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Effect of Blood Transfusion in an Experimental Sarcoma Model
Ho-Sheng Lin, MD;
Ravi N. Samy, MD;
Joanne Lum, BS;
Mary Jo Dorie, PhD;
David J. Terris, MD
Arch Otolaryngol Head Neck Surg. 2002;128:308-312.
ABSTRACT
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Objective To study the effect of allogeneic, syngeneic, and autologous blood transfusion
on the growth rate of the KHT tumor in a C3H murine model.
Design Prospective, randomized, and controlled animal study.
Subjects Sixty-one C3H female mice.
Interventions The C3H female mice were implanted with 2 x 105 cells
of KHT, a murine sarcoma. Ten days later, 0.3 mL of blood was removed from
a retro-orbital site to simulate surgical blood loss. This blood loss was
replaced by blood transfusion through a tail vein with the use of allogeneic
(major histocompatibility complex incompatible), syngeneic (major histocompatibility
complex compatible), or autologous blood. Tumor growth was measured daily
for 14 days. The tumor growth curve for each of the animals was constructed
and the mean slope of growth calculated for each group.
Results There were statistically significant differences in tumor growth rate
(P = .001) when the allogeneic group (mean slope
= 0.232, n = 14), the syngeneic group (mean slope = 0.190, n = 17), and the
autologous group (mean slope = 0.202, n = 14) were compared. A t test confirmed that there was no significant difference in the tumor
growth rate between the groups transfused with syngeneic and autologous blood
(P = .26). However, the rate of tumor growth in the
allogeneic group was found to be significantly higher when independently compared
with the syngeneic group (P<.001) and the autologous
group (P = .02).
Conclusions In this experimental model of a solid murine sarcoma, allogeneic blood
transfusion was associated with an increased rate of tumor growth compared
with syngeneic and autologous blood transfusion, likely reflecting immunomodulatory
effects incurred by the introduction of major histocompatibility complex–incompatible
antigens.
INTRODUCTION
TRANSFUSION OF BLOOD from allogeneic (heterologous) donors has a long
and interesting history in the practice of medicine. Before the ABO blood
group system was discovered by Landsteiner a century ago,1
transfusions were considered ineffective and dangerous. The recognition of
the ABO and Rh blood groups and development of preservation techniques for
storing allogeneic blood led to the increased application of transfusion therapy
with dramatic reductions in morbidity and mortality from hemorrhage during
surgery. The impact of blood transfusions on the recipient's cellular immune
system was appreciated in the 1970s, when the beneficial effect of pretransplant
allogeneic transfusion on renal transplant survival was described.2 Since the 1980s, the association between transfusion
and increased cancer recurrence and decreased survival has been reported in
a number of epidemiologic and experimental studies.3-8
Although the preponderance of data suggests an adverse association between
allogeneic blood transfusion and cancer outcome, this association remains
inconclusive. Nevertheless, this potential adverse effect combined with the
small risk of infectious transmission has heightened interest in the use of
autologous blood transfusion. The advantages of autologous blood transfusion
with respect to cancer outcomes have been suggested in clinical reports.3, 8
There is evidence to suggest that the deleterious effect of allogeneic
blood transfusion is due to immunosuppression caused by the infusion of incompatible
major histocompatibility complex (MHC) antigens.5, 9-11
The more dissimilar the MHC antigens between donor and recipient, the greater
the immunosuppression and, therefore, the greater the likelihood of adverse
effects. This may account for some of the inconsistencies in the effect of
blood transfusion seen in previous reports that varied in the degree of histocompatibility
of transfused blood because of the different animal models and strains used
and the amount and type of blood transfused. We investigated in a murine model
the effect of transfusing blood of various histocompatibility on the growth
rate of a sarcoma.
MATERIALS AND METHODS
EXPERIMENTAL PROTOCOL
Sixty-one C3H female mice were used in this study. The skin over the
posterior spine region of these mice was inoculated subcutaneously with 2
x 105 KHT murine tumor cells. Ten days after tumor implantation,
0.3 mL of blood was removed with the mouse under anesthesia from the plexus
of veins in the medial aspect of the orbit with a capillary tube containing
preservative-free heparin (1 U/mL) to simulate surgical blood loss. After
1 hour, this blood loss was replaced by blood transfusion through a tail vein.
These mice were randomized to receive 1 of 3 different types of transfusions,
with the investigator blinded to the identity of the transfusion. The first
group (n = 18) received 0.3 mL of blood removed from C57Bl, a different strain
of mice (allogeneic blood transfusion). The second group (n = 23) received
0.3 mL of blood removed from other C3H mice (syngeneic transfusion). The third
group of mice (n = 20) received the same 0.3 mL of blood that had been removed
from them 1 hour earlier (autologous transfusion). Tumor growth was then measured
daily. All animals were humanely killed 14 days after transfusion and a final
measurement was obtained.
ANIMALS
The C3H female mice and C57Bl mice (The Jackson Laboratory, Bar Harbor,
Me) ranged in age from 10 to 11 weeks. The mean weight of the mice was 24.2
± 1.8 g, which correlates with a total blood volume of 1.7 ±
0.1 mL. The surgical blood loss of 0.3 mL in these mice is therefore equivalent
to a blood loss of 864 ± 66 mL in a 70-kg human. The human MHC is equivalent
to the murine histocompatibility-2 (H-2) complex. The C3H mice have the major
H-2 complex antigens H-2k/H-2k, whereas the C57Bl mice have the major H-2
complex antigens H-2b/H-2b. They were housed in a climate-controlled, virus-free
environment with regulated light-dark cycles and permitted free access to
standard pellet chow and water.
The allogeneic blood transfusion was achieved by transfusion of major
H-2–incompatible blood (H-2b/H-2b) from C57Bl mice to C3H mice (H-2k/H-2k)
(Figure 1A). Syngeneic blood transfusion
was achieved by transfusion of major H-2–compatible blood from one C3H
mouse (H-2k/H-2k) to another C3H mouse (Figure
1B). These are inbred mice that have the same major H-2 complex
antigens but may differ in minor histocompatibility complex antigens. Finally,
autologous blood transfusion (transfusion of the mouse's own blood; Figure 1C) represented preservation of the
same major and minor histocompatibility complex antigens. This study was approved
by the Stanford University Administrative Panel on Laboratory Animal Care,
Stanford, Calif, and strict guidelines for the care and use of laboratory
animals were followed.
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Figure 1. Transfusion for the 3 groups of
mice. A, Allogeneic blood transfusion was achieved by transfusion of major
histocompatibility-2 (H-2)–incompatible blood (H-2b/H-2b) from C57Bl
mice to C3H mice (H-2k/H-2k). B, Syngeneic blood transfusion was achieved
by transfusion of major H-2–compatible blood (H-2k/H-2k) from one C3H
mouse to another C3H mouse. C, Autologous blood transfusion.
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TUMOR
The tumor cells were originally derived from the mouse KHT sarcoma line.12 They were maintained in culture in  -minimal
essential medium supplemented with 10% fetal calf serum, 1% L-glutamine, and
penicillin-streptomycin in 80-cm2 plastic tissue culture flasks
and cultured in a humidified incubator with 5% carbon dioxide at 37°C.
The cells were harvested by trypsinization for 3 to 5 minutes. The cell suspension
was washed in Hanks solution and centrifuged. The cell pellet was then resuspended
in Hanks solution and counted with a hemocytometer. The suspension was then
diluted to approximately 4 x 106 cells per milliliter. The
cell viability was checked by means of eosin exclusion. The cell solution
was maintained in ice until injection. A total of 2 x 105
KHT murine tumor cells (0.05 mL) were injected intradermally.
ASSESSMENT OF TUMOR GROWTH
After transfusion, the size of the tumor was measured daily for 14 days.
Since the tumor shape approximated a sphere (Figure 2), the volume was calculated by means of the method of Attia
and Weiss13: V = (0.4)
x (ab2), where V is tumor volume in cubic millimeters, a
represents the length of the major axis, and b represents
the length of the minor axis.
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Figure 2. Mouse used in the experiment,
demonstrating the spherical shape of the tumor on the back of the mouse.
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STATISTICS
The Microsoft Excel statistical program (Microsoft Corp, Redmond, Wash)
was used to generate the growth curve and to calculate the slope of tumor
growth in each animal. The slopes were then averaged to determine the mean
slope of growth for each of the 3 groups, and SDs were calculated. Any individual
slope more than 2 SDs away from the mean value of the slope was eliminated
from the final statistical calculation to minimize the incorporation of aberrant
values stemming from unrecognized cannibalization of tumors. An analysis of
variance was used to detect significant differences in tumor growth rates
among the 3 groups. Differences between pairs of groups were then evaluated
with a paired, 2-tailed t test.
RESULTS
Data from 45 of the 61 mice were able to be evaluated. Twelve mice (3
from the allogeneic group, 5 from the syngeneic group, and 4 from the autologous
group) died of various causes before the end point of the experiment was reached
(14 days after transfusion). The size of the tumor could not be determined
in 2 mice because of tumor cannibalism (1 from the allogeneic group and 1
from the autologous group). Finally, the data from another 2 mice (1 from
the syngeneic group and 1 from the autologous group) were eliminated from
evaluation because they were more than 2 SDs outside the mean of the growth
slope.
The mean slope of the growth curve was calculated to be 0.232 ±
0.035 for the allogeneic group (n = 14), 0.190 ± 0.025 for the syngeneic
group (n = 17), and 0.202 ± 0.032 for the autologous group (n = 14)
(Table 1 and Figure 3). The analysis of variance showed the difference in the
value of the mean slope between the 3 groups to be statistically significant
(P = .001). Further analysis with the t test showed no significant difference in mean slope when the syngeneic
and autologous transfusion groups were compared (P
= .26). Both of these groups were transfused with blood containing the same
major H-2 complex antigens. The allogeneic group, which was transfused with
blood containing incompatible major H-2 complex antigens, was associated with
a faster rate of tumor growth, with a mean growth slope of 0.232. This value
was significantly higher than the mean slope of 0.190 for the syngeneic group
(P<.001) and the mean slope of 0.202 for the autologous
group (P = .02).
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Individual Mouse Tumor Growth Slopes*
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Figure 3. Mean slope of growth for each
of the 3 transfusion groups. The mean slope is 0.232 for the allogeneic group,
0.190 for the syngeneic group, and 0.202 for the autologous group.
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COMMENT
A large body of clinical and experimental evidence supports an association
between allogeneic blood transfusion and poor cancer outcome.3, 6-8
However, the effect of autologous blood transfusion on patients undergoing
curative resection of cancer is less clear. The often contradictory literature
on this issue likely reflects the poor understanding of the complex immunomodulatory
processes associated with blood transfusion.
The first evidence pointing to the immunosuppressive effect of blood
transfusion was reported in the transplant literature when it was noted that
allogeneic transfusion improved renal transplant survival.1-2
Since then, multiple clinical and laboratory studies have demonstrated transfusion-induced
up-regulation of suppressor T-cell activity and down-regulation of natural
killer cell activity, cytotoxic T-lymphocyte antitumor activity, and T-cell
proliferative activity and decreased secretion of cytokines.1, 4-5,14-15
Evidence then accumulated that the immunosuppressive effect of allogeneic
blood transfusion may be due to the infusion of incompatible MHC antigens
present on the leukocytes.16 For example, suppression
of T-lymphocyte–mediated renal graft rejection occurred only when leukocytes
were present in the transfused blood. Since MHC antigens are not present on
erythrocytes, leukocyte-depleted blood failed to exert any immunosuppressive
effect in protecting grafts.16 Furthermore,
several studies have shown that the reduction or elimination of leukocytes
from allogeneic blood, or the use of autologous blood, prevents the negative
impact on cancer recurrence and survival associated with blood transfusion.3, 6-8
We set out in this study to investigate the effect of transfusing blood
of different histocompatibility complex antigens on the growth rate of tumor
in a murine model. Our data demonstrated an association between transfusion
of incompatible major H-2 complex antigens and faster rate of tumor growth
(allogeneic group). Conversely, transfusion of compatible major H-2 complex
antigens was associated with a slower rate of tumor growth (syngeneic and
autologous groups). In addition, our data confirmed the absence of a statistically
significant difference in tumor growth rate between the syngeneic and autologous
groups. Both groups received blood containing the same major H-2 complex antigens,
but with some differences in the minor H-2 complex antigens. This finding
suggests that the deleterious effect of blood transfusion is primarily due
to the infusion of incompatible major H-2 complex antigens. This is supported
by a study on the growth of MH 134 hepatoma, in which C3H mice were transfused
with either syngeneic blood from C3H mice (H-2k/H-2k) or allogeneic blood
from BDF mice (H-2b/H-2d) or AKR mice (H-2k/H-2k).11
The authors described a significant increase in the growth rate of hepatoma
in the group that was transfused with incompatible major H-2 blood from BDF
mice. On the other hand, the groups that received the blood with compatible
major H-2 complex antigens from C3H mice or AKR mice had slower tumor growth.
A number of clinical studies have concluded that the benefits of autologous
transfusion are maintained in the human model.3, 6-8
We previously published a retrospective analysis of the effect of blood transfusion
on recurrence rate in 165 patients with squamous cell carcinoma of the head
and neck treated surgically at Stanford University Medical Center.3 Patients who received allogeneic blood transfusion
had a 59% recurrence rate compared with 33% and 35% recurrence rates for patients
who received autologous blood and no transfusion, respectively. Similar findings
were reported with a prospective, randomized clinical trial comparing allogeneic
and autologous blood transfusions in 120 patients with colorectal cancer.17 The relative risk of recurrence was 0.96 in the group
transfused with autologous blood, in contrast to the group transfused with
allogeneic blood, which had a relative risk of recurrence of 7.01.
The conclusion that autologous blood transfusion does not impact cancer
outcomes is not uniformly accepted, however.18-20
Busch et al,20 for example, reported on a prospective,
randomized clinical trial involving 475 patients with colon cancer and concluded
that both autologous and allogeneic blood transfusions increased the risk
of cancer recurrence. Survival rates were 67% for the allogeneic transfusion
group, 62% for the autologous transfusion group, and 88% for the group of
patients without transfusion. Unfortunately, this study was confounded by
the fact that a third of the patients in the autologous group also received
allogeneic blood (since they required more than the 2 U of autologous blood
that was donated preoperatively). When these patients with a mixed transfusion
profile were excluded from consideration, the autologous recipients had a
15% lower recurrence rate and 23% lower death rate than the allogeneic group.
Further distorting the interpretation of clinical trials is the fact
that, although whole blood transfusion has been used in most animal studies,
the blood products used in prospective and retrospective clinical trials reported
from different institutions have been variable and sometimes undefined. The
blood components transfused to patients have changed during the evolution
of transfusion therapy and blood storage. Twenty years ago, transfusions were
often of whole blood, containing 2 x 109 to 3 x 109 white blood cells (WBCs) per unit.21
Patients now receive red blood cell transfusions that are partially leukocyte
depleted and vary in the amount of leukocyte concentration, depending on the
filtration method.4, 21-23
Low-performance leukodepletion methods such as the buffy-coat method widely
used in Europe reduce the residual WBC count to between 107 and
108 per unit (10- to 100-fold reduction of WBCs found in whole
blood). High-performance leukodepletion methods can reduce the residual WBC
count to between 1 x 106 and 5 x 106 per
unit (1000-fold reduction). The detrimental effect on cancer recurrence and
survival rates associated with MHC-incompatible blood transfusion suggested
a possible beneficial effect from reducing leukocyte content in allogeneic
blood transfusion.8 Recently, mainly because
of the fear of Creutzfeldt-Jakob disease, several European nations have implemented
universal WBC reduction to less than 1 x 106 WBCs per unit.23-24 Transfusion of this leukocyte-depleted
blood may ameliorate the potentially deleterious effect on tumor growth by
minimizing the infusion of incompatible MHC antigens.
CONCLUSIONS
Our data suggest that the adverse effect of blood transfusion on cancer
outcome is likely due to the infusion of incompatible MHC antigens. However,
caution must be exercised in extrapolating findings from animal studies to
clinical situations. Although animal studies have historically measured the
growth rate of implanted cancer cells, clinical trials typically assess recurrence,
metastasis, and survival rates after surgical resection. These end points
measured by clinical trials are obviously much more complex and can be confounded
by multiple variables. Nevertheless, the clinical and experimental data available
support the use of either autologous blood or leukocyte-depleted allogeneic
blood in place of the conventional allogeneic blood for transfusion in cancer
patients.
AUTHOR INFORMATION
Accepted for publication August 16, 2001.
This study was presented at the annual meeting of the American Head
and Neck Society, Palm Desert, Calif, May 14, 2001.
Corresponding author and reprints: David J. Terris, MD, Division
of Otolaryngology/Head & Neck Surgery, Edwards Building, R135, Stanford
University Medical Center, Stanford, CA 94305-5328 (e-mail: dterris{at}stanford.edu).
From the Divisions of Otolaryngology/Head & Neck Surgery (Drs Lin,
Samy, and Terris and Ms Lum) and Radiation Biology/Radiation Oncology (Dr
Dorie), Stanford University Medical Center, Stanford, Calif.
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