Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

310
Chapter | 10 Hemostasis
necessity to use a 96-well plate, and the limited shelf life
of reagents once the kit is opened limit extensive use of
this methodology for veterinary medicine unless samples
can be batched.
b. Fibrinogen
For the heat precipitation method, EDTA-anticoagulated
blood is centrifuged in a microhematocrit tube and heated
for 3 to 9 min at 56 ° C, and then the amount of precipitate
formed is quantified as a measure of fibrinogen. This method
is useful only as a crude estimate of an increased concentration
of fibrinogen and is no longer routinely used.
Derivation of fibrinogen concentration from the Clauss
thrombin clotting time ( Stockham and Scott, 2002 ) is more
often performed at veterinary labs, because routine fibrinbased
clotting assays are usually offered. Fibrinogen concentration
is inversely proportional to the time it takes for
thrombin to form fibrin from fibrinogen, so it depends on
the amount of fibrinogen present when excessive thrombin
is used to negate the effects of any inhibitors such as
heparin or fibrin degradation products ( Stockham and
Scott, 2002 ). The time to clot formation is recorded in seconds
and then converted to mg/dl or g/l based on a standard
curve. A fibrinogen antigen assay is available but is
not a functional test. Therefore, if the antigen assay is not
decreased but the TCT is abnormal, dysfibrinogenemia is
likely.
c. von Willebrand Factor
Assessment of von Willebrand factor (vWF) protein is
most commonly determined by detecting antigen concentration
in plasma, but additional tests are available if more
detailed information is needed to manage the animal. See
Section IV.A.1.b for more details.
d. Antithrombin
As an indicator of anticoagulant status, there are both functional
and antigenic assays for the detection of antithrombin
(AT) in plasma. Functional assays can be either thrombin or
FXa based, where the more AT there is, the less activity of
either of these enzymes there will be. A standard curve from
homologous pooled plasma must be prepared to determine
the test plasma activity. Both manual and automated methods
are available. The activity of AT is stable, and in one
study values remained relatively unchanged after 6 months
of storage of plasma at 70 ° C and after 6 weeks of storage
of whole blood at 4 ° C ( Green, 1988 ). The same study
suggested that FXa-based assays may be more appropriate
because heparin cofactor II activity may be measured in
some thrombin-based assays and falsely elevate the AT measurement.
Hemoglobin in the sample may interfere with the
ability to accurately determine plasma AT activity in some
chromogenic assays ( van der Merwe and Reyers, 2007 ).
C. Additional Assays
Several additional assays of activation of coagulation and
fibrinolysis have been examined on a limited basis in veterinary
medicine ( Roncales and Sancho, 2000 ). Most of
these tests are used widely in human medicine but need
more thorough evaluation and standardization before being
offered as routine tests for domestic animals. Some of the
proteins or complexes tested include activated clotting factors
such as FVIIa and FVIIa, activation peptides such as
fibrinopeptides A and B, and indicators of fibrinolysis such
as plasmin-antiplasmin complexes and PAI-1. Thrombinantithrombin
complexes (TAT) have been measured as an
indicator of coagulation activation in dogs with malignant
neoplasia ( Maruyama et al ., 2005 ) and horses with colic
( Topper and Prasse, 1996 ).
The Russell viper venom time (RVVT) test is an additional
assay used occasionally to provide information about
common pathway abnormalities ( Stockham and Scott,
2002 ). The time to clot formation depends on activation of
FX by the venom of a Russell’s viper. This test is not routinely
used.
Proteins induced by vitamin K antagonism or absence
(PIVKA) are inactive vitamin K-dependent clotting factors
produced by hepatocytes when this important cofactor is
unavailable (see Section IV.B.5) . The Thrombotest PT is a
modification of the prothrombin time that is sensitive, but
not entirely specific, to the presence of these abnormal proteins
( Center et al ., 2000 ).
Thromboelastography (TEG) provides a global assessment
of hemostasis and specifically provides general information
about the rate of fibrin polymerization and overall
clot strength. Analysis of the TEG tracing can give a summary
of platelet function, coagulation proteins, and fibrinolysis
( Luddington, 2005 ). TEG has traditionally been
used in human medicine to facilitate blood product use
decisions in patients undergoing liver transplant or cardiac
surgery. Its use has been minimally evaluated in veterinary
medicine ( Donahue and Otto, 2005 ). One study
used TEG to document hypercoagulability in dogs with
parvoviral enteritis ( Otto et al ., 2000 ). Routine use of TEG
suffers from lack of standardization, although a group
recently developed TEG values for clinically healthy dogs
( Wiinberg et al ., 2005 ).
IV. DISORDERS OF HEMOSTASIS
A. Hereditary Disorders
1. von Willebrand Disease
Although von Willebrand disease (vWD) is the most common
inherited bleeding disorder in both humans and dogs, it
has also been reported in swine and cats ( Denis and Wagner,
1999 ; Thomas, 1996 ). Human vWD was described initially