Laboratory Evaluation of Hemostasis

Laboratory Evaluation
of Hemostasis
Roger S. Riley, M.D., Ph.D., Ann R. Tidwell, MT(ASCP) SH, David
Williams, M.D., Ph.D., Arthur P. Bode, Ph.D., Marcus E. Carr, M.D., Ph.D.
Table of Contents
Table of Contents
CBC/Platelet Count/Blood Smear Examination ________________________
In Vivo Evaluation of Primary Hemostasis _____________________________
Platelet Aggregometry _____________________________________________
Automated Platelet Function Analysis ________________________________
Platelet Aggregation with Impedance Platelet Counting _________
Platelet Aggregation Under Flow Condition ____________________
Acceleration of Kaolin Activated Clotting Time by
Platelet-Activating Factor ________________________________
Automated Optical Platelet Aggregometry
Whole Blood Hemostatometry ______________________________________
Thromboelastography _____________________________________
Clot Retraction ___________________________________________
Clot-Based Assays _______________________________________________
Activated Clotting Time (ACT) ______________________________
Prothrombin Time (PT) _____________________________________
Activated Partial Thromboplastin Time _______________________
Thrombin Time ___________________________________________
Clotting Factor Assays ____________________________________
Fibrinogen Analysis _______________________________________
Plasma Mixing Studies ___________________________________
Reptilase Time __________________________________________
Dilute Russell Viper Venom Assay __________________________
Activated Protein C Resistance ____________________________
Chromogenic Analysis ___________________________________________
Latex Agglutination/Turbidimetry __________________________________
Enzyme Immunoassay ___________________________________________
Flow Cytometry _________________________________________________
Electrophoresis _________________________________________________
Genetic and Molecular Assays ____________________________________
Electron Microscopy _____________________________________________
Radioimmunoassay ______________________________________________
References ______________________________________________________
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Hemostasis
Introduction
Medical evaluation of the hemostasis system began with visual observation of the
clotting process. During the time of medical blood letting, observation of the size of
the clot in a basin (clot retraction) was
used to determine when blood letting had
to be decreased. In the early 20th century,
manual timing of whole blood clotting
(i.e., Lee-White Whole Blood Clotting
Time), and later plasma, in glass tubes
permitted a more accurate measurement
of blood clotting. Further discoveries
about hemostasis in the 1930’s and 1940’s
led to more sophisticated laboratory tests,
including the prothrombin time, activated
partial thromboplastin time, and specific
assays of platelet function and fibrinolysis.
The advent of the monoclonal antibody,
molecular analysis, and the microcomputer in the 1980s led to an explosion of
knowledge about hemostasis and hemostasis testing that is still growing. In the
Laboratory Evaluation of Hemostasis
hemostasis laboratory, automated assays
have replaced many of the manual procedure of the past, and there is increasing interest in rapid, point of care hemostasis assays for perioperative and critical care, as
well as self-testing to support the millions
of patients now receiving oral anticoagulation for hypercoagulable diseases. Interestingly, measurement of clot retraction is
still the focus of a variety of these techniques, a fact that would no doubt be appreciated by the early physicians. This paper presents a global overview of the techniques presently used in the hemostasis
laboratory, with the realization that many
of these may be quickly surpassed by new
information, developments, and applications in the near future.
3
Platelet Count, Bleeding Time
CBC/Platelet Count/Peripheral
Blood Smear Examination
The complete blood count (CBC), platelet count, and
peripheral blood smear examination are the most
fundamental assays of hemostasis and must be performed in all patients with suspected hemostatic abnormalities.
Peripheral Blood Smear Examination
Peripheral smear examination is the critical first step
in the investigation of any suspected hematologic
disease.(6) Peripheral smear examination reveals information about platelet size, gross morphology, and
granularity, as well as associated abnormalities in red
and white blood cells. It is also helpful for confirmation of the automated platelet count. An estimate of
the platelet count can be obtained by routine light
microscopy of a Wright's-stained peripheral smear by
multiplying the number of platelets per 1000x oil
magnification oil immersion field by 10,000, or more
accurately, by multiplying the sum of the number of
platelets counted in 8-10 fields under 1000 x oil magnification by 2000.(7) A visual platelet counting technique based on the white blood cell count (PCW,
platelet count based on WBC) has also been developed for thrombocytopenic samples.(8) Every peripheral blood smear should be carefully evaluated
for the presence of platelet clumps that may falsely
lower the platelet count. Platelet aggregates usually
indicate a poorly collected or anticoagulated blood
specimen of the presence of EDTA-induced
autoantibodies.(7)
Acquired thrombocytopenia secondary to leukemia,
myeloproliferative disorders, or other hematologic
diseases is more common than congenital platelet
disorders. In addition, peripheral smear examination
Laboratory Evaluation of Hemostasis
may reveal evidence of liver, renal, or other causes of
acquired platelet dysfunction. A predominance of
large platelets may be the initial clue to the diagnosis
of the Bernard-Soulier syndrome. The May-Hegglin
anomaly, Chediak-Higashi syndrome, and other diseases affecting platelets may be discovered by peripheral smear examination.(7)
Platelet Count
Modern hematology analyzers perform a platelet
count by electrical impedance or light scattering
techniques that are accurate to ±5% in the range of
1000 - 3,000,000 platelets/μL. A measurement of platelet volume (mean platelet volume, MPV) is provided
at the same time, as well as a platelet size distribution
curve. Automated platelet counts can be affected by
platelet aggregates due to spontaneous aggregation,
cold agglutinins, EDTA anticoagulants ("spurious
thrombocytopenia, pseudothrombocytopenia") or
particulate debris, such as red or white cell fragments
("spurious thrombocytosis").(2-4) In addition, hematology analyzers may overestimate the platelet count
in severe thrombocytopenia.(5) Therefore, confirmation of atypical platelet counts by manual inspection
of a peripheral smear is essential. If necessary, platelet counts can be performed in a hemocytometer by
phase contrast microscopy to an accuracy of ±1020%.
In Vivo Evaluation of
Primary Hemostasis
The Ivy skin bleeding time is an imprecise manual
screening assay of primary hemostasis that was
widely utilized in the past as a diagnostic assay for
patients with suspected bruising and bleeding disorders, as a therapeutic guide in actively bleeding patients, and as a predictor of hemorrhage in the gen-
Fig. 1. Photomicrograph of a normal peripheral
blood smear showing several platelets with normal
morphology (Arrows).
eral population of patients undergoing surgery or
invasive procedures.(9)
Bleeding times are performed directly on the patient
by phlebotomists or technologists who are trained
and experienced in this assay. A blood pressure cuff is
placed on the upper arm and inflated to 40 mm Hg to
provide uniform capillary pressure, and a standardized incision is made on the volar surface of the forearm with a standard cutting device, such as the Sur4
Bleeding Time
Fig 2. Example of optical and impedance platelet
counts with an automated hematology analyzer (CellDyne 4000). In the optical technique (upper histogram), platelets (arrow) are discriminated from other
cells by light scatter at 7o and 90o. An upper volume
threshold is used to separate platelets from microcytic red blood cells. In the impedance platelet count
(bottom histogram), platelets are differentiated from
other cells by electrical resistance. The mean platelet
volume (MPV) is determined from the platelet volume data provided by impedance measurements.
bleeding time has been entirely discontinued
at some medical institutions without a
measurable adverse affect on patient
care.(13)
from the incision with filter paper at 30-second intervals until bleeding ceases. The result is reported in
seconds as the bleeding time.(10; 11)
Fig 3. Performing the bleeding time. Upper photograph: A bleed pressure cuff was placed over the upper arm and the skin of the forearm cleaned with
alcohol. Middle photograph: Picture of skin incision
marks left after a template was applied. Blood is
starting to ooze from the wound. Bottom photograph: Wicking the wound with filter paper to determine the bleeding time.
gicut (International Technidyne Corp, Edison, NJ) and
the Triplett and Tip Tripper Bleeding Time Devices
(Helena Laboratories, Beaumont, TX). Blood is wicked
Laboratory Evaluation of Hemostasis
The bleeding time is determined by many physiologic
factors, including skin resistance, vascular tone and
integrity, and platelet adhesion and aggregation.
Thus, a prolonged bleeding time may reflect an intrinsic platelet function defect, von Willebrand disease, vascular anomaly, or medications that affects
platelet function, such as aspirin. If the actual bleeding time exceeds the expected bleeding time by five
minutes, a platelet function defect may be suspected.
Unfortunately, the precision, accuracy, and reproducibility of the bleeding time are severely impaired
by factors such as the thickness and vascularity of the
skin, the location of the incision, skin temperature,
wound depth, and patient anxiety. Because of its imprecision, the bleeding time must be used with extreme caution in a patient care setting. The US Food
& Drug Administration no longer accepts bleeding
time data in patients as a surrogate marker for the
evaluation of new hemostatic drugs, and it is no
longer indicated for the preoperative screening for
hemostatic defects.(12-15) The routine utilization of
the bleeding time for the diagnostic evaluation of
patients with von Willebrand disease, storage pool
disorder, and other hereditary mucocutaneous hemorrhagic diseases has been questioned.(16) The
5
Platelet Aggregometry
Conventional platelet aggregometry (light transmission aggregometry, turbidimetric aggregometry)
measures the in vitro response of platelets to various
chemical agents (i.e., aggregating agents, platelet agonists) that induce platelet functional responses.(17)
In the clinical laboratory, platelet aggregometry is
utilized for the diagnosis of inherited and acquired
platelet disorders, the assay of von Willebrand factor
activity (ristocetin cofactor assay) and for the diagnosis of heparin-induced thrombocytopenia.(18)
Conventional optical platelet aggregometers are
modified spectrophotometers that measure light
transmission through platelet-rich plasma (PRP).
Although the turbidity of fresh PRP limits light
transmission, transmission progressively increases as
platelet aggregation causes the formation of larger
and larger particles.(17) More recent innovations
include whole blood aggregometers and lumiaggregometers. Whole blood aggregometers require
less patient blood and provide faster turn-around
time than optical aggregometers. Lumiaggregometers simultaneously measure platelet aggregation and ATP secretion to provide a more accurate diagnosis of platelet function defects. The platelet agonists routinely used in the clinical laboratory
to differentiate various platelet function defects include adenosine diphosphate (ADP), epinephrine,
collagen, ristocetin, and arachidonic acid. Other agonists, such as thrombin, vasopressin, serotonin,
thromboxane A2 (TXA2), platelet activating factor,
and other agents are used by research and specialized
clinical laboratories.
Conventional platelet aggregation is a complex laboratory assay that is particularly sensitive to the assay
conditions, as well as drugs and other substances in
the blood.(19) Because of these influences, platelet
aggregometry is an advanced, manually intense,
Laboratory Evaluation of Hemostasis
Fig 4. Platelet aggregometry. The
curve shows the five stages of an
ideal response of platelets to the
addition of a platelet agonist. Following addition of the agonist, the
platelets undergo a shape change
after a short delay. This is followed by the release of stored
agents, resulting in primary aggregation. The synthesis and release of new agonists occurs after
another short delay, producing a
“second wave” of aggregation.
Eventually, maximal aggregation
has occurred and light transmission is at is lowest. In practice,
aggregation studies are performed with platelet-rich plasma
and a variety of agonists (i.e.,
ADP, epinephrine, arachidonic
acid, collagen, ristocetin, thrombin, etc.). A conventional commercial platelet aggregometer
(PACKS-4, Platelet Aggregation
Chromogenic Kinetics System-4)
is shown in the upper right.
Dilution
Primary
Aggregation
Light Transmission
Platelet Aggregometry
Shape
Change
costly assay restricted to specific clinical circumstances. A variety of commercial instruments and
reagents for platelet aggregometry are available from
Chrono-Log Corporation (Havertown, PA), Bio/Data
Corporation (Horsham, PA), and Helena Laboratories
(Beaumont, TX).
Glanzmann thrombasthenia and the Bernard-Soulier
syndrome are the best known inherited anomalies of
platelet surface receptors, although both diseases are
very rare. Glanzmann thrombasthenia arises from an
aberration in the most prevalent platelet surface receptor, GPIIbIIIa (specific binding site for fibrinogen), leading to moderate to severe bleeding prob-
Secondary
Aggregation
Maximal
Aggregation
Time
lems in affected individuals. Platelet aggregometry
reveals a lack of response to agonists requiring fibrinogen binding, including adenosine diphosphate
(ADP), epinephrine, arachidonic acid, and collagen. In
contrast, the aggregation response to ristocetin is
within normal limits. The Bernard-Soulier syndrome
is clinically similar, but arises from the absence of
another functionally important platelet surface receptor, GPIb-V-IX. However, platelets from patients with
the Bernard-Soulier syndrome show normal aggregation to agonists requiring fibrinogen binding, but
show a lack of response to agents requiring GPIb (i.e.,
thrombin, ristocetin plus von Willebrand factor). The
6
Table I
Platelet Aggregometry - Characteristic Findings in Different Diseases
-20
Disorder
0
Arachidonic acid
% Aggregation
20
40
Ep
in
ep
60
hri
ne
ADP
80
Co
lla
100
0
1
2
3
Minutes
ge
4
n
5
Fig. 5. Effect of aspirin on platelet function. Diagram
shows aggregation tracings (% aggregation vs. time)
for platelet-rich plasma from a donor who had recently
ingested aspirin. The aggregation response to aspirin
is markedly decreased to arachidonic acid (10% final
aggregation). Epinephrine (76%), ADP (79%), and collagen (103%) show essentially normal responses.
Bernard-Soulier syndrome is also characterized by
thrombocytopenia and large platelets, while the platelet count and morphology are normal in Glanzmann
thrombasthenia but clot retraction is absent. These
two separate but specific defects in essential platelet
surface components have provided valuable information on the role(s) of platelets in formation of the
initial hemostatic plug.
Laboratory Evaluation of Hemostasis
Collagen
Epinephrine
ADP
Arachidonic
Acid
Ristocetin
Bernard-Soulier
Disease
Normal
Normal
Normal
Absent
Glanzmann
thromboasthenia
Absent
Absent
Absent
Normal
Aspirin, many
drugs
Reduced or absent
Variable
Reduced or absent
Normal
Storage pool
disease
Reduced or absent
Variable
Variable
Normal
vWD, Type I
Normal
Normal
Normal
Reduced or absent
vWD, Type IIb
Normal
Normal
Normal
Increased
Heparin-induced, immune-mediated thrombocytopenia (HIT type II) is an unfortunate, but relatively
common complication of heparin therapy arising
from autoantibodies specific for a complex of heparin
and platelet factor 4 (PF4). The IgG/heparin/PF4 immune complexes bind to the FcyRIIA (CD32) receptor
on the platelet membrane, resulting in platelet activation, the release of additional PF4, new immune complexes, and rapid platelet consumption. The excess
PF4 also binds to glycosaminoglycans on endothelial
cells, leading to antibody-mediated endothelial damage, thrombosis, and disseminated intravascular coagulation. Since serum from patients with HIT can
aggregate normal platelets in the presence of heparin,
platelet aggregometry with heparin is often used to
confirm the clinical suspicion of HIT.(20; 21) However, due to the operational complexity of this assay
and its relatively low sensitivity, this assay has been
largely replaced by enzyme immunoassay and flow
cytometry. As a combinatorial strategy, the immunoassay can be used as a screening tool, with the aggregometry test for confirmation in patients that are
antibody-positive.
The ability of vWF to aggregate platelets in the presence of the antibiotic ristocetin is the basis for the
ristocetin cofactor assay, the most common laboratory method to measure vWF activity for the diagnosis and monitoring of von Willebrand disease.(22)
This assay is performed by incubating formalin-fixed
platelets with test plasma, adding ristocetin, and then
performing platelet aggregation. The results are interpolated from a standard curve prepared from aggregation slopes obtained with testing of dilutions of
normal pooled plasma. Due to the time consuming
manual nature of the classic ristocetin cofactor assay,
7
Automated Platelet Function Analysis
automated agglutination techniques are under evaluation(23; 24), as well as techniques using enzyme
immunoassay.(25-28) The aggregation test as currently performed has a large standard deviation,
which is unfortunate considering that von Willebrand
disease is the most common hemostatic disorder encountered in the hematology clinic.
Automated Platelet
Function Analysis
The manual, laborious nature of conventional platelet
aggregometry is unsuitable for many applications
where point of care and/or rapid testing is indicated.
Therefore, there is increasing interest in noncomplex, automated techniques of platelet function
analysis particularly suitable for the cardiovascular
suite, cardiovascular laboratory, dialysis, or intensive
care unit.(29) A number of innovative techniques are
presently available, and more are likely forthcoming
in the near future.
Platelet Aggregation
with Impedance Platelet Counting
Plateletworks (Helena Laboratories, Beaumont, Texas)
is a rapid in vitro point of care platelet aggregation
screening technique based on impedance platelet
counting and specifically developed for cardiopulmonary bypass and cardiac catheterization
settings.(30) The technique uses anticoagulated blood
to measure the change in platelet count due to platelet aggregation. Two separate samples of blood are
taken, including one containing ADP and collagen
platelet agonists. The platelet count is measured in
each tube using a small impedance hematology analyzer, and the percent aggregation is calculated. An
eight-profile hematology profile is provided at the
same time.(30) The Plateletworks assay has been recently used to monitor the reversal of platelet inhibi-
Laboratory Evaluation of Hemostasis
tion with clopidogrel or NSAISs in elective cardiac
surgery patients., monitoring the efficacy of therapy
with platelet GpIIb-IIIa antagonists in patients undergoing percutaneous coronary intervention or receiving medical therapy for non-ST elevation acute
coronary syndromes., and predicting post-operative
blooding and blood product utilization in patients
undergoing cardiac surgery with cardiopulmonary
bypass.
Platelet Aggregation
under Flow Conditions
The PFA-100 (DADE-Behring, Miami FL, USA) is a
rapid, automated laboratory instrument that is sensitive to quantitative and qualitative abnormalities of
platelets and von Willebrand factor (vWF). In the
PFA-100, citrated whole blood is aspirated from a reservoir under constant vacuum conditions through a
microscopic 150 um aperture.(31-36) This aperture is
cut into a biologically active nitrocellulose membrane
in a disposable cartridge device coated with a combination of platelet agonists. These agonists are either
collagen (fibrillar Type I equine tendon) and epinephrine (C/Epi) or collagen and adenosine-5’diphosphate (C/ADP). The blood is forced through
the aperture at a high shear rate (5000-6000 seconds1) that roughly corresponds to the flow conditions
present in small arteries.(32; 33) As the blood is
forced through the aperture, platelets undergo adherence, activation and aggregation on the membrane
surrounding the aperture and progressively form a
plug that finally occludes the aperture. The closure
time (CT) is the time required for the complete occlusion to occur.
The PFA-100TM is more rapid and less expensive than
the bleeding time for the evaluation of platelet
function.(35; 37) Since there is a good correlation
between the bleeding time and the PFA-100 in certain
patient populations, there, there is a trend to replace
Fig. 6. Schematic diagram of PFA-100 instrument.
Citrated blood is forced through a small membrane at high shear rate meant to simulate physiologic conditions. Platelet agonists on the membrane initiate platelet adhesion and aggregation
that eventually occlude the membrane and stop
the flow of blood (Closure time). Diagram from
DADE-Behring.
the bleeding time with the PFA-100TM for a first-line
screening test for platelet dysfunction in patients
undergoing preoperative evaluation. Other clinical
applications of the PFA-100 include the following:
• The non-specific identification of patients with
inherited platelet dysfunction, including
Bernard-Soulier syndrome, Glanzmann’s
thrombasthenia, and other diseases.(38)
• The evaluation of women with menorrhagia to
exclude platelet dysfunction.
• The determination of aspirin resistance, aspirin
hyperresponsiveness, and the assessment of
8
Automated Platelet Function Analysis
patient compliance with aspirin and other antiplatelet receptor agents during therapy.(39-41)
• Monitoring deamino-D-arginine (DDAVP) therapy in vWD patients belonging to subsets of
vWD that are responsive to DDAVP including
most type 1 and some type 2 patients.
There are several cavets in the clinical utilization of
the PFA-100. Strict adherence to specimen requirements, specimen transportation, and specimen processing is required, since the PFA-100 is affected by
critical pre-analytical variables such as hematocrit or
platelet count, blood collection technique, and transportation through pneumatic tube systems.(42) Since
the PFA-10 has been reported as insensitive to some
patients with platelet function defects, clinical correlation is critical, with follow-up with a different
screening technique in cases of high clinical
suspicion.(16; 38) The PFA-100TM is insensitive to alterations in the quantity or quality of fibrinogen and
therefore has not been shown to be useful in evaluating patients for the presence of dysfibrinogenemia or
hypofibrinogenemia. It is not sensitive to defects or
deficiencies in the classic coagulation factors and
appears to have little if any significant utility in assessing Hemophilia A and B.
The Clot Signature Analyzer (CSA, Xylum Corporation, Scarsdale, NY) is an automated in vitro instrument designed to simulate in vivo clotting and platelet function under physiological conditions using
unanticoagulated whole blood.(43-47) In the CSA,
blood flow is passed through two channels. In the
“punch” channel, shear-induced platelet activation is
simulated by two small (0.015 cm) holes punched in a
blood conduit, causing a pressure drop in the lumen
until closure of the punch holes occurs (platelet hemostasis time). The “collagen” channel incorporates a
small aperture with a collagen fiber immobilized at
the center of the aperture. Platelets adhere to the collagen and eventually close the aperture, representing
Laboratory Evaluation of Hemostasis
the end point (collagen-induced thrombosis formation). At the time of this writing, the CSA is no longer
being commercially developed but has important
features which are not found on other available instruments.
The Platelet-Stat (Precision Haemostatics, Inc., Clovis,
CA) is a physiologic in vitro simulation of the template bleeding time, using blood anticoagulated with
acid-citrate-dextrose (ACD). The device consists of a
membrane with a slit, similar to the template-induced
injury. Blood is forced at constant pressure from a
syringe through the slit, resulting in occlusion of the
slit as a platelet plug is formed. The time from the
start of blood flow through the slit until blood clotting at the slit is termed the bleeding time. Phase I
studies show that the in vitro bleeding time (PlateletStat®) is successful in predicting dysfunctional platelets. The Platelet-Stat has been successfully used to
diagnose TTP and monitor therapy with plasma
exchange.(48)
Acceleration of Kaolin Activated
Clotting Time by Platelet-Activating Factor
The hemoSTATUS (Medtronic, Minneapolis, MN) is
an automated system designed for whole blood
point-of-care platelet function testing, especially in
cardiovascular surgery. The assay principle is a comparison of the activated clotting time quantitated in
cartridges containing different concentrations of kaolin or kaolin and platelet-activating factor.
The system also provides quantitative analysis of
heparin concentration by heparin/protamine titration, as well as a base-line clotting time (plateletactivated clotting time). Clinical evaluation of the
instrument has been controversial, with several studies failing to demonstrate a correlation of results with
perioperative blood loss or an adequate sensitivity to
drugs affecting platelet function.(49-52)
Automated Optical Platelet
Aggregometry
A recent innovation is the development of optical
platelet aggregometry for point of care analysis using
microbead agglutination technology. The VerifyNow
System (Accumetrics, San Diego, CA) consists of a
small optical analyzer and disposable, single-use assay cartridges that contain all necessary reagents,
including fibrinogen-coated microbeads. The patient
sample of 3.2% citrated whole blood is automatically
dispensed from the blood collection tube into the
assay cartridge without operator intervention. Assay
devices for the monitoring of aspirin and anti-GP Iib/
IIIa receptor antagonists (i.e., abciximab and eptifibatide) are commercially available, and an assay to
monitor Clopidogrel (Plavix) therapy is under development. To date, the VerifyNow assay has been primarily used to measure aspirin resistance in patients
with coronary artery disease.(53; 54)
One instrument is especially marketed for the detection of GPIIb/IIIa receptor blockade in patients
treated with the platelet antagonist abciximab. The
Ultegra Accumetrics RPFA uses a turbidimetric optical detection system to measure the agglutination of
fibrinogen-coated microparticles in in anticoagulated
whole blood. In the assay, platelets with unblocked
GPIIbIIIa receptors are activated and cause microparticle agglutination with a change in optical light
transmission.(55; 56) However, a recent study did not
confirm the sensitivity of the Accumetrics RPFA in
comparison to conventional platelet aggregometry of
the Platelets assay.(57)
Whole Blood Hemostatometry
Thromboelastography, measurement of platelet contractile force, and related procedures are analytical
techniques to measure the global process of coagula9
Whole Blood Hemostatometry
tion (i.e., primary hemostasis to fibrinolysis) using
whole blood. Although this technology was originally
developed decades ago, there has been a recent resurgence of interest due to the increasing need for immediate information in critically ill patients and
those undergoing liver transplantation, cardiovascular surgery, and other procedures where rapid hemostatic changes occur.(58-63)
Thromboelastography
The conventional (rotational) thromboelastograph
uses a sample cuvette cup filled with native (unanticoagulated) whole blood to measure clot formation/
dissolution kinetics and the tensile strength of the
clot. A pin suspended from a torsion wire is lowered
into the cuvette and the cup is rotated through a 45o
angle over a period of time. Torque from the rotating
cup is transmitted from the pin and suspending rod
to a recorder. There is no initial torque, but this increases as the clot forms and decreases as fibrinolysis
occurs. More recent thromboelastographs use optical
detection systems to measure the movement of the
rotating pin, as well as computer hardware and software for data collection and analysis.(64) Commercial
thromboelastographs include the TEG® system Haemoscope Corporation (Niles, IL), and the ROTEG
(Pentapharm GmbH, Munich, Germany). Thromboelastography has been extensively used for interoperative cardiopulmonary and near-patient coagulation
monitoring to guide blood product utilization.(64)
Although thromboelastography can be measured in
citrated blood, the results are not compariable to
whole blood.(65)
Clot Retraction
A technology recently developed by Hemodyne, Inc.
(Richmond, VA) the Hemostasis Analysis System
permits direct measurement of the forces produced
in the sample during clot formation.(66; 67) The
Laboratory Evaluation of Hemostasis
sample is placed in a shallow cup and is trapped between parallel surfaces when an upper plate is lowered onto the upper surface of the forming clot (Fig.
1). The upper surface is attached to a strain gauge
transducer. As the clot forms and the platelets pull
within the network, a downward force is transmitted
to the upper plate and transducer. The downward
force stresses the transducer and a voltage proportional to the distance moved is generated. Since the
transducer actually measures distance moved, a calibration constant relating distance moved to force is
used to convert distance to force. Early work with this
device confirmed that the forces produced by platelets (platelet contractile force, PCF) in platelet rich
plasma or whole blood clots were significant (several
kilodynes in magnitude) and easily measured.(68)
The onset of force development occurred as soon as
the fibrin network was in place. Utilizing this new
technique, PCF was found to be directly dependent on
platelet count, to be sensitive to temperature and calcium concentration, but to be relatively independent
of fibrinogen concentration over the normal fibrinogen range of 100 to 400 gm/dL.(69) PCF is also a very
stable parameter, that persists in whole blood stored
at room temperature for as long as ten days. In contrast, platelet function by conventional aggregometry
must be performed within four to six hours. The robust nature of the parameter and its absolute dependence on platelet viability have led some groups
to examine the use of the PCF parameter as a marker
of platelet survival in stored and modified platelet
preparations.(70)
The thrombin generation time is another parameter
measurable by the Hemodyne. This is performed by
the use of Batroxobis, a snake venom proteolytic enzyme from the fer-de-lance that directly clots fibrinogen via cleavage of fibrinopeptide A. The addition of
batroxobin to citrated whole blood results in rapid
clot formation, but no initial PCF development. Although batroxobin does not activate platelets, after a
Fig. 7. A schematic illustration of the Hemodyne hemostasis analyzer used to measure platelet contractile force and clot elastic modulus. The test specimen
is placed in a sample space between a thermostated
cup and a parallel upper surface. During blood clotting, platelets pull fibrin strands inward, generating a
force that is detected by a displacement transducer
and converted to a voltage proportional to the
amount of force generated. Diagram used with permission of Hemodyne, Inc.
variable lag phase PCF development is noted. During
the lag phase, thrombin is generated as a consequence of sample re-calcification. Since the fibrin
network is in place prior to the generation of thrombin, PCF becomes apparent as soon as a small
amount of thrombin is generated. Thus, the inflection
or take off point in the PCF curve serves as a marker
of thrombin generation in the batroxobin mediated
assay. Assays of prothrombin fragment 1+2, reveal a
concurrent burst of activation fragment generation at
the moment of PCF upswing.(71) The lag phase is
thus the thrombin generation time (TGT). In normal
individuals, PCF developed by the addition of batroxobin differs only in the time of onset. However, if
thrombin generation is inhibited by the addition of
anticoagulants or by the presence of clotting factor
10
Clot-Based Assays
deficiencies, PCF in the batroxobin clots is dramatically delayed and deficient. TGT is sensitive to the
effects of heparin(72; 73), low molecular weight
heparins(74), dermatan sulfate(75), non-heparin antithrombins(76), inherited clotting factor deficiencies(77) and clotting factor deficiencies induced by
warfarin. In vitro studies indicate the potential for
documentation of the correction of deficient thrombin generation by hemostatic agents such as recombinant FVIIa.(78)
The Sonoclot Coagulation and Platelet Function Analyzer (Sienco Inc., Wheat Ridge, Colorado) is a versatile, whole blood point of care system that uses a viscoelastic clot detection mechanism to analyze the
global process of hemostasis, including coagulation,
fibrin gel formation, clot retraction (platelet function) and fibrinolysis.(79) The Sonoclot uses the oscillation of a tubular probe within a blood sample to
generate an analog electronic signal that reflects resistance to motion during clot formation and fibrinolysis. Data processing by a microcomputer generates a qualitative graph (Sonoclot Signature) as well
as quantitative results on clot formation kinetics and
the rate of fibrin polymerization. A variety of different reagent kits are available for general coagulation
monitoring, as well as more specific purposes, including heparin monitoring, hyperfibrinolysis screening,
hypercoagulable screening and platelet function
assessment.(80-84)
Each of these instruments has its own distinct features and advantages for the diagnostic laboratory,
but a full specific assessment of global hemostasis
defects requires multiple approaches.
Clot-Based Assays
Functional assays based on clot formation as the
endpoint are widely used in the clinical laboratory to
determine the integrity of the intrinsic or extrinsic
Laboratory Evaluation of Hemostasis
pathways of the coagulation system (Fig. 8). Similar
functional assays have been developed to measure
fibrinolysis and other coagulation pathways.
The clinical coagulation laboratory uses clotting assays (prothrombin time, activated partial thromboplastin time) in which tissue phospholipids are added
to platelet-poor plasma as full or partial thromboplastins to to initiate clotting for screening of hemophiliac defects or for specific factor assays (Fig. 9).
Instruments for automated performance of clotbased assays are available from several manufacturers, including Beckman Coulter, Inc. (Fullerton, CA),
Dade Behring (Deerfield, IL), Diagnostica Stago, Inc.
(Parsippany NJ), Global Medical Instrumentation, Inc.
(St. Paul, Minnesota), and Sysmex Corporation (Kobe,
Japan). Several similar assays using whole blood are
available for near-patient testing. The most widely
used of these assays is the activated clotting time
used to monitor clotting during cardiopulmonary
bypass.
Activated Clotting Time (ACT)
The ACT was developed in 1966 as a modification of
the Lee-White whole blood clotting time to monitor
coagulation status and heparinization in immediate
need situations.(52) The ACT uses tubes containing a
negatively-charged particulate activator of coagulation, such as kaolin, celite of diatomaceous earth.
When whole blood is drawn into the tube, the contact
system is activated and clotting occurs. The assay is
useful at high levels of heparin such as used in openheart surgery, but is also affected by platelets.(85-88)
The manual ACT has been replaced in recent years by
an increasingly sophisticated variety of
microprocessor-controlled instruments, exemplified
by those manufactured by Helena Laboratories Corp.
(Beaumont, Texas), ITC (Edison, NJ), Medtronics
(Minneapolis, MN), and Roche Diagnostics Corpora-
Contact
System
XII
XIIa
XI
XIa
VIII
PLT
IX
IXa
VIIIa
VII/VIIa
TF
PLT
Va
X
Xa
Prothrombin
Fibrinogen
V
XIII
Thrombin
Fibrin
XIII
X-Linked
Fibrin
Fig. 8. A color-coded schematic illustration of the
coagulation system. The diagram shows components
of the contact system (orange), extrinsic pathway
(blue), intrinsic pathway (magenta), and common
pathway (green). In vivo, platelets (yellow) are essential for contributing phospholipid and providing a
surface for the tenase and prothrombinase reactions
to occur.
tion (Indianapolis, IN). Many of these instruments
perform the PT, aPTT, thrombin time, fibrinogen
level, and other hemostatic assays in addition to the
activated clotting time. Some manufacturers also
provide ACT reagents containing heparinase so that a
patient’s baseline value can be established in the
presence of heparin. These instruments are increasingly being applied to the near-patient monitoring of
direct thrombin inhibitors and low molecular weight
heparins in critical situations.(89-91)
11
Clot-Based Assays
Clotting agent, Ca ++
Platelet-poor plasma
Incubation
Fibrin clot
Prothrombin Time (PT, Protime,
Quick’s time, Partial Prothrombin Time)
The PT provides a functional determination of the
integrity of the extrinsic (tissue factor) pathway of
coagulation and is sensitive to the vitamin-K dependent clotting factors (factors II, VII, IX, and X) as well
as to factors of the common pathway (fibrinogen,
prothrombin, factor V, factor X). The PT is a widely
used laboratory assay for the detection of inherited
or acquired coagulation defects related to the extrinsic pathway of coagulation, and is the standard test
for monitoring oral anticoagulation therapy
(coumadin).(92; 93)
In the PT an aliquot of test platelet-poor plasma is
incubated at 37oC with a reagent containing a tissue
factor, phospholipid (thromboplastin), and CaCl2. The
time required for clot formation is then measured by
Laboratory Evaluation of Hemostasis
Fig. 8. Basic principle of clot-based assays
of coagulation. A clotting activator, calcium, and a source of phospholipids is
incubated with platelet-poor plasma, resulting in activation of the extrinsic clotting system. The endpoint of the reaction
is the formation of a fibrin clot that can be
measured by visual, photo-optical, electromechanical means. The result is usually
reported as the time required for clot formation. Common clot-based assays used in
the clinical hemostasis laboratory include
the PT, aPTT, thrombin time, reptilase
time, dilute Russell Viper venom time, and
activated protein C resistance assay. Clotbased assays are also used for factor
analysis and to determine the presence of
factor deficiencies and anti-factor inhibitors.
one of a variety of techniques (photo-optical, electromechanical, etc.)(Fig. 8). The result is reported in
seconds (prothrombin time), or as a ratio compared
to the laboratory mean normal control (prothrombin
ratio, PTR). The PT is critically dependent on the
characteristics of the thromboplastin used in the assay, as well as manner of blood coagulation, the type
of container, the type of anticoagulant, specimen
transport and storage conditions, incubation time
and temperature, assay reagents, and the method of
end point detection. This means that patients on
coumadin will have different clotting times when
tested in different laboratories, so a means of standardization of results must be employed.
The International Normalized Ratio (INR) was introduced by the World Health Organization (WHO) in
the early 1980’s as a means of standardizing PT
results.(94) For this purpose, a very responsive batch
of human brain extract was designated as the first
International Reference Preparation (IRP), and a cor-
rection factor (International Sensitivity Index, ISI)
was developed to correlate the sensitivity of commercial thromboplastin preparations to the IRP. By definition, the ISI of the first IRP was 1.0. An additional
term, the INR, was introduced to compare a given
prothrombin ratio measurement to the IRP. Commercial vendors of thromboplastin preparations supply
the ISI with each reagent lot. If the ISI is known, the
INR for each clotting time is easily calculated. However, the ISI can be affected by instrumentation and
other laboratory factors and thus must be verified by
each testing site according to standards of the College
of American Pathologists. Unfortunately, even with
the INR, current prothrombin reagent/instrument
calibration techniques are insufficient to provide
good intralaboratory agreement.(95; 96)
There is great interest in point of care and patient
self-testing of oral anticoagulation status are popular
for patient convenience and to improve the efficiency
of medical care. Considering the 600,000 to 900,000
patients in the United States with heart valves, and
the millions requiring oral anticoagulation for hypercoagulability states, it is not surprising that several
small, user-friendly instruments are presently available for home testing by prescription from blood obtained by fingerstick. These instruments include the
Avocet PT-Pro (Avocet Medical, Inc. San-Jose, CA), the
CoaguChek (Roche Diagnostics, Basal, Switzerland),
the Harmony™ INR Monitoring System (LifeScan,
Inc., Milpitas, CA) , the INRatio Meter. (HemoSense,
Inc. San Jose, CA),and the HemosProTime Microcoagulation System (ITC, Edison, NJ), Presently, these
assays are CLIA waived and have been covered by
Medicare since late 2001. Point of care monitoring of
the PT and INR has been the subject of several recent
reviews.(97-103)
12
Clot-Based Assays
Activated Partial Thromboplastin Time
(aPTT, Activated Prothrombin Time)
The partial thromboplastin time (PTT) is the clotting
time obtained when “partial thromboplastin” is
added to plasma. Partial thromboplastin is the phospholipid fraction of a tissue extract, and differs from
a complete tissue extract (i.e., “thromboplastin”) by
the lack of tissue factor. The PTT is sensitive to the
intrinsic pathway of coagulation, but is most sensitive
to the contact factors (i.e., factor XII, prekallikrein,
high molecular weight kininogen) when a particulate
“activating agent” (i.e., silica, celite, kaolin, micronized silica, ellagic acid) is added to the reaction
(activated PTT, aPTT). Many different phosophlipid
reagents animal and plant origin, such as cephalin,
have been used as partial thromboplastins, and a variety of activating substances are in use.(104-110)
In the aPTT an aliquot of undiluted, platelet-poor
plasma is incubated at 37oC with an activator and
phospholipid (partial thromboplastin). CaCl2. is then
added, and the time required for clot formation is
measured by one of a variety of techniques (photooptical, electromechanical, etc.). The aPTT result is
reported as the time required for clot formation after
the addition of CaCl2. The aPTT is functional determination of the intrinsic (factors XII, XI, IX, VIII, V, II,
I,) and common pathways of coagulation.(111; 112)
The aPTT is utilized to detect congenital and acquired abnormalities of the intrinsic coagulation
pathway, monitor patients receiving heparin or coagulation factor replacement therapy, and to detect
inhibitors of the intrinsic and common
pathways.(113-120)
The aPTT clotting time may be influenced by many
pre-analytical and analytical variables and caution
must be used in the interpretation of the result. Preanalytical variables include slow or difficult specimen
collection, an improper blood:anticoagulant ratio,
Laboratory Evaluation of Hemostasis
failure to promptly mix the blood with the citrate
anticoagulant, improper transport or storage, or a
prolonged interval between specimen collection and
analysis. The sensitivity of the assay to factor deficiencies, inhibitors, and heparin also varies with the
reagents used in the assay. Because of these variables,
a normal aPTT result does not exclude a mild coagulation factor deficiency or the presence of a low-titer
or slow-reacting inhibitor. However, a significant prolongation of the aPTT indicates the presence of a factor deficiency (VIII, IX, XI, XII, prekallikrein, HMWK),
while prolongation of both the PT and aPTT suggests
a deficiency of factor I, II, V, or X. The aPTT is not
affected by deficiencies of factor VII or XIII.
Numerous modifications of the aPTT have been described for the functional analysis of specific
coagulation-related substances. Those routinely utilized in the coagulation laboratory at the present time
include the reptilase time, the Bethesda assay, protein
C and protein S activity, and several assays for lupus
anticoagulants (dilute Russell viper venom time
[dRVVT], platelet neutralization test, and hexagonal
phospholipid assay.
Specific anti-factor VIII antibodies (inhibitors) are a
serious medical problem for patients with hemophilia. Mixing studies can detect the presence of inhibitors, but other assays are required for the precise
measurement of antibody activity necessary for patient care.(121) The Bethesda assay is a modified
aPTT based on the ability of factor VIII inhibitors to
neutralize factor VIII activity in normal plasma. A
series of dilutions of patient plasma are added to a
standard amount of normal plasma and assayed for
factor VIII levels after two hours incubation at 37C:
the titer at which half of the FVIII activity remains is
used to calculate the “Betheda units” of inhibition.
Several modifications of the Bethesda assay have
been developed to improve its sensitivity.(122-124)
The new Oxford assay is similar, but uses factor VIII
concentrate as the source of factor VIII. Enzyme immunoassay, gel techniques, and other methods have
been also used to detect inhibitors.
The direct thrombin inhibitors are among the latest
form of anticoagulant drugs developed with the goal
of eliminating the side effects and improving the
therapeutic efficacy of anticoagulants which exert an
indirect antithrombin effect, including warfarin,
heparin, and low molecular weight heparin.(125) The
present generation of direct thrombin inhibitors includes recombinant hirudin (lepirudin), bivalirudin,
argatroban, and melagatran. Unfortunately, the direct
thrombin inhibitors present a problem for the hemostasis laboratory, since conventional coagulation assays such as the aPTT, thrombin time, and activated
clotting time show poor reproducibility and linearity
in the presence of these drugs.(126) Two modifications of the aPTT, the ecarin clotting time (ECT) and
prothrombinase-induced clotting time (PiCT) have
been developed for monitoring the direct thrombin
inhibitors, as well as chromogenic and enzyme
immunoassays.(127-129) There is presently no clear
concensus on the most optimal laboratory method
for direct thrombin inhibitor monitoring, although
the automated chromogenic assays and chromogenicbased point of care assays appear to offer adequate
sensitivity and precision and avoid interference problems by heparin and other substances.(126; 130-132)
Thrombin Time (Thrombin
Clotting Time, TCT, TT)
The thrombin time measures the thrombin-induced
conversion of fibrinogen to fibrin directly in patient
plasma, bypassing all other clotting factors. The
thrombin time is performed by the addition of a low
concentration of thrombin (usually bovine thrombin)
directly to the citrated plasma and measuring the
time required for the formation of fibrin monomers
by visual, mechanical, or opto-electronic
13
Clot-Based Assays
techniques.(133; 134) The thrombin time is prolonged
by thrombin inhibitors and inhibitors of fibrin formation and polymerization, but it is not affected by
problems with thrombin generation. Clinically, the
thrombin time is often used to monitor heparin therapy, and to differentiate heparin effect, hypofibrinogenemia, dysfibrinogenemia, elevated levels of fibrin
degradation products, and some paraproteins from
other coagulopathies as the cause of a prolonged PT
or aPTT.(135-138) It is also used to monitor heparin
reversal in following cardiothoracic surgery, and to
monitor thrombolytic therapy. Increased plasma fibrinogen may also prolong the thrombin time, possibly by interfering with fibrin assembly.(139) The reference range for the thrombin time is affected by the
source and concentration of thrombin and other
factors.(140)
human plasma deficient (<1%) in the coagulation
factor under study. When the factor-deficient plasma
is mixed with patient plasma in a 1:1 ratio, the PT or
aPTT of the mixture is dependent on the amount of
factor present in the patient plasma. The factor activity of the patient plasma is determined from a standard curve, prepared from the PT or aPTT values of
1:1 mixtures of factor deficient substrate and a
serially-diluted reference plasma with known factor
activity. Factors II, V, VII, and X are assayed with the
PT, while the assays for factors VIII, IX, XI, XII, and
the contact factors (i.e., prekappikrein, high molecular weight kininogen, Passovoy) use the aPTT. The
accuracy of a clotting factor determination is improved by using serial dilutions of patient plasma
and averaging the results. Factor inhibitors may interfere with assay results until sufficiently diluted out.
Heparin is the most common cause of a prolonged
thrombin time. This is confirmed by normalization of
the thrombin time or aPTT following in vitro heparin
neutralization with Heparinase, protamine sulfate,
Heptasorb, or other heparin-neutralizing agents, or
by the performance of the reptilase time. A fibrinogen assay, an inhibitor screen, or the dRVVT may be
indicated if heparin effect is not present.
The measurement of clotting factor activity is essential to determine the cause of an elevated PT or aPTT,
and to monitor the treatment of patients with known
factor deficiencies or inhibitors. In some patients, the
presence of a weak clotting factor inhibitor is sometimes initially suspected from “non-linearity” in the
dilution curves.
Clotting Factor Assays (Factors II – XII;
Contact factors)
The activity of individual coagulation factors are
usually determined in plasma using a one-stage clotting assay. Two-stage and amidolytic (chromogenic
substrate) methods for the determination of factor
activity exist but are rarely used in the United States.
In the past, aliquots of plasma obtained from patients
with hereditary deficiencies of clotting factors were
used for factor analysis, but the supply of some factor
deficient plasmas was very limited, and some contained HIV and/or hepatitis virus. Therefore, the onestage assays now use lyophilized, immunoadsorbed,
Laboratory Evaluation of Hemostasis
Fibrinogen Assay
Fibrinogen is the most abundant clotting protein in
the plasma, with a normal plasma level ranging rom
200-400 mg/dL. The quantitative determination of
plasma fibrinogen is essential in the diagnosis and
management of many coagulopathies. In addition,
since plasma fibrinogen levels are increased in some
patients who develop myocardial infarction and
stroke, there is interest in the measurement of fibrinogen for thrombotic risk assessment.(141) The
washed clot method (total clottable fibrinogen assay,
World Health Organization method) is the reference
technique for fibrinogen determination. In this technique, citrated plasma is incubated for an extended
period of time with thrombin in the presence of epsilon aminocaproic acid (EACA) to prevent digestion
of the fibrin clot by plasmin. Other serum proteins
are removed by washing, the clot is dissolved in concentrated urea, and the fibrinogen concentration is
measured colormetrically.(142) This technique is unsuitable for the determination of the large number of
specimens encountered in the clinical laboratory, but,
unfortunately, the accurate and precise measurement
of fibrinogen with the automated coagulometer has
proven difficult. Immunoassays (RID, ELISA, immunonephlometric) for fibrinogen quantitation are also
available but are rarely used.
In spite of their flaws, the von Clauss technique and
the Clotting Rate Assay (Kinetic Fibrinogen Assay,
Prothrombin Time Derived Method) are most widely
used in the clinical laboratories. The von Clauss technique is based upon the principle that when a high
concentration of thrombin is added to plasma diluted
in buffer (1:5 or 1:10), the effects of clotting inhibitors
are diminished and the clotting time is directly proportional to the level of clottable fibrinogen.(143)
Clotting times of patient plasma are read on a standard curve made with purified fibrinogen of known
concentration to interpolate a fibrinogen level in the
patient. The assay is accurate in the range of approximately 50 – 800 mg/dL. Since the von Clauss
technique requires a high level of technical skill, a
more recent prothrombin time-based kinetic assay is
preferred by many laboratories. In this assay, the rate
of increase in plasma turbidity is measured at 450 nm
during the thrombin-catalyzed conversion of fibrinogen to fibrin.(144; 145) This kinetic assay is rapid,
economical, and can be fully automated.(146) Generally, high levels of heparin or hirudin, but not therapeutic levels, can interfere with the clotting assays for
fibrinogen, and patients with known hyperfibrinolytic activity will continue to degrade the fibrinogen
in the collected blood sample before testing is completed unless a special tube is used containing apro14
Clot-Based Assays
tinin or other plasmin inhibitor. Many studies have
shown that fibrin degradation products cause an
overestimation of the fibrinogen level by the washed
clot and immunologic assays, and an underestimation by the clot-based techniques.(141) The kinetic
assay has also been reported to yield higher fibrinogen levels in patients receiving oral anticoagulation
than the von Clauss technique.
Plasma Mixing Studies (Clotting Factor
Inhibitor Screen, Circulating Anticoagulant
Screen)
A prolonged clotting test (i.e., PT, aPTT, and/or
thrombin time) indicates the presence of a factor deficiency or inhibitor of coagulation. The plasma mixing study is the initial step in the evaluation of a prolonged clotting time. The goal of a mixing study is to
determine if the prolonged clotting time is shortened
or “corrected” by mixing the test plasma with equal
volume of normal pooled plasma (NPP; also called
citrated normal plasma, CNP). Even a profound deficiency of a clotting factor, such as the 1% factor VIII
level encountered in severe hemophilia, will be corrected to the normal range by mixing with NPP, since
a 50% level of any factor will still yield a normal clotting time. “Factor assays” are then performed to identify the deficient clotting factor.
The failure of a prolonged clotting test to correct in
the mixing study indicates the presence of a “inhibitory” substance that is preventing clotting from occurring. Unfortunately, this is somewhat difficult to
accomplish since there are several different types of
inhibitors (also called “circulating anticoagulants”).
“Specific inhibitors” are immunoglobulins with specificity for phospholipid ("lupus anticoagulants") or a
specific clotting factor ("factor inhibitors"). “Global”
or “non-specific” inhibitors affect more than part of
the clotting process and include fibrin(ogen) degradation products, some pathologic antibodies such as
Laboratory Evaluation of Hemostasis
monoclonal paraproteins, and drugs such as heparin.
Clinical and other laboratory clues are necessary to
identify the inhibitor. For example, lupus anticoagulants are usually not associated with clinical bleeding,
while specific factor inhibitors frequently cause
bleeding. Generally, factor deficiencies produce a
complete correction of the prolonged clotting time
(i.e., corrected to within the normal range), specific
antibodies show very little, if any correction, and
non-specific may show a “partial correction,” (i.e.,
shortened clotting time but not to within the normal
range). The presence of heparin and other nonspecific inhibitors can be confirmed by other coagulation tests such as the thrombin clotting time and reptilase time, while lupus anticoagulants are identified
by a phospholipid-sensitive test such as the dilute
Russell Venom time (dRVVT). The last clue is provided by the effect of incubation on the activity of the
inhibitor.
An “immediate” mixing study is performed by mixing
equal amounts of the "test" plasma with NPP (1:1
mix) and immediately performing a clotting time
(i.e., PT, aPTT, or TT) on the mixed plasma
specimen.(147-149) Most factor inhibitors (except
factor VIII) and a most lupus anticoagulants (“fast
reacting inhibitors”) produce an immediate clotting
time inhibition and do not require incubation. In
contrast, most factor VIII inhibitors and some lupus
anticoagulants (15%) are weak and/or time dependent (“slow reacting inhibitors”), and require incubation of the 1:1 plasma mixture at room temperature
or 37oC for one or two hours (“incubated mix”) to
cause prolongation of the clotting time.(150-152) A
false diagnosis of a factor deficiency can result without incubation, since slow-reacting inhibitors may
correct the immediate mix. Some laboratories also
include a 4:1 aPTT mix (i.e., 4 parts patient plasma, 1
part NPP) to improve the detection of weak inhibitors
that minimally prolong the aPTT (usually 3-5 seconds
above baseline). The markedly prolonged aPTT of
plasma from a patient with hereditary prekallikrein
deficiency is normalized by prolonged preincubation
(i.e., 10 minutes) of the plasma with aPTT reagent
before the assay is performed. This unique feature of
prekallikrein deficiency is reportedly due to the
autoact ivat ion of factor XII dur ing
preincubation.(153)
Mixing studies are simple in principle, but can be
difficult to interpret. For example, if the laboratory
range for the aPTT is 24-35 seconds, and the patient
aPTT is 70 seconds, a 1:1 mixing study result of 34
seconds would clearly indicate a factor deficiency,
while a value of 69 seconds would indicate an inhibitor. However, what if the mixing study produced values of 39 seconds, 51 seconds, or 63 seconds? The
situation is made even more difficult because there
are no “official” criteria for determining if a correction has occurred. Furthermore, a number of patientspecific and laboratory-specific variable can affect
the result and are difficult to compensate for. These
include the biological heterogeneity of anti-factor
antibodies, the presence of drugs and other substances in the patient specimen, reagent and instrument sensitivity, the source of NPP, the validity of the
laboratory reference range, pre-analytical variables,
and other factors. Therefore, each laboratory presently establishes their own criteria for interpreting
mixing studies. As summarized by Ledford-Kraemer
(2004), these criteria generally fall into three categories:
• The use of the upper limit of the laboratory reference range as the “correction target”. A value,
such as ±2SD, ±3SD, or within 5 seconds of the
upper limit of the reference range is chosen as
the criteria for correction. A failure of correction is assumed if this value is not reached.
• The use of NPP tested in conjunction with the
patient 1:1 mix. This is particularly valuable to
correct for the decreased activity of the “labile”
15
Clot-Based Assays
coagulation factors, V and VIII, during incubated studies. Common criteria for correction
of the patient sample include to within 5 seconds, or to within 10% or 15% of the NPP value.
The Rosner index for the aPTT mixing study
quantitates the amount of correction to the patient plasma aPTT. A correction is assumed if
the Rosner index is ≤15.
• The Chang percentage, a formula that incorporates the degree of correction in relation to the
initial aPTT prolongation.
Chang and co-workers performed a detailed evaluation of the sensitivity and specificity of different
methods to define correction of the 1:1 mix.(148; 149)
They found that the three most widely used criteria
for a correction of the aPTT 1:1 mix (upper limit of
normal, NPP aPTT + 5 seconds, Rosner index ≤15) all
had high sensitivity (88-100%) but low specificity (713%) for detecting a factor deficiency, and low sensitivity (7-15%) and high specificity (88-100%) for detecting an anticoagulant. Using their correction formula and a % correction cutoff at 50%, the immediate
aPTT 4:1 mix had a 75% sensitivity for a factor deficiency and a 91% sensitivity for an anticoagulant. The
corresponding specificies were 91% and 75%. Using
an incubated aPTT 4:1 mix with a cutoff value of >
10% gave sensitivities and specificities of 100% for
both factor deficiencies and anticoagulants. Therefore, the authors recommend performing immediate
and incubated 1:1 aPTT mixes, with the interpretation
as follows:
Reptilase Time
The reptilase time measures the conversion of fibrinogen to fibrin clot by reptilase (Batroxobin,
Atroxin), a thrombin-like enzyme derived from the
venom of the fer-de-lance (barba amarilla, Bothrops
atrox).(135; 136; 154) In contrast to thrombin, which
cleaves fibrinopeptides A, AP, and B from the fibrino-
Laboratory Evaluation of Hemostasis
Fig. 9. Formulas for calculation of Chang
Percentage and
Rosner Index.
Chang Percentage
% Correction =
PP PT (or aPTT) - 1:1 (or 4:1) Mix PT (or aPTT)
PP PT (or aPTT) - CNP PT (or aPTT)
X 100
Rosner Index
Index =
1:1 Mix aPTT - CNP aPTT
gen molecule, reptilase cleaves only fibrinopeptides A
and AP. The resulting fibrin monomers polymerize
end-to-end to form a fibrin clot. Reptilase has no fibrinolytic activity, does not activate plasminogen,
and is not inhibited by antifibrinolytics, thrombin
inhibitors (heparin, hirudin, anti-thrombin antibodies) or antithrombin III.
The reptilase time is used in the evaluation of a prolonged aPTT, specifically to exclude the presence of
dysfibrinogenemia. Hypofibrinogenemia and dysfibrinogenemia are the usual causes of a prolonged
reptilase time. Prolongation of both the thrombin
time and reptilase time suggests hyopfibrinogenemia
or dysfibrinogenemia. A prolonged aPTT and normal
reptilase time indicates that heparin or other antithrombins is the cause of the prolonged aPTT. Myeloma proteins reactive with thrombin may prolong
the reptilase time. Fibrin degradation products
(FDPs) may slightly prolong the reptilase time.
Dilute Russell Viper Venom Assay (dRVVT)
The dRVVT is used to detect lupus anticoagulants
(LA), one type of autoantibody characteristic of patients with the antiphospholipid antibody
syndrome.(155-157) LA are autoantibodies of the IgG
PP aPTT
X 100
and IgM classes that interfere with the function of
anionic phospholipids and prolong phospholipiddependent clotting tests such as the aPTT and
dRVVT.(158-162) The dRVVT is more specific for LA
than the aPTT since it is not influenced by deficiencies of the contact or intrinsic pathway factors or antibodies to factors VIII, IX, or XI.(159; 163; 164) The
coagulant protein in Russell’s viper venom (RVV) is a
serine protease that directly activates factor X in the
presence of Ca++, bypassing the intrinsic and extrinsic pathways. The activated factor X then activates
prothrombin (factor II) in the presence of factor V
and phospholipid. In the dilute Russell’s viper venom
time (dRVVT), phospholipid is diluted to the point
that the clotting time becomes very sensitive to the
presence of substances blocking availability of the
phospholipid surface. The DVVtest is a commercial
reagent (American Diagnostica, Inc., Greenwich, CT)
developed to standardize the dRVVT. Similar reagents
are available from Precision Biologic (Dartmouth,
Nova Scotia) and other vendors. The DVVtest reagent
combines RVV, plant phospholipid, and calcium into
a single reagent. A second reagent, DVVconfirm, contains RVV, extra plant phospholipid, and calcium. The
extra phospholipid in the DVVconfirm reagent is
provided to see if it corrects a prolonged DVVtest
time (by overwhelming the LA). The finding of a pro16
Chromogenic Analysis
longed dRVVT with patient plasma is presumptive
evidence for the presence of a lupus anticoagulant.
This presumption is “confirmed” if the dRVVT is not
corrected with a mixture of normal and platelet
plasma, but is corrected by the substitution of platelets for phospholipid. With the DVVtest and DVVconfirm reagents, a DVVtest/DVVconfirm ratio >1.2 is
confirmatory for the presence of LA.
Activated Protein C Resistance
Assay (APCR)
The rapid screening assay for activated protein C resistance for (APCR) is another widely used modification of the aPTT. In 1993, Dahlback and coworkers
discovered a mutant clotting Factor V (Factor V Leiden) which results in the failure of Activated Protein
C to inactivate Va.(165-168) This defect in the protein
C pathway is associated with a significantly increased
risk of thromboembolic disease. The laboratory diagnosis of APCR begins with the rapid screening test,
followed by confirmation with a molecular assay if
the screening assay is positive. In the presently used
modification of Dahlback’s original aPTT-based
screening assay, the test plasma is first diluted with
factor V–deficient plasma to inactivate therapeutic
concentrations of heparin, correct for coagulation
factor deficiencies, and counteract the effect of some
lupus inhibitors. aPTT assays are then performed
with and without the addition of exogenous activated
protein C (APC).(169-171) The added APC significantly prolongs the aPTT in normal individuals, while
patients with APCR show less of an increase. The results are usually expressed as the ratio of the aPTT
with and without added APC. The modified APCR
screening assay is highly sensitive to factor V Leiden
and most other less common mutations of factor V,
can differentiate heterozygotes from homozygotes,
and is not influenced by heparin or warfarin at
therapeutic concentrations.(170)
Laboratory Evaluation of Hemostasis
Fig. 10. A chromogenic method for
the determination of factor VIII
activity. Test plasma is incubated
with calcium, phospholipid, and
excess amounts of purified factors
IX and X. The activated factor X
generated by the reaction hydrolyzes a chromogenic substrate,
generating a colored reaction
product that is measured by a
spectrophotometer. The amount of
generated factor Xa is directly
proportional to the concentration
of factor VIII activity.
Factor X
Factor IXa, Ca++, Phospholipid
Factor VIII
Peptide + pNA
Chromogenic Analysis
Chromogenic analysis is a technique of enzyme
analysis developed in the early 1970’s. Chromogenic
assays utilize synthetic substrates comprised of a
colored chemical substance (chromphore, chromagen) linked to a short amino acid residue specific for
the enzyme of interest.(172-174) Enzymatic action
releases the chromophore, which is quantitated by
spectrophotometry. The selectivity of chromogenic
substrates is similar to the native enzyme substrate,
but they are often more sensitive. Other advantages of
chromogenic assays include reagent stability and the
adaptability to a wide range of automated laboratory
instruments, including those used in the chemistry
and immunology laboratories. The selectivity of a
chromogenic substrate to the desired enzyme is affected by the relative concentrations of sample and
reagents, reaction conditions (i.e. pH, temperature,
buffer type and concentration, ionic strength, etc.),
the presence of inhibitors, substrate solubility and
stability, and other factors.(175) The best substrates
have high affinity for the enzyme and a high turnover
rate. The most common substrate is para-nitroaniline
(pNA), which has a maximum absorption spectrum at
405 nm.
Factor Xa
Chromogenic
substrate
The analysis of many coagulation factors utilize
chromogenic substrates for factor X.(176; 177) For
example, factor VIII is an enzymatic cofactor for factor IX. Activated factor IX causes the activation of
factor X, which then hydrolyzes the chromogenic substrate and releases the pNA chromophore that is read
spectrophotometrically at 405 nm (Fig. 10). If the assay conditions are properly controlled, the color intensity reflects the amount of factor VIII. In one comparative study of chromogenic analysis, an antigenic
assay, and the one-stage clotting assay for factor VIII,
the chromogenic factor VIII technique was the optimal method, with good precision and freedom from
interference by lupus inhibitors, heparin, or other
anticoagulant drugs.(178)
Chromogenic substrates for thrombin, tissue-type
plasminogen activator, urokinase, coagulation factors
IX, X, and XII, and other substances are commercially
available from Chromogenix (Orangeburg, NY),
(Trinity Biotech Plc, Wicklow, Ireland) and other
companies.
17
Latex Agglutination
Latex Agglutination
and Turbidimetry
Agglutination is a secondary immune phenomenon
that occurs when insoluble or particulate antigens
(cells or other particles) are cross-linked by an immune reaction. Agglutination occurs because antibodies have two or more antigen recognition sites
(bi- or multivalency). If multiple antigenic recognition sites are present on a particle, lattices can be
formed that grow in size and eventually become a
mass that is macroscopically visible. The major factors affecting the agglutination reaction include the
class, affinity and avidity of the antibody, the proximity and number of binding sites on the particle, the
relative concentrations of antibody and particles,
electrostatic interactions ("zeta potential"), and the
viscosity of the medium. Antibodies of the IgM class,
with ten antigen combining sites, are usually the best
“agglutinins,” and are more efficient than IgG in agglutination.
Agglutination assays are classified as direct or indirect, depending on whether the analyte is present in
its native state, or linked to a particle (carrier) to allow detection of the antigen-antibody reaction. Carriers vary in size from about 0.05 micron to 7 micron,
and may be red blood cells, latex particles, liposomes,
microcapsules, or other particles.(179)
The use of latex particles for immunoassay was first
reported by Singer and Plotz in 1956.(180) Latex particles are usually coated by passive means, with the
quantity of the adsorbed protein adjusted to provide
agglutination of the analyte in its biological range. In
addition, the use of latex particles avoids much of the
variability encountered with red blood cells. Even so,
the prozone phenomenon can still be significant, and
careful adherence to the manufacturer's instructions
is necessary during the performance of clinical assays
utilizing coated microspheres.
Laboratory Evaluation of Hemostasis
Turbidimetry and the related technique of nephelometry are extensively utilized in the clinical immunology for the quantitation of a large number of
medically important substances since they are precise, rapid, and automated. In the coagulation laboratory the use of these techniques is more limited. Light
scattering by particles in solution is the basic principle of turbidimetry and nephelometry. When an immune complex is formed under carefully controlled
conditions, measurement of light scatter can provide
information regarding the quantity of analyte
present.(181) Turbidimetric techniques determine the
reduction in the intensity of incident light from all
interactions of an immune complex with a light
beam, while nephelometric techniques measure light
scattered at a specific angle to the incident beam.
Nephelometry is more sensitive to small particles
than turbidimetry, while turbidimetry more accurately measures large complexes.(182)
Turbidimetry has been used since the early 1950's as
a method of quantitative analysis, particularly for
large immune complexes. In this technique, the
change in light intensity caused by interaction of a
light beam with a suspension of particles is determined spectrophotometrically. The major use of latex
particle agglutination and turbidimetry in the clinical
coagulation laboratory is the detection and semiquantitation of fibrin degradation products (FDPs)
and D-Dimers. Fibrin degradation products (FDPs)
are the result of plasmin degradation of fibrinogen,
fibrin monomers, fibrin polymers or cross-linked
fibrin, while D-dimers are degradation products that
arise specifically from the plasmin degradation of
fibrin crosslinked by Factor XIIIa activity.(183) Thus,
the measurement of cross-linked degradation products (XDPs), unlike total FDPs, is a specific measure
of fibrinolysis. Most turbidimetric assays for Ddimers utilize latex beads or other microparticles
coated with monoclonal antibody specific for fibrin
D-dimer or the fragment D of fibrin but not with in-
tact fibrinogen, permitting the analysis of whole human plasma. Elevated D dimers are seen in DIC, pulmonary embolism, arterial and venous thrombosis,
septicemia, cirrhosis, carcinoma, sickle cell crisis, and
following operative procedures. However, D-dimer
analysis is principally used in the evaluation of patients with suspected thromboembolic disease, especially pulmonary embolism and deep vein
thrombosis.(184-188) Both FDPs and XDPs are present during late pregnancy and for approximately 48
hours post-surgery. During fibrinolytic therapy the
FDP test is positive, while the D-dimer test is negative
in the absence of thrombolysis. Enzyme immunoassay has also been utilized for the detection of
D-dimers.(184; 189-192) Other turbidimetric reagents
are available for the analysis of von Willebrand factor,
free protein S, and other substances.
Nephelometric techniques have been applied very
successfully to the immunochemical measurement of
specific proteins, drugs and other substances.(182;
193) In nephelometry, a known amount of specific
antibody is added to a solution containing the antigen being measured. The intensity of light scattered
from the large antigen-antibody complexes formed
during the reaction is measured, and the rate signal is
transmitted to a microcomputer, where concentration
units are determined. Nephelometry is used by some
clinical laboratories for the quantitation of fibrinogen or factor VIII-related antigen.
Enzyme Immunoassay
The enzyme immunoassay (EIA) is a type of nonisotopic immunoassay in which enzymes, coenzymes,
fluorigenic substrates, or enzyme inhibitors are used
as labels.(194; 195) The major prerequisite for an EIA
is that an antigen or antibody must be linked to an
enzyme without destroying the immunologic or enzymatic activity of the antigen-antibody complex. In
solid-phase EIA techniques, the antigen or antibody
18
ELISA, Flow Cytometry
must be bound to a polystyrene test tubes or microtiter tray, a particle of polystyrene, latex, or agarose, a
magnetized bead, or another physical support.
Enzymes utilized in immunoassay systems must also
be stable, available in a highly purified state, have a
high turnover rate, and undergo minimal interference
by substances likely to be in the test solution, and be
specific for the substrate. The final reaction product
should be detected by a convenient means with a low
detection limit. The most widely utilized enzyme in
enzyme immunoassay is horseradish peroxidase
(HRP). The substrate of HRP is hydrogen peroxide
(H202) and the product is oxygen. This oxygen produced during the reaction is used to oxidize a reduced, colorless chromagen (usually reduced orthophenylenediamine, OPD). The final product, oxidized
OPD, has a brown color. Most EIA's utilize horseradish peroxidase or alkaline phosphatase as labels, although glucose oxidase, beta-D-galactosidase, and a
wide variety of other enzymes have also been used.
Utilizing fluorimetric techniques, the respective detection limits for HRP, beta-galactosidase, and alkaline phosphatase are 5, 0.2, and 10 attomol.(196) The
practical detection limit of the EIA is approximately
0.01 to 0.02 attomol of ligand.(197; 198) The enzymeLinked Immunosorbent Assay (ELISA) is the most
widely utilized type of enzyme immunoassay.
Enzyme immunoassay is a critical technique in the
clinical laboratory for a wide variety of analytes, including both antigens and antibodies. In the hemostasis laboratory, enzyme immunoassay is used for
the quantitation of antigen levels of most clotting
factors, fibrinolytic components, and regulatory substances.
Flow Cytometry
Flow cytometry is a technique of quantitative single
cell analysis. The flow cytometer was developed in the
Laboratory Evaluation of Hemostasis
1970’s and rapidly became an essential instrument for
the biologic sciences. Spurred by the HIV pandemic
and a plethora of discoveries in hematology, specialized flow cytometers for use in the clinical laboratory
were developed by several manufacturers. The major
clinical application of flow cytometry is diagnosis of
hematologic malignancy, but a wide variety of other
applications exist, such as reticulocyte enumeration
and cell function analysis. Presently, more than
40,000 journal articles referencing flow cytometry
have been published. This brief review of the principles and major clinical applications of flow cytometry may be supplemented by several recent review
articles and books. (199-203)
Prepared single cell or particle suspensions are necessary for flow cytometric analysis. Various immunoflurescent dyes or antibodies can be attached to the
antigen or protein of interest. The suspension of cells
or particles is aspirated into a flow cell where, surrounded by a narrow fluid stream, they pass one at a
time through a light beam. Light and/or fluorescence
scatter signals are detected and amplified. The resulting electrical pulses are digitized, and the data is
stored, analyzed, and displayed through a computer
system.(203; 204) The end result is quantitative information about every cell analyzed. Since large
numbers of cells are analyzed in a short period of
time (>1,000/sec), statistically valid information
about cell populations is quickly obtained.
The flow cytometer has been essential for the analysis of platelet structure and function in the research
laboratory. Although the small physical size and biovariability of the platelet creates inherent difficulties
for flow cytometric analysis, several clinical assays
are performed by specialized flow cytometry laboratories. These assays will achieve more widespread
practice in the near future as standardized techniques
and controls become available. These assays have
been classified by Bode and Hickerson to include
platelet surface receptor quantitation and distribution for the diagnosis of congenital platelet function
disorders, platelet-associated IgG quantitation for the
diagnosis of immune thrombocytopenias and for
platelet cross-matching in transfusion, reticulated
platelet assay to detect “stress” platelets, fibrinogen
receptor occupancy studies for monitoring the clinical efficacy of platelet-directed anticoagulation in
thrombosis, and the detection of activated platelet
surface markers, cytoplasmic calcium ion measurements, and platelet microparticles for the assessment
of hypercoagulable states.(205)
Flow cytometry is a critical research technique for
the study of diseases of platelet surface receptors, and
has been applied to clinical diagnosis by larger laboratories. The identification of Glanzmann thrombasthenia, the Bernard-Soulier syndrome, and even rarer
platelet receptor disorders is performed with panels
of monoclonal antibodies specific for the receptor
antigens under consideration. The use of monoclonal
antibody panels specific for different epitopes is particularly information in defining the heterogeneity
and extent of disease expression.
Flow cytometry has been utilized to detect both
platelet-associated immunoglobulins of autoimmune
and alloimmune origin. In general, this is performed
by incubating washed platelets with fluorochromelabeled antihuman immunoglobulin and quantitating
platelet surface fluorescence. Gating procedures are
used to exclude irrelevant cells and particles. The
differentiation of positive and negative results is dependent upon an adequate negative control, usually
platelets from normal individuals. Flow cytometry is
ideal for the study of low numbers of patients in
thrombocytopenic patients, since it is sensitive and
avoids the platelet activation, with the release of endogenous immunoglobulin molecules.(206) Although
procedural standardization and non-specificity have
limited the use of flow cytometric analysis for platelet
19
Electrophoresis, Genetic & Molecular Assays
associated immunoglobulin in idiopathic thrombocytopenia purpura (ITP), the technique appears more
clinically promising for the detection of alloimmune
antibodies arising from the transfusion of nonautologous platelets (Post-transfusion Purpura) or
the entrance of fetal cells into the maternal circulation (Neonatal Alloimmune Thrombocytopenia Purpura). The antibodies are typically formed against
alleles of the human platelet alloantigen system
(HPA), notably HPA-1a (Pl-A1) and HPA–1b
(Pl-A2).(207-209) These antibodies can be specifically
detected by flow cytometry, and flow cytometric
crossmatching can be performed to reveal HLA incompatibilities between donor platelets and sensitized recipients.(210-212)
Reticulated platelets are recently released into the
circulation, larger than average, and contain small
amounts of RNA remaining from the megakarocytic
process.(213) The quantitation of reticulated platelets
is valuable for the differentiation of accelerated destruction from impaired production in patients with
thrombocytopenia of unknown etiology(214-216)
Reticulated platelets can be detected by the utilization of thiazole orange, a brightly fluorescent dye that
binds to nucleic acid.(217; 218) The assay is somewhat difficult to perform, since the platelets must be
permeabilized and positive controls are difficult to
obtain.
Hypercoagulability is one of the most common medical problems, and it is not surprising that a wealth of
new discoveries have arisen from the application of
modern technology. Flow cytometry has been an essential technique for the elaboration of platelet function and understanding the contribution of platelet
activation to hypercoagulability. To date, many flow
cytometric studies have involved the detection of
platelet surface markers, the study of cytosolic calcium ion levels, and the detection of circulating platelet microparticles.(205)
Laboratory Evaluation of Hemostasis
The recent wealth of new discoveries in the field of
hemostasis and thrombosis has included a number of
antiplatelet agents of value in the therapy of patients
with inherited or acquired hypercoagulability and/or
those undergoing vascular interventional procedures.
As the most prevalent and functionally important
platelet surface receptor, the GPIIbIIIa complex is the
target of several of these agents. Abciximab is a
murine monoclonal antibody that blocks the GPIIbIIIa receptor to prevent platelet activation. Others,
such as Integrilin and Aggrastat are small peptides
that saturate the receptor. A variety of “receptor occupancy” assays have been developed to monitor the
clinical efficacy of these agents. Flow cytometry is the
most sensitive and versatile of these techniques, although it is not a point of care assay at the present
time.(219)
Electrophoresis
Historically, the Laurell rocket assay, radial immunodiffusion, and other gel-based procedures were
widely used in the coagulation laboratory for quantitative analysis of certain clotting factor protein levels,
especially the FVIII/vWF complex.. Today, these procedures have been largely replaced by enzyme immunoassay, and other more accurate and efficient methods, with the exception of von Willebrand factor multimer analysis. This is performed by electrophoresis
on agarose gel containing some acrylamide. The protein bands are then transferred to nitrocellulose for
Western blot with polyclonal anti-vWF antibody and
finally visualized by radiolabeling, enzymatic detection, or chemiluminescence.(220; 221) The pattern of
multimeric units and satellite bands differentiates
von Willebrand disease into type I, type IIA, B, M, and
N, and type III. Multimer analysis is a specialized
procedure only performed by a few coagulation reference laboratories, but it is sometimes critically important to subtype von Willebrand disease to avoid
inappropriate treatment (i.e., DDAVP is contraindicated in vWD Type IIb).
Genetic and Molecular Assays
The advances of genetic and molecular methods of
study during the past two decades have had a profound impact upon our understanding of hemostasis,
as well as other fields of medicine. The molecular
origin and function of many substances involved in
hemostasis are now understood, and the genetics and
molecular of the hemophilias and many diseases is
much clearer.(222-225) A diversity of new diagnostic
assays resulting from these discoveries is expected in
the near future, but at present the major clinical role
for molecular analysis is in the diagnosis of inherited
thrombophilia.
The majority of patients who develop recurrent venous thromboemboli (inherited thrombophilia) have
discernable abnormalities of the coagulation system,
including factor V Leiden, deficiencies of protein C,
protein S, antithrombin III, the prothrombin G20210A
gene mutation, homocysteinemia, elevated factor levels, dysfibrinogenemia, or abnormalities of the fibrinolytic system.(226) Most of these abnormalities
cause deficiencies of the regulatory substances of
clotting. Genetic abnormalities are especially common in individuals who develop thrombi at an early
age (< 40 years) and in those with a family history of
thrombosis. Although no genetic abnormality is detectable in about 15 percent to 20 percent of individuals with recurrent thromboembolic disease, research in this area is rapidly proceeding and new genetic abnormalities may be described in the near future.
Factor V Leiden, the major cause of APCR, first identified in February 1993, is the most common inherited cause of thrombosis known at this time.(167) It is
found in about 5 percent of the general population
20
Genetic & Molecular Assays
Table II
Molecular Techniques for the Evaluation of Hypercoaguable States*
Assay
Accuracy
Throughput
Present Clinical Applications
PCR/RFLP
Good
Limited
PCR/ARMS
Excellent
Intermediate
Light Cycler
Excellent
Intermediate
Factor V Leiden (1691G>A),
prothrombin 20210AG>A, MTHFR
677C>T
Array technology
Excellent
Very high
Under development
Invader assays
Excellent
Limited
Under development
Ligand-based
technologies
Excellent
Very high
Under development
Factor V Leiden (1691G>A),
prothrombin 20210AG>A, MTHFR
677C>T
*Modified from Nagy PL, Schrijver I, Zehnder JL. Molecular diagnosis of hypercoagulable states. Lab Med.
2004;35:214-221.
and is responsible for 20 percent to 50 percent cases
of inherited thrombosis. Approximately 50,000 individuals die yearly in the United States from complications caused by this abnormality.(154; 168; 170) Heterozygous individuals are at five to 10 times greater
risk of thrombosis than the general population, while
homozygotes are at 50-100 times greater risk. The
use of estrogen or oral contraceptives increases the
risk of thrombosis even further. In 90 percent to 95
percent of cases, APCR is a result of a single point
mutation (Arg506Gln) in the gene for factor V on
chromosome 1q23, inherited as an autosomal dominant trait. This mutation renders activated factor V
(Va) more resistant to inactivation by APC. The remaining five to 10 percent of APCR is due to other
genetic abnormalities in the factor V gene, including
Laboratory Evaluation of Hemostasis
the tR2 haplotype
(Arg306Thr).
and
factor
V
Cambridge
A mutation in the prothrombin gene that produces
elevated levels of prothrombin was discovered in
1996.(227-231) The mutation involves a single amino
acid substitution (20210G>A) in the 3’-UTR untranslated region of the prothrombin gene on chromosome 11, leading to more effective mRNA translation
and elevated plasma prothrombin. There is increasing evidence that the G20210A mutation is an important risk factor for deep venous thrombosis, myocardial infarction and stroke. The use of estrogen or oral
contraceptives increases the risk of thrombosis even
further in patients with the prothrombin 20210 mutation.
Hyperhomocysteinemia and homocysteinemia are
inherited abnormalities of homocysteine metabolism. Homocysteine is a naturally occurring substance involved in the metabolism of certain amino
acids, including cysteine and methionine. Abnormalities in at least three enzymes, methylenetetrahydrofolate reductase (MTHFR), cystathionine beta-synthase
(CBS) and methionine synthase (MS) associated with
homocysteine metabolism in the body can lead to
increased homocysteine levels in the body (hyperhomocysteinemia). Genetic abnormalities in these
enzymes, particularly homozygous defects in
MTHFR, are the common risk factors for thrombotic
disease, including heart disease and stroke.(232; 233)
Hyperhomocysteinemia also may be associated with
vitamin deficiency, advanced age, hypothyroidism,
impaired kidney function, systemic lupus erythematosus and the use of certain medications, including
nicotinic acid, theophylline, methotrexate and L-dopa.
Inherited abnormalities in antithrombin, protein C,
protein S, other regulatory of the coagulation system
are less common and more complex genetically. Inherited abnormalities in antithrombin, protein C,
protein S occur in two forms, leading to either low
plasma concentrations (Type I deficiency) or funtionally abnormal but quantitatively normal (Type II
deficiency) of the involved proteins. To date, more
than 250 mutations have been described in the antithrombin gene, together with more than 100 each in
the protein C and protein S genes. The likelihood of
clinically significant thrombotic disease or crises in
any one patient is greatly elevated when more than
one of these traits is present.
The molecular evaluation of the hypercoagulable
states was the subject of a recent review by Nagy,
Schrijver, and Zehnder (2004).(234) A variety of molecular techniques have been employed, but the field
is in a state of rapid development. The molecular
techniques presently employed in the evaluation of
21
Electron Microscopy, RIA, References
the inherited thrombophilia disorders are summarized in Table II.
Electron Microscopy
Ultrastructural examination of the platelet (platelet
electron microscopy) is performed in research studies of platelets, and to confirm the diagnosis of suspected storage pool disease or other diseases resulting from structural anomalies. Dr. James G. White of
the Department of Laboratory Medicine and Pathology, University of Minnesota has reported the majority of studies of platelet ultrastructure.(235-238)
Platelet ultrastructural examination is classically performed from thin sections taken from PRP fixed in
glutaraldehyde/osmium solutions and dehydrated,
plastic-embedded, sectioned, and stained by conventional techniques. However, Dr. White found that
dense bodies are better visualized by a simple wholemount procedure that involves the examination of
unfixed, unstained PRP fixed and dried on Formvarcoated, carbon-stabilized grids.(239) The procedure is
rapid and easy to perform, and the presence of dense
bodies excludes the diagnosis of the Hermansky–Pudlak syndrome and storage pool disease.
Other techniques, such as scanning electron microscopy, the freeze-fracture technique, histochemical
staining for platelet peroxidase, and staining with
labelled gold particles have been extensively utilized
in research studies of the platelet.
Radioimmunoassay
Most clinical laboratories would wish to avoid the
regulatory burden and safety hazards of use of radionuclides whenever possible. However, at this time,
the most sensitive and well-accepted laboratory assay
for heparin-induced thrombocytopenia is the 14Cserotonin release assay. In this assay, normal donor
Laboratory Evaluation of Hemostasis
platelets labelled with 14C-serotonin are incubated
with patient's serum in the presence and absence of
therapeutic and high concentrations of heparin. If >
20% of the 14C is released at a heparin concentration
of 0.1 U/ml heparin and < 20% is released by 100 U/
mL heparin, the test is positive for heparin antibodies. This was the original assay used by Sheridan et
al. to establish laboratory diagnosis of the HIT syndrome after observing spontaneous aggregation of
platelets incubated in HIT patient plasma with heaprin, and has remained the “gold standard” for validation of other diagnostic techniques.(240) Now, however, even more sensitive assays based on flow cytometry detection of circulating platelet microparticles are under development.(241)
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