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A novel approach for detecting hypercoagulability utilizing thromboelastography

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A novel approach for detecting hypercoagulability utilizing thromboelastography

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Content

A NOVEL APPROACH FOR DETECTING HYPERCOAGULABILITY UTILIZING
THROMBOELASTOGRAPHY

by

Richard Har Ko

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CLINICAL AND BIOMEDICAL INVESTIGATIONS)

May 2010

Copyright 2010 Richard Har Ko
ii
Table of Contents

List of Figures iii
Abbreviations iv
Abstract v
Chapter 1: Background 1
Chapter 2: Methods 4
Chapter 3: Results 7
Table 1. TEG values of our novel method and with thrombin addition in adult 7
volunteers

Chapter 4: Conclusions 10
References 12
iii
List of Figures

Figure 1: Mean R time v. [thrombin] 8
Figure 2: Mean K time v. [thrombin] 8
Figure 3: Mean angle v. [thrombin] 9
iv
Abbreviations

ALL: acute lymphoblastic leukemia
AML: acute myeloid leukemia
CTI: corn trypsin inhibitor
DVT: deep venous thrombosis
MA: maximum amplitude
PE: pulmonary embolus
PTS: post-thrombotic syndrome
TEG: thromboelastograph/thromboelastography
VTE: venous thromboembolic event
v
Abstract

Background: The thromboelastograph (TEG) is a point-of-care global hemostasis
assay that measures clot formation and their physical properties, and is licensed for use in
monitoring coagulation during complex surgical procedures, but has recently been
investigated to monitor patients with bleeding disorders. Although attempts have been made
to use the TEG to detect hypercoagulable states, the results have been inconsistent.
Objectives: To develop novel methods utilizing the TEG which are sensitive to detecting
hypercoagulability by demonstrating that baseline TEG parameters can be consistently
changed so that they will be sensitive to hypercoagulable conditions.
Patients/Methods: To make the TEG more sensitive to detecting hypercoagulability,
several pre-analytic modifications were tested on whole blood from healthy adult volunteers
with the goal of prolonging the standard clot initiation and clot propagation times. Methods,
which resulted in consistent and reliable desired changes, utilized corn trypsin inhibitor (CTI),
a contact pathway inhibitor, and blood samples that were not activated. In order to
demonstrate that these methods are sensitive to detecting hypercoagulability, increasing
concentrations of recombinant human thrombin were added.
Results: The novel methods were able to consistently and statistically significantly
change the baseline TEG parameters of R time, K time, and maximum amplitude (MA) in the
desired direction. In addition, the novel methods were able to detect increasing concentrations
of thrombin.
Conclusions: We describe novel TEG methods which are sensitive to the presence of
increasing concentrations of thrombin in vitro. Further studies are under way to determine if
these methods will be sensitive to detecting hypercoagulable states in vivo.
1
Background
Thromboelastography was developed during World War II by Dr. H. Hartert as a
research tool (1). Its main clinical use today is in the surgical settings of liver transplantation
and cardiac surgery, which commonly result in hemostatic derangements and increased
bleeding (2). Thromboelastography displays the dynamics of blood clot formation over time
rapidly, easily, and at relatively low cost, providing surgeons and anesthesiologists valuable
data, allowing for the safe and effective use of pro-hemostatic therapies, anticoagulation, and
blood products. Recently, the use of the TEG has expanded to hematology research
laboratories, particularly in patients with bleeding disorders (“hypocoagulable states”), such as
hemophilia. The currently utilized methods are geared towards assessing hypocoagulability,
with limited data regarding its use in detecting hypercoagulable states in diseases such as
sickle cell anemia and cancer (3, 4). A recent review of the literature regarding the use of the
TEG in predicting postoperative thrombotic events found that both the methods and results
were inconsistent (5).
Thromboembolic disease is the leading cause of mortality and a major cause of
morbidity in the developed world. This condition affects many adults and an increasing
number of children. While the exact rates specifically for venous thromboembolic events
(VTE), defined as deep venous thromboses (DVT) and/or pulmonary emboli (PE), in the
community are unknown, the estimated total annual number of symptomatic VTE

events in the
US exceeded 600,000 and VTE-related deaths were estimated at 296,370 annually (6). This
places the morbidity and mortality solely from VTE (not including other thromboembolic
disease such as myocardial infarction) among the top 10 health problems in the US. In fact,
two new objectives for Healthy People 2020 are to reduce the proportion of adults who
2
develop VTE during hospitalization and to reduce the proportion of persons who develop VTE
(7). These objectives emphasize the importance of preventing and treating VTE in patients.
Currently, there are no laboratory assays which can predict the occurrence of a VTE.
Assays for thrombophilia will detect conditions that predispose to VTE, but cannot on an
individual level predict if such an event will occur. Furthermore, the D-dimer has been shown
to be valuable for determining if patients presenting with symptoms of DVT or PE should
undergo additional testing, but does not globally evaluate the coagulation system nor has it
been proven to be predictive for thrombosis in asymptomatic patients. The sequelae of a
thrombotic event are often serious and occasionally life-threatening. For instance, thromboses
in the systemic vasculature can cause pulmonary emboli and those in the CNS can lead to
seizures, coma, and may result in permanent neurologic damage. In addition, deep vein
thrombosis in an extremity can cause the post-thrombotic syndrome (PTS), a clinical
condition of pain, edema, and ulcers which can be debilitating due to the inability to tolerate
prolonged use of the affected extremity. Thus a simple assay such as TEG, which would be
predictive of VTEs, would be an important advance as it would allow for the possibility of
preventive therapy in a manner similar to high cholesterol being predictive of vascular disease
and the subsequent institution of cholesterol lowering strategies, such as improved diet,
exercise, and HMG CoA reductase inhibitor therapy.
We believe that a global coagulation assay, such as thromboelastography, with pre-
analytical methods modified to detect a hypercoagulable state, will be able to distinguish
patients at high risk for developing a VTE. Were such an assay available, it could potentially
lead to a risk-stratification strategy whereby patients deemed to be at particularly high risk for
VTE could receive primary prophylactic anticoagulation. In this report, we describe novel
3
TEG methods which reliably and consistently change the baseline TEG curves/parameters
making them more sensitive to hypercoagulability.
4
Methods
Materials
Thromboelastography was performed with the TEG 5000® Thromboelastograph
Hemostasis Analyzer with TEG Analytical Software® Version 4 (Haemonetics Corporation,
Braintree, MA, USA). Thromboelastograph disposable pins and cups and 0.2M calcium
chloride also were purchased from Haemonetics Corporation. Four-and-a-half mL 3.2%
sodium citrate tubes (Becton, Dickson and Company, Franklin Lakes, NJ, USA), 1.7 mL
GeneMate microcentrifuge tubes (ISC BioExpress, Kaysville, UT, USA), and corn trypsin
inhibitor (Haematologic Technologies Incorporated, Essex Junction, VT, USA) were
purchased. Recombinant topical thrombin (RECOTHROM, ZymoGenetics, Inc., Seattle, WA,
USA) was provided by our hospital pharmacy.
Subjects
Blood was collected from 39 healthy adult volunteers who were identified via a
central Institutional Review Board-approved study for blood donation specifically for research
studies. All healthy subjects were without personal or first-degree family history of bleeding
or thrombosis, were not taking any medications, had no acute infection or chronic illness, and
provided written consent. Six were male and 33 were female. The mean age of volunteers
was 36.0±1.53 years (mean ± standard error; range 24-61 years). The mean age for females
was 35.03±1.65 years and the mean age for males was 45.17±3.64 years. There was no
statistically significant difference in age between males and females (p=0.17).

5
Thromboelastography Methods
Baseline Assays
Whole blood was obtained via atraumatic peripheral venipuncture. The first 3 mL of
blood were discarded, after which 4.5 mL of blood was collected in a syringe and immediately
transferred into 4.5 mL 3.2% sodium citrate tubes into which 300 μg of corn trypsin inhibitor
had been added (final concentration of CTI was 0.06 mg/mL). Three hundred forty μL of this
blood was pipetted into a TEG cup into which 20 μL of calcium chloride had been previously
added. All samples were run in quadruplicate until maximum amplitude (MA) was reached.
Of note, no activators such as kaolin or tissue factor were used.
Thrombin Addition
Blood was collected as described in the manner above. From this, 500 μL of blood
was pipetted into polypropylene microcentrifuge tubes. To each microcentrifuge tube of
blood, 11 μL of varying concentrations of recombinant human thrombin was added to the
blood to make the following final concentrations of thrombin in blood: 0, 10, 100, 500, 750,
1000, 2500, and 5000 picomolar. The tubes were then mixed by inverting them 3 times. The
samples were then run as described above.
Statistical Analysis
The R time (clot initiation), K time (clot propagation), angle (clot propagation), MA
(clot strength), and G (clot strength) from our novel methods (i.e., the addition of 300 mcg of
corn trypsin inhibitor and no activators) were compared to the standard TEG methods reported
in the literature using independent t-tests. For the comparison of the novel methods to the
published standards, each quadruplicate sample was treated as a separate sample and not
grouped together as a single sample. For the comparison of the samples with varying
concentrations of thrombin, linear mixed effect models were used to determine whether there
6
was a difference in the R time, K time, angle, MA, or G among blood samples. The effect
from thrombin concentrations was treated as fixed and patient effect treated as random (8).
Statistical analyses were performed using Microsoft Excel 2007 (Redmond, WA, USA) and
SAS version 9.2 (Cary, NC, USA). Statistical tests were 2-sided and conducted at the 0.05
overall level of statistical significance, with adjustments made as necessary.
7
Results
Comparing values from the newly described methods to the published values in the
literature using standard TEG methods, we were able to statistically significantly alter TEG
parameters in the desired direction (Table 1). Of the parameters investigated, it was found that
the R time (clot initiation), K time (clot propagation), and angle (clot propagation) are
statistically significantly altered towards less coagulable blood compared to published norms
(p<0.001). The parameters of MA (clot strength) and G (clot strength) were not statistically
significantly different from published methods. In addition, the data demonstrate that the
novel TEG methods are sensitive to increasing concentrations of thrombin (a surrogate for
hypercoagulability) in a dose-responsive manner for R time, K time, and angle (Table 1).
Table 1. TEG values of our novel method and with thrombin addition in adult
volunteers.

R time
(min.)
K time
(min.)
Angle (deg.) MA (mm) G (d/cm
2
)
Standard Method
(published values)
2-8 1-3 55-78 51-69 6-13
Novel Method (n=64) 37.63±1.06 10.13±0.53 24.42±0.93 55.00±0.75 6.34±0.22
THROMBIN
ADDITION

10 picomolar (n=16) 37.98±2.61 10.89±1.32 23.89±2.21 55.34±1.12 6.3±0.30
100 picomolar (n=19) 38.54±2.80 9.53±0.94 25.09±2.40 55.23±1.13 6.29±0.30
500 picomolar (n=16) 35.32±2.43 8.96±0.78 26.41±2.60 54.58±1.05 6.1±0.25
750 picomolar (n=14) 35.21±1.54 8.05±0.58 27.6±1.48 57.75±2.17 7.39±0.83
1 nanomolar (n=18) 27.98±3.43 6.7±1.00 33.39±3.82 59.81±1.77 7.88±0.64
2.5 nanomolar (n=13) 18.5±2.68 4.55±0.58 42.49±3.45 62.95±1.77 8.87±0.70
5 nanomolar (n=18) 2.35±0.19 3.28±0.16 51.26±1.12 66.04±2.11 10.59±0.87
*mean±standard error
There are no statistically significant differences in TEG parameters until the thrombin
concentration reaches 1000 picomolar. Figures 1-3 show the change in R time, K time, and
Angle with the addition of increasing concentrations of thrombin, respectively.
8
Figure 1. Mean R time v. [thrombin]

Figure 2. Mean K time v. [thrombin]

9
Figure 3. Mean angle v. [thrombin]

Using a mixed effects model of ANOVA (via the general linear model in SAS) and
the Bonferroni adjustment for multiple comparisons, we find that there are statistically
significant differences between baseline measurements utilizing our novel methods and blood
with 1000 picomolar of thrombin for the R time, K time, and angle (p=0.0005, p=0.0141, and
p=0.0074, respectively). We also find statistically significant differences in R time and angle
between 1 picomolar and 2500 picomolar of thrombin (p=<0.0001 and p<0.0001,
respectively) as well as between 2500 picomolar and 5000 picomolar of thrombin (p<0.0001
and p=0.009, respectively). Though we found a statistically significant difference in K time
between 1000 picomolar and 2500 picomolar of thrombin (p=0.0010), we did not find a
statistically significant difference between 2500 picomolar and 5000 picomolar of thrombin
(p=0.4147).
10
Conclusions
The thromboelastograph (TEG) is an approved, commercially available, point-of-care,
global hemostasis assay which provides a quantitative analysis of the dynamics of clot
formation over time. Thromboelastography has been mainly used to monitor bleeding
problems in the settings of cardiac and liver transplantation surgeries, but more recently it has
been used to try to predict hypercoagulability and venous thrombotic events in various
patients, though the results have been inconsistent most likely due to the use of activators in
the standard methods leading to a relatively short clot initiation time. In order to make the
assay more sensitive to identifying hypercoagulability, it is necessary to prolong the clot
initiation and propagation times. As such, we have modified the pre-analytical methods (no
activators and addition of CTI) resulting in consistently prolonged baseline clot initiation and
prolongation in healthy adults. Mann et al. also demonstrated the ability of CTI to affect the
TEG parameters in a similar way (9). While the novel methods demonstrate statistically
significant effects on R, K and angle, interestingly, they do not alter the MA or G. This is
consistent with the fact that although we are slowing the initiation and propagation times of
clot formation, we are ultimately not altering any mechanisms which would alter clot strength.
In order to demonstrate that these novel methods have the potential to be sensitive to
clinical thrombosis, experiments were then performed in which increasing concentrations of
thrombin were added in vitro to blood samples from healthy volunteers. Since thrombin
generation is key to clot formation, adding recombinant human thrombin serves as a surrogate
for hypercoagulability. The results demonstrate that the novel methods are able to distinguish
varying concentrations of thrombin though there seems to be a threshold for sensitivity.
Changes in the TEG parameters R, K, and angle can be appreciated beginning at a thrombin
concentration of 1000 picomolar. In addition, the novel methods are able to detect differences
11
between R and angle between 1000 picomolar, 2500 picomolar, and 5000 picomolar
concentrations of thrombin. There is evidence that these are clinically significant
concentrations of thrombin as well. Wolberg describes situations where the concentration of
free thrombin observed during a coagulation reaction ranges from less than 1000 picomolar to
greater than 100–500 nanomolar, depending on the conditions and detection method used (10).
In conclusion, we have developed a novel method for thromboelastography that
predictably and reliably alters the baseline TEG parameters for clot initiation and propagation
in the desired direction, essentially making blood from normal healthy volunteers appear very
hypocoagulable (i.e., like that of a hemophiliac). It was then demonstrated that this method is
sensitive to increasing and clinically relevant concentrations of thrombin. We plan on using
these methods to study patients who have risk factors for hypercoagulability.

12
References

8. Bryk AS, Raudenbush SW. Hierarchical linear models: applications and data analysis
methods. Newbury Park, CA: Sage; 1992.

5. Dai Y, Lee A, Critchley LA, White PF. Does thromboelastography predict
postoperative thromboembolic events? A systematic review of the literature. Anesth
Analg. 2009. 108(3): 734-42.

1. Hartert H. Blutgerinnung studien mit der thromboelastographie, einen Neuen
Untersuchingsverfahren. Klin Wochenschr. 1948. 1;26(37-38):577-83.

6. Heit JA, Cohen AT, Anderson FA. Estimated Annual Number of Incident and
Recurrent, Non-Fatal and Fatal Venous Thromboembolism (VTE) Events in the US.
Am Soc Hematology; 2005. p. 910.

9. Mann KG, Whelihan MF, Butenas S, Orfeo, T. Citrate anticoagulation and the
dynamics of thrombin generation. J Thromb Haemost 2007. 5: 2055–61.

4. Papa ML, Capasso F, Pudore L, Torre S, Mango S, Russo V, et al.
Thromboelastographic profiles as a tool for thrombotic risk in digestive tract cancer.
Exp Oncol. 2007. 29(2): 111-5.

2. Salooja N, Perry DJ. Thrombelastography. Blood Coagul Fibrinolysis. 2001. 12(5):
327-37.

7. Services USDoHH. 2009 [updated 2009; cited]; Available from:
http://www.healthypeople.gov/HP2020/Objectives/TopicArea.aspx?id=13&TopicArea
=Blood+Disorders+and+Blood+Safety.

10. Wolberg AS. Thrombin generation and fibrin clot structure. Blood Reviews. 2007.
21: 131–142.

3. Yee DL, Edwards RM, Mueller BU, Teruya J. Thromboelastographic and hemostatic
characteristics in pediatric patients with sickle cell disease. Arch Pathol Lab Med.
2005;129(6): 760-5.

.

Abstract (if available)

Abstract Background: The thromboelastograph (TEG) is a point-of-care global hemostasis assay that measures clot formation and their physical properties, and is licensed for use in monitoring coagulation during complex surgical procedures, but has recently been investigated to monitor patients with bleeding disorders. Although attempts have been made to use the TEG to detect hypercoagulable states, the results have been inconsistent.

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Asset Metadata

Creator Ko, Richard Har (author)

Core Title A novel approach for detecting hypercoagulability utilizing thromboelastography

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical and Biomedical Investigations

Publication Date 05/19/2010

Defense Date 04/05/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag hypercoagulability,OAI-PMH Harvest,thromboelastography

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Young, Guy (committee chair), Azen, Stanley Paul (committee member), Seeger, Robert C. (committee member)

Creator Email richarhk@usc.edu,rko@chla.usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3047

Unique identifier UC1280617

Identifier etd-Ko-3572 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-330535 (legacy record id),usctheses-m3047 (legacy record id)

Legacy Identifier etd-Ko-3572.pdf

Dmrecord 330535

Document Type Thesis

Rights Ko, Richard Har

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu