Scandinavian Journal of Clinical and Laboratory

Investigation

ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: www.tandfonline.com/journals/iclb20

Stability of 35 biochemical and immunological

routine tests after 10 hours storage and transport
of human whole blood at 21°C

Linda O. Henriksen, Nina R. Faber, Mette F. Moller, Ebba Nexo & Annebirthe
B. Hansen

To cite this article: Linda O. Henriksen, Nina R. Faber, Mette F. Moller, Ebba Nexo & Annebirthe
B. Hansen (2014) Stability of 35 biochemical and immunological routine tests after 10 hours
storage and transport of human whole blood at 21°C, Scandinavian Journal of Clinical and
Laboratory Investigation, 74:7, 603-610, DOI: 10.3109/00365513.2014.928940

To link to this article: https://doi.org/10.3109/00365513.2014.928940

© 2014 Informa Healthcare

Published online: 02 Jul 2014.

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Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 603–610

ORIGINAL ARTICLE

Stability of 35 biochemical and immunological routine tests after

10 hours storage and transport of human whole blood at 21°C

Linda O. Henriksen1, Nina R. Faber2, Mette F. Moller1, Ebba Nexo2 &

Annebirthe B. Hansen1
1Department of Clinical Biochemistry, Regional Hospital West Jutland, Herning and Holstebro, and
2Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark

Abstract
Background. Suitable procedures for transport of blood samples from general practitioners to hospital laboratories are
requested. Here we explore routine testing on samples stored and transported as whole blood in lithium-heparin or serum
tubes. Methods. Blood samples were collected from 106 hospitalized patients, and analyzed on Architect c8000 or Advia
Centaur XP for 35 analytes at base line, and after storage and transport of whole blood in lithium-heparin or serum tubes at
21  1°C for 10 h. Bias and imprecision (representing variation from analysis and storage) were calculated from values at
baseline and after storage, and differences tested by paired t-tests. Results were compared to goals set by the laboratory.
Results. We observed no statistically significant bias and results within the goal for imprecision between baseline samples and
10-h samples for albumin, alkaline phosphatase, antitrypsin, bilirubin, creatinine, free triiodothyronine, g-glutamyl transferase,
haptoglobin, immunoglobulin G, lactate dehydrogenase, prostate specific antigen, total carbon dioxide, and urea. Alanine
aminotransferase, amylase, C-reactive protein, calcium, cholesterol, creatine kinase, ferritin, free thyroxine, immunoglobulin
A, immunoglobulin M, orosomucoid, sodium, transferrin, and triglycerides met goals for imprecision, though they showed a
minor, but statistically significant bias in results after storage. Cobalamin, folate, HDL-cholesterol, iron, phosphate, potassium,
thyroid stimulating hormone and urate warranted concern, but only folate and phosphate showed deviations of clinical impor-
tance. Conclusions. We conclude that whole blood in lithium-heparin or serum tubes stored for 10 h at 21  1°C, may be
used for routine analysis without restrictions for all investigated analytes but folate and phosphate.

Key Words: Folic acid, laboratory test, phosphate, potassium, pre-analytical, sample transportation, storage temperature, storage time

Introduction tions are less variable, whole blood is centrifuged

soon after venipuncture and subsequent analysis per-
Centralized routine hospital laboratories perform an
increasing number of tests on blood samples pro- formed within a short time frame post-centrifugation
vided by general practitioners (GPs). It is important [3]. Furthermore primary tubes are used, in order to
that the results obtained on these samples, are com- ensure correct sample identity.
parable to those obtained when venipuncture is per- Focusing on cost-effectiveness it has in our region
formed at the hospital. In this context pre-analytical been suggested that centrifugation of blood samples
variations related to storage time and temperature as from GPs should be performed at the hospital labo-
well as transportation conditions of the blood samples ratories, provided that this does not compromise the
are of major importance [1,2]. quality of test results.
Transportation of blood samples from the GPs A number of studies have explored to which extent
office to the laboratories often relies on different it is possible to use whole blood samples, subject to
types of logistics, generally at ambient temperature prolonged storage, and thus allowing flexibility in the
and with variation in the time-frame of the entire requirements regarding transportation of blood sam-
process. In the hospital setting pre-analytical condi- ples to the hospital laboratories [4–15]. Results from

Correspondence: Linda O. Henriksen, PhD, Department of Clinical Biochemistry, Regional Hospital West Jutland, 7400 Herning, Denmark. Tel:  45
7843 5553. Fax:  45 7843 5580. E-mail: Linda.Oestervig.Henriksen@vest.rm.dk
This is an open-access article distributed under the terms of the CC-BY-NC-ND 3.0 License which permits users to download and share the article for
non-commercial purposes, so long as the article is reproduced in the whole without changes, and provided the original source is credited.

(Received 25 June 2013; accepted 25 May 2014)

ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare

DOI: 10.3109/00365513.2014.928940
604 L. O. Henriksen et al.

most studies indicate the necessity of more than one Holstebro (n  56), respectively, were included in
daily sample transportation from GPs office the study. Venous blood samples were collected
[4,6,8,9,11,13–15], but there are also studies sup- by experienced phlebotomists into two 7 mL
porting one daily transport [7,12]. However, the main lithium-heparin vacutainer tubes without gel sep-
part of the studies are relatively small covering up to arator for analysis on Architect c8000 (Holstebro)
30 analytes examined in five to 15 persons [4,6–8,12]. or two 5 mL serum vacutainer tubes without
Furthermore, most studies include blood from pre- gel separator for analysis on Advia Centaur XP
sumably healthy individuals [4,6–9,12,14,15]. (Herning) (Table I). Tubes were from Becton
Here we explore the feasibility of analyzing blood Dickinson, BD Diagnostics, Brøndby, Denmark
samples in lithium-heparin or serum tubes stored for (ref 368884 [lithium-heparin] and 368815
10 h at 21°C prior to centrifugation and cover 35 [serum]).
analytes with concentration both within and outside All samples for a specific analyte were collected
the reference interval. on the same day in September 2011. Sample
collection from each patient, otherwise undergoing
venipuncture, was performed during morning rou-
Methods tine, and samples were taken through the same
venipuncture site. Samples were subjected to
Sample collection
different settings of pre-analytical conditions
A total of 106 adult non-fasting patients admitted as described in the section entitled Storage
to regional hospitals in Herning (n  50) and conditions.

Table I. Analytes, number of samples and range of values.

IUPAC Baseline values Local reference

Short name Analyte Unit (NPU)† n Median (range) intervals (adults)

ALAT Alanine aminotransferase U/L 19651 56 18 (6–173) 10–70

ALB Albumin g/L 19673 56 37 (17–46) 34–48
AMYL Amylase U/L 19653 56 23 (5–218) 10–65
ATRYP Antitrypsin g/L 19692 34 1.83 (0.57–3.43) 0.97–1.68
B12* Cobalamin pmol/L 01700 49 406 (137–1476) 200–600
BASP Alkaline phosphatase U/L 19655 55 62 (33–503) 35–105
BILI Bilirubin mmol/L 01370 56 9 (4–87) 5–25
CA Calcium mmol/L 01443 56 2.33 (1.87–2.64) 2.20–2.55
CHOL Cholesterol mmol/L 01566 56 4.4 (2.0–7.2)  5.0‡
CK Creatine kinase U/L 19656 55 74 (7–551) 50–270
CO2 Total carbon dioxide mmol/L 01472 56 29 (16–41) 23–32
CREA Creatinine mmol/L 18016 56 69 (31–215) 45–105
CRP c-Reactive protein mg/L 19748 56 6.7 (0.2–217.5)  8.0
F-T3* Free triiodothyronine pmol/L 03625 50 3.7 (1.7–6.4) 3.9–6.8
F-T4* Free thyroxine pmol/L 03579 50 18.3 (11.7–33.1) 12.0–21.0
Fe Iron mmol/L 02508 55 12 (3–29) 9–34
FER* Ferritin mg/L 19763 50 309 (35–3716) 15–355
FOL* Folate nmol/L 02070 48 12 (2–39) 9
GGT g-glutamyl transferase U/L 19657 55 32 (11–442) 10–115
HAPT Haptoglobin g/L 19788 54 1.35 (0.20–4.33) 0.35–2.05
HDL-C HDL-cholesterol mmol/L 01567 56 1.2 (0.2–2.1)  1.0‡
IgA Immunoglobulin A g/L 19795 56 1.94 (0.86–5.21) 0.80–4.90
IgG Immunoglobulin G g/L 19814 56 9.9 (3.8–21.3) 6.1–15.7
IgM Immunoglobulin M g/L 19825 55 0.82 (0.13–2.27) 0.39–2.30
K Potassium mmol/L 03230 56 3.8 (3.1–4.8) 3.5–4.6
LD Lactate dehydrogenase U/L 19658 56 173 (95–609) 105–255
Na Sodium mmol/L 03429 56 139 (127–145) 137–145
OROS Orosomucoid g/L 19873 54 0.84 (0.28–2.45) 0.45–1.17
P Phosphate mmol/L 03096 56 1.02 (0.51–1.60) 0.71–1.53
PSA* Prostate specific antigen mg/L 08669 31 0.8 (0.1–11.6)  0.3
TGLY Triglycerides mmol/L 04094 56 1.1 (0.4–6.2)  2.0‡
TRANS Transferrin mmol/L 03607 55 27 (9–47) 24–41
TSH* Thyroid stimulating hormone mIU/L 03577 50 1.29 (0.020–8.72) 0.300–4.50
URATE Uric acid mmol/L 03688 56 0.25 (0.12–0.73) 0.15–0.48
UREA Urea mmol/L 01459 56 5.4 (2.6–25.7) 2.6–8.1

*Analysis performed on Advia Centaur XP using whole blood in serum tubes. For the other analytes analysis was performed on Architect
c8000 using whole blood in lithium-heparin tubes. †IUPAC, International Union of Pure and Applied Chemistry; NPU, Nomenclature
for Properties and Units. The NPU terminology is in national use in Denmark, for communication and recording of clinical laboratory
information. http://www.ssi.dk/EnglishNPU. ‡Decision limit.
 Stability of human whole blood at 21°C  605

Ethics and turbidimetry (ATRYP, CRP, HAPT, IgA,

IgG, IgM, OROS, TRANS). For HDL-C a direct
Procedures followed were in accordance with the eth-
Chol-HDL method using accelerator selective deter-
ical standards specified by the Helsinki Declaration
gent was used.
of 1975, revised in 1983. The study was presented to
Reliability of the analytical tests was ensured
and accepted by the Central Denmark Regional
through routine quality control determinations,
Science Ethics Committee as a technical and quality
including one daily measurement of quality control
investigation (Case nr. 1-10-72-2-12). Participants
materials in relevant levels and evaluating according
gave their informed consent.
to specified limits of acceptance. All analytical tests
were concluded reliable on the day of analysis as well
Storage conditions as on the following day. For PSA, only values  0.04
mg/L were included in the data evaluation (n  31).
Baseline samples were kept as whole blood (in lith-
ium-heparin or serum tubes) at room temperature Quality goals and related statistics
and centrifuged for 10 min at 2200 g and 20°C
within 30–90 min after sample collection. The The effect of sample storage including transportation
results for these samples were considered to repre- at 21°C was evaluated concerning bias and imprecision
sent the ‘true value’. 10 h samples, kept as whole (coefficient of variation [CV]). The mean deviation
blood (in lithium-heparin or serum tubes) were was calculated from the formula:
placed in racks in vertical upright position in trans- 100 ∗ ( X  X )
portation boxes and stored in the dark at a con- ∑ X0
10 0

trolled temperature of 21.0  1.0°C for 10 h. During Mean Deviation (%) 

N
the last 90 min of storage, 10-h samples were trans-
ported to the laboratory by car, air-conditioned to where: N Total number of pairs; X10  10-h value;
meet a temperature of 21.0  1.0°C. and X0  Baseline value.
At all times storage temperature was monitored The mean deviation was then compared to the
using temperature loggers (ICU Scandinavia AB, acceptable systematic difference (bias goal). We used
Aalborg, Denmark), which displayed a temperature values at baseline as compared to values obtained
span of 20.4–22.0°C during the described storage of after 10 h of storage in order to calculate the CV of
10 h samples. replicate measurements as previously described by
Ten-hour samples were centrifuged as described Rodbard [16], and compared the results to the
for the baseline samples after the relevant storage analytical- and the goal-CV. CV was calculated from
period. Following centrifugation both baseline sam- the following formula.
S
ples and 10 h samples were kept in the primary blood CV (%) 100 ∗ .
collection tubes at room temperature without light X
—
protection until analysis. where: X  mean of all measurements (both baseline
and 10-h values)

Biochemical analysis
S
∑(X 10  X 0 )2
Samples (n  34–56, depending on analyte) were ana- 2N
lyzed for 35 analytes (Table I) by single determination The precision we calculated thus represents variation
on automated analyzer systems Architect c8000 from both analysis and storage. Analytical CV denotes
(Abbott Diagnostics, Copenhagen, Denmark) at the the CV determined as the average analytical variation
regional hospital in Holstebro, or on Advia Centaur in laboratory routine quality control data in the year
XP (Siemens Healthcare Diagnostics, Ballerup, 2011. Bias- and CV goals refer to limits used in
Denmark) at the regional hospital in Herning accord- evaluating differences in laboratory routine quality
ing to the manufacturers’ recommendations, and fol- control and are generally determined from within-
lowing the routine procedure for the analysis. Both and between-subject biological variation [17,18] or
baseline samples and samples processed after 10 h of empirically. Data for goal bias as well as analytical-
storage were analyzed between 15 min and 3 h after and goal-CV used in this study are indicated for each
centrifugation dependent on the analyte. 10 h samples analyte in Table II.
were analyzed in random order as compared to the For all analytes differences between baseline values
order of baseline samples. The 35 analytes are listed in and values obtained after 10 h of storage of whole
Table I, which also lists the short names used. blood were assessed for normality and concluded
Analysis methods were: Chemiluminescense to meet the requirements for parametric statistical
immunoassay (B12, FER, FOL, F-T3, F-T4, PSA analysis. Student’s t-tests (Paired t-tests [two-tailed])
and TSH), photometry (ALAT, ALB, AMYL, BASP, were used, the level of significance set at p  0.05. All
BILI, CA, CHOL, CK, CO2, CREA, Fe, GGT, LD, statistical analyses were performed using STATA
P, TGLY, URATE, UREA), potentiometry (K, Na) (StataCorp LP, TX, USA) version 12.1.
606 L. O. Henriksen et al.

Table II. Imprecision (CV) and bias for 35 analytes. Results obtained after storage of whole blood at
21°C was compared to baseline results.

CV (%) Bias (%)

Paired t-test
Analyte CV† Analytical‡/goal§ Mean deviation (95% CI)|| Goal¶ p value††

ALAT 2.4 2.7/9.0  1.7 ( 2.9; 0.4) 12.0** 0.0034

ALB 1.5 0.9/3.2** 0.2 (0;0.5) 2.2** 0.067
AMYL 1.1 2.4/5.9  0.8 ( 1.2; 0.3) 8.0  0.0001
ATRYP 2.9 0.9/4.5  0.7 ( 1.5;0.1) 6.5 0.12
B12* 6.4 6.4/7.5  3.2 ( 5.6; 0.8) 17.7 0.015
BASP 1.2 2.2/4.8 0.2 ( 0.3;0.7) 6.4 0.31
BILI 2.1 3.2/6.4** 1.0 (0.2;1.9) 10.0** 0.11
CA 1.1 1.0/2.5** 1.0 (0.7;1.3) 2.0**  0.0001
CHOL 1.3 0.5/3.0** 0.6 (0.2;1.1) 4.0 0.0085
CK 1.4 0.8/5.7  0.7 ( 1.3; 0.1) 11.5 0.0023
CO2 3.4 5.0/6.0** 1.2 (0.0;2.4) 5.0** 0.16
CREA 1.1 1.3/2.7**  0.1 ( 0.6;0.3) 3.8** 0.50
CRP 1.8 1.8/5.0** 2.6 ( 0.5;5.7) 5.0** 0.046
F-T3* 2.4 2.2/7.5** 0.8 ( 0.3;1.8) 7.2 0.28
F-T4* 4.6 4.6/7.5** 2.1 (0.3;3.9) 3.6** 0.039
Fe 3.7 1.6/6.7** 6.8 (5.8;8.2) 8.8  0.0001
FER* 7.0 4.3/7.1  1.1 ( 2.7;0.5) 5.2 0.039
FOL* 20.5 6.6/12.0  13.7 ( 19.1; 8.3) 19.2 0.0007
GGT 2.6 1.6/3.5  0.8 ( 2.1;0.6) 5.4 0.62
HAPT 1.7 1.4/5.1 0.1 ( 0.4;0.6) 10.4 0.72
HDL-C 2.9 1.8/3.6 4.6 (3.7;5.4) 5.2  0.0001
IgA 2.1 1.3/2.7 1.9 (1.4;2.3) 9.1  0.0001
IgG 1.0 0.9/2.3 0.2 ( 0.2;0.5) 4.3 0.75
IgM 1.5 2.5/4.5** 1.7 (0.9;2.5) 11.9 0.0001
K 2.9 0.8/2.4 1.2 (0.1;2.2) 1.8 0.040
LD 4.5 3.1/5.0** 1.0 ( 1.3;3.1) 4.3 0.55
Na 0.4 0.7/1.0** 0.3 (0.1;0.4) 0.6** 0.0003
OROS 1.5 0.9/5.7 0.8 (0.1;1.5) 6.8 0.017
P 9.0 1.8/4.3  11.2 ( 12.4; 10.0) 3.2  0.0001
PSA* 3.4 3.4/9.1  0.4 ( 2.0;1.2) 18.7 0.083
TGLY 2.0 1.0/5.3**  0.9 ( 1.7; 0.1) 5.4** 0.018
TRANS 1.5 0.9/2.3 1.8 (1.4;2.2) 2.6**  0.0001
TSH* 9.2 4.9/9.7 6.0 (3.4;8.7) 7.8 0.0040
URATE 1.6 1.3/4.3** 1.5 (0.9;2.1) 4.8**  0.0001
UREA 1.0 1.9/6.2  0.2 ( 0.6;0.2) 5.5 0.072

*Analysis performed on Advia Centaur XP using whole blood in serum tubes. For the other analytes
analysis was performed on Architect c8000 using whole blood in lithium-heparin tubes. †CV calculation
based on measurements at baseline and after 10 h of storage. ‡Annual (2011) average CV of laboratory
routine quality control samples. §CV goals used in evaluating analytical variation in laboratory routine
quality control. Goals were generally based on biological variation from http://westgard.com/biodatabase1.
htm and calculated as described by Fraser et al. [18]. **denotes goals where empirically based modifications
to the three stated levels of performance were used. ||Mean deviation % between baseline and 10-h values
and 95% confidence interval (shown as confidence limits). ¶Bias goals used in evaluating systematic
differences in laboratory routine quality control. See §CV goals for further details. ††p-values for testing
baseline values against values obtained after 10 h of storage of whole blood.

Fractional Bland-Altman plots were depicted in whole blood (in lithium-heparin or serum tubes) at
order to evaluate study results, e.g. concentration 21°C. Our choice was to use samples from hospital-
dependent alterations. Baseline results were depicted ized patients in order to cover a wide range of values,
as X-values, and fractions [(10-h storage values)/ including both normal as well as pathological levels
(baseline values)] as the Y-values. (Table I).
Calculations were executed using Microsoft We used the upper or lower measurement range
Office Excel 2003 software, and GraphPad version values for the few results for ALAT, B12, CK, CRP
4.0 was used for depicting the results. and HAPT reported as above or below this range.
For CK, CRP and HAPT values were below the
lower measurement range for both baseline and 10-h
Results
samples (one sample set affected for each analyte).
We examined the stability of 35 biochemical and For ALAT three sets of samples showed results below
immunological analytes as a function of storage of the lower measurement range for 10-h samples, two
 Stability of human whole blood at 21°C  607

of these also at baseline. For B12 two sets of samples increase was observed. Thus, for values below
showed results above the upper measurement range 10 mmol/L, the average increase was approximately
for baseline samples, one of these also for the 10-h 11% after 10 h of storage. FOL decreased around
sample. 14%, whereas for HDL-cholesterol, a concentration-
Thirteen of the analytes showed no statistically dependent relative increase was seen, the average
significant bias and mean CVs below or close to the increase being 7% for values below 1.0 mmol/L. For
analytical CV upon storage of whole blood for 10 h K, we observed a tendency to increased values after
(ALB, ATRYP, BASP, BILI, CO2, CREA, F-T3, 10 h of storage. P showed an average of around 11%
GGT, HAPT, IgG, LD, PSA, UREA), and thus will decrease in values throughout the measurement
not be subject to any further discussion (Figure 1 range. For TSH, we found a relative increase in
and Table II). For most of the remaining analytes values, especially in the lower measurement range,
bias was statistically significant though quantitatively where values  1.25 mU/L showed an average
small (ALAT, AMYL, CA, CHOL, CK, CRP, F-T4, increase of 8% after 10 h of storage. This was how-
FER, IgA, IgM, K, Na, OROS, TGLY, TRANS and ever caused by few samples with a very high bias. For
URATE). A more pronounced bias was observed for URATE, we observed a concentration dependent
B12, Fe, FOL, HDL-C and TSH. Bias exceeding the relative increase after 10 h of storage. For values in
bias goal was observed only for P (Figure 1 and Table the lower part of the reference interval and below
II) and the 95% confidence interval (95% CI) of bias (values  0.24 mmol/L) the average increase was 3%.
was below the bias goal for all but CRP, FT-4,
HDL-C, K and TSH. Despite the changes in bias all
Discussion
but three analytes (FOL, K and P) met the criteria
set forward for imprecision – the goal-CV (Figure 1 We investigated the effect of storage of venous whole
and Table II). blood in lithium-heparin or serum tubes at a tem-
We also analyzed the results using fractional perature of 21°C for 10 h, including 90 min of trans-
Bland-Altman plots, and present plots for B12, Fe, portation by car. Our study included the 35 most
FOL, HDL-C, K, P, TSH, and URATE (Figure 2). frequently requested biochemical and immunological
B12 showed an average of around 3% decrease analytes from GPs. The results obtained covered a
in values. For Fe, a concentration-dependent relative broad range of analyte concentrations, since samples

Figure 1. Imprecision and bias calculated for results obtained at baseline and after 10 h of storage of whole blood (in lithium-heparin or
serum tubes). Data on imprecision (A) shows CV calculated from results obtained at baseline and after storage of whole blood in lithium-
heparin or serum tubes at 21°C for 10 h. The analytical imprecision is indicated as grey columns and the goal imprecision as the total
height of the columns. Data on bias (B) shows the mean bias between baseline and results obtained after storage of whole
blood for 10 h. The columns indicate bias goal. Data for analytical- and goal-CV as well as bias goal are indicated for each analyte in
Table II.
608 L. O. Henriksen et al.

Figure 2. Bland-Altman plots of results obtained after storage of whole blood (in lithium-heparin or serum tubes) for 10 h prior to analysis
as compared to results at baseline. Fraction: Ratio between baseline results and results at 10 h. Indicated on each figure is the line of
identity, i.e. baseline/10 h  1.0 (bold dashed line), the analytical CV (thin dashed line), goal-CV (full drawn thin line) and the reference
interval (shaded area). Data for analytical- and goal-CVs are indicated for each analyte in Table II.

were obtained from hospitalized patients rather than decrease in concentration upon storage to be of
from volunteers (Table I). To the best of our know­ limited importance.
ledge this is the first study to include data on ATRYP, We estimated the mean CV for all analytes
IgA, IgG, IgM, and OROS evaluating stability of studied based on comparison of baseline values
whole blood at 21°C. with values obtained after 10 h of storage. Obviously
In our study some of the analytes showed a sta- CVs calculated in this manner will mirror not
tistically significant but quantitatively small bias not only the analytical imprecision but also any observed
depending on the concentration of the analyte. Only bias. The fact that the mean CV exceeded the
few analytes showed more pronounced changes (B12, goal-CV only for FOL, K and P underscored
Fe, FOL, HDL-C, P, and TSH); however, the that the observed bias was unlikely to be of clinical
observed bias were smaller than the bias goal for all importance.
but P. Previous relatively small studies covering up to
In addition we reported concentration-dependent 30 analytes, have shown that prolonged (up to 24 h)
statistically significant relative changes for Fe, storage of blood samples from presumed healthy
HDL-C, TSH, and URATE. For these analytes we individuals introduces considerable changes for sev-
found spuriously high results in the lower level of the eral analytes, both when samples of whole blood
reference interval and below (Figure 2).The increased are stored at room temperature (20–25°C) and espe-
level for Fe was likely to be caused by leakage from cially when stored at temperatures well above room
the red blood cells, while the deviations for HDL-C, temperature [4–8]. Particular critical analytes are
TSH, and URATE remained unexplained. Though ALAT, K, LD, and P, which in most studies are
these differences may be of importance in the research reported as stable only for a few hours at room tem-
setting, we judged them to be of no importance in perature and above (up to 12 h depending on analyte
the clinical setting. For B12 we judged the observed and study) [4,8–10,12,15,19].
 Stability of human whole blood at 21°C  609

The studies were undertaken on serum [5–9,15,19] showing more than a ∼10% deviation to baseline after
or plasma [7–10,12], in tubes with [5,7,10,15] or 8 h of storage. One possible explanation for the dis-
without gel separator [6,8,9,19]. The most extensive crepancies was a difference in the design of our study
study performed so far was based on whole blood (in as compared to the others. The three studies
lithium heparin tubes with gel) drawn from a large [11,21,23] all using lithium-heparin whole blood
cohort of patients (n  406) at GPs offices [11]. Ana- samples separated plasma after centrifugation of
lyzing samples from 100 patients, the authors con- baseline samples, whereas we followed daily routine
cluded that all but two (K and P) out of 21 and performed the analysis on primary tubes soon
analytes tested were stable for 8 h at 21°C pre- after centrifugation without separation of plasma.
centrifugation, while K and P met quality goals only Based on our data, we concluded that K can be ana-
after storage for up to 4 h. The study included a lyzed on blood stored for up to 10 h at 21  1°C.
transportation process of 10–15 min duration, which In agreement with others, studying the stability
was shorter than the relevant transportation time of P in lithium-heparin whole blood stored at
for most GPs. A recent study on patient samples 21  1°C for periods between 4 and 12 h [8,10–12],
(n  100) stored as whole blood (in serum-separation we observed a considerable mean decrease to baseline
tubes with gel) at 15–25°C for 6 h, including a 2-h samples (11.2%) in P after 10 h of storage.
transportation procedure, concluded that K, LD Storage at room temperature has shown a biphasic
and P did not meet quality goals set by the authors, change in the concentration of P. Decreased levels
whereas FOL did [13]. are reported after storage up to 8–16 h, and increased
Combined with the results obtained in our study, concentrations after 24–48 h of storage [4,7,9,14].
all of these results suggest that whole blood can be The decrease in the concentration of P is likely to be
transported for analysis at the hospital laboratory. caused by a consumption of inorganic P during intra-
However, as evident from the above, some concern cellular phosphorylation of glucose, whereas an
is still warranted for some analytes. increase could be ascribed to hydrolysis of organic
An increased level of K caused by leakage from phosphates and leakage from the red blood cells, red
blood cells has over the years been a major concern blood cells having a 7-fold higher concentration of
[20,21]. Also, the changes in K levels caused by the P as compared to plasma [4,14,19].
activity of the temperature-dependent NaK- We observed a large average decrease (13.7%) in
ATPase has to be considered. Transporting samples the FOL level after storage of whole blood in serum
at low temperatures leads to increased K levels, and tubes. It is well known that FOL is unstable, under-
at high temperatures K levels are typically decreased going oxidative cleavage in stored blood samples
[12,14,22]. [24]. In addition, FOL has been described to be
Tanner et al. [14] studying serum obtained from light-sensitive [25]. Our results on FOL are in accor-
gel tubes reported temperature-dependent changes dance with previous studies on the stability of human
in K concentrations, and concluded that storage at whole blood stored at 21°C and above for periods
25°C was superior to 15°C and 35°C. However, between 2 and 96 h [9,26,27].
accepting a 7% deviation, K only remained stable for The current study was planned to mirror the rou-
4 h. Temperature-dependent changes in K concen- tine setting of the clinical biochemistry laboratory,
trations in lithium-heparin whole blood stored for and thus did not allow refined conclusions concern-
2–12 h at temperatures ranging from 17–25°C have ing the influence of 10 h storage of whole blood. To
also been presented by Stahl et al. [12].They observed do so, several issues should be taken into account.
the mean difference from initial values to be lowest Plasma/serum should be separated as soon as possible
at 20°C, and accepting a 10% deviation, concluded after blood collection and analysis of the baseline
K being stable in whole blood for 12 h at 20°C. sample should be done not only at baseline but also
Our study showed less deviations in K concentra- at the same time as the 10-h sample. In addition, in
tions upon storage of lithium-heparin whole blood, a refined study, it may be of relevance to use goals
than observed in other studies on patient whole for bias and CV defined solely by biological variation
blood samples [11,21,23]. In our study, only one out rather than based on a combined knowledge of
of 56 samples showed K values with more than 10% biological variation and analytical performance in
deviation (10.1%) to baseline samples – a criteria daily practice.
used by both Jensen et al. [11] and Nybo et al. [23]. Based on the results, it is our conclusion that
Nybo et al., however, observed approximately 40% storage of whole blood in lithium-heparin or serum
of samples deviating more than ∼10% after 6 h of tubes at 21  1°C for up to 10 h induces changes
storage at room temperature, and applying the same that are of no importance in the clinical setting,
criteria to the data presented by Seamark et al. [21] and that caution is warranted only for FOL and P.
only 70–80% of the samples showed acceptable val- In the research setting time- and concentration-
ues for K after storage at temperatures between 15 dependent changes may be of importance, notably
and 30°C for up to 12 h. Jensen et al., who stored if samples are to be retrieved both in the GPs office
samples at 21  1°C, reported 12–13% of samples and at the hospital. For daily clinical practice, we
610 L. O. Henriksen et al.

believe that the use of whole blood samples is supe- [10] Leino A, Koivula MK. Stability of chemical and immuno-
rior to the alternative, involving centrifugation and chemical analytes in uncentrifuged plasma samples. Ann
Clin Biochem 2009;46:159–61.
further handling of the blood samples in the GPs [11] Jensen EA, Stahl M, Brandslund I, Grinsted P. Stability of
office.­­­­ heparin blood samples during transport based on defined
pre-analytical quality goals. Clin Chem Lab Med 2008;
46:225–34.
[12] Stahl M, Brandslund I. Controlled storage conditions pro-
Acknowledgements
long stability of biochemical components in whole blood.
We wish to thank our phlebotomy staff for their assis- Clin Chem Lab Med 2005;43:210–5.
tance with the specimen collection and analysis. [13] Dialma P, Piaulenne S, Baty S, Zeitoun T. [Preanalytical
phase and accreditation: acceptance criteria for samples of
We also thank Karin Biering, MHSc, Department of multisite laboratory]. Ann Biol Clin (Paris) 2013;71:121–8.
Occupational Medicine, Regional Hospital West [14] Tanner M, Kent N, Smith B, Fletcher S, Lewer M. Stability
Jutland, Herning, Denmark, for assistance regarding of common biochemical analytes in serum gel tubes subjected
statistical analyses. Financial support from the to various storage temperatures and times pre-centrifugation.
Central Denmark Region is acknowledged. Ann Clin Biochem 2008;45:375–9.
[15] Kang HJ, Jeon SY, Park JS, Yun JY, Kil HN, Hong WK,
Lee MH, Kim JW, Jeon JP, Han BG. Identification of
Declaration of interest: The authors report no clinical biomarkers for pre-analytical quality control of
blood samples. Biopreserv Biobank 2013;11:94–100.
conflict of interest. The authors alone are responsible [16] Rodbard D. Statistical quality control and routine data
for the content and writing of the paper. processing for radioimmunoassays and immunoradiometric
assays. Clin Chem 1974;20:1255–70.
[17] Fraser CG, Petersen PH, Ricos C, Haeckel R. Proposed
quality specifications for the imprecision and inaccuracy of
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