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

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Chapter | 10 Hemostasis
2. Assessment of Platelet Quality
a. Buccal Mucosal Bleeding Time
The BMBT is an in vivo test of platelet function, which also
depends on adequate platelet concentration and properly
functioning endothelium. The incision is small enough that
secondary hemostasis is not required to stop blood flow;
formation of a platelet plug alone will stop the bleeding. A
standardized incision using a commercially available device
is made on the buccal mucosa of the lip and the time to clotting
recorded in seconds or minutes. Time to clotting can
vary substantially within and between observers, making this
a less than ideal test, but most healthy dogs clot in less than
4 min (Sato et al ., 2000 ). It is, however, useful as a quick and
inexpensive screening test for congenital or acquired thrombopathia,
which can be followed up by more specific testing
if the result is prolonged. Skin and toenail bleeding time
tests have also been described but are even less standardized
than the BMBT and are not recommended.
b. Platelet Aggregometry
The propensity of platelets to bind to each other in the
presence of certain agonistic substances can be exploited
as a method to determine appropriate platelet function.
Platelet aggregometers trace the optical density of a standardized
platelet preparation after the addition of an aggregating
substance such as ADP, epinephrine, or collagen.
There are marked species differences with respect to concentration
and type of agonist required to promote aggregation.
Platelet aggregation is technically demanding, time
consuming, and therefore generally limited to specialized
or research laboratories.
c. PFA-100
The Platelet Function Analyzer-100 (Bayer) has revolutionized
assessment of platelet function in human medicine.
The ability to quickly assess aperture closure time in
a whole blood sample in a clinical setting has eliminated
all the limitations of time-consuming and technically
demanding platelet aggregometry. The PFA-100 simulates
high shear flow blood vessel conditions and, with the addition
of either epinephrine or ADP as an agonist on a collagen
membrane, determines how quickly a platelet plug can
close an aperture and arrest blood flow.
The method has been evaluated for use in the dog
(Couto et al ., 2007), and the collagen/ADP cartridge has
been found to be useful to screen for platelet dysfunction
in von Willebrand disease ( Mischke and Keidel, 2003 ),
NSAID administration ( Gaal et al ., 2007 ; Neilsen et al .,
2007), and during endotoxemia ( Yilmaz et al ., 2005 ).
There are single reports evaluating the instrument for
horses where it was determined it was sensitive enough to
detect decreased platelet function related to ASA administration
( Segura et al ., 2005 ) and in a pig model of severe
hemorrhagic shock ( Arnaud et al ., 2006 ).
3. Screening Tests of Hemostasis
a. OSPT
The one-stage prothrombin time (OSPT) assesses the
plasma activity of prothrombin, FV, FVII, and FX after
addition of calcium and activator known as tissue thromboplastin.
The test therefore gives information about the
tissue factor (extrinsic) pathway and the common pathway.
Different sources of thromboplastin will result in different
clotting times, and although the use of homologous
tissue is ideal, human source reagents can be used effectively
( Hall, 1970 ; Mischke and Nolte, 1997 ; Mischke
et al ., 2003 ). Clotting times tend to be rapid in domestic
animals, often less than 10sec, which reduces the sensitivity
of the assay for detecting minor abnormalities. Dilution of
reagents has been advocated as a way to extend clotting
times to 10 to 15sec.
As a screening test, the OSPT is not sensitive to minor
coagulation abnormalities, and it generally takes loss of
70% activity of one or more factors to prolong the clotting
time. Modification of the test to assess individual factor
activity can be achieved using specific factor deficient
plasma, and several assays have been shown to have similar
ability to detect factor deficiencies ( Mischke, 2002 ).
The effects of common interferences (lipid, hemoglobin,
bilirubin) on the PT assay have been studied ( Moreno and
Ginel, 1999 ). These authors found that bilirubin caused a
statistically significant prolongation of the OSPT but that
clinical significance associated with this prolongation was
unlikely. Point-of-care analyzers are now available that can
quickly perform the OSPT at the animal’s cage side and
have been evaluated in dogs ( Tseng et al ., 2001 ).
The International Normalized Ratio (INR) is frequently
used in place of the OSPT in human medicine for monitoring
people on warfarin anticoagulant therapy because
different thromboplastins have variable sensitivities to
warfarin-induced changes in factor activity ( Stockham
and Scott, 2002 ). The International Sensitivity Index (ISI),
which is specific for an individual reagent, is incorporated
into the INR calculation and is meant to correct for reagent
variation. ISI values are not widely available for domestic
animal species, so the INR is not frequently used in veterinary
medicine.
b. aPTT
The activated partial thromboplastin time (aPTT) assay
detects fibrin clot formation after addition of an activating
agent, partial thromboplastin (phospholipids that lack
TF), and recalcification of plasma. It assesses the activity
of prothrombin, FV, FVIII, FIX, FX, FXI, and FXII, therefore
providing information about the integrity of the contact
activation (intrinsic) and common pathways (see Section
II.C.3). Activating agents include substances such as kaolin
and ellagic acid, and clotting times can vary between species