Clinica Chimica Acta

CCA-12242; No of Pages 3
Clinica Chimica Acta xxx (2011) xxx–xxx
Contents lists available at ScienceDirect
Clinica Chimica Acta
journal homepage: www.elsevier.com/locate/clinchim
Letter to the editor
Elimination of the venous stasis error for routine coagulation
testing by transillumination☆
Dear editor
The preanalytical phase is responsible for more than two-thirds of
all errors attributed to the clinical laboratory [1–4] and there are only
a few routine procedures for the detection of nonconformities in this
field of activity [5,6]. In this phase the procedures involving
phlebotomy, critical to the obtainment of diagnostic blood specimens,
are poorly studied as regards the major sources of errors and the
procedures related to quality control process [7,8]. The collection of
diagnostic blood specimens for routine coagulation tests are traditionally
performed by phlebotomists using a tourniquet [9]. The
Clinical and Laboratory Standards Institute (CLSI) recommends the
use of the tourniquet for localizing suitable veins for ≤60 s. When
performing specimen collection for diagnostic purposes such an
interval of time both allows easy localization of vein paths and
concomitantly circumvents possible problems due to excess venous
stasis [10–12]. Although the venous stasis can influence the
concentration and/or the activity of several blood analytes, the
tourniquet time is rarely regarded as a potential source of laboratory
variability [13–17]. Reportedly, the mean tourniquet application
times by phlebotomists were 98 s in public laboratories and 70 s in
private laboratories respectively, thus raising some issues about
proper specimen collection [18]. The use of transillumination devices,
based on cold near infrared light-emitting diodes (LEDs) whose
lightsare absorbed by intra-erythrocyte hemoglobin flowing along the
veins, has been initially proposed in order to ease the vein puncture in
children [19]. The efficacy of palm transillumination for establishing
venous access in small infants has already been assessed [20].
Moreover, the transillumination has been proposed for mapping
veins to be cannulated prior to ambulatory phlebotomy because it
allows accurate visualization of the vein course [21]. Reliability in
coagulation testing is pivotal to the appropriate diagnosis and
treatment of patients with hemostasis disturbances [17,22]. In this
context some preanalytical details/procedures appear critical such as
a) adequate fasting time before blood collection [23], b) use of
appropriate tubes [24–26] and additives [27], c) appropriateness of
blood collection, storage and centrifugation[28–30], and d) strict
conformity to the recommendations regarding tourniquet time.
Clinical laboratory results are estimated to be able to influence 60%
to 70% of medical decisions and thus affect diagnostic outcome and/or
patient treatments [31], such as oral anticoagulant therapy employed
in patients at risk of thrombosis [32,33] or blood transfusion
components prescription [34] recommended in bleeding patients
with disseminated intravascular coagulation (DIC) and prolonged
☆ We are grateful to Mrs. Nadia Keder Assan for her dedication to collect all
diagnostic blood specimens for coagulation routine tests in this work.
both PT and APTT [35,36]. In this article we explored a way to improve
the quality process of the preanalytical phase for coagulation sample
laboratory work-flow. In order to completely eliminate the venous
stasis, a source of possible errors in coagulation tests, we evaluated
whether a transillumination device can advantageously replace the
tourniquet usually employed during the procedure for the collection
of diagnostic blood specimens.
Two hundred adult ambulatory patients of both sexes, volunteers for
this study, from Dante Pazzanese Cardiology Institute, São Paulo City,
Brazil, were evaluated. This study was submitted to the Internal Review
Board and approved by our Human Research Ethics Committee. All
volunteers signed informed consent. The collection of diagnostic blood
specimens was realized by a single expert phlebotomist, following the
CLSI standard [10–12]. Wefirst studied 50 patients (G1). All patients,
who fasted for 8 h, were seated for 15 min to eliminate possible
interferences of blood distribution due to different posture [37]. After
this interval, a vein was located on the left forearm by a subcutaneous
tissue transilluminator device (Venoscópio IV, Duan do Brasil, Brazil),
and the diagnostic blood sample was collected using a 20 G straight
needle (BD Vacuntainer®, Becton Dickinson Diagnostics, Brazil) directly
into 4.5 ml siliconized vacuum tubes containing citrate solution, 0.5 ml;
sodium citrate, 12.35 mg and citric acid, 2.21 mg—equivalent to 3.2%
sodium citrate (BD Vacuntainer®, Becton Dickinson Diagnostics, Brazil).
All the tubes used in this study were of the same lot. In sequence a
tourniquet was applied on the right forearm during 30 s (accepted
standard time for tourniquet application) [10–12], and another sample
was collected into the same type of vacuum tube. The study was then
continued by evaluating the other 3 groups (G2, G3 and G4) of 50
different volunteers each, and the same methodology for diagnostic
blood sample collection was applied, but tourniquet time was varied as
follows: in G2 the tourniquet was applied for 90 s, in G3 for 120 s and in
G4 for 180 s.
All samples were processed for routine coagulation tests in
triplicate immediately after collection (b30 min) on the same
instrument, Sysmex CA 1500 Coagulation Analyzer (Sysmex Corporation,
Kobe, Japan). The parameters tested included: fibrinogen
(Multifibren U®, Siemens Healthcare Diagnostics Products GmbH,
Germany), prothrombin time (PT-Thromborel® S “lyophilized human
placental thromboplastin,” Siemens Healthcare Diagnostics Products
GmbH) and activated partial thromboplastin time (APTT-Pathromptin®
SL “vegetable phospholipid with micronized silica,” Siemens
Healthcare Diagnostics Products GmbH). The instrument was calibrated
against appropriate proprietary reference standard material
and verified with the use of proprietary controls.
The significance of the differences between samples was assessed
by paired Student's t-test after checking for normality. The values
obtained on samples collected using the subcutaneous tissue
transilluminator device were considered as the reference ones. The
level of statistical significance was set at pb0.05. Finally, the biases
from G1, G2, G3 and G4 were compared with the current desirable
quality specifications for bias (B), derived from biological variation
according to the formula Bb0.25 (CV w 2 +CV g 2 ) 1/2 where CV w and CV g
are within- and between-subject CVs [38].
0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.cca.2011.04.008
Please cite this article as: Lima-Oliveira G, et al, Elimination of the venous stasis error for routine coagulation testing by transillumination,
Clin Chim Acta (2011), doi:10.1016/j.cca.2011.04.008

2 Letter to the editor
Table 1
Effects of tourniquet application vs. transilluminator device in blood sample collection on coagulation parameters.
G1 (N=50)
G2 (N=50)
Desirable
bias (%)
Mean value±SD
of transilluminator
Mean value±SD
of 30 s tourniquet
p-Value Mean %
difference
Mean value±SD
of transilluminator
Mean value±SD
of 90 s tourniquet
p-Value Mean %
difference
FIB (mg/dl) 4.8 384.9±94.9
[240.0–604.8]
PT (s) 2.0 12.32±3.20
[10.30–14.21]
APTT (s) 2.3 29.86±4.28
[22.50–33.80]
385.1±94.9
[240.0–605.0]
12.30±3.23
[10.31–14.19]
29.80±4.30
[22.50–33.77]
N0.05 0.05 358±72
[190–512]
N0.05 −0.2 14.47±7.94
[10.9–38.9]
N0.05 −0.2 31.80±7.90
[25.8–36.7]
364±72
[199–525]
14.41 ±8.00
[10.8–37.4]
31.70 ±7.80
[25.3–35.1]
0.002 1.7 ⁎
N0.05 −0.4
N0.05 −0.3
Desirable
bias (%)
G3 (N=50)
Mean value±SD
of transilluminator
FIB (mg/dl) 4.8 288±89
[173–540]
PT (s) 2.0 12.89±1.61
[11.0–18.4]
APTT (s) 2.3 31.70±4.50
[24.1–44.3]
Mean value±SD
of 120 s tourniquet
296±93
[189–545]
12.67±1.57
[10.9–18.0]
31.20±4.40
[20.1–38.7]
p-Value Mean %
difference
G4 (N=50)
Mean value±SD
of transilluminator
0.001 3.0 ⁎⁎ 361±85
[218–495]
0.003 −1.7 ⁎⁎ 12.65±3.29
[11.0–15.1]
0.009 −1.5 ⁎⁎ 30.20±4.40
[22.5–42.3]
Mean value±SD
of 180 s tourniquet
385±95
[240–605]
12.30 ±3.23
[8.7–14.2]
29.80 ±4.30
[22.5–22.8]
p-Value Mean %
difference
0.001 6.7 ⁎⁎
0.001 −2.8 ⁎⁎
0.002 −1.4 ⁎⁎
The values were mean ±SD [range: minimum–maximum]. The bold p-values are statistically significant (pb0.05) and bold mean % differences represent clinically significant
variations, when compared with desirable bias [38].
⁎ pb0.05 vs. G1.
⁎⁎ pb0.05 vs. G1 and G2.
The results of this investigation are shown in Table 1. InG1no
significant increases were observed in coagulation parameters
evaluated after 30 s of the tourniquet application. In G2, after 90 s of
the tourniquet application, a significant increase was observed in
fibrinogen. In G3, significant increases in FIB, PT and APTT were
observed after 120 s of the tourniquet application. In G4 significant
increases were observed in all coagulation tests evaluated after 180 s
of the tourniquet application. However, a clinically significant variation,
as compared with the current desirable quality specifications
[38], was only observed for fibrinogen and PT after 3 min of stasis
(G4).
Our results demonstrate that the prolonged stasis consequent on
tourniquet application for 120 s causes significant reduction of PT and
APTT values in both tests (Table 1), a well known phenomenon of in
vitro activation, thus paving the way for possible errors in critical
patient management and follow-up. As reported, rarely the expert
phlebotomist concludes the collection of diagnostic blood specimens
within 60 s of tourniquet application [18]. Several concurrent causes
might contribute to lengthen the tourniquet time even over 3 min,
such as a difficult location of an appropriate venous access, selection
of the most suited blood collection system, needle insertion into the
vein, collection of many tubes, etc. [3,5,6,9,15–17]. As regards
fibrinogen, the significant increase observed in groups G2, G3, and
G4 appears on overall modest and could be considered not clinically
significant. Nevertheless, even modest increases of fibrinogen, a
marker of inflammation, have been associated with future coronary
heart disease and with unstable and stable coronary artery disease
and coronary complications after interventions [39]. Thus the use of
tourniquet could generate false positives and prospectively induce the
caring physicians to adopt undue treatments. On the contrary,
transillumination devices appear able to eliminate such risk [40]. In
order to deal with some of the above issues, the quality laboratory
manager might devise and adopt improved blood collection procedures
with no or very reduced tourniquet times, e.g., by implementing
transillumination devices after accurate re-evaluation of the
whole blood collection procedures employed by phlebotomist [7].
Obviously such a process of change of blood collection procedures
should be supported by adequate practical instructions provided by
expert professionals. In conclusion, the results we obtained demonstrate
that by employing the recommended procedure of tourniquet
application with gold standard time (30 s) [10–12] in blood collection,
the performance of the coagulation routine testing is identical to that
shown by blood collected with the aid of transillumination. Probably
the errors induced by venous stasis on specialized coagulation tests
like D-dimer, Factors VII, VIII and XII [17] would be eliminated too
using the transillumination. Further insight has to be made in this
respect. Moreover, the whole results demonstrate that transillumination
devices can eliminate the venous stasis and improve the
quality process in preanalytical phase especially in phlebotomy
procedures, mostly when considering that inappropriately prolonged
tourniquet application is rather widespread and no common rules
and/or guidelines appear applied in this respect. These results do
apply to our experimental design, even though they represent a viable
basis for the governance of the preanalytical variability related to
sample stasis.
No potential conflicts of interest relevant to this article were
reported.
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Clin Chim Acta (2011), doi:10.1016/j.cca.2011.04.008

Letter to the editor
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G. Lima-Oliveira
Laboratory of Clinical Biochemistry,
Department of Life and Reproduction Sciences,
University of Verona, Verona, Italy
Post Graduate Program of Pharmaceutical Sciences,
Department of Medical Pathology Federal University of Parana,
Curitiba, Parana, Brazil
MERCOSUL: Sector Committee of Clinical Analyses and In Vitro
Diagnostics, CSM 20, Rio de Janeiro, Brazil
Brazilian Society of Clinical Analyses on São Paulo State,
São Paulo, Brazil
Corresponding author at: Rua Vicente Licínio, 99 Tijuca,
Rio de Janeiro, RJ 20.270-902, Brazil.
Tel.: +55 11 7800 5868; fax: +55 11 27810712.E-mail address:
dr.g.lima.oliveira@gmail.com.
G.L. Salvagno
Laboratory of Clinical Biochemistry,
Department of Life and Reproduction Sciences,
University of Verona, Verona, Italy
G. Lippi
U.O. Diagnostica Ematochimica,
Azienda Ospedaliero-Universitaria di Parma, Parma, Italy
M. Montagnana
Laboratory of Clinical Biochemistry,
Department of Life and Reproduction Sciences,
University of Verona, Verona, Italy
M. Scartezini
G. Picheth
Post Graduate Program of Pharmaceutical Sciences,
Department of Medical Pathology Federal University of Parana,
Curitiba, Parana, Brazil
G.C. Guidi
Laboratory of Clinical Biochemistry,
Department of Life and Reproduction Sciences,
University of Verona, Verona, Italy
20 December 2010
Available online xxxx
Please cite this article as: Lima-Oliveira G, et al, Elimination of the venous stasis error for routine coagulation testing by transillumination,
Clin Chim Acta (2011), doi:10.1016/j.cca.2011.04.008