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.
Article: Storage of whole blood before separation: the effect of temperature on red cell 2,3 DPG and the accumulation of lactate.[show abstract] [hide abstract]
ABSTRACT: Although whole blood intended for component preparation is commonly left to cool at ambient temperature, knowledge is insufficient as to the effects this may have on red cell quality, in particular after a prolonged hold. Whole blood collected in ACD-A (7% wt/wt) and CPD (12% wt/wt) was incubated at 4, 10, 15, 20, 25, and 30 degrees C for 24 hours. Blood gases, pH, bicarbonate, glucose, lactate, and red cell 2,3 DPG were investigated. When the blood was stored at 30 degrees C, the 2,3 DPG concentration decreased within 4 hours from 858 +/- 106 to 316 +/- 172 mmol per mol of hemoglobin (a 63% decrease); 99 percent was lost within 18 hours. At 25 degrees C, 46 percent was lost within 4 hours and 94 percent within 18 hours; at 20 degrees C, the decrease at 18 hours was 62 percent and that at 15 degrees C was 24 percent. No loss of 2,3 DPG was observed at 4 degrees C and 10 degrees C storage. No difference was attributable to the anticoagulant used. After 24 hours, the lactate concentration at 15 degrees C was 2.9 times the original, that at 20 degrees C was 3.8 times the original, that at 25 degrees C was 7.0 times, and that at 30 degrees C was 9.2 times. With current anticoagulants, storage of whole blood at temperatures of 25 to 30 degrees C before separation causes a great and rapid loss of 2,3 DPG and an accumulation of acid metabolites. In a hold of blood for >4 hours, rapid cooling is desirable to avoid initial loss of 2,3 DPG.Transfusion 05/1999; 39(5):492-7. · 3.22 Impact Factor
Article: Changes in automated complete blood cell count and differential leukocyte count results induced by storage of blood at room temperature.[show abstract] [hide abstract]
ABSTRACT: To delineate changes that occur in various parameters of automated complete blood cell count (CBC) and differential leukocyte count (differential) on prolonged storage of blood at room temperature. A CBC and an automated differential were performed on the Coulter Gen.S on 40 K(3) (tripotassium ethylenediamine-tetraacetate) EDTA-anticoagulated blood specimens once daily, specimen volume permitting, for 3 to 7 days. Specimens were kept at room temperature throughout the study. The results were tabulated using a personal computer with Excel software. Percent change or absolute difference from the initial value for each parameter for each subsequent day of the study period was calculated. Among the CBC parameters, hemoglobin, red blood cell count, and mean corpuscular hemoglobin were stable for the duration of the study (7 days), white blood cell count was stable for at least 3 days (up to 7 days, if the count was within or above the normal range), and platelet count was stable for at least 4 days (up to 7 days, if the count was within or above the normal range). The mean corpuscular volume, mean platelet volume, hematocrit, and red blood cell distribution width each increased, and the mean corpuscular hemoglobin concentration decreased from day 2 onward. Among the differential parameters, the relative percentages and absolute numbers of neutrophils, lymphocytes, and eosinophils tended to increase, whereas those of monocytes trended downward over time. Limited data on basophils did not reveal an appreciable change. Blood specimens stored at room temperature for more than 1 day (up to 3 days or possibly longer) were found to be acceptable with some limitations for CBC but not for the differential.Archives of pathology & laboratory medicine 04/2002; 126(3):336-42. · 2.58 Impact Factor
Article: Pre-separation storage of whole blood: the effect of temperature on red cell 2,3-diphosphoglycerate and myeloperoxidase in plasma.[show abstract] [hide abstract]
ABSTRACT: Although whole blood intended for component preparation is commonly left to cool at ambient temperature, knowledge is insufficient concerning what effects this may have on red blood cell (RBC) quality, in particular after a prolonged hold. Whole blood collected in CPD was incubated at 20 degrees C and 28 degrees C for 6 h designed as a paired study. Blood components were prepared and the red blood cell concentrates (RBCs) were stored for 28 days at 4 degrees C +/- 2 degrees C. Blood gases, pH, glucose, lactate, adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG) and plasma myeloperoxidase (MPO) were investigated. After 6 h the 2,3-DPG concentrations had lowered to 88% (20 degrees C) and 54% (28 degrees C) of initial levels, respectively. The difference was significant and was maintained for 28 days, although, at low levels from day 7 (28 degrees C) and day 14 (20 degrees C) of storage. ATP was maintained at the initial level in both groups during the first 6 h of storage but after component separation the levels were significantly higher in the 28 degrees C group during the first 5 days. The release of myeloperoxidase (MPO) was significantly higher in the non-cooled group than in the cooled group. Pre-separation holding for 6 h of whole blood at temperatures of 28 degrees C causes a great and rapid loss of 2,3-DPG and considerable formation of acid metabolites resulting in clearly subnormal 2,3-DPG levels even on day 1. Active pre-separation cooling to 20 degrees C is to be recommended.Transfusion Science 11/1999; 21(2):111-5.
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
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.
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
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
J Ayub Med Coll Abbottabad 2006;18(1)
old were collected. The volume of blood sample was
9ml for each subject that was collected in vials
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.
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
Table 1: Comparison of the mean of MCV (fmol/l), in
different times at 250C
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.
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.
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.
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
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
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
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.
J Ayub Med Coll Abbottabad 2006;18(1)
Hogman CF, Knutson I. Storage of whole blood before
separation: the effect of temperature on red cell 2,3-DPG and
the accumulation of lactate. Transfusion 1999; 39(5): 422-7
Gulati GL, Hyland LJ. Changes in automated complete blood
cell and differential leukocyte count results induced by
storage of blood at room temperature. Arch patted lab Med
2002; 126(3): 336-42
Shimizut L, Ishikawa Y. Biochemical and membrane
functional alterations in red cell during preparation and
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Knuston I, Loof H. Pre-separation storage of whole blood:
the effect of temperature on red cell 2,3-DPG and
myeloperoxidase in plasma, Transfusion 1999; 21(2): 111-15
Vogelaar SA, Posthuma D, Boomsma D, Kluft C. Blood
sample stability at room temperature for counting red and
white blood cells and platelets. Vascul Pharmacol 2002;
Hirase Y, Makatsuka H, Kawai T, Horiguchi S, Ikeda M.
Stable blood cell counts after one-week storage at room
temperature., Bull Environ Contam Toxicol 1992; 49(4):
Address for Correspondence:
Dr. Mehdi Mahmoodi, Department of Biochemistry and Biophysics, Faculty of Medicine, Rafsanjan, Iran.
Wood BL, Andrews J, Miller S, Sabath DE. Refrigerated
storage improves the stability of the complete blood cell
count and automated differential. Am J Clin Pathol 1999;
Zhang JN, Wood J, Bergeron AL, McBride L, Ball C, Yu Q,
Pusiteri AE, Holcomb JB, Dong JF. Effects of low
temperature on shear-induced platelet aggregation and
activation. J Trauma 2004; 57(2): 216-23
Qi R, Yatomi Y, Ozaki Y. Effects of incubation time,
temperature, and anticoagulants on platelet aggregation in
whole blood. Thromb Res 2001; 101(3): 139-44
10. Shortland AP, Rhodes NP, Rattray A, Black RA, Williams
DF. The effect of temperature and shear rate on platelet
aggregation. Mater Sci Mater Med 1997; 8(12): 887-90
11. Uchida K, Sakai K, Ito E, Kwon OH, Kikuchi A, Yamato M,
Okano T. Temperature-dependent modulation of blood
platelet movement and
isopropylacrylamide)-grafted surfaces. Biomaterials 2000;
12. Ho CH, Chan IH. The influence of time of storage,
temperature of storage, platelet number in platelet-rich
plasma, packed cell, mean platelet volume, hemoglobin
concentration, age, and sex on platelet aggregation test. Ann
Hematol 1995; 71(3): 129-33.
morphology on poly(N-