This document discusses pre-analytical variables that can affect coagulation testing. It emphasizes the importance of proper sample collection, including using the correct anticoagulant (sodium citrate), collection technique (slow draw with mixing), and storage requirements. Errors in collection such as short draws, improper mixing or storage, excessive mixing, or hemolysis can falsely affect test results. Proper centrifugation is also needed to obtain platelet-poor plasma for certain tests.

1. Preanaly(cal
Variables
in
Coagula(on
Tes(ng
• Pre-­‐analy(cal
variables
in
coagula(on
tes(ng
1. Sample
Collec(on
2. Site
Selec(on
3. Storage
Requirements
4. Transporta(on
of
Specimen
5. Venipuncture
vs
line
draws
6. Tube
sequence
7. Unintended
drug
effects
1

2. • Ideally
Sample
Collec(on
the
results
of
blood
tests
should
accurately
reflect
the
values
in
vivo
• Many
misleading
results
in
blood
coagula(on
arise
not
from
errors
in
tes(ng
but
from
carelessness
in
the
preanaly)cal
phase.
– Labeling
– Improper
collec(on
– Fas(ng/non-­‐fas(ng
– Sample
collec(on
• Slow
draw,
vigorous
mixing,
an(coagulant
• When
blood
is
withdrawn
from
a
vessel,
changes
begin
to
take
place
in
the
components
of
blood
coagula(on
• Some
occur
almost
immediately,
such
as
platelet
ac(va(on
and
the
ini(a(on
of
the
cloTng
mechanism
dependent
on
surface
contact

3. Sample
Collec(on
• An(coagulant
of
choice
– 3.8%
or
3.2%
Sodium
Citrate
• 3.2
%
Preferred
as
the
standard
measure
due
to
stability
and
closeness
to
the
plasma
osmolality
– An(coagulant/blood
ra(o
is
cri(cal
(1:9)
• Exact
amount
of
blood
must
be
drawn.
No
short
draws
are
acceptable,
this
will
falsely
increase
results
due
to
presence
of
too
much
an(coagulant
• CLSI
guideline
is
+/-­‐
10
%
of
fill
line
– Purpose
of
the
an(coagulant
is
to
bind
or
chelate
calcium
to
prevent
cloTng
of
specimen

4. • Other
Sample
Collec(on
an(coagulants,
including
oxalate,
heparin,
and
EDTA,
are
unacceptable.
• The
labile
factors
(factors
V
and
VIII)
are
unstable
in
oxalate,
whereas
heparin
and
EDTA
directly
inhibit
the
coagula(on
process
and
interfere
with
end-­‐point
determina(ons
• Addi(onal
benefits
of
trisodium
citrate
are
that
the
calcium
ion
is
neutralized
more
rapidly
in
citrate,
and
APTT
tests
are
more
sensi(ve
to
the
presence
of
heparin

5. Storage
Requirements
— Prothrombin
Time:
PT
◦ Uncentrifuged
or
centrifuged
with
plasma
remaining
on
top
of
cells
in
unopened
tube
kept
at
2-­‐4
oC
or
18-­‐24
oC
must
be
tested
within
24
hours
of
collec(on
— Ac(vated
Par(al
Thrombin
Time:
APTT
◦ Uncentrifuged
or
centrifuged
with
plasma
remaining
on
top
of
cells
in
unopened
tube
kept
at
2-­‐4
oC
or
18-­‐24
oC
must
be
tested
within
4
hours
of
collec(on
◦ Other
assays
– Fibrinogen,
Thrombin
Time,
Factor
Assays
– Centrifuged
with
plasma
remaining
on
top
of
cells
in
unopened
tube
kept
at
2-­‐4
oC
or
18-­‐24
oC
must
be
tested
within
4
hours
of
collec(on

6. • Other
Storage
Requirements
general
notes
– Perform
coagula(on
tests
ASAP
• Specimen
may
deteriorate
rapidly
(especially
factors
V
and
VIII)
– If
the
tes(ng
is
not
completed
within
specified
(mes,
plasma
should
be
removed
from
the
cells
and
placed
in
a
frost
free
freezer
• -­‐
20
oC
for
two
weeks
• -­‐70
oC
for
six
months

7. • Platelet
Centrifuga(on
–Poor
plasma
(PPP)
– <10
x
10
9
/L
– Specimen
has
been
centrifuged
for
15
minutes
@
2500
x
g
– Why
is
PPP
essen(al?
1. Contains
platelet
factor
4(heparin
neutralizer)
2. Contains
phospholipid
(affects
lupus
an(coagulant
and
factor
assay
tes(ng)
3. Contains
proteases
(affect
tes(ng
for
vWF)

8. Common
Collec(on
Problems
Error
Consequence
Comment
Drawing
coagula(on
tube
PRIOR
to
other
an(coagulant
tubes
PT/PTT
falsely
affected
Contamina(on
Probing
the
vein
PT/PTT
falsely
shortened
Tissue
thromboplas(n
is
released
ac(va(ng
coagula(on
Heparin
contamina(on
from
line
draw
PTT
falsely
prolonged
Heparin
keeps
the
blood
from
cloTng
Lipemia
Test
may
not
work
Photo-­‐op(cal
methods
affected

9. Common
Collec(on
Problems
Error
Consequence
Comment
Short
draw
<2.7
mL
PT/PTT
falsely
prolonged
An(coagulant
to
blood
ra(o
exceeds
1:9
Failure
to
mix
specimen
aher
collec(on
PT/PTT
falsely
prolonged
Blood
clots
form
when
an(coagulant
&
blood
do
not
mix
Excess
vigorous
mixing
PT/PTT
falsely
shortened
Hemolysis
and
platelet
ac(va(on
cause
start
of
cascade
Hemolysis
PT/PTT
falsely
shortened
Reject
specimen
Improper
storage:
wrong
temperature
or
held
too
long
PT/PTT
falsely
prolonged
Must
follow
storage
requirements
Chilling
in
refrigerator
or
placing
on
ice
PT
falsely
shortened
Chilling
to
4
oC
ac(vates
factor
VII.
Inadequate
centrifuga(on
PTT
loses
sensi(vity
for
lupus
an(coagulants
and
heparin.
Factor
assays
inaccurate
Desire
platelet
poor
plasma
Prolonged
tourniquet
applica(on
Falsely
elevates
vWF,
factor
VIII
Tourniquet
causes
venous
stasis,

10. Analy(cal
variables
• Instrumenta(on
– Op(cal
versus
mechanical
clot
detec(on
• Affected
by
interferences
– lipemia,
bilirubinemia,
etc.
• Reagents
– PT
source
(recombinant,
brain
extract,
etc)
– aPTT
• ac(vator
• phospholipid
source
and
concentra(on
• designed
for
heparin,
factor
VIII
&
IX,
possible
LA