Journal of Clinical Anesthesia (2008) 20, 186–190

Original contribution

Measurement of functional fibrinogen levels using the

Thrombelastograph☆
Roger C. Carroll PhD (Professor and Director of Research)a,⁎,
Robert M. Craft MD (Associate Professor)a , Jack J. Chavez MD (Associate Professor)a ,
Carolyn C. Snider MT (Medical Technologist)a ,
Ryan K. Kirby BS (Senior Research Technician)a ,
Eli Cohen PhD (Chief Executive Officer)b
a
Department of Anesthesiology, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville,
TN 37920, USA
b
Haemoscope Corporation, 6231 West Howard Street, Niles, IL 60714-3404, USA

Received 3 January 2007; revised 10 September 2007; accepted 29 September 2007

Keywords:
Abstract
Thrombelastograph;
Study Objective: To validate a Thromboelastograph (Haemoscope Corporation, Niles, IL) assay for
Fibrinogen;
functional fibrinogen.
Hematocrit
Design: Correlation study of the Thromboelastograph assay with two conventional fibrinogen assays by
the standard Clauss method.
Setting: Research laboratory of a university medical center.
Participants and Interventions: Blood samples were obtained from 19 healthy volunteers.
Measurement and Main Results: Thromboelastograph assays, using heparinized whole blood from
19 healthy donors, indicated that reptilase-XIIIa mixture (Activatorf)–generated clot shear elasticity in
dynes per square centimeter (Gf) correlated with fibrinogen (mg/dL). Blood from four donors was used
to define the contribution of hematocrit (Hct) to Gf by titration with platelet-rich plasma. The Gf versus
Hct gave linear correlations (r 2 = 0.746) with Gf = 1258 − 17.8 × % Hct. A commercial collection of
19 normal, 10 borderline, and one deficient for functional fibrinogen-citrated plasmas was assayed for
Gf after recalcification using Activatorf. Of the 30 plasma samples, four were from factor X– or factor
VII–deficient donors and one was from a coumadin-treated donor. There was a linear correlation of
Activatorf Gf with functional fibrinogen (r 2 = 0.940) with Gf = −730 + 9.21 × fibrinogen (mg/dL).
Conclusion: Thrombelastography with Activatorf may be used to determine fibrinogen levels in
whole blood.
© 2008 Elsevier Inc. All rights reserved.

☆
Funding was provided by the Anesthesiology Research Fund, 1. Introduction
University of Tennessee, Knoxville, TN, and Haemoscope Corporation,
Niles, IL. The Thrombelastograph (TEG; Haemoscope Corp, Niles,
⁎ Corresponding author. Department of Anesthesiology, University of
Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville,
IL) system measures the clot's physical properties by use of a
TN 37920, USA. Tel.: +1 865 544 9469; fax: +1 865 525 5143. cup and pin assembly, as shown in Fig. 1. The cup holds an
E-mail address: rccarrol1@mc.utmck.edu (R.C. Carroll). aliquot of blood and is oscillated 6 times per minute through a

0952-8180/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.jclinane.2007.09.017
Measuring functional fibrinogen 187

exogenously activated factor XIIIa because there is no

thrombin generation to activate endogenous factor XIII. No
platelet activation without the addition of agonists such as
adenosine diphosphate or arachidonic acid was verified by
the lack of effect of glycoprotein IIb/IIIa inhibitors.
Because fibrin is formed by added reptilase and factor
XIIIa, deficiencies of other coagulation factors should have
no impact on the Gf in contrast to celite or tissue factor (TF)–
generated clots [1]. In this study we tested the correlation of
functional fibrinogen with the Gf in whole blood generated
by Activatorf without platelet activation. We also separately
examined the relative effects of functional fibrinogen levels
Fig. 1 Thrombelastograph cup and pin design. and Hct on Gf.

4°45′ angle. A pin is suspended in the cup by a torsion wire,

which is monitored for motion. As thrombin forms, it
converts fibrinogen to fibrin polymers, which are cross-
2. Materials and methods
linked together by thrombin-activated factor XIIIa. Thrombin
also maximally activates platelets that interact via glycopro- 2.1. Materials
tein IIb/IIIa with the fibrin network to further strengthen the
clot. These fibrin-platelet bonds transmit the motion of the Activatorf was obtained as single-use lyophilized vials
cup to the pin. The strength of the clot determines the degree from Haemoscope Corporation.
of pin motion such that a strong clot will make the pin move in
synchronization with the cup. The pin movement is converted 2.2. Human subjects
to a TEG tracing, as shown in Fig. 2. Reaction time (R) is the
time from start of the reaction until a measurable clot is The institutional review board of the University of
detected. K time is from the R point until a certain clot Tennessee Medical Center Hospital, Knoxville, approved
firmness is achieved. The angle (α) reflects the rate of clot this protocol. Informed consent was obtained from
formation. The maximum amplitude (MA) of clot shear 19 healthy donors. No special criteria in terms of diet or
elasticity reflects the contribution of fibrin and platelets to clot drugs were used.
strength. The MA may be converted by the formula: Gf =
5000 × MA/(100 − MA) to a shear elasticity value (Gf) in 2.3. Thrombelastograph assays
dynes per square centimeter. The Gf of a platelet-free plasma
clot is proportionate to the functional fibrinogen concentra- Blood was drawn into Vacutainer tubes (Vacutainer
tion (from 75 to 345 mg/dL) [1]. This study also showed that Systems, Becton-Dickinson, Franklin Lakes, NJ), either
other coagulation factor deficiencies such as thrombin or anticoagulated with 14.7 U/mL heparin or one-tenth volume
fibrin cross-linking factor XIII had an impact on the Gf as of 105 mmol/L sodium citrate. Blood samples were obtained
well as on other TEG parameters. It has been suggested that in from healthy donors by standard antecubital vein phlebot-
children, younger than 12 months, not all of the fibrinogen is omy. The heparinized blood was used for TEG assays by
completely functional [2,3]. In addition, adults with certain
genetic variants have dysfunctional fibrinogen [4-9]. The
TEG measures functional fibrinogen as opposed to fibrinogen
antigen levels, which are measured by immunoassay. In
whole blood the Gf is a combination of functional fibrinogen,
hematocrit (Hct), and activated platelet interacting with fibrin
[10-12]. To distinguish the contribution of functional
fibrinogen from platelets, glycoprotein IIb/IIIa antagonists
have been used [11,12]. These studies also suggested a
correlation of functional fibrinogen, minus the platelet
contribution, with Gf.
We recently tested a TEG Platelet Mapping assay that
Fig. 2 Thrombelastograph tracing parameters. R = reaction time,
isolates platelet function by forming a cross-linked clot with or time from start of the reaction until a measurable clot is detected;
a mixture of reptilase and factor XIIIa, Activatorf, obtained MA = maximum amplitude of clot shear elasticity, reflecting the
from Haemoscope Corporation [13]. The reptilase converts contribution of fibrin and platelets to clot strength; K = time from R
fibrinogen to fibrin without activating platelets. These fibrin until a certain clot firmness is achieved; α = angle that reflects the
polymers are cross-linked together by the addition of rate of clot formation; A30 = amplitude at 30 minutes.
188
mixing 350 μL whole blood three times by pipette with
10 μL of reconstituted Activatorf in the TEG cup. Assays
were done in duplicate, and the TEG monitored the reaction
until a stable MA was obtained. The Gf value in dynes per
square centimeter was calculated from MA using the formula:
Gf = 5000 × MA/(100 − MA). A collection of 30 citrated
platelet-free plasma samples was also obtained from George
King Bio-Medical, Inc (Overland Park, KS). The fibrinogen
levels provided with the collection had been determined by
the standard Clauss method using the Diagnostica Stago
Compact Instrument (Diagnostica Stago, Asnières s/Seine,
France). For TEG assays of these plasmas, we used 340 μL of
plasma and either 20 μL of Activatorf reconstituted with
0.2 mol/L CaCl2 or 20 μL TF (Innovin, Dade Behring
Marburg GmbH, Marburg, Germany) reconstituted per
manufacturer's directions and then diluted 50-fold with
0.2 mol/L CaCl2, 0.02 mol/L HEPES, 0.14 mol/L sodium
chloride, 2% bovine serum albumin, pH 7.4 buffer.
2.3.1. Hematocrit
Hematocrits were determined on an Ichor-PlateletWorks
(Helena Laboratories, Beaumont, TX) using 1 mL of
heparinized blood.
2.3.2. Platelet-rich plasma isolation
Platelet-rich plasma (PRP) was isolated from heparinized
whole blood by centrifugation at 100g for 20 minutes at
room temperature.
2.3.3. Plasma fibrinogen levels
Plasma was isolated from either heparinized or citrate
anticoagulated blood by centrifugation at 1000g for
15 minutes. Functional fibrinogen (mg/dL) was determined
in citrated plasma by an independent Dynacare Core Lab
using the standard Clauss method with an MD180 analyzer
(Organon Teknika Co, Durham, NC). These latter fibrinogen
values were corrected for the 10% dilution by citrate before
data analysis of heparinized whole blood.
Fig. 3 Linear regression plot of Activatorf TEG whole blood clot
shear elasticity (Gf) versus plasma fibrinogen (mg/dL) for 19
healthy donors.
R.C. Carroll et al.
Table 1 Linear regression slopes and intercepts of hematocrit
(Hct) titration of the Activatorf TEG Gf in four healthy donors
Gf slope Intercept r2
−21.7 1369 0.957
−17.1 1216 0.994
−15.8 1001 0.950
−19.0 1494 0.984
2.4. Statistics
Linear regressions were performed with StatView 4.0
software (SAS Institute, Cary, NC).
3. Results
Heparinized whole blood from 19 healthy subjects was
assayed to determine the relationship of functional fibrino-
gen levels to Activatorf TEG Gf. Fig. 3 presents the linear
regression of Gf to functional fibrinogen (mg/dL). There was
a good correlation (r2 = 0.605) with Gf = −250 + 2.15 ×
fibrinogen (mg/dL).
The results above indicate that the Activatorf TEG Gf
was sensitive to functional fibrinogen without platelet
interaction in whole blood. We then used blood from four
donors to determine the relative contribution of Hct to the
Gf for the whole blood Activatorf assay. Autologous PRP
was used to dilute the Hct, keeping the functional plasma
fibrinogen level constant for each individual, as confirmed
by the Core Lab. Table 1 summarizes the individual slopes,
intercepts, and r2 (N0.95) data for all four donors. A
composite set of data for the Activatorf TEG Gf assay for
these four donors is shown in Fig. 4. Linear regression of
the composite data indicates Gf = 1258 − 17.8 × % Hct.
Thus, the effect of Hct at a fixed plasma concentration of
Fig. 4 Typical linear regression plot of Activatorf TEG clot
shear elasticity (Gf) versus % hematocrit (Hct) using four healthy
donors' whole blood samples diluted with autologous platelet-rich
plasma (PRP).
Measuring functional fibrinogen
fibrinogen through the reference Hct range of 36% to 55%
reduces Gf by 338 dynes/cm2.
A commercially available collection of 30 platelet-free
plasma samples was used to determine the correlation of
plasma fibrinogen (mg/dL) to Gf , without the effect of Hct
or platelets. Included were 4 plasma samples from factor X–
or factor VII–deficient (b1%) individuals and one from a
coumadin-treated person. In addition, one plasma sample
was deficient in fibrinogen with a level of 21 mg/dL. These
plasma samples were assayed in duplicate on the TEG using
either Activatorf or TF with 0.2 mol/L CaCl2 to initiate
clotting. The plasma deficient in fibrinogen did not give a
detectable Gf by either clotting method and was not included
in the data analysis. The TF did not clot the 5 samples with
clotting factor deficiencies. Fig. 5 presents a plot of
Activatorf Gf versus functional fibrinogen (mg/dL) for this
plasma collection, excluding the fibrinogen-deficient sam-
ple. There is a good linear regression correlation (r2 = 0.940)
with Gf = −730 + 9.21 × fibrinogen (mg/dL). The effect of
the reference range of fibrinogen from 200 to 400 mg/dL
would be an increase of Gf from 1,112 to 2,954 dynes/cm2.
This difference of 1,842 dynes/cm2 is much larger than the
negative contribution of the reference Hct range. The Gf
obtained with TF also gave a good correlation (r2 = 0.929)
with plasma fibrinogen (mg/dL) for the 24 samples that
could be assayed. Fig. 6 shows the good correlation (r 2 =
0.920) of the Gf as determined by TF for the 24 samples that
clotted. The slope of 0.82 indicates a lower Gf with TF
relative to Activatorf .
4. Discussion
These studies indicate that when platelet interaction is
absent, the TEG shear elasticity Gf is proportionate to
functional fibrinogen with some smaller effect of Hct. The
Hct effect possibly reflects a reduction in the absolute
Fig. 5 Linear regression plot of Activatorf TEG Gf versus plasma
fibrinogen (mg/dL) for the George King Biomedical collection of
29 plasma samples.
189
Fig. 6 Linear regression plot of tissue factor (TF) Gf versus
Activatorf Gf for the George King Biomedical Collection of
24 coagulable plasma samples.
amount of fibrinogen per milliliter of blood, although the
plasma concentration remains constant. Alternatively, there
may be interference with the shear elasticity of the fibrin
network by red blood cells entrapped in the fibrin network.
From these results we derived a formula to calculate the
contribution of plasma functional fibrinogen levels to
whole blood Activatorf TEG Gf , ignoring the relatively
smaller effect of Hct. Because the coagulation is controlled
by added Activatorf rather than endogenous coagulation
factors, the assay would not be affected by coagulation
factor deficiencies other than functional fibrinogen. The
finding that the Activatorf gives higher Gf than TF formed
clots may indicate a more complete clot formation or some
subtle difference in clot structure with fibrin polymers
formed by reptilase rather than thrombin. The clotting
efficiency of Activatorf was optimal even with plasma that
was deficient in functional clotting factors. The availability
of a point-of-care test for quick determination of functional
fibrinogen in either whole blood or plasma should prove
useful to the clinician. In cardiopulmonary bypass
surgeries, this assay, along with Platelet Mapping, could
rapidly assess the coagulation status of the patient both in
terms of fibrinogen and platelet functionality.
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