CE Update

Submitted 6.3.11 | Revision Received 7.4.11 | Accepted 7.25.11

Pre-analytical Variables in Coagulation Testing

Associated With Diagnostic Errors in Hemostasis
Emmanuel J. Favaloro, PhD, MAIMS, FFSc (RCPA),1 Dorothy M. (Adcock) Funk, MD,2 Giuseppe Lippi, MD3
(1Department of Haematology, ICPMR, Westmead Hospital, Westmead, NSW, Australia, 2Esoterix Inc., Englewood, CO,
3Clinical Chemistry and Hematology Laboratory, Academic Hospital of Parma, Parma, Italy)
DOI: 10.1309/LM749BQETKYPYPVM

Abstract

The use of modern laboratory instrumentation

with high levels of test reliability and
appropriate quality assurance measures
will lead to very few analytical errors within
hemostasis testing. Nevertheless, incorrect
or inappropriate test results are still reported,
often due to events outside the control of the
laboratories performing the tests. This is due
primarily to pre-analytical events associated

with sample collection and processing, as

well as post-analytical events related to the
reporting and interpretation of test results.
This review focuses on the pre-analytical
phase, highlighting contributory elements and
providing suggestions on how problems can
be minimized or prevented, thereby improving
the likelihood that reported test results
actually represent the true clinical status of
the patient rather than that of an inappropriate

After reviewing this article, readers should be able to describe preanalytical variables affecting various coagulation tests and discuss how
these problems can be avoided in order to ensure the accurate reporting
of patient results.

Modern instrumentation is generally capable of providing

highly accurate test results. Utilized with appropriate internal
quality control and external quality assurance measures, analytical errors within hemostasis testing are generally minimal.
Nevertheless, incorrect or inappropriate test results will on occasion be reported to clinicians, most often due to circumstances
beyond the control of the laboratories performing these tests.
Overall, a significant impact on patient care arising from

sample. This review should be of value to both

laboratory personnel and clinicians because
an appreciation of these issues will enable the
optimal clinical management of patients.
Keywords: pre-analytical variables, extraanalytical variables, diagnostic errors,
hemostasis, coagulation

Coagulation exam 51202 questions and corresponding answer form

are located after this CE Update on page 59.

diagnostic errors has been estimated to arise in around 9% to

15% of errors, while the likelihood of inappropriate care has
been described in 2% to 7% of such cases.1 Many of these errors will originate due to the inappropriate collection, handling,
or processing of samples referred for testing and sometimes
because testing has been initiated on the wrong patient or at the
wrong timepoint. In these instances, test results will accurately
reflect the status of the test sample, but conversely they will not
accurately reflect the clinical status of the patient being investigated. These issues are referred to as pre-analytical variables.

Corresponding Author
Emmanuel J. Favaloro, PhD, MAIMS, FFSc (RCPA)
emmanuel.favaloro@swahs.health.nsw.gov.au

Abbreviations
LA, lupus anticoagulant; APS, antiphospholipid (antibody) syndrome; VWD, von Willebrand disease; VWF, von Willebrand factor;
PT, prothrombin time; INR, international normalized ratio; APTT,
activated partial thromboplastin time; TT, thrombin time; DIC, disseminated intravascular coagulation; F, factors; CLSI, Clinical and
Laboratory Standards Institute; ISI, international sensitivity index;
EDTA, ethylenediaminetetraacetic acid; aPL, antiphospholipid; aCL,
anticardiolipin; aB2GPI, anti-beta-2-glycoprotein I; NRR, normal
reference range; MNPT, mean normal PT; APCR, activated protein
C resistance

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Consequences of Pre-analytical Issues

It is inherently challenging to establish a direct relationship between spurious test results and patient outcomes since
laboratory errors do not always and necessarily translate into
serious harm for the patient as may occur in cases of mishandled surgery or inappropriate drug therapy. Nevertheless,
the consequences of incorrect test results might still be clinically
meaningful and lead to several unwanted clinical outcomes
or adverse economical consequences, and place laboratories at
risk.2 The seriousness of the potential consequences relates to
the test being performed, the extent of the difference between
the reported result and the true result, as well as the ability
of laboratory personnel and clinicians to recognize the issues.3,4
Consequences may be particularly serious for errors related to specialized hemostasis tests because these assays are often considered
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diagnostic. Thus, a patient might be diagnosed with a particular disorder, when he/she does not have it (ie, false-positive
test result obtained) or a patient with a true disorder might be
missed (ie, false-negative test result obtained). Both situations
will cause adverse consequences for both patients and the health
care system. As examples, 1) a false-negative antiphospholipid
(aPL) antibody or lupus anticoagulant (LA) test result in a patient with the aPL antibody syndrome (APS) may lead to lack
of appropriate treatment with anticoagulant therapy to prevent
a future thrombosis; and 2) a false diagnosis of von Willebrand
disease (VWD) may lead to inappropriate treatment with factor concentrates or to a lifelong label of a congenital disorder
affecting quality of life. Serious consequences may also result
due to errors in routine coagulation testing. For example, 1) a
falsely prolonged screening coagulation test might influence a
clinical decision to undertake further costly and time consuming (eg, specific diagnostic) investigations, unnecessarily delay
invasive procedures, and raise unnecessary anxiety in the patient
being investigated; 2) a false-normal screening test result might
prevent further evaluation of factor assays, thus incorrectly discounting hemophilia and possibly placing a patient at an unjustified risk of bleeding with invasive procedures (ie, surgery, dental extraction, biopsies); and 3) a false low or high coagulation
test time in a patient being monitored for anticoagulant therapy
may lead to subsequent incorrect dosing of anticoagulant
therapy with a risk of thrombosis or bleeding depending on the
direction of the error.

Overview of Hemostasis, Laboratory Testing,

and Pre-analytical Issues
Hemostasis is commonly thought of in terms of coagulation pathways or as a surrogate of coagulation. However,
hemostasis is far more complex than coagulation, which
in essence reflects clot formation, as it incorporates several
components unrelated to the coagulation process. The components of hemostasis can be considered within the context

of Virchows Triad, or as a composite of primary hemostasis

(platelet plug formation, von Willebrand factor [VWF]/platelets/subendothelial components), secondary hemostasis (fibrin
clot formation, procoagulant clotting factors, and the natural
anticoagulants) and fibrinolytic pathways.5,6
The modern hemostasis laboratory performs a large number of distinct tests, often using a variety of methodologies
(Table 1). All hemostasis laboratories perform routine coagulation tests comprising the prothrombin time (PT)/international
normalized ratio (INR) and the activated partial thromboplastin time (APTT), sometimes supplemented by specific fibrinogen assays, and occasionally thrombin time (TT) assays. Most
routine test laboratories also perform D-dimer assays. These
tests are variably performed to investigate hemostasis in patients
suspected of having a potential dysfunction in the secondary
hemostasis pathway, either congenital (eg, hemophilia) or acquired (eg, disseminated intravascular coagulation [DIC]).5,710 This is because PT/INR, APTT, and TT are sensitive to
deficiencies or defects in various procoagulant factors. Thus,
the PT/INR is sensitive to factors (F) I, II, VII, V, and X, and
the APTT to F I, II, V, VIII, IX, X, XI, and XII. The single or
compound deficiency or absence of most of these factors will
lead to an increased tendency to bleeding and will occasionally define hemophilia (eg, deficiency in FVIII, hemophilia
A; deficiency in FIX, hemophilia B). In contrast, an excess of
some procoagulant factors (eg, FVIII, FIX, and FXI) may lead
to thrombophilia.11 Although the PT/INR and APTT are not
highly sensitive to elevations in procoagulant factors, a short
APTT is sometimes indicative of this, and hence may reflect
an increased risk of thrombosis.8,12,13 The PT/INR is also used
to monitor vitamin K antagonists such as warfarin,7,14 and the
APTT to monitor unfractionated heparin.8 Indeed, these tests
are more often performed for monitoring anticoagulant therapy
than for assessing secondary hemostasis. Other tests performed
by hemostasis laboratories in general comprise a battery of specific diagnostic assays (largely listed in Table 1).
The large number of distinct tests involving a variety of
methodologies may result in significant problems when unsuitable

Table 1_Summary of Hemostasis Tests and Sample Requirements

Comprise

Usually Performed Via

Sample Type

A. Routine coagulation tests

Citrate anticoagulated plasma post single centrifugation
PT/INR, APTT, TT, fibrinogen
Clot-based tests, automated instrument, primary
collection tube (sometimes separated plasma)
D-Dimer (D-D)
ELISA or ELFA or agglutination (primary or
secondary tube)
B. Specialized Hemostasis Tests

Factor assays (ie, II, V, VII, VIII, IX, X, XI, XII), factor Clot-based tests, automated instrument
Separated citrate anticoagulated plasma, post single
inhibitor assessments, protein S, protein C
centrifugation (usually post freezing)
VWF tests
ELISA, immunoassay, or agglutination
Protein C, protein S, antithrombin
ELISA, immunoassay, clot based, or chromogenic assays
Heparin (anti-Xa) assay
Chromogenic assays
Separated citrate anticoagulated plasma, post single (or
preferably double) centrifugation (usually post freezing)
APCR
Clot-based tests, automated instrument
LA
Clot-based tests, automated instrument
Separated citrate anticoagulated plasma, post double
centrifugation (usually post freezing)
Solid phase aPL tests including aCL
ELISA or immunoflourescent assay
Separated serum preferred; separated citrate anticoagulated
and ab2GPI plasma post single centrifugation sometimes acceptable.
Usually post freezing.
Platelet function tests
Specialized instrumentation
Citrate anticoagulated whole blood or special processing
required.
Genetic thrombophilia tests
Specialized instrumentation
EDTA or citrate anticoagulated whole blood or special
processing required

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samples are submitted for testing. Although guidelines are available for how to manage and when to reject unsuitable specimens,15,16 it is not always clear when unsuitable samples have
been received. Pre-analytical problems can arise at any point
prior to sample testing, including (but not limited to) sample
collection, handling, transportation, processing, and storage.
Whereas analytical errors are largely avoided or intercepted by
using appropriate test methodologies and by incorporation of
appropriate control measures, pre-analytical issues present a
more difficult scenario for laboratories as they are often outside
the control of the laboratory performing the tests, and often
the laboratory is unaware that the adverse pre-analytical event
has occurred. Thus, the laboratory may issue a test result with
the best of intentions as reflecting an accurate patient-related
result, but this may not be the case. Clinicians would be even
less aware of the issue of pre-analytical variables than the laboratory, and would base their clinical actions on the test result
received (as reflecting a true and correct result). For this reason,
guidelines for specimen collection and handling must be strictly
followed and deviations avoided unless their impact, or lack
thereof, on coagulation testing is known.

Appropriate Sample Collection, Processing,

and Storage
These are critical to the attainment of appropriate test
results but are often neglected, overlooked, or poorly applied.
Positive Patient and Sample Identification
The importance of proper patient identification cannot
be overemphasized. In an outpatient setting, the principle of
double identifiers should be used, specifically; the conscious
patients should be asked to identify themselves and also produce some form of identification. Within the hospital, positive
patient identification should follow institutional rules and will
typically entail electronic or bar-code methods to reduce the
risk of patient misidentification. Other guidelines to ensure
positive patient and sample identification include those related
to printing tube labels; matching patient identification with the
patients full name, and an additional identifier, such as date of
birth or medical record number; and identification of collection
date and time.16,17
Sample Collection
All tests have specific collection requirements. Sample collection issues might arise because of inexperience or time pressures when collectors are faced with a busy clinic and multiple
collection requirements. 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). The current Clinical
and Laboratory Standards Institute (CLSI) guidelines16 favor the
use of the lower citrate concentration, except for specific applications.4,16 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.18 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. Sometimes
there is no apparent difference in relation to testing (eg, anti-Xa
[heparin] testing) based on citrate concentration. The major
recommendation therefore is that laboratories standardize to 1
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citrate concentration and develop normal ranges appropriate

for that concentration. This standardization should include all
components of the assay (eg, including determination of patient
PT, mean normal PT [NMPT], and international sensitivity
index [ISI] for the INR).
Coagulation samples should preferably be collected before
other test samples are drawn, if these contain stronger anticoagulant agents such as ethylenediaminetetraacetic acid (EDTA)
(for a complete blood count), lithium-heparin (for clinical
chemistry testing), as well as clot activators (ie, thrombin), since
these materials may contaminate a subsequent coagulation test
sample. A specific sequence of tube collections (so-called order
of draw) is provided by the CLSI.19 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.20 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.16,21 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
tubes.22,23 Sample dilution will also lead to under-estimation of
quantitative test results (eg, clotting factor levels).
Blood should never be transferred from 1 collection tube
to another in an effort to provide the required complete fill volume. This is true even if 2 sodium citrate tubes are combined,
as this may lead to doubling up of anticoagulant citrate levels
and further dilution of the plasma sample. The introduction
of stronger anticoagulants (eg, EDTA or lithium-heparin) or
clot activators (eg, thrombin) must also be avoided, and this
will occur if blood from non-citrate collection tubes is added to
citrate tubes.
Samples should be mixed thoroughly (but gently) by 3 to
6 end-over-end tube inversions to ensure adequate mixing of
test sample with anticoagulant19 and to prevent sample clotting. Insufficient mixing may have a greater effect on specialized hemostasis assays performed some time after collection
than on basic coagulation tests performed soon after collection.
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).
Some tests referred to hemostasis laboratories may require
sample matrices other than sodium citrate anticoagulated
plasma, leading to additional scope for pre-analytical error. For
example, while the test sample for LA testing must be citrate
anticoagulated plasma, the preferred test sample for solid phase
testing of aPL antibodies, such as anticardiolipin (aCL) antibody and anti-beta-2-glycoprotein I (aB2GPI), in serum. As all
of these different tests might be requested for a patient being
investigated for APS, problems may arise should the laboratory
inadvertently perform LA testing using the serum sample.
Other issues arising from blood collections include difficult
collections, or those derived from central venous lines, leading
to partially clotted, hemolyzed, or activated samples, or samples
diluted by saline or contaminated with heparin. Collections
from venous lines should include a process for flushing and/
or discarding the initial collection volumes. Size and type of
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needle used may also influence results and too large (less than
16 gauge) or too small a needle bore (greater than 25 gauge)
should be avoided, and heparinized needles (sometimes used for
blood gas collection) not used.24
Sample Transport
Samples should be transported as per current guidelines,16
non-refrigerated at ambient temperature (15-22C) in as short
a time as possible. 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.25,26 However, APTT testing for unfractionated heparin monitoring should preferably be processed within
1 hour due to the potential for heparin neutralization by platelet releasates.16,27,28 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.29 In such cases,
local centrifugation and separation of plasma followed by freezing and frozen transport of the plasma should be considered.
Sample Processing and Storage
This should also in general proceed as per current CLSI
guidelines,16 noting limitations according to which test is being
performed. Most coagulation-based tests, including PT, APTT,
and clotting factor assays, are performed on plasma derived
from once-centrifuged samples (Table 1). Some samples, such
as those for LA testing, should be double centrifuged to ensure
platelet-free preparations prior to freezing.30 Centrifugation
should essentially be at an ambient temperature (15C-22C),
but this is sometimes difficult to control. Non-refrigerated
centrifuges are adequate, providing they do not overheat.
Alternatively, refrigerated centrifuges may be used but should
be set to maintain ambient temperatures, rather than low temperatures, which can lead to platelet activation and adverse effects. Nevertheless, refrigerated centrifugation does not appear
to affect routine coagulation tests when testing is performed
soon after centrifugation. Centrifugation should ideally be at
1500 g for a minimum of 10-15 minutes.16 Shorter centrifuge times might be acceptable for routine coagulation tests
performed immediately post-centrifugation when there are no
subsequent test requirements (ie, plasma not to be frozen or
processed for additional assays). 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 or monitored to avoid remixing of test
samples, particularly if plasma is to be frozen, since there is a
potential for hemolysis and platelet contamination, which may
subsequently affect most hemostasis assays.
Testing generally proceeds using the once-centrifuged test
sample in the primary collection tube or on the once-centrifuged
separated plasma sample before or after freezing (Table 1). For
some assays, samples should be double centrifuged (doublespun), which entails the re-centrifugation of the separated
plasma, and re-separation of this double-spun plasma from any
residual cellular pellet prior to freezing. Since all plasma-based
hemostasis tests can safely be performed on double-spun material, it might be prudent to institute this process as a general laboratory policy for any plasma that will be frozen prior to testing.
Use of filtered plasma is no longer recommended for LA testing,
since this might produce spurious test results with some assays,
as highlighted later.30 Lastly, some tests require additional special
differential processing (eg, platelet function testing).31
4

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The stability of coagulation samples varies depending

on a number of variables such as the blood collection system,
whether the samples are stored as whole blood or centrifuged,
the temperature at which samples are maintained during storage, the reagent/instrument system used for analysis, and the
test parameter to be analyzed. For example, whole blood stored
up to 24-48 hours prior to centrifugation has been reported
as acceptable for many hemostasis tests (although not for FV,
FVIII, and protein S),25,32 but other studies have reported significant changes in some test results over such time periods.4
Moreover, storage of refrigerated whole blood is now actively
discouraged and leads to activation events affecting FVII,
FVIII, VWF, and possibly others.16,33
In general, to afford the greatest sample integrity, samples
should be processed as quickly as possible (ideally within 1 hour
of collection) and testing performed within 4 hours of procurement (or else be processed by centrifugation and plasma frozen).
During this short-term storage, whole blood samples should be
kept capped and maintained at room temperature. If testing is
not to be performed within about 4 hours for the APTT and 24
hours for the PT, the plasma should be separated from the cellular fraction of the once or twice-centrifuged sample, without disturbing the cell pellet. For many tests of hemostasis, the separated
plasma can be safely frozen for later testing. Separated plasma can
generally be maintained at room temperature or refrigerated for
a few hours without an adverse effect on coagulation. Otherwise,
separated plasma samples should be frozen. Frost-free freezers
with automatic defrost cycles are generally unsuitable, since they
cycle freeze-thaw events to maintain the frost-free environment,
and this adversely affects subsequent coagulation tests. However,
the use of frost-free freezers for patient samples is acceptable
where freezers are monitored by a continuous-monitoring temperature recording device, or a minimum-maximum thermometer, enabling the laboratory to show the acceptable temperature
range is never exceeded. When storing plasma, the lower the
freezer temperature, the longer the specimens can be maintained
for future testing. As a general rule of thumb, testing for samples
maintained at around -20C should be finalized within 2-4 weeks
of storage, whereas testing for samples maintained at around
-80C can occur several months and sometimes years later (useful
for research studies and prospective trials).34
Controlled Thawing of Frozen Plasma Samples
Previously frozen samples should be rapidly thawed in a
37C water bath for 5-10 minutes or until completely thawed.4,16
Close monitoring during this time is necessary to avoid inadequate or excessive incubation at 37C. Sample integrity may be
compromised if samples are either not completely thawed or if
maintained too long at 37C. Furthermore, water baths must be
properly maintained to make certain they are not inadvertently
maintained at a higher temperature because this may lead to
deterioration of coagulation factor activities and spurious coagulation test results. Once samples are thawed, it is imperative they
are thoroughly and adequately mixed prior to testing.
Typical Issues Related to Inappropriate
Sample Collection, Processing, and Storage
Incorrect Patient Collected or Wrong Label Attached
Patient misidentification errors are potentially associated
with the worst clinical outcome due to the potential for misdiagnosis and inappropriate therapy. Whenever misidentification
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CE Update
is suspected, the laboratory might be able to identify this as
being the case by investigation, but the safest approach is generally recollection and retesting.

plasma may derive plasma-like test results for D-dimer and

some VWF tests but a false impression of absent FVIII activity
by 1-stage clotting assays.

Incorrect Anticoagulant Matrix Collected or

Provided to the Laboratory
Although serum, heparin, or EDTA samples provided as a
primary collection tube can be quickly identified as unsuitable
by the laboratory, collection into the wrong anticoagulant may
be missed when samples are provided in secondary tubes, or if
samples have been mixed or transferred or added to a primary
citrate tube.4,16 The potential consequences will differ according to the sample type received and the tests being performed as
will the ability of laboratory personnel to recognize an incorrect
sample. For example, testing for routine coagulation tests such
as the PT and APTT will result in no clot or prolonged clotting times, but effects on other test results may be more subtle.
Thus, testing of normal serum for VWF tests will not provide
extreme changes but might instead give rise to patterns consistent with Type 2 VWD. Similarly, testing of heparin or EDTA

Serum or Clotted Samples

Samples in which the blood is slow to fill the collection
container, where there is prolonged use of a tourniquet, or
considerable manipulation of the vein by the needle may be
prone to develop a clot in vitro. Clots may also develop when
samples are incompletely mixed immediately following collection or in under-filled tubes. Although modern laboratory
instrumentation is increasingly being equipped with various
additional sensors (eg, bubble/volume/clot), samples yielding
long clotting times should routinely be checked for the presence
of a clot, either visually or preferably by inserting 2 wooden
applicator sticks into a whole blood sample. The presence of a
clot is a cause for rejection of the specimen. Serum will lead to
loss of fibrinogen and many other coagulation factors (notably
FII, FV, and FVIII) as well as differential loss of high molecular
weight VWF (Tables 2 and 3).35,36 Serum may also yield high

Table 2_Summary of Differential Effects of Testing Different Sample Types on Select Hemostasis Tests

Sample Type
Routine Coagulation Tests

Potential Consequences
On Factor Assays

Potential Consequences On
Other Hemostasis Tests

EDTA plasma
Prolongs PT and APTT, and occasionally TT.
False low levels (especially
False impression of inhibitors to FV and
Might influence fibrinogen and FV and FVIII) FVIII, and may show time dependence
D-dimer assays (ie, enhanced with incubation); false
LA feasible
Serum or fully clotted
No fibrinogen, so no clot in PT, APTT, or TT. False low levels (especially FII, FV, False impression of factor inhibitors or VWD;
coagulation sample False impression of afibrinogenemia. and FVIII); false high FVII false LA feasible
D-dimer assays can be affected especially
if testing delayed
Partially clotted
Depending on relative extent of platelet
False low factor levels or false
Flow obstructions in PFA-100 testing
coagulation sample activation, hemolysis and loss of fibrinogen high factor VII
might lead to false prolongation of PT,
APTT, and TT, or false shortening of APTT
Underfilled primary citrate
Will typically prolong PT, APTT, and TT. May False low factor levels likely
False low levels of most hemostasis tests
anticoagulant tube underestimate fibrinogen and D-dimer likely
Vitamin K-deficient plasma,
Prolongs PT and APTT (PT raised >APTT
False low factors (especially
False low protein C (potentially different
patient on vitamin K antagonist raised) FII, FVII, FIX, FX) effect with clot-based assays vs
therapy, liver disease sample chromogenic assays); false low protein S;
false APCR; false LA feasible
Heparin contamination (either
Prolongs PT, APTT, and TT (usually TT raised
Reduced factors (especially
False low Antithrombin; false LA feasible
ex-vivo or due to collection >APTT raised >PT raised), false low FVIII, FIX, FXI, FXII)
tube error) fibrinogen
False impression of factor inhibitors
Table has been adapted and updated from reference 4.

Table 3_Summary of Effects of Inappropriate Sample Processing Issues on Select Hemostasis Tests
Issue

Effect on Hemostasis Tests

Whole blood refrigerated

Platelet activation and loss of FVIII and VWF; can lead to false diagnosis of hemophilia or VWD
prior to centrifugation
Filtered plasma
Loss of fibrinogen, FVIII, and VWF; can lead to false diagnosis of dysfibrinogenemia, hypofibrinogenemia, hemophilia, or
VWD; prolongs routine coagulation test times (PT, APTT, and TT); false LA feasible
Delayed transport, delayed
1. Loss of labile factors (especially FV and FVIII); can lead to false impression of hemophilia; prolongs routine coagulation test
testing, poor storage, several times (PT, APTT); 2. Samples with unfractionated heparin can yield lower than expected APTTs and lower anti-FXa (heparin)
freeze-thaw events; storage test levels; 3. Potential activation of FVII
in frost-free freezer
Poor centrifugation, heavy braking,
Platelet contamination, hemolysis, and platelet disruption post freezing; activation, false low APTT, false low heparin levels,
sample remixing prior to freezing false negative LA, false high factor levels
Table has been adapted and updated from reference 4.

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values for some factors (eg, FVII) due to activation. Testing of
serum will therefore lead to non-coagulation in tests such as the
PT, APTT, and TT, possible diagnosis of coagulation factor
deficiencies, false diagnosis of certain subtypes of VWD, and
problems with LA identification. Alternatively, test results using
serum might be normal with some other tests. Testing of partially clotted blood may lead to prolongation or shortening of
coagulation tests depending on the extent of fibrinogen/factor
loss vs activation events, and is often harder to identify.
To determine if the sample is serum, a TT can be performed. Non-clotting will suggest either serum or heparin contamination, which can then be differentiated by mixing studies
(ie, TT performed on sample mixed 1:1 with normal plasma; if
the mixed plasma clots, then serum is confirmed, whereas if the
mixed plasma does not clot, this would suggest heparin contamination). The presence of heparin can also be determined by
use of a heparin anti-FXa assay or by measuring a TT or APTT
before and after the addition of a heparin-neutralizer.37
EDTA Plasma
This will raise coagulation test times such as PT and
APTT and reduce FV and FVIII, leading to the potential false
identification of factor deficiencies and/or factor inhibitors.35,36
If normal plasma mixing studies are performed on EDTA
plasma, lack of correction is seen, suggesting the presence of
an inhibitor. In some cases it may also lead to false identification of weak LA. As before, some test results might be normal
(eg, VWF:Ag, D-dimer), and so, the ability of a laboratory to
recognize an EDTA plasma sample will depend on the tests
performed. Assessment of potassium (extremely increased) or
calcium (very low to absent) will usually identify the presence
of an inappropriate EDTA collection.37
Heparin
Within a hospital setting heparin contamination is much
more common than hemostasis assays incorrectly collected in
either EDTA or submitted as serum. Heparin is used as therapy
for treating patients (eg, post thrombosis), in heparin flushes
to maintain flow in central lines, within heparinized needles
(eg, blood gas collection), and for many surgical applications.
Effects on hemostasis tests depend on the heparin concentration
(or level of contamination) and the test performed. In general,
clotting times (APTT and especially the TT) are prolonged,
and fibrinogen and clotting factors (especially APTT based,
viz FVIII, FIX, FXI, FXII) reduced.35,36 This might lead to
false identification of dysfibrinogenemia/hypofibrinogenemia
and certain factor deficiencies. There is also a potential for
false identification of LA and factor inhibitors and reduced
antithrombin. Sometimes, test reagents (eg, for PT or LA detection) include heparin neutralizers, at levels sufficient to neutralize about 1 U/mL unfractionated heparin. Although useful, this
can lead to complex patterns of test results and laboratories are
sometimes falsely reassured that these tests are not influenced
by heparin. Thus, a normal PT but abnormal APTT, or an
abnormal PT and APTT, can both arise, depending on the
contaminating level of heparin and whether the PT reagent
contains heparin neutralizers. Results might be normal with
some other tests (eg, D-dimer, VWF:Ag), and so whether the
laboratory recognizes a heparin-contaminated sample as such
will again depend on the tests performed.
Heparin contamination can be provisionally identified by
testing of select clot-based assays (especially APTT and TT),
and then by mixing studies (see end of serum section above),
6

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and confirmed by using an anti-FXa assay or by repeat APTT

or TT testing after addition of a heparin neutralizer.37
Processing Issues
Badly processing samples can lead to hemolysis or platelet activation, falsely prolonging or shortened clotting times,
depending on the extent of hemolysis vs platelet activation.
Alternatively, inadequate mixing may lead to clotting or partial
clotting and prolongation or shortening of clotting times and
elevated or diminished clotting levels. Freezing of plasma contaminated with cellular material may also lead to hemolysis or
activation events, as well as the potential for false-negative LA.
Hemolysis
This results from cellular destruction within whole blood
and the release of cellular lysis products including hemoglobin into the plasma. Although in vitro hemolysis might be a
byproduct of a problematic collection or the result of poor
handling of blood post collection, hemolysis can also derive
from in vivo blood cell lysis (eg, from hereditary, acquired, and
iatrogenic conditions such as autoimmune hemolytic anemia,
severe infections, intravascular disseminated coagulation, or
transfusion reactions).38 Hemolysis increases the spectrometric
absorbance of the plasma sample and leads to high background
absorbance readings, which may compromise clot detection by
some instruments and thus affect the accuracy of test times.
Instruments utilizing mechanical means of clot detection are
not affected by this interference, but the test result may still be
compromised since cell lysis products include tissue factors that
may activate coagulation. The net effect is that detected fibrinogen levels may fall with increasing hemolysis, whereas D-dimer
levels may increase. Prothrombin time values may fall in line
with decreasing fibrinogen, whereas APTTs may increase or
decrease depending on the net effect of activation vs the loss of
fibrinogen. Hemolysis may also influence other test results (eg,
decrease antithrombin levels).
If possible, grossly hemolyzed specimens should be rejected. If testing must be pursued (eg, if in vivo hemolysis is
present), testing using a mechanical end point detection system
is recommended, although the potential effect of activation
should also be noted. Samples appearing hemolyzed due to the
presence of a hemoglobin substitute are not a cause of specimen
rejection, and these samples should be evaluated using a mechanical or electromechanical method for clot detection.
Hematocrit
The presence of significant anemia has not been shown to
influence test results.39 Too high a hematocrit will influence the
anticoagulant to plasma ratio and thus test results.4,16 An adjustment in the ratio of anticoagulant solution/volume of blood
at different packed cell volume when hematocrit values are
above 55% may be undertaken using CLSI recommendations,
although a simplified method is to remove 0.1 mL of sodium
citrate from a 5 mL 3.2% sodium citrate evacuated tube prior
to collection.40
Lipemia
It is not easy to dichotomize the biological and analytical
effect of lipemia on coagulation tests.4,16 Acute elevation of
the coagulant activity of FVII is observed after consumption
of high-fat meals, mostly due to an increase in the concentration of activated FVII (FVIIa). High-fat meals also have
a substantial, acute effect on platelet function and may also
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CE Update
induce a lowering of some clotting factor activities (eg, FII,
FIX, FX, FVII, FVIIa, FXIIa). Analytical interferences in some
laboratory assays (especially those based on optical clot detection) also occur but are minimized using mechanical or electromechanical-based procedures or using analyzers comparing the
absorption of samples at 2 wavelengths or performing coagulation assays at alternative wavelengths.3,4 Nevertheless, regardless
of the potential source of interference (biological or analytical),
the best approach might be recollection of blood samples at
fasting, provided that metabolic problems (ie, dyslipidemia)
are absent.
Freeze-thawing Events
These result in the loss of some labile factors, notably
FV and FVIII. Since it is not always clear how many times a
sample has been thawed and refrozen prior to testing, retesting
using a fresh sample is always indicated should an unexpected
low factor result be obtained.

Under-recognized Pre-analytical Issues

Normal Reference Range Derivations and Related
Issues
Laboratories use normal reference ranges (NRRs) to identify whether a test result is within the normal range or outside
this range (and to thus identify an abnormal result). Use of
an inappropriate NRR may mean some normal individuals
will yield apparently abnormal test results. However, even the
use of a typical and potentially appropriate NRR, generated
as the mean +/- 2 standard deviations, will identify 5% of
the normal population as outside this range, simply based on
the statistical model used (ie, to capture 95% of the normal
population). Another way to consider this is to recognize that
a standard laboratory NRR will correctly identify only 95 out
of every 100 normal test results. Put into clinical context, 5
in every 100 (or 1 in every 20) tests a clinician orders (using
such NRR estimates) will likely reflect a false abnormal test
result, again simply based on the statistical model used to
generate the NRR. The relative false positive to true positive
rate increases substantially for rare disorders and is a particular
problem with congenital disorders such as protein C, protein
S, and antithrombin, especially when patient cohorts are inappropriately selected for testing.41,42
Miscellaneous Variables
Age, gender, ethnicity, and blood group might influence
reference values for certain parameters of laboratory hemostasis,
and/or generate variable test results for some tests.4,43 For example, FVIII and VWF and platelet function tests are generally
influenced by such factors. Thus, interpretation of test results
should consider these issues to prevent misdiagnosis.
International Normalized Ratio (INR)
The INR is the most common test performed by coagulation laboratories. The INR derives as a mathematical calculation, viz: INR=(patient PT/MNPT)ISI where MNPT=mean
normal PT, and ISI=international sensitivity index. The
patients PT is an analytical event and is derived from the instrument. However, the ISI and MNPT are derived separately
and might be considered as pre-analytical variables within the
context of inaccurate INRs.4,7
labmedicine.com

Filtered Plasma
Plasma for LA testing must be essentially platelet free (<10
109/L) if frozen prior to testing.4,16,30 Platelet-free preparations can be achieved by a process of double centrifugation,
high-speed (ultra-) centrifugation, microfiltration, or combinations thereof; however, microfiltration, commonly used in the
past because of its ease, is no longer recommended. This is
because the process leads to loss of other plasma components,
including FVIII and VWF, and may cause potential problems
in LA detection because it artificially elevates the baseline clotting times observed using some LA clot-based assays, and may
also lead to a false conclusion of an elevated APTT. In extreme
cases, microfiltration may even generate false (weak) positive LA
findings. Moreover, in many routine hemostasis laboratories,
LA testing is requested not only for specific investigation of
APS but also for investigation of unexplained prolongation of
APTT test times, a common incidental finding in a laboratory
practice. The most common explanations are low levels of FXII
or (unless the laboratory uses an LA-insensitive reagent) the
presence of (usually asymptomatic) LA. However, since an elevated APTT may also define a clinically significant event, such
as hemophilia or VWD, prolonged APTTs should be further
investigated to determine the underlying cause. It is therefore
not uncommon to receive requests including test combinations
for LA, and FVIII and/or VWF to exclude LA, hemophilia or
VWD, respectively. The dilemma is that should the laboratory
process the sample for LA testing by filtration, and then unwittingly test that sample for FVIII and VWF, a false diagnosis of
hemophilia or VWD is then quite feasible. Accordingly, the
double centrifugation approach for preparation of hemostasis
samples prior to freezing is now strongly favored.
Physical Activity, Illness, and Stress
Excess physical activity in patients immediately prior to
collection leads to certain in vivo events (eg, plasma volume
expansion and increased basal metabolism), which may in turn
lead to significant effects on hemostasis. However, perhaps the
best-known acute effects are related to acute phase reactants,
which may rise due to physical activity, illness or stress, and
include fibrinogen, VWF, and FVIII. In the worse case scenario, these elevations may result in a misdiagnosis of (mild)
hemophilia A or VWD Type 1 patients as a non-hemophilia or
non-VWD (false negatives). Blood collection may sometimes
be stressful for some patients (particularly children) leading to
acute phase changes in proteins secondary to the phlebotomy
itself.
Circadian and Diurnal Rhythms
Levels of some hemostasis components follow a circadian
or diurnal rhythm, with differential levels detectable at different
times of the day.4,44 For example, fibrinogen and plasminogen
activator inhibitor-1 levels tend to be higher in the early morning hours. PFA-100 closure times and possibly VWF may also
provide different values throughout a 24-hour period. Although
most changes tend to be fairly subtle, in the worse case scenario
this might also lead to some clinically significant differences.
Patients on Anticoagulant Therapy
Testing for thrombophilia is often performed in patients
who have recently suffered a thrombotic event. Patients are
placed on anticoagulant therapy after a thrombosis. Testing
while on anticoagulant therapy will affect (both biologically
and analytically) many of the tests undertaken in this context,
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CE Update
including LA, activated protein C resistance (APCR), antithrombin, protein C, and protein S. Thus, false-positive and
false-negative diagnoses can both occur, depending on the extent of the anticoagulant effect, and the test performed.41,42
Other Medications That May Interfere With
Coagulation Testing
A variety of therapeutic agents may cause spurious coagulation results due to variable mechanisms. This effect is not
always intuitive based on the pharmaceutical product.4
Clinical Ordering and Inappropriate Requests
as a Pre-analytical Issue
The concept of clinical ordering patterns as a pre-analytical
issue is also worth mentioning, in particular for the case of
inappropriate clinical orders.4 There is heightened concern currently with respect to thrombophilia tests, which comprise an
area of investigation that is growing rapidly within hemostasis,
and perhaps leading to over or inappropriate ordering.41,42
The proper timing of test orders is an important but poorly
recognized issue. Following a thrombotic event, some loss (consumption) of the natural anticoagulants might arise; hence, testing too soon after a thrombosis might lead to false conclusion
of a deficiency. FVIII may also be elevated post thrombosis,
leading to a missed LA diagnosis if only APTT-based screening
tests are used. Alternatively, anticoagulant therapy will affect
the detected levels of the natural anticoagulants, as mentioned
previously (viz, heparin therapy may influence antithrombin
detection, warfarin therapy may influence protein C and protein S levels, and heparin and warfarin therapy may influence
APCR testing). Heparin and warfarin therapy may also influence the appropriate identification of LA. Recent audits of
clinical practice indicate that up to 1/3 of samples destined for
thrombophilia investigations are from patients on warfarin and/
or heparin therapy, or the sample is otherwise heparin contaminated, and thus representing high potential for diagnostic error.
Considered another way, upwards of 80% of abnormal thrombophilia test results may be a reflection of inappropriate testing
while on anticoagulant therapy.42
Test Methodology and Test Panel Selection
While test results and methodologies comprise analytical
issues, the choice of which particular methodologies or test panels to use might best be considered as pre-analytical variables.
The presence of LA or APCR, seen in about 2%-5% of the

general Caucasian population, may interfere with some clotbased protein C and protein S assays, and lead to false identification of such deficiencies.4 Insufficient laboratory test panels
may also miss significant disease. For example, VWD may be
misdiagnosed or missed if the test panel does not include tests
for VWF activity, such as a collagen-binding assay.45 Some
methodologies are also poor at identifying low levels of VWF,
so type 3 VWD may be misidentified as type 1. In another
example, different laboratories and even experts use different
tests (or methodologies) and test panels for the identification
(or exclusion) of APS.46 Moreover, there are wide variations in
the detection of solid phase aPL by different commercial assays,
and different perceptions will arise among practitioners regarding general sensitivities and specificities of different tests and
panels for APS, and different perceptions of positive or negative
aPL for any given patient will arise among clinicians, depending
on both the methodologies, as well as the test panels, used to
identify APS.

Conclusion
Pre-analytical issues in hemostasis testing are an important
cause of diagnostic error (summarized in Tables 2 and 4) and
can lead to significant adverse clinical events. However, the burden of laboratory errors is estimated to remain globally modest
(ie, 1 in every 900-2074 patients or every 214-8316 laboratory
results).47 Notably, a large number of errors are likely to be intercepted before they are released to the clinician and, thereby,
before they translate into real harm for the patient. The ultimate aim of laboratory practice would be to have no errors or
to at least detect and correct all errors before the test result is
released. Accordingly, several tools might assist in their identification, including a comprehensive education of all personnel
regarding types and sources of errors, the accurate evaluation of
sample quality (ie, volume, blood to anticoagulant ratio, presence of potential interferents, or contaminants), and the systematic recording of suspect results along with pertinent clinical
information.48 When data are considered clinically questionable, the original test request should be checked and the specimen inspected and retested, sometimes with different assays and
instruments. The most reliable approach to deal with laboratory
errors is to establish a total quality management system.49-51
This would entail the elimination or strict supervision of the
most vulnerable activities, the implementation of customized

Table 4_Summary of Misdiagnosis and/or Misidentification in Hemostasis Possibly Arising From Inappropriate
Sample Types
Misdiagnosis/Misidentification

Can Arise From Testing Of

False-positive LA
Normal EDTA plasma, normal serum, vitamin K deficiency patient, anticoagulated patient, heparin-contaminated sample,
plasma containing factor inhibitors
False diagnosis of VWD:
Filtered normal plasma, normal serum, normal plasma derived from refrigerated whole blood sample
False subtype identification (Type 2
Type 1 VWD plasma derived from refrigerated whole blood sample, testing of filtered plasma or serum
diagnosis in Type 1 VWD patient)
False diagnosis of hemophilia A
Filtered normal plasma, normal serum, normal plasma derived from refrigerated whole blood sample, aged sample,
sample post several freeze-thaw events, heparin-contaminated sample, EDTA sample, normal serum sample, underfilled
primary citrate collection tube
False identification of factor inhibitors
Heparinized normal sample, EDTA sample, normal serum sample, lupus anticoagulant
Table has been adapted and updated from reference 4.

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CE Update
Table 5_Important Issues for Laboratories and Clinicians to Consider Within the Context of Pre-analytical Issues
in Hemostasis Testing, and Some Recommendations
Issue Consideration/Recommendation
Test selection
Population to be tested and
clinical condition/medication
at time of testing
Sample collection





Sample transport

Sample processing



Sample storage
Sample testing

Result interpretation

Select/order the best tests/test processes/test panels for the condition being investigated
Select the appropriate population/methodology to determine the normal reference range
Only order the test(s) when clinically appropriate and in the right patient at the right time
Proper patient and sample identification
Atraumatic phlebotomy with minimal tourniquet use
Draw blue stopper tube (citrate anticoagulant) first or only after a non-additive tube
Fill tube adequately (no less than 90% fill)
Adequately and thoroughly mix with tube anticoagulant
Transport promptly at room temperature
Ideally centrifuge within 1 hour of phlebotomy to obtain platelet-poor plasma (most tests)
Double centrifuge plasma for some tests, namely LA, APCR, and heparin (anti-Xa) assays
Aliquot (in a non-activating secondary tube) immediately following centrifugation for those tests to be performed later
Special requirements for some tests such as platelet function and PFA-100
Test plasma within appropriate timeframe; store as required samples to be tested subsequently
Select the best test/methodology/test panel for the analyte/parameter being tested
Perform test in timely manner and according to best practice
Laboratory: Provide clinician with appropriate guidance/test interpretation
Clinician: Recognize test limitations/extra-analytical issues that may influence test results and follow local expert laboratory advice

Table has been adapted and updated from reference 4.

informatics systems for error identification and recording, and

for highlighting collection requirements as related to specifically
ordered tests, and facilitate the continuous education of operators (both inside and outside the laboratory) by dissemination
of best practice recommendations.49-51 This would include providing adequate training and guidance to blood collectors.
Additional useful advice to laboratories and clinicians is
be vigilant of all these issues; select/order the best tests and test
panels available, undertake testing only when necessary, at the
correct point in time for the condition under investigation;
incorporate as much clinical information as possible into the
diagnostic approach; follow the recommendations of local laboratory experts/specialists; repeat tests when not in keeping with
clinical expectations or when an abnormal finding is reported;
implement restrictive specimen acceptance policies and tolerance criteria for inappropriate specimens; put quality practices
into place where possible to aid in identifying problem samples;
and establish a mutually beneficial clinical-laboratory interface
where both parties discuss the problems within meetings or
teaching moments as well as actively collaborate to achieve the
best possible patient outcome (Table 5). We also strongly recommend that laboratories use appropriate post-test guidance to
assist clinicians in the interpretation of test results, as well as to
guide when repeat, confirmatory, and follow-up testing may be
required.4,52
There have been several new oral anticoagulants recently
released onto the market (most notably Dabigatran and
Rivaroxaban) for a variety of clinical indications including prevention of venous thromboembolism after major orthopaedic
surgery or secondary prevention in atrial fibrillation. These
agents will variously affect coagulation and hemostasis tests as
described elsewhere,53,54 but should now also be considered
within the context of preanalytical problems associated with
hemostasis testing. LM

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