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Preanalytical and Postanalytical Variables: The Leading Causes of Diagnostic

Error in Hemostasis?

Article in Seminars in Thrombosis and Hemostasis · November 2008

DOI: 10.1055/s-0028-1104540 · Source: PubMed

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 Preanalytical and Postanalytical Variables:
The Leading Causes of Diagnostic Error
in Hemostasis?
Emmanuel J. Favaloro, Ph.D., M.A.I.M.S.,1 Giuseppe Lippi, M.D.,2
and Dorothy M. Adcock, M.D.3

ABSTRACT

The advent of modern instrumentation, with associated improvements in test

reliability, together with appropriate internal quality control and external quality assurance
measures, has led to substantial reduction in analytical errors within hemostasis laborato-
ries. Unfortunately, the reporting of incorrect or inappropriate test results still occurs,
perhaps even as frequently as in the past. Many of these cases will arise due to a variety of
events largely outside the control of the laboratories performing the laboratory tests and
primarily comprise preanalytical events related to patient collection and sample processing
and postanalytical events related to the reporting and interpretation of test results. The
current article provides an overview of these events and provides some suggestions on how
they can be minimized or prevented to ensure that the test results the clinician receives
actually represent the true clinical status of the patient under investigation rather than just
reflecting the status of an (inappropriate) clinical sample received and tested. This article
should be of interest to both laboratory scientists working in hemostasis and the clinicians
that request such tests. The former, because these are ultimately responsible for the test
results they provide to clinicians, and there is a duty of care to provide both accurate and
precise results to enable clinicians to manage patients appropriately and to avoid the need to
recollect and retest. The latter because unless clinicians gain an appreciation of these issues,
they will not be in a position to best manage their patients.

KEYWORDS: Preanalytical variables, postanalytical variables, extra-analytical variables,

diagnostic errors, hemostasis

T he advent of modern instrumentation, usually to a considerable reduction in analytical errors within

interfaced to the laboratory information system and hemostasis laboratories. Unfortunately, the reporting of
capable of providing improvements in test reliability, incorrect or inappropriate test results still occurs, perhaps
together with appropriate internal quality control (IQC) even as frequently as in the past. Many of these cases can
and external quality assurance (EQA) measures, has led be related to inappropriate collection or processing of

1
Department of Haematology, ICPMR, Westmead Hospital,
Westmead, Australia; 2Sezione di Chimica Clinica, Università di
Verona, Verona, Italy; 3Esoterix Coagulation, Englewood, Colorado.
Address for correspondence and reprint requests: Emmanuel J.
Favaloro, Ph.D., M.A.I.M.S., Department of Haematology, Institute
of Clinical Pathology and Medical Research (ICPMR), Westmead
Hospital, SWAHS, Westmead, NSW 2145, Australia (e-mail:
emmanuel.favaloro@swahs.health.nsw.gov.au).
612
Laboratory Diagnostics in Thrombosis and Hemostasis: The Past, the
Present, and the Future; Guest Editors, Emmanuel J. Favaloro, Ph.D.,
M.A.I.M.S., Giuseppe Lippi, M.D., and Massimo Franchini, M.D.
Semin Thromb Hemost 2008;34:612–634. Copyright # 2008 by
Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY
10001, USA. Tel: +1(212) 584-4662.
DOI 10.1055/s-0028-1104540. ISSN 0094-6176.

samples referred for laboratory testing. In these situa-
tions, the test result might ‘‘accurately’’ reflect the status
of the test sample being tested, but this test sample
might not ‘‘accurately’’ reflect the clinical status of the
patient being investigated. These events can lead to
several unwanted clinical consequences, as well as place
laboratories at some risk.1 To some extent, the serious-
ness of the consequences relates to the test being per-
formed and the experience of laboratory scientists and
clinicians in terms of recognizing these issues.2 Within
hemostasis, consequences are often more serious for
errors related to specialized tests, as these are often
considered as ‘‘diagnostic.’’ Thus, a patient might be
diagnosed with a particular disorder, when in fact he or
she does not have this disorder (i.e. false-positive test
result is obtained), or else a patient with a true disorder
might be missed (i.e. false-negative test result is ob-
tained). Both situations will cause adverse consequences
for both patients and the health care system. As an
example, a false-negative antiphospholipid antibody or
lupus anticoagulant (LA) test result in a patient with the
antiphospholipid antibody syndrome (APS) may lead to
lack of appropriate treatment with anticoagulant therapy
to prevent a future thrombosis. An alternative example is
a false diagnosis of von Willebrand disease (VWD)
leading to inappropriate treatment with factor concen-
trates or to a lifelong label of VWD affecting quality of
life.
Nevertheless, consequences for errors related to
routine (‘‘screening’’) coagulation tests might also be
serious, as these might influence the clinical decision
regarding whether or not to undertake further (e.g.
‘‘specific diagnostic’’) testing, unnecessarily delay oper-
ative procedures, and may also affect anticoagulant
therapy decisions. Thus: (i) a false-normal screening
test result might prevent further testing for factor assays
and incorrectly discount a hemophilia, thus placing the
patient at an unjustified risk of bleeding when under-
going operative procedures (i.e., surgery, dental extrac-
tion); (ii) a false-abnormal screening test result might
lead to fruitless additional costly and time-consuming
investigations, accompanied by unnecessary anxiety in
the patient being investigated; (iii) 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 risk of thrombosis
or bleeding depending on the direction of change.
PREANALYTICAL ISSUES
The modern hemostasis laboratory performs a large
number of distinct tests, often using a variety of method-
ologies (Table 1). This leads to considerable problems
when samples are provided in a nonideal presentation.
There are guidelines available for how to manage un-
suitable specimens, and for deciding when to reject
EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 613
‘‘poor’’ specimens.3–5 Unfortunately, it is not always clear
when a sample referred to the laboratory is ‘‘poor’’ or
unsuitable. Problems arising from pretest sample collec-
tion, processing, transportation, and storage can broadly
be placed within the category of preanalytical factors.
Whereas analytical errors can be avoided by using
appropriate test methodologies and by incorporation of
appropriate internal control measures, preanalytical is-
sues present a more difficult scenario as they are often
outside the control of the laboratory performing the
tests. To date, no reliable quality indicators have been
broadly implemented to monitor performance of this
essential part of the total testing process. Indeed, pre-
analytical issues are often difficult to detect, and the
laboratory may not be aware that an inappropriate
sample is being tested. The laboratory would issue the
test result with the best of intentions as reflecting an
accurate ‘‘patient-related’’ result, but this may not be the
case. The clinician would be even less aware of the issue
of preanalytical variables than would the laboratory and
would base their clinical response on the test result that
they received. For this reason, guidelines for specimen
collection and handling must not only be widely avail-
able but also should be strictly followed and deviations
avoided unless their impact, or lack thereof, on coagu-
lation testing is known.
POSTANALYTICAL ISSUES
As previously highlighted, a laboratory test result often
leads to some clinical action, and this action might have
serious adverse consequences if not appropriate to the
clinical situation. Whereas laboratories should not direct
clinical action, they should provide as much guidance as
possible to enable clinicians to make the best, most
informed choices for patient management.6 In this
respect, how the laboratory reports their test results
can also have significant adverse consequences if clini-
cians base treatment on the test report and this report
fails to appropriately identify the significance or non-
significance of test results. An example here is where a
laboratory merely reports a test result of a weakly positive
IgM anticardiolipin antibody (aCL) without advising
that this has a low clinical significance, and the clinician
otherwise places too much importance upon the test
result, diagnoses APS, and then applies anticoagulant
therapy without further testing or verification.
PREANALYTICAL VARIABLES
IN HEMOSTASIS TESTING
Appropriate Sample Collection, Processing,
and Storage
These are critical to the attainment of appropriate test
results but are often neglected, overlooked, or poorly
614 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7 2008

Table 1 Summary of Hemostasis Tests and Sample Requirements

Category of
Testing Sample Type Comprise Usually Performed via

Routine Citrate-anticoagulated plasma
coagulation tests after single centrifugation
Specialized Separated citrate-anticoagulated
hemostasis tests plasma, after single centrifugation
and after freezing
Separated citrate-anticoagulated
plasma, after single
(or preferably double) centrifugation
and after freezing
Separated citrate-anticoagulated
plasma, after double centrifugation
and after freezing
Separated serum preferred; separated Solid-phase antiphospholipid
citrate-anticoagulated plasma after antibody (aPL) tests including
single centrifugation and after
freezing sometimes acceptable
Citrate-anticoagulated whole blood or
special processing required
EDTA- or citrate-anticoagulated whole Genetic thrombophilia tests
blood or special processing required
EDTA, ethylenediaminetetraacetic acid; VWF:Ag, VWF antigen; VWF:RCo, VWF ristocetin cofactor activity; VWF:CB, VFW collagen binding
activity.
Prothrombin time (PT)/ international
normalized ratio (INR), activated
partial thromboplastin time (APTT),
thrombin time (TT), fibrinogen
D-dimer (D-D)
Factor assays (i.e., II, V, VII, VIII,
IX, X, XI, XII), factor inhibitor
assessments, protein S, protein C
von Willebrand factor (VWF) tests
including VWF:Ag, VWF:RCo,
VWF:CB; protein C, protein S
Protein C, antithrombin
Heparin (anti-Xa) assay
Activated protein C resistance
(APCR)
Plasminogen activator inhibitor
(PAI-1)
Lupus anticoagulant (LA)
anticardiolipin antibody
(aCL) and anti-b-2-glycoprotein
I antibody (ab2GPI)
Platelet function tests
Clot-based tests, automated
instrument, primary collection
tube (sometimes
separated plasma)
Enzyme-linked immunosorbent
assay (ELISA) or enzyme-linked
fluorescent assay (ELFA) or
agglutination (primary or
secondary tube)
Clot-based tests, automated
instrument.
ELISA (VWF:Ag, VWF:CB,
protein S, protein C) or
immunoassay (VWF:Ag,
protein S) or agglutination
(VWF:RCo)
Chromogenic assays
Chromogenic assays
Clot-based tests, automated
instrument
ELISA
Clot-based tests, automated
instrument
ELISA
Specialized instrumentation
Specialized instrumentation

applied.7 We therefore provide an overview of appro-
priate procedures and note some of the problems that can
arise when appropriate procedures are not followed.
Positive Patient and Sample Identification
The importance of positive patient identification and
proper sample labeling cannot be overemphasized. To
ensure proper patient identification, the conscious pa-
tient should be asked to identify himself or herself and
should also be asked to produce some form of identi-
fication, preferably according to the principle of ‘‘double
identifiers,’’ as recommended by the Joint Commission
(JC) on Accreditation of Healthcare Organizations.8,9 In
the hospital situation, positive patient identification
should strictly follow the scheme provided by the in-
stitution, and this may entail a variety of electronic or
bar-code methods that reduce the risk of patient mis-
identification. This information must match that on the
test request. The JC provides other clear guidelines:
Preferably, the labels for the tubes should be printed
by a device located near the patient, just before blood

collection, activated by an identification worn (e.g., a
wrist-bracelet with patient data) or else directly linked to
his or her physical body. Alternatively, if labeled blood
collection tubes are prepared in advance, patient identi-
fication reported on the tubes should be automatically
matched at the time of venipuncture, with patient data
reported on a wrist-bracelet or other device attached to
the patient’s body. This transaction must be automati-
cally recorded. Each label should contain the patient’s
full name and an additional identifier such as date of
birth or medical record number. Date and time of
collection should also be identified, particularly in pa-
tients undergoing some form of therapy.
Sample Collection
After proper patient and sample identification, subse-
quent potential problems may arise during the sample
collection process.7 There are several pathology tests
performed these days, and the number continues to
increase. Each test has specific collection requirements,
and most tests have (sometimes subtly) different require-
ments. Sample collection issues might arise because of
inexperience or time-pressures when collectors are faced
with a busy collection clinic and multiple collection
requirements. Most samples referred for coagulation
testing must be drawn into citrate-based anticoagulant
and are generally collected into 105 to 109 mM or
129 mM sodium citrate (sometimes referred to as 3.2%
or 3.8%, respectively) tubes. Although there appears to be
no difference in relation to testing of anti-Xa (heparin)
levels,10 our own preferences match the current Clinical
and Laboratory Standards Institute (CLSI) guidelines,5
which are in favor of the lower citrate concentration.
Rarely and mostly for select studies, the higher citrate
concentration might still be used or preferred. Specimens
collected in 129 mM (3.8%) buffered sodium citrate may
overestimate the prothrombin time (PT) and activated
partial thromboplastin time (APTT) if the normal
range is based on a 3.2% citrated sample and also under-
estimate fibrinogen.11–13 On the other hand, samples
collected into 129 mM (3.8%) citrate may provide a
more stable sample for assessing aspirin response using
Q1 the PFA-100Q1 (Siemens Healthcare Diagnostics,
Deerfield, IL).14,15 The major recommendation here is
that laboratories should standardize to one citrate con-
centration and develop normal ranges appropriate for that
concentration. It is particularly important that all com-
ponents of the assay (e.g., for the international normal-
ized ratio [INR] equation, including determination of
patient PT, mean normal PT, and international sensi-
tivity index), be standardized to one citrate concentration
to avoid introducing result variability.12
Although mostly based on anecdotal evidence of
cross-contamination between tubes, coagulation samples
should preferably be collected before other pathology
EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 615
test samples are drawn, if these contain stronger anti-
coagulant agents such as ethylenediaminetetraacetic acid
(EDTA) (for a complete blood count) or lithium-
heparin (for clinical chemistry testing), as these materials
may possibly contaminate later coagulation test samples.
This is also in agreement with the so-called order of
draw, a specific sequence of tube collections issued by the
CLSI.16 The old dogma that the first collection tube be
discarded may not generally be required, as evidence for
differential effects on coagulation are lacking.17,18
Nevertheless, a discard tube is needed if the sample is
drawn with a winged collection set that has variable
length of tubing. This is because the air in the tubing of
the winged system will be introduced into the collection
tube and may lead to underfilling.5 Tubes should be
adequately filled, that is, no less than 90% of the total
volume or to the mark noted on the tube (if provided).
Underfilling may cause significant sample dilution and
tends to 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 testing performed.19,20 For example, the effect of
underfilling appears to be more pronounced with 3.8%
citrate anticoagulant test tubes, providing additional
support for the preferential use of 3.2% citrate collection
tubes.19 This effect is also more pronounced when small-
volume (pediatric) collection tubes are used.20 Sample
dilution itself will also lead to some underestimation of
quantitative test results (e.g., clotting factor levels).
Using primary tubes, the blood should never be
transferred from one collection tube to another to provide
the required complete fill volume for any tube. This is
critical when using noncitrate anticoagulant collection
tubes, to prevent stronger anticoagulants such as EDTA
or lithium-heparin from contaminating the citrated co-
agulation sample, but is also important when using citrate
anticoagulant tubes, as this will double up on the anti-
coagulant (i.e., exaggerate effects related to high citrate
levels) in the final tube and act to further dilute the
plasma sample. Samples should also be mixed thoroughly
(but gently) by three to six end-over-end tube inversions
to provide adequate mixing of test sample with anti-
coagulant.16 This is particularly important with evalua-
tion of some specialized tests such as prothrombin
fragment 1 þ 2 and thrombin antithrombin (TAT) com-
plex, both measures of thrombin generation. For exam-
ple, in a patient with a normal baseline TAT result,
inadequate mixing may lead to a 2.5-times increase in the
test result.21 In practice, there is a potential for a clot to
form if samples are not properly mixed, although such
clot formation may develop slowly over time and may not
always be apparent when tests are performed soon after
collection. Thus, ‘‘undermixing’’ will not generally lead to
test problems with basic coagulation tests performed soon
after collection22 but may affect downstream specialized
hemostasis assays performed some time after collection.
616 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7
On the other hand, too vigorous a mixing (e.g., by
shaking of tubes) might lead to in vitro hemolysis or
spurious test activation and false shortening of test
clotting times and even possible false elevation of clotting
factor activity (e.g., factor VII).
Because not all tests referred for hemostasis labo-
ratories would by necessity be performed on sodium
citrate anticoagulated samples, there is some scope for
additional preanalytical error. For example, the preferred
test sample for solid-phase testing of antiphospholipid
antibodies (aPL) such as aCL and anti-b2-glycoprotein I
(ab2GPI) is serum. In contrast, the test sample for LA
testing must be citrate anticoagulated plasma. All of these
different tests might be requested for a patient being
investigated for APS. Accordingly, if these tests are
performed in the same laboratory, problems may arise
should the laboratory inadvertently perform LA testing
using the serum sample. Alternatively, testing for LA and
solid-phase aPL assays such as aCL and ab2GPI might
be performed in different laboratories, but the same
situation (incorrect sample type received/tested) might
still occur if the sample-processing laboratory inappropri-
ately distributes the samples.
Other issues can arise when blood collections are
difficult or derive from central venous lines instead of
direct collections. In the case of the former, samples
might end up being partially clotted, hemolyzed, or
activated. In the case of the latter, samples might be
diluted by saline or contaminated with heparin used to
flush lines or keep them pliant. If samples are collected
from a vascular access device, it is recommended that the
line be flushed with saline and that six dead-space
volumes of the tubing be discarded.23 If the sample is
drawn from a capped intravenous port such as a saline
lock, two dead-space volumes of the catheter extension
set should be drawn and discarded.24 The size and type
of the needle used may also influence appropriate col-
lection. Both too large (less than 16 gauge) and too small
a bore needle (greater than 25 gauge) should be avoided,
and heparinized needles (sometimes used for blood gas
collection) should not be used. However, clinically
meaningful effects might not eventuate for most routine
tests using needle sizes between 21 gauge and 25 gauge25
or a butterfly device.26 The effect of blood stasis might
also be considered; accordingly, the tourniquet should be
applied only when necessary and always for less than
1 minute.27
Sample Transport
This should also proceed as per current guidelines.5,7
Samples should be transported nonrefrigerated at am-
bient temperature (158C to 228C) 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. However, tolerances are probably
2008
better than this, particularly for the PT.5,28 On the other
hand, testing with the APTT for unfractionated heparin
monitoring should preferably be processed within 1 hour
due to the potential for heparin neutralization by platelet
factor 4 (PF4) released in vitro by the platelets.5,29
Extremes of temperature during transport should be
avoided. Samples should neither be transported refriger-
ated (e.g., on ice) nor at high temperature (e.g., as might
occur during vehicular transportation), and considera-
tion should be made to include temperature and humid-
ity data loggers and recorders in transport containers to
monitor transport conditions. Delays in transport may
affect in particular the labile factors (e.g., factors V and
VIII), leading to prolonged clotting times and potential
false identification of deficiencies. If there are expected
delays in transport, consideration should be given to
local centrifugation and separation of plasma followed by
freezing and frozen transport of the plasma should the
test warrant this.
Sample Processing, Transport, and Storage
In general, this should also proceed as per current CLSI
guidelines,5 except that we should also recognize certain
limitations here, depending on which test is being
performed. Most coagulation-based tests, including
PT, APTT, clotting factor assays, and so forth, are
performed on plasma derived from once-centrifuged
test samples. Some tests, such as LA, should be double
centrifuged to ensure platelet-free preparations prior to
freezing. According to CSLI guidelines,5 centrifugation
should essentially be at ambient temperature (158C to
228C), but this is sometimes difficult to control. Non-
refrigerated centrifuges are adequate but can overheat if
used excessively, and this may adversely raise the temper-
ature of test samples. Alternatively, refrigerated centri-
fuges, if used, should in general be set to maintain
ambient temperatures, as low temperatures can lead to
platelet activation and other adverse effects as high-
lighted later. Nevertheless, refrigerated centrifugation
does not appear to affect routine coagulation tests
when performed soon after centrifugation.30 Centrifu-
gation should ideally be at 1500
g for a minimum of
10 to 15minutes,5,31 although shorter centrifuge times
might be acceptable for routine coagulation tests per-
formed immediately after centrifugation in primary
collection tubes and without any further test require-
ments (i.e., plasma not to be frozen). Using relative
centrifugal forces greater than 1500
g is not recom-
mended because this may induce platelet activation and
lysis of red blood cells.32 The use of centrifuge breaks
should also be avoided or at least monitored to avoid
overt remixing of test samples, especially if the plasma is
to be frozen before testing. If this happens, hemolysis
and platelet contamination will affect most hemostasis
assays performed subsequent to freezing.
 EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 617

In some cases, testing can proceed using the once-
centrifuged test sample still in the primary collection
tube. Most modern hemostasis instruments can perform
routine coagulation tests on these primary tubes, and
provided that the tests proceed soon after centrifugation,
without disturbing the cell pellet, there will rarely be a
problem. Tests often performed immediately using pri-
mary tubes (Table 1) primarily comprise routine coagu-
lation tests and represent the largest numerical
proportion of tests performed by most laboratories
worldwide these days. Other tests can also be performed
on once-centrifuged plasma samples, even after freezing
(Table 1). In other test cases, samples should be double
centrifuged (‘‘double-spun’’). This entails (prior to freez-
ing) the recentrifugation of the separated plasma, and
reseparation of this double-spun plasma from any resid-
ual cellular pellet (Fig. 1). Tests preferably performed
using double-spun plasma are also listed in Table 1.
Because all plasma-based hemostasis tests can safely be
performed on double-spun material, it might be better to
instigate this process as a general laboratory policy for
any plasma to be frozen, to avoid inappropriate testing
issues that may otherwise occur when using single-spun
plasma inappropriately for some tests. We also recom-
mend against the use of filtered plasma, as this might
produce spurious hemostasis test results, as highlighted
later.33–35
Lastly, some tests performed within hemostasis
laboratories require special differential processing. For
example, testing using the PFA-100 and other platelet
function screening tests may require citrate-anticoagu-
lated whole blood, and such samples have limited
stability (e.g., PFA-100 tests must be performed within
4 to 5 hours of collection). These samples must not be
centrifuged, and if centrifuged, the samples become no
longer suitable for testing (even if remixed to obtain
reconstituted whole blood) because of platelet activa-
tion events. Testing of platelet function by classic
aggregometry requires more complex processing, in-
cluding differential centrifugation steps to differentially
provide ‘‘platelet-rich’’ and ‘‘platelet poor’’ plasma, the
description of which is beyond the scope of this article.
The stability of coagulation samples varies de-
pending on several variables such as the blood collection
system, whether the samples are stored as whole blood or
centrifuged, the temperature that samples are maintained
at during storage, the reagent/instrument system used for
analysis, and the test parameter to be analyzed. Published
studies vary in their recommendations due to the many
variables just listed. A recent study reported that whole
blood could be stored up to 24 to 28 hours prior to
centrifugation without observing clinically relevant
changes in most hemostasis parameters with the excep-
tion of factor V (FV) and FVIII coagulant activity and
total protein S antigen.28 Conversely, van Geest-Daal-
derop et al,36 in an earlier study, observed a significant
change (> 10%) in PT during the first 6 hours after blood
collection in one of two laboratories evaluated, when
uncentrifuged blood samples were stored at room tem-
perature, 48C to 68C, or 378C. It was hypothesized that
such variation may be related to the difference in the
blood collection systems, thromboplastin reagents, and
coagulometers used. Twenty-four-hour stability was
demonstrated when these samples were centrifuged im-
mediately after blood collection and stored at room
temperature. Across several different laboratories, storage

Figure 1 The process of ‘‘double centrifugation.’’ This simply involves the primary citrate-anticoagulated blood tube (labeled
‘‘1’’) being centrifuged as per normal (e.g., 1500
g, 15 minutes, no brake), the subsequent transfer of the top (e.g., 90%)
plasma fraction (i.e., without disturbing the cell pellet; labeled ‘‘2’’) into a second centrifuge tube (labeled ‘‘3’’), and then the re-
centrifugation (e.g., 1500
g, 15 minutes, no brake), and the subsequent retransfer of the top plasma fraction (labeled ‘‘4’’) to a
third patient-identity-labeled plastic tube (labeled ‘‘5’’), which is then capped and frozen until tested.
618 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7
for 24 hours at 378C has proved unacceptable.36 A
significant reduction, although within the range of ‘‘de-
sirable bias,’’ was also observed by Salvagno et al37 for PT
and fibrinogen in uncentrifuged specimens stored at
room temperature for 3 and 6 hours. Tests for von
Willebrand factor (VWF) seem somewhat stable when
samples are stored for several days at ambient temperature
as either whole blood or separated plasma, although small
rises in levels are observed with whole blood storage using
some tests, perhaps reflecting cellular release.38 On the
other hand, storage of refrigerated whole blood is now
actively discouraged and leads to activation events that
affect FVII, FVIII, VWF, and possibly others.5,39–41
Because the results of the available studies on
sample stability are strongly dependent upon the test, the
instrument and the reagents used, laboratories should
locally test the impact of a delay greater than 4 hours, for
each combination of materials and conditions, testing
both normal and abnormal samples. In general, to afford
the greatest sample integrity, samples should be proc-
essed 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 freezing). During this short-term storage, whole
blood samples should be kept capped and at room
temperature. If testing is not to be performed within
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 disturb-
ing the cell pellet. For many tests of hemostasis, the
separated plasma can now be safely frozen for later
testing. Most specialized tests of hemostasis are per-
formed using frozen samples, usually by batch testing.
Finally, separated plasma samples can generally be
maintained at room temperature or refrigerated for a few
hours without adverse effect on coagulation. This is
convenient for repeat testing needs (e.g., clinician adds
a test request after initial sample testing, or when a test
repetition is advisable to confirm a previous [e.g., abnor-
mal] test result). Otherwise, separated plasma samples
should be frozen in non–frost-free freezers. Frost-free
freezers are unsuitable, because they cycle freeze-thaw
events to maintain the frost-free environment, and this
will adversely affect subsequent coagulation tests. The
lower the freezer temperature, the longer that the speci-
mens can be maintained for future testing. As a general
rule of thumb, testing for samples maintained at around –
208C should be finalized within 2 to 4 weeks of storage,
whereas testing for samples maintained at around –808C
can occur several months and sometimes years later
(useful for research studies and prospective trials).42
Controlled Thawing of Frozen Plasma Samples
Previously frozen samples should be rapidly thawed in a
378C water bath for 5 to 10 minutes, or until com-
2008
pletely thawed. Close monitoring during this time is
necessary to avoid inadequate or excessive incubation in
the heated chamber. Sample integrity may be compro-
mised if samples are either not completely thawed or if
maintained too long at 378C. Furthermore, water baths
must be properly maintained to make certain that they
are not inadvertently maintained at a higher temper-
ature because this may lead to degradation of coagu-
lation factor activity and spurious coagulation test
results. Once samples are thawed, it is imperative that
they are thoroughly and adequately mixed prior to
testing.
Some Issues Related to Inappropriate Sample
Collection, Processing, and Storage
There are a variety of possible preanalytical problems
related to inappropriate sample collection, processing,
and/or storage that may arise and make life difficult for
both laboratory scientists and the clinicians that manage
the patients being tested by the laboratory. The major
issues can be listed as follows:Q20 Q20
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. This would
rarely occur with current collection standards and labo-
ratory practices (summarized in the previous section) but
is still a possibility to consider when faced with an
unexpected test result. Despite general progress in this
area, appropriate patient identification is a goal that must
be achieved by all laboratories and is the first among the
JC National Patient Safety Goals from 2003 throughout
2008.9 Whenever misidentification 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.
Incorrect Anticoagulated Sample Collected
or Provided to Laboratory
As previously mentioned, the correct test sample for
most coagulation tests is citrate anticoagulant plasma.
Sometimes, the wrong sample is collected and the
laboratory quickly identifies this. For example, a serum
sample or EDTA sample provided to the laboratory for
coagulation testing as the primary collection tube will
quickly be identified by the laboratory as ‘‘unsuitable.’’
However, inappropriate collections may be missed if
the sample (e.g., serum or EDTA) is provided as a
separated (‘‘seemingly’’ citrated plasma) sample in a
secondary container or if the samples have been mixed
or transferred (i.e., collected into one anticoagulant or
 EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 619

Table 2 The Effect of Different/Incorrect Sample Types on Common Hemostasis Assays

Tube Type
3.2% Citrate EDTA Sodium Heparin Serum
Assay (Mean/Range) (Mean/Range) (Mean/Range) (Mean/Range)

APTT (s) 29/25–33 68/45–92 > 180 > 180

PT (s) 12.4/11.5–13.2 23/19–27 > 60 > 60
dRVVT (s) 34.6/27–43 55/45–64 > 150 > 150
FV Act (%) 113/84–142 71/39–103 81/59–103 23/13–33
FVII Act (%) 115/50–180 116/51–182 77/43–107 308/80–437
FVIII Act (%) 141/80–202 7.5/2–19 <1 4.5/1.3–7.7
FIX Act (%) 122/97–148 115/63–168 <1 350/135–565
VWF:Ag (%) 122/50–194 143/59–228 70/42–98 101/32–169
VWF:RCo (%) 114/41–188 131/46–215 37/13–60 74/25–124
PC Ag 97/60–134 115/97–159 125/94–156 120/71–169
PC Act (%) 111/66–155 152/100–205 <1 21.6/0–70
PS Act (%) 96/73–119 30/17–42 <1 15.3/0–39.5
Free PS Ag (%) 108/72–144 131/91–171 126/94–159 131/97–164
AT Act (%) 102/86–118 121/105–138 126/108–143 47/30–65
AT Ag (%) 110/83–138 121/92–150 100/83–118 114/79–148
APTT, activated partial thromboplastin time; PT, prothrombin time; dRVVT, dilute Russell viper venon time; Act, activity; Ag, antigen; FV, factor
V; FVII, factor VII; FVIII, factor VIII; FIX, factor IX; VWF:Ag, von Willebrand factor antigen; VWF:RCo, von Willebrand factor ristocetin cofactor
activity; PC, protein C; PS, protein S; AT, antithrombin.
Samples were collected into different collection tubes containing additives as identified in each column. Ranges reflect the actual range of
values obtained (n ¼ 10 healthy volunteers).
Source: Unpublished data from Valcour A and Marshall T (2007), Laboratory Corporation of America, Inc; used with permission.

primary tube [e.g., serum or EDTA] and transferred or
added to a second primary tube [e.g., citrate]). Should
this be the case, the laboratory may or may not judge
the sample as inappropriate and might issue a test result
that reflects the true status of the test sample provided
(e.g., serum or EDTA) but that obviously might not
reflect the true status of the patient from which the
sample derived. The consequences will differ according
to the sample type received and the tests being per-
formed (Tables 2 and 3). Furthermore, the ability of a
laboratory scientist or technician to recognize whether
or not a correct sample has been received will also

Table 3 Differential Effect of Testing Different Sample Types on Select Hemostasis Tests: A Summary
Sample Type Effect on Hemostasis Tests

EDTA plasma
Serum
Vitamin K–deficient plasma,
patient on vitamin K antagonist
therapy, liver disease sample
Heparin ‘‘contamination’’
(either ex vivo or due to
collection tube error)
Partially clotted sample
Routine coagulation tests: prolongs PT and APTT and occasionally TT. Might influence
fibrinogen and D-dimer assays; factor assays: false low especially factors V and VIII.
Other: leads to false impression of inhibitors to factors V and VIII and may show time
dependence (i.e., enhanced with incubation); false LA feasible.
Routine coagulation tests: no fibrinogen, so no clot in PT, APTT, or TT. False
impression of afibrinogenemia. D-dimer assays can be affected especially if test
delays; factor assays: false low especially factors II, V, and VIII; false high factor VII.
Other: may lead to false impression of factor inhibitors or VWD; false LA feasible.
Routine coagulation tests: prolongs PT and APTT (PT raised > APTT raised); factor assays:
false low factors (especially II, VII, IX, X). Other: false low protein C (potentially
different effect with clot-based assays vs. chromogenic assays), false low protein S,
false APCR, false LA feasible.
Routine coagulation tests: prolongs PT, APTT, and TT (usually TT raised > APTT
raised > PT raised), false low fibrinogen; factor assays: reduced factors
(especially VIII, IX, XI, XII). Other: false low antithrombin, false LA feasible.
Routine coagulation tests: depending on relative extent of platelet activation,
hemolysis, and loss of fibrinogen, might lead to false prolongation of PT, APTT,
and TT, or false shortening of APTT; factor assays: false low factor levels or false high
factor VII. Other: flow obstructions in PFA-100.
620 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7
depend on the sample type received, the tests being
performed, and, last but not least, the skills of the
operator.
For example, a serum, heparinized, or EDTA
sample provided to the laboratory and tested for
routine coagulation tests such as the PT and APTT
will result in noncoagulable or poorly coagulable test
mixtures and will usually generate suspicion of ‘‘serum’’
or other ‘‘inappropriate sample or problem’’ in the
mind of the scientist performing the test. However,
the effects on other test results may be subtler. For
example, testing of normal serum for VWF tests will
not provide extreme changes and might instead give
rise to patterns consistent with type 2 VWD.34 The
laboratory will need to be aware of this possibility, or
they might (for this example) otherwise report these
VWF test results per se, and the clinician looking after
the patient may inadvertently identify VWD as a
result. EDTA plasma or heparinized plasma can lead
Q2 to other issues (see later sectionQ2 and Tables 2 and 3).
Sometimes, inappropriate samples will provide essen-
tially ‘‘citrated-plasma-like’’ test results with some
tests, but not with others, and this might be method
related. Hence, recognition of inappropriate samples
may be achieved for some tests but not for other tests.
As another example, testing of an EDTA plasma
sample will derive ‘‘plasma-like’’ test results for some
VWF tests performed by ELISA, whereas the same
EDTA plasma will lead to a false impression of absent
Q3 FVIII:CQ3 by clotting tests. Alternatively, lithium-
heparin or EDTA specimens might still be suitable
for some immunoassays, such as those for measuring
D-dimer43 and immunologic fibrinogen,44 provided
that results are adjusted for dilution factors. Although
the tested sample can be assessed for EDTA (e.g.,
perform a potassium test, which will be very high if the
sample is EDTA anticoagulated, and/or perform a
calcium test, which is essentially undetectable in an
EDTA tube) or assessed for heparin contamination, it
is not always easy to define when a sample is actually an
inappropriate one and thus requires specific investiga-
tion (Tables 2 and 3).45,46
It is also important to recognize that occasionally,
inexperienced or negligent collectors will do some odd
things. As an example, when one medical intern was
requested to collect a range of samples for pathology
testing by a consultant, she first collected a large number
of blood tubes corresponding with the number of tests
requested but using only EDTA blood collection tubes.
She subsequently consulted the test requirement man-
uals, which naturally identified differential collection
requirements, but instead of recollecting the blood, the
intern just poured the EDTA blood into the correct
tubes according to test requirements, and this then
caused some havoc in the various laboratories later
consigned to perform testing!
2008
Serum
Although serum is the preferred sample matrix for solid-
phase tests of aPL, it is inappropriate for most other tests
of hemostasis. In brief, testing of serum will lead to loss
of fibrinogen and some other coagulation factors (most
notably II, V, and VIII), as well as differential loss of
high-molecular-weight VWF (Tables 2 and 3).34,45,46
Serum will also yield very high values for factor VII and
sometimes other factors (due to activation). Testing of
serum will therefore lead to noncoagulation in tests such
as the PT, APTT, and thrombin time (TT), possible
diagnosis of coagulation factor deficiencies, false diag-
nosis of certain subtypes of VWD, as well as problems
with LA identification. On the other hand, test results
using serum might be ‘‘normal’’ with some other tests
(e.g., VWF antigen [VWF:Ag], certain factor assays).
Furthermore, while testing of a completely clotted
sample will often be identified if tested in a clotting-
based assay (e.g., PT, APTT, TT, fibrinogen, factor
assays), testing of a sample obtained from partially
clotted blood will be harder to identify and may lead to
prolongation or shortening of coagulation tests depend-
ing on the extent of fibrinogen/factor loss versus activa-
tion events. Whether the laboratory recognizes that it is
testing serum (instead of citrate plasma) thus depends on
the tests it performs on this sample. To determine if the
sample type is serum (in a situation where the plasma or
serum component of the sample has been aliquoted into
a secondary container), a TT can be performed to see if a
clot forms (suggesting that the sample is plasma) or a clot
does not form (suggesting that the sample is serum or
heparin contaminated). The latter (serum vs. heparin)
can then be differentiated by mixing studies (i.e., sample
mixed 1:1 with normal plasma and then thrombin added;
if the mixed plasma clots, then serum is confirmed,
whereas if the mixed plasma still 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 before and after
the addition of a heparin-neutralizing enzyme such as
Hepzyme (Dade Behring, Marburg, Germany).
EDTA Plasma
This will raise coagulation test times such as PT and
APTT, which at a minimum will lead to additional
unnecessary investigations, factor profiles, an so on
(Tables 2 and 3).45,46 EDTA plasma also exhibits
reduced factor V and VIII and inhibits clotting factor
activity in coagulation tests, leading to the potential false
identification of factor deficiencies and/or factor inhib-
itors.45,46 In some cases it may also lead to false identi-
fication of weak LA.45,46 EDTA would rarely be
received by the laboratory for performance of hemostasis
tests, but it is possible for reasons previously identified,
and thus should be considered, especially when the
 EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 621

laboratory observes the above test patterns. Again, test
results might be normal with some other tests (e.g.,
VWF:Ag, D-dimer), and so, whether the laboratory
recognizes that it is testing an EDTA ‘‘contaminated’’
sample or not will depend on the tests it performs on this
sample. As mentioned previously, a test for potassium
(extremely increased) or calcium (very low to absent) will
usually identify the presence of an inappropriate EDTA
collection.
Heparin
Heparin contamination is a much more common issue
than is either EDTA or serum. Heparin is used ubiq-
uitously within the hospital setting, as therapy for treat-
ing patients (e.g., postthrombosis), in ‘‘heparin flushes’’ to
maintain flow in central lines, and as ‘‘heparinized nee-
dles’’ (e.g., blood gas collection). The effect of heparin on
hemostasis tests depends on the heparin concentration
(or level of ‘‘contamination’’) within the sample and the
test being performed. In general, a test sample containing
heparin will show prolonged clotting times (APTT and
especially the TT), and reduction in the level of detected
fibrinogen and clotting factors (especially APTT-based
factors, VIII, IX, XI, XII; Tables 2 and 3).45,46 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 (Tables 2–4).45,46 Gross heparin contamina-
tion or heparin therapy itself may also influence the level
of antithrombin detected.47 Sometimes, test reagents
(e.g., for PT or LA detection) include heparin neutral-
izers, such as protamine or polybrene, at levels usually
sufficient to neutralize 1 U/mL unfractionated heparin
and generally no more. Although generally beneficial for
testing, this can also lead to false reassurance within
laboratories that these tests are therefore not influenced
by heparin, and/or can lead to complex patterns of test
results. For example, a heparin-contaminated sample
might yield a normal PT but abnormal APTT, or an
abnormal PT and APTT, depending on the contaminat-
ing level of heparin and whether or not the PT reagent
contains heparin neutralizers. Again, results of testing
might be normal with some other tests (e.g., D-dimer),
and so, whether the laboratory recognizes that it is testing
a heparin ‘‘contaminated’’ sample will therefore depend
on the tests it performs on this sample. Heparin con-
tamination 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), and confirmed by repeat APTT or TT testing
after addition of a heparin neutralizer or using an anti-
FXa assay.
Right Anticoagulated Sample, but Badly
Processed
Problems can also arise in the case where badly processed
citrate-anticoagulated samples are provided to the labo-
ratory. We have already mentioned the example where
the sample may have been mixed too vigorously, leading
to hemolysis or platelet activation. This might lead to
either falsely prolonged or falsely shortened clotting
times, depending on the extent of hemolysis versus
platelet activation. Alternatively, the sample may not
have been mixed adequately enough, in which case
partial clotting may eventuate. This may lead to prolon-
gation of clotting times and diminished clotting factor
activities if a significant clot has formed, or activation of
the sample. Other consequences might include falsely
lowered fibrinogen levels, or false low or false high
coagulation factor assay results. As mentioned previ-
ously, freezing of plasma contaminated with cellular
material may also lead to hemolysis or activation events
and to several consequences including the potential for
false-negative LA.
Given the previous background, it should now be
obvious that provision of appropriate samples for testing
is critical for ensuring the appropriateness of test results.
However, even when the type of sample (e.g., citrate-
anticoagulated plasma) received by the laboratory is
appropriate, other events may still transpire to yield

Table 4 Summary of Misdiagnosis and/or Misidentification in Hemostasis Arising from Inappropriate Sample Types
Misdiagnosis/Misidentification Can Arise from Testing of:

False-positive LA
False diagnosis of VWD
False subtype identification
(type 2 diagnosis in type 1 VWD patient)
False diagnosis of hemophilia A
False identification of factor inhibitors
Normal EDTA plasma, normal serum, vitamin K deficiency patient, anticoagulated
patient, heparin-contaminated sample, plasma containing factor inhibitors
Filtered normal plasma, normal serum, normal plasma derived from refrigerated
whole blood sample
Type 1 VWD plasma derived from refrigerated whole blood sample, testing of
filtered plasma or serum
Filtered normal plasma, normal serum, normal plasma derived from refrigerated
whole blood sample, aged sample, sample after several freeze-thaw events,
heparin-contaminated sample, EDTA sample, normal serum sample
Heparinized normal sample, EDTA sample, normal serum sample, LA
622 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7
test results that do not reflect the patient’s true clinical
status. The following section provides an overview of the
usual and not so usual circumstances that can be iden-
tified to provide inappropriate test results or otherwise
lead to events that compromise test accuracy and patient
management.
PREANLYTICAL ISSUES: THE OTHER
USUALLY MENTIONED CULPRITS
Clotted Sample
It has already been noted that the presence of a clot in
the specimen may significantly alter results of coagula-
tion assays. This may occur even when the clot is not
visible to the naked eye. Samples in which the blood is
slow to fill the collection container, where there is
prolonged use of a tourniquet or considerable manipu-
lation of the vein by the needle may be prone to develop a
clot in vitro. Clots may also develop in vitro when
samples are incompletely mixed immediately after col-
lection or in underfilled tubes. Samples that yield long
clotting times should routinely be checked for the
presence of a clot. This can be done visually although a
more thorough technique would entail inserting two
wooden applicator sticks into a whole blood sample.
The presence of a clot is a cause for rejection of the
sample.
Hemolysis
At a basic level, hemolysis results from cellular destruc-
tion within whole blood and the release of cellular lysis
products into the plasma. Although in vitro hemolysis
might be a by-product of a problematic collection or
result from poor handling of blood post collection,
hemolysis can also derive from in vivo blood cell lysis
(e.g., from hereditary, acquired, and iatrogenic condi-
tions such as autoimmune hemolytic anemia, severe
infections, intravascular disseminated coagulation, or
transfusion reactions).48 Hemolysis presents as a deep-
ening redness in the plasma resulting from red blood cell
destruction and release of hemoglobin into the sur-
rounding plasma, but damage to other cellular types is
frequently evident. There are a variety of schemes
evident in different laboratories to gauge the degree of
hemolysis depending on the color of the plasma. The
subjective nature of this determination makes this a
poorly reproducible exercise. 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 that
use mechanical means of clot detection are not affected
by this interference. In addition, cell lysis products
include tissue factors that may activate coagulation.
2008
The net effect is that detected fibrinogen levels may
fall with increasing hemolysis, whereas D-dimer levels
may increase.49 PT values may fall in line with decreas-
ing fibrinogen, whereas APTTs may increase presum-
ably because the net effect of activation exceeds the loss
of fibrinogen.
The presence of hemolysis in the specimen might
also strongly influence other special coagulation tests.
For example, a significant decrease of antithrombin
assayed by a chromogenic method has also been reported
in hemolyzed specimens50 and has also been observed by
some of us (unpublished data). Given the potential
impact of hemolysis on coagulation assays, it is best to
reject hemolyzed specimens upon recognition by objec-
tive means (i.e., free hemoglobin quantification or meas-
urement of the ‘‘index of plasma,’’ as increasingly
available by laboratory instruments and coagulometers).
If testing of the hemolyzed sample must be pursued (e.g.,
when the presence of in vivo hemolysis has been ascer-
tained), analysis of the plasma using a mechanical end
point detection system is recommended. Nevertheless,
the potential effect of activation should also be noted.
Samples that appear 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 CLSI currently recommends an adjustment in the
ratio of anticoagulant solution/volume of blood at differ-
ent packed cell volume using the following equation:
volume of anticoagulant required ¼ (100 – PCV)/(595 –
PCV), where PCV is the packed cell volume expressed in
percentage (%).5 This recommendation mainly applies to
subjects with hematocrit values above 55%, whereas
there is no current data available to support a recom-
mendation for those with hematocrit values < 20%. A
simplified method to overcome the excess citrate effect
in most samples with an elevated hematocrit is to remove
0.1 mL of sodium citrate from a 5-mL 3.2% sodium
citrate evacuated tube prior to collection.51 If the sodium
citrate is removed from the evacuated tube using a sterile
tuberculin syringe inserted through the stopper, the
vacuum on the tube can be retained and normal fill
volume obtained. Because most samples with an elevated
hematocrit have values that fall between 0.55 (55%) and
0.65 (65%), removing this constant volume of sodium
citrate is generally sufficient.5
Lipemia
The interference from lipemia is an old issue in coagu-
lation testing, first reported by O’Brien in 1955.52 It is
not easy to dichotomize the biological and analytical

effect of lipemia on coagulation tests. Acute elevation of
the coagulant activity of coagulation factor VII (FVIIc)
is observed after consumption of high-fat meals, mostly
due to an increase in the concentration of activated FVII
(FVIIa).53 High-fat meals also have a substantial, acute
effect on platelet function.54 It has also been reported
that high-fat diets might induce a significant lowering of
some clotting factor activities (e.g., FII:C, FIX:C, FX:C,
FVII:C, FVIIa, FXIIa), as well as lowering plasminogen
activator inhibitor (PAI-1), plasma viscosity, and plate-
let activity.55
In a study on healthy dogs, lipemia significantly
interfered with PT and TT, and both tests shortened
when lipemia increased.56 Analytical interferences in
some laboratory assays (including coagulation tests based
on optical clot detection) are also reported, due to light
scattering (triglycerides-rich particles interfere with lab-
oratory instrument systems based on light detection or
scatter, such as turbidimetry and nephelometry, inducing
an artificial prolongation of clotting times) and volume
displacement. Mechanical or electromechanical-based
assays are more robust in handling interference from
lipemia.2,57 Moreover, analyzers that compare the ab-
sorption of samples at two wavelengths or perform
coagulation assays at alternative wavelengths, such as
570 nm, can obviate this problem.58 However, regardless
of the potential source of ‘‘interference’’ (biological or
analytical), it is still recommended that re-collection of
blood samples at fasting be arranged, provided that the
interference does not result from metabolic problems
(i.e., dyslipidemia).
Freeze-Thaw Events
Freezing-thawing of plasma samples results in the loss of
some labile factors, notably FV and FVIII. It is not
always clear how many times a sample has been frozen,
thawed, and refrozen prior to testing in a specific assay.
Accordingly, a low FVIII:C, for example obtained by a
referral laboratory, might simply be an artifact of several
preceding freeze-thaw events. Retesting using a fresh
sample is always indicated, should a low factor result be
obtained. It is also important for laboratories to warm
samples to 378C for at least 5 to 10 minutes before
testing, to ensure reversal of any cryoprecipitate formed
during freezing. This may affect testing of FVIII and
VWF tests, as these proteins are known to precipitate at
cold temperatures.
PREANALTICAL ISSUES: SPECIFIC,
UNUSUAL, OR SPECIAL CASES
The following section details some special issues for
consideration within the context of preanalytical varia-
bles. This includes the selection of tests, test method-
ologies, and test panels. Although laboratory test results
EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 623
comprise an analytical process, which is beyond the scope
of this article, the selection of the test methodologies and
test panels used within the laboratory might be consid-
ered a preanalytical variable, and the effect of such test
selection on outcome, and the eventual reported results,
should be considered within the postanalytical phase.
Different test methodologies will lead to differences in
test results due to differences in analyte detection. A
sampling of some common tests to highlight this issue is
provided below, although it should be recognized that
similar situations might exist with respect to other assays
and diseases of hemostasis.
Special consideration should also be given to
specific clinical investigations including patient selection
and timing of the investigation. The estimation of the
normal reference range and of the population and data
processing method is also important and mentioned
here.
Normal Reference Range Derivations
and Related Issues
Laboratories derive normal reference ranges (NRRs) by a
multitude of methods. A common approach is to use the
mean  2 standard deviations (SDs) of a set of results
obtained from otherwise healthy or normal individuals.
Sometimes normal values follow Gaussian distributions
(i.e., equal distributions below and above the mean),
which enable direct calculation of data. In this case, the
calculated range will capture 95% of the normal pop-
ulation values. Notably, around 5% of the normal pop-
ulation data will therefore lie outside this range (i.e.,
some 2.5% of results will lie below the NRR and 2.5% of
results above, and these will actually therefore represent
unavoidable false-positive/false-negative events in test-
ing). Sometimes (as is actually often the case in tests of
hemostasis), the NRR distribution in non-Gaussian, and
data need to be transformed (e.g., logarithmically) to
make the distribution (near-)Gaussian prior to calcula-
tion of the NRR. In this case, somewhat different
proportions of data will lie above and below the cutoff
limits. Sometimes, laboratories will need to use other
procedures to derive NRR values (examples are 95% or
99% confidence intervals). It is also possible that the
laboratory may not be aware of the best way of generat-
ing NRR for each and every test and may not (for
example) use the correct process for such derivation for
some tests. Alternatively, the laboratory may have used
an inappropriate group or an insufficient number of
individuals to derive the NRR.
The clinical importance of these issues depends
on the test performed and whether appropriate meth-
ods have been used to generate the NRR, although
problems may still arise even when a laboratory has used
the appropriate method. Take for example a NRR of
50 to 200% for factor VIII; does 48% represent a true
624 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7
deficiency or a false low value based on the NRR
variation event, and does 52% represent a true normal
result? And is there really any difference between 48%
and 52% FVIII:C, and should clinicians define the
former as abnormal and the latter as normal? As
Q4 another example, consider a cutoff value of 20 GPLQ4
units for aCL testing; does a value of 21 GPL units
represent a true-positive aCL result, or a false-positive
result because of a NRR variation event, or does 19
GPL units represent a true- or false-negative aCL
result? Again, is there any real difference in the two
results of 19 versus 21 GPL, and should the former
exclude and the latter conclude a diagnosis of APS? It is
also important to keep in mind that in some circum-
stances, as in the detection of protein C, protein S, and
antithrombin deficiencies (see later), the likelihood of a
false-positive result based on NRR issues actually ex-
ceeds the probable rate of detection of a true-positive
result as true congenital deficiencies are rare.59–61 How
do we overcome these problems? The solution is chal-
lenging, but the introduction of laboratory-specific
quality specifications (e.g., the coefficient of variation
[CV] for each specific assay) would be a valuable aid for
interpreting test results, especially those lying at the
cutoff. These could be provided in the laboratory
report, together with provision of other useful inter-
pretive guidance (see next section).
It is also important to mention that other varia-
bles such as age, sex, and blood group might influence
reference values for certain parameters of laboratory
hemostasis.59,62–66 As an example, both VWF and
platelet function tests (e.g., PFA-100) are influenced
by many of these, and it is well-established that indi-
viduals with blood group O have lower values of the
former and prolonged closure times in the latter com-
pared with those with non-O blood. Interpretation of
test results should consider these issues, especially to
prevent misdiagnosis of VWD in blood group O indi-
viduals, which is a well-known phenomenon that might
be prevented, for instance, by including interpretative
comments on laboratory reports. It is also important to
mention that diagnosis of VWD should rely on the
clinical signs and symptoms of the patient and family
members in relation to the absolute levels of VWF.
Finally, the manner in which tests are performed,
and the sample dilution used versus the way in which
assay calibration curves are generated, can also influence
assay results and thus the eventual clinical ‘‘diagnosis.’’67
International Normalized Ratio
This test is the most common test performed by
coagulation laboratories. Given current instrumenta-
tion, satisfactory ‘‘precision’’ in terms of analytical
performance is usually guaranteed. However, given
preanalytical issues, ‘‘accuracy’’ is sometimes more per-
2008
ceived than granted. The international normalized ratio
(INR) derives as a mathematical calculation, viz.:
INR ¼ (patient PT/MNPT)ISI, where PT ¼ prothrom-
prothrombin time, MNPT ¼ mean normal prothrom-
bin time, and ISI ¼ international sensitivity index. The
patient’s PT is an analytical event and derived from the
instrument. However, the ISI and MNPT are derived
separately and might be considered as preanalytical
variables within the context of inaccurate INRs. The
MNPT is typically derived using the World Health
Organization (WHO) recommended procedure, by
testing a minimum of 20 individual plasmas from
healthy individuals, avoiding those with confounding
variables (e.g., oral contraceptives, pregnancy).68 Un-
fortunately, testing of such a small number of individual
samples will not define a conclusive MNPT, and indeed
testing of different sets of 20 individual normal plasmas
will generally derive different MNPT values.69 The ISI
is provided by the reagent manufacturer for some (but
not all) reagent/instrument combinations or for a type
of clot detection system employed (e.g., optical vs.
mechanical). Where the ISI is not available for a
specific reagent/instrument combination or appropriate
methodology, the laboratory needs to establish its own
ISI value. There are various options available to enable
this, including the WHO-recommended procedure,68
an onerous procedure that very few laboratories can
perform.69 The currently more commonly applied ap-
proach is that of using commercial calibration plasma
sets. This process is certainly feasible, but the use of
different sets may lead to different ISI values.69,70 The
combination of the ‘‘wrong’’ MNPT estimate and
‘‘wrong’’ ISI estimate will lead to some significant
variation in derived INR values that could feasibly
cover a range of INRs from 2.0 to 6.0 for the same
plasma sample tested, depending on differentially ap-
plied ISI and MNPT values. The consequences of
reporting an INR of 2.0 when it is really 6.0, or vice
versa, could indeed be clinically serious.
Filtered Plasma Provided to Laboratory
There is a requirement that plasma, if destined for LA
testing, be essentially platelet free (< 10
109/L) if it is
to be frozen prior to testing.5,71–74 This requirement may
not be necessary if the sample is tested for LA immedi-
ately (i.e., prior to freezing), and if freezer storage and
retesting is not planned,5,75 but this is not the manner in
which LA testing is typically applied (i.e., LA testing is
usually performed in batches using frozen-stored patient
plasma). Current guidelines indicate that platelet-free
preparations can be achieved by a process of double
centrifugation, high-speed (ultra-)centrifugation, micro-
filtration, or combinations thereof.5,71–74 Indeed, micro-
filtration is very commonly used for this purpose
because it is so easily applied by laboratories. However,

this process also leads to loss of other plasma compo-
nents, including FVIII and VWF.33–35 The loss of FVIII
causes problems in LA detection because it artificially
elevates the baseline clotting times observed using some
of the LA clot-based assays, and for example may itself
lead to a false conclusion of an elevated APTT. In
extreme cases, microfiltration may even generate false
(weak) positive LA finding. 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. Per-
formance of routine coagulation test times such as PT
and APTT are ubiquitous in clinical practice.76,77 Ele-
vated APTTs, in particular, are a common incidental
finding in laboratory practice. The most common ex-
planations are low levels of factor XII, or (unless the
laboratory uses an LA-insensitive reagent) the presence
of (usually asymptomatic) LA. However, because a raised
APTT may also define a clinically significant event, such
as hemophilia or VWD, raised APTTs should be further
investigated to determine the underlying cause.78
Clinicians may request further testing for inves-
tigation of elevated APTTs in patients with vague
clinical histories. It is therefore not uncommon to
receive requests that include test combinations for
LA, and FVIII and/or VWF to exclude LA, hemo-
philia 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.33–35 This possibility is
made more likely by the current common practice of
sample preparation in one area by technical assistants or
other staff (e.g., in remote collection/processing sites or
in a large primary centralized specimen reception area),
followed by the actual testing of that material in
another area or secondary (core or specialized) labora-
tory by different (technical or scientific) personnel. To
avoid the loss of sample integrity related to micro-
filtration, the double-centrifugation (Fig. 1) approach
for preparation of LA samples is strongly favored. Such
double-centrifuged (‘‘double-spun’’) samples are also
suitable for all other coagulation tests including FVIII
and VWF.
Physical Activity
Excess physical activity in patients immediately prior to
collection leads to certain in vivo events (e.g., plasma
volume expansion and increased basal metabolism),
which may in turn lead to significant effects on hemo-
stasis. Perhaps one of the best-known acute effects is on
VWF and FVIII, which are elevated immediately after
exercise.79 In the worse-case scenario, this might result
in a misdiagnosis of a (mild) VWD type 1 patient as a
non-VWD case (‘‘false-negative’’).
EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 625
Circadian and Diurnal Rhythm
Similarly, levels of some hemostasis components follow a
circadian or diurnal rhythm, with differential levels
detectable at different times of the day.80–82 For exam-
ple, fibrinogen and PAI-1 levels tend to be higher in the
early morning hours. PFA-100 closure times and possi-
bly 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
Patient testing in hemostasis is often applied within
the context of the investigation of thrombotic events.
The situation often goes like this: a patient presents to
the emergency ward of the hospital with a suspected
thrombosis (e.g., deep vein thrombosis or pulmonary
embolism). The physician orders the usual battery of
investigative tests and at some stage, should a thrombosis
be confirmed or strongly suspected, the patient will be
placed on anticoagulant therapy. Often, while on this
anticoagulant therapy, the clinical investigation into the
reasons for the thrombosis ensues or continues, and the
clinical request may then include a full thrombophilia
investigation. However, the patient is now on antico-
agulant therapy, and this will affect (both biologically
and analytically) many of the tests that will be under-
taken in this context, including LA, activated protein C
resistance (APCR), antithrombin, protein C, and pro-
tein S.
Other Medications May Interfere
with Coagulation Testing
A variety of therapeutic agents may cause spurious
coagulation results due to variable mechanisms, and
this effect is not always intuitive based on the pharma-
ceutical product. For example, the addition of poly-
ethylene glycol (PEG) groups to proteins and peptides,
in an effort to prolong the circulating half-life and
bioavailability, may cause a dose-dependent prolonga-
tion of the APTT with certain APTT reagents but not
others.83 Likewise, dose-dependent, clinically significant
prolongation of PT may occur with specific types of
antibiotic agents effective against methicillin-resistant
Staphylococcus aureus, and the degree of this effect is
dependent on the thromboplastin reagent used.84
Clinicians as a Preanalytical Issue
The concept of clinical ordering patterns as a preanalyt-
ical issue is also worth mentioning, in particular for the
case of inappropriate clinical orders.4 This may be a
particular concern with respect to thrombophilia tests,
which comprise an area of investigation that is growing
626 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7
rapidly within hemostasis, and perhaps leading to ‘‘over’’
or ‘‘inappropriate’’ ordering by clinicians without due
consideration. For example, the proper timing of test
orders is an important but obviously poorly recognized
issue. After a thrombotic event, some loss (‘‘consump-
tion’’) of the natural anticoagulants might arise; hence,
testing too soon after a thrombosis might lead to false
conclusion of a deficiency. Also after thrombosis, factor
VIII may be elevated and lead to missed LA diagnosis if
APTT-based screening tests only are used. Alterna-
tively, anticoagulant therapy affects detected levels of
natural anticoagulants. As mentioned above, 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. Internal audits
from one of our laboratories indicate that up to one
third of test samples destined for thrombophilia inves-
tigations are from patients on warfarin and/or heparin
therapy or that the sample is otherwise heparin ‘‘con-
taminated.’’59 Additional audits also suggest wide use
of thrombophilia investigations in patients without ap-
propriate need (e.g., protein C and protein S requests in
first thrombosis presenting at age > 60 years!) or per-
formance of protein S activity assays in women during
pregnancy or the postpartum period.
Potential solutions to the inappropriate ordering
of tests by clinicians include development of simple
clinical guidelines and possibly computerized ordering
assistance programs such as the Computerized Physician
Order Entry (CPOE) systems, which have been pro-
moted in Australia and internationally for their potential
to improve the quality of care.85 As for other areas of
testing, the impact of such systems might be particularly
evident for improving adherence to guidelines, decreas-
ing costs, and increasing the overall organizational
efficiency and usability.
Deficiencies in Protein C, Protein S,
and Antithrombin
True well-defined familial protein C and protein S
deficiency is very rare (< 5% of thrombophilic popula-
Q5 tion; < < 0.5%Q5 normal population), and true well-
defined familial antithrombin deficiency is rarer still
(< 2% of thrombophilic population; < 0.2% normal
population). Normal range estimation effects means
that 2% of test samples will give a ‘‘low’’ level of
protein C, 2% of test samples will give a ‘‘low’’ level
of protein S, and 2% of test samples will give a ‘‘low’’
level of antithrombin (but not necessarily the same test
samples in each test case). To this can be added false low
levels detected in patients on heparin and/or warfarin
therapy (potentially up to 33% of test cases). Clearly, the
risk of false-positive identification also worsens for
2008
poorer patient selection (i.e., nonthrombophilic popula-
tion). It can be hypothesized, then, that the level of false
identification of protein C, protein S, and antithrombin
deficiency exceeds, by several orders of magnitude, the
true rate of such deficiencies (e.g., 10 times false-
positive to true-positive identification in pathology prac-
tice is not impossible).59–61
Test Methodology and Test Panel Selection
The presence of LA or APCR, seen in 2% and 5% of
the general Caucasian population, respectively, may
interfere with some clot-based protein C and protein S
assays and lead to false identification of such deficien-
cies.86
Activated Protein C Resistance
In stark contrast with protein C, protein S, and antith-
rombin, APCR is fairly ‘‘common’’ within the Caucasian
population, just like its major ‘‘cause’’ (factor V Leiden;
FVL). Indeed, although APCR can be detected in
25% of the thrombophilic population, it can also be
detected in 5% of the normal Caucasian population.
Like the case for protein C, protein S, and antithrombin,
internal audits suggest wide use of thrombophilia inves-
tigations in patients without due need. Further, the
percent ‘‘hit rate,’’ if we were testing a true thrombophilic
population, would be 25% of test cases, but internal
audits indicate that values are approaching numbers
closer to 10%.59–61,87 When testing finally reaches a
5% ‘‘hit rate,’’ we can be assured that we will simply be
testing the general population for thrombophilia! So,
what are the dangers? At a simple level, such indiscrimi-
nate testing costs the health care service millions of
dollars. At a clinically relevant level, such indiscriminant
testing elevates the risk of false-positive cases of clinically
presumptive thrombophilia. As most individuals with
APCR (or FVL) will never have a thrombosis, this begs
the question of whether clinicians are actively treating
these patients (with low to no risk for thrombosis) that
they are identifying with APCR/FVL if all they are
identifying are background cases of APCR/FVL?
Test Methodology and Test Panel Selection
The most common tests used for assessment of APCR
are based on either the APTT or the Russell viper venom
time (RVVT). The latter will generally detect FVL
without the need for predilution in factor V–deficient
plasma, whereas the former will only consistently detect
FVL if the assay incorporates this predilution step.86–88
On the other hand, the RVVT assay and the APTT
assay incorporating predilution step will generally fail to
identify ‘‘acquired’’ APCR, such as those arising from
the presence of elevated FVIII values. This has led one of

us to initiate a two-test system for APCR, incorporating
a RVVT assay to identify FVL-related APCR and an
APTT-based assay (without predilution with factor
V–deficient plasma) to identify non–FVL-related
(‘‘acquired’’) APCR.87
Assessment of VWD
Several examples have already been provided regarding
VWD testing. The main issues are ABO blood group
(lower levels in O group); stress, exercise, pregnancy, and
age (all of which raise VWF levels); and possibly men-
strual cycle (lowest levels during menstrual phase), estro-
gen replacement therapy, and circadian rhythm.
Test Methodology and Test Panel Selection
VWF ristocetin cofactor (VWF:RCo) assays measure a
different functional property of VWF than either VWF
antigen (VWF:Ag) or collagen binding (VWF:CB)
assays.65 Hence, test results will not correlate in all test
cases. In general, there are fewer differences in meas-
urable values in normal individuals and in patients with
type 1 VWD, and so values generated by these tests will
tend to be fairly concordant in these test cases. In
contrast, type 2 VWD patients display VWF dysfunc-
tion characterized by a wide range of possible defects;
hence, VWF values tend to be discordant when meas-
ured by different assays.65 Different results can be
obtained even when presumably dealing with the same
assay. Sometimes these differences can be ascribed to
methodological differences. For example, VWF:Ag
measured by latex immunoassay will often be different
than values obtained by ELISA, and the diagnostic
performances of ELISA D-dimer assays can vary from
those of the immunoturbidimetric assays.
An example of the scope of the problem relating to
misdiagnosis of VWD because of limited or inappropri-
ate test selection is worth mentioning. Within Australia,
Q6 data from the RCPAQ6 external quality assurance
program (QAP) indicates that of some 55 participant
laboratories, the breakdown of test panels for VWD
investigation varies widely. About one third of lab-
oratories perform FVIII:C, VWF:Ag, and VWF:RCo,
about one quarter FVIII:C, VWF:Ag, VWF:RCo, and
VWF:CB, and the rest perform 10 other varying test
combinations.89–91 Reports from this QAP have consis-
tently shown that some test panels (notably incorporating
a VWF:CB) will result in substantially fewer diagnostic
errors than will one restricted to VWF:RCo as the sole
functional VWF assay.89–91
The problem is not restricted to this geographic
area or to general pathology laboratories. Several recent
genetic/phenotypic studies have recently been reported,
and these have identified error rates of around 20% for
presumed ‘‘expert’’ VWD diagnostic laboratories in
EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 627
terms of misidentifying type 2 VWD as type 1
VWD.92,93
Assessment of Antiphospholipid Antibodies
We gave the example previously where different VWF
test results might be obtained using different method-
ologies. However, different test results can also be
obtained using the supposed ‘‘same methodology.’’ A
good example here is the detection of aCL by ELISA,
where different laboratories testing the same sample may
obtain widely different test results, despite all test
methods purporting to test aCL.94,95 Whereas the test
results and methodologies comprise analytical issues, the
choice of which particular methodologies to use might
best be considered as a preanalytical variable. Further,
depending on the test methodologies and test panels in
place within particular laboratories, different test results
might be reported, and this might influence the clinical
perception regarding the presence of disease or not (i.e.,
postanalytical), and in this case whether or not the
patient has APS.
Test Methodology and Test Panel Selection
Different laboratories and even experts within the field
use different tests (or methodologies) and test panels for
the identification (or exclusion) of APS.94 Moreover,
there are wide variations in the detection of solid-phase
aPL by different commercial assays.94,95 Thus, 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.
Different tests also have different sensitivities and
specificities for ‘‘liquid-phase’’ aPL (i.e., LA testing).
According to the current International Society on
Thrombosis and Haemostasis (ISTH) SSCQ7 criteria Q7
for identification of LA, there is a need to (i) show
prolongation of clotting time in a LA-sensitive test,
(ii) demonstrate the presence of an inhibitor, usually
by plasma mixing studies, (iii) show that such prolonga-
tion/inhibitor is phospholipid (PL)-dependent (i.e., test
corrects with addition of PL), (iv) perform at least two
tests with both being negative before excluding LA, and
(iv) exclude other potential causes of coagulopathy that
might explain abnormal coagulation test results (e.g.,
factor inhibitors).71–74,94 In actual clinical and pathology
practice, however, different clinicians may order LA tests
in different ways (e.g., they may request an LA screen, a
KCT, or a dRVVTQ8). Laboratories are often only able Q8
to perform tests that the doctor actually orders. Thus, if a
doctor only orders (for example) an ‘‘RVVT’’ test, and if
LA is negative by this RVVT test, then the ISTH SSC
628 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7 2008

Q9 Table 5 SampleQ9 Interpretative Comments for Inclusion on Test Reports to Guide Appropriate Clinical Action
Test Test Result Sample Comment

Antithrombin Low level Reduced AT level detected. Congenital deficiencies of AT are

very rare. Low levels of AT may occur immediately after a
thrombotic episode, during heparin therapy, in liver disease,
or from a consumptive coagulopathy, hemodilution, in nephrotic
syndrome, after L-asparaginase therapy, or a blood collection
artifact (including hemolysis). Please exclude these events
and repeat the test 1 week after cessation of any
anticoagulant therapy.
Protein C and/ Low level Reduced protein C (and/or S) level detected. Congenital
or protein S deficiencies of protein C (and/or S) are very rare. Low levels of
protein C (and/or S) can occur immediately after a thrombotic
episode, with anticoagulant or vitamin K antagonist therapy
(e.g., warfarin), vitamin K deficiency, or liver disease, on hormone
replacement/oral contraceptive therapy/during pregnancy/with
nephrotic syndrome (protein S), or from a consumptive coagulopathy,
hemodilution, or a blood collection artifact. Please exclude these
events and repeat the test 6 weeks after cessation of any
anticoagulant therapy.
Protein C and/ Low level: some Might need comment regarding possibility of interference by APCR
or protein S methods (e.g., some depending on assay/reagents used.
clot-based assays)
Protein C and/ Elevated: clot-based assay Might need comment regarding possibility of interference from LA.
or protein S
Activated protein C Any result Individuals with LA, factor inhibitors, factor deficiencies,
resistance or on anticoagulant therapy may not provide reliable assay results.
Anticardiolipin Negative Some patients with antiphospholipid antibody syndrome have
antibody undetectable aCL. LA testing may be indicated.
Anticardiolipin Low/equivocal/positive The risk of clinical symptoms in the antiphospholipid antibody
antibody syndrome appears to rise with increasing levels of IgG aCL.
Repeat testing (after 12 weeks) is recommended, as is
LA testing. Transient low level/positive results generally are of
questionable clinical significance.
Anticardiolipin IgG negative/IgM positive IgM aCL are less specific than are IgG aCL for the antiphospholipid
antibody antibody syndrome. Transient IgM aCL may be found in a range of
other inflammatory, infectious, and malignant disorders,
and rheumatoid factors may also produce false-positive results.
Repeat testing (after 12 weeks) is recommended, as is LA testing.
von Willebrand Pattern suggestive of Results suggestive of type 2A or 2B or pseudo/platelet-type von
factor functional discordance Willebrand disease. Further studies may be indicated; please contact
between VWF:Ag and VWF:CB laboratory for advice, or else send repeat sample for retesting and
and/or VWF:RCo, or loss of confirmation. Please note: the following can all provide a false
high-molecular-weight type 2 VWD test pattern: testing of filtered plasma or serum sample,
VWF multimers or testing of plasma after the refrigeration or storage of whole blood
sample at low temperature.
von Willebrand Pattern suggestive of functional Results suggestive of hemophilia A, hemophilia A carrier,
factor discordance between acquired deficiency, or type 2N von Willebrand disease. Further
VWF:Ag and FVIII:C studies may be indicated.
Lupus anticoagulant Prolongation in screen LA (lupus inhibitor) not detected. However, screening test suggested
test test/mixing study, but negative potential presence of other inhibitor type. If patient on anticoagulant
for lupus anticoagulant by therapy (vitamin K antagonist or heparin), please repeat testing when
confirmation test therapy ceased. Otherwise, might indicate other inhibitor
(e.g., FV or FVIII); please discuss with laboratory as further testing
may be required.
 EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL 629

Table 5 (Continued )
Test Test Result Sample Comment

PFA-100 Prolonged closure time (CT) result Prolonged CT result with C/Epi, normal with C/ADP. Results consistent with
with C/Epi, normal with C/ADP any of the following: low platelet count, low hematocrit, recent antiplatelet
medication (e.g., aspirin), mild platelet dysfunction, and/or mild von
Willebrand disorder. Suggest medication review and full blood count.
Other studies may be indicated; please discuss with laboratory if required.
PFA-100 Prolonged closure time (CT) Prolonged CT result with both C/Epi and C/ADP. Results consistent with any
result with both C/Epi and C/ADP of the following: very low platelet count, very low hematocrit, recent
antiplatelet medication (e.g., aspirin), moderate to severe platelet dysfunction,
and/or moderate to severe von Willebrand disorder. Suggest medication
review and full blood count. Other studies may be indicated; please discuss
with laboratory if required.
PFA-100 Normal closure time (CT) result Normal CT result with both C/Epi and C/ADP. This result will not always
with both C/Epi and C/ADP discount a primary hemostasis disorder. If patient being investigated
for mucocutaneous bleeding, please discuss with laboratory as further
testing may be required.
Any test Additional add-on comment to Please contact the laboratory for further advice.
any of above comments.
Note: Some of these textual examples are overlong; the intention is that they be adapted for specific use in laboratories as per suitability. For
example, text comments related to pregnancy may be irrelevant to a pediatric hospital.

criteria are not being followed, and a false-negative LA is
feasible.73,94
New Emerging Tests: Thrombin Generation
and Detection of Microparticles
General coagulation function tests, principally the
thrombin-generation assay (TGA), enable the global
assessment of the hemostatic system. Such tests, how-
ever, are not as simple as the conventional clotting times
and require specific features that should be standardized
to achieve reliable and reproducible results. There are
several variables that can influence the tests, including
issues related to the sample collection (procedure, equip-
ment) and the sample matrix (defibrinated platelet-poor
plasma, nondefibrinated platelet-rich plasma, and even
whole blood).7,96 Another emerging test of relevance to
thrombosis is that of microparticle detection, and these
assays also pose a large number of potential preanalytical
considerations.97,98
POSTANALYTICAL ISSUES
Clinicians usually initiate some clinical action in re-
sponse to a test report. This action might have serious
adverse consequences if not appropriate to the clinical
situation. Whereas laboratories should not direct clinical
action, they should provide as much guidance as possible
to enable clinicians to make the best, most informed
choices for patient management. In this respect, how the
laboratory reports test results can have significant adverse
consequences if clinicians base their treatments on the
test report and this report fails to appropriately identify
the significance or non-significance of test results. An
example given at the start of this article is where a
laboratory merely reports a test result of a weakly positive
IgM aCL without advising that this has a low clinical
significance, and the clinician otherwise places too much
clinical significance upon the test result and makes a
diagnosis of APS and applies anticoagulant therapy
without further testing. Indeed, the situation with
APS is so potentially serious that within the geographic
locality of Australasia, a working party was formed and
has since published several guidelines for the appropriate
reporting of aCL and ab2GPI assays.99,100
Other hemostasis tests could also benefit from
specific guidance. For VWD, it is important that labo-
ratories advise on the significance or non-significance
of certain test results to ensure that clinicians do not
overdiagnose type 1 VWD or underdiagnose type
2 VWD.65,89 The former might feasibly occur when
test values fall below the normal reference range, and the
latter might feasibly occur because the laboratory is using
a limited test panel that may miss some forms of VWD.
For thrombophilia tests, it is important to highlight that
low test results for protein C, protein S, and antithrom-
bin does not necessarily mean that the patient has a
congenital deficiency, as the risk of a false positive (low
value) exceeds by several orders of magnitude the like-
lihood of a true positive.59–61 The situation is made
worse by poor patient test selection and inadequate
sampling (e.g., patient on anticoagulant therapy). There-
fore, the inclusion of interpretative comments on labo-
ratory reports is always an add-value, which enhances
visibility and competency of laboratory activity, as al-
ready mentioned.101 Such interpretative comments
630 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7 2008

Table 6 Effect of Inappropriate Sample Processing Issues on Select Hemostasis Tests

Issue Effect on Hemostasis Tests

Whole blood refrigerated prior to
centrifugation
Filtered plasma
Delayed transport, delayed testing,
poor storage, several freeze-thaw events;
storage in frost-free freezer
Poor centrifugation, heavy braking,
sample remixing prior to freezing
Platelet activation and loss of factor VIII and VWF; can lead to false diagnosis
of hemophilia or VWD.
Loss of fibrinogen, factor VIII, 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.
(1) Loss of labile factors (especially factors V and VIII); can lead to false
impression of hemophilia; prolongs routine coagulation test times (PT, APTT).
(2) Samples with unfractionated heparin can yield lower than expected APTTs
and lower anti-FXa (heparin) test levels. (3) Potential activation of FVII.
Platelet contamination, hemolysis and platelet disruption after freezing;
activation, false low APTT, false low heparin levels, false-negative LA,
false high factor levels.

might be particularly advisable when reporting results of
hemostasis testing. The laboratory and clinician also
need to reflect on the appropriateness of the NRR listed
on the test report. Some sample interpretative laboratory
comments are provided in Table 5.
CONCLUSION
Preanalytical and postanalytical issues in hemostasis test-
ing are a significant cause of diagnostic error and can lead
to significant adverse clinical events. This article has
detailed the situation with respect to a large number of
the more common tests employed within hemostasis, but
we would be foolish to assume that these issues are
limited to those described in this article. Tables 1–4
and 6 provide some useful summary data regarding
preanalytical issues, Table 5 provides sample interpreta-
tive laboratory comments to assist clinicians in the post-
analytical phase, and Table 7 provides a useful overall
summary of the main issues to consider, as well as some
simple recommendations. In the end, the most useful
advice we can offer is this: be aware of these issues, select/

Table 7 Important Issues for Laboratories and Clinicians to Consider within the Context of Extra-analytical Issues in
Hemostasis Testing, and Some Recommendations
Issue Consideration/Recommendation

Test selection Select/order the best tests/test processes/test panels for the condition being investigated
Population to be tested and Select the appropriate population/methodology to determine the normal reference range
clinical condition/medication
at time of testing
Only order the test(s) when clinically appropriate and in the right patient at the right time
Sample collection Proper patient and sample identification
Atraumatic phlebotomy with minimal tourniquet use
Draw 3.2% blue stopper tube first or only after a nonadditive tube
Fill tube adequately (no less than 90% fill)
Adequately and thoroughly mix with tube anticoagulant
Sample transport Transport promptly at room temperature
Sample processing Centrifuge within 1 hour of phlebotomy to obtain platelet-poor plasma (most tests)
Double-centrifuge plasma for some tests, namely LA and APCR
Aliquot (in a nonactivating secondary tube) immediately after centrifugation for those tests
to be performed later on
Special requirements for some tests such as platelet function and PFA-100
Sample storage Test plasma within appropriate time frame; store as required samples to be tested subsequently
Sample testing Select the best test/methodology/test panel for the analyte/parameter being tested
Perform test in timely manner and according to best practice
Result interpretation 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
 EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL
order the best tests and test panels available, undertake
testing only if necessary and at the correct point in time
for the condition under investigation, collect as much
clinical information as possible, follow the recommenda-
tions of local laboratory experts/specialists, repeat the
tests when not in keeping with clinical expectations or
when an abnormal finding is reported, and establish a
mutually beneficial clinical-laboratory interface where
both parties actively collaborate to achieve the best
possible patient outcome.
REFERENCES
1. Lippi G, Guidi GC. Risk management in the preanalytical
phase of laboratory testing. Clin Chem Lab Med 2007;45:
720–727
2. Lippi G, Guidi GC, Mattiuzzi C, Plebani M. Pre-analytical
variability: the dark side of the moon in laboratory testing.
Clin Chem Lab Med 2006;44:358–365
3. Lippi G, Banfi G, Buttarello M, et al.for the Italian
Intersociety SIBioCSIMeL-CISMEL Study Group on
Extraanalytical Variability Recommendations for detection
and management of unsuitable samples in clinical laborato-
ries. Clin Chem Lab Med 2007;45:728–736
4. Lippi G, Fostini R, Guidi GC. Quality improvement in
laboratory medicine: extra-analytical issues. Clin Lab Med
2008;28:285–294
5. Adcock DM, Hoefner DM, Kottke-Marchant K, Marlar
RA, Szamosi DI, Warunek DJ. Collection, Transport, and
Processing of Blood Specimens for Testing Plasma-Based
Coagulation Assays and Molecular Hemostasis Assays:
Approved Guideline-Fifth Edition. CLSI document H21–
A5. Wayne, PA: Clinical Laboratory Standards Institute;
2008
6. Plebani M. The clinical importance of laboratory reasoning.
Clin Chim Acta 1999;280:35–45
7. Lippi G, Franchini M, Montagnana M, Salvagno GL, Poli
G, Guidi GC. Quality and reliability of routine coagulation
testing: can we trust that sample?. Blood Coagul Fibrinolysis
2006;17:513–519
8. Kiechle FL, Adcock DM, Calam RR, Davis C, Schwartz
JG. So You’re Going to Collect a Blood Specimen. An
Introduction to Phlebotomy. 12th ed. Northfield, IL:
College of American Pathologists; 2007
9. Joint Commission on Accreditation of Healthcare Organ-
izations. 2008 National Patient Safety Goals. Available at:
http://www.jointcommission.org/PatientSafety/National
PatientSafetyGoals/08_amb_npsgs.htm. Accessed August
16, 2008
10. Payne S, MacKinnon K, Keeney M, Morrow B, Kovacs
MJ. Effect of 3.2 vs. 3.8% sodium citrate concentration
on anti-Xa levels for patients on therapeutic low
molecular weight heparin. Clin Lab Haematol 2003;25:
317–319
11. Danielson CF, Davis K, Jones G, Benson J, Arney K,
Martin J. Effect of citrate concentration in specimen
collection tubes on the international normalized ratio. Arch
Pathol Lab Med 1997;121:956–959
12. Adcock DM, Kressin DC, Marlar RA. Effect of 3.2% vs
3.8% sodium citrate concentration on routine coagulation
testing. Am J Clin Pathol 1997;107:105–110
631
13. Lippi G, Salvagno GL, Montagnana M, Guidi GC.
Influence of two different buffered sodium citrate concen-
trations on coagulation testing. Blood Coagul Fibrinolysis
2005;16:381–383
14. Jilma B. Platelet function analyzer (PFA-100): a tool to
quantify congenital or acquired platelet dysfunction. J Lab
Clin Med 2001;138:152–163
15. Crescente M, Di Castelnuovo A, Iacoviello L, Vermylen J,
Cerletti C, de Gaetano G. Response variability to aspirin as
assessed by the platelet function analyzer (PFA)-100. A
systematic review. Thromb Haemost 2008;99:14–26
16. Ernst DJ, Balance LO, Calam RR, et al. Procedures for the
Collection of Diagnostic Blood Specimens by Venipuncture.
Approved Standard-Sixth Edition. CLSI document H3–
A6. Wayne, PA: Clinical and Laboratory Standards
Institute; 2007
17. Adcock DM, Kressin DC, Marlar RA. Are discard tubes
necessary in coagulation studies?. Lab Med 1997;28:530–
533Q10 Q10
18. Lippi G, Guidi GC. Effect of specimen collection on
routine coagulation assays and D-dimer measurement. Clin
Chem 2004;50:2150–2152
19. Adcock DM, Kressin DC, Marlar RA. Minimum specimen
volume requirements for routine coagulation testing.
Dependence on citrate concentration. Am J Clin Pathol
1998;109:595–599
20. Chuang J, Sadler MA, Witt DM. Impact of evacuated
collection tube fill volume and mixing on routine coagu-
lation testing using 2.5 mL (pediatric) tubes. Chest 2004;
126:1262–1266
21. Adcock DM. Sample integrity and preanalytical variables.
In: Preston E, Olsen J, Kitchen S, eds. Quality in
Laboratory Hemostasis and Thrombosis. In pressQ11 Q11
22. Lippi G, Salvagno GL, Montagnana M, Guidi GC.
Influence of primary sample mixing on routine coagulation
testing. Blood Coagul Fibrinolysis 2007;18:709–711
23. Laxson CJ, Titler MG. Drawing coagulation studies from
arterial lines. An integrative literature review. Am J Crit
Care 1994;3:16–22
24. Powers JM. Obtaining blood samples for coagulation studies
from a normal saline lock. Am J Crit Care 1999;8:250–253
25. Lippi G, Salvagno GL, Montagnana M, Poli G, Guidi GC.
Influence of the needle bore size on platelet count and
routine coagulation testing. Blood Coagul Fibrinolysis
2006;17:557–561
26. Lippi G, Salvagno GL, Guidi GC. No influence of butterfly
device on routine coagulation assays and D-dimer measure-
ment. J Thromb Haemost 2005;3:389–391
27. Lippi G, Salvagno GL, Montagnana M, Guidi GC. Short-
term venous stasis influences routine coagulation testing.
Blood Coagul Fibrinolysis 2005;16:453–458
28. Zürcher M, Sulzer I, Barizzi G, Lämmle B, Alberio L.
Stability of coagulation assays performed in plasma from
citrated whole blood transported at ambient temperature.
Thromb Haemost 2008;99:416–426
29. Adcock D, Kressin D, Marlar RA. The effect of time and
temperature variables on routine coagulation tests. Blood
Coagul Fibrinolysis 1998;9:463–470
30. Lippi G, Salvagno GL, Montagnana M, Poli G, Guidi GC.
Influence of centrifuge temperature on routine coagulation
testing. Clin Chem 2006;52:537–538
31. Lippi G, Salvagno GL, Montagnana M, Manzato F, Guidi
GC. Influence of the centrifuge time of primary plasma tubes
632 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7
on routine coagulation testing. Blood Coagul Fibrinolysis
2007;18:525–528
32. Aursnes I, Vikholm V. On possible interaction between
ADP and mechanical stimulation of platelet activation.
Thromb Haemost 1984;51:54–56
33. Favaloro EJ, Mohammed A, Coombs R, Mehrabani PA.
Filtered plasma as a potential cause of clinical misdiagnosis:
inappropriate testing in a haematology laboratory. Br J
Biomed Sci 1995;52:243–248
34. Favaloro EJ, Facey D, Grispo L. Laboratory assessment of
von Willebrand factor: use of different assays can influence
the diagnosis of von Willebrand’s disease, depending on
differing sensitivity to sample preparation and differential
recognition of high molecular weight VWF forms. Am J
Clin Pathol 1995;104:264–271
35. Favaloro EJ. Pre-analytical variables in coagulation testing.
Blood Coagul Fibrinolysis 2007;18:86–89
36. van Geest-Daalderop JH, Mulder AB, Boonman-de Winter
LJ, Hoekstra MM, van den Besselaar AM. Preanalytical
variables and off-site blood collection: influences on the
results of the prothrombin time/international normalized
ratio test and implications for monitoring of oral anti-
coagulant therapy. Clin Chem 2005;51:561–568
Q12 37. Salvagno GL, Q12Lippi G, Montagnana M, Franchini M,
Poli G, Guidi GC. Influence of temperature and time before
centrifugation of specimens for routine coagulation testing.
Int J Lab Hematol 2008;(Mar): 25 [Epub ahead of print]
38. Favaloro EJ, Mehrabani PA. Laboratory assessment of von
Willebrand factor: differential influence of prolonged
ambient temperature specimen storage on assay results.
Q13 Haemophilia 1996;2:218–223Q13
39. Favaloro EJ, Nair SC, Forsyth CJ. Collection and transport
of samples for laboratory testing in von Willebrand’s disease
(VWD): time for a reappraisal?. Thromb Haemost 2001;86:
1589–1590
40. Favaloro EJ, Soltani S, McDonald J. Potential laboratory
misdiagnosis of haemophilia and von Willebrand disorder
due to cold activation of blood samples for testing. Am J
Clin Pathol 2004;122:686–692
41. Böhm M, Täschner S, Kretzschmar E, Gerlach R, Favaloro
EJ, Scharrer I. Cold storage of citrated whole blood induces
drastic time-dependent losses in factor VIII and von
Willebrand factor: Potential for misdiagnosis of haemo-
philia and von Willebrand’s disease. Blood Coagul
Fibrinolysis 2006;17:39–45
42. Woodhams B, Giradot O, Blanco MJ, Colesse G,
Gourmelin Y. Stability of coagulation proteins in frozen
plasma. Blood Coagul Fibrinolysis 2001;12:229–236
43. Lippi G, Salvagno GL, Rossi L, Montagnana M, Franchini
M, Guidi GC. Analytical performances of the D-dimer
assay for the Immulite 2000 automated immunoassay
analyser. Int J Lab Hematol 2007;29:415–420
44. Babson AL, Opper CA, Crane LJ. Kinetic latex agglutin-
ometry I. A rapid, quantitative immunologic assay for
fibrinogen. Am J Clin Pathol 1982;77:424–429
45. Favaloro EJ, Bonar R, Duncan E, et al. on behalf of the
RCPA QAP in Haematology Haemostasis Committee).
Identification of factor inhibitors by diagnostic haemostasis
laboratories: a large multi-centre evaluation. Thromb Hae-
most 2006;96:73–78
46. Favaloro EJ, Bonar R, Duncan E, et al. on behalf of the
RCPA QAP in Haematology Haemostasis Committee).
Mis-identification of factor inhibitors by diagnostic haemo-
2008
stasis laboratories: recognition of pitfalls and elucidation of
strategies. A follow up to a large multicentre evaluation.
Pathology 2007;39:504–511
47. Kottke-Marchant K, Duncan A. Antithrombin deficiency:
issues in laboratory diagnosis. Arch Pathol Lab Med 2002;
126:1326–1336
48. Lippi G, Blanckaert N, Bonini P, et al. Haemolysis: an
overview of the leading cause of unsuitable specimens in
clinical laboratories. Clin Chem Lab Med 2008;46:764–772
49. Lippi G, Montagnana M, Salvagno GL, Guidi GC.
Interference of blood cell lysis on routine coagulation
testing. Arch Pathol Lab Med 2006;130:181–184
50. van der Merwe L, Reyers F. The effect of hemolysis on
plasma antithrombin activity as determined by a chromo-
genic method. Vet Clin Pathol 2007;36:55–59
51. Marlar RA, Potts RM, Marlar AA. Effect of routine and
special coagulation testing values of citrate anticoagulant
adjustment in patients with high hematocrit values. Am J
Clin Pathol 2006;126:400–405
52. O’Brien JR. Relation to blood-coagulation to lipaemia.
Lancet 1955;269:690–693
53. Larsen LF, Bladbjerg EM, Jespersen J, Marckmann P.
Effects of dietary fat quality and quantity on postprandial
activation of blood coagulation factor VII. Arterioscler
Thromb Vasc Biol 1997;17:2904–2909
54. Freese R, Mutanen M. Postprandial changes in platelet
function and coagulation factors after high-fat meals with
different fatty acid compositions. Eur J Clin Nutr 1995;49:
658–664
55. Junker R, Pieke B, Schulte H, et al. Changes in hemostasis
during treatment of hypertriglyceridemia with a diet rich in
monounsaturated and n-3 polyunsaturated fatty acids in
comparison with a low-fat diet. Thromb Res 2001;101:
355–366
56. Moreno P, Ginel PJ. Effects of haemolysis, lipaemia and
bilirubinaemia on prothrombin time, activated partial
thromboplastin time and thrombin time in plasma samples
from healthy dogs. Res Vet Sci 1999;67:273–276
57. Law RC, Willatts SM. Lipaemia affecting automated
clotting results. Anaesthesia 1993;48:1011
58. Junker R, Käse M, Schulte H, Bäumer R, Langer C,
Nowak-Göttl U. Interferences in coagulation tests—evalua-
tion of the 570-nm method on the Dade Behring BCS
analyzer. Clin Chem Lab Med 2005;43:244–252
59. Favaloro EJ, Soltani S, McDonald J, Grezchnik E, Easton
L. Laboratory identification of familial thrombophilia: do
the pitfalls exceed the benefits? A reassessment of ABO-
blood group, gender, age and other laboratory parameters on
the potential influence on a diagnosis of protein C, protein S
and antithrombin deficiency and the potential high risk of a
false positive diagnosis. Lab Hematol 2005;11:174–184
60. Favaloro EJ. Diagnostic issues in thrombophilia: a
laboratory scientist’s view. Semin Thromb Hemost 2005;
31:11–16
61. Favaloro EJ. More on ‘universal’ versus ‘selected’ screening
for thrombophilia: the hidden costs of false-positive
diagnosis. Br J Haematol 2006;134:239–240
62. Lippi G, Salvagno GL, Rugolotto S, et al. Routine
coagulation tests in newborn and young infants. J Thromb
Thrombolysis 2007;24:153–155
63. Favaloro EJ, Soltani S, McDonald J, Grezchnik E, Easton
L, Favaloro JWC. Reassessment of ABO-blood group,
gender and age on laboratory parameters used to diagnose
 EXTRA-ANALYTICAL VARIABLES IN HEMOSTASIS/FAVALORO ET AL
von Willebrand disorder (VWD): potential influence on the
diagnosis versus the potential association with risk of
thrombosis. Am J Clin Pathol 2005;124:910–917
64. Monagle P, Barnes C, Ignjatovic V, et al. Developmental
haemostasis. Impact for clinical haemostasis laboratories.
Thromb Haemost 2006;95:362–372
65. Favaloro EJ. Laboratory identification of von Willebrand
disease: technical and scientific perspectives. Semin Thromb
Hemost 2006;32:456–471
66. Lippi G, Franchini M, Brocco G, Manzato F. Influence of
the ABO blood type on the platelet function analyzer PFA-
100. Thromb Haemost 2001;85:369–370
67. Favaloro EJ, Mehrabani PA, Koutts J. Laboratory assess-
ment of von Willebrand factor: altered interpretation of
laboratory data, and altered diagnosis of von Willebrand’s
disease, as influenced by the use of different vWF assays
and assay conditions. Clin Appl Thromb Hemost 1997;
Q14 3:110–118Q14
68. Adcock DM, Brien WF, Duff SL, et al. Procedures for
Validation of INR and Local Calibration of PT/INR
Systems; Approved Guideline. H54-A, Vol. 25 No. 23
(replaces H54-P, Vol. 24 No. 30). Wayne, PA: Clinical and
Laboratory Standards Institute; 2005
69. Favaloro EJ, Hamdam S, McDonald J, McVicker W, Ule V.
Time to think outside the box? Prothrombin time (PT),
international normalized ratio (INR), international sensi-
tivity index (ISI), mean normal prothrombin time (MNPT)
and measurement of uncertainty (MU): a novel approach to
standardisation. Pathology 2008;40:277–287
Q15 70. Favaloro EJ, Q15Adcock DM. Standardization of the INR:
how good is your laboratory’s INR and can it be improved?
Semin Thromb Hemost 2008;34: current issue
71. Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for
the diagnosis of lupus anticoagulants: an update. On behalf
of the Subcommittee on Lupus Anticoagulant/Antiphos-
pholipid Antibody of the Scientific and Standardisation
Committee of the ISTH. Thromb Haemost 1995;74:1185–
1190
72. Greaves M, Cohen H, Machin SJ, Mackie I. Guidelines on
the investigation and management of the antiphospholipid
syndrome. Br J Haematol 2000;109:704–715
73. Ledford-Kraemer MR. Laboratory testing for lupus anti-
coagulants: pre-examination variables, mixing studies and
diagnostic criteria. Semin Thromb Hemost 2008;34:380–388
74. Tripodi A. Laboratory testing for lupus anticoagulants:
diagnostic criteria and use of screening, mixing and
confirmatory studies. Semin Thromb Hemost 2008;34:
373–379
75. Carroll WE, Wollitzer AO, Harris L, Ling MC, Whitaker
WL, Jackson RD. The significance of platelet counts in
coagulation studies. J Med 2001;32:83–96
76. Watson HG, Greaves M. Can we predict bleeding? Semin
Thromb Hemost 2008;34:97–103
77. Chee YL, Crawford JC, Watson HG, Greaves M. Guide-
lines on the assessment of bleeding risk prior to surgery or
invasive procedures. British Committee for Standards in
Haematology. Br J Haematol 2008;140:496–504
78. Lippi G, Franchini M, Brazzarola P, Manzato F. Preop-
erative screening: the rationale of measuring APTT in risk
assessment. Haematologica 2001;86:328
79. Lippi G, Salvagno GL, Montagana M, Guidi GC. Chronic
influence of vigorous aerobic training on hemostasis. Blood
Coagul Fibrinolysis 2005;16:533–534
633
80. Banfi GCommentStCommentEndCommentBody, Del Fab-
bro M. Biological variation in tests of haemostasis. Semin
Thromb Hemost 2008;34: In pressQ16 Q16
81. Spencer FA, Becker RC. Circadian variations in acute
myocardial infarction. J Am Coll Cardiol 2003;41:2143–2146
82. Dalby MC, Davidson SJ, Burman JF, Davies SW. Diurnal
variation in platelet aggregation iwth the PFA-100 platelet
function analyser. Platelets 2000;11:320–324
83. Bethel M, Adcock D, Zalevsky J, Young M, Fagrell B.
Polyethylene glycol-induced prolongation of aPTT in two
biopharmaceuticals. Int J Lab Hematol 2007;29(Suppl 1):69
(Abst)
84. Webster PS, Oleson FBJr, Paterson DL, et al. Interaction of
daptomycin with two recombinant thromboplastin reagents
leads to falsely prolonged patient prothrombin time/interna-
tional normalized ratio results. Blood Coagul Fibrinolysis
2008;19:32–38
85. Georgiou A, Westbrook JI. Computerized order entry
systems and pathology services—a synthesis of the evidence.
Clin Biochem Rev 2006;27:79–87
86. Favaloro EJ, Bonar R, Sioufi J, et al. (on behalf of the
RCPA QAP in Haematology). Multi-laboratory testing of
thrombophilia: current and past practice in Australasia as
assessed through the Royal College of Pathologists of
Australasia Quality Assurance Program in Haematology.
Semin Thromb Hemost 2005;31:49–58
87. Favaloro EJ, Orsag I, Bukuya M, McDonald D. A nine-year
retrospective assessment of laboratory testing for activated
protein C resistance: evolution of a novel approach to
thrombophilia investigations. Pathology 2002;34:348–355
88. Favaloro EJ, Mirochnik O, McDonald D. Functional
activated protein C resistance assays: correlation with factor
V DNA analysis is better with RVVT- than APTT-based
assays. Br J Biomed Sci 1999;56:23–33
89. Favaloro EJ, Bonar R, Kershaw G, et al. (on behalf of the
RCPA QAP in Haematology). Laboratory diagnosis of von
Willebrand disorder: Use of multiple functional assays
reduces diagnostic error rates. Lab Hematol 2005;11:91–97
90. Favaloro EJ, Bonar R, Kershaw G, et al. (on behalf of the
RCPA QAP in Haematology). Reducing errors in identi-
fication of von Willebrand disease: the experience of the Royal
College of Pathologists of Australasia Quality Assurance
Program. Semin Thromb Hemost 2006;32:505–513
91. Favaloro EJ, Bonar R, Meiring M, Street A, Marsden K.
(on behalf of the RCPA QAP in Haematology). 2B or not
2B? Disparate discrimination of functional VWF discord-
ance using different assay panels or methodologies may lead
to success or failure in the early identification of type 2B
VWD. Thromb Haemost 2007;98:346–358
92. Favaloro EJ. An update on the von Willebrand factor
collagen binding (VWF:CB) assay: 21 years of age and
beyond adolescence, but not yet a mature adult. Semin
Thromb Hemost 2007;33:727–744
93. Favaloro EJQ17. Detailed von Willebrand factor multimer Q17
analysis in patients with von Willebrand disease in the
European study, molecular and clinical markers for the
diagnosis and management of type 1 von Willebrand disease
(MCMDM-1VWD) – a rebuttal. J Thromb Haemost 2008;
In press
94. Favaloro EJ, Wong RCW. Laboratory testing and identi-
fication of antiphospholipid antibodies and the antiphospho-
lipid syndrome: a potpourri of problems, a compilation of
possible solutions. Semin Thromb Hemost 2008;34:389–410
634 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER 7
95. Reber G, Boehlen F, de Moerloose P. Technical aspects in
laboratory testing for antiphospholipid antibodies: is stand-
ardization an impossible dream? Semin Thromb Hemost
2008;34:340–346
96. Berntorp E, Salvagno GL. Standardization and clinical
utility of thrombin generation assays. Semin Thromb
Q18 Hemost 2008;34: In pressQ18
97. Enjeti AK, Lincz LF, Seldon M. Detection and measure-
ment of microparticles: an evolving research tool for
vascular biology. Semin Thromb Hemost 2007;33:771–
779
2008
98. Enjeti AK, Lincz L, Seldon M. Microparticles in health and
disease. Semin Thromb Hemost 2008;34: In pressQ19 Q19
99. Wong RC, Gillis D, Adelstein S, et al. Consensus
guidelines on anti-cardiolipin antibody testing and report-
ing. Pathology 2004;36:63–68
100. Wong RC, Favaloro EJ, Adelstein S, et al. Consensus
guidelines on anti-beta2 glycoprotein I testing and report-
ing. Pathology 2008;40:58–63
101. Plebani M. What information on quality specifications
should be communicated to clinicians, and how?. Clin Chim
Acta 2004;346:25–35
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As an added benefit to all contributing authors, a discount is offered on all Thieme books.
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As a Thieme author you are entitled to a 25% discount for new books and a 35%
discount for forthcoming books.
We selected two books that might be of interest for you:
new! 25%
Thurlbeck's
Pathology of the Lung
rd
3 Edition
Andrew M. Churg, M.D. Ph. D.
Professor of Pathology, University of British
Columbia; Pathologist, Vancouver Hospital &
Health Sciences Center, Vancouver, BC,
Canada
Thurlbeck's cornerstone textbook and reference on pulmonary
pathology returns in a brand new edition! Updated with the latest
advances in the field, you will save time with all-inclusive coverage of
neoplastic, non-neoplastic, infectious, occupational/environmental, and
developmental pathologies in one book, learn how molecular biology
provides a greater understanding of lung development, gain new
insights into the diagnosis of neoplastic and non-neoplastic lung
disease, find pertinent information on clinical features, epidemiology,
and pathogenetic mechanisms of lung disease and much more!
Comprehensive in its scope and authoritative in its scholarship,
Thurlbeck's Pathology of the Lung is a virtual one-volume encyclopedia
written by a ''who's who'' list of specialists. It is the one text that no
pathologist, pulmonologist, or resident in either specialty can afford to
be without.
forthcoming! 35%
Vascular Diagnosis with
Ultrasound
Cerebral and Peripheral
Vessels
Michael Hennerici, M.D.
Professor and Chairman, Department
of Neurology, University of Heidelberg,
Mannheim, Germany
Covering the entire venous and body circulation as examined
by vascular ultrasound, this unique text/atlas is invaluable for
diagnosing arterial and venous disease. It includes
comprehensive chapters on vascular ultrasonography in the
arteries and veins of the cerebral circulation and the
peripheral upper and lower limb circulation, systematic
coverage of all available ultrasound technologies, including
continuous and pulsed-wave Doppler mode, b-mode, and
conventional and color-coded duplex analysis in frequency
and amplitude power modes, anatomy and physiology,
normal and abnormal findings, test accuracy and sensitivity,
pitfalls, and comparison with other diagnostic tests in each
vascular region and special, difficult-to-interpret cases
discussed in a separate section

2005, app.1032 pp., app.1064 illus., hardcover $249.95 $187.46 2006, 336 pp., 530 illus., hardcover, $149.95 $97.47
ISBN 1-58890-288-9 ISBN 1-58890-144-0

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