Review article 513
Quality and reliability of routine coagulation testing: can we
trust that sample?
Giuseppe Lippia, Massimo Franchinib, Martina Montagnanaa, Gian Luca
Salvagnoa, Giovanni Polia and Gian Cesare Guidia
Poor standardization of preanalytic variables exerts
a strong influence on the reliability of coagulation
testing, consuming valuable health care resources and
compromising patient outcome. Most uncertainties
emerge from patient misidentification and the procedures
for specimen collection and handling. Location of
unsuitable venous access or problematic phlebotomies
may produce spurious activation of the hemostatic system
and hemolytic specimens. Prolonged venous stasis is
associated with hemoconcentration and spurious variations
of most coagulation assays. Additional pitfalls can be
introduced by inappropriate phlebotomy tools
and small-gauge needles. Inappropriate filling and
mixing of the tube, unsuitable procedures for centrifugation
and storage of the specimens are additional aspects that
need accurate standardization. Besides traditional
preanalytic variables affecting routine coagulation testing,
thrombin-generation assays require specific criteria to be
accurately fulfilled. These aspects include the type of
specimen (platelet-poor plasma, platelet-rich plasma or
whole blood), blood collection tubes, storage conditions
and the presence of residual platelets. Compliance
with new international quality assessment programs,
Introduction
Routine coagulation testing, traditionally comprised of
prothrombin time (PT), activated partial thromboplastin
time (APTT), fibrinogen and D-dimer assessment, is an
integral part of the clinical decision-making process, as it
often strongly influences diagnosis and therapy [1–3]. In
analogy with other areas of laboratory diagnostics, the
activity is traditionally defined as a three-part process,
which develops within the preanalytic, analytic and post-
analytic phases. There is consolidated evidence that lack
of standardization and monitoring of several preanalytic
variables, including procedures for patient identification,
sample collection, handling, and processing before
analysis, has an adverse influence on the reliability of test
results, consuming valuable health care resources and
compromising the patient’s outcome [4]. Considerable
advances in analytic techniques, laboratory instrumenta-
tion, automation and organization have granted an excep-
tional degree of analytic quality over the past 50 years.
Moreover, total analytic quality systems, along with
accreditation and certification programs, have started to
arise in all sectors of laboratory diagnostics, reducing the
burden of unnecessary expenditure on health care systems
0957-5235 ß 2006 Lippincott Williams & Wilkins
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
which will also involve coagulation laboratories,
encompasses the adoption of suitable strategies for
reducing undue variability throughout the whole
testing process. Such strategies would not entail
extraordinary costs and are affordable with a
structured outlay of existing resources,
educational policies and compliance with reliable
guidelines. Blood Coagul Fibrinolysis 17:513–519 ß 2006
Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2006, 17:513–519
Keywords: coagulation testing, preanalytic variability, quality
a
Istituto di Chimica e Microscopia Clinica, Dipartimento di Scienze Morfologico-
Biomediche, Università degli Studi di Verona, Verona and bServizio di
Immunematologia e Trasfusione, Azienda Ospedaliera di Verona, Verona, Italy
Correspondence and requests for reprints to Prof. Giuseppe Lippi, MD, Istituto di
Chimica e Microscopia Clinica, Dipartimento di Scienze Morfologico-Biomediche,
Università degli Studi di Verona, Ospedale Policlinico G.B. Rossi, Piazzale Scuro
10, 37121 Verona, Italy
Tel: + 39 45 8074517; fax: +39 45 8201889; e-mail: ulippi@tin.it
Received 12 March 2006 Accepted 20 May 2006
and improving consumer confidence and safety. Never-
theless, reliability and quality of routine coagulation test-
ing performances might still be substantially enhanced
[5,6]. There is consolidated evidence that most testing
errors fall outside the analytic phase, involving operations
that escape the direct control or supervision of the
laboratory staff and that are mostly concerning blood
sample collection and handling [7–9]. Most unsuitable
testing samples result from hemolysis, insufficient
quantity, clotting, being lost or not received in the labora-
tory, inadequately labeled or misidentified [7–9]. The
preanalytic phase therefore seems to present the greatest
potential for quality improvement, once reliable strategies
are identified and properly applied [4]. There are several
occasions for decreasing reliability and quality of routine
coagulation testing in this phase, some of which are mutual
with other areas of laboratory diagnostics — others are
peculiar.
Specimen collection
Phlebotomy is as yet one of the most neglected pro-
cedures in healthcare, although it suffers from a high
degree of preanalytic variability and still involves serious
 514 Blood Coagulation and Fibrinolysis 2006, Vol 17 No 7
health risks for both patient and operator [10]. Shortages
of skilled staff and overloaded phlebotomy facilities have
amplified the potential for easily preventable errors. The
introduction of disposable straight needles and evacuated
tube systems has allowed collection of consistent quality
blood specimens, with additional advantages from both a
safety and a practical point of view. However, the whole
procedure and the success rate itself are as yet influenced
by several aspects. Identification errors can occur during
any part of the test cycle, mostly involving phlebotomy
facilities where a great deal of specimens are drawn daily
and the turnover of phlebotomists is particularly high [7].
Proper patient identification therefore remains a central
issue. Specimen collection policies are supposed to com-
ply with accurate verification of the patient identity,
and the widespread adoption of bar-coded wristbands
would entail a great potential for decreasing patient
identification errors [11].
Location of an appropriate vein is an essential requisite to
ensure successful drawing and quality specimens. The
antecubital area is the most suited for phlebotomy, and
the vein of choice for venous blood drawing should be the
median cubital vein. Unfortunately the antecubital area is
not always easily accessible for venipuncture, and sec-
ondary venous accesses should be regarded as altern-
atives. Veins of the legs, ankles and feet should
preferably not be accessed, as blood in the inferior limb
veins can undergo changes in coagulability due to contact
with atherosclerotic plaques in arteries [12]. Fistulas,
shunts, arterial lines, locks, arteries, femoral and varicose
veins, and veins of the arm or hand from the side of a
mastectomy are not recommended, unless traditional
sites have been ruled out and the permission of the
primary care physician can be obtained [13]. An improper
choice of site, such as drawing venous blood from a site
distal to the antecubital region of the arm, has also
resulted in more unsuitable samples for hemolysis, as
well as cleansing the venipuncture site with alcohol
detergent and not allowing it to dry properly [14].
The association of straight needles with evacuated tube
collection systems has revolutionized the blood collection
technique, yielding substantial advances over ordinary
syringes [15]. Nevertheless, venous blood is frequently
collected by alternative techniques, employing infusive
routes or butterfly devices. These disposals, originally
developed for administration of infusive therapies, would
also be applied to draw blood in some cases, such as
patients with permanent subcutaneous venous cannula-
tion who undergo permanent dialytic processes or receive
long-term infusive therapies, anesthesia or sedation
before surgery, or noxious clinical and diagnostic pro-
cedures. Blood collection by butterfly systems might also
be advisable in newborns, children and patients with
small, difficult and secondary venous access [16]. Finally,
the less intimidating butterfly device is seldom used in
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
nervous or anxious patients, who suffer from so-called
‘needle phobia’ [17]. There are several disadvantages
when using butterfly devices for collecting venous blood,
such as the greater cost, the major chance of obtaining
unsuitable samples (incomplete filling of the tube, hemo-
lysis, activated or clotted samples) and the increased
health risk for the operators. Although little scientific
evidence so far exists on the reliability of routine coagu-
lation testing using alternative techniques to draw blood
rather than straight needles associated with evacuated
tubes, infusive devices do not apparently result in
dramatic activation of the hemostatic system within
the long plastic tubing. As these variations are globally
modest and clinically negligible, butterfly or winged
infusion devices might be proposed as suitable altern-
atives, when properly used and within certain limitations
[18], provided that heparin contamination is accurately
prevented [19,20] and a minimum amount of blood is
discarded after initial flushing [21].
Venipuncture using needles is unavoidable when collect-
ing venous blood specimens. Needles are basically cali-
brated by gauge (G), which refers to the diameter of the
needle in millimeters. The larger the gauge number,
the smaller the diameter of the bore. Venipunctures
are usually performed with needles ranging from 19 to
25 G; 19–21 G needles are used primarily for large ante-
cubital veins, and 23 G or smaller for secondary veins.
Although there are no definitive data on the influence of
the needle bore size on results of routine coagulation
testing, there is general consensus that blood should be
drawn carefully to avoid excessive pressure or shear
stress, which is associated with damage or rupture of
blood cells, especially erythrocytes. In fact, the major
problem encountered when using small-bore needles in
association with evacuated tubes is the large vacuum
force applied to the blood, which may cause shear stress
on the erythrocytes, enhancing the risk of in-vitro hemo-
lysis [22] and blood clotting, causing needle occlusion and
increasing coagulation activity [23,24], especially when
needleless connectors are used [25]. Although both the
PT and APTT display a trend towards lower values
in specimens collected by smaller needles ( 23 G),
results are not significantly or clinically different from
those assayed in samples collected by a reference larger
needle (21 G). A significant increase of D-dimer values
can be observed in specimens collected by very small
needles (25 G), although such a bias should not
be considered clinically meaningful [26]. Small-bore
needles, along with butterfly devices, may therefore be
used to draw venous blood for routine coagulation testing
in selected clinical settings.
Tourniquet placing is commonplace before routine venous
venipuncture to assist the phlebotomist in locating a
suitable vein. The tourniquet should ideally be tight
enough to obstruct venous but not arterial flow, without

causing discomfort to the patient. It should be removed as
soon as the blood flow is established. Phlebotomy is
expected to be a fast process, although several circum-
stances might contribute to lengthening considerably the
time since tourniquet tying (location of an appropriate
vein, selection of the blood collection system, collection of
many tubes). Regardless of anecdotal evidence that pro-
longed venous stasis influences the concentration and/or
the activity of several analytes in blood, the tourniquet
time is rarely regarded as a potential source of variability.
The pattern of changes observed after a prolonged venous
stasis depends mostly upon the length of the stasis, the size
and the protein-binding characteristic of the analyte. The
increased intravascular pressure is the major mechanism
responsible for increments of larger constituents and
protein-bound substances in blood (hemoconcentration),
whereas a consistent decrease is usually observed for
smaller analytes, especially electrolytes [27,28]. Following
a 1–3 min standardized venous stasis, reproducing the
tourniquet placement, significant differences are observed
for all parameters of the routine coagulation testing,
achieving clinical significance for the PT, fibrinogen and
D-dimer [28]. Venous stasis during specimen collection
should therefore be regarded as a considerable source of
variability, which should be anticipated and potentially
corrected, on the basis of standardized procedures for
blood drawing (nonapplication of the tourniquet in
patients with easy and prominent veins, early release after
the needle insertion, standardization of the external pres-
sure by easy-to-apply, re/de-inflatable electronic devices,
or precise records establishing the time elapsed between
sample collection and tourniquet placing). Finally, if many
tubes are to be collected, a rigorous and uniform sequence
should be respected [28].
Specimen handling and storage
Assay interferences from improper handling of blood
collection tubes represent challenges to coagulation
laboratories, because they are not detected by conven-
tional quality-control or proficiency testing programs.
There is still debate on the most appropriate sequence
of drawing tubes for routine coagulation testing. Devel-
oped in the mid-1970s, the so-called ‘order of draw’ was
aimed to prevent the effects on test results that tube
additives could introduce in a sequence, with the needle
carrying on some contamination. In the following years,
the order has undergone radical changes, including sep-
arate indications when tubes are filled by syringe [29]. In
1998, the standards organization recommended a single
order of draw for both tube-holder collection and syringe
draws. Since then, the widespread use of plastic serum
tubes containing clot activators required the develop-
ment of a modified order. Accordingly, two distinct sets
of instructions have been developed for glass and plastic
tubes [30]. In 2003, the National Committee for Clinical
Laboratory Standards announced further changes affect-
ing the procedures of the collection of diagnostic blood
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Preanalytic variability and coagulation testing Lippi et al. 515
specimens by venipuncture, including a new and simpli-
fied order that could be applied to both glass and plastic
tubes [31]. A thorough sequence is hence recommended:
blood culture tubes should first be collected, followed by
nonadditive, coagulation and additive tubes. If coagu-
lation analyses are the only tests ordered, a single non-
additive tube should be drawn first and discarded to
remove potential contamination by tissue thromboplastin
released by the trauma of venipuncture, which could
eventually interfere with coagulation assays by activating
the coagulation pathway. An additional guideline, especi-
ally developed for coagulation testing and updating the
prior edition, was released by the Clinical and Laboratory
Standards Institute (CLSI), on the basis that tissue
thromboplastin does not influence results even when
the coagulation tube is the first or the only drawn [32].
This is consistent with the evidence that no statistically
and clinically meaningful differences in results of routine
coagulation testing can be observed between the first and
the second test tubes sequentially collected within the
same venipuncture, nor during separate consecutive
venipunctures on veins of different arms [33]. The guide-
line emphasizes, however, that the necessity for drawing
a discard tube for other coagulation tests is ‘circumstantial
at best’, although reliable information recommending
such a practice is not so far available.
The collection of inappropriate containers for testing is an
major source of concern for clinical laboratories, account-
ing for as much as 13–16% of unsuitable specimens
[7–9], along with inappropriate filling and mixing. Incor-
rect anticoagulant to blood ratios and clotted specimens
are, respectively, the second and third most frequent
reasons for rejection, behind hemolysis [9,34]. Current
guidelines recommend that anticoagulant-containing
blood collection tubes for coagulation testing must be
filled to the proper level (usually to complete the vacuum
volume) and gently inverted several times to allow effec-
tive mixing, without inducing hemolysis or clotting [32]. In
fact, underfilling of coagulation tubes may significantly
affect the APTT and PT results, producing spurious
prolongations of these tests. Unfortunately, there are no
univocal indications on the minimum tube filling that
should be ensured, and results might be influenced by
the nature of the vials, the final concentration of the
anticoagulant in the specimen and the sensitivity of
the reagents employed [35]. There is still open debate
on the influence of the vacuum tube material on coagu-
lation testing. It has occasionally been reported that PT
values for samples in plastic tubes might be significantly
lower than for samples in glass tubes [36]. Two additional
studies on this subject, however, highlighted that no
clinically significant differences have been observed on
samples collected in tubes made from either plastic or
glass, suggesting that plastic tubes can be conveniently
used in place of traditional glass tubes for a wide variety of
coagulation assays [37,38].
 516 Blood Coagulation and Fibrinolysis 2006, Vol 17 No 7
Reneke et al. demonstrated that accurate PT values can
be obtained from normal specimens when tubes are filled
up to 65% more of capacity. For pathological specimens,
accurate PT results in the therapeutic range are achieved
when tubes are filled up to 80% (using moderately
sensitive thromboplastin reagent) and up to 90% (using
highly sensitive thromboplastin reagent) or more of
capacity. Prolonged APTT values can be observed only
in specimens filled to less than 90% of capacity [39]. By
using 3.8% citrate anticoagulated tubes, significant differ-
ences in PT and APTT are observed in samples filled less
than 80 and 90%, respectively [40]. The effect is less
pronounced when samples are drawn into 3.2% sodium
citrate. Pediatric blood collection tubes should be filled at
least 90% to grant accurate PT test results [41]. The
current CLSI document recommends that coagulation
samples should be discarded if the evacuated tube con-
tains less than 90% of the expected fill volume [32]. This
is the most suitable recommendation to be provided to
phlebotomists, who are not necessarily informed on the
final concentration of the anticoagulant in the test tube or
on specific test characteristics, such as reagents compo-
sition and sensitivity.
Specimen stability following blood collection is a further
matter of concern for the coagulation laboratory. Gener-
ally, PT results are stable for up to 24 h, remaining constant
regardless of storage conditions [42]. In some cases, the PT
and APTT values for plasma that had been left on spun-
down blood cells at room temperature tend to vary with
time, with PT values shortening and APTT values increas-
ing. Nevertheless, such biases are modest and with little
clinical significance. Within an 8-h period and with plasma
on spun-down cells at room temperature, therefore, add-on
tests for the PT and APTT could be performed with
results similar to what would be obtained from testing
unstored specimens [43], except for the APTT measured
on samples of patients receiving unfractionated heparin
therapy [42]. The APTT and PT values are significantly
increased for samples stored frozen when compared with
refrigerated and room-temperature conditions at 6 h [44].
Therefore either plasma or whole blood samples can be
accepted for most routine and second-line coagulation
testing from 6 to 12 h, when stored either at room tempera-
ture or under refrigeration [5,42,45].
The CLSI guideline contains the specific recommenda-
tion to centrifuge capped specimens for routine coagula-
tion testing within 1 h from collection at 1500  g for no
less than 15 min at room temperature [32]. There is no
definitive evidence, however, that alternative tempera-
tures for whole blood centrifugation might significantly
influence results of coagulation testing or generate ana-
lytically or clinically meaningful biases. In particular,
results of recent investigations are not in support of
the current recommendation and testify that whole blood
specimen centrifugation at different temperatures than
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
that currently recommended are not likely to generate
significant analytical or clinical biases [5,46]. Although
cold activation of factor VII, factor VIII and von
Willebrand factor might occur, this is not likely to influ-
ence results of PT, APTT, fibrinogen and D-dimer
testing. Therefore, although control and standardization
of several aspects of the preanalytic phase is essential to
achieve accurate and reliable results, a temperature-
controlled centrifuge appears not strictly required for
the determination of routine coagulation tests.
Hemolysis interference
Hemolyis is the leading source of unsuitable specimens
for both clinical chemistry and routine coagulation testing
[47]. Problems from troublesome specimen collection or
handling, such as wet alcohol transfer into the blood
specimen, small-bore needles, difficult phlebotomies,
partial obstruction of catheters and other collection
devices, excessive shaking or mixing of the blood after
collection, exposure to excessively hot or cold tempera-
ture, or centrifugation at too high a speed for a prolonged
period of time, frequently compromise the integrity of
blood and vascular cells, causing leakage of intracellular
components and producing significant biological and
analytical interference [47,48]. Reduction in the likeli-
hood of membrane survival under stress was acknowl-
edged nearly a half century ago by Deryagin and Gutop
[49]. An unstressed fluid–lipid membrane, such as the
plasma membrane, can survive for a very long period of
time in the form of a solid supported film or closed
vesicle. When stressed, however, some level of mem-
brane tension will cause an unstable hole to open rapidly
and rupture the membrane, which is usually an extremely
fast and invisible event on the scale of light microscopy.
Lysis of blood cells has been observed at steady shear
stresses in the range of 1500 dyn/cm2 for erythrocytes, at
300 dyn/cm2 for leukocytes and at 100–150 dyn/cm2 for
platelets, although the relative threshold values for lysis
depend upon the nature and duration of shear [22].
Visible hemolysis, as a hallmark of a more generalized
process of blood cell damage, is usually not apparent
until the separation of serum or plasma has occurred and
it is commonly defined for extracellular hemoglobin
concentrations greater than 0.3 g/l, resulting in a detect-
able pink-to-red hue of serum or plasma with a visible
appearance in specimens containing as low as 0.5%
hemolysate [14]. Slightly hemolyzed specimens might
yet be analyzable; nevertheless, a moderate blood cell
lysis, as low as 0.9%, strongly influences the reliability of
routine coagulation testing [50]. Although in a problem-
free phlebotomy activity it is unlikely for one to observe
hemolyzed samples, each laboratory should clearly docu-
ment the test procedures that are influenced by blood cell
lysis and to what extent they are affected. Moreover, as
such interference displays a wide interindividual bias,
which does not always allow lysis correction, the most
appropriate corrective measure should be warning

physicians, free hemoglobin quantification and even-
tually sample recollection [50].
Preanalytic variability and thrombin
generation assay
The recent introduction of general coagulation function
tests, namely the thrombin-generation assay (TGA), has
enabled a global assessment of the hemostatic system [51].
By using a fluorogenic substrate, the TGA produces throm-
bin-generation curves in a fully automated manner that
may be useful and sensitive enough to screen for either
hypercoagulable states or hemorrhagic diatheses [52].
The test, however, requires specific features and criteria
that should be accurately standardized to achieve reliable
results, besides traditional preanalytic variables. Basi-
cally, the test can be performed in defibrinated plate-
let-poor plasma [53], nondefibrinated platelet-rich
plasma (PRP) [54] and whole blood [55]. Although the
main parameters and curves of thrombin generation
obtained on platelet-poor plasma, PRP and whole blood
specimens are globally comparable in terms of diagnostic
efficiency, the absolute values are obviously not compar-
able or interchangeable [54]. In whole blood specimens
the total amount of thrombin activity generated and the
maximal concentration of thrombin achieved are pro-
portional to the hematocrit throughout a wide range of
clinically relevant cell concentrations. Red blood cell
lysate also augments the thrombin generation, due to
phospholipid release from damaged cells, highlighting
the need to achieve consistent quality specimens during
blood collection or to achieve lysate elimination by
filtration [55].
Although the integral amount of thrombin generated in
time expressed by the endogenous thrombin potential
appears substantially unmodified in frozen–thawed PRP,
the lag time is decreased and the thrombin generation is
augmented and accelerated, apparently due to cold-
induced platelet activation, membrane damage, and pro-
coagulant phospholipid exposure [54,56]. As there is a
linear relationship between most parameters of the
thrombin-generation curve and the number of residual
platelets in thawed specimens, it was concluded that
frozen–thawed PRP induces a substantial bias on several
TGA parameters [57]. Platelets are probably not the ideal
surrogate for exogenous phospholipids in TGA, as the
fatty acid composition of membrane phospholipids in
platelets might be rather heterogeneous among different
patients [58]. Therefore, although TGA should be ideally
evaluated on whole blood or PRP, it is recommended that
fresh plasma or frozen plasma only are suitable, provided
that they are filtered prior to testing to eliminate the
unwanted effect of residual platelets [59]. The blood
collection tube may also influence results [60]. The
clotting times for blood collected in Vacutainer or Vacu-
ette plastic tubes decreases with time, with maximal
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Preanalytic variability and coagulation testing Lippi et al. 517
effect after 30 min. Conversely, blood from Monovette
plastic tubes displays longer clotting times, which does
not decrease with time. These effects are probably due to
a rapid activation of factor XII and factor XI in Vacutainer
or Vacuette tubes following blood contact with foreign
surfaces [60]. The differences for clinical routine analyses
with clotting times in the range of seconds may be
negligible, but they may still be relevant in samples
for which clotting times are prolonged. Finally, cold
activation of hemostasis, which is not usually detected
in routine hemostasis assays, would produce more sub-
stantial influences on chromogenic assays based on
thrombin generation.
Conclusions
While the costs of laboratory testing continue to be the
dominant issue within the healthcare service worldwide,
the quality, effectiveness and impact on outcomes are
also emerging as critical value-added features [61]. Rou-
tine coagulation testing is commonplace in laboratory
diagnostics to identify coagulation disorders and monitor
anticoagulant therapies [1–3]. Over the past decades
several efforts have been directed towards improving
analytic quality and standardization of coagulation tests
[62]. Nevertheless, poor control and standardization of
the preanalytic phase still plague result reliability, regard-
less of the continuous efforts for identifying and prevent-
ing errors. Full efficiency of coagulation testing cannot be
achieved through control of the analytic phase alone, but
it requires adequate policies for global quality assessment
targeted at reducing the burden of uncontrollable pre-
analytic sources of variability, from patient identification
to specimen collection and handling. Compliance with
the new ISO 15189 standard, which will also be applied to
coagulation laboratories [63], encompasses the adoption
of suitable strategies for reducing undue variability
throughout the testing process. These strategies would
not entail extraordinary costs and are affordable with a
structured outlay of existing resources, educational and
feedback policies with nonlaboratory professionals, and
compliance with reliable operative guidelines. Never-
theless, we should be aware that the application of rigid
certification or accreditation procedures may also gener-
ate obstacles for introduction of innovative and promising
coagulation tests, which would otherwise improve the
efficiency of the whole diagnostic process and the
patient’s outcome.
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