Survey of changes in complete blood count and red cell indices of whole blood incubated in vitro at different temperatures up to 48 hours.

Abstract

Complete blood count (CBC) is one of the most common and conventional blood test that physicians usually request. However the results of this test are affected by different factors such as, the temperature and duration of incubation, therefore the aim of this survey was to evaluate the effect of temperature and time of incubation on CBC, red blood cells (RBC) indices and white blood cells (WBC) differential count.
In a cross-sectional study, blood samples were taken from 30 healthy medical students of Rafsanjan University (15 males and 15 females). The samples divided into three parts; CBC were done on the samples up to 48 hours incubation at temperature of 25, 30, and 370 C at the time of sampling, and after 2, 8, 24 and 48 hours. Data were statistically analyzed and the following results were obtained.
RBC count, hematocrit, MCH, percent of monocytes and eosinophils were constant in different temperatures, WBC count, MCHC, hemoglobin, platelets count, the percent of lymphocytes and neutrophils were constant up to 24 hours and then tend to increase with increasing temperature except lymphocytes percent that tend to decrease. MCV decreased with increasing temperature up to 8 hours and then significantly increased (from 83.89 to 87.50 fmol/l, p < 0.001). WBC, hematocrit, MCV, platelets count, and neutrophils' percent tend to increase by the time of incubation, but RBC count, MCHC, lymphocytes' percent decreased. Hemoglobin, MCH, and the percent of monocytes and eosinophils were constant.
The finding of this survey showed that some of CBC parameters can be changed with the incubation, therefore it is better to do the CBC test after blood taking as soon as possible.

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J Ayub Med Coll Abbottabad 2006;18(1)

SURVEY OF CHANGES IN COMPLETE BLOOD COUNT AND RED
CELL INDICES OF WHOLE BLOOD INCUBATED IN VITRO AT
DIFFERENT TEMPERATURES UP TO 48 HOURS
Mehdi Mahmoodi*, Mohammareza Hajizadeh*, Hamidreza Rashidinejad**, Gholamreza
Asadikaram*, Mohammad Khaksari***, Mohammadreza Mirzaee*,
Naser Seyedi†, Amir Rahnema††, Ahmadreza Sayadi†††
*Department of Biochemistry and Biophysics, **Internal Medicine, Physiology and Pharmacology, Faculty of Medicine, Rafsanjan, Iran.
†General Practitioner, †† Department of Pathology, †††Department of Psychology, Faculty of Medicine, Rafsanjan, Iran
Background: Complete blood count (CBC) is one of the most common and conventional blood
test that physicians usually request. However the results of this test are affected by different
factors such as, the temperature and duration of incubation, therefore the aim of this survey was to
evaluate the effect of temperature and time of incubation on CBC, red blood cells (RBC) indices
and white blood cells (WBC) differential count. Methods: In a cross-sectional study, blood
samples were taken from 30 healthy medical students of Rafsanjan University (15 males and 15
females). The samples divided into three parts; CBC were done on the samples up to 48 hours
incubation at temperature of 25, 30, and 370 C at the time of sampling, and after 2, 8, 24 and 48
hours. Data were statistically analyzed and the following results were obtained. Results: RBC
count, hematocrit, MCH, percent of monocytes and eosinophils were constant in different
temperatures, WBC count, MCHC, hemoglobin, platelets count, the percent of lymphocytes and
neutrophils were constant up to 24 hours and then tend to increase with increasing temperature
except lymphocytes percent that tend to decrease. MCV decreased with increasing temperature up
to 8 hours and then significantly increased (from 83.89 to 87.50 fmol/l, p<0.001). WBC,
hematocrit, MCV, platelets count, and neutrophils’ percent tend to increase by the time of
incubation, but RBC count, MCHC, lymphocytes’ percent decreased. Hemoglobin, MCH, and the
percent of monocytes and eosinophils were constant. Conclusion: The finding of this survey
showed that some of CBC parameters can be changed with the incubation, therefore it is better to
do the CBC test after blood taking as soon as possible.
Key Words: CBC, RBC Indices, Temperature of storage, Time of storage
INTRODUCTION
In clinical laboratories, the cycles of each test starts
with the preparation of patient, and continues with
biologic sample collection,
incubation the sample in suitable conditions and
finally finishes by reporting the result. It is
sometimes necessary to redo a test to obtain a reliable
result. Sometimes there is a period of time between
sample collection and doing or redoing a test. In this
situation the satiability of the samples is very
important. Since blood tests are more common than
the other biologic fluids, therefore using the standard
methods for sample collection, incubation and the
role of environmental factors that affect the blood’s
indices should be considered.
Complete blood count (CBC) is one of the
most common and routine laboratory tests that is the
first step to diagnose an illness and since this test is
become easy and quick, it can give valuable
information to the physicians.
The results of CBC can be affected by
different factors such as the temperature and
incubation period. Sometimes it takes time between
blood samples collection and doing the test.
preparation and
The study of Hogman and Knutson in 1999
showed that the level of 2,3- bisphosphoglycerate
(2,3-BPG) could be changed by incubation the blood
sample.1 Gulati and Hyland in 2002 determined that
the long-term incubation of blood in room
temperature changes some blood indices.2 In another
study it has been demonstrated that incubation of
blood samples in different temperature leads to
considerable alteration in blood cells and red blood
cells (RBcs) indices.3 It has been also shown that
RBCs are concentrated by incubation and in different
times and temperatures changes will occur in blood
cells.4
In Hogman and Knutson report has been
mentioned that the studies in this field are not
sufficient1 therefore the aim of this study was to
evaluate the simultaneous effect of different
incubation times and temperatures on changes of
CBC and RBC indices and also the differential count
of white blood cells (WBC).
MATERIAL AND METHODS
In this sectional study the blood samples of 30 (15
male and 15 female) of healthy students of Rafsanjan
University of Medical Sciences, with age 18-26 years

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old were collected. The volume of blood sample was
9ml for each subject that was collected in vials
containing EDTA.
CBC was carried out on blood samples by
cell counter (Coulter T890) immediately after mixing
and then each sample divide into three parts of 3ml.
The samples were incubated in three different
temperatures (25, 30 and 370C) and after 2, 4, 8, and
24 hours the CBC was done again on samples. The
samples were mixing during the test and returned to
the incubators immediately after CBC. The slides of
blood smears for WBC differential count were
prepared and stained by Gimsa staining solution for
each temperature and time of incubation.
Repeated measure of ANOVA and Tukey’s
test were used to analyze the data.
RESULTS
The finding of this study showed that in different
times and temperatures, a number of RBC indices
changed significantly. The changes in MCH, RBC
and WBC counts were not significant, but with
increasing the temperature and time MCV decreased
significantly (tables 1 to 3).
MCHC after 8 hours decreased significantly.
The change in Hct was not significant up to 24 hours
incubation but after 48 hours increased with
increasing the temperature. In WBC differential
count lymphocytes and neutrophils counts after 48
hours of incubation
Hemoglobin concentration increased significantly
with raising the temperature after 48 hours of
incubation.
Table 1: Comparison of the mean of MCV (fmol/l), in
different times at 250C
2
Hours
83.93 83.89 Mean
4.95 5.07 SD
0.90 0.93 SEM
The differences were significant (p<0.001) in different times.
Table 2: Comparison of the mean of MCV (fmol/l), in
different times at 300C.
2
Hours
83.88 83.89 Mean
5.07 5.07 SD
0.91 0.93 SEM
The differences were significant (p<0.05) in different times.
Table 3: Comparison of the mean of MCV (fmol/l), in
different times at 370C.
2
Hours
83.41 83.89 Mean
5.09 5.07 SD
0.93 0.93 SEM
The differences were significant (p<0.0001) in different times.
decreased significantly.
48
Hours
86.99
5.34
0.97
24
Hours
86.42
5.28
0.96
8
Hours
84.99
5.12
0.94
Initial Time
48
Hours
87.61
5.01
0.91
24
Hours
86.92
5.35
0.98
8
Hours
84.82
5.23
0.95
Initial Time
48
Hours
87.50
5.36
0.99
24
Hours
86.69
5.34
0.97
8
Hours
84.89
4.88
0.89
Initial Time
DISCUSSION
In this study the effects of temperature and
incubation time on blood indices in CBC test and
differential counts of WBC was evaluated.
In WBC counts we found that after 2 and 8
hours of incubation there was an increase in the
numbers of WBC but was not significant.
Vogelaar et al. that evaluated 304 blood
samples of 17-70 years old individuals have reported
that incubation blood samples in room temperature
for 48 hours did not change the number of RBC,
WBC and platelets5. According to the study of
Hirase, the blood cells were stable after one week of
incubation6, but in Wood's survey the incubation of
samples for 24 hours resulted in the increase of WBC
counts.7
The WBC counts at 300C in our survey was
similar to the research of Gulati who has reported that
WBC counts would not change in first 3 days of
incubation2, but the WBC counts at 25 and 370C in
our study was not in consistent with Gulati's study the
difference between our results and the results of
Gulati was due to the role of different temperatures,
since Gulati studied the WBC counts in only one
temperature.
The number of RBC at 370C in our study
decreased significantly (p=0.048) after 48 hours
incubation that is against the results of Gulati who
has reported that the RBC counts was constant up to
7 days in room temperature.2
The change in hemoglobin concentration in
this experiment was in consistent with Gulati study
that found that Hb concentration was constant up to 7
days at room temperature2. Our results in MCV and
MCH were similar to the results of Gulati2.
Our findings showed that platelets counts
after 48 hours incubation increased with raising
temperature significantly (p=0.007), but Gulati has
reported that platelets counts did not change up to 4
days incubation at room temperature2. In Zhang study
the cause of platelets increasing explained as increase
in whole blood viscosity.8 Uchida in 2000, Shortland
in 1997 and Qi in 2001 have reported that raising the
temperature leads to changes in platelets' morphology
and movement.9-11 It has also been reported in a
comprehensive study by Ho et al. that different
temperature and time of incubation can affect in
platelets counts and hemoglobin concentrations .12
CONCLUSION
Overall the results of this study showed that delay in
doing the CBC test can leads to changes in some
parameters, therefore the blood samples should not
be leaved in the laboratory and the test should be
done on blood samples as soon as possible.

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_____________________________________________________________________________________________________________________
Address for Correspondence:
Dr. Mehdi Mahmoodi, Department of Biochemistry and Biophysics, Faculty of Medicine, Rafsanjan, Iran.
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