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The Importance of Pre-Analytical

in Hemostasis Testing
Dr. Yetti Hernaningsih, dr., Sp.PK(K)
Clinical Pathology Department, Faculty of
Medicine, Universitas Airlangga

17th ASEAN Congress of Clinical Laboratory Medicine

April 25th, 2018

Types and potential outcomes of laboratory

2 2
Lippi G & Favaloro EJ. Quality in laboratory hemostasis and thrombosis. 2 ed., 2013
Types and rates of error in the three stages of the laboratory
testing process

3 3
Plebani M. Clin Chem Lab Med 2006;44(6):750-759
The laboratory cycle

Olson JD. Quality in laboratory hemostasis and thrombosis. 2nd ed., 2013
Salvagno GL, Lippi G, Bassi A, Poli G, Guidi GC.
(J Eval Clin Pract 2008;14:351–353)

Recent data have determined that preanalytical

problems can be identified in up to 5.5% of
coagulation specimens,

the more frequent being samples not received in the

laboratory after physician’s order (49%), hemolysis
(20%), sample clotting (14%), and inappropriate
blood-to-anticoagulant ratio (14%)

5 5
Leading causes of preanalytical errors

Lippi G & Favaloro EJ. Quality in laboratory hemostasis and thrombosis. 2nd ed., 2013
Pre-Analytical Factors

Sample Collection
• Tubes should be made of non-activating surfaces
(e.g. siliconized glass or polypropylene)
• A winged-device, syringe, and vacutainer system
can be used to draw blood samples for coagulation
• Syringe  small volume (<20 ml) is recommended
and blood should be added immediately into the
citrated tube and mix well
• Winged needle non-activating and non-additive
discard tube is needed to fill the dead space
Sample Collection
•Clean venipuncture with minimal stasis.
Do not prolonged tourniquet application. The
tourniquet may be applied for up to 1 minute.
Tourniquet causes venous stasis, which falsely
elevates the concentration of large molecules

•Prolonged use of a tourniquet or considerable

manipulation of the vein by needle, may develop a
clot in vitro, which the blood is slow to fill the
collection container

About citrate?
•Most samples referred for coagulation testing must
be drawn into citrate-based anticoagulant tubes
(generally 105-109 mM or 129 mM sodium citrate,
also referred to as 3.2% or 3.8%), respectively

•CLSI guidelines favor the use of the lower citrate

concentration, except for specific applications.

About citrate?
• Specimens collected in 129 mM (3.8%) buffered sodium
citrate may overestimate the PT and APTT and
underestimate fibrinogen if the normal range is based on
3.2% citrated samples

• Conversely, samples collected into 129 mM (3.8%) citrate

may provide a more stable sample for assessing antiplatelet
(eg, aspirin) therapy response using the PFA-100

About citrate?
• Ensure that citrate tubes are completely filled and
comply with a 9:1 blood to anticoagulant ratio. A
minimum 90% is recommended unless otherwise
indicated by manufacturer or internal validation

• The APTT test is the most sensitive to variations in

the final citrate concentration
Variables Lengthens (%) Underfilling (%)
APTT 3 10
10 20
PPT 5 20
15 30 12
Anticoagulant/Blood Ratio
•The presence of significant anemia has not been
shown to influence test result
•Adjust the volume of the citrate anticoagulant
when collecting samples from patients with
hematocrit of >0.55 L/L (55%)
•As recommended by the NCCLS,

Anticoagulant volume(ml) =
blood volume (ml) x [100 – hematocrit(%)]
[595 – hematocrit (%)]

Needle gauge
•Use 19 – 21 gauge needle. Avoid smaller (> 25
gauge) or bigger needles (< 16 gauge)

•>25 gauge:
As the slower rate of blood flow through the small
bore may induce clotting or activation of the

•Very large bore needle (<16 gauge):

May induce hemolysis of the sample due to
turbulence of flow through the needle

The way to mix
• Samples should be mixed thoroughly (but gently) by 3 to 6
end-over-end tube inversions to ensure adequate mixing of
test sample with anticoagulant and to prevent sample

• Conversely, too vigorous mixing (eg, by shaking of tubes)

might lead to in vitro hemolysis or spurious factor activation
resulting in false shortening of test clotting times and even
possible false elevation of clotting factor activity (eg, FVII).

How the sample ordering?
• If multiple evacuated tubes will be collected, the
coagulation tube should be the first tube drawn
• If only one evacuated tube will be collected, the
blue stopper tube can be the only tube drawn and
a discard tube is not necessary
• If using an evacuated tube collection system, the
tube for hemostasis testing should not be collected
following collection of an additive tube
About drawing sample?
• The old dogma that the first collection tube should be
discarded may not generally be required, as evidence for
differential effects on coagulation assays are lacking

• Nevertheless, a discard tube is needed if the sample is

drawn using a winged collection with variable tubing
length so air in the tubing is not introduced into blood
collection tubes leading to under-filling

About drawing sample?

• Tubes should be adequately filled (to the mark noted on the

tube if provided) or to no less than 90% of this total volume.
Under-filling may cause significant sample dilution and may
also provide falsely prolonged clotting times due to the excess
calcium-binding citrate present
• This effect depends on the citrate concentration, the tube
size, and the test performed being more pronounced with
3.8% citrate tubes and small volume (pediatric) collection
The effect of tourniquet
• Application of a tourniquet for 3 minutes vs 1
Value (%) Effect Variables

3.1 shortening PPT

10.1 increase fibrinogen

13.4 increase D-dimer

10.6 increase FVII activity

10.2 increase FVIII activity

Influence of stress
• In situation of stress (a child is very tearful):
Factor VIII
may increase, as these are acute phase reactan

•This may cause spurious shortening of the APTT or

bring low levels of these factors into the normal
range resulting in a missed diagnosis, for example
of hemophilia or von Willebrand disease

Pre-Analytical Factors

• Sample should be preferably delivered non
refrigerated, at room temperature (18 – 25oC) as
short a time as possible

• Whenever delay in transport is expected , local

centrifugation and separation of plasma followed by
freezing and frozen transport of the plasma should
be considered

• The effect of freez—thawing of plasma might

determine the loss of some labile factor (FV, FVIII)
• Transportation of whole blood sample in ice should
NOT be done due to possible cold activation of
platelet and FVII, loss of FVIII and vWF

• When transporting via a pneumatic system, protect

tubes from excessive agitation or vibration

• Ideally, testing for routine coagulation tests like the
PT and the APTT should be accomplished within 4
hours of collection, although allowable tolerances
may be greater than this

• However, APTT testing for unfractionated heparin

monitoring should preferably be processed within 1
hour due to the potential for heparin neutralization
by platelet releasates.

• Extremes of temperature (ie, both refrigerated or

high) should be avoided

• Delays in transport may affect in particular the

labile factors (FV, FVIII), leading to prolonged
clotting times and in vitro loss of factor activity

Pre-Analytical Factors

• To maintain the highest level of specimen integrity, citrated
blood samples should ideally be processed within the first
hour after collection

• Check for the presence of clot either by gentle inversion or by

using wooden sticks

• Centrifuge the citrated whole blood samples to produce a

platelet poor plasma with platelet count of <10,000/ul (<10 x
109/L). Residual platelet count can be verified using an
automated hematology cell counter

• The use of filters to obtain platelet poor plasma is NOT


• Coagulation tests when testing is performed soon
after centrifugation. Centrifugation should ideally be
at 1500 g for a minimum of 10-15 minutes, with
rotor swing out bucket

• Double centrifugation can be performed to ensure

the plasma is platelet-poor

• Using centrifugal forces greater than 1500 g are not

recommended as this may induce platelet activation
and lysis of RBCs

• The use of centrifuge breaks should also be
avoided to avoid remixing of test samples, since
there is a potential for hemolysis and platelet
contamination, which may subsequently affect
most hemostasis assays

• Samples that cannot be tested within 4 hours

should be centrifuged and the plasma aliquot

Pre-Analytical Factors

Variables Stability (hours) Temperature (0C) Sample can be Additional
stored information
PT/INR 24 room WB or in capped tube
centrifuged with and either
plasma on top of uncentrifuged or
the cellular centrifuged
APTT do not 4 room
contain UFH
Sample 1 or Release PF4 from
containing UFH 4 (after platelet in vitro
centrifuge within will neutralize of
1 hour) heparin
Vit K dependent 24 room

Protein S activity 8

Fibrinogen, 7 days
protein C,
activity 31
• If samples for coagulation testing are stored frozen,
they should not be maintained in freezer that has
automatic defrost cycles. The use of frost-free
freezers for patient samples is acceptable

• Storage as whole blood at cold temperatures (2-8o C)

is NOT recommended. Refrigeration of
separated/aliquoted plasma for up to 4 hours is
acceptable for APTT and other coagulation assays but
NOT for PT assay

• If testing cannot be completed as described above,

platelet-poor plasma (platelet-free plasma for LA
testing) should be removed and frozen at ≤ -20o C for
two weeks or at ≤ -70o C for longer storage
• Prior to testing, previously frozen plasma samples
should be thawed in 370C water bath for
approximately 5 minutes or until completely
• The vWF activity assay tends to decrease to a
greater extent than the antigen assay in response
to cold whole blood storage

Causes for Sample Rejection

1. Clotted Samples
2. Samples in wrong anticoagulant
3. Inappropriate blood fill volume
4. Mislabeled or unlabeled samples

Take home messages
• Pre-analytical issues in hemostasis testing are an important cause
of diagnostic error and can lead to significant adverse clinical

• Sample collection must be proper in vein puncture, needle bore,

blood: anticoagulant ratio, tourniquet application

• Transportation should be in room temperature, as short time as


• Samples were processed for centrifugation in 1500 g not less than

15 minutes, at room temperature

• Storage stability depends on variables and temperature. Plasma

frozen at ≤ -20o C for two weeks or at ≤ -70o C for longer storage
• Olson JD. General quality planning in the hemostasis laboratory. In: Kitchen S, Olson JD,
Preston FE (eds.). Quality in laboratory hemostasis and thrombosis. 2nd ed. Wiley-
Blackwell. New Delhi, India, 2013
• John D. Olson1,2Lippi G & Favaloro EJ. Causes of errors in medical laboratories. In: Kitchen
S, Olson JD, Preston FE (eds.). Quality in laboratory hemostasis and thrombosis. 2nd ed.
Wiley-Blackwell. New Delhi, India, 2013
• Funk D (Adcock). Sample integrity and preanalytical variables. In: Kitchen S, Olson JD,
Preston FE (eds.). Quality in laboratory hemostasis and thrombosis. 2nd ed. Wiley-
Blackwell. New Delhi, India, 2013
• Favaloro EJ, Funk DM (Adcock), Lippi G. Pre-analytical variables in coagulation testing
associated with diagnostic errors in hemostasis. Labmedicine. Februaru 2012;43(2)
• Plebani M. Errors in clinical laboratories or errors in laboratory medicine? Clin Chem Lab
Med 2006;44(6):750-759
• Salvagno GL, Lippi G, Bassi A, Poli G, Guidi GC. Prevalence and type of pre-analytical
problems for inpatients samples in coagulation laboratory. J Eval Clin Pract