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Chapter 129 Laboratory Evaluation of Hemostatic and Thrombotic Disorders 1929

LABORATORY EVALUATION OF FIBRINOLYSIS bleeding risk and efficacy of inhibitor bypassing than traditional
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tests. TGAs are not yet used broadly in the clinical context; test
Disorders of fibrin cross-linking and fibrinolysis are not recognized procedures are not yet well standardized, and validated reference
by routine coagulation protein and platelet testing (see Table 129.1). ranges for specific conditions have not been developed.
Congenital factor XIII deficiency, a rare disease with a prevalence of Viscoelastic assays like thromboelastography and thromboelas-
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1 : 2 or 3 million people worldwide, is one example. Screening tests tometry are increasingly used in the clinical context. Viscoelastic tests
for factor XIII deficiency aim to dissolve fibrin clots in acetic acid, are based on the premise that the end result of normal hemostasis is
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monochloracetic acid, or urea. In the urea clot solubility test, to rapidly create a strong, stable clot; changes in the speed of clot
patient plasma and a normal plasma control are induced to clot with creation, or its strength or stability, may indicate abnormal hemostatic
or without added thrombin. Both samples are exposed to a 5 M urea function. Thromboelastography was first described in 1948 by
solution and observed over time. The control clot should remain Hartert, who presented a method in which clotting was triggered in
undissolved at 24 hours, as fibrin clots cross-linked in the presence fresh whole blood with the addition of celite (an activator of the
of thrombin and factor XIII are stable. If the patient is factor XIII– intrinsic pathway). The blood was then put into a continuously
deficient, the clot will dissolve rapidly. The urea clot solubility test, rotating cup, and a torsion wire was introduced into the system. As
and related assays, are not standardized and have poor sensitivity; the clot gradually became stronger, the movement of the torsion wire
their detection limit is generally less than 5% of factor XIII activity. dampened and ultimately stopped. A tracing of the torsion wire’s
Suspected factor XIII deficiency must be confirmed by functional movement over time reflected the velocity of clot formation, its
assays (which measure the release of ammonia during the transgluta- maximal stability, and its gradual dissolution. This tracing was
minase reaction, or incorporation of radioactive amines into proteins), recorded onto light-sensitive photographic paper by a mirror-
or immunologic factor XIII antigen assays. 22 galvanometer; the image developed over hours to days.
Tests that give a global picture of fibrinolysis can also be per- Currently, two semi-automated commercial viscoelastic devices on
formed; they measure the combined effect of plasminogen activators the market allow results to be obtained in 10–15 minutes: the
(e.g., urokinase, tissue plasminogen activator) and inhibitors (e.g., ROTEM-analyzer (TEM International, Munich, Germany), which
α 2 -antiplasmin defects, plasminogen activator inhibitor 1). Defects uses a fixed cup with a rotating pin and optical detector, and the
in the latter group of proteins can produce hyperfibrinolytic states TEG-analyzer (Haemonetics Corp., Braintree, MA, USA), which
and increased bleeding potential. In the euglobulin clot lysis assay, uses a torsion wire and a rotating cup. Both are kinetic tests, and their
plasma is diluted and acidified. This causes precipitation of the acid tracings measure clot formation over time. They also measure maximal
insoluble or euglobulin fraction of plasma, which includes fibrinogen, clot strength, clot elasticity, and clot lysis. Both use citrated whole
plasminogen, plasminogen activators, and plasminogen activator blood that is recalcified and added to a cuvette. An activator (e.g.,
inhibitor-1. The precipitate is redissolved, and the fibrinogen is tissue factor) is commonly used to standardize the test, and speed up
clotted by adding calcium. The time to spontaneous lysis of the fibrin the assay. These analyzers can perform additional tests (using different
clot is then monitored. Shortened lysis times are observed in hyper- activators or additives) to provide information similar to that of the
fibrinolytic states such as DIC, or when there is a deficiency of APTT or PT, to neutralize heparin, to inhibit fibrinolysis, and to
plasminogen activator inhibitor-1; however, like the urea clot solubil- qualitatively analyze the functional fibrinogen component.
ity test, the euglobulin clot lysis time is hampered by a lack of Viscoelastic methods are increasingly used at the bedside. They
standardization and susceptibility to interference by other factors. have shown some promise in detecting coagulopathies and guiding
Specialized coagulation laboratories may offer specific assays for the use of blood products and other prohemostatic therapy intraop-
plasminogen inhibitors as well. eratively, in obstetric hemorrhage, in the trauma setting, and in the
intensive care unit. 24,25 They may also be helpful in screening for
hypercoagulable states, as some individuals with a history of throm-
OTHER ACTIVITIES FOR HEMOSTASIS LABORATORIES boembolism demonstrate accelerated clot propagation. Viscoelastic
methods do have some drawbacks. They are insensitive to platelet
Global Hemostasis Assays and vWF dysfunction, as well as factor XIII problems. Assay stan-
dardization and variability of results continue to be a challenge. 26,27
Standard coagulation tests are capable of measuring the individual
components of hemostasis. There is tremendous interest in evaluating
hemostasis more globally, to accurately evaluate in vivo hemostatic Evaluation of Prothrombotic States
function, sensitively and specifically diagnose hemostatic disorders,
monitor treatment, and better predict clinical manifestations of dis- In addition to the diagnosis of bleeding disorders, the hemostasis
ordered hemostasis. Global hemostasis assays aim not only to measure laboratory can help evaluate acquired and inherited prothrombotic
components of hemostasis, but also determine how they interact with states (see Chapter 140). It is known that certain prothrombotic states
each other. They do so by assessing the rate of thrombin generation, (e.g., antithrombin, protein C, and protein S deficiencies; antiphos-
the quantity of thrombin generation, the formation of clots in whole pholipid antibody syndrome) may confer a higher risk for recurrent
blood, and/or the polymerization of fibrin. thrombosis. Patients with these abnormalities may benefit from long-
Measurement of thrombin generation was first described in the term anticoagulation after their first episode of thrombosis. Less
early 1950s by MacFarlane and Biggs (who used whole blood), and severe prothrombotic states (e.g., heterozygous factor V Leiden and
Pitney and Dacie (who used plasma). Over the years, methods were prothrombin 20210 polymorphisms, functional defects in fibrinogen
refined; ultimately, continuous measurement of thrombin generation and plasminogen) do not appear to impact the risk of recurrence.
was accomplished by using a thrombin-chromogenic substrate in Clinicians often consider testing for prothrombotic states in patients
defibrinated plasma, or a fluorogenic substrate in whole plasma. In who develop thrombosis at a young age, who have a strong family
general, modern thrombin generation assays (TGAs) use recalcified history of thrombosis, who have thrombosis at an unusual site, or
platelet-rich or platelet-poor plasma, and then trigger clotting using who experience recurrent or unexplained thrombosis. However the
tissue factor. TGAs can capture not only the rate and amount of utility of this testing is limited, as the results usually do not affect
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thrombin generated, but also the role of platelets (which act as an patient management or result in improved patient outcomes.
amplifying surface for thrombin activity). TGAs have been widely Testing can also result in potential harm, including inappropriate use
used in research. Clinically, they may play a role in thrombophilic of anticoagulation and undue patient anxiety. For these reasons,
states (e.g., ATII deficiency, protein C or S deficiency, activated testing for prothrombotic states is not routinely recommended in
protein C resistance, cancer-associated thrombosis) and in hemor- unselected patients with thrombosis. Testing should only be under-
rhagic tendencies (hemophilia, other factor deficiencies, cardiac taken with patients whose clinical presentation strongly suggests
surgery). In hemophilia, TGAs might more accurately predict an underlying prothrombotic condition. Before testing is done,