Effects of storage duration and temperature of human blood on red cell deformability and aggregation.

Abstract

Blood samples used in hemorheological studies may be stored for a period of time, the effects of storage have yet to be fully explored. This study evaluated the effects of storage temperature (i.e., 4 degrees C or 25 degrees C) and duration on RBC deformability and aggregation for blood from healthy controls and from septic patients. Our results indicate that for normal blood, RBC deformability over 0.3-50 Pa is stable up to six hours regardless of storage temperature; at eight hours there were no significant differences in EI but SS1/2 calculated via a Lineweaver-Burk method indicated impaired deformability. Storage temperature affected the stable period for RBC aggregation: the safe time was shorter at 25 degrees C whereas at 4 degrees C aggregation was stable up to 12 hours. Interestingly, blood samples from septic patients were less affected by storage. Blood can thus be stored at 25 degrees C for up to six hours for deformability studies, but should be limited to four hours for RBC aggregation; storage at 4 degrees C may prolong the storage period up to 12 hours for aggregation but not deformability measurements. Therefore, the time period between sampling and measurement should be as short as possible and reported together with results.

Full text

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Clinical Hemorheology and Microcirculation 41 (2009) 269–278
DOI 10.3233/CH-2009-1178
IOS Press
269
Effects of storage duration and temperature
of human blood on red cell deformability and
aggregation
Mehmet Uyuklua, Melike Cengiza, Pinar Ulkera, Timea Heverb, Julien Tripettec,
Philippe Connesc, Norbert Nemethb, Herbert J. Meiselmandand Oguz K. Baskurta,∗
aDepartment of Physiology, Akdeniz University Faculty of Medicine, Antalya, Turkey
bDepartment of Operative Techniques and Surgical Research, Auguszta Surgical Center, Institute of
Surgery, Medical and Health Science Centre, University of Debrecen, Hungary
cLaboratoire ACTES (EA 3596), Département de Physiologie, Université des Antilles et de la Guyane,
97159 Pointe à Pitre, Guadeloupe, French West Indies
dDepartment of Physiology and Biophysics, Keck School of Medicine, Los Angeles, CA, USA
Abstract. Blood samples used in hemorheological studies may be stored for a period of time, the effects of storage have
yet to be fully explored. This study evaluated the effects of storage temperature (i.e., 4◦C or 25◦C) and duration on RBC
deformability and aggregation for blood from healthy controls and from septic patients. Our results indicate that for normal
blood, RBC deformability over 0.3–50 Pa is stable up to six hours regardless of storage temperature; at eight hours there were
no significant differences in EI but SS1/2calculated via a Lineweaver–Burk method indicated impaired deformability. Storage
temperature affected the stable period for RBC aggregation: the safe time was shorter at 25◦C whereas at 4◦C aggregation was
stable up to 12 hours. Interestingly, blood samples from septic patients were less affected by storage. Blood can thus be stored
at 25◦C for up to six hours for deformability studies, but should be limited to four hours for RBC aggregation; storage at 4◦C
may prolong the storage period up to 12 hours for aggregation but not deformability measurements. Therefore, the time period
between sampling and measurement should be as short as possible and reported together with results.
Keywords: Aggregation, blood storage, deformability, temperature
1. Introduction
Clinical and basic studies in the field of hemorheology include the measurement of blood and plasma
viscosities as well as red blood cell (RBC) deformability and aggregation. The results of the measure-
ments of such hemorheological parameters are known to be affected by a variety of factors, including
sampling technique, storage of the samples, pre-analytical handling and the measurement conditions, as
well as the instrument specifications. These factors have been the subject of several investigations [2,8,
14,19,23] and, in part, were the basis for guidelines for good laboratory practice (GLP) in hemorheology
made by the Expert Committee on Blood Rheology [13]. However, there have been significant techni-
cal developments in the field of hemorheology since the 1986 date of these guidelines, and the newer
*Correspondingauthor:OguzK.Baskurt,MD,PhD,ProfessorandChairman,DepartmentofPhysiology,AkdenizUniversity
Faculty of Medicine, Antalya, Turkey. Tel.: +90 242 310 1560; Fax: +90 242 310 1561; E-mail: baskurt@akdeniz.edu.tr.
1386-0291/09/$17.00 © 2009 – IOS Press and the authors. All rights reserved

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methods and instruments have improved sensitivities for alterations in hemorheological parameters (e.g.,
RBC deformability and aggregation), which necessitated the revision of the above mentioned guidelines.
Hence a new expert panel is currently working on such a revision [3] and various laboratory studies are
also being conducted for determining the best laboratory practice in hemorheology.
Blood samples used in hemorheological studies may need to be stored for a period of time prior to
being measured. This period may sometimes include shipping to a remote laboratory and thus the stor-
age might be prolonged for 24 hours or longer. It is well known that RBC properties are affected by
prolonged storage, with these effects potentially related to metabolic depletion, disturbed ion homeosta-
sis, protein and lipid modifications (e.g., oxidation, degradation, cross-linking), and volume changes
accompanied by alterations in intracellular hemoglobin concentrations [12,21]. These alterations may
have different time courses at cooler temperatures compared to room or body temperatures, and the time
course of these alterations may also be altered for RBC obtained from patients with various diseases.
RBC mechanical properties have also been shown to be altered during the time period between the
sampling and measurement [2,23]. While it was previously recommended that the storage duration for
the blood samples should be less then four hours [13], these recommendations were mostly related to
previous hemorheological methods which may not be relevant to current technology.
The present study was designed to explore the effects of storage temperature and storage duration on
RBC deformability and aggregation parameters for human blood samples obtained from healthy individ-
uals and septic patients. Its overall objective was to determine the time period after the blood sampling
that could be allowed without significant hemorheological alterations due to storage; mechanisms of
alterations during the storage period were not considered.
2. Materials and methods
2.1. Blood samples
Venous blood samples were obtained from 10 healthy human male volunteers, aged between 25 to 52
years. A tourniquet was briefly applied to locate the antecubital vein and 50 ml blood samples were ob-
tained and anticoagulated with sodium heparin (15 IU/ml). Additionally, blood samples from 10 patients
hospitalized in the intensive care unit of Akdeniz University Hospital with the diagnosis of severe sep-
sis (5 male, 5 female; aged between 18 to 55 years) were obtained using the same sampling technique.
Note that all septic patients had respiratory insufficiency, with the presumed sources of sepsis being
the lower respiratory tract, blood or urinary tract. Vasoactive drugs were used in six of the patients for
maintenance of hemodynamic stability, and only one patient died because of adult respiratory distress
syndrome before discharge from ICU.
After saving a 2 ml aliquot for immediate measurements, the remaining blood was divided into
20 aliquots of 2 ml each. Ten of these aliquots were stored in a refrigerator at 4 ± 2◦C and the other
10 aliquots were kept at room temperature (25 ± 2◦C). RBC deformability and aggregation were mea-
sured for both healthy and septic samples immediately following blood sampling and at 1, 2, 3, 4, 6, 8,
12 and 24 hours after sampling for the two series of aliquots kept at 4 or 25◦C.
In a separate series of experiments to test the effects of re-warming duration, aliquots of blood samples
from the group of healthy individuals described above were stored at 4 ± 2◦C for 4 hours and then re-
warmed at 37◦C for periods between 0–30 min prior to measuring RBC deformability and aggregation.

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2.2. RBC deformability measurements
RBC deformability was determined at various fluid shear stresses by laser diffraction analysis using an
ektacytometer (LORCA, RR Mechatronics, Hoorn, The Netherlands). The system has been described
elsewhere in detail [10]. Briefly, a low hematocrit suspension of RBC in an isotonic viscous medium
(4% polyvinylpyralidone 360 solution, MW 360 kD) is sheared in a Couette system composed of a glass
cup and a precisely fitting bob, with a gap of 0.3 mm between the cylinders. A laser beam is directed
through the sheared sample and the diffraction pattern produced by the deformed cells is analyzed by a
microcomputer. Based upon the geometry of the elliptical diffraction pattern, an elongation index (EI)
is calculated as: EI = (L − W)/(L + W), where L and W are the length and width of the diffraction
pattern. An increased EI at a given shear stress indicates greater cell deformation and hence greater RBC
deformability. All measurements were carried out at 37◦C.
EI values were obtained for nine separate shear stresses between 0.3–50 Pascal (Pa). Additionally, the
maximum EI at infinite shear stress (EImax) and the shear stress required for half-maximal deformation
(SS1/2) were calculated using this data set for each measurement using a Lineweaver–Burk analysis as
described elsewhere [4]. Briefly, the shear stress–EI curve was linearized by plotting the reciprocal of
EI versus the reciprocal of shear stress. The x-intercept of the resulting line corresponds to the negative
reciprocal value of shear stress causing half-maximal deformation (SS1/2). Obviously, impairment in
RBC deformability leads to increased SS1/2values.
2.3. Determination of RBC aggregation
RBC aggregation was assessed using a custom-built photometric aggregometer interfaced to a digital
computer via monitoring light transmittance through a blood sample during the aggregation process [5].
The shearing portion of the system consists of two parallel glass plates with a gap of 0.3 mm between
them; a stepper motor, controlled by the computer, rotates one of these plates. The blood sample under
investigation is placed between the glass plates, and is first sheared at 500 s−1for 10 s to disperse
RBC aggregates. After a sudden stop of the motor, the infrared light transmission through the blood
sample is monitored for 10 s and recorded by the computer. The computer then calculates the area under
the light transmission curve and reports a dimensionless index (M) which increases with the extent
of RBC aggregation. Measurements were done in triplicate for each sample and the mean of the three
measurements assigned to that sample. Measurements were carried out at 37◦C.
2.4. Red blood cell properties and morphology
Mean RBC Volume (MCV) for the blood aliquots was determined using an electronic hematology
analyzer (Cell-Dyn 3500R, Abbott Diagnostic Division, Illinois, USA). RBC shape was examined in
wet-mount, diluted and unstained preparations under light microscopy.
2.5. Statistics
Results are expressed as mean ± standard error (SE). Statistical comparisons between groups were
done by “repeated measures ANOVA” followed by “Newman–Keuls post test”, with p values <0.05
accepted as statistically significant.

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3. Results
3.1. Samples from healthy donors
MCV values were 82–88 fl in the samples immediately after the sampling and were not changed
during first 12 hours of storage regardless of the temperature. However, after 24 hour storage at room
temperature, MCV had slightly but significantly increased to 86.2±0.9 fl compared to the control value
measured immediately after sampling (85.3 ± 0.7). MCV remained unchanged in the samples stored at
4◦C. No significant changes in RBC morphology were detected.
3.1.1. Red blood cell deformability
There were no significant changes of red cell EI values over the entire range of shear stress (i.e., 0.3–
50 Pa) for blood samples from healthy donors stored for up to 6 hours at either room temperature or
4◦C. However, at eight hours and beyond, some EI values differed from that for the initial measurement:
(1) for room temperature storage at eight hours, only EI values measured at shear stresses between 1.1
and 3.9 were significantly decreased (i.e., −22% at 1.1 Pa, −6% at 3.9 Pa); (2) at 12 hours, EI for room
temperature stored blood was 5% lower over a stress range of 7.4–50 Pa and about 7% lower over 7.4–
26 Pa for blood stored at 4◦C; (3) at 24 hours only blood stored at 4◦C exhibited decreased EI values
(i.e., 7% decrease at 26.4 and 50 Pa).
Figure1presentsEImaxvaluescalculatedusingthedataobtainedattheninelevelsofshearstress.EImax
values remained unchanged during eight hours of storage at room temperature and were significantly
decreased at 12 hours. However, if the blood was stored at 4◦C, this significant decrement in EImaxwas
only seen at 24 hours. SS1/2values were found to be significantly increased following eight hours of
storage for both room temperature and 4◦C (Fig. 2). Interestingly, SS1/2returned to control level after
12 hours of storage at room temperature, while the return of SS1/2to control was delayed to 24 hours in
the samples stored at 4◦C.
3.1.2. Red blood cell aggregation
Values for the RBC aggregation index (M) measured during the 24 hour period after sampling are
presented in Fig. 3. At room temperature, RBC aggregation remained stable for at least four hours;
Fig. 1. Maximum EI at infinite shear stress (EImax) calculated using EI measured at nine shear stresses between 0.3–50 Pa for
normal blood samples stored up to 24 hours after sampling. (A) At room temperature (25 ± 2◦C). (B) At 4 ± 2◦C. Data are
presented as mean ± standard error. Difference from control (0 time),∗p < 0.05.

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Fig.2.Shearstressforhalf-maximaldeformation (SS1/2)calculated usingEImeasuredatnine shearstressesbetween 0.3–50Pa
fornormalsamplesstoredupto24hoursaftersampling.(A)Atroomtemperature(25±2◦C).(B)At4±2◦C.Dataarepresented
as mean ± standard error. Difference from control (0 time),∗p < 0.05;∗∗p < 0.01.
Fig. 3. Red blood cell aggregation indexes for normal blood samples stored up to 24 hours after sampling. (A) At room
temperature (25 ± 2◦C). (B) At 4 ± 2◦C. Data are presented as mean ± standard error. Difference from control (0 time),
∗∗p < 0.01.
aggregation started to decrease after six hours and was only 50% of control after 24 hours (Fig. 3A). In
contrast, aggregation values weremorestable if the blood wasstored at4◦C,withasignificant decrement
only detected after 24 hours (Fig. 3B).
3.1.3. Effect of 37◦C re-warming time after storage at 4◦C
Figure 4 presents the RBC deformability parameters (SS1/2and EImax) and the aggregation index M
measured after various periods of re-warming at 37◦C following four hours of storage at 4◦C. Note
that: (1) the parameters measured immediately after removing the sample from the storage at 4◦C (i.e.,
0 minutes re-warming) were essentially identical to those obtained prior to cooling; (2) there was no
effect of re-warming over the entire 5–30 min periods (Fig. 4).

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Fig. 4. Shear stress for half-maximal deformation (SS1/2), maximum EI at infinite shear stress (EImax) and RBC aggregation
indexes for normal blood samples measured before storage (BS) and after various periods of pre-warming at 37◦C following
storage at 4◦C for four hours. No significant alterations were observed for any of the parameters.
3.2. Samples from septic patients
3.2.1. Red blood cell deformability
Unlike the data obtained for samples from healthy subjects that showed some decreases of EI with
storage time at both room temperature and 4◦C, EI for septic subjects tended to increase with storage at
room temperature. The most prominent increases were observed at 24 hours: the significant increases of
EI at 24 hours were inversely related to shear stress and, on average, ranged from 35% at 0.6 Pa to 2%
at 50 Pa. Storage at 4◦C essentially eliminated alterations of EI. There were no significant alterations of
EImaxvalues during the 24 hour storage period regardless of the storage temperature (data not shown).
SS1/2in septic patients was found to be decreased significantly only after 24 hours of storage at room
temperature, with no alterations observed if the kept at 4◦C (Fig. 5).
3.2.2. Red blood cell aggregation
RBCaggregationforbloodfromsepticpatientsremainedstableatroomtemperatureforupto12hours
after the sampling, with a significant decrement in aggregation index observed only after 24 hours
(Fig. 6A). Storage at 4◦C prevented this alteration (Fig. 6B).

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Fig.5.Shearstressforhalf-maximaldeformation (SS1/2)calculated usingEImeasuredatnine shearstressesbetween 0.3–50Pa
for samples from septic patients stored up to 24 hours after sampling. (A) At room temperature (25 ± 2◦C). (B) At 4 ± 2◦C.
Data are presented as mean ± standard error. Difference from control (0 time),∗p < 0.05;∗∗p < 0.01.
Fig. 6. Red blood cell aggregation indexes for blood samples from septic patients stored up to 24 hours after sampling. (A) At
room temperature (25 ± 2◦C). (B) At 4 ± 2◦C. Data are presented as mean ± standard error. Difference from control (0 time),
∗∗p < 0.01. The open squares in Fig. 5A, B indicate M values measured for healthy subjects immediately following sampling
(i.e., no storage at either temperature).
4. Discussion
Our results indicate that the deformability of RBC from normal human blood, as measured by an
ektacytometer over a shear stress range of 0.3–50 Pa, is stable up to six hours after sampling regardless
of the storage temperature (i.e., 4 or 25◦C). Although at 8 hours there were no significant differences
in EI as tested by “repeated measure ANOVA”, SS1/2values calculated via a Lineweaver–Burk method
were significantly increased at this time point, indicating impaired deformability; EImaxvalues were
stable up to 12 hours.
The ektacytometer used in this study is a sensitive device with good reproducibility [11]. Therefore,
any deterioration in RBC deformability during the six hours after the sampling should have been de-
tected using this device. Based on the findings summarized above, it can be argued that RBC in whole
blood can be safely stored up to 6 hours either at room temperature or at 4◦C for the measurement of

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deformability. However, RBC deformability, indicated as an elevated SS1/2, was found to be impaired
in samples stored for eight hours at either room temperature or 4◦C. Interestingly, RBC deformability
returned to values for fresh samples with further storage at room temperature, and at 24 hours at room
temperature had a SS1/2lower than control value, thus indicating an improvement of deformability. The
small but significant increase of the average MCV in the room temperature samples stored for 24 hours
(i.e., 85.3–86.2 fl) may provide a partial explanation for this improved RBC deformability. However,
measurements made 12 hours after sampling indicated significantly decreased EImax, probably indicat-
ing a structural change in the RBC membrane: these alterations were slower (i.e., no change in EImax
after 12 hours) and milder (Fig. 1) if the samples were stored at 4◦C. Note that as a sub-study, some of
the blood samples from healthy donors were stored up to 72 hours at room temperature: SS1/2values
were very significantly decreased (1.92±0.09 Pa versus 3.96±0.28 Pa measured immediately after the
sampling); storage at 4◦C partly prevented this decrement in 72 hours (data not shown). These observa-
tions at 72 hours can be interpreted as reflecting changes in membrane mechanical behavior, probably
due to disintegration of the membrane skeletal network [18]. Therefore, the biphasic pattern of RBC
deformability changes might reflect two separate processes: the first phase yielding impairment of RBC
deformability due to altered metabolic activity and/or disturbed ion homeostasis, and the second which
becomes effective later interfering with membrane mechanical properties. It appears that the second
effect becomes operative sooner if the blood samples are stored at room temperature.
In contrast to the behavior of samples from normal individuals, no significant alterations of EI were
found during the first 12 hours in the samples from septic patients. It can be speculated that the above
mentioned second phase of the storage effect might be shifted to earlier periods for septic blood, thus
offsetting the mechanisms that might have caused impairment of RBC deformability.
Storage temperature affected the stable period for RBC aggregation in normal healthy samples: the
safe storage time was shorter if the samples were kept at room temperature (Fig. 3), with aggregation
indexes stable at least up to 12 hours if the samples were stored at 4◦C. It was again interesting to
observe that the samples from septic patients seemed to have better stability compared to the samples
from normal subjects (Fig. 3 versus Fig. 5). Both plasmatic and cellular factors affect RBC aggregation
[17]. Alterations in plasmatic factors play a significant role in the enhanced aggregation seen in sepsis
[6], whereas RBC properties should be more sensitive to prolonged storage and hence the contribution
of cellular factors seem to be more prominent in the normal samples versus the septic samples. At
room temperature, RBC aggregation tended to decrease after 12 hours of storage, with aggregation
indexes approaching very low values in the samples stored for 72 hours (1.32±0.32 versus 11.35±1.02
measured immediately after the sampling). Conversely, aggregation indexes decreased to only 8.24 ±
1.28 if the samples were stored for 72 hours at 4◦C. These findings are in agreement with the previously
published effects of prolonged storage on RBC aggregation [16] and the greater stability of low shear
blood viscosity with storage at 4◦C [1].
In this study, we have also evaluated whether a pre-warming time is required for the samples stored for
four hours at 4◦C. It seems reasonable that all RBC metabolism-related mechanisms (e.g., ion pumps)
might be suppressed by cooling, and that a certain time period at 37◦C prior to the measurement might be
needed to restore disturbed cellular homeostasis. However, re-warming at 37◦C up to 30 min had no ef-
fect on the measured RBC deformability and aggregation parameters, indicating that such a re-warming
may not be necessary. This finding is in contrast with the report by Bartoli et al., which indicated a
significant effect of re-warming time on whole blood filtration [2]. However, whole blood filtration is
known to be very sensitive to leukocyte alterations and the dependence on re-warming time might have
reflected the influence on leukocyte function [22].

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This study did not include any measured parameters specifically designed to investigate the mecha-
nisms of alterations during the storage. However, the biphasic nature of the effects of storage on RBC
deformability (Fig. 2) was unexpected and clearly warrants further studies. It can be hypothesized that
the first phase of this response to prolonged storage (i.e., increased SS1/2indicating impaired deforma-
bility) likely reflects metabolic depletion that results in: (1) slowing of ion pumps [15]; (2) lipid and
protein oxidation and hence increased oxidant damage due to insufficient generation of antioxidant co-
factors (NADH and NADPH) leading to [7]; (3) alterations of RBC membrane skeleton structure (e.g.,
increased protein crosslinking) [20]. The later phase of the storage-induced changes is characterized by
very significant decrements in SS1/2. This decrease may reflect degradation of oxidized-damaged pro-
teins [9], resulting in abnormalities of the viscous and/or elastic rheologic behavior of the cell membrane
rather than an improvement of RBC function.
In summary, blood samples can be stored at room temperature for up to six hours for the measurement
of RBC deformability, but this period should be limited to four hours if the study also includes RBC
aggregation. Storageat4◦Cmayprolong thestorageperiod upto12hoursforaggregation measurements
but not for deformability measurements. However, it should be kept in mind that these “safe storage
periods” apply to samples obtained from normal, healthy individuals. The time course of changes in
RBC deformability and aggregation might be significantly different in the blood samples from patients
with various pathologies (e.g., hemoglobinopathies) as observed herein for the samples from septic
patients. Therefore, the time period between sampling and measurement should be as short as possible
and, together with storage temperature, should be reported with results.
Acknowledgements
This study was supported by NIH Research Grants HL15722, HL 70595 and FIRCA IR03 TW01295,
and by the Akdeniz University Research Projects Unit.
References
[1] T.Alexy,R.Wenby,E.Pais,L.J.Goldstein,W.HogenauerandH.J.Meiselman,Anautomated tube-type blood viscometer:
validation studies, Biorheology 42 (2005), 237–247.
[2] V. Bartoli, B. Albanese, P.G. Manescalchi, L. Mannini and G. Pasquini, Influence of blood storage conditions and antico-
agulants on results of blood cell filtration test, Clin. Hemorheol. 6 (1986), 137–149.
[3] O.K. Baskurt, M. Boynard, G.R. Cokelet, P. Connes, B.M. Cooke, M.R. Hardeman, F. Liao, H.J. Meiselman, G.B. Nash,
N. Nemeth, B. Sandhagen, S. Shin, G.B. Thurston, J.L. Wautier and S. Yedgar, New guidelines for the assessment of
hemorheological parameters, Biorheology 45 (2008), 82.
[4] O.K. Baskurt and H.J. Meiselman, Analyzing shear stress-elongation index curves: comparison of two approaches to
simplify data presentation, Clin. Hemorheol. Microcirc. 31 (2004), 23–30.
[5] O.K. Baskurt, H.J. Meiselman and E. Kayar, Measurement of red blood cell aggregation in a “plate–plate” shearing system
by analysis of light transmission, Clin. Hemorheol. Microcirc. 19 (1998), 307–314.
[6] O.K. Baskurt, A. Temiz and H.J. Meiselman, Red blood cell aggregation in experimental sepsis, J. Lab. Clin. Med. 130
(1997), 183–190.
[7] O.K. Baskurt and S. Yavuzer. Some hematological effects of oxidants, in: Environmental Oxidants, J.O. Nriagu and M.S.
Simmons, eds, Wiley, New York, 1994, pp. 405–423.
[8] P. Connes, M. Uyuklu, J. Tripette, J.H. Boucher, E. Beltan, T. Chalabi, O. Yalcin, R. Chout, O. Hue, M.D. Hardy-
Dessources and O.K. Baskurt, Sampling time after tourniquet removal affects erythrocyte deformability and aggregation
measurements, Clin. Hemorheol. Microcirc. 41 (2009), 9–15.
[9] K.J.A. Davies and A.L. Goldberg, Proteins damaged by oxygen radicals are rapidly degraded in extracts of red blood cells,
J. Biol. Chem. 262 (1987), 8227–8234.

Page 10

278
M. Uyuklu et al. / Effects of storage on red cell deformability and aggregation
[10] M.R. Hardeman, P.T. Goedhart, J.G.G. Dobbe and K.P. Lettinga, Laser-assisted optical rotational cell analyzer (LORCA).
I. A new instrument for measurement of various structural hemorheological parameters, Clin. Hemorheol. 14 (1994),
605–618.
[11] M.R. Hardeman, P.T. Goedhart and N.H. Schut, Laser-assisted optical rotational cell analyzer (LORCA): II. Red blood
cell deformability elongation index versus cell transit time, Clin. Hemorheol. 14 (1994), 619–630.
[12] J. Ho, W.J. Sibbald and I.H. Chin-Yee, Effects of storage on efficacy of red cell transfusion: When is it not safe?, Critical
Care Medicine 31 (2003), S687–S697.
[13] ICSH Expert Panel on Blood Rheology, Guidelines for measurement of blood viscosity and erythrocyte deformability,
Clin. Hemorheol. 6 (1986), 439–453.
[14] A.J. Keidan, S.S. Marwah and J. Stuart, Evaluation of phosphate and hepes buffers for study of erythrocyte rheology, Clin.
Hemorheol. 7 (1987), 627–635.
[15] N. Mohandas and S.B. Shohet, The role of membrane-associated enzymes in regulation of erythrocyte shape and deforma-
bility, Clin. Hematol. 10 (1981), 223–237.
[16] V. Nagaprasad and M. Singh, Sequential analysis of the influence of blood storage on aggregation, deformability and
shape parameters of erythrocytes, Clin. Hemorheol. Microcirc. 18 (1998), 273–284.
[17] M.W.Rampling,H.J.Meiselman,B.NeuandO.K.Baskurt,Influenceofcell-specificfactorsonredbloodcellaggregation,
Biorheology 41 (2004), 91–112.
[18] B.D. Riquelme, P.G. Foresto, J.R. Valverde and R.J. Rasia, Alterations to complex viscoelasticity of erythrocytes during
storage, Clin. Hemorheol. Microcirc. 22 (2000), 181–188.
[19] R.S. Rosenson and C.C. Tangney, Effects of tourniquet application on plasma viscosity measurement, Clin. Hemorheol.
Microcirc. 18 (1998), 191–194.
[20] N. Uyesaka, S. Hasegawa, N. Ishioka, R. Ishioka, H. Shio and A.N. Schechter, Effect of superoxide anions on red cell
deformability and membrane proteins, Biorheology 29 (1992), 217–229.
[21] R. van Wijk and W.W. van Solinge, The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis,
Blood 106 (2005), 4034–4042.
[22] J.L. Wautier, G.W. Schmid-Schonbein and G.B. Nash, Measurement of leukocyte rheology in vascular disease: clinical
rationale and methodology, Clin. Hemorheol. Microcirc. 21 (1999), 7–24.
[23] J. Zhang, X. Zhang, N. Wang, Y. Fan, H. Ju, J. Yang, J. Wen and X. Qu, What is the maximum duration to perform the
hemorheological measurement for the human and mammals, Clin. Hemorheol. Microcirc. 31 (2004), 157–160.