Beruflich Dokumente
Kultur Dokumente
DOI: 10.1093/labmed/lmx050
Abbreviations syndrome; dRVTT, dilute Russell viper venom time; PCC, prothrombin
HS, heparin sulfate; AT, antithrombin; PS, protein S; PC, protein C; VWF, complex concentrate; RIPA, ristocetin-induced platelet aggregation; LA,
von Willebrand Factor; FVIII, factor VIII; GPIb, glycoprotein receptor; lupus anticoagulant; DOAC, direct-acting oral anticoagulant; NOAC, novel
TF, tissue factor; RBC, red blood cell; DVT, deep venous thrombi; PE, oral anticoagulant
pulmonary embolism; tPA, tissue plasminogen activator; APC, activated
PC; HMWK, high molecular weight kininogen; aPTT, activated partial Department of Pathology, Immunology & Laboratory Medicine, University
thromboplastin; PT, prothrombin time; ISI, international sensitivity index; of Florida, Gainesville, FL
WHO, World Health Organization; INR, international normalized ratio;
PTT, partial thromboplastin time; TT, thrombin time; ELISA, enzyme linked *To whom correspondence should be addressed.
immunoabsorbant; NPP, normal pooled plasma; APLS, antiphospholipid harris@pathology.ufl.edu
© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 295
Review
Pseudopodia
VWF binds to 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.
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
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.
D E D
D E D
D E D D E D D E D
D E D D E D
D E D D E D D E 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 bottom). This lattice provides initial strength to the thrombus;
in vivo ultimately leads to the conversion of factor IX (FIX) however, fibrin crosslinking does not occur until early tertiary
to FIXa (Figure 6B). In contrast, we will see that in vitro, hemostasis, resulting in greater strength of the thrombus.
the major action of TF-FVIIa is to convert factor X (FX)
to FXa. Besides its action in converting fibrinogen to fibrin (Figure 8),
thrombin 1) converts FV to FVa (with increased activity over
Returning to in vivo events, FIXa plus its cofactor factor FV), 2) converts FVIII to FVIIIa (with increased activity over
VIII (FVIII) now acquires the ability to activate FX to FXa FVIII), 3) feeds forward converting factor XI to FXIa, 4) acti-
(Figure 6C). This action of FIXa-FVIII is that of a “Xase.” Just vates factor XIII (FXIII, fibrin stabilizing factor) to FXIIIa and
as FIX has FVIII as a cofactor, FX has a cofactor: factor V 5) stimulates platelets involved in primary hemostasis.
(FV). Indeed the structure of FV and FVIII are similar. FXa-FV
acquires prothrombinase activity converting prothrombin The action of thrombin to convert FXI to FXIa allows throm-
(factor II, FII) to thrombin (FIIa) (Figure 6D). Thrombin has bin and fibrin formation to continue until otherwise inhibited
numerous actions. Collectively these actions are sometimes (eg, inhibited by tissue-factor-pathway-inhibitor [TFPI]). The
described as the “thrombin explosion.” Subsequently throm- action of FXIa is to convert FIX to FIXa. Therefore, FIX can
bin converts fibrinogen to fibrin (Figure 6E). be converted in vivo to FIXa in 2 ways: 1) via TF-FVIIa or 2)
via FXIa.
Fibrinogen is a symmetrical molecule composed of 2 sets of
alpha, beta, and gamma chains (one set of the alpha-beta- If after successful primary hemostasis, no further proco-
gamma chains forms one half of fibrinogen). At the ends of agulant events occur (eg, there is a failure of secondary
the molecule are D domains, while in the middle of the mol- hemostasis), delayed bleeding is likely. Therefore, the role
ecule is an E domain. Thrombin converts fibrinogen to fibrin of secondary hemostasis is to strengthen the thrombus
by cleaving fibrinopeptides A (FPA) and B (FPB) respectively through the development of a fibrin matrix around and
from the alpha and beta chains of fibrinogen (Figure 7, top). among the platelets. It is important to also point out that
Subsequently fibrin monomers form a noncovalent, over- red blood cells (RBCs) are caught in the process of throm-
lapping lattice by associating one with the other (Figure 7, bus formation and contribute to the mass of the thrombus.
It is believed that factors II, VII, IX, and X interact with the
surface of the platelet, localizing and concentrating the
FXI FXIa
events of secondary hemostasis (Figure 10). This changes
the events from 3 dimensions to 2 dimensions concen-
FVa
FIX FIXa + FVIII FIXa-FVIII trated on the platelet surface. In a sense, the surface of
the activated platelet provides a physical “stage” for the
FX FXa + FV FXa-FV formation of fibrin. As more and more fibrin is produced, the
fibrin lattice is formed, which contributes to the thrombus.
Therefore, very severe anemia might impair normal hemo- In the modern world, where death from hypovolemic shock
stasis. Additional clinical manifestations of defects in sec- is unusual, the most common causes of death involve
ondary hemostasis include bruising, soft tissue hematomas, pathologic or physiologic thrombi. PE and thromboembolic
and, in children, hemarthrosis. stroke are consequences of pathologic thrombi, whereas
thrombosis of a medium-sized artery is the expected (phys-
Tertiary Hemostasis iologic) consequence of the fracture of an atherosclerotic
plaque in a coronary or cerebral artery. Although hyperco-
Tertiary hemostasis begins with the action of FXIIIa agulability can contribute to atherosclerosis and accen-
crosslinking D domains of adjacent fibin monomers in the tuate thrombus formation, the major pathologic process
lattice forming a polyfibrin meshwork (Figure 9). This adds in coronary artery disease, cerebrovascular disease, and
to the strength of the thrombus preventing short-term dis- peripheral vascular disease is the endothelial pathology of
solution of the thrombus. The later stages of tertiary hemo- the atherosclerotic plaque. In a sense, thrombus formation
stasis conclude with the resolution of the thrombus. following rupture of an atherosclerotic plague is expected
and physiologic. Unfortunately, this “physiologic” thrombus
Temporally, primary hemostasis and secondary hemostasis formation can lead to acute ischemia or infarction.
actually occur concurrently. Furthermore, the events of pri-
mary hemostasis and secondary hemostasis interact in the To understand the balance between bleeding and throm-
bosis, we now review the factors that prevent excessive
formation of the thrombus. To understand this interaction,
thrombus formation. The continuous flow of blood through
recall that factors II, VII, IX and X are vitamin K-dependent
arteries, capillaries, and veins modulates the time allowed
factors. The role of vitamin K is to assist in the addition of
for the interaction of the blood and the endothelium. Stasis
gamma carboxylate groups to glutamic acids in the GLA
predisposes to thrombosis; flow opposes thrombosis. The
domains of these factors. Gamma carboxylation is neces- vascular endothelium creates a physical barrier between
sary for proper synthesis of the factor and proper function the blood and subendothelial collagen and TF. This pre-
of the synthesized factors. Without adequate levels of vents thrombus formation in the absence of endothelial
vitamin K, the synthesis of these factors is reduced and the injury. Normal endothelium also produces prostacyclin,
function of the synthesized factors is also reduced. which inhibits platelets (Figure 11). Therefore prostacyclin
D E D D E D
D E D D E D
D E D D E D
D E D D E D
D E D D E D
D E D D E D
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 While thrombin is an extremely powerful and important
produced by activated platelets. procoagulant factor at the beginning of thrombus forma-
tion, later in this process, thrombin becomes a relative
Expressed on endothelial surfaces, HS activates AT to anticoagulant when it binds to a normal endothelial
impair thrombus formation (Figure 11). The action of AT plasma membrane protein termed “thrombomodulin”
is to impair thrombin and FX, and, to a lesser extent, FIX (Figure 12). The binding of thrombin to thrombomod-
and FXI. Physicians take advantage of the actions of AT ulin extinguishes the procoagulant action of thrombin
when heparin is therapeutically administered. During ongo- and initiates its anticoagulant action. Thrombin-
ing coagulation, at least 3 further processes can prevent thrombomodulin activates PC producing activated PC
excessive thrombosis: 1) the activation of protein S, 2) the (APC). APC plus protein S (PS) inactivates FVa and
release of tissue plasminogen activator (tPA), and 3) the FVIIIa. It is worth noting that thrombin stimulates the
release of TFPI. formation FVa and FVIIIa early in coagulation, whereas,
Collagen
Basement
membrane
Prostacyclin (–)
AT activation
Collagen
HS
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 keep coagulation running until a factor is exhausted or the
and FVIIIa. process is otherwise inhibited (eg, by the actions of AT and
APC plus PS).
The release of tPA from injured tissues may be inter-
preted as “tissues requiring perfusion despite the pos- In Vitro Coagulation
sibility of hemorrhage.” tPA catalyzes the conversion of
plasminogen to plasmin (Figure 12). Plasmin degrades We now explore coagulation in vitro, beginning with a discus-
the crosslinked fibrin strands, producing a variety of sion of 3 pathways: 1) intrinsic, 2) common, and 3) extrinsic
fibrin-split (or degradation) products. One of these (Table 5). The intrinsic pathway is initiated when fresh whole
products is the D-dimer subunit that was earlier formed blood is placed in a glass tube. The negative charge of the
during FXIIIa-initiated fibrin crosslinking. In early tertiary glass initiates the contact pathway, converting factor XII
hemostasis, FXIIIa normally catalyzes the crosslinking of (FXII) to FXIIa. FXIIa then converts prekallikrein (PK; bound
D-domains between adjacent fibrin monomers present to high molecular weight kininogen [HMWK]) to kallikrein (K).
in the nascent lattice. An elevation in plasma (or serum) Kallikrein in turn converts more FXII to FXIIa, triggering a pos-
D-dimers is evidence of thrombus formation and subse- itive feedback loop for further generation of FXIIa (Figure 14).
quent thrombus breakdown. Not shown in Figure 12 is FXIIa catalyzes the conversion of FXI to FXIa (Figure 15).
alpha-2 antiplasmin, a normal plasma protein that inhibits
plasmin. As in the in vivo pathway, FXIa cleaves FIX to FIXa (Figure
15). Thereafter an Xase develops (FIXa plus FVIII or FVIIIa)
Lastly, TFPI is a physiological “brake” on the activity cleaving FX to FXa and the process continues as described
of TF-FVIIa (Figure 13). However even if the activity of for the in vivo events leading to the formation of thrombin
the TF-FVIIa complex is extinguished by TFPI, the feed- and fibrin. In summary the intrinsic pathway (named such
forward loop of thrombin converting FXI to FXIa, etc, will because blood will intrinsically clot when added to a glass
TM
FIIa
PC APC
+PS
tPA Plasminogen
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
[–]
FXII
Coagulation Testing
Surface
PK-HMWK
FXIIa FXII Clinical tests of coagulation are functional assays that evalu-
ate the rate of clot formation from the time that the coagula-
Kallikrein-HMWK
tion cascade is activated. These tests are commonly used to
FXIIa
identify defects of the extrinsic, intrinsic, and final common
Positive feedback loop pathways of the coagulation cascade so that more advanced
FXII
pathway
Contact
FXIIa
FXI FXIa
FX FXa + FV FXa-FV
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
calibrated using the WHO standard and assigned an ISI testing process, resulting in an increase in the PT. The anti-
value to indicate their relative sensitivity. A commercial coagulant should be adjusted, according to the patient’s
thromboplastin with a low ISI, near 1.0, is very sensitive hematocrit, prior to collection to prevent false elevation
to the presence or absence of functional clotting factors. of the PT. Similarly, underfilled tubes in other patients can
A low ISI thromboplastin will be more useful in the detec- result in prolongation of the PT. Severe anemia has not been
tion of clotting factor deficiencies, in the extrinsic and demonstrated to commonly interfere with the PT.
common pathways, than a thromboplastin with a high
ISI. The ISI also is specific for the instrument used for PT If heparin is present in the patient sample, in addition to
Figure 17
cascade (Figure 17). The TT was first developed by Jim and
In the measurement of the thrombin time, thrombin and calcium are
Goldfein in 1957. It is used clinically to detect hypofibrino-
added to citrated plasma. The time to the appearance of a clot in
genemia, dysfibrinogenemia (abnormal fibrinogens), and the
the reaction tube is measured to tenths of a second.
presence of thrombin inhibitors, such as certain therapeutic
drugs (eg, heparin) and antibodies (Table 8).
vary according to the type of instrumentation, anticoagulant,
tube type, reagent type and reagent lot. The TT measures the time for clot formation when thrombin
is added to citrated plasma and is measured in tenths of
Pre-analytic variables seconds similar to the PT and aPTT. Thrombin catalyzes
the conversion of fibrinogen to fibrin, in the last stage of
Pre-analytic variables that may affect the aPTT include
the final clot formation, by cleaving fibrinopeptides A and
difficult venipuncture, which leads to in vivo activation of
B. Polymerization of fibrin to from a clot occurs. By adding
the extrinsic pathway and results in a shortened aPTT. Of
exogenous thrombin, the phospholipid-dependent pathways
interest, a persistently shortened aPTT result with repeat
(extrinsic, intrinsic, and common) are bypassed. Sources
venipuncture may be a risk factor for hypercoagulability.
of reagent thrombin include human and bovine sources,
which vary in thrombin concentration and heparin sensitivity.
Prolongation of the aPTT may result when blood is obtained
Lower concentrations of thrombin are more sensitive to hep-
from intravenous catheters that have been flushed with hep-
arin, dysfibrinogenemias, and other abnormalities than are
arin. In order to prevent heparin contamination of the sam-
preparations containing higher amounts of thrombin. The TT
ple, an initial volume of blood is typically discarded. Other
normal reference interval will thus vary according to the type
medications, if infused in the same catheter as a blood
and concentration of the thrombin preparation used in the
sample for the aPTT is obtained, may have variable effects
assay and must be established by the testing laboratory.
on the aPTT results. Similar to the PT, aPTT results may be
adversely prolonged by alterations of the ratio of blood to
Preanalytic Variables
anticoagulant in polycythemic patients or with underfilled
blood tubes, leading to an excess of anticoagulant. Causes Reagents containing bovine thrombin may result in elevated
of a prolonged aPTT are listed in Table 7. TTs in patients who develop bovine thrombin antibodies,
often following exposure to topical hemostatic agents. The
Thrombin Time TT may be prolonged in patients receiving thrombin inhibi-
tors, such as hirudins, or in patients with elevated fibrin-deg-
The TT is used to evaluate the conversion of fibrin to fibrin- radation products (FDPs). The presence of FDPs inhibits the
ogen, in the final common pathway of the coagulation conversion of fibrinogen to fibrin. The TT is sensitive to the
presence of heparin and is often used to screen samples clot formation. Hyperbilirubinemia and hyperlipidemia
with a prolonged aPTT result for heparin contamination. High are 2 such conditions that can result in increased plasma
levels of immunoglobulin paraproteins can interfere with turbidity.
fibrin formation and result in prolongation of the TT.
Very high concentrations of unfractionated heparin may lead
Tests to Measure Fibrin Formation to underestimation of the true fibrinogen level.
Factor inhibitors can also appear in the form of sponta- Specific Tests for Von Willebrand Disease
neous autoantibodies in individuals who were previously
well and had no prior bleeding problems. This is an auto- In von Willebrand disease (VWD), there is defective syn-
immune condition called acquired hemophilia. It is seen thesis or release of functional von Willebrand factor (VWF),
in both men and women who are often middle aged or which results in defective primary hemostasis. The con-
older. Acquired hemophilia is associated with very severe dition can be associated with decreased antigenic and
bleeding, usually within skin, soft tissue, muscles, and the functional activity (Types 1 and 3) or with a relatively normal
Normal
Patient Normal plasma –
specimen + 100% FVIII
Loss of high and
intermediate molecular
weight multimers
This is the basic setup for a Bethesda Inhibitor titer assay. Dilutions
of the patient specimen in buffer are shown on the left. These VWD are associated with the lack of intermediate and high
samples are mixed with an equal volume of normal pooled plasma. molecular weight multimers.
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 von Willebrand antigen concentrations and/or activity
made by mixing the diluting buffer with an equal volume of normal
assays should be performed in tandem. When interpreting
plasma, and this is also incubated at 37°C for 2 hours.
results, one must be aware that individuals with blood group
O have the lowest mean VWF antigen levels as compared to
antigen concentration in the setting of reduced functional other blood groups (AB has the highest mean level). Other
activity (Type 2). ancillary tests include a FVIII activity determination because
low VWF is frequently associated with a low FVIII activity
Most coagulation laboratories can measure the plasma (VWF is the carrier protein for FVIII). If the FVIII activity is
concentration of VWF protein (VWF antigen) by an immu- sufficiently decreased, the aPTT may be prolonged.
noturbidimetric technique. Testing the functional activity
of VWF utilizes the drug ristocetin. Ristocetin facilitates
In summary, the laboratory workup for VWD should include
receptor (GPIb) and ligand (VWF) interaction in this agglu-
the following evaluations (von Willebrand Disease. Leebeek
tination (GPIIb-IIIa independent) reaction. VWF activity is
FW, Eikenboom JC):
determined by using patient’s plasma (the source of VWF)
in combination with formalin-fixed lyophilized platelets (the 1. Platelet count
source of GPIb) and ristocetin. This procedure is called the 2. Platelet function analyzer-100 (PFA-100 or equivalent),
ristocetin cofactor activity. This is in contrast to ristoce- which tests the ability of platelets in flowing blood to
tin-induced platelet aggregation (RIPA), which utilizes living obstruct an aperture in a collagen-coated cartridge
patient-derived platelets and patient plasma. The latter test 3. aPTT
is used in rare variants (Type 2B) that produce an unusually 4. Factor VIII activity
brisk aggregation response in the presence of low concen- 5. VWF antigen determination
trations of ristocetin. 6. VWF activity determination (ristocetin cofactor activity
or additional collagen-binding activity)
The state of multimerization of VWF is important and is
7. VWF multimer analysis
assessed by electrophoresis on agarose gels (Figure 19).
Normal plasma produces an extended “ladder” of multim- The RIPA may be used in very selected cases where Type
ers including high molecular weight forms. Type 2A and 2B 2B VWD is suspected.
1) Reagent 1) Reagent
Synthetic chromogenic substrate Synthetic chromogenic substrate
The Anti-Xa Assay The laboratory definition of the APLS requires the presence
of either an LA or a persistent titer of antiphospholipid anti-
Heparin activity in plasma can be directly assessed by a bodies. Persistent is defined as repeatedly positivity after a
chromogenic procedure commonly referred to as an anti-Xa minimum of 12 weeks interval between tests.
assay (Figure 3). Generally, the reaction mixture contains
exogenous FXa as well as a chromogenic substrate for FXa. Lupus Anticoagulant Testing
Some versions of the assay utilize the patient’s own AT,
while others supplement with exogenous AT. Irrespective, in One of the more commonly used LA assays is the dilute
both methods, heparin, present in the specimen, complexes Russell viper venom time (dRVVT) in which the common
with AT, and this complex inhibits FXa. Any residual FXa coagulation pathway is activated through the action of a
cleaves the synthetic chromogenic substrate, releasing a snake venom that converts FX to FXa. The advantage of the
yellow-colored chromophore (p-nitroaniline), which is read dRVVT is that it is not affected by hemophilia or by antibod-
optically at 405 nm. The amount of chromophore released ies to factors VIII and IX or by an elevated FVIII level.
is inversely proportional to the concentration of heparin
present. Results are expressed as units per mL of anti-Xa The dRVVT assay requires an initial baseline reaction
activity. The assay can be automated and can be used to (referred to as screening) followed by a confirmatory step
measure both unfractionated heparin and low molecular in which the reaction is supplemented by exogenous phos-
weight heparin, as well as fondaparinux when properly pholipid, revealing correction of the previously prolonged
calibrated. aPTT clotting time. A true LA effect is characterized by
shortening of the clotting time on phospholipid supplemen-
Testing for the Antiphospholipid Syndrome tation, while factor deficiencies are impervious to this step.
Many dRVVT reagents contain a cationic heparin neutralizer
The APLS is an acquired autoimmune phenomenon asso- that binds and inactivates unfractionated heparin when
ciated with an increased incidence of both venous and present in the therapeutic range.
arterial thromboses, as well as fetal loss or premature
birth. The diagnosis of this syndrome requires that both In order to assess if the clotting time of the confirmatory
clinical signs and laboratory features be met. Typically, step is shortened relative to the screening step, it is com-
there is a paradoxical prolongation of the aPTT in the mon practice to establish a ratio of screening-to-confirm
absence of any clinical features of bleeding. This is the times. These ratios are typically accepted as positive for
so-called lupus anticoagulant (LA) effect. Most often, the a lupus anticoagulant effect if they are greater than 1.2.
prolonged aPTT does not correct on a 1:1 mix; however, it Recently, this approach has been modified by several
is not that uncommon to encounter correction in the pres- national and international organizations. The important
ence of a weak LA. modification is that both the screening and confirm times
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