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Clinica Chimica Acta 440 (2015) 164–168

Contents lists available at ScienceDirect

Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Postural change during venous blood collection is a major source of bias

in clinical chemistry testing
Giuseppe Lippi a,⁎, Gian Luca Salvagno b, Gabriel Lima-Oliveira b, Giorgio Brocco b,
Elisa Danese b, Gian Cesare Guidi b
a
Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy
b
Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University of Verona, Verona, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Background: To investigate the influence of different phlebotomy postures on clinical chemistry testing.
Received 16 November 2014 Materials and methods: Nineteen volunteers were recruited from the laboratory staff. A first set of samples was
Accepted 24 November 2014 drawn after 25 min of resting in supine position, a second after 20 min in sitting position, and a third after
Available online 29 November 2014 20 min in upright position. Clinical chemistry testing was performed on Roche Cobas C501.
Results: The plasma volume change (PVC) was −3.4% from supine to sitting, −14.1% from supine to standing and
Keywords:
−9.7% from sitting to standing. Compared to quality specifications for bias, hemoglobin, hematocrit, albumin and
Preanalytical variability
Posture
total proteins exhibited meaningful increases from supine to sitting, whereas meaningful increases were
Plasma volume change observed for hemoglobin, hematocrit, albumin, alkaline phosphatase (ALP), amylase, aspartate aminotransferase
Laboratory testing (AST), total bilirubin, calcium, total and high-density lipoprotein (HDL) cholesterol, gamma-glutamyl transferase
Clinical chemistry (GGT), glucose, lactate dehydrogenase (LDH), magnesium, total protein and triglycerides from sitting to stand-
ing. The parameters with meaningful bias from sitting to upright were hemoglobin, hematocrit, albumin, ALP,
total bilirubin, calcium, total and HDL cholesterol, glucose, LDH and total protein.
Conclusions: These results provide further support to the need of standardizing patient's posture during
phlebotomy.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
According to the historical definition coined with felicitous intuition
by George D. Lundberg in the early 1980s [1], the total testing process
can be divided into three discrete parts, that are the preanalytical, analyt-
ical and postanalytical phases. This thoughtful picture of laboratory diag-
nostics can then be further clustered in more specific activities, which
include test ordering, patient preparation and identification, sample col-
lection, transportation, preparation and analysis, test reporting and
interpretation [2]. Several lines of evidence now attest that most errors
in laboratory diagnostics emerge from inappropriate, incorrect or
mishandled procedures during collection of diagnostic samples [3].
More specifically, problems in the preanalytical phase account for as
many as 70% of all errors throughout the total testing process [4,5].
Major focus has been placed in harmonizing sample collection over
the past decades, by development and implementation of national and
international guidelines [6]. Reference documents such as the Clinical
and Laboratory Standards Institute (CLSI) H3-A6 standard [7], or the
⁎ Corresponding author at: U.O. Diagnostica Ematochimica, Azienda Ospedaliero-
Universitaria di Parma, Via Gramsci, 14, 43126 Parma, Italy. Tel.: +39 0521 703050;
fax: +39 0521 703791.
E-mail addresses: glippi@ao.pr.it, ulippi@tin.it (G. Lippi).
World Health Organization (WHO) guidelines [8], contain a large num-
ber of recommendations, which are aimed to standardize the procedure
of collecting venous blood, from patient preparation to sample treat-
ment prior to analysis. Specific indications are also provided regarding
patient posture during venipuncture. In the CLSI H3-A6 standard, the
item “patient position” indicates that “specimens should be drawn
with the patient seated comfortably in an appropriate chair or lying
down” [7]. The WHO guidelines on drawing blood contain a different in-
dication, that is “make the patient comfortable in a supine position (if
possible)” [8]. As such, two major drawbacks seemingly emerge from
these indications. First, no clear distinction is made between supine or
sitting position during venipuncture, thus assuming that the two pos-
tural positions may be virtually interchangeable and the shift from
one posture to the other may not generate meaningful bias of laboratory
testing. This may be a major issue for hospitalized patients, especially
when blood is drawn at different times of the day (e.g., in sitting posi-
tion during the day or in supine position during the night) [9]. Then, it
is not clearly stated for how long the patient should maintain a stable
position (either supine or sitting) before venipuncture. This is notewor-
thy, because blood may be occasionally drawn from subjects (especially
outpatients) who have walked or remained in upright position for long
and had seated for a very short time before phlebotomy. Since it is now
clearly acknowledged that either hemoconcentration and hemodilution

http://dx.doi.org/10.1016/j.cca.2014.11.024
0009-8981/© 2014 Elsevier B.V. All rights reserved.
 Author's personal copy
G. Lippi et al. / Clinica Chimica Acta 440 (2015) 164–168
may be associated with different postural positions (i.e., supine, sitting
or standing) [10], this study was aimed to establish to what extent the
concentration of a large number of clinical chemistry analytes may be
influenced by phlebotomy posture.
2. Materials and methods
The study population consisted in 19 ostensibly healthy volunteers
(mean age 44 ± 11 years; 7 males, 12 females; mean height 1.68 ±
0.11 m; mean weight 68 ± 12 kg), recruited from the staff of the labo-
ratory of the University Hospital of Verona (Italy). Venous blood sam-
pling was performed after an overnight fast, between 9 and 12 AM, by
the same experienced phlebotomist and without venous stasis. Three
separate sets of samples were collected from each subject. The first
was drawn after the volunteer had rested for 25 min in supine position,
the second after 20 min in comfortable sitting position, and the last after
20 min of permanence in upright position. At each time point one serum
vacuum tube with clot activator and gel separator (Venosafe, Terumo
Europe N.V., Leuven, Belgium) and another vacuum tube containing
5.9 mg K2EDTA (Venosafe, Terumo Europe N.V.) were collected from a
forearm vein and immediately transported to the laboratory. The
serum samples were separated with standard centrifugation, according
to manufacturer's instructions (i.e., 1300 ×g for 15 min, at room tem-
perature). No samples ought to be discarded for unsuccessful venipunc-
tures or spurious hemolysis. The following analytes were measured on a
Cobas c501 (Roche Diagnostics GmbH, Mannheim, Germany), using
proprietary reagents: albumin, alkaline phosphatase (ALP), alanine ami-
notransferase (ALT), alpha-amylase, aspartate aminotransferase (AST),
bilirubin total and conjugated, calcium, cholesterol total and high-
density lipoprotein (HDL), chloride (mmol/L), creatine kinase (CK), cre-
atinine, C reactive protein (CRP), gamma-glutamyl transferase (GGT),
glucose, iron, lactate dehydrogenase (LDH), lipase, magnesium, phos-
phate, potassium, total protein, sodium, triglycerides, urea and uric
acid. Hematocrit and hemoglobin were also assessed on K2EDTA blood,
using an Advia 2120 (Siemens Healthcare Diagnostics, Deerfield, IL).
The plasma volume change (PVC) was then calculated with the refer-
ence formula of Dill and Costill [11]. Results of measurements were fi-
nally reported as median and interquartile range (IQR). The
significance of differences was assessed with Wilcoxon's signed rank
test, and potential associations between variables were explored by uni-
variate and multivariate analysis, using Analyse-it (Analyse-it Software
Ltd, Leeds, UK). The degree of statistical significance was set at p b
0.05. The percentage variation calculated from the different postural po-
sitions was also compared with the desirable quality specifications for
bias, as provided by Ricos et al. [12]. Each patient provided a written con-
sent for being enrolled in the study, which was performed in accord with
the ethical standards established by the institution in which the experi-
ments were performed and the Helsinki Declaration of 1975.
3. Results
The main findings of this study are reported in Table 1. The change
from supine to sitting posture generated a median PVC of − 3.4%,
whereas the PVC from supine to standing posture was − 14.1%, and
that from sitting to standing posture was −9.7% (Fig. 1). No significant
correlation was found between PVC (at any time point) and weight,
height, body mass index, sex and age in both univariate and multivari-
ate analysis (all interactions, p N 0.05). Statistically significant differ-
ences from supine to sitting posture were found for hemoglobin,
hematocrit, albumin, ALP, ALT, amylase, AST, total and HDL cholesterol,
CK, GGT, iron, LDH, magnesium, total protein, triglycerides, and urea.
When compared to the quality specifications for bias [12], only hemo-
globin, hematocrit, albumin and total proteins exhibited meaningful in-
creases (Table 1). Statistically significant differences from supine to
standing posture were found for all analytes except chloride, CRP, phos-
phate, potassium, sodium and uric acid. When compared with the
165
quality specifications for bias [12], meaningful increases were observed
for hemoglobin, hematocrit, albumin, ALP, amylase, AST, total bilirubin,
calcium, total and HDL cholesterol, GGT, glucose, LDH, magnesium, total
protein and triglycerides (Table 1). The statistically significant differ-
ences from sitting to standing posture were identical to those observed
between supine and standing position, with the exception of magnesium,
which remained unchanged (Table 1). When compared with the quality
specifications for bias [12], the number of analytes exhibiting meaning-
ful variation included hemoglobin, hematocrit, albumin, ALP, total bili-
rubin, calcium, total and HDL cholesterol, glucose, LDH, and total
protein.
4. Discussion
The plasma volume reacts dynamically to substantial modifications
of gravitational force and hydrostatic pressure [13]. It is hence obvious
that postural changes may also exert a strong influence on plasma vol-
ume distribution, especially in bipedal species such as humans and
other primates, that can assume an erect position. This is mainly attrib-
utable to the fact that the venous pressure in the lower parts of the body
increases after a prolonged standing position, thus generating an
enhancement of capillary pressure, which ultimately leads to ultrafiltra-
tion of plasma in the interstitial space [14]. In this process of plasma
extravasation, larger and nondiffusible plasma components remain
entrapped within the blood vessels, whereas smaller and filtrable ele-
ments migrate along with water in the interstitial space. This is clearly
reflected by the results of our study, wherein the concentration of virtual-
ly all analytes except non protein-bound ions were significantly modified
by postural changes and the relative gap of gravitational force (Table 1).
Some studies have previously addressed the influence of posture on
some laboratory parameters. The first ever human investigation about
the effect of postural position on laboratory testing was published by
Stoker et al. in 1966 [15], who investigated 13 healthy subjects and 4
patients with hypercholesterolaemia. In brief, mean increases of 12.9%
for plasma cholesterol, 8.5% for plasma protein and 8.6% for hematocrit
were recorded after 15 min permanence in upright posture compared to
a reference supine position.
In 1974, Statland et al. studied 11 healthy men (ages 20–25 years),
who had their blood collected in three separate days, after being supine
for 30 min, remaining seated for 30 min and standing for 30 min [16].
When the sitting posture was used as a reference, a significant increase
was observed after 30 min standing for albumin (+3.0%), ALP (+4.6%),
total cholesterol (+ 2.5%), phosphate (+ 6.1%) and total protein
(+2.5%), whereas the values of AST, ALT, total bilirubin, calcium, chlo-
ride, creatinine, iron, potassium, sodium, urea and uric acid remained
unchanged. When the sitting posture was instead compared with
30 min in supine position, a significant decrease was observed for albu-
min (− 6.7%), AST (− 4.7%), calcium (− 3.1%), total protein (− 6.1%),
potassium (− 3.9%), whereas the remaining analytes tested remained
unchanged. Interestingly, these findings seem rather different from
those reported in other investigations, and also differ from data obtained
in our experiments. This is probably attributable to the different study de-
sign, since Statland et al. performed sample collection in separate days, so
that the final results may have been at least partially biased by a greater
degree of inter-day biological and analytical variation.
In 1978, Dixon and Paterson studied 12 healthy students (8 men and
4 women, with age comprised between 20 and 25 years) [14], and
reported that the values of albumin (+12%), ALP (+12%), total biliru-
bin (+ 17%), calcium (+ 5%), cholesterol (+ 18%), and total protein
(+ 11%) significantly increased when the subjects changed position
from supine to standing (for at least 20 min). No significance difference
was instead observed for creatinine and phosphate. Interestingly,
despite our study population was older than that studied by Dixon
and Paterson, all changes were reproduced rather similarly in our inves-
tigation (Table 1), including the invariability of phosphate. At variance
with Dixon and Paterson, we observed a modest increase of creatinine,
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166
Table 1
Variation of clinical chemistry parameters in different postural positions before and during venipuncture.

Desirable bias Supine Sitting Standing

Value p vs supine Bias (%) vs supine Value p vs supine Bias (%) vs supine p vs sitting Bias (%) vs sitting

Hemoglobin (g/L) ±1.8% 131 (127–145) 134 (129–150) b0.001 2.3 (1.9 to 2.9) 141 (134–154) b0.001 7.1 (5.3 to 8.9) b0.001 4.8 (3.9 to 6.0)
Hematocrit ±1.7 0.41 (0.40–0.44) 0.42 (0.41–0.44) 0.009 1.7 (1.3 to 2.5) 0.44 (0.43–0.47) b0.001 6.9 (5.0 to 8.9) b0.001 5.3 (3.3 to 6.3)
PVC (% variation) – – −3.4 (−4.3 to −1.5) b0.001 – −14.1 (−15.7 to −9.1) b0.001 −9.7 (−11.1 to −6.6) b0.001 –
Albumin (g/L) ±1.4 40.9 (39.9–43.7) 42.4 (41.1–44.2) 0.001 2.0 (0.8 to 3.9) 45.2 (43.4–45.9) b0.001 10.3 (4.1 to 12.2) b0.001 6.4 (2.9 to 10.0)
ALP (U/L) ±6.7 54 (47–58) 55 (49–59) b0.001 3.6 (1.8 to 4.4) 58 (50–65) b0.001 11.1 (7.3 to 12.5) b0.001 7.8 (4.8 to 9.6)

G. Lippi et al. / Clinica Chimica Acta 440 (2015) 164–168

ALT (U/L) ±11.5 16 (12–19) 17 (13–20) 0.035 2.0 (0.0 to 5.0) 19 (13–21) b0.001 9.1 (5.3 to 11.0) b0.001 8.3 (2.4 to 10.8)
Amylase (U/L) ±7.4 56 (41–69) 57 (43–70) b0.001 2.6 (1.7 to 4.1) 59 (45–74) b0.001 8.7 (6.0 to 10.6) b0.001 6.3 (2.3 to 7.7)
AST (U/L) ±6.5 17 (15–19) 18 (16–20) 0.001 4.5 (0.0 to 6.9) 19 (17–22) b0.001 11.1 (4.7 to 14.3) 0.003 5.6 (2.4 to 6.5)
Bilirubin, conjugated (μmol/L) ±14.2 2.4 (2.0–3.2) 2.5 (2.0–3.3) 0.055 – 2.8 (2.0–3.4) b0.001 7.2 (2.8 to 16.0) 0.003 6.2 (1.1 to 11.7)
Bilirubin, total (μmol/L) ±8.9 8.1 (5.3–10.3) 8.1 (5.5–11.2) 0.139 – 8.4 (6.5–12.3) b0.001 13.3 (6.0 to 21.9) 0.001 12.0 (7.8 to 19.1)
Calcium (mmol/L) ±0.8 2.32 (2.25–2.42) 2.34 (2.29–2.42) 0.290 – 2.40 (2.35–2.49) b0.001 3.4 (1.4 to 4.2) b0.001 3.1 (1.7 to 4.5)
Cholesterol, HDL (mmol/L) ±5.6 1.4 (1.2–1.6) 1.5 (1.2–1.7) b0.001 3.1 (2.3 to 5.2) 1.6 (1.3–1.8) b0.001 9.7 (7.2 to 13.1) b0.001 5.7 (4.6 to 9.5)
Cholesterol, total (mmol/L) ±4.1 4.9 (4.1–5.2) 5.0 (4.2–5.3) b0.001 2.5 (0.7 to 4.9) 5.3 (4.7–5.5) b0.001 8.9 (5.3 to 12.3) b0.001 5.7 (3.4 to 10.2)
Chloride (mmol/L) ±0.5 104 (103–105) 104 (102–105) 0.194 – 104 (102–104) 0.096 – 0.070 –
CK (U/L) ±11.5 83 (61–105) 84 (64–108) b0.001 2.5 (0.6 to 3.9) 86 (67–116) b0.001 8.0 (4.7 to 9.9) b0.001 5.3 (2.7 to 7.9)
Creatinine (μmol/L) ±4.0 64 (57–75) 64 (57–64) 0.126 – 66 (56–74) 0.016 1.9 (−1.4 to 4.6) 0.028 1.4 (−0.9 to 4.5)
CRP (mg/L) ±21.8 0.4 (0.3–1.0) 0.5 (0.3–1.0) 0.253 – 0.5 (0.3–1.0) 0.058 – 0.074 –
GGT (U/L) ±11.1 15 (11–20) 16 (12–21) 0.001 4.2 (0.0 to 7.9) 17 (12–22) b0.001 11.1 (8.9 to 16.0) 0.002 9.5 (2.1 to 11.6)
Glucose (mmol/L) ±2.3 4.6 (4.4–4.9) 4.7 (4.5–5.0) 0.417 – 4.9 (4.6–5.1) 0.001 5.4 (1.0 to 7.6) 0.022 3.8 (−0.9 to 6.6)
Iron (μmol/L) ±8.8 14.1 (12.0–17.7) 14.3 (12.1–18.9) 0.008 1.4 (0.1 to 3.5) 15.0 (12.9–19.9) 0.002 7.0 (2.8 to 11.8) 0.015 5.2 (2.1 to 9.9)
LDH (U/L) ±4.3 288 (276–319) 296 (283–311) 0.045 2.3 (−0.3 to 4.3) 319 (289–334) b0.001 9.8 (3.4 to 12.8) 0.001 7.2 (4.3 to 11.3)
Lipase (U/L) ±11.3 27 (24–35) 27 (23–34) 0.432 – 28 (24–35) 0.016 3.4 (0.0 to 6.4) 0.004 4.0 (1.4 to 4.7)
Magnesium (mmol/L) ±1.8 0.79 (0.76–0.83) 0.81 (0.77–0.87) 0.001 1.4 (0.6 to 3.4) 0.83 (0.78–0.86) b0.001 3.5 (1.3 to 6.5) 0.197 –
Phosphate (mmol/L) ±3.4 0.99 (0.95–1.11) 0.99 (0.94–1.08) 0.445 – 1.02 (0.93–1.10) 0.235 – 0.364 –
Potassium (mmol/L) ±1.8 4.1 (3.9–4.4) 4.2 (4.0–4.5) 0.127 – 4.2 (4.0–4.4) 0.194 – 0.350 –
Protein, total (g/L) ±1.4 67.0 (64.4–70.6) 68.8 (67.2–70.8) 0.003 2.9 (1.1–3.6) 73.3 (69.8–76.3) b0.001 10.7 (5.1 to 12.4) b0.001 8.0 (5.8 to 10.3)
Sodium (mmol/L) ±0.2 141 (139–142) 141 (139–142) 0.432 – 141 (140–142) 0.236 – 0.081 –
Triglycerides (mmol/L) ±9.6 0.7 (0.6–1.2) 0.8 (0.7–1.3) 0.021 5.1 (0.8 to 8.4) 0.9 (0.8–1.3) b0.001 11.5 (6.8 to 18.4) 0.002 6.6 (2.4 to 11.7)
Urea (mmol/L) ±5.6 5.08 (3.97–5.69) 5.06 (4.09–5.48) 0.012 −1.4 (−2.4 to −0.4) 5.01 (3.95–5.53) b0.001 −3.1 (−1.4 to −4.0) 0.005 −1.9 (−0.6 to 2.6)
Uric acid (μmol/L) ±4.9 255 (201–301) 256 (199–304) 0.237 – 254 (200–303) 0.125 – 0.182 –

Significant differences in bold.

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G. Lippi et al. / Clinica Chimica Acta 440 (2015) 164–168
5%
0%
Plasma Volume Change
-5%
-10%
-15%
-20%
-25%
Supine Sing Standing
Fig. 1. Estimated variation of plasma volume induced by postural changes (◆, median
variation).
although the bias was only half than the value corresponding to a clini-
cal significant change.
Renoe et al. also studied 11 healthy men recruited form the laborato-
ry personnel [17], and measured calcium and related variables before
and after the subjects changed from the supine to upright posture.
After changing posture, calcium (+4.6%), total protein (+11.5%), albu-
min (+ 12.2%) and magnesium (+ 3.8%) increased significantly, with
variations highly comparable to those observed in our study.
In a subsequent study, Felding et al. studied 40 subjects [18], and
found an increase of approximately 6.5% in the serum values of proteins,
enzymes and lipids from lying to sitting position, reporting values mar-
ginally lower than those observed in our study. No significant changes
were instead observed in the values of potassium and sodium.
Miller et al. also examined the effects of postural change on the con-
centration of lipids and lipoproteins in 6 healthy volunteers (age 19 to
34 years) [19], and concluded that triglycerides, total and HDL choles-
terol increased by 8–12% when the subjects changed from the supine
to the standing posture. These changes were also very similar to those
recorded in our investigation.
In a more recent investigation, Campbell et al. studied 25 dyslipid-
emic subjects (15 women and 10 men; mean age 59 ± 9 years), whose
blood was drawn twice, with subjects seated for a mean time of 1 and
6 min, respectively [20]. Interestingly, hemoglobin (+3.4%), albumin
(+0.9%) and total protein (+1.1%) were significantly increased when
blood was collected with the shorter seated time. Conversely, triglycer-
ides, total and HDL cholesterol remained virtually unchanged, and this
is plausible since the difference between one procedure (1 min sitting)
and the other (6 min sitting) was minimal compared to previous studies.
To the best of our knowledge, the present study is the largest pro-
spective trial that has ever assessed the impact of postural changes
(i.e., from supine, to sitting or standing) on a very large panel of clin-
ical chemistry analytes in a middle-age population, thus extending
the findings of previous studies which only investigated a discrete
number of parameters in limited or different study populations.
Taken together, our results suggest that changing posture from seated
to supine or upright is associated with substantial hemodilution and
hemoconcentration, which are both attributable to the varying influ-
ence of gravitational force on the compartmental distribution of body
fluids, but are seemingly independent from demographical variables
such as age, sex, weight, height and BMI. These variations of plasma
volume were effective to generate a clinically significant bias in a num-
ber of parameters, especially hemoglobin, hematocrit, albumin, ALP,
amylase, AST, total bilirubin, calcium, total and HDL cholesterol, GGT,
glucose, LDH, magnesium, total protein and triglycerides. The variation
167
of these analytes was greater than the desirable quality specifications
for bias derived from biological variation [12], and can hence influence
the clinical decision making for a variety of reasons. First, the reference
ranges of most laboratory parameters are calculated by drawing blood
from patients in a comfortable sitting position, as currently advocated
by the CLSI [7]. It is hence obvious that the application of these reference
ranges to patients whose blood has been collected in supine position, or
after long permanence in standing position and without adequate time
of sitting, may be misleading and may potentially generate false positive
(hemoconcentration) or false negative (hemodilution) results. This
consideration is even more straightforward using the longitudinal com-
parison of patient's data, in which the bias is only compared against the
intra-individual biological variability, that is typically lower that the
inter-individual variation [21]. Another important aspect is the fact
that even subtle statistical differences of selected analytes emerging
from changing phlebotomy posture may have a notable impact on the
outcome of rigorous controlled trials, in which modest but significant
changes may still have an impact on surrogate endpoints such as
biomarkers.
In conclusion, the results of this investigation provide further sup-
port to the notion that major focus should be placed for achieving a
widespread standardization of phlebotomy practice. Clear indications
should be given that patient posture during venous blood sampling
must be uniformed to a reference position, either sitting or supine. Irre-
spective of the chosen criterion, a recommendation should be given that
a minimum period (i.e., 15 to 20 min) of resting in the reference position
should be observed before collecting venous blood. This would allow
to overcome the undue variability attributed to venous hydrostatic
changes that can emerge when changing position from supine to either
upright or sitting.
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