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

III. Laboratory Assessment of Hemostasis
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There is controversy whether a citrated plasma sample
obtained directly from a peripheral catheter is suitable
for hemostatic testing. Advocates of this technique argue
that the ability to get the sample from a previously placed
access port minimizes patient discomfort, allows for more
accurate volume extraction, and ensures no contamination
with tissue factor. If this approach is used, the catheter
should be flushed with saline, and then blood volume equal
to six times the catheter dead space should be removed and
discarded before collecting the sample for analysis ( Arkin
et al ., 1998 ). A study by Mills et al . (1995) documented
that this procedure did not result in significant differences
compared with direct venipuncture in healthy dogs.
Several reports have compared the effect on various
hemostatic parameters with respect to the concentration
of citrate used and the volume of blood to citrate obtained.
Historically, 3.8% sodium citrate tubes only were available,
but recently 3.2% sodium citrate tubes have become the standard
in Europe and are now available in the United States
and elsewhere ( Morales et al ., 2007 ). One study determined
that for some coagulation assays there can be significant
differences between the two types of tubes ( Stokol et al .,
2000a ), whereas another more recent study ( Morales et al .,
2007 ) suggests that there is no difference in any common
coagulation assay between the two concentrations. It is recommended
that each laboratory specify the citrate concentration
preferred and develop reference intervals based on
results from samples collected in the defined manner.
The time a sample is stored before assessment may also
have an impact on the test result. Most often samples are
analyzed relatively quickly (i.e., within a few hours or at
least the same day), but there may be an occasion when a
sample cannot be assessed until the following day or two,
or, depending on the assay, samples may be stored until
sufficient numbers are collected and then run as a batch.
Although longer than generally considered acceptable, a
recent paper ( Furlanello et al ., 2006 ) found that samples
could be kept at room temperature for up to 48 h without
any significant change from immediate analysis, whereas
storage at either room temperature or refrigeration was not
recommended beyond 48 to 72 h. The typical recommendation
is to perform assays within 4 h of sampling, after
centrifuging and removal of plasma or to freeze the sample
until analysis can be performed. Plasma samples can
be safely stored at 70 ° C for up to 6 months with minimal
change in results except for the aPTT ( Bateman et al .,
1999a ; Iazbik et al ., 2001 ).
B. Testing of Hemostasis
1. Assessment of Platelet Quantity
a. Platelet Concentration and Volume
Platelets can be enumerated from either an EDTA or
sodium citrate-anticoagulated blood sample. Heparinized
blood is not suitable as marked platelet clumping often
occurs. Assessment is best performed within 4 to 5 h, but
the count may remain relatively stable for up to 24 to 48 h
if the sample is refrigerated ( Stockham and Scott, 2002 ).
Manual or automated methods can be used for platelet
quantity assessment. Manual methods include estimation
of platelet quantity from a stained blood smear and hemacytometer
chamber counting. Most automated hematology
analyzers using impedance or light scattering technology
can also enumerate platelets.
Platelet clumping must be avoided for any platelet
quantification method, or underestimation of the platelet
count will occur. This can happen in any species with traumatic
sampling and tends to occur most frequently in cats
and cattle. Therefore, it is critical that a peripheral blood
smear be examined for the presence of clumps; this also
provides an opportunity to ensure appropriate cell morphology
and to perform an estimate of platelet numbers as
a cross-check of an automated determination.
The mean platelet volume (MPV) is frequently determined
by automated analyzers and reflects the average
volume of all cells that are enumerated as platelets. For
an accurate determination, platelet clumping needs to be
avoided, and the cells should be unactivated and circular in
shape without pseudopod formation. Variables such as time
to analysis, storage temperature, and anticoagulant used
can all impact MPV assessment. Generally, there tends to
be an inverse relationship between platelet concentration
and MPV; the value is higher when platelet concentration
is low, and it is lower when numerous platelets are present.
This has been documented in healthy cats ( Weiser and
Kociba, 1984 .) An increase in MPV is a favorable finding
in an animal with thrombocytopenia, as it is usually
a reflection of active thrombopoiesis. Excessively large
platelets are often observed in Cavalier King Charles spaniels,
but the corresponding platelet concentration also tends
to be lower, making overall platelet mass similar to other
breeds ( Smedile et al ., 1997 ).
b. Clot Retraction
Retraction of a blood clot depends on platelets, so defects
in platelet quantity or quality will prolong the time required
for whole blood to clot in a tube. Measurements of clot
retraction are available, but this test is limited by subjectivity
and is not often performed.
c. Buccal Mucosal Bleeding Time
Although typically assessed as an indicator of platelet
function, a sufficient quantity of platelets also needs to be
present to form a platelet plug in a standardized mucosal
incision. Once thrombocytopenia is present, the BMBT
may be prolonged simply from lack of platelets. See
Section III.B.2.a for further details.