International Journal of Laboratory Hematology

The Official journal of the International Society for Laboratory Hematology

ORIGINAL ARTICLE INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Guidance for storing blood samples in laboratories performing

complete blood count with differential
E. CORNET* , † , 1 , C. BEHIER* ,1 , X. TROUSSARD* , †

*CHU de Caen, Laboratory of S U M M A RY

Hematology, Caen, France
†
University of Caen, Medical
School, Caen, France
Correspondence:
Edouard Cornet, Laboratory of
hematology, CHU de Caen,
Avenue Côte de Nacre, 14033
Caen Cedex 9, France.
Tel.: +33 2 31 06 33 15;
Fax: +33 2 31 06 50 99;
E-mail: cornet-e@chu-caen.fr
doi:10.1111/j.1751-553X.2012.01452.x
Received 1 February 2012;
accepted for publication 8 May
2012
Keywords
Blood cell count, laboratories/
standards, reproducibility of
results, blood preservation, tem-
perature
Introduction The complete blood count (CBC) with differential leu-
kocyte count (DIFF) is an important part of clinical laboratory anal-
yses and provides crucial data for clinicians. Delivery time after
blood collection and conditions of storage is known to affect the
reliability of results of some hematologic parameters. The aim of
this study was to assess the variations of hematologic parameters
over time and the influence of storage temperature.
Methods Blood samples were randomly selected from hospitalized
patients and stored at room temperature and at 4 °C. CBC and
DIFF were performed on an automated hematology analyzer and
the results between the two groups were compared.
Results Samples stored at room temperature showed an important
increase in mean corpuscular volume and hematocrit and a
decrease in mean corpuscular hemoglobin concentration. Neutro-
phil counts tended to increase, whereas monocyte counts tended to
decrease.
Conclusion Storing samples at 4 °C improved reproducibility over
time of all quantitative and qualitative parameters. We also
observed that NEUT-X, a routine parameter useful in detecting
myelodysplastic syndrome, became unreliable when analyzed 24 h
after sample collection. Our results led us to recommend that sam-
ples should be analyzed within 6 h, particularly if samples are
transported at room temperature. We also recommend storing sam-
ples at 4 °C in case of remote CBC analysis, especially in the con-
text of clinical trials.

formed laboratory tests in our institution, with about

INTRODUCTION
The complete blood count (CBC) with differential leu-
kocyte count (DIFF) is one of the most routinely per-
1
These authors contributed equally to this work.
190 000 analyses performed in 2011. It is a valuable
screening tool for a wide variety of medical conditions
and is an essential routine in the optimal management
of patients. Compliance with International Organiza-
tion for Standardization (ISO) 15 189, the international

© 2012 Blackwell Publishing Ltd, Int J Lab Hem. 2012, 34, 655–660 655
656 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES
standard for the accreditation of medical laboratories
(1), is becoming progressively accepted as the optimal
approach to assure quality in medical testing.
The main objective of any clinical laboratory is to
produce results that are accurate and precise enough
for clinical use, and therefore, the correct pre-analyti-
cal procedures are essential. Many external variables
can influence laboratory results, including conditions
of blood collection, storage and delivery time of sam-
ples to the laboratory. It is particularly critical when
blood samples are collected at the patients’ home.
Delivery time to the laboratory can exceed 8 h.
The development of clinical trials and centralized
analyses will impose adoption of strict pre-analytical
rules. Healthcare laboratories must establish limits
for delivery time and conditions of storage and the
impact of pre-analytical conditions on hematologic
parameters must be known for accurate interpre-
tation of all parameters provided by an automated
analyzer.
This study confirms the impact of pre-analytical
conditions on variations of CBC-DIFF parameters and
also highlights new data to interpret NEUT-X, which
is a routine parameter useful in detecting myelodys-
plastic syndrome (MDS). Performing CBC/DIFF analy-
sis within 6 h after sample collection guarantees good
reliability of the results.
M AT E R I A L S A N D M E T H O D S
Samples and analyzer
We studied 64 venous peripheral blood samples,
randomly selected among hospitalized patients in
Caen University Hospital. Samples were collected in
vacutainers containing dipotassium ethylene diamine-
tetraacetate (K2EDTA – BD Vacutainer; Becton and
Dickinson, Le Pont-De-Claix, France), which is the
anticoagulant of choice in specimen collection for
blood cell counting and sizing, as recommended by
the International Council for Standardization in
Hematology (2). This salt also has been shown to
affect red blood cell size less than tripotassium salt
(3). Thirty-two samples were stored at room tempera-
ture (RT) (Group 1) and 32 at +4 °C (Group 2). CBC/
DIFF was processed at different time points during a
period of 3 days, that is, at delivery (t0) then 6, 12,
24, 48 and 72 h (t6, t12, t24, t48 and t72). The maxi-
mum time interval between blood collection and sam-
ple analysis at t0 (reference values) was 4 h. All
samples were normal blood samples, without any
qualitative abnormalities.
Laboratory methods
Analyses were performed in automatic mode on a
Sysmex XE-2100 automated multiparameter hematol-
ogy analyzer (Sysmex Corporation, Kobe, Japan) in
accordance with manufacturer’s recommendations.
The analyzer was calibrated by the manufacturer.
Two levels of controls (internal quality control) were
run on a daily basis (e-check® XE control) according
to manufacturer’s recommendations. Only the data
from the CBC, DIFF and Immature Myeloid Informa-
tion channels were taken into account, we did not
consider reticulocyte count or nucleated red blood
cell count (NRBC) in this analysis. Parameters of
CBC measured in this study were the following:
white blood cell count (WBC), red blood cell count
(RBC), hemoglobin concentration (HB), hematocrit
(HCT), mean corpuscular volume (MCV), mean corpus-
cular hemoglobin (MCH), mean corpuscular hemo-
globin concentration (MCHC) and platelet count (PLT).
The study assessed relative percentages of clusters: neu-
trophil (NEU%), eosinophil (EOS%), basophil (BASO
%), lymphocyte (LYMPH%), monocyte (MONO%)
and immature granulocyte (IG%). All presented per-
centages were automatically generated parameters.
Statistical analysis
As previously described in the literature, stability of
an analyte was defined in relation to the imprecision
of the respective analytical method. A parameter was
considered stable when its average change was smaller
than one coefficient of variation (CV, %) of the
assessed method, allowing a 5% risk of error (4, 5).
CV was obtained with daily controls. Except for EOS
%, BASO% and IG%, we evaluated the variability of
each parameter at each time point by calculating the
mean percent change from the initial value (t0). We
evaluated the variability of EOS%, BASO% and IG%
by calculating the mean absolute difference from the
initial value rather than percent change because some
initial values were at zero. Statistical analyses were
performed for the two groups. Mean, standard
© 2012 Blackwell Publishing Ltd, Int J Lab Hem. 2012, 34, 655–660
 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES 657

deviation (SD) and 95% confidence interval (95% CI)

Table 1. Initial characteristics (t0) of the 32 samples
were calculated with MicrosoftTM Excel® software. stored at room temperature (Group 1) and the 32
samples stored at +4 °C (Group 2)
R E S U LT S Group 2 (+4°
Group 1 (RT) C)
Samples characteristics Mean SD Mean SD P

All samples were normal blood samples. Characteris- White blood cell 9.75 3.83 8.50 4.83 0.25575 (NS)
tics of the two groups were comparable for all mea- count (109/L)
Red blood cell 4.04 0.83 3.86 0.76 0.38470 (NS)
sured parameters (P > 0.05) (Table 1). We considered count (1012/L)
values at t0 as reference values. Hemoglobin (g/dL) 12.30 2.58 11.66 2.15 0.28535 (NS)
Hematocrit (%) 36.3 7.2 34.7 6.0 0.34182 (NS)
Mean corpuscular 90.3 5.9 90.4 5.5 0.94268 (NS)
CBC parameters volume (fl)
Mean corpuscular 30.6 2.3 30.4 2.4 0.72081 (NS)
Many parameters of CBC were stable throughout the hemoglobin (pg)
Mean corpuscular 33.8 1.3 33.5 1.4 0.39222 (NS)
study period. No significant difference was observed hemoglobin
for WBC, RBC, HB, MCH and PLT in Group 1 and concentration (g/
Group 2 until 72 h. Conversely, there was a signifi- dL)
Platelet count 224 87 225 87 0.97345 (NS)
cant MCV difference from t0 to t6 in Group 1. At 6 h, (109/L)
the percent change was 4.2% (95% CI, 1.91; 6.42) Neutrophils (%) 67.73 13.56 62.65 16.46 0.18334 (NS)
and regularly increased, reaching 17.5% (16.6418.42) Eosinophils (%) 2.22 1.79 3.33 2.85 0.06595 (NS)
Basophils (%) 0.34 0.26 0.50 0.47 0.11342 (NS)
at 72 h (CV for MCV was 0.96%) (Figure 1). In Lymphocytes (%) 18.22 10.84 22.36 14.12 0.19332 (NS)
Group 2, percent changes at t6 and t72 were, respec- Monocytes (%) 10.75 4.28 10.15 5.33 0.61772 (NS)
tively, 1.2% (1.08; 1.31) and 1.5% (1.14; 1.85). Immature 0.74 0.92 1.01 1.49 0.38757 (NS)
granulocytes (%)
Hence, MCV increased at both RT and +4 °C but this
change was more limited in case of refrigerated stor- Results are expressed as mean and standard deviation
age. As a consequence, we also observed a significant (SD). NS: Not significant.
variation of MCV-related parameters at RT with a pro-
gressive decrease in MCHC and a progressive increase temperature (4, 6–9). All of these studies described an
in HCT. increase in MCV and a variation of its related
parameters (HCT and MCHC). These observations
were independent of the type of automated analyzer.
DIFF parameters
Similarly, our study demonstrated a significant varia-
No significant variations of the WBC count were tion of many hematologic parameters over 3 days
detected between t0 and t72. Only samples from related to storage conditions of blood. The largest vari-
Group 1 showed an increase in NEU% from t48 ation concerned erythrocyte parameters. MCV
(Figure 1). Percent change at t72 was +8.2% (5.84; increased dramatically from 6 h if samples were kept
10.54) (CV for NEU% was 1.62%). In contrast, at RT, but was limited in case of refrigerated storage
MONO% decreased from t48 ( 28.4%, 36.87; (+4 °C). This observation could be related to a swell-
19.88) (CV for MONO% was 7.08%). No significant ing of red blood cell because of a decrease in mem-
variation was detected in Group 2. Both groups brane resistance and an increase in cell permeability.
showed no LYMPH% variation, and no significant This phenomenon seemed to depend on storage dura-
variation was observed for EOS%, BASO% and IG%. tion and temperature. As expected, these modifica-
tions also affected MCV-related parameters, such as
HCT and MCHC, which are calculated from the MCV,
DISCUSSION
with an increase in HCT and a decrease in MCHC. As
A few studies have analyzed variations of CBCDIFF previously described (6), RBC, HB, MCH and PLT
with regard to the storage of blood specimens at room measurements were unmodified until 72 h at RT.

© 2012 Blackwell Publishing Ltd, Int J Lab Hem. 2012, 34, 655–660
658 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES

HB MCH
from reference value (t0)

RBC

from reference value (t0)

from reference value (t0)

1

% - Mean (CI95%)
1

% - Mean (CI95%)
% - Mean (CI95%)

Percent change

Percent change
Percent change

±1 CV% 1
±1 CV% 0
0 (±0.68%)
(±0.62%) –1 ±1 CV%
0
–1 RT (±0.81%)
RT –2
+4 °C –1 RT
+4 °C
–2 –3 +4 °C
t6 t12 t24 t48 t72 t6 t12 t24 t48 t72 t6 t12 t24 t48 t72

WBC LYMPH% PLT

from reference value (t0)

from reference value (t0)

from reference value (t0)

RT
6 15 RT
% - Mean (CI95%)

% - Mean (CI95%)

% - Mean (CI95%)
+4 °C
Percent change

Percent change

Percent change
RT 10
10
+4 °C
4 +4 °C 5 5
2 0
±1 CV%
(±2.91%) 0
±1 CV%
±1 CV% –5
0 (±2.81%)
(±2.15%) –10 –5
–2
–15
t6 t12 t24 t48 t72 t6 t12 t24 t48 t72 t6 t12 t24 t48 t72

HCT MCV MCHC

from reference value (t0)

from reference value (t0)

from reference value (t0)

* 20 * 5
% - Mean (CI95%)

% - Mean (CI95%)

% - Mean (CI95%)
Percent change

Percent change
Percent change

15 * RT * RT ±1 CV%
0
+4 °C 15 +4 °C (±1.25%)
10 * 10
* –5
* * * * * *
–10
5 5 * RT
±1 CV% ±1 CV% –15
* +4 °C
0
(±1.05%)
0
(±0.96%) –20
*
t6 t12 t24 t48 t72 t6 t12 t24 t48 t72 t6 t12 t24 t48 t72

NEU% MONO%
from reference value (t0)

from reference value (t0)

* RT 20
% - Mean (CI95%)
% - Mean (CI95%)
Percent change

Percent change

10
+4 °C ±1 CV%
5
* 0
(±7.08%)
0
±1 CV% –20
(±1.62%) RT
–5 –40
* +4 °C
–10 –60 *
t6 t12 t24 t48 t72 t6 t12 t24 t48 t72

Figure 1. Average percent changes from reference value (t0) of complete blood count and differential leukocyte
count (DIFF) parameters for Group 1 (RT) and Group 2 (+4 °C). Percent changes were calculated from reference
values at t0. Each data point represents the mean percent change and each bar represents the 95% confidence
interval (95% CI). A parameter was considered as unstable if its average change (with 95% CI) was higher than
the coefficient of variation (CV) of the method. Results are presented for samples stored at room temperature (RT)
and under refrigeration (+4 °C). CV% was obtained with daily controls. WBC indicates white blood cell count;
RBC, red blood cell count; HB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume;
MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet count;
NEU%, neutrophils (%); LYMPH%, lymphocytes (%); MONO%, monocytes (%). *Significant variation as
compared to CV of the method. All parameters were stable until 72 h at +4 °C. Significant variations were
observed at RT from t6, for erythrocyte volume-related parameters, such as HCT, MCV and MCHC. Significant
variations were observed at RT for NEUT% (increase) and MONO% (decrease) from t48.

© 2012 Blackwell Publishing Ltd, Int J Lab Hem. 2012, 34, 655–660
 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES 659

In our study, leukocyte count (WBC) was
unchanged until 72 h at both RT and +4 °C. Modifica-
tions of DIFF were exclusively observed at RT, with
an increase in NEU% and a decrease in MONO%.
Indeed, modification of cellular structure over time
could alter detection and identification of cells, partic-
ularly if blood specimens are stored at RT. To evaluate
this progressive cellular deterioration, we tested sec-
ondarily eight normal blood samples at RT (data not
shown). In addition to the usual DIFF parameters, we
also considered the neutrophil structural and matura-
tion parameters provided by our analyzer: NEUT-X
(10). NEUT-X is the direct measurement of side scatter
diffraction and is representative of neutrophil struc-
ture. We compared NEUT-X with manual differential
and cell morphology analyzed by microscopic meth-
ods. Microscopic DIFF count became unreliable from
48 h, with a decrease in monocyte count and an
increase in neutrophil count. We observed a progres-
sive decrease in NEUT-X from 48 h, which was corre-
lated with an increase in neutrophil cellular
deterioration on blood smears (karyorrhexis)
(Figure 2). Analysis of the DIFF scattergram confirmed
that the position of neutrophils on the x-axis had
shifted to the left (NEUT-X) and showed that position
of monocytes on the y-axis tended to shift down into
the neutrophil cluster (Figure 2). Lymphocyte mor-
phology seemed to be less affected by this cellular
deterioration. As recently demonstrated (11) that
NEUT-X value is correlated with hypogranularity of
neutrophils and therefore is useful in detecting MDS.
Our observations indicated the unreliability of NEUT-
X when samples were analyzed 24 h after collection.
Checking the time between sample collection and
analysis should be given high priority before reporting
results, because significant variations were observed for
RBC volumetric indices from 6 h and from 48 h for dif-
ferential WBC. These variations may have conse-
quences on diagnostic classification and clinical
strategies (12). For instance, a false increase in MCV,
especially if associated with a false decrease in NEUT-X,
could be interpreted as a sign of MDS. Such a mistake
could result in unnecessary complementary tests, espe-
cially invasive procedures such as medullar biopsy. In

Figure 2. Influence of time on differential leukocyte count (DIFF) parameters and neutrophils cellular structure for
a sample stored at RT. DIFF parameters and neutrophils cellular structure were analyzed by automated method
(NEUT-X) and microscopic method. Analyzer graphs, automated and manual DIFF are represented. We observed a
progressive increase in neutrophils (NEU%), a progressive decrease in monocytes (MONO%). Microscopic analysis
revealed a progressive neutrophil cellular deterioration on blood smears from t48 (karyorrhexis at t96), correlated
with a degradation of neutrophils structure (side scatter on x-axis) identifiable by the progressive decrease in
NEUT-X. LYMPH%, lymphocytes (%).

© 2012 Blackwell Publishing Ltd, Int J Lab Hem. 2012, 34, 655–660
660 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES

contrast, this false increase in MCV could hide a real
microcytosis in case of iron deficiency anemia.
Limits for delivery time and storage condition could
be defined with goals for desirable bias for each
parameter. Quality specifications have been published:
(13) proposed recommendations for maximum allow-
able storage time on EDTA-anticoagulated blood: 6 h
for CBC and DIFF at RT and 24 h at +4 °C. Various
systems were used to propose tolerance limits for
analytic bias (13). The method based on variations in
population test distribution seemed to be the best,
even though it is based on relatively complicated
calculations. It is the method that best meets clinical
needs (14). For example, they proposed a desirable
bias of 0.7% for MCV (15). Our MCV results did not
comply with these recommendations from 6 h at RT
and +4 °C. For MONO%, the decrease did not comply
with specifications from 48 h only at RT (desirable
specification for bias is 3.5%). Regarding the other
parameters for which percent changes were calculated,
the bias was not higher than that recommended.
To conclude, we recommend that CBC and DIFF
analysis should be performed within 6 h after blood
sample collection. In samples analyzed 24 h after col-
lection, a decreased NEUT-X, which is associated with
morphological degradation of neutrophils, has to be
interpreted cautiously and, in particular, should not
be used for detecting MDS. This recommendation has
to be taken into account when blood samples are col-
lected at home or in clinical trials, where analysis is
sometimes performed in remote analytical platforms.
In cases where the time between collection and analy-
sis is longer than 6 h, we recommend that samples
should be stored at +4 °C to maintain the stability of
CBC/DIFF parameters and a good reliability of results.
Complying with ISO requirements, especially for con-
trolling the pre-analytical phase, appears to be imper-
ative to avoid erroneous conclusions.
DISCLOSURES
The authors disclosed no potential conflicts of interest.

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