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Comparison of the effects of radiographic contrast media on dehydration and filterability of red blood cells from donors homozygous for hemoglobin A or hemoglobin S.

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American Journal of Hematology 68:149±158 (2001)
Comparison of the Effects of Radiographic Contrast
Media on Dehydration and Filterability of Red Blood
Cells From Donors Homozygous for Hemoglobin A
or Hemoglobin S
Patricia Losco,1* Gerard Nash,2 Phil Stone,2 and John Ventre3
1
Schering-Plough Research Institute, Lafayette, New Jersey
Department of Physiology, University of Birmingham Medical School, Birmingham, United Kingdom
3
Nycomed Amersham Imaging, Princeton, New Jersey
2
Iodinated radiographic contrast media have traditionally been contraindicated in patients
with sickle cell disease because their high osmolality may induce osmotic shrinkage of
red blood cells, impair blood ¯ow through the microcirculation, and precipitate or exacerbate a sickle cell crisis. This study investigated that concept by comparing the hematological and rheological effects in vitro of four X-ray contrast media of differing
osmolalities: Visipaque (290 mOsm/kg), Hexabrix (600 mOsm/kg), Omnipaque (844
mOsm/kg), and RenoCal-76 (1940 mOsm/kg). Blood was tested from 10 normal and 10
sickle cell donors at drug concentrations of 0, 1, 10, and 30% w/v in an attempt to
approximate the relative concentrations of contrast medium to blood that might occur
during the bolus-injection and circulation-diluted phases of drug administration. Parameters evaluated included hematology, red cell morphology, and red cell ¯ow resistance through a micropore ®lter to approximate the microcirculatory effects. Signi®cant
hematological effects for both normal and sickle cell donors included a concentration
dependent decrease in hematocrit and MCV, and increase in MCHC, all of which varied
directly with the osmolality of the contrast media in the order of RenoCal-76 > Omnipaque
> Hexabrix > Visipaque. The contrast media had minor effects on red blood cell morphology except for RenoCal-76, 10 30% in which marked echinocytosis was observed.
There was no signi®cant increase in the number of irreversibly sickled cells in donors
with hemoglobin S. Filterability of red cell suspensions through capillary size pores was
impaired in both normal and sickle cell samples in direct proportion to the osmolality of
the contrast media, as listed above. Filterability effects were greater for sickle cells than
for normal red cells. Visipaque, which was closest to isotonicity, had little effect on red
cell volume and had no signi®cant effect on ®lterability of normal or sickle cells. These
results suggest that microcirculatory impairment following infusion of contrast media
may occur in sickle patients because of the unusual rheological sensitivity of HbSS red
cells, and may be avoided by choice of an isotonic medium. Am. J. Hematol. 68:149±158,
2001. ã 2001 Wiley-Liss, Inc.
Key words: radiographic contrast media; red blood cells; sickle cell disease; microcirculation; blood osmolality
INTRODUCTION
Previous studies of iodinated water-soluble radiographic contrast media (RCM) have demonstrated adverse effects on blood viscosity, erythrocyte morphology
and aggregation behavior [1 3], intracellular pH of red
cells [4], and blood rheology [5,6]. Most effects have been
attributed to either the viscosity of the media and/or to
their high osmolality, up to eight times greater than blood.
ã 2001 Wiley-Liss, Inc.
Contract grant sponsor: Nycomed Amersham Imaging, Princeton,
New Jersey.
*Correspondence to: Patricia Losco, V.M.D., Schering-Plough Research Institute, P.O. Box 32, Lafayette, New Jersey 07848. E-mail:
pat.losco@spcorp.com
Received for publication 28 September 2000; Accepted 15 June 2001
150
Losco et al.
The latter is due to their high iodine content necessary for
radiographic contrast [7]. Hypertonic solutions induce
dehydration and crenation of red blood cells and can
impede their flow through the microcirculation [8,9]
Studies of blood from sickle cell (HbSS) donors show
similar effects of crenation of red cells and increased
viscosity, which impair rheology in hypertonic media
[10 13]. Clinically, hypertonic RCM have been implicated in severe adverse reactions in sickle cell patients
including precipitation of vascular occlusive crisis
[14 19]. Although hypertonicity alone does not induce
morphological sickling of HbS red cells [11], the resultant
dehydration and increase in intracellular Hb concentration does promote polymer formation during physiologic
fluctuations in pO2. In fact, some polymer may form in
cells with high HbS concentration even when fully oxygenated with consequent reduced filterability [20]. Dehydrated sickle cells do not necessarily take on classical
elongated forms in response to HbS polymerization, but
may have irregular outline and increased rigidity due to
formation of shorter HbS filaments [12]. Such cells are,
nevertheless, highly rheologically compromised [21].
Hypertonicity, plus the low oxygen saturation and acidosis that occurs during dosing with some RCM or can
preexist in ill patients, may contribute to polymerization
and sickling and increase the probability of occlusion in
the microcirculation [14,22 23].
In recent years, development of newer contrast media
has focused on reducing the osmolality of the RCM and,
thereby, some of the toxic effects [24 29]. However,
there have been no comparisons of the effects of different agents on the rheology of sickle cells. Visipaque,
formulated at 290 mOsmol/kg of water, is the first
contrast agent developed for broad intravascular application that has the same osmolality as whole blood. It
Abbreviations
RCM
ISC
HbSS
HbAA
HbS
RBC
Hb
Hct
MCV
MCH
MCHC
SEM
PBS
IRFR
CR
CP
b
radiographic contrast media
irreversibly sickled cells
hemoglobin genotype of sickle cell donors
hemoglobin genotype of normal donors
hemoglobin S
red blood cell count
hemoglobin
hematocrit
mean corpuscular volume
mean corpuscular hemoglobin
mean corpuscular hemoglobin concentration
scanning electron microscopy
phosphate buffered saline
initial relative flow rate
clogging rate
clogging particles
a calculated value that represents mean cellular resistance to
flow through a pore
TABLE I. Contrast Media Data
Contrast media
Iodixanol nonionic
dimer (Visipaque)
Ioxaglate ionic dimer
(Hexabrix)
Iohexol nonionic
monomer (Omnipaque)
Diatrizoate sodium
meglumine ionic
monomer (RenoCal 76)
Concentration of
iodine (mgI/ml)
Osmolality
(mOsmol/kg water)
320
290
320
600
350
844
370
1,940
might, therefore, be expected to have minimal effects on
red cell parameters and should not cause the marked
deterioration in the flow properties of HbS cells that is
associated with red cell dehydration. This study was
performed to compare the iso-osmolar contrast agent,
Visipaque, with other contrast media, covering a range
of increasing osmolalities, with regard to hematological
parameters, morphology of normal and HbS erythrocytes, and their resistance to flow.
MATERIALS AND METHODS
Blood Donors
Venous blood (15 ml) was collected in EDTA from
ten adults with homozygous (HbSS) sickle cell disease in
the steady state and from 10 adults with normal (HbAA
blood). Donors gave informed consent. HbS donors had
not received a blood transfusion within the four months
prior to donating blood or received treatment with any
drugs that would alter their concentration of HbS.
Contrast Media
The contrast media studied are listed in Table I. They
were selected to provide a range of osmolalities with
minimal differences in viscosity. Although the concentration of iodine (mgI/ml) in the different contrast media
was not identical, the maximum difference in iodine
concentration between test samples was approximately
15%, which was not considered significant in affecting
study results.
Aliquots of 500 ll of whole blood or red cell suspension (109 cells/ml) were mixed with contrast media
and phosphate buffered saline (PBS) in the proportions
shown in Table II. The final mixtures contained contrast
media at 0, 1, 10, or 30% v/v, and red cells at half their
original concentration. Thirty percent was considered a
reasonable concentration to represent the bolus-injection
phase of arteriography necessary to achieve successful
imaging [30]. The blood and erythrocyte suspensions
Effects of X-Ray Contrast Media on Hemoglobin S Red Blood Cells
TABLE II. Volumes of Blood or Red Cell Suspension Mixed
With Contrast Media before Analysis
Volume of blood
or red cell
suspension
…l1†
500
500
500
500
Volume of
contrast
media
…l1†
0
10
100
300
Volume of
PBS
…l1†
Final concentration
of contrast media
(%v/v)
500
490
400
200
0
1.0
10
30
were incubated at 37°C for 10 minutes with agitation
before measurement of hematological and rheological
parameters.
Hematological Measurements
Osmolality (mOsmol/kg) was measured for treated
and untreated samples of blood and red cell suspensions
by freezing point depression after centrifugation of a
small aliquot to remove cells.
Red blood cell count (RBC) was measured by Coulter
counter (Coulter Electronics Ltd, Luton, UK). Hemoglobin concentration (Hb) was measured by the
cyanmethemoglobin method, and hematocrit (Hct) was
measured using microhematocrit tubes centrifuged at
15,000 g for three minutes. Derived parameters were
mean corpuscular volume (MCV = Hct/RBC), mean
corpuscular hemoglobin (MCH = Hb/RBC), and mean
corpuscular hemoglobin concentration (MCHC = Hb/
Hct). An automated counter was not used to determine
MCV directly because sample-diluting media would
reverse effects of hypertonic contrast media. Hematocrit
(and thus MCV) may be overestimated slightly for rigid
cells, which pack less well than normal cells on centrifugation, but this effect is small [31].
Red Cell Morphology
Morphology was assessed using a small aliquot of
blood, fixed by dilution 1:10 with 1% glutaraldehyde
solution made up in 2/3 strength PBS containing appropriate volumes of contrast media to match osmolality
to test samples. Wet films were examined by light microscopy, and 400 red cells were classified by shape as
discocytes, stomatocytes, echinocytes, or irreversibly
sickled cells (ISC). Changes in morphology were confirmed by examining treated and untreated samples from
one normal donor and two sickle cell donors (selected at
random) by scanning electron microscopy (SEM). The
same glutaraldehyde-fixed blood suspensions were used.
Electron microscopy specimens were prepared as follows: round (13 mm) coverslips were dipped into a so-
151
lution containing 10% gelatin dissolved in distilled water
and were air dried. On each of the coverslips was placed
100 ll of the glutaraldehyde fixed RBCs. An additional
100 ll of 2% glutaraldehyde in PBS was added, and
allowed to fix for 30 minutes at room temperature. The
prepared coverslips were washed in PBS and were then
fixed in 1% OsO4 in PBS for 30 minutes at room temperature. The samples were dehydrated in an ascending
series of ethanol concentrations and dried with CO2 at
38°C and 1,200 psi. The glass coverslips were coated
with gold using a Pelco SC-6 sputter coater and viewed
on a Hitachi 2460N scanning electron microscope. Images from selected samples were digitally recorded using
Quartz PCI software.
Rheological Measurements
Resistance of red cells to flow through capillarysized pores was measured using suspensions of purified
red cells. Leukocytes and platelets were removed from
blood by filtration through Imugard IG 500 cotton wool
(Terumo Corporation, Tokyo, Japan), and red cells
were washed twice with PBS. Cells were counted in a
Coulter counter and adjusted to109/l with PBS. The
deformability of red cells in suspension (final concentration = 5108 cells/ml) was assessed by measuring
flow rate through 5 lm pore filters in a St. George's
filtrometer [32]. The device measures the time taken
for three equal, sequential volumes of red cells to pass
through a vertically mounted filter. The flow rate is
expressed relative to the flow rate of suspending medium alone (PBS with the appropriate concentration of
contrast media added). Linear regression of the relative
flow rate versus volume filtered allows calculation of
the initial relative flow rate (IRFR) and the clogging
rate (CR), or rate of decrease of flow per milliliter.
IRFR and cell concentration are used to calculate the
parameter b, which represents a measure of the average
resistance of individual cells to flow through the pores
[33,34]. CR and the known number of filter pores allow
calculation of the clogging particles (CP/ml), which
gives a measure of the concentration of cells that
blocked the pores. The viscosities of suspending media
were measured by a capillary viscometer (Coulter
Electronics, Ltd.).
Statistical Methods
Treatment related effects, associated with red blood
cell shrinkage, were observed in the hematocrit and the
calculated values of MCV and MCHC. MCV was selected as the best representative parameter to test the
effect statistically. Treatment related effects on red cell
suspension filterability were also observed and tested
statistically.
152
Losco et al.
For each agent, pairwise comparisons between 0% and
each of the remaining concentrations (1, 10, and surrogate 30%) were made for MCV, and the filterability
parameters of b and CP. A two-way analysis of variance
(ANOVA) was used, with factors for concentration and
donor (blocking factor) included in the model [35].
Pairwise comparisons were made using linear contrasts
comparing the specified levels of concentration. Each
contrast was tested at the Bonferroni corrected level of a
= 0.0167 (0.05 divided by the number of comparisons).
Linear regression was used to determine the relationship
between MCV and osmolality. The model regressed the
parameter 1/osmolality onto MCV. In the case of sickle
blood treated with RenoCal-76, the 30% concentration
was not included in the analyses on b and CP due to
stopped flow.
For comparisons of the four media within each concentration, the two-way ANOVA was again used, including factors for medium and donor. All pairwise
comparisons between the four mediums were performed
using linear contrasts. Each contrast was tested at the
a = 0.01 level. These analyses were modified if heterogeneity of variance between the concentrations or media
was noted. In this case, paired t-tests were performed to
compare either the media or concentrations.
RESULTS
Osmolality
Osmolality was measured using the plasma after
centrifuging blood/contrast agent mixtures to remove the
red cells. However, it was not possible to reliably separate red cells for any of the contrast media at 30%
concentration. The red cells either floated to the top or
remained suspended throughout the liquid column with
portions at the top and/or bottom. This indicated that
either the density of the fluid phase was greater than that
of the red cells, or the red cells had a density spanning a
range including the fluid density. If cells floated to the
top, osmolality could be measured for the fluid column,
but in many cases this was not possible. In no case could
the Hct be measured reliably. To obtain representative
values for the 30% concentration, a surrogate sample
was generated by adding concentrated saline with the
same osmolality as the contrast media to the blood. This
sample was then used to measure the hematological
parameters for the 30% concentration. It should be noted
that red cell morphology could be measured in all
samples, as centrifugation was not required. Filtration
measurements were also unaffected, as they used samples with known cell count rather than hematocrit.
Table III shows the data for osmolality. Values for
30% mixtures are shown where possible (generally
n < 10), along with values for the surrogate samples (n =
10). The undiluted media had osmolality in the order
TABLE III. Mean Values for Osmolality of Blood With Contrast
Media Added
Agent
0%a
A. Normal blood
Visipaque 295  3
Hexabrix
295  3
Omnipaque 295  3
RenoCal-76 295  3
B. Sickle blood
Visipaque 291  2
1%
10%
Osmolality (mOsmol/Kg)
296  1 311  3 331  7
…n ˆ 10†
302  3 342  6 429  6
…n ˆ 2†
302  2 357  6 491  3
…n ˆ 7†
314  5 439  18 801  15
…n ˆ 6†
294  3
309  3
291  2
298  4
337  3
Omnipaque 291  2
300  1
354  5
RenoCal-76 291  2
311  4
428  8
Hexabrix
30%b
330
…n ˆ 1†
…n ˆ 0†
484
…n ˆ 1†
778
…n ˆ 2†
Surrogate 30%
339  2
480  18
543  6
813  10
337  3
474  4
531  4
793  9
*Data are mean  SD from 10 measurements except where stated.
a
The same control sample was used for the comparisons of all contrast
media.
b
Data are from n samples where red cells separated from medium upon
centrifugation (other samples were not measurable).
Visipaque < Hexabrix < Omnipaque < RenoCal-76
(Table I), and the samples diluted in blood retained this
order. At 30% suspension, all media were hypertonic,
including Visipaque. Suspensions containing RenoCal76 had by far the greatest osmolality.
Hematological Parameters
The sickle donors had RBC and Hb approximately
half those of the normal donors. However, Hb, RBC, and
MCH were not significantly altered by exposure to any
of the contrast media (data are not shown).
Values for Hct (Table IV) and MCV (Table V) were
decreased, and values for MCHC (data not shown) were
increased in direct proportion to the osmolality of the
contrast media added. Because all three parameters
equally reflect the degree of cell shrinkage, MCV was
chosen for discussion here.
For normal blood, all media induced significant reduction in MCV at 30% v/v (P < 0.0167). Omnipaque,
Hexabrix, and RenoCal-76 also caused significant
shrinkage at 10%. For sickle blood, Omnipaque, Hexabrix, and RenoCal-76 induced significant changes at 10
and 30% v/v (P < 0.0167), but Visipaque caused no
significant changes at any concentration. No agent
caused significant shrinkage at 1% for either normal or
sickle blood. For both types of red cells, the degree of
shrinkage was consistently ordered among the four media, regardless of concentration, as RenoCal-76 > Omnipaque > Hexabrix > Visipaque. Significant differences
Effects of X-Ray Contrast Media on Hemoglobin S Red Blood Cells
153
TABLE IV. Mean Values for Hct of Blood With Contrast Agents Added*
0%a
01%
10%
Surrogate 30%
A. Normal blood
Visipaque
Hexabrix
Omnipaque
RenoCal-76
0:220  0:013
0:220  0:013
0:220  0:013
0:220  0:013
0:219  0:013
0:218  0:012
0:219  0:012
0:211  0:012
0:215  0:014
0:208  0:013
0:203  0:010
0:182  0:014
0:204  0:012
0:173  0:011
0:166  0:005
0:144  0:009
B. Sickle blood
Visipaque
Hexabrix
Omnipaque
RenoCal-76
0:128  0:018
0:128  0:018
0:128  0:018
0:128  0:018
0:130  0:016
0:127  0:017
0:128  0:016
0:125  0:015
0:129  0:016
0:121  0:015
0:118  0:016
0:110  0:013
0:121  0:014
0:103  0:012
0:099  0:011
0:088  0:010
Agent
*Note that each blood sample has been diluted by 50% (500 ll blood/ml) and, therefore, values are half the actual patientÕs value for Hct.
a
The same control sample was used for the comparisons of all agents.
TABLE V. Mean Values for MCV of Red Cells in Blood With
Contrast Agents Added
0%a
TABLE VI. Summary of Signi®cant Pairwise Comparisons of
MCV Among the Four Tested Media
1%
10%
Surrogate 30%
A. Normal blood
Visipaque
87  4
Hexabrix
87  4
Omnipaque
87  4
RenoCal-76
87  4
MCV (fl)
89  4
87  2
86  4
86  3
Concentration of
agent % v/v
Significant differences in levels
of MCV
86  4
84  4b
80  4b
72  4b
83  4b
69  3b
65  3b
59  3b
A. Normal blooda
1%
10%
Surrogate 30%
No differences
V>R, V>O, H>R, O>R
V>H, V>O, V>R, H>O, H>R, O>R
B. Sickle blood
Visipaque
Hexabrix
Omnipaque
RenoCal-76
91  13
89  12
88  12
88  13
91  12
84  14b
81  13b
77  10b
87  12
72  10b
68  10b
62  9b
B. Sickle blooda
1%
10%
Surrogate 30%
V>O
V>O, V>H, V>R, H>R
V>H, V>O, V>R, H>O, H>R, O>R
Agent
90  15
90  15
90  15
90  15
a
The same control sample was used for the comparisons of all agents.
b
P < 0.0167.
(P < 0.01) between the mediums depended on the level
of concentration and generally supported the order of
osmolalities of the contrast media (Table VI).
Values for MCV showed a close inverse correlation
with osmolality (R2 = 0.96 for linear regression of MCV
vs. 1/Osmolality for normal blood, (R2 = 0.98 for sickle
blood). The regression line for normal blood was MVC/
MCVo = 0.43 + 0.58 (295/osmolality), where MCVo is
the volume in plasma alone (295 mOsmol/kg). The regression line for sickle blood was MVC/MCVo = 0.49 +
0.52 (291/osmolality), where MCVo is the volume in
plasma alone (291 mOsmol/kg).
Red Cell Morphology
Morphology results are presented in Table VII, sections A and B, for normal and sickle blood, respectively.
Evaluation of red blood cells by SEM confirmed the
results of the light microscopic examination of the cells.
In control normal donor samples, the vast majority of red
cells were discocytes. The control sickle samples displayed a variety of red cell forms, including stomato-
a
R = RenoCal-76, O = Omnipaque, H = Hexabrix, V = Visipaque.
cytes, reticulocytes, and discocytes, as well as elongate
boat shaped cells (irreversibly sickled cells), which
predominated over the classical ÔÔsickleÕÕ shape. None of
the contrast media produced significant changes in red
cell shape at 1%, and only RenoCal-76 caused a noticeable change at 10%, with an increase in echinocytes
(shrunken cells). At 30%, Visipaque produced a slight
increase in stomatocytes, but showed no change in the
incidence of echinocytes for either normal or sickle
blood (Fig. 1). At 30%, Hexabrix and Omnipaque produced a slight increase in echinocytes (4 and 9%, respectively, for normal donors), whereas RenoCal-76
caused a marked increase in echinocytes for both normal
and sickle donors (Figs. 2 and 3, respectively).
The effects of RCM on morphology of discocytes
were similar for normal and sickle blood, although sickle
blood contained a greater variety of red cell shapes before media were added and showed a wider range of
morphologic changes when exposed to contrast media.
Notably, shrinkage of sickle cells at high RCM concentrations was not associated with an increase in classical sickling. Also, the ISC or sickled component of
blood from HbS donors usually did not show echinocytosis.
154
Losco et al.
TABLE VII. Morphology of Red Cells in Blood/Contrast Media Mixtures
Agent
A. Normal blooda
None
Visipaque
Hexabrix
Omnipaque
RenoCal-76
B. Sickle blooda
None
Visipaque
Hexabrix
Omnipaque
RenoCal-76
(%)
Stomatocytes (%)
Discocytes (%)
Echinocytes (%)
0
1
10
30
1
10
30
1
10
30
1
10
30
65
85
85
13  9
74
53
22
64
33
11
54
11
0
93  5
92  5
91  5
86  9
92  3
92  2
94  2
93  3
94  2
91  10
93  3
67  17
0
11
11
11
11
11
32
42
11
33
9  10
21
33  17
100
0
1
10
30
1
10
30
1
10
30
1
10
30
43
53
53
76
32
32
11
43
22
21
33
11
11
65  10
66  10
66  11
63  10
63  11
61  10
61  12
66  11
63  11
57  15
63  10
44  16
33
11  6
10  7
11  9
12  9
14  8
17  9
18  8
11  7
15  9
22  13
13  8
37  18
74  13
Sickled cells (%) (ISC)
20  8
20  11
22  16
18  9
21  10
20  10
20  11
19  9
20  10
19  10
21  10
18  9
21  11
a
Mean values for 10 patients each.
Fig. 1. Scanning electron micrograph of red cells from a
sickle donor exposed to 30% Visipaque. Red cell appearance is similar to 0% control.
Fig. 2. Red cells from a normal donor exposed to 30%
RenoCal-76 showing 100% echinocyte formation.
Filterability of Red Cell Suspensions
Filtration measurements allowed calculation of the
average resistance of individual cells to flow through the
pores (b) and the concentration of pore blocking particles
(CP). The filterability measurements were made relative
to suspending medium alone, so that changes in viscosity
were taken into account in calculation of the filtration
parameter b. Viscosity of the contrast media in PBS was
measured independently. Each RCM caused marked and
comparable increase in viscosity (Table VIII).
Effects of X-Ray Contrast Media on Hemoglobin S Red Blood Cells
155
Fig. 3. Red cells from a sickle donor exposed to 30%
RenoCal-76. Most nonsickled red cells are echinocytes, but
sickled cells show little change.
TABLE VIII. Values for the Viscosity of PBS/Contrast
Media Mixtures
Viscosity (mPa.s)
Agent
0%a
1%
10%
30%
Visipaque
Hexabrix
Omnipaque
RenoCal-76
0.90
0.90
0.90
0.90
0.91
0.91
0.92
0.92
1.03
1.01
1.05
1.03
1.50
1.42
1.53
1.49
a
The same control sample was used for the comparisons of all constrast
media.
Data for b are presented in Fig. 4 and for CP in Fig. 5.
Values for b and CP were higher for sickle than normal
red cells. There was much greater variation in filtration
parameters and responses to media for sickle blood
compared with normal blood. Although the trends were
often the same, statistical significance was not reached
as often for sickle blood, even though the magnitude of
changes induced by media was generally larger. Visipaque did not significantly alter filterability of either
normal or sickle cells. For normal cells, Hexabrix significantly increased b and CP, but only at 30%.
Omnipaque significantly increased b at 10 and 30%, and
CP at 30%. RenoCal-76 significantly increased b and CP
at 10 and 30%. For sickle cells, Hexabrix and Omnipaque significantly increased b and CP only at 30%.
RenoCal-76 significantly increased CP, but not b, at
10%. Neither b nor CP could be evaluated for RenoCal76 at 30%, due to stoppage of flow. However, there was
clearly massive increase in flow resistance. For either
type of red cell, results for b and CP were consistently
ordered as RenoCal-76 > Omnipaque > Hexabrix >
Visipaque. Statistical comparisons between contrast
Fig. 4. Effect of increasing concentration of radiographic
contrast media on mean resistance to ®ltration (b) of red
cells from (A) HbAA donors and (B) HbSS donors. Data are
mean ‹ the standard error of the mean from measurements
on 10 donors. For 30% RenoCal-76, ¯ow rate was too slow
to allow measurement (*).
media for b and CP showed significant variation depending on the level of concentration (P < 0.01), but the
changes were inconsistent relative to defined parameters
of osmolality, and did not provide useful insight in interpreting the data (data not shown).
DISCUSSION
Patients with sickle cell disease characteristically
suffer from periodic painful crises caused by occlusion
of microvessels. HbSS red cells are abnormally resistant
156
Losco et al.
Fig. 5. Effect of increasing concentration of radiographic
contrast media on the number of clogging particles during
®ltration of red cells from (A) HbAA donors and (B) HbSS
donors. Data are mean ‹ the standard error of the mean
from measurements on 10 donors. For 30% RenoCal-76,
¯ow rate was too slow to allow measurement (*).
to flow even when oxygenated, and this abnormality is
exacerbated on deoxygenation when polymerization of
HbS causes formation of elongated rigid cells [36 39].
The impaired flow of oxygenated HbSS red cells is
largely attributable to the existence of a population of
unusually dense cells, including both dense discocytes
containing HbS polymer and boat-shaped irreversibly
sickled cells [20,36]. In the present study, the preexisting
rheological abnormality was evident as increased values
for b and CP for untreated sickle cells compared with
normal red cells.
The likelihood of new polymer formation occurring
during transit through the microcirculation in vivo is
increased if the intracellular concentration of HbS is
increased, because the delay time for gelation is highly
dependent on the HbS concentration [22]. Thus, infusion
of hypertonic RCM may cause adverse reactions in
sickle patients, both because of deterioration in the flow
properties of oxygenated cells and also because decrease
in MCV and associated increase in MCHC will increase
the chance of sickling and further impairment.
Hypertonic RCM also caused dehydration of normal
red cells and impairment of flow properties, in agreement with previous reports [3 6]. The increase in flow
resistance was not so dramatic as with HbSS cells, and
probably arose from an increase in viscosity of the cytoplasmic HbA solution. At MCHC around 500 g/l, the
cytoplasm of HbAA cells shows transformation to solidlike behavior, but the rheological effect is not so dramatic as that caused by sickling of HbS [40]. The implications of these studies are, therefore, that dehydration
of red cells by RCM is undesirable in all blood because it
can increase flow resistance, and it is especially to be
avoided in sickle blood where more dangerous changes
in flow properties may ensue.
Osmolality was the major determinant of changes in
most of the parameters studied, judging from the relative
magnitude of changes in osmolality, and in hematological
and rheological parameters for the different contrast
media. Changes in MCV were linearly, inversely related
to changes in osmolality, so that shrinkage was fully explained by the increases in osmolality caused by the
contrast media. Linear regression of MCV vs. l/Osmol-1
ality for normal red cells yielded an equation that agreed
well with literature reports obtained using saline [41]. The
equation for sickle cells indicated that they shrank less
than normal cells in hypertonic medium. This suggests
that there is slightly less osmotically active or exchangeable water in sickle cells. Hemoglobin, red cell
counts, and MCH were not affected by addition of contrast media in either normal or sickle blood. Hemoglobin
levels of samples were not expected to vary on addition of
media, because equal volumetric dilutions were used for
all samples in this protocol. Constancy of red cell counts
indicated that there was negligible hemolysis of red blood
cells due to any RCM. Nor was there any discoloration of
the plasma that might indicate free Hb. MCH, calculated
from Hb/RBC, would also not be affected.
Of the agents tested here, only Visipaque had no
significant effect on filterability parameters for either
normal or sickle red cells. The other RCM caused large
changes in filterability at 30%, whereas RenoCal-76 also
caused marked changes at 10%. In the circulation, RCM
concentrations in the range of 30% concentration exist
only for brief periods during the bolus injection phase of
drug administration before being diluted by the circulating blood. Clinical studies have shown, however, that
achieving high concentrations of 20 65% in the target
organ being radiographed is common and considered
Effects of X-Ray Contrast Media on Hemoglobin S Red Blood Cells
necessary for achieving good vascular imaging [30].
Therefore, the effects of high RCM concentrations on
flow resistance constitute a valid concern for adverse
effects on the patient.
RCM also caused modification of morphology of the
red cells. However, only RenoCal-76 induced marked
changes, with high levels of echinocytes formed. This
may be partly an effect of high ionic strength. However,
it was notable that even at comparable osmolality, 30%
Hexabrix or Omnipaque did not induce the same level of
echinocytosis as 10% RenoCal-76. It is uncertain how
great the effect of the echinocytosis was on flow resistance in this study. In general, spheroechinocytes have
high resistance to deformation, but lesser degrees of
echinocytosis have little effect [41]. Sickle blood was
slightly more resistant to formation of echinocytes than
normal blood. This may reflect resistance to transformation of rigid dense cells, including sickle discocytes
containing HbS polymer. It may also be related to the
fact that the irreversibly sickled population (about 20%
of RBCs in our cohort) were already dehydrated cells.
Shrinkage did not increase the percentage of ISC, consistent with the finding that dense cells (sickle discocytes) do not form classical elongated shapes when
dehydrated [12].
This study addressed only the physical properties of
RCM and their interactions with red blood cells. Significant additional effects on flow in large conduit
blood vessels and the microcirculation may arise in
response to RCM administration secondary to the release of cytokines from white blood cells, platelets, and
the vascular endothelium [42 45]. The effects of abnormalities in pH and oxygen saturation, both of which
may be compromised in sickle cell patients in crisis,
also were not investigated here. Nevertheless, this study
supports the concept that hypertonic contrast media
may increase the risk of microcirculatory occlusion in
sickle cell patients. Use of RCM with nearly physiologic osmolality may not eliminate all adverse effects
of contrast arteriography, but it is likely to reduce interference with red cell passage through the microcirculation, particularly for sickle cell patients.
ACKNOWLEDGMENTS
The authors wish to thank J. Suhan for technical assistance in electron microscopy and V. Sansone and T.
Santillo for technical assistance in the planning and
preparation of this manuscript. We are grateful to S.
Stevens for liasing with blood donors and carrying out
phlebotomy, and to Drs. D. Bareford and J. Wilde for
enabling recruitment of donors with homozygous sickle
cell disease.
157
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