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Oak Ridge Conference |
1
Sanguinex, P.O. Box 98385, Raleigh, NC 27624-8685.
2
Cardiovascular Diagnostics, Inc., 5301 Departure Dr.,
Raleigh, NC 27616.
a Author for correspondence. Fax 919-676-5436; e-mail bjo{at}ntwrks.com
Abstract
Point-of-care (POC) coagulation tests with paramagnetic iron oxide
particles have provided alternatives to testing previously done only in
the laboratory. With this technology, POC fibrinolytic tests have
followed quietly the trail blazed by POC clotting tests and have found
specific applications. These include rapid verification of in vivo
thrombolytic drug action by in vitro testing with subsequent
quantitative drug monitoring of the systemic lytic state, and also the
determination of in vitro thrombolytic drug response before the drug is
actually administered, to individualize therapy by selection of the
most appropriate drug. Other applications include POC coagulation
factor assays associated with fibrinolysis, and most recently the POC
screening of patients with fibrinolytic defects. In this latter
application, plasma from cardiac catheterization (n = 19) and
venous thrombosis (n = 47) patient groups were tested. Controls
consisted of two independent donor pools (n = 10, n = 21) as
negatives and two plasma samples with known genetic defects in the
fibrinogen molecule (A
554 Arg 
Cys) as positives.
Blood coagulation testing at the point of care (POC) now exists in many hospitals.3 POC testing has been driven by economic factors and by improved technology (1). In particular, dry chemistry coagulation testing at the POC with paramagnetic iron oxide particles (PIOP) has provided alternatives to testing previously done only in the laboratory and has opened the door to convenient fibrinolytic testing (2)(3)(4). With this technology, POC fibrinolytic tests have followed quietly the trail blazed by POC dry chemistry clotting tests and have found specific applications. For example, during and after thrombolytic drug administration, a quantifiable measure of a patient's systemic lytic response can be determined rapidly at the POC to verify drug action (5). Furthermore, the response of a patient's in vitro-generated clot to a thrombolytic drug can be determined before the drug is actually given, allowing therapy to be individualized by selecting the most appropriate drug (6). Both of these fibrinolytic applications are being pursued in multicenter clinical trials in several countries. Two additional potential clinical applications of POC fibrinolytic testing are the ability to perform fibrinolytic factor assays4 and to screen for fibrinolytic defects, which will be discussed further in this report.
The PIOP/dry chemistry technology may be used with whole blood or
citrated plasma, yielding similar results for most tests. After
placement of the test card that contains appropriate dry reagents and
PIOP in the instrument, the card is warmed by a 37 °C heat source.
The sample is then added to the test card. The liquid sample dissolves
the reagents and frees the PIOP. The PIOP then organize into ensembles
or "stacks" under the influence of orthogonal magnetic fields. The
field perpendicular to the test card surface oscillates, causing the
particles to oscillate (3)(7). The motion of
PIOP is monitored in the near infrared throughout the test. When the
sample clots, the PIOP become entrapped in the gel clot, and their
motion diminishes gradually. The peak PIOP oscillation signal before
the decay in amplitude is the typical end point for clotting time
measurements that correlates well with conventional laboratory methods.
A variety of conventional and new clotting tests are possible with this
technology. For example, a PIOP/dry chemistry test [ecarin clotting
time (ECT)] was developed for antithrombin drugs. Fig. 1
shows a plot of clotting time vs the concentration of the
thrombin inhibitor polyethylene glycol (PEG)–hirudin, a therapeutic
drug being developed as a replacement for heparin in patients with
conditions such as heparin-induced thrombocytopenia. This test is based
on ECT (8)(9). Ecarin is a snake venom product
from Echis carinatus (EC 3.4.99.27) that provides a test
specific for thrombin inhibitors.
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Ecarin, a protein prothrombin activator, causes coagulation of citrated whole blood or citrated plasma by the calcium-independent activation of prothrombin. Ecarin catalyzes the hydrolytic cleavage of the 323Arg–324Ile bond in the human prothrombin molecule, whereby thrombin activity is generated without the release of a zymogen fragment. This form of active prothrombin has been termed meizothrombin and is inhibited by r-hirudin or PEG–hirudin, but surprisingly, not efficiently by the heparin–ATIII complex. Ecarin has been used as the basis to develop a sensitive analytical method for the determination of thrombin inhibition by antithrombin drugs such as r-hirudin and PEG–hirudin and is based on the method of Nowak and Bucha (8)(9). The PIOP/dry chemistry ECT test card is intended to determine the concentration of an antithrombin drug in citrated whole blood and can be used to monitor patients receiving antithrombin drugs such as r-hirudin or PEG–hirudin.
The data shown in Fig. 1
are for citrated whole-blood samples from
healthy donors, supplemented with increasing concentrations of
PEG–hirudin (Knoll) and (or) porcine heparin (Sigma) and tested in the
dry chemistry format. The ECT test cards were prepared from ecarin
(Centerchem), calcium, PIOP, buffers, and stabilizers. As the in vivo
concentration of antithrombin drug increases, the clotting time
increases in a linear dose-dependent manner with essentially no effect
of heparin on the clotting time end point, a potentially important
clinical consideration when determination of only the anticoagulant
effects of the direct thrombin inhibitor is desired.
For fibrinolysis (thrombolysis) measurements, where the clot
subsequently dissolves, a new end point known as lysis onset time (LOT)
was established
(10).5
LOT is the point at which the PIOP oscillation
amplitude starts to increase again (Fig. 2
), eventually resulting in full restoration of movement and is
detected well before the clot dissolves, which provides the earliest
indication of fibrinolysis. LOT can be precisely measured and is
inversely proportional to the concentration of plasminogen activator
present in the system (3). A mathematical model for LOT
was developed that is supportable by experimental data
(7). Fig. 2
shows a PIOP oscillation curve vs time for a
lytic test. The peak event corresponding to the induced clotting time
is observed along with the subsequent rise event corresponding to LOT.
Increased concentrations of thrombotic agent incorporated in the test
card dry chemistry decrease the clotting time in the oscillation curve.
For high concentrations of a thrombotic agent such as thrombin or
batroxobin, clotting time can be reduced to a few seconds. Increased
concentrations of thrombolytic agent in the test card or in the sample
being tested shorten the LOT in the oscillation curve. For high
concentrations of thrombolytic agent, LOT can be <90 s. In this
report, data are shown to demonstrate that two new potential
applications of LOT may be achievable: fibrinolytic factor assays and
fibrinolytic defect screening.
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Materials and Methods
plasminogen assay
Plasminogen-free citrated plasma was prepared by passing pooled
normal citrated plasma (Specialty Assayed Reference Plasma, George
King) through a lysine–Sepharose column (Pharmacia). This treatment
selectively removes plasminogen without affecting other coagulation
proteins. Plasminogen (Sigma) was then added to the
plasminogen-depleted citrated plasma to yield the different
concentrations used in the assay. Dry chemistry plasminogen test cards
were prepared by assembling the test card device and injecting 24 µL
of a solution [containing 1500 U/L thrombin, 500 000 U/L tissue
plasminogen activator (t-PA), 500 000 U/L urokinase, PIOP, salts,
buffers, and stabilizers] into the card chamber, freezing the
card/reagent mixture, and lyophilizing the test card to yield the dry
chemistry formulation. Test cards were placed in a Thrombolytic
Assessment System (TAS) analyzer (Cardiovascular Diagnostics), and 30
µL of the various plasminogen-supplemented citrated plasmas were
added to the test card. The TAS analyzer was programmed to detect the
LOT and display this value to the user.
screening for fibrinolytic defects
Citrated plasma samples were obtained from stockpiled venous
thrombosis patients and patients undergoing cardiac catherization.
CHIII and Dusart are samples from well-studied thrombosis-prone
families with abnormal fibrinogens known to exhibit impaired
fibrinolysis (12)(13)(14)(15)(16). The defect in both families
consists of the same amino acid substitution of cysteine at the A554
arginine site (15). To provide "normals," samples were
obtained from local donors at Cardiovascular Diagnostics, and from
donors at a major blood center. All citrated plasma samples were
maintained frozen at -85 °C before analysis and thawed at 37 °C
before use. Dry chemistry test cards suitable for screening patient and
normal samples were prepared as follows: 24 µL of a solution
containing 1500 U/L thrombin, t-PA at a concentration of 200 000,
400 000, 800 000, or 1 000 000 U/L, PIOP, salts, buffers, and
stabilizers were injected into an assembled test card device, frozen,
and lyophilized to yield the dry chemistry formulation. The formulation
included heparin neutralizers, which negated the effects of heparin, if
present. Results shown are for a t-PA concentration of 200 000 U/L.
The test cards were placed in the TAS analyzer, and 30 µL of patient
or normal donor citrated plasma were added to the test card. The TAS
analyzer was programmed to detect and display the LOT value.
Results
plasminogen assay
A set of increasing responses forming a curve resulted when LOT
values were plotted vs plasminogen concentration in the sample (Fig. 3
). The resulting LOT values ranged from 90 s at the higher
plasminogen concentrations (120 to 160 mg/L) to 250 s at 45 mg/L.
Plotting the LOT results of the plasmin(ogen) assay vs the inverse of
the plasminogen concentration results in a linear LOT response (Fig. 4
).
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screening for fibrinolytic defects
When the low-concentration t-PA (200 000 U/L) test data were
obtained, we observed that the venous thrombosis, cardiac
catheterization, and two normal groups exhibited a wide range of
individual responses. The mean LOT values and their standard deviations
(in s) were: 432 ± 102 for the venous thrombosis group (n =
47), 329 ± 88 for the cardiac catheterization group (n =
19), 283 ± 76 for the local "normal" donors (n = 10),
and 319 ± 54 for the blood center "normal" donors (n =
21). The LOT values obtained for the Dusart and CHIII samples were 875
and 670 s, respectively. All measurements are means of duplicates.
LOT measurements, with low-concentration t-PA, are generally
within ± 10%. Precision approaches ± 5% as the amount of
t-PA reaches 500 000 U/L, the concentration used to generate the data
in Fig. 2
.
Discussion
The calibration curve for plasminogen indicates that it should be possible to provide a POC test for low-range plasminogen in patient samples. This test would actually measure total available plasmin and provide the composite fibrinolytic effect of plasminogen and free plasmin, if any. Typically, plasminogen would be measured. In the case of a patient undergoing extended thrombolytic therapy, e.g., for deep vein thrombosis, it may be important to determine periodically that the patient has sufficient plasmin(ogen) reserves for the therapy to continue to be effective. A low-range test for total available plasmin is well suited to meet this objective. Normal- and higher-range plasminogen (concentrations >200 mg/L) is not a critical care issue and can be determined with a laboratory test in a routine fashion.
A linear LOT response is obtained by plotting LOT vs the reciprocal of
the plasminogen concentration (Fig. 4
). This linearization had
previously been established for plasminogen activators
(3). This depiction of the LOT response allows easier
conversion of LOT into plasmin(ogen) concentration. Our test may be
suitable for use with thrombolytic therapy patients to assess
plasmin(ogen) reserves at the POC.
In screening for fibrinolytic defects, it is important to establish a
normal range. Extensive testing was not undertaken to do this, but
selected random samples were used from two different donor pools.
Whether all of the samples in each pool are "normal" was not
addressed by our study. Similarly, some of the cardiac catheterization
patients are probably normals. For example, patients with heart valve
problems but without vessel disease might have normal LOT values.
However, when the means of each "normal" pool and their standard
deviations are different, a general range of probable normals may be
inferred from the data, as depicted graphically in Fig. 5
. Similarly, the known abnormal samples, Dusart and CHIII, fall
well outside of this general range of probable normal values. In fact,
LOT values for both Dusart and CHIII are more than 5 SD from the mean
of either normal group. Therefore, other samples falling well beyond
the normal ranges would be expected to be abnormal as well. It is
remarkable, therefore, that the venous thrombosis group contains two or
four individuals whose LOT values are >5 SD from the mean value of the
"normals," depending upon which "normal" group is used.
Therefore, we have been able to demonstrate impaired fibrinolysis in
vitro for some of the venous thrombosis patients. Specific fibrinolytic
defects were not detected and correlated to clinical outcomes, as that
was beyond the scope of the present feasibility study.
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Venous thrombosis is caused by a variety of defects, at least one subset of which are fibrinolytic defects. Finding or identifying this subset of patients is not easily accomplished. Two methods that have been used are euglobulin lysis time and whole-blood clot lysis time. Both methods are labor intensive and difficult to standardize. For the individual samples tested here, the origin of the fibrinolytic defects is unknown. However, the simple, rapid test methodology, as described, should enable convenient screening and identification of abnormal individuals who could undergo further testing to determine the exact cause of the fibrinolytic defect.
Table 1
tabulates POC clotting time measurements and LOT measurements
for different end uses. For the end use of lytic state monitoring,
clotting time end points, such as in fibrinogen and thrombin time
tests, provide an indirect measure of the systemic lytic state. LOT
provides a direct measure (3)(5). For drug
response, clotting time end points provide a measure of in vitro
anticoagulant effects after administration of an anticoagulant drug.
With appropriate test chemistry, LOT provides a measure of in vitro
fibrinolytic effects before the administration of a thrombolytic drug
(6). In factor assays, clotting time and LOT end points
(as discussed previously) may be used to determine concentrations of
clotting and fibrinolytic factors, respectively. Finally, clotting time
end points in tests such as prothrombin time and activated partial
thromboplastin time enable screening for clotting defects, and
fibrinolytic tests such as the low-concentration t-PA response test
described in this report enable screening for fibrinolytic defects.
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Acknowledgments
The contributions of the following individuals are also acknowledged: Omar Iqbal, Zaid Y. Alkadi, Stuart Lind, William H. Bingham, Susan T. Lord, Gregory J. Dehmer, Sharon C. Dermott, David R. Musselman, Jawed Fareed, David C. Sane, Gregory Goslen, David A. Tate, Nancy J. Gresalfi, and Aldo A. Tabares. Support of NIH of this research, in part, through the grants R43 HL 47223, R44 HL 47223, R43 HL 52412, and R44 HL 52412 is gratefully acknowledged.
Footnotes
3 Nonstandard abbreviations: POC, point of care; PIOP,
paramagnetic iron oxide particles; ECT, ecarin clotting time; PEG,
polyethylene glycol; LOT, lysis onset time; t-PA, tissue plasminogen
activator; and TAS, Thrombolytic Assessment System. 
4 Normally the term "coagulation factor" refers to protein factors that, when activated, cause both clotting and lysis. We prefer to separate the coagulation factor, plasminogen, as a "fibrinolytic factor" that, when activated, causes lysis of the clot.
5 LOT differs from clot lysis time, an older term found in hematology textbooks. Clot lysis time is related to the time required to dissolve an in vitro clot in tests such as whole-blood clot lysis time. Whole-blood clot lysis time is used as a simple functional screening test to determine lytic tendency and consists of allowing a tube of whole blood to clot and examining the tube periodically to determine how long it takes for the clot to dissolve. In healthy individuals, the clot remains undissolved, even after 24 h. In individuals with abnormal antiplasmin activity or in some fibrinolytic states, the clot is observed to dissolve in a few hours [11]. It should be recognized that the visual determination of clot lysis time cannot be made until a substantial amount of lysis of the thrombus occurs. Euglobulin clot lysis time is a variation of this test.
References
Cys) in two unrelated kindred, Dusart and Chapel Hill III. Blood 1994;84:3709-3714.
The following articles in journals at HighWire Press have cited this article:
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  T. W. Stief and J. Fareed Point of Care: Diagnostics in Hemostasisthe Wrong Direction? Clinical and Applied Thrombosis/Hemostasis, July 1, 2003; 9(3): 191 - 195. [Abstract] [PDF]
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