Coagulation Tests
When damage to small blood vessels and capillaries occurs, the body controls blood loss via
physiological processes referred to as hemostasis. In vivo, hemostasis depends on an
interaction between the plasma–based coagulation cascade, platelets, and the endothelium of
blood vessels. In the clinical laboratory, in vitro analytical assays are capable of measuring
only the first two components of this system. Consequently, laboratory measurements of
blood coagulation represent only a close approximation of the body's hemostatic system.
Clinicians frequently order coagulation tests, such as the prothrombin time (PT), activated
partial thromboplastin time (aPTT), and thrombin time (TT), to assess blood clotting function
in patients. While these laboratory tests may be helpful in elucidating the cause of
unexplained bleeding, they are not helpful in predicting if bleeding will occur. In fact, no
single test can predict bleeding in the perioperative or post-operative period. Furthermore,
these common laboratory tests are of little help in predicting blood clotting or thrombosis in
the absence of vessel injury. Well-described assays are available to test for hereditary
predisposition to thrombosis, but the majority of thrombophilic states cannot be quantified by
any current laboratory tests.Clearly, laboratory assessment of hemostasis presents many
challenges for laboratorians and the clinicians who interpret the results. This review briefly
explains the common tests used to assess hemostasis, as well as their clinical context, and
provides a guide for clinical chemists to assess unexplained bleeding.
Clearly, laboratory assessment of hemostasis presents many challenges for laboratorians and
the clinicians who interpret the results. This review briefly explains the common tests used to
assess hemostasis, as well as their clinical context, and provides a guide for clinical chemists
to assess unexplained bleeding.
The ABCs of Coagulation Tests
Laboratory tests for hemostasis typically require citrated plasma derived from whole blood.
Specimens should be collected into tubes containing 3.2% sodium citrate (109 mM) at a ratio
of 9 parts blood and 1 part anticoagulant. The purpose of the citrate is to remove calcium ions
that are essential for blood coagulation; however, failure to fill the draw tube adequately
causes the final citrate concentration of the patient sample to be too high. This is important
because PT and aPTT tests require the addition of calcium. If the specimen contains excess
citrate, addition of calcium may be inadequate, and the low plasma calcium will lead to an
artificial prolongation of PT or aPTT.
A similar but more subtle problem might arise if the patient's hematocrit is unusually high,
typically ≥55%. Normally, 10 mL of blood with a hematocrit of 40% contains 6 mL of
plasma. If the hematocrit is abnormally elevated, for example 65%, the specimen will contain
only 3.5 mL of plasma, effectively under-filling the draw tube with plasma, leading to
overcitration of the plasma.
For the PT test, adding a thromboplastin reagent containing a tissue factor, calcium, and
phospholipids initiates coagulation of the pre-warmed specimen via the extrinsic coagulation
pathway (Figure 1). Similarly, the aPTT test is initiated by adding a negatively charged
surface such as silica to the plasma, as well as a phospholipid extract that is free of tissue
factor. The coagulation pathway that occurs in the aPTT test represents the intrinsic
coagulation pathway (Figure 1).
Coagulation Cascade
The coagulation cascade is a series of enzymatic reactions that turn inactive precursors into
active factors. The end result of the cascade is the production of fibrin, a protein that binds
platelets and other materials in a stable clot. The cascade has two initial pathways: the
extrinsic (tissue factor-mediated) and the intrinsic (contact system-initiated). These two
pathways converge to become the common pathway with the activation of factor X. The steps
in the cascade that are measured by the three common coagulation assays, PT, aPTT, and TT,
are indicated.
When a patient has an abnormally prolonged PT or aPTT, laboratories should perform a
mixing study of the specimen (Table 1). To perform the test, the technologist mixes an equal
volume of the patient's citrated plasma with normal pooled plasma (NPP) and repeats the PT
and/or aPTT. If the clotting assay time now falls within the PT and/or aPTT reference
intervals, the initial abnormal result was due to one or more clotting factor deficiencies. In
contrast, the presence of inhibitors in patient plasma interferes with the clotting factors in the
NPP, but the mixing study results will not produce normal clotting times. Another common
assay used to assess hemostasis is TT (Figure 1). This test measures the ability of fibrinogen
to form fibrin strands in vitro. To perform the test, the technologist adds exogenous thrombin
to pre-warmed plasma. This step ensures that the result is independent of endogenous
thrombin or any of the other clotting factors. TT is particularly sensitive to heparin
Table 1
Assessment of Prolonged aPTT
This table shows how coagulation assays can be combined to elucidate the possible causes
of a prolonged aPTT.
Clinical Increased Increased Increased No Thrombophilia,
Features Bleeding Bleeding Bleeding Haemostatic DVT, PE
Problems
1:1 Corrects Corrects No correction Corrects No correction
Mixing
Study
PT Normal Prolonged Normal Normal Normal
Pathology Deficiency of Deficiency Autoantibodies Deficiency of Ant phospholipid
factors VIII, of factors II, to factor VIII factor XII syndrome
IX and some V, X, (acquired
cases of fibrinogen haemophilia) Deficiency of
factor XI other contact
deficiency Warfarin Rx factors such as
prekallikrein
Some cases of
factor XI
deficiency
Abbreviations: DVT, deep vein thrombosis; PE, pulmonary embolism; Rx, treatment.
Antiphospholipid syndrome. An autoimmune prothrombotic acquired condition,
antiphospholipid syndrome (APLS) is frequently associated with a markedly prolonged
aPTT, leading to a concern that the affected individual might be at risk for a major
haemorrhage. Not only is this highly unlikely, but as a prothrombotic state, APLS is typically
associated with venous thromboembolism and/or arterial thrombosis. The condition may also
present with fetal loss or stillbirth, which likely occurs as a result of placental inflammation
or thrombosis.
Individuals with APLS have antibodies known as lupus anticoagulants (LA). These
antibodies are directed to complexes of beta-2-glycoprotein I/phospholipid or
prothrombin/phospholipid, and they interfere with and prolong in vitro clotting assays. In the
body's vascular system, however, the presence of endothelial cells and leukocytes, as well as
many other components that are absent from the simplified in vitro clotting assay, increase
the likelihood of clotting.
The classic laboratory findings in APLS patients are prolonged aPTT, normal PT, and no
correction of the aPTT 1:1 mixing study. Adding excess phospholipid to the aPTT assay,
however, reduces the clotting time. This is the basis for the so-called LA assay. One version
of the LA assay is the dilute Russell's viper venom time (dRVVT). The assay components
activate only the common coagulation pathway via factor X, and they are independent of
factor VIII or antibodies to factor VIII. Laboratories also can confirm APLS by detecting IgG
or IgM antibodies to cardiolipin or to beta-2-glycoprotein I in an ELISA-type assay.
Factor V Leiden. A variant of factor V, factor V Leiden causes a hereditary
hypercoagulability disorder. Individuals with the disorder have a point mutation in the factor
V gene that produces a single amino acid switch (arginine to glutamine, R506Q) that makes
the protein resistant to inactivation by activated protein C. Heterozygosity for factor V Leiden
increases the risk for venous thromboembolism about two- or three-fold.
Laboratory results for this genetic condition include PT and aPTT within the normal range. In
addition, aPTT will be resistant to activated protein C, and in normal individuals, adding
activated protein C to a fresh plasma specimen will cause prolongation of aPTT. This effect is
blunted for individuals with factor V Leiden. DNA studies definitively confirm the G1691A
nucleotide switch.
Prothrombin G20210A. Another hereditary thrombophilia, the G20210A polymorphism in
the prothrombin gene elevates the plasma concentrations of prothrombin (FII) without
changing the amino acid sequence of the protein.
Patients with this mutation have PT and aPTT results that fall within the normal range, as
well as normal functional clot-based studies. DNA studies will show a G-to-A substitution in
the 3'-untranslated region of prothrombin gene at nucleotide 20,210.
Protein C and S deficiency. These two vitamin K-dependent factors interrupt the activity of
clotting factors V and VIII. Activated protein C is a proteolytic enzyme, while protein S is an
essential co-factor.
Antithrombin deficiency. AT, formerly called AT III, is a vitamin K-independent
glycoprotein that is a major inhibitor of thrombin and other coagulation serine proteases,
including factors Xa and IXa. AT forms a competitive 1:1 complex with its target but only in
the presence of a negatively charged glycosaminoglycan, such as heparin or heparin sulfate.
Patients with AT deficiency will have little-to-no AT III activity as measured in a
chromogenic assay.
Interpretation of Coagulation Tests
As with any laboratory test, our goal as laboratorians is to assist clinicians with utilization
and interpretation of tests that assess hemostasis. Unlike an elevated serum creatinine or a
high thyroid stimulating hormone that indicate impaired renal function and hypothyroidism
respectively, tests of hemostasis have to be interpreted in the context of clinical findings as
well as other laboratory findings. Given the dire consequences of unexplained bleeding,
clinical laboratorians should actively advise clinicians on use and interpretation of
coagulation tests. A shotgun approach to hemostatic disorders is rarely successful and can
result in delayed diagnosis and treatment for patients.
Blood clotting factors are needed for blood to clot (coagulation). Prothrombin, or factor II, is
one of the clotting factors made by the liver. Vitamin K is needed to make prothrombin and
other clotting factors. Prothrombin time is an important test because it checks to see if five
different blood clotting factors (factors I, II, V, VII, and X) are present. The prothrombin time
is made longer by:
Blood-thinning medicine, such as warfarin.
Low levels of blood clotting factors.
A change in the activity of any of the clotting factors.
The absence of any of the clotting factors.
Other substances, called inhibitors that affect the clotting factors.
An increase in the use of the clotting factors.
An abnormal prothrombin time is often caused by liver disease or injury or by treatment with
blood thinners.
