 (95% confidence interval [CI]: 1.51–9.27).
This same pattern was also observed when exclusively looking at mortality among the
129 nonsepsis patients, OR = 3.85 (95% CI: 1.33–9.88). SOFA-score and SAPS-score had
the best overall predictive ability for 30-day mortality (area under receiver operator
characteristics [ROC] curve 0.79 and 0.77), whereas ROTEM®-tPA parameters yielded
very moderate areas under ROC curve, highest for t-AUCi (0.67).
Table 4
Odds ratios for 30-day mortality with univariate logistic regression
|
Parameter
|
OR (95% CI)
|
p
|
AUROC
|
|
Age, per 1-y increase
|
1.06 (1.02–1.09)
|
<0.01
|
0.70
|
|
Female sex
|
0.82 (0.37–1.77)
|
0.62
|
0.52
|
|
SAPS III, pr. 1-point increase
|
1.07 (1.04–1.10)
|
<0.0001
|
0.77
|
|
SOFA score, pr. 1-point increase
|
1.43 (1.25–1.67)
|
<0.0001
|
0.79
|
|
ISTH DIC score, pr. 1-point increase
|
1.62 (1.19–2.27)
|
<0.01
|
0.62
|
|
Impaired fibrinolysis[a]
|
3.52 (1.51–9.27)
|
<0.01
|
0.63
|
|
LT, pr. 1-min increase
|
1.05 (1.01–1.09)
|
0.01
|
0.62
|
|
LOT, pr. 1-min increase
|
1.03 (1.01–1.05)
|
0.01
|
0.63
|
|
ML, pr. 10% increase
|
0.91 (0.83–0.99)
|
0.03
|
0.66
|
|
FS, pr. 1-mm/min increase
|
0.74 (0.55–0.99)
|
0.05
|
0.62
|
|
LI45, pr. 1% increase
|
1.01 (1.00–1.02)
|
0.01
|
0.66
|
|
MCF, pr. 1-mm increase
|
1.04 (1.00–1.08)
|
0.07
|
0.61
|
|
MaxV, pr. 1-mm/min increase
|
1.02 (0.97–1.08)
|
0.45
|
0.54
|
|
t-AUCi, pr. 1-min increase
|
1.09 (1.04–1.15)
|
<0.001
|
0.67
|
Abbreviations: AUROC, area under receiver operator characteristics curve; CI, confidence
interval; DIC, disseminated intravascular coagulation; FS, fibrinolysis speed; ISTH,
International Society on Thrombosis and Haemostasis; LI45, lysis index at 45 minutes;
LOT, lysis onset time; LT, lysis time; MaxV, maximum velocity; MCF, maximum clot formation;
ML, maximum lysis; OR, odds ratio; SAPS, Simplified Acute Physiology Score; SOFA,
Sequential Organ Failure Assessment; t-AUCi, time to attain maximal clot amplitude
after reaching maximal clot formation velocity.
a Defined as LT > 50 minutes.
In multivariate analysis including SAPS-III score at admission, SOFA-score, DIC-score,
and impaired fibrinolysis (LT > 50 minutes; [Table 5]), impaired fibrinolysis was still associated with 2.3 times higher risk of 30-day
mortality, although the association was no longer statistically significant (OR = 2.26
[95% CI: 0.83–6.69]). Areas under ROC curve were similar for a model including SAPS + SOFA + DIC
score (0.83 [95% CI: 0.75–0.89]), a model including SAPS + SOFA + DIC + impaired fibrinolysis
(0.83 [95% CI: 0.75–0.90]), and a model including SAPS + SOFA + DIC + t-AUCi (0.83
[95% CI: 0.75–0.90]). A Kaplan–Meier analysis revealed distinct differences in survival
(p = 0.009), as shown in [Fig. 4], with a 30-day survival probability of 69% in the impaired fibrinolysis group and
89% in the normal fibrinolysis group.
Table 5
Odds ratio for 30-day mortality with a multivariate logistic regression model
|
Parameter
|
OR (95% CI)
|
p
|
|
SAPS III score, pr. 1-point increase
|
1.04 (1.01–1.08)
|
0.01
|
|
SOFA score, pr. 1-point increase
|
1.28 (1.08–1.54)
|
<0.01
|
|
ISTH DIC score, pr. 1-point increase
|
0.94 (0.61–1.44)
|
0.77
|
|
Impaired fibrinolysis[a]
|
2.26 (0.83–6.69)
|
0.12
|
Abbreviations: CI, confidence interval; DIC, disseminated intravascular coagulation;
ISTH, International Society on Thrombosis and Haemostasis; LT, lysis time; OR, odds
ratio; SAPS, Simplified Acute Physiology Score; SOFA, sequential organ failure assessment.
a Defined as LT > 50 minutes.
Fig. 4 Probability of survival in 62 patients with normal lysis times (≤50 minutes) and
97 patients with prolonged lysis times (>50 minutes). ICU, intensive care unit.
Furthermore, patients with impaired fibrinolysis on day 1 had a higher incidence of
symptomatic VTE within 30 days of ICU admission (n = 11/97, 11%) than those with normal fibrinolysis (n = 2/62, 3%), with an OR of 3.84 (95% CI: 0.87–17.8), p = 0.08. Impaired fibrinolysis was also associated with longer ICU stays and with
more interventions during ICU admission. A higher incidence of bleeding episodes with
the WHO bleeding score ≥ 1 was observed in patients with impaired fibrinolysis than
in patients with normal fibrinolysis (75 [77%] vs. 35 [57%]).
Discussion
The main finding of the present study was a significant impairment of whole-blood
fibrinolytic capacity in sepsis patients compared with nonsepsis ICU patients measured
with ROTEM®-tPA. Further, ICU patients generally had impaired fibrinolysis compared
with healthy individuals, regardless of the presence of sepsis. In the entire ICU
cohort, patients with impaired fibrinolysis on day 1 of their ICU admission had a
three times increased risk of venous thrombosis and close to three times higher 30-day
mortality compared with patients with normal fibrinolysis. Our findings underline
the importance of fibrinolysis in critical illness and establish ROTEM®-tPA as a clinically
relevant and feasible assay for assessment of fibrinolysis in critically ill patients.
Recent studies utilising plasma-based clot lysis assays have reported an impaired
fibrinolytic capacity in the majority of sepsis patients compared with nonsepsis patients
or healthy individuals.[13]
[15]
[32] Our findings expand previous studies by using a whole-blood assay with a large nonsepsis
ICU control group. Apart from the current study, three other studies have used modified
viscoelastic tests in patients with sepsis. Kuiper et al used ROTEM® modified with
tPA in 21 sepsis patients but excluded those with DIC and did not investigate associations
with clinical endpoints. They reported impaired fibrinolytic capacity in sepsis patients
compared with healthy individuals, pregnant women, and cirrhotic liver disease patients.[22] Panigada et al used a urokinase (uPA) and kaolin-activated thromboelastography (UK-TEG)
assay to compare fibrinolysis in 40 sepsis patients with healthy individuals and found
a higher TEG maximal amplitude and impaired fibrinolysis indicated by lower TEG lysis
index at 30 minutes in sepsis patients. They also investigated associations between
fibrinolysis impairment and adverse outcomes in sepsis patients and reported higher
SOFA scores, mortality, and longer ICU stays in patients classified as “low responders”
to urokinase.[21] A recent paper by the ISTH SSC (Scientific and Standardization Committee) subcommittee
on fibrinolysis by Scarlatescu et al[23] evaluated the utility of t-AUCi parameter (time between MaxV and maximum clot formation)
in ROTEM® with different concentrations of tPA. In 30 sepsis patients, they found
that t-AUCi could discriminate between sepsis patients and healthy controls and was
associated with LI45. We also found higher t-AUCi in sepsis patients than in healthy
controls, but t-AUCi did not differ significantly between sepsis and nonsepsis ICU
patients.
The present study and Kuiper et al[22] found impaired fibrinolytic capacity in the majority of sepsis patients, whereas
Panigada et al[21] reported that more than half of sepsis patients exhibited a normal response to uPA
in their UK-TEG assay. This discrepancy may stem from differences in assays, most
importantly the use of kaolin instead TF and uPA instead of tPA, or use of different
parameters and cutoff points for the definition of hypofibrinolysis.
Since tPA- or uPA-modified viscoelastic assays are relatively new, the most appropriate
parameters for describing fibrinolytic capacity with these assays have not yet been
defined, nor have normal ranges or reference intervals been established. The significant
differences in outcomes observed in the present study suggest that our definition
of hypofibrinolysis has clinical relevance. However, other parameters than LT may
yield more details—for instance LOT, ML, or the calculated FS. Scarlatescu et al proposed
t-AUCi to assess sepsis-related hypofibrinolysis, but since we found no significant
difference between sepsis and nonsepsis ICU patients, other parameters might be better
suited for this. However, the discrepancy between our results may be due to different
tPA concentrations between our assays or differences between our included sepsis populations.
We observed that impaired fibrinolytic capacity correlated with organ dysfunction
and decreased survival in the composite ICU population. Of note, we found that the
association between hypofibrinolysis and mortality persisted even after excluding
sepsis patients from the analysis, indicating that the association cannot be explained
by the higher mortality rates observed in the sepsis group. However, the addition
of LT to a model including SAPS-III, SOFA score and DIC score did not improve area
under ROC curve. Associations between mortality and impaired fibrinolysis measured
with tPA-modified TEG have also been reported in other patient groups such as haemorrhagic
shock patients[20] and liver transplant recipients,[33] which supports the clinical relevance of viscoelastic tests with tPA.
To the best of our knowledge, we are the first to investigate the association between
venous thrombosis and hypofibrinolysis using a modified viscoelastic assay. Previous
studies have reported an association between thrombosis and hypofibrinolysis assessed
with plasma-based assays.[34] Our findings revealed an OR of more than 3 for VTE development in ICU patients with
impaired fibrinolysis compared with those with normal fibrinolysis. Although the wide
confidence interval limits the precision of our estimate, our findings indicate a
substantial increase in risk of VTE associated with hypofibrinolysis and highlight
the potential of ROTEM®-tPA as a marker for identifying patients at increased risk
of VTE.
One unexpected result was a higher 7-day incidence of bleeding episodes with a WHO
bleeding score ≥ 1 in patients with impaired fibrinolysis compared with normal fibrinolysis.
A possible explanation for this observation is that the impaired fibrinolysis group
experienced significantly longer ICU stays, underwent more invasive procedures, and
received more heparins during ICU admissions.
Few patients showed increased fibrinolysis compared with healthy individuals, which
may in part be ascribed to the exclusion of those who were treated with tranexamic
acid due to a possible hyperfibrinolytic state.
Strengths of the present study include a large and diverse ICU group and systematic
collection of detailed clinical and biochemical information. However, this study has
certain limitations that should be considered. First, this was a single-center study
with a relatively small sepsis group (n = 30) and to increase the generalisability of our findings, validation in larger
cohorts is needed. Further, due to the relatively infrequent event rates for mortality,
VTE, and other outcomes, it was not possible to perform statistical analysis to adjust
for possible confounding factors. As the blood sample was taken on the morning round,
the timing of blood sampling in relation to ICU admission was variable between 1 and
23 hours. Despite this variability, we still found a clear association between ROTEM®-tPA
results and clinical outcomes. Furthermore, this design had the advantage of minimising
circadian variation.
Lastly, although our findings demonstrate associations between impaired fibrinolysis
and adverse outcomes in ICU patients, we have not demonstrated causality. Further
research should focus on underlying mechanisms behind lysis resistance assessed in
whole blood and the specific contributions from both pro- and antifibrinolytic proteins
and the formed elements of blood to identify potential therapeutic targets.
In summary, our findings demonstrate that ROTEM®-tPA is a sensitive and feasible tool
for investigation of fibrinolysis in critically ill patients. Impaired fibrinolysis
assessed with ROTEM®-tPA was associated with both mortality and VTE risk in ICU patients.
This method holds the potential to improve early diagnosis and management of disturbed
fibrinolysis with the perspective of more individualised therapy.
What is Known on this Topic?
-
Dysregulated fibrinolysis may play a pivotal role in the development of sepsis-induced
coagulopathy.
-
Routine laboratory tests for fibrinolysis are currently very limited.
-
Impact of fibrinolytic capacity on clinical outcome in intensive care patients is
poorly investigated.
What Does this Paper Add?
-
We used rotational thromboelastometry® modified with tissue plasminogen activator
((ROTEM®-tPA), a novel fibrinolysis assay, to investigate fibrinolysis in 159 intensive
care (ICU) patients.
-
ROTEM®-tPA showed impaired fibrinolysis in sepsis patients compared with non-sepsis
patients.
-
Impaired fibrinolysis was associated with higher 30-day mortality and venous thromboembolism
risk in the entire ICU cohort.
