1818
Quantitation of Venous Clot Lysis With the
D-Dimer Immunoassay During Fibrinolytic
Therapy Requires Correction for Soluble
Fibrin Degradation
Benjamin Brenner, MD, Charles W. Francis, MD, Saara Totterman, MD,
Craig M. Kessler, MD, A. Koneti Rao, MD, Ronald Rubin, MD, Hau C. Kwaan, MD,
K. Ruben Gabriel, PhD, and Victor J. Marder
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Plasma cross-linked fibrin-degradation products were analyzed using a D-dimer (DD) immunoassay in patients with deep vein thrombosis (DVT) or acute myocardial infarction (MI)
treated with fibrinolytic therapy, and the results were correlated with clot lysis documented
angiographically. In 13 patients with DVT, the mean DD concentration increased 10-fold
(1,074+252 to 10,333+±1,004 ng/ml) during therapy, but neither the peak level nor the DD
concentration integrated over the course of therapy correlated with clot lysis. Since plasma DD
can derive from degradation of soluble plasma fibrin as well as from thrombi, the contribution
of the former was estimated by in vitro incubation of the pretreatment plasma with plasminogen
activator. Subtraction of this value from the measured posttreatment DD concentration
provided a "corrected" level that represented DD originating from lysis of thrombi. This
modification resulted in improved correlation of DD levels with clot lysis. The mean corrected
peak DD was higher in patients with successful thrombolysis (8,780±1,352 ng/ml) compared
with patients without lysis (3,075±589 ng/ml, p<0.001). There was a moderate correlation
between the volume of clot lysed and the corrected peak DD (r=0.62) and a higher correlation
with the corrected DD integrated over the course of treatment (r=0.97). By contrast, the
corrected DD concentrations were near zero in patients treated for MI with or without
thrombolytic reperfusion, suggesting that fibrin in small coronary thrombi did not contribute
significantly to total plasma DD during therapy. These findings indicate that the elevation in
cross-linked fibrin-degradation products during fibrinolytic therapy results from degradation
of soluble fibrin as well as from lysis of thrombi. Adjustment of plasma DD concentrations for
the contribution from degradation of soluble fibrin offers an approach to noninvasive
monitoring of venous clot lysis during fibrinolytic therapy. (Circulation 1990;81:1818-1825)
enographic assessment of therapeutic
response in patients with deep vein thrombosis (DVT) treated with thrombolytic
therapy is accurate and quantitative, but its invasive
nature makes it unsuitable for routine follow-up.1'2
Noninvasive approaches rely on clinical observation
or evaluation of venous flow using plethysmography
or Doppler ultrasound but may be inaccurate. Clinical changes such as symptomatic improvement or
reduction in leg swelling are nonspecific, often
reflecting the effects of bed rest, leg elevation, and
analgesics rather than a reduction in thrombus size.
Venous flow measurements may be insensitive to
lysis of calf vein thrombi or incomplete resolution of
proximal leg vein clots.' 3 A potential alternative
V
From the Hematology Unit, Department of Medicine (B.B.,
C.W.F., V.J.M.), Radiology (S.T.), and Biostatistics (K.R.G.),
University of Rochester School of Medicine and Dentistry, Rochester, New York, Department of Medicine (H.C.K.), Northwestern University Medical School, Chicago, Hematology Unit
(C.M.K.), George Washington University Medical Center, Washington, D.C., and Thrombosis Research Center (A.K.R., R.R.),
Temple University School of Medicine, Philadelphia.
Supported in part by grant HL-30616 from the National Heart,
Lung, and Blood Institute, National Institutes of Health,
Bethesda, Maryland, and a grant from Smith, Kline & French,
Philadelphia. C.W.F. is the recipient of an Established Investigator Award from the American Heart Association.
Address for correspondence: Charles W. Francis, MD, Hematology Unit, P.O. Box 610, University of Rochester Medical
Center, 601 Elmwood Avenue, Rochester, NY 14642.
Received June 27, 1989; revision accepted February 1, 1990.
Brenner et al Noninvasive Quantitation of Clot Lysis
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
approach to monitoring clot lysis is by measurement
of soluble products of fibrin lysis such as D-dimer
(DD) in the blood. Because the fibrin matrix of
thrombi is cross-linked by factor XIII,45 plasmin
liberates soluble degradation products that are structurally distinct from fibrinogen degradation
products6-8 and could potentially be used as markers
of clot lysis. Application of the DD assay9 to patients
with DVT, pulmonary embolism, or myocardial
infarction has shown elevated concentrations at
presentation10-15 with further elevations during
fibrinolytic therapy.16-19 However, there has been
poor correlation between DD concentration and
angiographic improvement, suggesting that circulating cross-linked fibrin-degradation products derived
from thrombus dissolution may be masked by lysis of
fibrin from other sources.1620
We have previously suggested that soluble crosslinked fibrin polymers may represent an important
source of DD during fibrinolytic therapy.20'21 Soluble
fibrin containing y-chain cross-links is found in normal
subjects and in increased concentrations in patients
presenting with acute myocardial infarction.21 These
fibrin polymers degrade during fibrinolytic therapy16
and may thereby contribute to elevated DD levels. In
the present study, we evaluated the contribution of
soluble cross-linked fibrin polymers to DD elevations in
patients treated with fibrinolytic therapy for DVT or
myocardial infarction. The results indicate that soluble
fibrin is an important source of DD during therapy and
that analysis of DD levels after adjustment for soluble
fibrin degradation may represent a useful approach to
noninvasive monitoring of clot lysis.
Methods
Patients
Patients with DVT included nine men and four
women with symptoms of venous thrombosis for less
than 7 days who were enrolled in an open, risingdose, safety, and dose-ranging study of recombinant
tissue-type plasminogen activator (rt-PA) (Smith,
Kline & French, Philadelphia). Men and postmenopausal or surgically sterilized women between the
ages 18 and 75 years were eligible for enrollment if
they had a venographically demonstrated DVT
involving popliteal, femoral, iliac, or axillary veins.
Patients were excluded if they had surgery within 10
days, severe hypertension, abnormal renal function
precluding contrast venography, recent gastrointestinal bleeding, significant central nervous system vascular disease, or abnormal hemostasis. After obtaining
informed consent, treatment was begun with rt-PA at
4 ,ug/kg/min i.v. during 4 hours (five patients), 22
hours (three patients), or 34 hours (five patients). The
total rt-PA dose was 55 ± 3 mg for the short, 121 ± 8 mg
for the intermediate, and 181± 11 mg for the long
regimen. Plasma fibrinogen concentrations were monitored during therapy, and intravenous heparin
administered concurrently with rt-PA in a dose to
1819
prolong the aPTT to twice control values if the fibrinogen concentration did not fall by 25% or more.
The 27 patients with myocardial infarction were
part of a randomized double-blind trial of 373
patients comparing therapy with intravenous APSAC
(Beecham Laboratories, Bristol, Tennessee) to intravenous streptokinase (Hoechst-Roussel, Somerville,
New Jersey). Requirements for enrollment included
continuous ischemic chest pain for at least 20 minutes beginning less than 4 hours from beginning
therapy, ST elevation of 0.1 mV or more in one or
more standard leads, or 0.2 mV in precordial leads
and age less than 75 years. Patients were excluded if
they had an excessive risk of bleeding, cardiogenic
shock, streptokinase therapy within the previous 6
months, prosthetic valve, or dilated cardiomyopathy.
Women of child-bearing potential were also
excluded. After giving informed consent, patients
were treated with either 30 units APSAC i.v. given by
bolus injection during 2-5 minutes or with 1.5 x 106
units streptokinase by continuous intravenous infusion during 60 minutes. Heparin was administered as
an intravenous bolus of 5,000-10,000 units before
catheter insertion. The subset of patients analyzed in
this study were chosen randomly from those who had
adequate blood sampling and angiography.
Venography and Angiography
Ascending venography22 was performed before and
6-16 hours after termination of the rt-PA infusion. All
venograms were evaluated by one radiologist (S.T.)
without knowledge of the DD assay results. Venous
clots were diagnosed when a constant intraluminal
filling defect was present in more than one projection
or when the contrast media did not opacify the deep
venous system above the knee despite adequate venographic technique. Clot lysis was quantitated by comparison of the pretreatment and posttreatment venograms by a modification of the technique of Marder et
al,23 in which the volume (ml) of lysed clot was
calculated by multiplying the cross-sectional area of
the recanalized vessel by its length. Coronary artery
patency was determined by angiography at 90-240
minutes after the start of fibrinolytic therapy using the
Sones or Judkins technique to determine the degree of
stenosis and patency of the infarct-related vessel. The
flow was graded according to TIMI criteria,24 in which
grade 0 or 1 was considered occlusion and grade 2 or
3 was termed "patency."
Blood Samples
Venous blood samples from DVT patients were
obtained with a 19-gauge needle before and 1, 2, 6,
12, 36, 48, and 72 hours after starting therapy;
samples from patients with acute myocardial infarction were obtained before and 90 minutes after the
start of fibrinolytic therapy. Samples were collected
into sodium citrate (0.4% final concentration) without inhibitor or with aprotinin (Mobay Chemical, New
York) (200 KIU/ml), placed immediately on ice, centrifuged within 1 hour at 3,000 rpm for 20 minutes, and
1820
Circulation Vol 81, No 6, June 1990
TABLE 1. Observed and "Corrected" Plasma D-Dimer Concentrations After Fibrinolytic Therapy
Volume
of lysed
clot (ml)
3
0
30
4
4
0
0
0
45
24
18
15
0
Peak concentration (ng/ml)
Observed
13,500
Total integrated amount (units)
Observed
Induced
Corrected
2
8.5
6.5
9
9
0
10,000
56
27
29
20,000
11,500
37
32.5
4.5
12,500
60
58.5
1.5
31
29.5
1.5
9,500
16
13.5
2.5
19,000
30
26
4.0
11,000
7,000
4,000
56
13
43
11,000
2,400
8,600
46
21
25
15,500
6,300
9,200
31
14
17
2,400
8,600
11,000
26
8,500
4,000
4,500
16.5
9.5
6
7,000
3,200
3,800
4.5
1.5
Peak observed D-dimer (DD) concentrations noted during therapy with recombinant tissue-type plasminogen
activator (rt-PA). "Induced" DD concentration represents the total amount measured after in vitro incubation of
pretreatment plasma with rt-PA, including that amount detected before rt-PA incubation (1,074+252 ng/ml) (see
'Methods"). "Corrected" concentrations of peak DD are obtained by subtracting the induced values from observed
peak values, and corrected integrated DD values are obtained as indicated in Figure 1 and the text.
Patient
1
2
3
4
5
6
7
8
9
10
11
12
13
Induced
8,250
8,050
7,000
9,000
11,800
8,000
14,000
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
frozen at -35° C. Citrate plasma samples with aprotinin were used for all studies unless otherwise specified.
In Vitro Digests
Pretreatment citrate plasma samples without
inhibitor were incubated with 200 ,tg/ml rt-PA
(100,000 IU/mg) (Burroughs Wellcome, Research
Triangle Park, North Carolina) for 20 minutes at
370 C, after which further degradation was inhibited
by addition of aprotinin to 300 MU/mi and immediate freezing at -70° C.
DD ELISA
Cross-linked fibrin-degradation products were
measured with an ELISA (Dimertest, American
Diagnostica, Greenwich, Connecticut) using plates
precoated with monoclonal antibody DD/3B6.
Results were calculated using a standard curve prepared with purified DD (provided by the manufacturer) at concentrations from 40 to 5,000 ng/ml.
Time-integrated DD values were expressed in arbitrary graph units by plotting DD concentrations
versus time on arithmetic graph paper, including all
measurements from the beginning of therapy to the
second venogram.
Statistical Analysis
Student's t test and linear regression analysis were
performed with standard computer software. Classification methods were based on normal distribution
theory.25 For classification of patients into groups
with or without lysis, a cutoff between groups was
chosen midway between the sample means of
patients with lysis or without lysis on a In (x +0.5)
scale. Estimates were corrected for overoptimism,
and confidence intervals were calculated by bootstrap
methods.26 A total of 1,000 subsamples (with replace-
Corrected
5,250
1,950
13,000
2,500
700
1,500
5,000
ment) of 13 of the 13 patients were used, recalculating the classification characteristics for each subsample and using these results to obtain the sampling
distribution of the estimated characteristics.
Results
Pretreatment DD concentrations were increased
in the DVT patients (1,074±252 ng/ml) compared
with normal ambulatory adults (75+3 ng/ml). After
initiation of rt-PA therapy, plasma DD levels rose
rapidly to 6,112+1,001 ng/ml at 2 hours, then more
slowly during the next 4 hours to a mean of
10,333-+ 1,004 ng/ml at 6 hours. After rt-PA was
stopped, DD concentrations gradually decreased to
5,314±1,293 ng/ml at 48 hours and eventually
reached the pretreatment levels at 72 hours.
Peak DD concentrations observed during rt-PA
therapy ranged from 7,000 to 20,000 ng/ml (Table 1).
To obtain an estimate of the contribution of soluble
fibrin to DD levels during therapy, the pretreatment
plasma samples were incubated in vitro with rt-PA.
This resulted in an approximately sixfold increase in
DD levels to a mean of 7,030±986 ng/ml. For each
plasma sample, a corrected DD was calculated by
subtracting the "induced" DD concentration of the
in vitro digest of the pretreatment sample (representing lysis of soluble plasma fibrin) from the DD
concentration measured in the postthrombolytic
therapy samples (Table 1). To adjust for different
durations of therapy, an integrated DD was calculated as the area under the curve connecting the DD
concentrations measured from the beginning of therapy until the posttreatment venogram. To correct for
the contribution of soluble fibrin degradation, the
integrated area for each patient was divided by a
horizontal line at the level of "induced" DD concentration. The corrected integrated area was obtained
Brenner et al Noninvasive Quantitation of Clot Lysis
rt - PA REGIMEN
41sglkg/min
1i,g/kg/min
0
IN VITRO
:c
DIGEST
I.-C
L. E
)
0
cj
'
-r---.
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O
0
qcrI.-:
E
0'
Q
C
IN
D
I4
64
TIME (hours)
by subtraction of the area below the line, representing the contribution from soluble fibrin lysis, from the
total integrated area (Figure 1).
After therapy, repeat venograms showed substantial response in five patients, with lysis of 15-45 ml of
clot, minimal lysis in three patients (3-4 ml), and no
lysis in five patients (Table 1). Peak and integrated
DD levels before and after correction for soluble
fibrin lysis were compared in patients with substantial, minimal, or no clot lysis (Figure 2). Measured
peak DD levels did not differ in patients with ot
without lysis (Figure 2A, left). However, after correction for the contribution of soluble fibrin degradation, the DD concentrations were higher in
patients with lysis compared with patients with minimal or no lysis (8,780+ 1,352 vs. 3,075±589 ng/ml;
p<0.001) (Figure 2A, right). The time-integrated DD
levels were higher in patients with substantial lysis,
but values overlapped with those of patients whose
thrombi did not lyse (Figure 2B, left), and mean
values were not significantly different (43.6±6.3 vs.
22±6.7 units; p=0.06). However, the difference was
more striking when the corrected time-integrated
72
72
1821
FIGURE 1. Plots of D-dimer concentrations in two patients with deep vein thrombosis treated with recombinant tissue-type
plasminogen activator (rt-PA). There is a
sharp increase in D-dimer concentration
during the first 6 hours of therapy, maintenance ofpeak levels for a variable interval,
and a gradual decrease in concentration
after termination of rt-PA infusion. The
treatment regimen in the two patients shown
here was 4 pg/kg/min for 2 hours followed
by],1g/kg/min for an additional 34 hours.
The horizontal interrupted line represents
DD immunoreactivity obtained by in vitro
incubation of the pretreatment plasma with
rt-PA (see "Methods"). The time-integrated
DD concentration is calculated from the
total area under the concentration curve
from the start of treatment until the time of
repeat venography. The corrected integrated
area is represented by the area above the
interrupted horizontal line, which subtracts
an amount that could be derived from degradation of soluble fibrin present in plasma.
In panel A, most of the D-dimer immunoreactivity is explained by degradation of
plasma soluble fibrin, whereas panel B indicates that most of the total derives from
sources other than soluble fibrin. The patient
shown in panel A (Table 2, patient 5)
represents a patient in whom minimal
thrombus (4 ml) was dissolved by treatment,
whereas the follow-up venogram of the
patient presented in panel B (Table 2,
patient 11) showed a significant volume of
clot dissolved (18 ml).
DD values were compared (24.7+5.7 vs. 2.2±0.5;
p<O.OOO5) (Figure 2B, right). All five patients with
lysis had a corrected value of 9.5 or more, and all
eight without lysis had a value of 4.5 or less, suggesting that the corrected integrated DD could be used
to classify patients into those with or without substantial clot lysis. Using an integrated corrected DD
of 6.6 as a cutoff between patients with or without
substantial lysis, statistical analysis suggests a median
estimate of 0.055 for the probability of misclassification, with a 95% confidence interval of 0.00-0.08,
indicating a high degree of confidence that no more
than 8% of patients would be misclassified.
Correlations of DD concentrations with each
patient's venographic response (ml of lysed clot) are
shown in Figure 3. While there was no correlation
between measured peak DD concentration and clot
lysis, a moderate correlation (r= 0.62) existed
between the corrected peak DD value and volume of
lysed clot (Figure 3A). Similarly, the correlation
demonstrated between the measured time-integrated
DD and clot lysis (r= 0.47) was considerably
improved after correction for DD derived from soluble fibrin degradation (r=0.97) (Figure 3B).
Circulation Vol 81, No 6, June 1990
1822
A
B
60-
2000016000-
i
z
;~50c_
40-
d
30-
LU
0
cfi
12000-
LU
2
0
8000-
31 *i
Q
a
20-
CE
C])
n
10-
0
4000
.
(
A4
o
-
Lysis
Lysis No Lysis
CORRECTED
No Lysis
Lysis
MEASURED
No Lysis
MEASURED
Lysis
FIGURE 2. Scatterplot of
measured and corrected peak
D-dimer (panel A) and integrated D-dimer (panel B) in
patients with deep vein thrombosis showing substantial lysis
(lysis) or minimal or no lysis
(no lysis). Corrected values are
obtained from measured values
as described in the text. Mean +
SEM are indicated by open
symbols and brackets.
No Lysis
CORRECTED
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
ml) patent coronary arteries did not differ significantly. Both were close to zero, indicating that there
was no significant increase in DD resulting from lysis
of the small coronary artery thrombus (Figure 4).
Discussion
These results demonstrate a correlation between
the amount of venous thrombus lysed during thrombolytic treatment and the increase in plasma concen-
For comparison, a similar analysis was performed
on samples obtained from 27 patients with myocardial infarction who received fibrinolytic therapy. Of
this group, 21 showed substantial patency of the
infarct-related artery (TIMI grade 2 or 3) at 2.4+0.9
(±SD) hours after treatment, whereas six still
showed vascular occlusion (grade 0 or 1). The corrected postinfusion DD concentration in patients
with (-120±134 ng/ml) or without (-213±413 ng/
A
E
r =0.03
c
r=0.62
0)
2
CE:
O 20000-
0
o-
LUZ
2 150 00
,,C
Q
0
0
0
2v
0: 2-
*
~~~~~~~C)000
c)
CS
0
0 30
0
cc)
Q
40 L 50i
LU 5 0~~~~~~~~~~~~
~~~~~~CE
0
10
0
20
30'
4'0
10
50
20
30
40
50
40
s0
LYSED CL OT (ml)
B
r
=
0.47
a
000
r_
CE
50-
r = 0.97
E 50-
WU
0:
40 -
400
v
CS
304
0
20-
'/
cc 30O
(D
20
:2
CO)
LUi1
LU--0 100CC
50
0
LYSED CLOT (ml)
10
20
30
FIGURE 3. Scatterplot of correlation of D-dimer concentrations with clot lysis in patients with deep vein
thrombosis. Volume of clot lysed during therapy was
estimated by comparison ofpretreatment and posttreatment venograms. Correlations of volume of clot lysed
with measured and corrected peak concentrations (panel A) and with measured and corrected time-integrated
D-dimer concentrations (panel B) are presented.
Brenner et al Noninvasive Quantitation of Clot Lysis
E
2000-
0
2000 -
0
13A
`U
a
41-*
0
-1000
.
cc
0
C
-2000
0-1
2-3
ANGIOGRAM GRADE
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
FIGURE 4. Scatterplot of correlation of angiographic findings and corrected D-dimer concentrations after treatment for
myocardial infarction. Patients presenting with clinical and
electrocardiographicfindings of myocardial infarction received
fibrinolytic therapy with APSAC or streptokinase. The corrected D-dimer concentration was obtained by subtracting the
D-dimer concentration of pretreatment plasma incubated in
vitro with tissue-type plasminogen activator from the plasma
concentration 90 minutes after completion of therapy. The
mean ±SEM of each group is shown.
tration of cross-linked fibrin-degradation products.
While measured plasma DD levels did not correlate
with venous clot lysis, adjustment of these values for
degradation of soluble fibrin (Table 1) resulted in
strong correlations between volume of clot lysed and
both peak DD (Figure 2) and time-integrated DD
(Figure 3). During therapy for myocardial infarction,
the corrected DD levels were near zero and did not
differ in patients with or without successful lysis
(Figure 4). This suggests that the increase in plasma
DD observed during therapy in patients with myocardial infarction may result from degradation of
soluble plasma fibrin and that the additional DD that
originates from lysis of the small coronary artery
thrombus is too small to be detected.
The pretreatment sample of each patient's plasma
was incubated with rt-PA in vitro to determine the
amount of DD that could derive from soluble fibrin.
The high rt-PA concentration (200 ,ug/ml) used to
treat pretreatment plasma results in maximum elevation of DD concentration in vitro, with preferential
degradation of soluble fibrin polymers compared to
fibrinogen.20 The DD potentially derived from soluble fibrin was subtracted from the measured DD
concentration in posttreatment plasma samples to
provide corrected DD concentrations (Figure 1).
Elevated corrected DD levels were interpreted as
deriving from lysis of thrombi and were correlated
with angiographic findings. Three technical considerations impact on this approach. The first concerns
the specificity of the DD assay for cross-linked fibrindegradation products rather than fibrinogen derivatives. The reaction of monoclonal antibody DD3B6
with fibrinogen fragment D in Western blots27 has
1823
raised questions about its specificity for cross-linked
fibrin derivatives. Also, the panspecific antifibrinogen
secondary antibody could theoretically react with
fibrinogen-degradation products captured in association with cross-linked fibrin derivatives such as DD.
However, no appreciable crossreactivity with fibrinogen degradation products has been found with the
ELISA9,28 or latex agglutination assays.29 Second, the
formation of soluble cross-linked fibrin polymers
during storage would increase the in vitro correction
and mask DD derived from thrombolysis. If this
occurred, the corrected levels in patients without clot
lysis should be negative; this was not seen in patients
with DVT, and values in patients with myocardial
infarction were within experimental error of zero.
Last, fibrinolysis occurring in vitro would also interfere with the results, causing excessively elevated DD
levels, but the use of aprotinin effectively inhibits
plasmin action after sample collection.30,31
Several studies have reported an increase in
plasma DD in patients with DVT,10,11'13,15 perhaps
reflecting physiological thrombolysis. Mirshahi et al5
found increased plasma concentrations at presentation but no correlation with extent of thrombosis
shown venographically. They further observed a
decrease in concentration during heparin therapy in
patients whose follow-up venogram showed a
decrease in thrombus size but persistently high levels
in patients with little or no venographic resolution.
Elevated concentrations have also been reported at
presentation with pulmonary embolism,10,12 and
Goldhaber et al12 found that a DD level of more than
145 ng/ml had a diagnostic sensitivity of 89% but a
specificity of only 44%. Limited diagnostic specificity
was also found by Greenberg et al,29 who found
elevations of plasma DD in hospitalized patients with
numerous conditions including arterial and venous
thrombosis, septicemia, carcinoma, and the postoperative state.
Greater elevations in DD levels have been found
during fibrinolytic therapy, with concentrations
increasing fivefold to 25-fold compared with pretreatment values during therapy for myocardial
infarction,14'1617 DVT,19 or pulmonary embolism.'8
Despite the consistent increase during lytic therapy,
no correlation between the degree of thrombolysis
and the elevation in DD levels has been found during
treatment for myocardial infarction14'1617 or DVT.19
A potential explanation that has been proposed for
the lack of correlation is the presence of multiple
sources of fibrin that may degrade during therapy.
For example, patients with myocardial infarction may
have concurrent DVT, left ventricular mural
thrombi, and fibrin on atherosclerotic plaques, while
patients with DVT may also have clinically unsuspected pulmonary emboli. Whatever the extraneous
source of fibrin, it has been of sufficient quantity to
obscure the specific detection of thrombus-derived
DD, thereby limiting the application of this assay for
noninvasive quantitation of therapeutic thrombolysis.
1824
Circulation Vol 81, No 6, June 1990
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These results support the hypothesis that a major
source of cross-linked fibrin-degradation products
during fibrinolytic therapy is soluble plasma fibrin.
Electrophoretic analyses in normal subjects indicates
that approximately 0.8% of total fibrinogen is present
as cross-linked fibrin dimer and that this percentage
is increased more than fourfold in patients with
myocardial infarction.20 After incubation of plasma
with rt-PA in vitro, these polymers are degraded in a
manner and extent similar to that induced by fibrinolytic therapy,20 associated with an increase in DD
reactivity from a mean of 1,074+252 to 7,030±986
ng/ml (Table 1). Considering that 29% of crosslinked fibrin dimer is DD, this increase in immunoreactivity would reflect degradation of approximately
23 ,ug/ml of fibrin dimer, representing 0.6% of an
average plasma fibrinogen concentration (3.5 mg/
ml). The DD concentration after in vitro incubation
with rt-PA (Table 1) represents the sum of DD
present in the plasma before treatment plus that
produced by degradation of soluble fibrin. Higher
concentrations during therapy may therefore result
from lysis of fibrin in thrombi, and this was observed
with successful treatment of DVT with a good correlation between the degree of elevation and extent
of lysis. DD levels during treatment for myocardial
infarction were close to those resulting from digestion of the pretreatment plasma in vitro, suggesting
that DD elevations during therapy resulted from
degradation of soluble plasma fibrin and consistent
with the small size of a coronary thrombus in comparison to a leg vein DVT.
While this method corrects for the contribution of
soluble fibrin to elevated plasma DD concentrations
during fibrinolytic therapy, there may be additional
intravascular or extravascular sites of fibrin deposition. However, the finding of a strong correlation
between the DD levels and quantitative venous clot
lysis after correction for soluble fibrin degradation
suggests that other fibrin deposits do not contribute
significantly to the results. Furthermore, the net
corrected DD value of close to zero in patients with
myocardial infarction argues against the presence of
unknown, noncoronary thrombi in these patients. An
improved correlation may be possible by mathematically correcting the time-integrated values for clearance of DD from the blood, but this approach is not
yet feasible because a multitude of degradation products are present and little information is available on
clearance rates during thrombolytic therapy.
This approach offers promise for more accurate
noninvasive monitoring of lytic therapy for venous
thrombosis, a possibility that would require assessment by a prospective study. If DD levels measured
during therapy do not increase above the in vitro
digest level of the pretreatment sample, our analysis
would suggest that little or no lysis is occurring. On
the other hand, if concentrations do exceed the in vitro
digest level, the degree of elevation should correlate
with the amount of clot lysis. Potentially, the course of
therapy over time could be followed by measuring serial
DD values and predicting continued clot lysis if the in
vivo level remained above that of the in vitro digest,
thereby guiding the duration of therapy.
Acknowledgments
The authors acknowledge the technical assistance
of Kristina Klingbeil and Janice White and the help
of Carol Weed in preparation of the manuscript.
References
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KEY WORDS * venous thrombosis * thrombolysis * fibrin
tissue plasminogen activator
Quantitation of venous clot lysis with the D-dimer immunoassay during fibrinolytic
therapy requires correction for soluble fibrin degradation.
B Brenner, C W Francis, S Totterman, C M Kessler, A K Rao, R Rubin, H C Kwaan, K R
Gabriel and V J Marder
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Circulation. 1990;81:1818-1825
doi: 10.1161/01.CIR.81.6.1818
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