Review
Coagulation Testing in the Core Laboratory
William E. Winter, MD, Sherri D. Flax, MD, Neil S. Harris, MD*
Laboratory Medicine 48:4:295-313
DOI: 10.1093/labmed/lmx050
ABSTRACT
Primary hemostasis begins with endothelial injury. VWF, produced by
endothelial cells, binds to platelets and links them to subendothelial
collagen. Platelet-derived ADP and thromboxane activate non-adhered
platelets via their GPIIb/IIIa receptors, allowing these platelets to
participate in platelet aggregation. Secondary hemostasis is initiated
with the binding of factor VII to extravascular tissue factor (TF). Factors II,
VII, IX and X are vitamin K-dependent factors. The role of vitamin K is to
assist in the addition of gamma carboxylate groups to glutamic acids in
the “GLA” domains of these factors.
In vitro the intrinsic pathway is initiated when fresh whole blood is
placed in a glass tube. The negative charge of the glass initiates the
“contact pathway” where FXII is activated and then FXIa cleaves FIX to
FIXa. The extrinsic pathway is triggered when tissue factor, phospholipid
and calcium are added to plasma anticoagulated with citrate. In vitro,
FVII is activated to FVIIa, and TF-FVIIa preferentially converts FX to FXa
activating the common pathway.
The prothrombin time is commonly used to monitor warfarin
anticoagulant therapy. To correct for differences in reagent and
instrument, the international normalized ratio was developed to
improve standardization of PT reporting globally. The activated partial
thromboplastin time (aPTT) is used to evaluate the intrinsic and common
pathways of coagulation. The aPTT is useful clinically as a screening
test for inherited and acquired factor deficiencies as well as to monitor
unfractionated heparin therapy although the anti-Xa assay is now the
preferred measure of the effects of unfractionated heparin. The Clauss
assay is the most commonly performed fibrinogen assay and uses
diluted plasma where clotting is initiated with a high concentration of
reagent thrombin.
The mixing study assists in the assessment of an abnormally prolonged
PT or aPTT. An equal volume of citrated patient plasma is mixed with
normal pooled plasma and the PT or aPTT are repeated on the 1:1 mix.
Factor activity assays are most commonly performed as a one-stage
assay. The patient’s citrated plasma is diluted and mixed 1-to-1 with a
Abbreviations
HS, heparin sulfate; AT, antithrombin; PS, protein S; PC, protein C; VWF,
von Willebrand Factor; FVIII, factor VIII; GPIb, glycoprotein receptor;
TF, tissue factor; RBC, red blood cell; DVT, deep venous thrombi; PE,
pulmonary embolism; tPA, tissue plasminogen activator; APC, activated
PC; HMWK, high molecular weight kininogen; aPTT, activated partial
thromboplastin; PT, prothrombin time; ISI, international sensitivity index;
WHO, World Health Organization; INR, international normalized ratio;
PTT, partial thromboplastin time; TT, thrombin time; ELISA, enzyme linked
immunoabsorbant; NPP, normal pooled plasma; APLS, antiphospholipid
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single factor-deficient substrate plasma. A PT or aPTT is performed on
the above mix, depending on the factor being tested.
Factor inhibitors are antibodies that are most commonly diagnosed in
male patients with severe hemophilia A (FVIII deficiency) where they are
induced by factor replacement therapy.
Factor inhibitors can also appear in the form of spontaneous
autoantibodies in both male and female individuals who were previously
well. This is an autoimmune condition called “acquired hemophilia.”
Most coagulation laboratories can measure the plasma concentration of
VWF protein (VWF antigen) by an immunoturbidimetric technique. Testing
the functional activity of VWF, utilizes the drug ristocetin.
The state of multimerization of VWF is important and is assessed by
electrophoresis on agarose gels. Type 2a and 2b VWD are associated
with the lack of intermediate- and high molecular weight multimers.
The antiphospholipid syndrome (APLS) is an acquired autoimmune
phenomenon associated with an increased incidence of both venous and
arterial thromboses, as well as fetal loss. Typically, there is a paradoxical
prolongation of the aPTT in the absence of any clinical features of
bleeding. This is the so-called “lupus anticoagulant (LA) effect.” The
laboratory definition of the APLS requires the presence of either a “lupus
anticoagulant” or a persistent titer of antiphospholipid antibodies.
There are now 2 broad classes of direct-acting oral anticoagulants
(DOACs): [1] The oral direct thrombin inhibitors (DTIs) such as dabigatran;
and [2] The oral direct factor Xa inhibitors such as rivaroxaban and
apixaban. The PT and aPTT are variably affected by the DOACs and are
generally unhelpful in monitoring their concentrations. Most importantly, a
normal PT or aPTT does NOT exclude the presence of any of the DOACs.
Keywords: coagulation, prothrombin time, partial thromboplastin time,
von Willebrand factor, intrinsic pathway, extrinsic pathway, thromboembolism
syndrome; dRVTT, dilute Russell viper venom time; PCC, prothrombin
complex concentrate; RIPA, ristocetin-induced platelet aggregation; LA,
lupus anticoagulant; DOAC, direct-acting oral anticoagulant; NOAC, novel
oral anticoagulant
Department of Pathology, Immunology & Laboratory Medicine, University
of Florida, Gainesville, FL
*To whom correspondence should be addressed.
harris@pathology.ufl.edu
295
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Table 1. Definitions of Blood Coagulum

The Physiology of Coagulation Thrombus Blood coagulum in a blood vessel in a
living person
Following endothelial injury, a thrombus will normally form Clot Blood coagulum that forms 1) after
(Table 1). A thrombus is a blood coagulum formed in the death, 2) ex vivo, or 3) in a living person
outside of a blood vessel
blood vessel of a living person. The function of the thrombus
Thromoembolism An embolism that originated from a
is to stem the loss of blood to secure vascular integrity, there- thrombus
fore, maintaining blood volume. Elements of the thrombus,

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such as platelets, can then contribute to healing. It is rather
amazing that flowing blood does not readily coagulate; how-
ever, upon removal of blood from the body when placed into Table 2. Hemostatic Balance
a glass tube, the blood immediately begins to clot. Prothrombotic Antithrombotic
Stasis Flowing blood
In contrast to a thrombus, a clot is a blood coagulum that Endothelial injury Endothelial integrity
develops 1) in blood vessels in the postmortem state, Hypercoagulability Heparan sulfate and
2) when blood is removed from the body and is not anti- antithrombin
coagulated, or 3) in a living person outside of their blood Thrombomodulin and
proteins S and C
vessels (Table 1). Nevertheless, in practice, the terms blood Alpha 2-antiplasmin Tissue plasminogen
clot and thrombus are often used interchangeably when, activator that converts
indeed, the terms are actually quite distinct. That said, we plasminogen to
discuss the biology of blood coagulation. plasmin
Platelet-produced Endothelium-
TxA2 produced PGI2
The first challenge is to recognize that the coagulation events
PGI2, Prostacyclin; TxA2:,Thromboxane A2.
in vivo and in vitro are not identical. In order to properly
interpret coagulation testing, the similarities and differences
between in vivo and in vitro events must be understood.
[VWF]), 2) secondary hemostasis that concludes with the
Ultimately, coagulation in vivo is the physiologically important
formation of a fibrin lattice and 3) tertiary hemostasis that
event, and therefore we first examine in vivo coagulation.
begins with fibrin crosslinking and ends with thrombus dis-
solution (which includes fibrinolysis).
In Vivo Coagulation
Primary Hemostasis
Hemostasis is the balance between factors that favor and
factors that oppose thrombosis. Factors that favor thrombo- Coagulation begins with endothelial injury. In terms of primary
sis are endothelial injury, hypercoagulability. and stasis. This hemostasis, VWF binds to exposed subendothelial collagen
thrombotic triad was described more than 100 years ago (Figure 1). Similar to factor VIII (FVIII) and unlike other plasma
by Virchow (Table 2). Opposing thrombus formation are the protein clotting factors, VWF is produced by endothelial cells
following factors: 1) the continuous flow of blood through of the body. VWF monomers are 2813 amino acids in size.
the blood vessels, 2) the vascular endothelium, 3) prostacy- VWF monomers bind head-to-head and tail-to-tail, producing
clin production by endothelial cells, 4) heparan sulfate (HS) VWF multimers of 500 to 20,000 kDa. In VWF, amino acids
expression on endothelial surfaces, and 5) circulating plasma 1 to 272 are involved in FVIII binding; amino acids 449 to
proteins that regulate coagulation including plasminogen, 728 bind to the glycoprotein receptor Ib (GPIb) on plate-
antithrombin (AT), protein S (PS), protein C (PC) and alpha-2 lets (Table 3); amino acids 911 to 1365 bind collagen; and
antiplasmin (Table 2). Once we examine coagulation in vivo, amino acids 1744 to 1746 bind to the GPIIb/IIIa receptor on
we return to the actions of plasminogen, AT, PC, PS and platelets.
alpha-2 antiplasmin, and then discuss blood clotting in vitro.
Via the GPIb receptor, platelets bind to VWF (bound to colla-
Coagulation is frequently divided into 3 stages: 1) primary gen) forming a monocellular cover over the site of endothelial
hemostasis (a function of platelets and von Willeband factor injury (Figure 2). This constitutes platelet adhesion. In the

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Loss of endothelial integrity GPIIb/IIIa

Endothelium
Change in
confirmation of
Basement membrane GPIIb/IIIa
ADP and TxA2
Collagen Activation

Pseudopodia
VWF binds to collagen

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Basement membrane
Collagen Collagen

Figure 1  Figure 3 
In most capillaries, endothelial cells form a continuous barrier With platelet binding to VWF, platelets become activated, and
separating blood from the basement membrane, subendothelial 3 events occur: 1) platelets develop pseudopodia that allow
space (that includes collagen), and subendothelial cells. A break platelets to interlock with one another providing strength to the
in this barrier exposes subendothelial collagen (upper panel). The thrombus; 2) platelets degranulate, releasing ADP, and activated
initial event in primary hemostasis is the binding of von Willebrand platelets synthesize thromboxane A2 (TxA2); and 3) the molecular
factor to collagen (lower panel). confirmation of the GPIIb/IIIa receptor changes allowing it to bind
to fibrinogen. The release of ADP and TxA2 activates nonadhered
platelets in the microenvironment. This will foster their involvement
in aggregation.
Table 3. Platelet Receptors and Their Functions
Receptor Function
pseudopodia, 2) the platelet granules are discharged
GPIb Binds to VWF
via the platelet canalicular system, and thromboxane A2
GPIa/IIa Binds to collagen under low shear-stress
conditions (TxA2) is synthesized and released, and 3) the GPIIb/IIIa
GPIIb/IIIa Binds to fibrinogen; lesser binding to VWF receptor conformation changes, allowing it to bind plasma
VWF, von Willebrand factor. fibrinogen. The open canalicular system is the surface-con-
nected channels that afford 1) transport into platelets and
2) the release of alpha granule contents from platelets.
GPIIb/IIIa Functioning similarly to the sarcotubules of skeletal mus-
Platelets bind to VWF
cle, the dense tubular system regulates platelet activity by
GPIb
Adhesion releasing or sequestering calcium.

Regarding platelet granules, the platelet’s alpha granules

Collagen
Figure 2 
Once VWF has bound to collagen, platelets bind to VWF via the
platelet’s GPIb receptor. This event defines platelet adhesion. GPIb is
part of a complex that also includes GPV and GPIX (ie, GPIb-V-IX).
venous system where shear stress is lower, platelets can bind
directly to exposed collagen via the GPIa/IIa receptor.
contain various proteins including VWF, factors V and XIII,
fibrinogen, fibronectin, P-selectin, platelet-derived growth
factor, beta-thromboglobulin, platelet factor-4, and throm-
bospondin. Individual platelets have 40 to 80 alpha granules
of 200 to 500 nm diameter that are round to oval in shape.
Table 4 details the functions of these proteins. The platelet’s
delta (dense) granules contain ADP, ATP, pyrophosphate,
calcium and serotonin. There are usually 4 to 8 such elec-
tron-dense granules per platelet. These granules contain
60% to 70% of the calcium in the platelet.

Once platelets have adhered to the injured endothelium, via During platelet activation, ADP and TxA2 act in autocrine and
the collagen-VWF-GPIb interaction, the platelet becomes paracrine fashions. The paracrine effect is to activate non-ad-
activated, leading to three important events (Figure 3): hered platelets in the microenvironment. Via their activated
1) the platelet changes shape, extending interlocking GPIIb/IIIa receptors, these platelets can now participate in

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Loss of endothelial integrity

Table  4. Constituents of Alpha Granules and Endothelium
Their Function
TF
Constituent Function Basement membrane
TF
TF
VWF Tethers platelets to collagen; binds to collagen
and GPIb on platelets
Factors V and VIII Clotting factors FVII binds to tissue factor (TF)
Fibrinogen Clotting factor FVII
Fibronectin Involved in cell adhesion and migration

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processes FVIIa
P-selectin Adhesion molecule; binds to sialyl Lewis-X TF TF TF
PDGF Stimulates fibroblast chemotaxis & proliferation
Beta-thromboglobulin Low molecular weight (36 kDa) heparin-binding
protein
PF4 A heparin-binding protein Figure 5 
Thrombospondin High molecular weight (450 kDa) heparin-
Secondary hemostasis in vivo is activated when plasma gains
binding protein
access to TF on subendothelial cells that were exposed as a
PDGF: platelet-derived growth factor; PF4: Ppatelet factor 4; VWF: von Willebrand consequence of injury to the vascular endothelium. FVII binds to TF
factor.
and undergoes autoactivation to FVIIa.

Fibrinogen A) FVII TF TF-FVIIa

Aggregation
B) FIX FIXa + FVIII FIXa-FVIII

C) FX FXa + FV FXa-FV

D) Prothrombin Thrombin
Collagen

Figure 4 
E) Fibrinogen Fibrin
Aggregation occurs when platelets are crosslinked via fibrinogen
binding to GPIIb/IIIa on platelets. Figure 6 
As a consequence of formation of the TF-FVIIa complex, a plasma
protein cascade ultimately leads to the conversion of fibrinogen to
platelet aggregation as they bind to adhered platelets and fibrin. The individual steps are described in detail in the text.
other aggregated platelets via fibrinogen (Figure 4). In a cas-
cade-like event, platelets further aggregate one to another
via GPIIb/IIIa and fibrinogen to build the mass and strength of primary hemostatic defects involving platelets or VWF
the thrombus. This completes primary hemostasis. include petechiae and mucosal bleeding.

The thrombus formed in primary hemostasis can produce Secondary Hemostasis

an immediate reduction in blood loss, assuming that the
size of the blood vessel lumen is not excessive. If a named
blood vessel is cut (meaning that the vessel is large enough
to be “named” by anatomists), thrombus formation alone
will not usually re-establish hemostasis, and external
mechanical compression and/or suturing will be required to
halt continued blood loss.
Clinically a failure to establish primary hemostasis after
injury results in immediate bleeding. Manifestations of
Secondary hemostasis is initiated with the binding of fac-
tor VII (FVII) to exposed subendothelial tissue factor (TF).
TF (CD142) is a transmembrane protein that is expressed
on subendothelial cells. However, TF is not normally
expressed on vascular endothelial membranes. An archaic
name for TF is “coagulation factor III.” Once FVII binds to
TF, FVII undergoes autocatalysis to FVIIa (a = activated;
Figure 5 and Figure 6A). Although the process of the initi-
ation of plasma clotting factor activation is quite complex,

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D E
Thrombin
D E
D E D D
D E D D E D D
D E D D
D E D D
Figure 7 
With the release of fibrinopeptide A and fibrinopeptide B, fibrinogen is converted to fibrin via the action of thrombin-activated FXIIIa. Fibrin
monomers initially stack, noncovalently forming a lattice.
it is sufficient for our purposes to understand that TF-FVIIa
in vivo ultimately leads to the conversion of factor IX (FIX)
to FIXa (Figure 6B). In contrast, we will see that in vitro,
the major action of TF-FVIIa is to convert factor X (FX)
to FXa.
Returning to in vivo events, FIXa plus its cofactor factor
VIII (FVIII) now acquires the ability to activate FX to FXa
(Figure 6C). This action of FIXa-FVIII is that of a “Xase.” Just
as FIX has FVIII as a cofactor, FX has a cofactor: factor V
(FV). Indeed the structure of FV and FVIII are similar. FXa-FV
acquires prothrombinase activity converting prothrombin
(factor II, FII) to thrombin (FIIa) (Figure 6D). Thrombin has
numerous actions. Collectively these actions are sometimes
described as the “thrombin explosion.” Subsequently throm-
bin converts fibrinogen to fibrin (Figure 6E).
Fibrinogen is a symmetrical molecule composed of 2 sets of
alpha, beta, and gamma chains (one set of the alpha-beta-
gamma chains forms one half of fibrinogen). At the ends of
the molecule are D domains, while in the middle of the mol-
ecule is an E domain. Thrombin converts fibrinogen to fibrin
by cleaving fibrinopeptides A (FPA) and B (FPB) respectively
from the alpha and beta chains of fibrinogen (Figure 7, top).
Subsequently fibrin monomers form a noncovalent, over-
lapping lattice by associating one with the other (Figure 7,
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D
FPA and FPB
D
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E D
E D
E D
E D D E D
bottom). This lattice provides initial strength to the thrombus;
however, fibrin crosslinking does not occur until early tertiary
hemostasis, resulting in greater strength of the thrombus.
Besides its action in converting fibrinogen to fibrin (Figure 8),
thrombin 1) converts FV to FVa (with increased activity over
FV), 2) converts FVIII to FVIIIa (with increased activity over
FVIII), 3) feeds forward converting factor XI to FXIa, 4) acti-
vates factor XIII (FXIII, fibrin stabilizing factor) to FXIIIa and
5) stimulates platelets involved in primary hemostasis.
The action of thrombin to convert FXI to FXIa allows throm-
bin and fibrin formation to continue until otherwise inhibited
(eg, inhibited by tissue-factor-pathway-inhibitor [TFPI]). The
action of FXIa is to convert FIX to FIXa. Therefore, FIX can
be converted in vivo to FIXa in 2 ways: 1) via TF-FVIIa or 2)
via FXIa.
If after successful primary hemostasis, no further proco-
agulant events occur (eg, there is a failure of secondary
hemostasis), delayed bleeding is likely. Therefore, the role
of secondary hemostasis is to strengthen the thrombus
through the development of a fibrin matrix around and
among the platelets. It is important to also point out that
red blood cells (RBCs) are caught in the process of throm-
bus formation and contribute to the mass of the thrombus.
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FXI FXIa
FVa
FIX FIXa + FVIII FIXa-FVIII
FX FXa + FV FXa-FV
Prothrombin Thrombin
“Feed-forward” =Thrombin
actions
Fibrinogen Fibrin
FXIIIa
Figure 8  thrombi are deep venous thrombi (DVT) in the legs and pel-
The actions of thrombin are detailed in this figure (large arrows).
This figure also explains the role of thrombin in the conversion of
FXI to FXIa, which explains the role of FXI in normal hemostasis.
FVa is more active than FV. FVIIIa is more active than FVIII. FXIII is
fibrin-stabilizing factor.
Therefore, very severe anemia might impair normal hemo-
stasis. Additional clinical manifestations of defects in sec-
ondary hemostasis include bruising, soft tissue hematomas,
and, in children, hemarthrosis.
Tertiary Hemostasis
Tertiary hemostasis begins with the action of FXIIIa
crosslinking D domains of adjacent fibin monomers in the
lattice forming a polyfibrin meshwork (Figure 9). This adds
to the strength of the thrombus preventing short-term dis-
solution of the thrombus. The later stages of tertiary hemo-
stasis conclude with the resolution of the thrombus.
Temporally, primary hemostasis and secondary hemostasis
actually occur concurrently. Furthermore, the events of pri-
mary hemostasis and secondary hemostasis interact in the
formation of the thrombus. To understand this interaction,
recall that factors II, VII, IX and X are vitamin K-dependent
factors. The role of vitamin K is to assist in the addition of
gamma carboxylate groups to glutamic acids in the GLA
domains of these factors. Gamma carboxylation is neces-
sary for proper synthesis of the factor and proper function
of the synthesized factors. Without adequate levels of
vitamin K, the synthesis of these factors is reduced and the
function of the synthesized factors is also reduced.
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It is believed that factors II, VII, IX, and X interact with the
surface of the platelet, localizing and concentrating the
events of secondary hemostasis (Figure 10). This changes
the events from 3 dimensions to 2 dimensions concen-
trated on the platelet surface. In a sense, the surface of
the activated platelet provides a physical “stage” for the
formation of fibrin. As more and more fibrin is produced, the
fibrin lattice is formed, which contributes to the thrombus.
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Crosslinking of fibrin via FXIIIa provides increased and nec-
essary strength to maintain hemostasis.
Regulation of Coagulation
FXIII
The regulation of coagulation is essential to prevent both
bleeding and pathologic thrombi. Examples of pathologic
vis, and atrial thrombi that can occur in the setting of atrial
fibrillation. The danger posed by DVT is thromboembolism
to the lung producing a potentially fatal pulmonary embo-
lism (PE). The danger posed by an atrial thrombus is throm-
boembolism to the brain producing acute ischemic stroke.
In the modern world, where death from hypovolemic shock
is unusual, the most common causes of death involve
pathologic or physiologic thrombi. PE and thromboembolic
stroke are consequences of pathologic thrombi, whereas
thrombosis of a medium-sized artery is the expected (phys-
iologic) consequence of the fracture of an atherosclerotic
plaque in a coronary or cerebral artery. Although hyperco-
agulability can contribute to atherosclerosis and accen-
tuate thrombus formation, the major pathologic process
in coronary artery disease, cerebrovascular disease, and
peripheral vascular disease is the endothelial pathology of
the atherosclerotic plaque. In a sense, thrombus formation
following rupture of an atherosclerotic plague is expected
and physiologic. Unfortunately, this “physiologic” thrombus
formation can lead to acute ischemia or infarction.
To understand the balance between bleeding and throm-
bosis, we now review the factors that prevent excessive
thrombus formation. The continuous flow of blood through
arteries, capillaries, and veins modulates the time allowed
for the interaction of the blood and the endothelium. Stasis
predisposes to thrombosis; flow opposes thrombosis. The
vascular endothelium creates a physical barrier between
the blood and subendothelial collagen and TF. This pre-
vents thrombus formation in the absence of endothelial
injury. Normal endothelium also produces prostacyclin,
which inhibits platelets (Figure 11). Therefore prostacyclin
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Fibrin lattice without polymerization

D E D D E D

D E D D E D

D E D D E D

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FXIIIa

D E D D E D

D E D D E D

D E D D E D

Fibrin lattice with polymerization

Figure 9 
The top image shows the noncrosslinked fibrin lattice. Via FXIIIa, D and E domains become crosslinked, adding strength to the thrombus
(bottom image).

Fibrinogen Fibrin

FVIIa
TF
FIX FIXa FX FXa FII FIIa

Subendothelial cell

Figure 10 
Primary hemostasis and secondary hemostasis occur concurrently and do interact. This drawing depicts the binding of factors VIIa, IX/IXa,
X/Xa, and II/IIa to cell surfaces, localizing secondary hemostasis to a 2-dimensional framework, allowing active clotting factors to become
concentrated.

provides a counterbalance to the stimulatory effects of TxA2
produced by activated platelets.
Expressed on endothelial surfaces, HS activates AT to
impair thrombus formation (Figure 11). The action of AT
is to impair thrombin and FX, and, to a lesser extent, FIX
and FXI. Physicians take advantage of the actions of AT
when heparin is therapeutically administered. During ongo-
ing coagulation, at least 3 further processes can prevent
excessive thrombosis: 1) the activation of protein S, 2) the
release of tissue plasminogen activator (tPA), and 3) the
release of TFPI.
While thrombin is an extremely powerful and important
procoagulant factor at the beginning of thrombus forma-
tion, later in this process, thrombin becomes a relative
anticoagulant when it binds to a normal endothelial
plasma membrane protein termed “thrombomodulin”
(Figure 12). The binding of thrombin to thrombomod-
ulin extinguishes the procoagulant action of thrombin
and initiates its anticoagulant action. Thrombin-
thrombomodulin activates PC producing activated PC
(APC). APC plus protein S (PS) inactivates FVa and
FVIIIa. It is worth noting that thrombin stimulates the
formation FVa and FVIIIa early in coagulation, whereas,

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Endothelium
Prostacyclin
AT activation
Figure 11 
Hemostatic balance is provided by the barrier action of endothelial cells, endothelial cell production of prostacyclin, which inhibits platelets,
and heparin sulfate that activates antithrombin.
later in coagulation, thrombin triggers inactivation of FVa
and FVIIIa.
The release of tPA from injured tissues may be inter-
preted as “tissues requiring perfusion despite the pos-
sibility of hemorrhage.” tPA catalyzes the conversion of
plasminogen to plasmin (Figure 12). Plasmin degrades
the crosslinked fibrin strands, producing a variety of
fibrin-split (or degradation) products. One of these
products is the D-dimer subunit that was earlier formed
during FXIIIa-initiated fibrin crosslinking. In early tertiary
hemostasis, FXIIIa normally catalyzes the crosslinking of
D-domains between adjacent fibrin monomers present
in the nascent lattice. An elevation in plasma (or serum)
D-dimers is evidence of thrombus formation and subse-
quent thrombus breakdown. Not shown in Figure 12 is
alpha-2 antiplasmin, a normal plasma protein that inhibits
plasmin.
Lastly, TFPI is a physiological “brake” on the activity
of TF-FVIIa (Figure 13). However even if the activity of
the TF-FVIIa complex is extinguished by TFPI, the feed-­
forward loop of thrombin converting FXI to FXIa, etc, will
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Collagen
Basement
membrane
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Platelet
(–)
Collagen
HS
keep ­coagulation running until a factor is exhausted or the
­process is otherwise inhibited (eg, by the actions of AT and
APC plus PS).
In Vitro Coagulation
We now explore coagulation in vitro, beginning with a discus-
sion of 3 pathways: 1) intrinsic, 2) common, and 3) extrinsic
(Table 5). The intrinsic pathway is initiated when fresh whole
blood is placed in a glass tube. The negative charge of the
glass initiates the contact pathway, converting factor XII
(FXII) to FXIIa. FXIIa then converts prekallikrein (PK; bound
to high molecular weight kininogen [HMWK]) to kallikrein (K).
Kallikrein in turn converts more FXII to FXIIa, triggering a pos-
itive feedback loop for further generation of FXIIa (Figure 14).
FXIIa catalyzes the conversion of FXI to FXIa (Figure 15).
As in the in vivo pathway, FXIa cleaves FIX to FIXa (Figure
15). Thereafter an Xase develops (FIXa plus FVIII or FVIIIa)
cleaving FX to FXa and the process continues as described
for the in vivo events leading to the formation of thrombin
and fibrin. In summary the intrinsic pathway (named such
because blood will intrinsically clot when added to a glass
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TM

FIIa
PC APC
+PS

tPA Plasminogen

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FVa Inactive
and
FVIIIa
Plasmin
Injured
cell

Fibrinolysis

Figure 12 
In the later stages of coagulation, thrombin (FIIa) binds to thrombomodulin. This quenches the procoagulant actions of thrombin.
Furthermore, thrombin now acquires the ability to activate protein C (forming APC). APC plus protein S inactive FVa and FVIIIa inhibiting
further thrombin generation. Injured cells release tissue plasminogen activator (tPA). tPA converts plasminogen to plasmin. Plasmin degrades
cross-linked fibrin (eg, fibrinolysis) releasing fibrin degradation products such as D-dimers (not shown).

TFPI
[–]
Table 5. In Vitro Pathways: Plasma Proteins
Start here
FXI FXIa TF FVIIa TF + FVII Intrinsic pathway
  Prekallikrein (PK)
FIX FIXa + FVIIIa FIXa-FVIIIa   High molecular weight kininogen (HMWK)
 FXII
 FXI
FX FXa + FVa FXa-FVa
 FIX
 FVIII
Prothrombin Thrombin Extrinsic pathway
 FVII
Common pathway
Fibrinogen Fibrin  FX
 FV
FXIIIa FXIII
  FII (prothrombin)
  FI (fibrinogen)

Figure 13 
An overview of in vivo coagulation and the inhibitor role of tissue-
and I (fibrinogen), essentially identical to the events involv-
pathway-factor inhibitor.
ing factors X, V, II, and I that occur in vivo.

tube) includes PK, HMWK, and factors XII, XI, IX and VIII. The extrinsic pathway is triggered when tissue factor, phos-
The common pathway includes factors X, V, II (prothrombin), pholipid, and calcium are added to plasma anticoagulated

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Review
[–]
FXII
Surface
PK-HMWK
FXIIa FXII
Kallikrein-HMWK
FXIIa
Positive feedback loop
Figure 14  aPTT, TT, and fibrinogen measurements are commonly used
The contact pathway is outlined that initiates the in vitro intrinsic
pathway.
with citrate. In essence, to make citrated plasma clot,
extrinsic factors must be added. In vitro, FVII is activated to
FVIIa and TF-FVIIa preferentially converts FX to Fxa, acti-
vating the common pathway. In contrast in vivo, the major
action of TF-FVIIa is to convert FIX to FIXa.
In Vitro Pathways and Clotting Factor Tests
As we discusse below, the activated partial thromboplastin
time (aPTT; including the intrinsic and common pathways)
is triggered when a negatively charged substance (eg,
silica, kaolin, celite, or elargic acid), partial thromboplas-
tin (aka phospholipid) and calcium are added to citrated
plasma. The aPTT consists of the intrinsic and common
pathways.
The prothrombin time (PT; including the extrinsic [TF and
FVII] and common pathways) is triggered when complete
thromboplastin (aka phospholipid plus tissue factor) and
calcium are added to citrated plasma. The PT consists of
the extrinsic and common pathways. Both the PT and aPTT
are recorded in seconds. These tests detect the generation
of fibrin, which causes a change in resistance, light scatter-
ing, or viscosity of the sample. Neither the PT nor the aPTT
include the events of early tertiary hemostasis: FXIII activity
causing fibrin covalent crosslinking is not included in the PT
or aPTT measurements. Therefore, people with FXIII defi-
ciency have normal PT and aPTT (and thrombin time [TT])
values.
Upon reviewing the in vivo events, considering the in vitro
extrinsic and intrinsic pathways, the in vivo activation of
FIX to FIXa by TF-FVIIa constitutes a crossover pathway
between the in vitro extrinsic pathway (TF+FVIIa) and the in
vitro intrinsic pathway (FIX and FVIII) (Figure 16).
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Coagulation Testing
Clinical tests of coagulation are functional assays that evalu-
ate the rate of clot formation from the time that the coagula-
tion cascade is activated. These tests are commonly used to
identify defects of the extrinsic, intrinsic, and final common
pathways of the coagulation cascade so that more advanced
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testing can be done to identify specific defects. The PT,
to detect abnormalities of secondary hemostasis. The meas-
urement of fibrinogen is commonly used to detect fibrinogen
deficiencies and defects in the fibrinogen activity of plasma.
Prothrombin Time
PT measures the function of the extrinsic and final common
pathways of coagulation (Figure 15). Dr. Armand Quick,
an American physician, described the test in 1935. The PT
is sensitive to factors of the extrinsic (FVII) and common
(factors II, V and X) pathways as well as fibrinogen (Table 5).
The PT is commonly used to monitor warfarin anticoagu-
lant therapy. This is often performed as a point of care test.
Very high levels of heparin, high levels of antiphospholipid
antibodies and elevated levels of fibrin split products may
prolong the PT (Table 6).
The PT is performed as a 1-stage (quick) assay. The
patient’s plasma is added to a pre-warmed commercial
thromboplastin/CaCl2 reagent. The thromboplastin/CaCl2
is pre-warmed to enhance the activation of the enzymatic
coagulation factors. Sodium citrate is the preferred anti-
coagulation for patient sample collection. This places the
coagulation cascade in a state of stasis until testing can be
performed. The addition of calcium chloride restores the
calcium required for coagulation and effectively replaces
the calcium that was bound by sodium citrate. The result-
ing clot formation is detected by increased impedance or
turbidity (mechanical clot detection) or decreased optical
clarity (optical clot detection), based on the instrumentation
used. The time to clot formation is then measured to the
nearest 0.1 second. The reference interval for the PT is gen-
erally 10 to 13 seconds and varies with the type of thrombo-
plastin used in the testing procedure and the method of clot
detection.
Several point-of-care instruments are available for PT
testing. Amperometric (electrochemical) clot detection
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 Review

FXII
pathway
Contact

FXIIa

FXI FXIa

FXI FXIa + FVIII FXIa-FVIII TF-FVIIa TF + FVII

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Common pathway

FX FXa + FV FXa-FV

Prothrombin (FII) Thrombin (FIIa)

Fibrinogen (FI) Fibrin (FIa)

Figure 15 
The aPTT includes the intrinsic and common pathways. The PT includes the extrinsic and common pathways.

TF-FVIIa TF + FVII
Table  6. Causes of Prolongation of the
Prothrombin Time (PT)
“Crossover” pathway
Deficiencies, dysfunction or inhibition of factors VII, X, V, II (prothrombin)
and/or I (fibrinogen)
FXI FXIa
Liver failure
Disseminated intravascular coagulation
Elevated fibrin degradation products
FIX FIXa + FVIII Vitamin K deficiency or antagonist
Heparin (high doses)
Lupus anticoagulant (high levels)*
FX FXa + FV *An uncommon cause of an increased PT

Prothrombin Thrombin International Normalized Ratio (INR)

“Feed-forward”
Fibrinogen
Figure 16 
In vivo, a cross-over pathway normally exists where TF-FVIIa (of the
extrinsic pathway) activates FIX to FIXa (of the intrinsic pathway).
system after the activation of coagulation with human
recombinant thromboplastin is the basis of the CoaguChek
(Roche Diagnostics, Indianapolis, IN) instrumentation. The
Hemachron Signature Elite (Accriva Diagnostics San Diego,
CA) uses a mechanical-optical method to detect a true
end-point clot.
Prior to the development of commercial thromboplas-
Fibrin tins, individual laboratories somewhat crudely prepared
thromboplastin using a variety of techniques and animal
sources, most often brain tissue. These preparations
differed widely in their sensitivity to decreased levels of
clotting factors. Commercial preparations of thromboplas-
tin are derived from a variety of tissue sources, including
rabbit brain, rabbit brain-lung mixtures, human placenta,
and recombinant human tissue sources. The relative differ-
ences in the sensitivity of commercial thromboplastin rea-
gents are indicated by the International Sensitivity Index
(ISI). The World Health Organization (WHO) thromboplastin
is assigned an ISI of 1.0. Commercial thromboplastins are

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Review
calibrated using the WHO standard and assigned an ISI
value to indicate their relative sensitivity. A commercial
thromboplastin with a low ISI, near 1.0, is very sensitive
to the presence or absence of functional clotting factors.
A low ISI thromboplastin will be more useful in the detec-
tion of clotting factor deficiencies, in the extrinsic and
common pathways, than a thromboplastin with a high
ISI. The ISI also is specific for the instrument used for PT
testing.
The PT is commonly used to monitor anticoagulation with
warfarin therapy, which antagonizes vitamin K. The sensitiv-
ity of the PT varies according to the source of thromboplas-
tin, but the assay also varies according to the instrument
used. To correct for these differences, the international nor-
malized ratio (INR) was developed to improve standardiza-
tion of PT reporting globally. The INR represents the PT ratio
that would be obtained if the international reference throm-
boplastin had been used to test the patient. The INR is cal-
culated by plotting the logarithms of PT results obtained on
normal individuals and patients on warfarin using a primary
international reference thromboplastin, against the results
the logarithms of PT results obtained using the testing labo-
ratory’s thromboplastin. The resulting slope of the compar-
ison is the ISI for the particular thromboplastin-instrument
combination. The PT ratio is derived by dividing the patient
PT result by the geometric mean normal PT for the testing
laboratory, raised to the power of the ISI of the thrombo-
plastin/instrument combination used in the assay.
ISI
INR =(patient PT in seconds / mean normal PT in seconds)
The ISI must be specified for each new lot of thromboplas-
tin, in addition to identifying the laboratory instrument used
for PT testing.
Preanalytical Variables
Factor VII can be activated by prolonged cold storage (4ºC
or lower). Therefore, PT results can be shortened with pro-
longed plasma storage.
The PT is often prolonged with patients with polycythemia
as a result of the change in the ratio of the anticoagulant
to plasma. The blood to sodium citrate ratio is 9:1 for most
coagulation tests. Elevation in the polycythemic patient’s
hemoglobin concentration lowers the relative amount of
plasma in the patient’s blood, resulting in a relative increase
in the anticoagulant in the collection tube. The increase
in citrate binds the CaCl2 added to the system during the
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testing process, resulting in an increase in the PT. The anti-
coagulant should be adjusted, according to the patient’s
hematocrit, prior to collection to prevent false elevation
of the PT. Similarly, underfilled tubes in other patients can
result in prolongation of the PT. Severe anemia has not been
demonstrated to commonly interfere with the PT.
If heparin is present in the patient sample, in addition to
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warfarin, then the PT will reflect the combined effect of
heparin and warfarin. Heparin can be either be removed or
neutralized, prior to testing, to improve test accuracy.
Activated Partial Thromboplastin Time
aPTT is used to evaluate the intrinsic and common path-
ways of coagulation (Table 5; Figure 15). The aPTT is useful
clinically as a screening test for inherited and acquired fac-
tor deficiencies as well as to monitor unfractionated heparin
therapy, although the anti-Xa assay (see below) is the pre-
ferred measure of the effects of unfractionated heparin.
The assay was originally developed as a modification of the
PT. The partial thromboplastin time (PTT) was described by
Langdell, Wagner, and Brinkous in 1953 at the University of
North Carolina. When thromboplastin was added to the PT,
available at the time, the plasma from hemophiliac patients
clotted as rapidly as normal plasma. By altering the thrombo-
plastin used in the assay, Langdell and his associates were
able to develop an assay that was sensitive to the defect in
hemophiliac plasma. The altered thromboplastin or partial
thromboplastin was prepared by ultracentrifugation of tissue
extracts. In 1961, Rapaport and colleagues described the
addition of kaolin to stabilize and shorten the PTT, which maxi-
mally activates plasma. This assay was described as the aPTT.
Current commercial aPTT reagents contain a partial thromo-
plastin, which is a contact activator and a platelet phospholipid
substitute. Thromboplastins, such as cephalin or phosphatide,
are combined with a contact activator such as kaolin, clite,
micronized silica and ellagic acid, which eliminate the variability
associated with glass activators. A platelet phospholipid sub-
stitute is included in commercial aPTT reagents as well to elimi-
nate variability caused by altered platelet number or function.
The aPTT is performed by adding the commercial aPTT rea-
gent to the citrated patient plasma and incubating for several
minutes, which results in factor activation in the extrinsic and
common pathways. Calcium chloride is added, resulting in
clotting with the time from activation to clot detection meas-
ured in tenths of seconds. The aPTT reference interval will
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Table 7. Causes of Prolongation of the aPTT
Deficiencies, dysfunction or inhibition of prekallikrein, high-molecular
weight kininogen, factors XII, XI, IX, VIII, X, V, II (prothrombin) and/or
I (fibrinogen)
Disseminated intravascular coagulation
Elevated fibrin degradation products
Vitamin K deficiency or antagonists (high doses)*
Heparin
Lupus anticoagulant
*The PT is the more sensitive test for vitamin K deficiency or antagonists
Thrombin
Fibrinogen Fibrin
Figure 17 
In the measurement of the thrombin time, thrombin and calcium are
added to citrated plasma. The time to the appearance of a clot in
the reaction tube is measured to tenths of a second.
vary according to the type of instrumentation, anticoagulant,
tube type, reagent type and reagent lot.
Pre-analytic variables
Pre-analytic variables that may affect the aPTT include
difficult venipuncture, which leads to in vivo activation of
the extrinsic pathway and results in a shortened aPTT. Of
interest, a persistently shortened aPTT result with repeat
venipuncture may be a risk factor for hypercoagulability.
Prolongation of the aPTT may result when blood is obtained
from intravenous catheters that have been flushed with hep-
arin. In order to prevent heparin contamination of the sam-
ple, an initial volume of blood is typically discarded. Other
medications, if infused in the same catheter as a blood
sample for the aPTT is obtained, may have variable effects
on the aPTT results. Similar to the PT, aPTT results may be
adversely prolonged by alterations of the ratio of blood to
anticoagulant in polycythemic patients or with underfilled
blood tubes, leading to an excess of anticoagulant. Causes
of a prolonged aPTT are listed in Table 7.
Thrombin Time
The TT is used to evaluate the conversion of fibrin to fibrin-
ogen, in the final common pathway of the coagulation
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Review
Table 8. Causes of a Prolonged TT
Congenital disorders
 Afibrinogenemia
 Hypofibrinogenemia
 Dysfibrinogenemia
Acquired disorders
 Hypofibrinogenemia
  Liver disease
   Consumption (disseminated intravascular coagulation or
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thrombolytic therapy)
 Dysfibrinogenemia
  Liver disease
  Hepatic malignancy
  Elevated fibrin-degradation products
  Pharmacologic thrombin inhibitors (eg, heparin, dabigatran)
  Thrombin-induced anti-thrombin antibodies
 Paraproteinemias
cascade (Figure 17). The TT was first developed by Jim and
Goldfein in 1957. It is used clinically to detect hypofibrino-
genemia, dysfibrinogenemia (abnormal fibrinogens), and the
presence of thrombin inhibitors, such as certain therapeutic
drugs (eg, heparin) and antibodies (Table 8).
The TT measures the time for clot formation when thrombin
is added to citrated plasma and is measured in tenths of
seconds similar to the PT and aPTT. Thrombin catalyzes
the conversion of fibrinogen to fibrin, in the last stage of
the final clot formation, by cleaving fibrinopeptides A and
B. Polymerization of fibrin to from a clot occurs. By adding
exogenous thrombin, the phospholipid-dependent pathways
(extrinsic, intrinsic, and common) are bypassed. Sources
of reagent thrombin include human and bovine sources,
which vary in thrombin concentration and heparin sensitivity.
Lower concentrations of thrombin are more sensitive to hep-
arin, dysfibrinogenemias, and other abnormalities than are
preparations containing higher amounts of thrombin. The TT
normal reference interval will thus vary according to the type
and concentration of the thrombin preparation used in the
assay and must be established by the testing laboratory.
Preanalytic Variables
Reagents containing bovine thrombin may result in elevated
TTs in patients who develop bovine thrombin antibodies,
often following exposure to topical hemostatic agents. The
TT may be prolonged in patients receiving thrombin inhibi-
tors, such as hirudins, or in patients with elevated fibrin-deg-
radation products (FDPs). The presence of FDPs inhibits the
conversion of fibrinogen to fibrin. The TT is sensitive to the
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Review
presence of heparin and is often used to screen samples
with a prolonged aPTT result for heparin contamination. High
levels of immunoglobulin paraproteins can interfere with
fibrin formation and result in prolongation of the TT.
Tests to Measure Fibrin Formation
Fibrin formation can be evaluateted by the fibrinogen assay
and the reptilase time.
Fibrinogen Assay
Activation of fibrinogen by thrombin leads to the formation
of an effective clot. Fibrinogen assays are useful for the
diagnosis of hypofibrinogenemia, dysfibrinogenemia, dis-
seminated intravascular coagulation, primary fibrinolysis,
and other clinical conditions. Several fibrinogen assays
are available, including gravimetric, immunological (anti-
gen-based), optical, and functional assays.
The Clauss assay is the most commonly performed fibrin-
ogen assay and is a modified TT. The assay uses diluted
plasma where clotting is initiated with a high concentration
of reagent thrombin. A calibration curve is plotted using
serial dilutions of a reference plasma standard. The test
plasma is diluted, incubated, and then phospholipid and
thrombin are added, followed by calcium. Timing begins
with the addition of calcium. The time taken for a clot to
form is compared to the reference plasma calibration curve
to derive the fibrinogen concentration (eg, mg/dL) of the
test sample. Most laboratories use an automated method
in which the optical density of the mixture exceeds a deter-
mined threshold, as a means of clot detection.
Immunological mass assays are based on enzyme linked
immunoabsorbant (ELISA), radial immunodiffusion and elec-
trophoresis techniques. The immunological assays measure
protein (antigen) concentration rather than functional activ-
ity. Discrepancy between the fibrinogen activity and antigen
level are characteristic of dysfibrinogenemias.
Gravimetric assays are performed by adding thrombin and
calcium to dilute patient plasma. The resultant clot is com-
pressed to extrude plasma and unused reagent, dried and
weighed. This assay is technically difficult.
Preanalytical Variables
Increased turbidity of patient plasma may produce falsely
low results for assays that use optical density to detect
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clot formation. Hyperbilirubinemia and hyperlipidemia
are 2 such conditions that can result in increased plasma
turbidity.
Very high concentrations of unfractionated heparin may lead
to underestimation of the true fibrinogen level.
Reptilase Time
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The reptilase time is similar to the TT except that snake
venom is used to activate the coagulation cascade.
Snake venom, from Bothrops atox, a South American pit
viper, contains reptilase, an enzyme that is thrombin-like
in nature and hydrolyzes fibrinopeptide A from intact
fibrinogen molecules, in contrast with thrombin, which
cleaves fibrinopeptides A and B from fibrinogen. The
resulting clot is weaker than a clot formed by the action
of thrombin on fibrinogen. The advantage of the reptilase
time is that is not affected by the presence of heparin and
only minimally affected by fibrin-degradation products.
The reptilase time is useful, when compared to the TT
for a given patient, to detect the presence of thrombin
inhibitors.
Special Coagulation Studies
Mixing Studies: The 1:1 Mix
The mixing study assists in the assessment of an abnor-
mally prolonged PT or aPTT. An equal volume of citrated
patient plasma is mixed with normal pooled plasma (NPP),
and the PT or aPTT are repeated on the 1:1 mix. If the
assay time corrects (ie, the clotting time is now within PT
or aPTT reference interval), the prolongation is likely due
to either single or multiple clotting factor deficiencies or
dysfunctions. In contrast, the presence of inhibitors in the
specimen will result in a lack of correction following mix-
ing of the patient plasma and the NPP. Even if the result
decreases significantly, but the clotting time is still above
the reference interval, it is deemed a lack of correction.
Inhibitors include heparin, direct-acting oral anticoagulants
and autoantibodies directed against coagulation factors
or autoantibodies directed against phospholipid-binding
proteins such as occurs in the antiphospholipid syndrome
(APLS).
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Factor Activity Assays
These assays are most commonly performed as a 1-stage
assay. An essential component of the assay is a factor-de-
ficient substrate plasma that lacks the specific factor being
evaluated.
1. The patient’s citrated plasma is diluted 1:10, 1:20, and
1:40. The diluted specimen is then mixed 1 to 1 with the
factor-deficient substrate plasma. The patient’s speci-
men therefore supplies the missing factor.
2. A PT or aPTT is performed on the above mix, depend-
ing on the factor being tested. For testing factors VIII
and IX, for example, one would run an aPTT (intrinsic
pathway and common pathway), while for a FVII assay,
a PT (extrinsic pathway and common pathway) would
be performed.
3. The assay is calibrated using a standard reference
plasma with a known concentration of the factor being
evaluated.
4. A standard curve is plotted, with the factor concentra-
tions on the x-axis and the aPTT (or PT) clotting times
on the y-axis. The x- and y-axes are usually displayed
as a semilog plot.
5. The standard curve is used to interpolate or translate
the clotting times of the unknown specimen into spe-
cific factor concentrations.
Factor Inhibitors
Factor inhibitors are most commonly diagnosed in male
patients with severe hemophilia A (FVIII deficiency) where
the inhibitors occur as a result of factor replacement ther-
apy. The prevalence of FVIII inhibitors is about 10% to
15% in such patients. These inhibitors are alloantibodies
that result from exposure to human FVIII in a setting of an
absence or near absence of endogenous FVIII. These anti-
bodies produce resistance to the replacement factor and
may manifest as bleeding, despite adequate dosing of the
factor.
Factor inhibitors can also appear in the form of sponta-
neous autoantibodies in individuals who were previously
well and had no prior bleeding problems. This is an auto-
immune condition called acquired hemophilia. It is seen
in both men and women who are often middle aged or
older. Acquired hemophilia is associated with very severe
bleeding, usually within skin, soft tissue, muscles, and the
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Review
gastrointestinal tract. Hemarthroses (seen in congenital
hemophilia) are rare. Although FVIII activity is low, the
bleeding often appears out of proportion to the reduced
FVIII level. There is some association with other autoim-
mune diseases, as well as with lymphoproliferative disor-
ders and solid tumors. However, such associations are not
seen in every case.
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If a FVIII inhibitor is present, the aPTT will be prolonged
with a normal PT, and FVIII activity will be low, especially
in acquired hemophila. The TT is normal as is the dilute
Russell viper venom time (dRVVT). The 1:1 aPTT mix either
shows no correction, or initially corrects, but then the clot-
ting time prolongs after incubation for 1 to 2 hours at 37°C.
This later prolongation is the result of catalytic antibodies
that degrade FVIII.
It is important to measure the titer (concentration) of the
inhibitor. This is achieved by incubating the diluted patient
plasma with NPP for two hours at 37°C (Figure 18). At the
end of the incubation, the residual FVIII activity in the mix
is determined by comparing it with a similarly diluted and
incubated control specimen. If the titer of the inhibitor is
1 Bethesda unit (1 BU), the residual factor activity is 50%;
at 2 BU, 25% activity remains; and 12.5% residual activity
means that the titer is 3 BU. A titer less than 5 BU indicates
that the inhibitor can be overcome by the administration of
additional FVIII. For higher titers (≥5 BU), this therapy is no
longer effective. At this point, therapy includes some type
of bypassing agent. This may be either recombinant FVIIa
(Novoseven RT, Novo Nordisk Inc, Plainsboro, New Jersey)
or a prothrombin complex concentrate (PCC). The latter is a
4-factor preparation. Factors II, IX, and X in the PCC prepa-
ration are mainly nonactivated, while factor VII is mostly
in the activated form. For adults with acquired hemophilia
A, an alternative therapy is a recombinant porcine Factor
VIII (Obizur, Baxalta Incorporated, Bannockburn, Illinois).
In addition to these alternative FVIII therapies or bypassing
agents, an important treatment is the introduction of immu-
nosuppression to reduce the antibody response against
FVIII.
Specific Tests for Von Willebrand Disease
In von Willebrand disease (VWD), there is defective syn-
thesis or release of functional von Willebrand factor (VWF),
which results in defective primary hemostasis. The con-
dition can be associated with decreased antigenic and
functional activity (Types 1 and 3) or with a relatively normal
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Review
Patient Normal plasma –
specimen + 100% FVIII
Patient Normal plasma –
specimen 1:5 + 100% FVIII
Patient Normal plasma –
+
specimen 1:10 100% FVIII
Patient + Normal plasma –
specimen 1:50 100% FVIII
Figure 18  loss of those multimers that are essential to normal VWF function.
This is the basic setup for a Bethesda Inhibitor titer assay. Dilutions
of the patient specimen in buffer are shown on the left. These
samples are mixed with an equal volume of normal pooled plasma.
The mix is incubated at 37°C for 2 hours, and the residual factor
activity is determined relative to a control. The control (not shown) is
made by mixing the diluting buffer with an equal volume of normal
plasma, and this is also incubated at 37°C for 2 hours.
antigen concentration in the setting of reduced functional
activity (Type 2).
Most coagulation laboratories can measure the plasma
concentration of VWF protein (VWF antigen) by an immu-
noturbidimetric technique. Testing the functional activity
of VWF utilizes the drug ristocetin. Ristocetin facilitates
receptor (GPIb) and ligand (VWF) interaction in this agglu-
tination (GPIIb-IIIa independent) reaction. VWF activity is
determined by using patient’s plasma (the source of VWF)
in combination with formalin-fixed lyophilized platelets (the
source of GPIb) and ristocetin. This procedure is called the
ristocetin cofactor activity. This is in contrast to ristoce-
tin-induced platelet aggregation (RIPA), which utilizes living
patient-derived platelets and patient plasma. The latter test
is used in rare variants (Type 2B) that produce an unusually
brisk aggregation response in the presence of low concen-
trations of ristocetin.
The state of multimerization of VWF is important and is
assessed by electrophoresis on agarose gels (Figure 19).
Normal plasma produces an extended “ladder” of multim-
ers including high molecular weight forms. Type 2A and 2B
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Normal
Loss of high and
intermediate molecular
weight multimers
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Figure 19 
Schematic representation of von Willebrand factor (VWF) multimers
as illustrated by western blotting and VWF detection with VWF
antibodies. The distribution of VWF multimers is assessed by
agarose gel electrophoresis, which separates the multimers
according to their molecular weight, with the larger multimers being
at the top of the figure. The western blot on the right represents the
VWD are associated with the lack of intermediate and high
molecular weight multimers.
von Willebrand antigen concentrations and/or activity
assays should be performed in tandem. When interpreting
results, one must be aware that individuals with blood group
O have the lowest mean VWF antigen levels as compared to
other blood groups (AB has the highest mean level). Other
ancillary tests include a FVIII activity determination because
low VWF is frequently associated with a low FVIII activity
(VWF is the carrier protein for FVIII). If the FVIII activity is
sufficiently decreased, the aPTT may be prolonged.
In summary, the laboratory workup for VWD should include
the following evaluations (von Willebrand Disease. Leebeek
FW, Eikenboom JC):
1.  Platelet count
2. Platelet function analyzer-100 (PFA-100 or equivalent),
which tests the ability of platelets in flowing blood to
obstruct an aperture in a collagen-coated cartridge
3. aPTT
4.  Factor VIII activity
5.  VWF antigen determination
6.  VWF activity determination (ristocetin cofactor activity
or additional collagen-binding activity)
7. VWF multimer analysis
The RIPA may be used in very selected cases where Type
2B VWD is suspected.
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No heparin
1) Reagent
Synthetic chromogenic substrate
2) Exogenous Xa
FXa Release of
colored product
Figure 20 
An outline of the anti-Xa assay which can be adapted to measure plasma concentrations of heparin, including unfractionated heparin, low
molecular weight heparin, and fondaparinux, an ultra-low molecular weight synthetic heparin. The basis for the assay is the cleavage of a
synthetic peptide by exogenous (ie, not from the specimen) FXa. This cleavage releases a colored product. In the presence of heparin and
antithrombin, the exogenous Xa is inhibited, as is the color development.
The Anti-Xa Assay
Heparin activity in plasma can be directly assessed by a
chromogenic procedure commonly referred to as an anti-Xa
assay (Figure 3). Generally, the reaction mixture contains
exogenous FXa as well as a chromogenic substrate for FXa.
Some versions of the assay utilize the patient’s own AT,
while others supplement with exogenous AT. Irrespective, in
both methods, heparin, present in the specimen, complexes
with AT, and this complex inhibits FXa. Any residual FXa
cleaves the synthetic chromogenic substrate, releasing a
yellow-colored chromophore (p-nitroaniline), which is read
optically at 405 nm. The amount of chromophore released
is inversely proportional to the concentration of heparin
present. Results are expressed as units per mL of anti-Xa
activity. The assay can be automated and can be used to
measure both unfractionated heparin and low molecular
weight heparin, as well as fondaparinux when properly
calibrated.
Testing for the Antiphospholipid Syndrome
The APLS is an acquired autoimmune phenomenon asso-
ciated with an increased incidence of both venous and
arterial thromboses, as well as fetal loss or premature
birth. The diagnosis of this syndrome requires that both
clinical signs and laboratory features be met. Typically,
there is a paradoxical prolongation of the aPTT in the
absence of any clinical features of bleeding. This is the
so-called lupus anticoagulant (LA) effect. Most often, the
prolonged aPTT does not correct on a 1:1 mix; however, it
is not that uncommon to encounter correction in the pres-
ence of a weak LA.
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Review
With heparin
1) Reagent
Synthetic chromogenic substrate
FXa Heparin
AT
No
colored product
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The laboratory definition of the APLS requires the presence
of either an LA or a persistent titer of antiphospholipid anti-
bodies. Persistent is defined as repeatedly positivity after a
minimum of 12 weeks interval between tests.
Lupus Anticoagulant Testing
One of the more commonly used LA assays is the dilute
Russell viper venom time (dRVVT) in which the common
coagulation pathway is activated through the action of a
snake venom that converts FX to FXa. The advantage of the
dRVVT is that it is not affected by hemophilia or by antibod-
ies to factors VIII and IX or by an elevated FVIII level.
The dRVVT assay requires an initial baseline reaction
(referred to as screening) followed by a confirmatory step
in which the reaction is supplemented by exogenous phos-
pholipid, revealing correction of the previously prolonged
aPTT clotting time. A true LA effect is characterized by
shortening of the clotting time on phospholipid supplemen-
tation, while factor deficiencies are impervious to this step.
Many dRVVT reagents contain a cationic heparin neutralizer
that binds and inactivates unfractionated heparin when
present in the therapeutic range.
In order to assess if the clotting time of the confirmatory
step is shortened relative to the screening step, it is com-
mon practice to establish a ratio of screening-to-confirm
times. These ratios are typically accepted as positive for
a lupus anticoagulant effect if they are greater than 1.2.
Recently, this approach has been modified by several
national and international organizations. The important
modification is that both the screening and confirm times
Lab Medicine 2017;48;295–313   311
DOI: 10.1093/labmed/lmw049
Review
themselves be converted to normalized ratios by dividing
the dilute Russell viper venom clotting time of the unmodi-
fied patient specimen by that of normal plasma.
Antiphospholipid antibodies are those antibodies that inter-
act with phospholipid-binding proteins or phospholipids
themselves (eg, cardiolipin, which is present primarily in
mitochondrial inner membrane). International guidelines
recommend detection of either IgG or IgM antibodies to
cardiolipin (aCL) or to beta-2 glycoprotein I (aβ2GP1).
Antiphospholipid antibodies are identified by ELISA. The
specimen from the patient is added to the ELISA well, and if
the appropriate antibodies are present, they will bind to the
target antigen coating the well. The laboratory definition of
antiphospholipid syndrome requires that a significant titer of
aCL or aβ2GP1 persist for at least 12 weeks. International
guidelines recommend that positive antibody titers are
titers above the 99th percentile of the population reference
interval.
Direct-Acting and Novel Anticoagulants
The direct-acting novel, oral anticoagulants (DOACs or
NOACs) are intended to replace warfarin as orally admin-
istered agents. There are two broad classes of DOACs
(Figure 21): 1) the oral direct thrombin inhibitors (DTIs), such
as dabigatran; and 2) the oral direct factor Xa inhibitors,
such as rivaroxaban and apixaban. Therapeutic monitoring
is usually not necessary; however, in certain circumstances
monitoring becomes vital, including in patients with renal
impairment, patients with significant bleeding, patients with
extremes of body weight, and when there is apparent
treatment failure.
There are 2 broad groups of laboratory assays: 1) quantita-
tive assays designed to monitor plasma concentrations of
the drug; and 2) qualitative, or noncalibrated, assays, which
can indicate if the drug is present or absent in a plasma
sample.
All of these direct-acting medications can be measured
quantitatively by liquid chromatography/mass spectroscopy.
Such analytical methods are time consuming and not easily
transferrable to the majority of clinical diagnostic laboratories.
Furthermore, quantitative therapeutic ranges are not well
established. Quantitative measurements of the direct Xa inhib-
itors are possible using anti-Xa chromogenic assays calibrated
with either rivaroxaban or apixaban. For the direct thrombin
inhibitors, the most practical quantitative assay is the dilute
312  Lab Medicine 2017;48;295–313
DOI: 10.1093/labmed/lmw049
FXa Thrombin (FIIa)
Direct Xa
inhibitors
A1 A2
s
FVa
A3
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s Direct
C1 thrombin
inhibitors
C2
GLa
Figure 21 
On the left, is the factor Xa/Va prothrombinase complex. Direct Xa
inhibitors block the action of FXa without the need for antithrombin.
Direct factor Xa inhibitors are able to inhibit free, prothrombinase-
bound (as shown here), and clot-associated FXa. Thrombin (FIIa)
is shown on the right. The direct thrombin inhibitors (DTIs) inhibit
thrombin without the need for antithrombin.
TT. This differs from the standard TT because the specimen is
diluted 1:4 in normal plasma prior to being assayed.
Qualitatitive, or noncalibrated, assays are best used for their
negative predictive value. Firstly, for dabigatran there is the
standard TT measurement. A normal TT can be used to
exclude the presence of an oral direct thrombin inhibitor. For
rivaroxaban and apixiban, one can use a heparin-calibrated
anti-Xa assay designed for measuring either unfractionated
or low molecular weight heparin. An anti-Xa value of less than
0.1 u/mL excludes the presence of rivaroxaban and apixaban.
The PT and aPTT are variably affected by the NOACs and
generally unhelpful in monitoring their concentrations.
Most importantly, a normal PT or aPTT does not exclude
the presence of any of the NOACs. In general, dabigatran
prolongs the aPTT more than the PT; the opposite is true
for rivaroxaban, where the PT is the test that is mostly pro-
longed; however, this depends on the PT reagent in use.
Summary
Appropriate test ordering and interpretation are based on a
thorough understanding of normal and pathologic coagula-
tion, and the similarities and differences between in vivo and
in vitro coagulation. The basic tests used to assess coagu-
lation include platelet count, PT, and aPTT. More sophisti-
cated testing (Figure 20) is then undertaken depending on
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the results of these antecedent tests guided by the patient’s
initial assessment consisting of the patient’s history and
physical examination. LM
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DOI: 10.1093/labmed/lmw049