Journal of Veterinary Diagnostic
The online version of this article can be found at:
2011 23: 698 originally published online 13 June 2011 J VET Diagn Invest
Benjamin M. Brainard, Janan M. Abed and Amie Koenig
thromboelastography and the PFA-100® platelet function analyzer system
The effects of cytochalasin D and abciximab on hemostasis in canine whole blood assessed by
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Journal of Veterinary Diagnostic Investigation
23(4) 698 –703
© 2011 The Author(s)
Reprints and permission:
The in-vitro addition of platelet-inhibiting substances to com-
mon coagulation tests can aid in the diagnosis of hypercoagu-
lable diseases. Specifically, the addition of platelet inhibitors
to whole blood aggregation tests such as thromboelastography
(TEG) and the PFA-100® platelet function analyzer systema
can give information about the contribution of platelets to
hypercoagulable states, and potentially allow clinicians to
customize anti-thrombotic therapy. This approach has been
investigated in human patients following major abdominal
and cardiac surgery,12,17 as well as in healthy rabbits.14 Many
diseases in dogs have been associated with hypercoagulability
and formation of venous or arterial thrombi,11 and although
antiplatelet medications (such as aspirin or clopidogrel) or
anticoagulant medications (e.g., heparin) are frequently rec-
ommended, there are no data to delineate whether factor-
related hypercoagulability, platelet hyperactivity, or both
contribute to the development of thrombi in a given patient.
Knowledge of the primary component of hypercoagulability
may allow more tailored thromboprophylactic treatments.
Thromboelastography refers to a whole blood viscoelastic
coagulation monitoring protocol that depicts the combined
activity of the cellular and plasmatic aspects of coagulation, in
addition to fibrinolysis.9 Thromboelastography has been used
to demonstrate hypercoagulable tendencies in dogs with a
variety of conditions.7 Primary TEG measurements include the
reaction time (R), the alpha angle (Ang), and the maximum
amplitude (MA). Reaction time primarily evaluates the intrin-
sic pathway of coagulation, and thus represents interactions
between coagulation factors VIII, IX, XI, and XII.7Alpha
angle represents the rate of clot formation, and is influ-
enced by concentrations of coagulation factors II and VIII
and fibrinogen, in addition to platelet number and function.
Maximum amplitude represents the maximum clot strength,
and reflects contributions from soluble factors and fibrinogen,
in addition to platelets. Mathematical transformation of the
MA results in a value known as the G value, or the elastic
shear modulus, which occasionally is reported in lieu of MA.32
The PFA-100 system evaluates platelet aggregation under
conditions of simulated shear, and has been evaluated and
validated for use in dogs.5,20 The PFA-100 system uses citrated
ard et al.Abciximab and cytochalasin D in canine platelet function
From the Department of Small Animal Medicine and Surgery, College
of Veterinary Medicine, University of Georgia, Athens, GA.
1Corresponding Author: Benjamin M. Brainard, Department of Small
Animal Medicine and Surgery, College of Veterinary Medicine, University
of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602. firstname.lastname@example.org
The effects of cytochalasin D and
abciximab on hemostasis in canine
whole blood assessed by
thromboelastography and the
PFA-100® platelet function analyzer system
Benjamin M. Brainard,1 Janan M. Abed, Amie Koenig
Abstract. The selective inhibition of platelet function in whole blood coagulation testing may allow insights into the nature
of hypercoagulability in dogs with critical illness. To determine the effects of cytochalasin D and abciximab on hemostatic
parameters in canine citrated whole blood, an in-vitro study was designed using thromboelastography (TEG) and a platelet
function analyzer (PFA-100®). 8 clinically healthy mixed breed dogs donated blood that was anticoagulated with 3.2% sodium
citrate in a 9:1 blood-to-citrate ratio. Addition of cytochalasin D to citrated whole blood from 6 dogs at concentrations ranging
from 0 µg/ml to 10 µg/ml caused a maximal reduction of TEG maximum amplitude (MA) at a concentration of 7.5 µg/ml (52.7
± 4.3 to 14.3 ± 7.8 mm). Addition of abciximab to canine citrated whole blood at concentrations of either 20 µg/ml or 40 µg/
ml did not affect the TEG tracing; however, addition of abciximab to citrated canine whole blood at concentrations of 10 µg/
ml and 20 µg/ml significantly prolonged PFA-100 closure times (72.5 ± 15 to 149.2 ± 91 sec and 275.6 ± 54 sec, respectively,
P < 0.04). Inhibition of canine platelet function by cytochalasin D is demonstrated by TEG, but abciximab did not change TEG
tracings. Abciximab does, however, inhibit platelet aggregation under shear stress as measured by the PFA-100. Inhibition of
canine platelet function with cytochalasin D may allow further TEG studies in dogs with clinical disease.
Key words: Anti-platelet agents; GPIIb/IIIa; PFA-100.
Abciximab and cytochalasin D in canine platelet function 699
whole blood aspirated at high shear rates through disposable
plastic cartridges coated with collagen and adenosine diphos-
phate (ADP) as activators (a cartridge with collagen and epi-
nephrine is also available, but is not frequently used in
evaluation of dogs).5 The end point of the assay is the time to
occlusion of blood flow through the aperture of the membrane,
expressed in seconds and reported as closure time (CT), or
nonclosure if CT exceeds 300 sec.5 In dogs, CT prolongations
are seen with aspirin administration, von Willebrand disease,
and thrombocytopenia.5,20 The PFA-100 system is also sensi-
tive for platelet function disturbances in dogs caused by
exogenous substances such as hetastarch.31
Cytochalasin D is a fungal metabolite that prevents micro-
tubule assembly within the platelet and thus prevents cyto-
skeletal reorganization in response to platelet activation.27
The platelet cytoskeleton is responsible for platelet shape
change and secretion of platelet granule contents.8 The platelet
granules contain procoagulant and proaggregatory substances
that promote platelet recruitment and contribute to secondary
hemostasis at sites of tissue injury.2 Because of the global
effects on platelet activation, the expected effects of cytocha-
lasin D on TEG tracings are a decrease in both MA and Ang.
In-vitro studies that have used TEG or the related technology
rotational thromboelastometry (ROTEG) to evaluate the
effects of cytochalasin D on whole blood coagulation have
been performed in human beings, rabbits, and pigs, and have
confirmed these expectations.14,22,30
Abciximab is a monoclonal Fab-fragment directed against
the human platelet GPIIb/IIIa receptor.29 The presence of
abciximab prevents fibrinogen binding by the human GPIIb/
IIIa receptor. As such, it is a potent inhibitor of platelet aggre-
gation, and is used clinically in human patients undergoing
coronary procedures such as stent placement.26 The effects
of abciximab on TEG tracings in human beings include a
decreased Ang and decreased MA, presumably due to inhibi-
tion at the GPIIb/IIIa receptor.14 Of note, results of a study in
rabbits failed to demonstrate a change in the TEG tracing
after ex vivo addition of abciximab.22 Because abciximab is
a monoclonal antibody directed against the human fibrino-
gen receptor, a significant receptor homology is necessary
for the drug to have similar effects in other species, and the
drug may not work as well in nonhuman species, including
The objectives of the current study were to investigate the
effect of different in-vitro concentrations of cytochalasin D
and abciximab on the TEG tracings of healthy dogs. The
hypothesis was that the addition of cytochalasin D would
result in a dose-dependent decrease in MA and Ang, while
abciximab would not change TEG parameters due to receptor
variations between species. A third objective was to investi-
gate the effect of similar concentrations of in-vitro abciximab
on the platelet function analyzer (PFA) CTs of healthy dogs.
The hypothesis was that abciximab would not prolong closure
times by use of collagen/ADP cartridges, again secondary to
receptor differences amongst species.
Materials and methods
Cytochalasin Db was diluted with sterile 0.9% salinec from a
stock solution (90 mg/ml) in dimethyl sulfoxide (DMSO)b to a
concentration of 180 µg/ml and added to TEG reaction cups
to achieve serial concentrations (see below). A control solu-
tion was made with diluted DMSO. The commercially avail-
able injectable abciximabd was diluted with sterile 0.9% salinec
and added to the whole blood samples 5 min prior to analysis
to final concentrations of 10, 20, and 40 µg/ml.
Eight adult mixed-breed dogs deemed healthy by physical
exam, complete blood cell counts, and coagulation profiles
(prothrombin and activated partial thromboplastin times) were
studied. Blood samples were obtained by jugular venipuncture
by use of a 20-gauge needle and vacutainer adapter. Blood
was collected by vacuum into 3-ml plastic tubese containing
3.2% sodium citrate, for a final citrate-to-blood ratio of 1:9.
These samples rested at room temperature (20–22°C) for
30 min prior to TEG analysis. The first tube obtained by this
method was not analyzed, to limit the effect of venipuncture
artifact on the tracings.
Thromboelastography analysis was performed on calibrated
channels on 4 TEG analyzers.f Twenty µl of 0.2 M calcium
chloride solutiong was placed in a TEG cuvette warmed to
37°C for 1 min prior to addition of whole blood. For test runs,
an additional 1–10 µl of cytochalasin D or 10 µl of dilute
DMSO was added and mixed with the warmed calcium chlo-
ride solution by gentle aspiration with the pipette. Dilute cyto-
chalasin D (180 µg/ml, as described above) was added to the
reaction cup to result in final concentrations of 0.5, 1.0, 2.5,
5.0, 7.5, or 10 µg/ml. Dilute abciximab was added directly to
the tubes of citrated whole blood to result in final concentra-
tions of 10 and 20 µg/ml in 3 dogs, and 20 and 40 µg/ml in
6 dogs (1 dog had all 3 concentrations evaluated, in addition
to a control sample). Abciximab was added 5 min prior to
initiation of analysis, and the tube was inverted 5 times to dis-
tribute the drug. To all mixtures of drug and calcium chloride
or calcium chloride alone, 340 µl of citrated whole blood was
added to start the reaction, without the use of additional acti-
vators or mixing of the sample. Thromboelastography trac-
ings were recorded on a computer with specialized software,h
which also calculated and displayed all measured TEG
To evaluate the effect of abciximab on canine platelet function,
blood was collected from 6 of the previously studied dogs using
the same collection protocol. The citrated blood was divided
into three 800-µl aliquots to which either nothing, 10 µg/ml
of abciximab, or 20 µg/ml of abciximab were added. As with
the samples for TEG analysis, abciximab was added 5 min
700 Brainard et al.
prior to analysis. Cytochalasin D was not evaluated in this
system based on pilot data that showed a general insensitiv-
ity to the different concentrations tested in the current study
(i.e., results were frequently >300 sec).
Samples were run on the PFA-100a using collagen/ADP
cartridges, as previously described.5 Eight hundred µl of citrated
whole blood with or without abciximab at the tested concen-
trations was placed into the cartridge, and the machine was
allowed to record the CT without any modification. If CT was
greater than 300 sec, the machine only reported >300 sec as a
CT; for statistical analysis, these samples were treated as if
they were 300 sec. The CT was evaluated at 37°C for all sam-
ples. Any samples that generated flow errors were repeated
within 10 min.
Thromboelastography data for measured parameters (R, Ang,
MA) at each concentration of inhibitor were analyzed with a
commercial statistical analysis program.i Parametric data were
analyzed by use of a one-way analysis of variance, while
nonparametric data were analyzed with a Kruskal–Wallis one-
way analysis of variance on ranks. The same testing method-
ology was used to compare the TEG tracings that resulted
from different concentrations of abciximab, as well as the
PFA data from different abciximab concentrations. When
significant differences were found, post-hoc testing by
means of either the Holm–Sidak or Dunn method was con-
ducted to identify significant comparisons. Significance was
set at P < 0.05.
Baseline TEG parameters and complete blood cell counts for
all dogs were within the reference intervals for the authors’
institution. Mean platelet count was 296.7 ± 35 × 103/µl (refer-
ence interval: 242–407 × 103/µl). Mean hematocrit of all dogs
was 46.9 ± 4.5% (reference interval: 36.6–59.6%). Cytochalasin
D caused a significant decrease in MA in canine citrated whole
blood at all concentrations (P < 0.02; Table 1). While a strong
trend toward a decreasing angle was seen (P = 0.01), this
change failed to maintain significance when corrections
were made for multiple comparisons. There was no change in
R-values at any dose of cytochalasin D (P = 0.438). There were
no significant differences between the baseline TEG values
and the DMSO control samples (Table 1). The addition of any
concentration of abciximab to canine citrated whole blood did
not significantly change TEG parameters compared to baseline
(Table 2). Abciximab significantly prolonged the PFA CT at
both 10 µg/ml and 20 µg/ml (P = 0.033 and P = 0.001, respec-
tively; Table 3). Only 2 samples generated flow errors, and
thus the tests for those samples were repeated.
Table 1. Thromboelastography (TEG) values (median (range) or mean ± standard deviation) for 6 dogs after in-vitro exposure of
citrated whole blood to varying concentrations of cytochalasin D.*
Cytochalasin D concentration (µg/ml)
0 0.5 1.0 2.55.07.5 10.0 0 (DMSO)
R (2.1–11.0 min)
MA (44.5–61.7 mm)
3.6 3.6 3.2
3.6 4.8 3.5 5.1
52.7 ± 4.3
41.4 ± 8.9†
34.8 ± 12.8†
24.1 ± 13.1†
15.4 ± 9.4†
14.3 ± 7.8†
19.1 ± 0.9†
53.0 ± 6.7
*R = reaction time; Ang = alpha angle; MA = maximum amplitude; DMSO = dimethyl sulfoxide.
†P < 0.02, significantly different from baseline.
Table 2. Thromboelastography (TEG) values (mean ± standard
deviation) for 6 dogs after in-vitro exposure of citrated whole
blood to varying concentrations of abciximab.*
Abciximab concentration (µg/ml)
reference interval010 2040
R (2.1–11.0 min)
MA (44.5–61.7 mm) 52.1 ± 5.7 55.0 ± 3.0 54.1 ± 4.1 53.1 ± 4.6
4.4 ± 1.6
58.6 ± 9.0 60.4 ± 3.6 58.6 ± 4.7 59.8 ± 2.9
3.5 ± 1.44.5 ± 1.45.0 ± 1.2
*R = reaction time; Ang = alpha angle; MA = maximum amplitude. There
were no significant differences between the values for any concentration
range, for any parameter (all P > 0.05).
Table 3. PFA-100® system closure times (mean ± standard
deviation) using collagen–adenosine diphosphate cartridges
in 6 dogs after in-vitro exposure of blood samples to varying
concentrations of abciximab.
Abciximab concentration (µg/ml)
0 10 20
Closure time (reference
interval: 58–86 sec)72.5 ± 15 149.2 ± 91*275.6 ± 54†‡
*P = 0.033, significant difference from baseline closure time indicated.
†P = 0.001, significant difference from baseline closure time indicated.
‡P = 0.004, significant difference from 10 µg/ml concentration closure
Abciximab and cytochalasin D in canine platelet function 701
Cytochalasin D added to canine citrated whole blood resulted
in a significant decrease in MA at all concentrations, with a
maximal decrease at 7.5 µg/ml. Cytochalasin D suppresses
platelet shape change, phosphatidylserine externalization,
granule release, and clot retraction as a result of effects on the
platelet cytoskeleton.27 These platelet functions are important
to initiate and sustain coagulation in vivo, and are expected
to contribute to in vitro whole blood coagulation as well.2,21
The inhibition of platelet function in these samples likely
contributed to the significant effects on the TEG tracing.
The addition of cytochalasin D to canine citrated whole
blood did not change the R time from baseline values. This was
expected, as the platelet contribution to coagulation is not eval-
uated by R, which is primarily dependent on contact activation
in the recalcified sample (Ralph AG, Koenig A, Pittman JR,
et al.: 2010, The effect of corn trypsin inhibitor on thrombe-
lastography endpoints of citrated canine whole blood. J Vet
Emerg Crit Care 20(Suppl s1):A6. Abstract). The effects of
cytochalasin D on TEG parameters are consistent with prior
reports in human patients.6 While abciximab at a number of
doses extrapolated from human studies did not alter the
TEG tracing in dogs, the effect of these same (and lower)
doses on the ability of platelets to aggregate in the PFA-100
assay was profound. The difference between these 2 assays
most likely illustrates the difference between the role of the
GPIIb/IIIa receptor in platelet function under conditions of
high and low shear.
The expression of the GPIIb/IIIa receptor is the final com-
mon pathway for many different platelet agonists. Expression
of this receptor allows the platelet to anchor fibrin or fibrino-
gen and to be integrated within a clot. The GPIIb/IIIa receptor
is also critically important for binding small molecular weight
von Willebrand factor (vWF) molecules that allow attachment
of additional platelet aggregates to the basal layer of platelets
that interacts directly with subendothelial collagen.25 Without
adequate GPIIb/IIIa function, platelets cannot bind together
to form a strong clot under high shear conditions. By contrast,
when there is low shear, such as in the TEG assay, the effects
of abciximab are much less obvious, as the formation of a
clot can proceed without a need for strong platelet binding to
fibrinogen. Platelets, in the presence of GPIIb/IIIa inhibition,
are still able to be activated, and the shape-change reaction,
granule secretion, and membrane changes (i.e., exposure of
phosphatidylserine) are able to occur and support the initia-
tion and propagation of a clot.26 Thromboelastography anal-
ysis can be performed with citrated plasma alone, if there is
sufficient fibrinogen concentration and a strong coagulation
activator.33 This may explain the lack of effect of abcix-
imab; initiation of clot formation was able to proceed as
normal, and the plasma components were able to form a clot
in this low shear environment, regardless of platelet binding
There is strong homology between the canine and human
GPIIb/IIIa receptors, especially in the area of the fibrinogen-
binding site on the IIIa portion, and abciximab is expected
to bind to similar epitopes as on the human receptor.24 The
lack of full sequence homology may, however, result in
altered binding of this drug, and incomplete inhibition of the
binding of either fibrin or vWF. There is also evidence that
abciximab has affinity for the human vitronectin receptor
(alphavbeta3), which plays a role in cell adhesion, migration,
and proliferation.16 Additional studies are required to describe
the kinetics of abciximab association with canine GPIIb/IIIa
receptor and other cellular components.
The results of other studies in dogs support the importance
of abciximab in attenuating platelet adherence under high
shear conditions. With a maximum intravenous dose of
0.8 mg/kg, abciximab resulted in a dose-dependent inhibition
of thrombosis of a nitinol stent placed in an ex-vivo arteriove-
nous shunt.18 In a canine model of coronary artery thrombosis,
the use of abciximab at intravenous doses of 0.6 mg/kg and
0.8 mg/kg slowed thrombosis of coronary arteries exposed to
an electrical thrombotic stimulus.28
The kinetics of abciximab in dogs may be different from
those in human beings. Human platelets contain approximately
70–80% of GPIIb/IIIa receptors on their surface with the
remaining 30% located within alpha-granules and the open
canalicular system.10 This internal pool is only expressed as a
functional surface receptor upon platelet activation, and thus,
the exposure of this pool of GPIIb/IIIa may counteract the
effects of the initial abciximab dose. In addition, there is some
evidence that 80% of the GPIIb/IIIa receptors on human
platelets become bound with abciximab after 30 min1; a lon-
ger incubation time (especially if the epitopes are slightly dif-
ferent between species) in the current study may have resulted
in more profound effects on TEG analysis. The effects of the
PFA analysis, however, would argue against this hypothesis.
Researchers pursuing a similar study14 using in vitro addi-
tion of abciximab to human whole blood at similar concentra-
tions did report a significant effect on automated ROTEG
activated with Integj (a proprietary contact activator for the
intrinsic system containing ellagic acid and phospholipids),
and specifically described a decrease in Ang and MA. An
additional decrease in MA was seen with the addition of cyto-
chalasin D to these mixtures. Similar results were also seen
in a study in which investigators evaluated the effects of in
vitro abciximab and cytochalasin D on tissue-factor–activated
TEG.13 A more recent study to evaluate the effect of in vitro
addition of abciximab on kaolin-activated TEG in human
blood failed to duplicate these results,1 although the results of
clinical studies using abciximab–TEG have shown a strong
correlation of MA with fibrinogen concentration (compared
to a weaker correlation between routine TEG MA and fibrin-
ogen concentration in the same patient population).12 The
results of a study in rabbits also failed to show an effect of
abciximab on TEG analysis, which was thought to be due to
702 Brainard et al.
species differences in receptors, although further study was
not pursued.22 It is unclear why there was a difference between
the species, and between the results of conspecific studies, but
differences in incubation times, activators of coagulation, and
other analytic and preanalytic variables may have contributed.
The study22 protocols also did not evaluate aggregation under
high shear conditions. In the current study, a validated TEG
protocol23 failed to show effects of abciximab in canine
citrated whole blood.
Thromboelastography may not be an appropriate or reli-
able modality for evaluation of platelet inhibitors such as
abciximab in dogs. It is known that platelet antagonists
such as aspirin and clopidogrel do not affect TEG tracings in
dogs,3,4 and that TEG tracings may be generated in the absence
of any functional platelets,33 which indicates that the use of a
GPIIb/IIIa inhibitor may be an unreliable method to isolate the
platelet contribution to the TEG tracing in dogs. One con-
cern about using cytochalasin D rather than abciximab to
evaluate the platelet component of a TEG tracing is that
the aspects of platelet activation that support coagulation
(e.g., exposure of phosphatidylserine) are maintained with
abciximab, but are not present with the addition of cytochala-
sin D due to inhibition of microtubule assembly, although
cytochalasin E has been reported to have no effect on platelet
Genetic polymorphisms in human beings result in vari-
ability of surface expressed P-selectin, GPIIb/IIIa–bound
fibrinogen, and activated GPIIb/IIIa in response to low-dose
ADP.19 Human platelets with more surface bound ligands
were found to have an enhanced response to abciximab.19
Given the existence of this polymorphism in human beings, a
similar scenario may exist in dogs, and may explain some of
the variability seen in the current study population, although
these were unrelated mixed-breed dogs. Further investigation
is needed to characterize the surface expression of canine
platelet surface ligands.
Hypercoagulable disease states are diagnosed with increas-
ing frequency in veterinary medicine.11The diagnosis may be
made with TEG, but a true diagnosis of hypercoagulability
requires corroborating evidence such as fibrinogen concentra-
tions, antithrombin levels, or gross evidence of thrombosis.11In
veterinary medicine, platelet inhibitors and other anticoagu-
lant medications are used based upon clinician preference,
generally without knowledge of the underlying basis of the
hypercoagulable state. Removal of the contribution of the
platelet component of the TEG analysis may allow the cli-
nician to extrapolate (based on comparison with an uninhib-
ited tracing) the platelet contribution to hypercoagulability.
With this information (and an appropriately generated refer-
ence interval for cytochalasin D–inhibited TEG), the clinician
may be able to more accurately choose therapies for hyperco-
agulable patients (i.e., the choice between a platelet inhibi-
tor such as clopidogrel and a coagulation inhibitor such
Limitations of the current study include the small number
of dogs tested, and the in-vitro nature of the drug application.
If polymorphisms exist in canine platelet receptors, some of
the results and conclusions in the present study may have
been biased. The use of mixed-breed dogs was chosen to limit
the possibility for breed-specific variability, but this is always
a possibility. Although coagulation profiles were evaluated
in the dogs, fibrinogen concentrations were not evaluated
(although all dogs had previously tested within the reference
interval). If alterations in plasma fibrinogen concentrations
were present, actual effects of the tested drugs on TEG tracings
may have been minimized. In the absence of advanced studies
of the interaction of the abciximab antibody with the canine
GPIIb/IIIa receptor (and other receptors), it is impossible to
derive the exact site of action of this drug in the canine plate-
let. Cytochalasin D may be added for in-vitro inhibition of
platelet function to allow TEG analysis of the relative contri-
butions of coagulation factors and platelets to clot formation.
The authors acknowledge the assistance of Drs. Curtis Cathcart
and Amy Dixon-Jimenez for PFA analyses.
Sources and manufacturers
a. Siemens Healthcare Diagnostic, Deerfield, IL.
b. Sigma Chemical, St. Louis, MO.
c. Hospira Inc, North Chicago, IL.
d. ReoPro, Eli Lilly, Indianapolis, IN.
e. Vacutainer, BD, Franklin Lakes, NJ.
f. TEG 5000, Haemoscope (now Haemonetics), Niles, IL.
g. Haemoscope, Niles, IL.
h. TEG Software v.4.3, Haemoscope, Niles, IL.
i. Sigma Stat 3.5, Systat Inc., Chicago, IL.
j. Integ-LS Activator, NOBIS, Endingen, Germany.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This work
was supported by the UGA Critical Care research fund.
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