REVIEW ARTICLE
Biologic Effects of Leukocytes Present in Transfused
Cellular Blood Products
By Jose 0. Bordin, Nancy M . Heddle, and Morris A. Blajchman
D ESPITE CAREFUL donor selection and extensive test-
ing, the transfusion of allogeneic cellular blood prod-
ucts is associated occasionally with adverse reactions and
other complications. The latter include febrile nonhemolytic
transfusion reactions (FNHTR), refractoriness to platelet
transfusions, graft-versus-host disease (GVHD), immuno-
modulation, and transmission of infectious agents. There is
increasing awareness that some of these may be attributable
to the presence of leukocytes in donor blood. The use of
leukodepleted blood components has thus been recom-
mended as a preventative intervention for selected groups
of patients who are at high risk of r e a ~ t i 0 n . IThis
~ review
will focus on the data currently available relating to the
pathogenesis of the various biologic effects associated with
the presence of allogeneic leukocytes in transfused cellular
blood products. In addition, available methods to prevent
such complications will be discussed.
NORMAL PHYSIOLOGIC PROPERTIES OF LEUKOCYTES
Formation, Kinetics, and Function
For the purpose of this discussion, peripheral blood leuko-
cytes will be divided into two functional groups: the phago-
cytes and the immunocytes. Granulocytes (neutrophils, eo-
sinophils, and basophils) and the monocytes will be
considered among the phagocytes, whereas the various types
of lymphocytes, along with their precursors, and the plasma
cells will be discussed as immunocytes. Both classes of leu-
kocytes originate from a common pluripotent hematopoietic
stem cell and develop into specific cell types under the influ-
ence of various growth factors generally known as cytokines.
These hormone-like glycoproteins, produced by a wide vari-
ety of cells, are involved in intercellular communication.
The cytokines act hierarchically and synergistically at low
concentration to induce leukocyte proliferation, differentia-
tion, maturation, and specific f~nction.~” The cytokines thus
influence the development and maintenance of immunity,
and the inflammatory processes, by regulating the biologic
interaction among the different cell types.’”’
The cytokines are biologically active at various specific
steps during the differentiation and proliferation of hemato-
poietic stem cells, acting as either lineage-specific factors,
progenitor cell-cycling factors, or inhibitory factors.6.” Thus,
erythropoietin (EPO) regulates erythropoiesis; macrophage
colony-stimulating factor (M-CSF) and interleukin-5 (IL-5)
control the growth of the eosinophil and monocyte/macro-
phage lineages; granulocyte-CSF (G-CSF) acts on neutrophil
progenitors; whereas IL-6, IL-11, IL-3, IL-1, stem cell factor
(SCF), leukemia-inhibitory factor (LIF), and granulocyte-
macrophage-CSF (GM-CSF) have been shown to stimulate
the formation of neutrophils, macrophages, and megakaryo-
cytes.‘2”6IL-3, IL-4, and GM-CSF act on intermediate pro-
genitors after they leave the resting state, termed G0.17,18
Several factors, including IL-3 and GM-CSF, have been
shown to act synergistically to cycle dormant human hemato-
Blood, Vol84, No 6 (September 15), 1994: pp 1703-1721
poietic progenitors to promote colony formati~n”~’~; whereas
IL-6, G-CSF, IL-1 l , steel factor (SF), and IL-12 are active in
dormant murine hematopoietic progenitors.6 The inhibitory
cytokines include tumor necrosis factor-a (TNF-a), trans-
forming growth factor-@ (TGF-P), macrophage inflamma-
tory protein l a ( M I P ~ c ~and,
) , ~possibly, IL-4.”
Recently, it has been recognized that there are stem cells
that are progenitors for all hematopoietic lineages, including
immunocytes.6 These stem cells are not only in the bone
marrow but also in the lymphoid tissues, but those in the
latter tissue appear to produce both B and T lymphocytes.
B lymphocytes, originating from the bone marrow, represent
approximately 20% of circulating lymphocytes. Circulating
T lymphocytes also arise from the bone marrow, but these
are processed in the thymus before they are distributed to
the lymph nodes and spleen. Thus, approximately 80% of
circulating lymphocytes are T cells that remain in the circula-
tion for approximately 10 hours.
Dendritic leukocytes, which also originate in the hemato-
poietic system, participate in the immune response by acting
as antigen-presenting cells (APC). T lymphocytes are able
to recognize foreign antigens associated with the major histo-
compatibility complex (MHC) molecules (in humans, the
HLA antigens), presented by the APC, to initiate the immune
response. Thus, APC both present peptide-MHC complexes
to T lymphocytes and deliver activation signals initiating
the T-dependent immune response.21,22 This T-cell-antigen
interaction induces the production of various cytokines by
bothAPCand T cells to cause the rapid expansion and
differentiation of alloantigen-specific T lymphocytes. The
production of specific cytotoxic T lymphocytes is thus de-
pendent on specific cytokines, especially IL-2 and IL-4. The
IL-2 drives the differentiation of naive T cells toward the T
helper 1 (TH1)subset, which, in turn, controls cell-mediated
immunity. IL-4 directs naive T lymphocytes to become T
helper 2 (TH2)cells, which, in turn, mediate antibody produc-
tion by B lymphocytes. The B cell is then primed to respond
to a variety of cytokines released by T helper cells that
include IL-2, IL-4, IL-6, and interferon-y (INF-y) IL-2 to-
gether with IL-5 induces B-cell proliferation, whereas IL-6
participates in B-cell maturation.’ Some cytokines are thus
involved, directly and indirectly, in both the differentiation
and the activation of the various immunocytes, whereas other
cytokines inhibit these events to prevent uncontrolled B-cell
activation.
Fromthe Departments of Pathology and Medicine, McMaster
University; and The Canadian Red Cross Society, Hamilton, On-
tario, Canada.
Submitted February 14, 1994; accepted May 10, 1994.
Address reprint requests to Morris A. Blajchman, MD, FRCP(C),
Department of Pathology, Room 2N31, McMaster University Medi-
cal Centre, 1200 Main St W, Hamilton, Ontario, U N 325 Canada.
0 1994 by The American Society of Hematology.
0006-4971/94/8406-0013$3.00/0
1703
1704 BORDIN. HEDDLE, ANDBLAJCHMAN

Table 1. Approximate Total Leukocyte and Lymphocyte Count concentrates has been shown to be associated with a signifi-
of NonleukodepletedBlood Components Currently cant reduction of the occurrence of FNHTR in mostpa-
Available for Clinical Use tients,+uh.in-~s
Such observations lend credence to the argu-
Approximate Approximate mentthat multitransfused patients should receive only
No. of No. of leukodepleted blood components to prevent or minimize the
Leukocytes Lymphocytes
Blood Component per Product per Product severity of such reactions.z.32.76 Althoughthereduction of
the number of allogeneic leukocytes can decrease the overall
Whole blood 1 o9 5 x lo8
RBC concentrate 5 x 108 108
FNHTR ratecompared with that seen with nonleukodepleted
Washed RBC concentrate 107 5 x 106 products, the use of leukodepleted platelet concentrates has
Frozen-deglycerolized RBCs 5 x 106 106 not been totally effective in preventing such reaction^.^"^^^^^
Random donor platelet Moreover, it is reasonable to expect that even a pure suspen-
concentrate 5 X 10' 107 sion of platelets could cause FNHTR because platelets are
Apheresis single-donor platelet known to have HLA antigens on their surface membranes.
concentrate 5 x 10s 1 08 However, it has been shown that FNHTR also occur in pa-
Apheresis single-donor tients transfused onlywith the supernatant plasma from
granulocyte concentrate 10'0 5 X 109 platelet concentrates that contain very low numbers of plate-
Fresh plasma 5 X 105 1 o5 lets and leukocytes (< lo6 total cells per pr~duct).'~" In addi-
Fresh frozen plasma Undetectable Undetectable
tion, a significantly higher overall transfusion reaction rate
Cryoprecipitate Undetectable Undetectable
has been demonstrated with increasing storage age of both
RBC and platelet product^.'^."
A correlation between increased levels of TNFa, IL- 1 p,
andIL-6 in platelet concentrates andthe frequency of
There are two important requirements for immune system
FNHTR has been reported, providing evidence that such
activation. First, the interaction between receptor molecules
reactions may not always be the result of an antigen-antibody
and alloantigens is necessary to prime the immunocompetent
event, but may result from the transfusionof soluble biologic
cells to respond to the cytokines. Second, cytokine release
mediators, such as cytokines, that are known to be actively
is determined by the type of cellular interactions that take
synthesized by the leukocytes present in a blood component
place. Cytokines produced byAPC also contribute to the
and that accumulate during ~torage.~"~" Thus, the removal of
recruitment of host defense cells, even though, in some in-
leukocytes that actively synthesize and release such cytokines
stances, the overproduction of these cytokines may be harm-
frombloodcomponentsshortlyaftercollection(prestorage
ful to the host. The consequence of the latter include the
leukodepletion) may reduce the incidence of such reactions.
production of fever, chills, rigors, and increased capillary
Relevantly, it has been shown that the prestorage leukodepletion
permeability. These biologic effects may contribute to cellu-
of platelet concentrates significantly reduces the level of L I P ,
lar damage, particularly to damage of capillary endothelial TNF-a, IL-6,andIL-8 in thesupematant p l a ~ m a ? ' ~ .It~is~ ~ l
cells by TNF.Z',23,24
also possible that FNHTR in some patients are caused by
the release of cytokines from recipient macrophages that
BIOLOGIC EFFECTSOF TRANSFUSED might be activated by antigen-antibody-complement com-
ALLOGENEIC LEUKOCYTES plexes formed during or immediately after a tran~fusion.~'
The total number of leukocytes present in a unit of donor Moreover, it has been suggested recently that cytokines par-
whole blood is approximately lo9.There is a smaller number ticipate in the pathogenesis of some of the signs and symp-
in the various blood components, because some are lost dur- toms associated with hemolytic transfusion reaction^.".^
ing their preparati~n.'~ Table 1 summarizes the approximate Fever is a hallmark of ETTHTR. It is likely that the fever
total number of leukocytes and lymphocytes in the various is caused by the release of IL-l and IL-6 by the leukocytes
cellular blood products available in transfusion practice. contaminating cellular blood products. There are specific
cellular receptors for each cytokine present on the mem-
FNHTR branes of various cells and it isthis cytokine cellular receptor
interaction that triggers a specific biologic response. It ap-
An FNHTR is defined as an increase in temperature, of pears that the febrile response to IL-l and IL-6 is caused by
1°C or more, occurring in association withan allogeneic the production of prostaglandin E' (PGE2) by cells in the
blood transfusion. FNHTR are estimated to occur in approxi- hypothalamus causing the fever. In addition to causing fever,
mately 1% of red blood cell (RBC) transfusions and in ap- IL-1 can stimulate the recruitment of neutrophils from the
proximately 30% of platelet concentrate t r a n s f ~ s i o n s . ~ ~ .bone
~ ~ marrow, a phenomenonthat may cause the further
Most FNHTR have been attributed to the presence, in the elaboration of cytokines to induce other biologic activi-
recipient's plasma, of alloantibodies reactive with HLA or The biologic responses to the various cytohnes can
other alloantigens present on donor leukocytes and/or plate- thus be very complex and all these complexities remain to
l e t ~ . ~ ~Single-donor
,** HLA-unmatched nonleukodepleted be elucidated."
apheresis platelet concentrates are associated with a lower
incidence of FNHTR than that seen withpooled random Transfusion-Related Acute Lung Injury (TRALI)
donor platelet concentrate^.^^.^^ TRALI is a potentially lethal complication of allogeneic
The removal of 75% to 90% of the leukocytes from RBC bloodproduct transfusions characterized by chills, fever,
BIOLOGIC EFFECTS OF LEUKOCYTES
acute respiratory distress, bilateral pulmonary edema, and
severe hypoxemia, occurring 1 to 6 hours after an allogeneic
blood transfusion. Although the clinical features of T R A L I
are indistinguishable from those associated with the adult
respiratory distress syndrome, the pulmonary infiltrates in
most patients with TRALI resolve rapidly, usually within 48
to 96 hours, with no long-term sequelae. The mortality rate
associated with TRALI has been reported to be approxi-
mately 5 %:5
TRALI can be associated with the transfusion of whole
blood, RBC concentrates, fresh-frozen plasma, or cryopre-
cipitate. The pathogenesis of TRALI has been attributed to
the passive transfer of donor antileukocyte antibodies that
react with doantigens on the leukocytes of the recipient.65o
TRALI thus appears to be an immune-mediated disorder
associated with the passive transfer of donor HLA-A or
HLA-B alloantibodies, and/or granulocyte-specific alloanti-
bodies, including anti-NB2,49anti-NA2;' and a11ti-5b.~~ The
pulmonary vascular leakage associated with TRALI has been
shown to be preceded by complement system activation, with
neutrophil and endothelial cell injury representing additional
factors in the pathogenesis of the acute lung pathology>8
TRALI has been reported to occur even with the infusion of
small quantities of plasma containing leukocyte alloantibod-
i e ~ .It~ is
' not clear how the transfusion of only small quanti- blood products are at lower risk for platelet refractoriness
ties of plasma can trigger TRALI, but, as the result of such
reports, it has been proposed that only the cells of blood
donors implicated in such reactions be used for patient care.
It has been suggested also that allogeneic blood product units
from multiparous donors be screened for HLA or neutrophil-
specific antibodies before using such units for transfusion;
however, evidence for the efficacy of such a practice is un-
a~ailable.~' The role of the various cytokines in the pathogen- siderably higher than that observed in patients who received
esis of TRALI is unclear at the present time.
Platelet Refractoriness and Alloimmunization
Approximately 50% of multiply transfused patients be-
come refractory to pooled random platelet concentrates, lim-
iting the clinical effectiveness of such the rap^.^^.^^ Most
platelet transfusion refractoriness appears to be caused by
alloimmunization to HLA and/or platelet-specific antigens,
but the presence of bleeding, concurrent use of drugs, fever,
sepsis, disseminated intravascular coagulation, splenomeg-
aly, and A B 0 blood group incompatibility all may contribute
to inadequate allogeneic platelet recovery after transfusion.57m nization would nolonger occur is yet to be determined. Thus,
It has been estimated that HLA and/or platelet-specific allo-
antibodies can be detected in 20% to 50% of the patients
receiving multiple random donor platelet^.^^.^^.^' A recent
prospective study showed that HLA antibodies were detected
in 13 (26%) of 50 multitransfused patients, with platelet-
specific alloantibodies detectable in only 2 and platelet auto- reduction of RBC suspensions provided no significant advan-
antibodies in 2.62The exact incidence of platelet alloimmuni-
zation causing refractoriness is difficult to ascertain because 30% of the rabbits that received either 2 or 3 log,, leukode-
of the possible presence, in the patients evaluated, of con-
founding factors that could contribute to inadequate platelet
recovery. Moreover, the definition of platelet refractoriness
versus that of platelet alloimmunization has been a contro-
versial issue because the detection of alloantibodies does not
always parallel the refractory state, and vice versa. In this
1705
context, it has been shown that, despite the presence of plate-
let alloantibodies, some patients continue to have good plate-
let count increments after allogeneic platelet transfusion^.^^
The precise mechanism of platelet alloimmunization is
unknown. Because platelets express only HLA class I anti-
gens, it has been postulated that functional APC bearing both
class I and class I1 HLA antigens, present in donor blood
components, are required to initiate an immune response in
a r e ~ i p i e n t Thus,
. ~ ~ it has been postulated that APC present
antigen peptides in conjunction with donor HLA class I1
antigens to recipient's CD4 (TH2) cells to induce cytokine
production and thus stimulate B cells to produce alloantibod-
ies. Class I HLA peptides may also be recognized by recipi-
ent CD8 (TH1) T cells, initiating a cytotoxic immune re-
sponse. This T-cell-mediated immune response thus
requires interactions between the APC, the CD4 lymphocyte,
and the CD8 T
A variety of methods have been used to reduce or prevent
platelet alloimmunization. These include the use of single
donor cross-match compatible platelets:' the administration
of single-donor HLA-matched platelets,@ UV-B irradia-
ti0n,6~and the leukodepletion of allogeneic cellular blood
components intended for transf~sion.'~ Recent studies indi-
cate that patients receiving leukodepleted random donor
than are recipients of nonleukodepleted Table
2 summarizes the results of 10 studies comparing the inci-
dence of platelet alloimmunization in patients who had re-
ceived unmodified blood products with those who had been
transfused with leukodepleted blood products. The mean fre-
quency of platelet alloimmunization in patients transfused
with nonleukodepleted blood components appears to be con-
leukodepleted blood products. Despite these results, many
issues remain unresolved concerning the clinical impact of
leukodepletion to prevent platelet refractoriness. These in-
clude the extent and timing of leukodepletion; the correlation
between alloimmunization, refractoriness, and the risk of
morbidity due to bleeding; and the cost-effectiveness of such
an interventi~n.~~
Several investigators have postulated that platelet alloim-
munization occurs only with leukocyte counts greater than
lo6 per transfused allogeneic blood ~omponent,6~*"*~~.'~ but
the threshold number of leukocytes below which alloimmu-
in an experimental animal model, 50% of mice receiving
transfusions of platelets with even a very low number of
residual leukocytes (52/pL) become alloimm~nized.~~ In an-
other experimental animal model of refractoriness to alloge-
neic donor platelets, it was shown that 3 log,, leukocyte
tage over 2 log,, leukocyte removal, and that approximately
pleted allogeneic blood still became refractory. Interestingly,
the extent of platelet refractoriness could be reduced by com-
bining plasma removal with le~kodepletion.'~ Moreover, it
has been shown that the leukodepletion of allogeneic whole
blood before its storage (prestorage leukodepletion) was as-
sociated with a reduced frequency of platelet refractoriness
1706 BORDIN, HEDDLE, AND BLAJCHMAN

Table 2. Summary of Published Studies of Platelet teins Ib and IIb/IIIa." Such changes may result in the loss
Alloimmunization in Recipients of Either Leukodepleted or of platelet responsiveness to ristocetin and thrombin, but
Nonleukodepleted Allogeneic Blood Products they do not appear to affect in vivo platelet survivdx'
Nonleukodepleted Leukodepleted Another approach to prevent platelet alloimmunization is
the use of UV-B light. Basically, UV-B irradiation appears
Total No. Total No.
of No. 1%) of No. 1%) to modify allogeneic antigen presentation by depressing the
Investigators Patients Alloimmunized Patients Alloimmunized degree of expression of both class I and class I1 HLA anti-
Eernisse and gens on immunocytes. Changes in adhesion molecules such
Brand" (92.8)
26 28 68 (23.5) 16 as ICAM- 1 and CD14 have been reported with such irradia-
Schiffer et 12 4 15
(33.3) 4 (26.7) tion interfering with the production of IL-1 and IL-6, which,
Fisher et ala - - 24* 5 (20.8) in turn, might impair antigen presentation by B cells." Addi-
Murphy et al" (48.4)
1531
19 3 ( 15.8) tionally, UV-B light has been shown to enhance the number
Sniecinski et al" (50.0)
1020
20t 3 (15.0) of CD8 T-cell population in allogeneic blood product recipi-
Andreu et al" 35 11 (31.4)
34* 4 (11.71
ents affecting their secondary immune response." Most im-
Saarinen et a172 (70.6)
18
1217 0 (0)
van Marwijk Kooy
portantly, doses of UV-B that have been shown to reduce
et 27
(46.1)
12 26 0 3 (11.1) the reactivity of mixed lymphocyte reactions do not seem to
Oksanen et a174 15 1 (6.7) 16n 0 (0) affect in vitro platelet function or posttransfusion platelet
Handa75 21 8 (38.0) 32 2 (6.0) Studies in dogs that had received UV-B-irradi-
Total 20599/205(48.3) 273 401273 (14.7) ated allogeneic platelets have been shown to have a much
Percentages are in parentheses.
lower alloimmunization rate than control anirnal~.'~ Rele-
*All 24 subjects in this study received leukodepleted blood prod- vantly, in two small clinical studies, the rate of platelet allo-
ucts. Twelve patients received platelet concentrate transfusions, with immunization was lower in patients transfused withUV-
a mean leukocyte contamination of 15 x lo6 per transfusion, whereas B-treated allogeneic platelet concentrates thanincontrol
the other 12 patients were transfused with platelet concentrates con- patients who received nonirradiated platelet^.^^.^'
taining less than 5 X lo6 leukocytes per transfusion. All 5 alloimmu-
nized patients belonged to the first group. Transfusion-AssociatedGVHD ( T A - G V H D )
t The mean number of leukocytes per leukodepleted platelet con-
TA-GVHD is a potentially lethal immunologically medi-
centrate product transfused was 6 x lo6.
* The leukodepletion of RBC concentrates, pooled platelet concen-
ated disorder that results from the engraftment of viable
immunocompetent allogeneic donor T lymphocytes into the
trates, and single-donor platelet concentrates resulted in 97%, 92%,
and 76% leukocyte removal, respectively. tissues of a blood transfusion Whole bloodor
§ T h e mean leukocyte count after leukodepletion was less than 5 x RBC concentrate transfusions have been implicated in the
lo6 per pooled platelet concentrate consisting of six random donor majority of the cases,*5but granulocyte concentrate^,'^ plate-
units. let concentrates,'" and fresh plasma9' containing viable lym-
YThe mean numbers of residual leukocytes per unit of RBC and phocytes have also been implicated in TA-GVHD. Frozen
platelet concentrate was 0.1 X IO6 and 0.04 x lo6,respectively. blood products have not beenreported to cause TA-GVHD.9Z
The diagnosis of TA-GVHD is usually clinically based, but
histopathologic findings of theskin showing lymphocytic
and higher in vivo platelet survival, compared with that seen infiltration with satellite dyskeratosis may help to distinguish
in animals that received poststorage leukodepleted allogeneic TA-GVHD from drug toxicity or a skin infection. More
whole blood." These experimental animal data suggest that recently, genetic fingerprinting and the polymerase chain
alloimmunization may be related not only to the number of reaction have been used to confirm HLA chimerism in the
white blood cells present in an allogeneic blood component, peripheral blood lymphocytes of affected individual^.^^,"
but also to the presence of MHC or other antigens, either in Recently, TA-GVHD has been shown to be caused by
the soluble form or as microparticles, that escape leukocyte a network of interactions involving effector cells, multiple
filtration. Relevantly, it has been reported that the quantity cytokine pathways, and target cells."5 Epithelial and hemato-
of soluble HLA class I substance present in human whole poietic stem cells represent the host target cells, whereas
blood or platelet concentrates does not increase during stor- cytotoxic T lymphocytes and natural killer (NK) cells act as
age." It is probable, therefore, that particulate HLA class I the primary effector cells of the allogeneic donor. Although
antigens, in the form of microparticles present in allogeneic activated NK cells may cause cytolysis by direct cell contact,
plasma, contribute to the alloimmunization associated with host tissue damage can occur due to the release of TNF-a,
the transfusion of blood components. The recent demonstra- TNF-P, and IL-I by donor cytotoxic T lymphocytes and NK
tion that the combination of leukodepletion and plasma re- cells.96GVHD may be exacerbated by associated herpes or
moval abolishes platelet refractoriness supports such an hy- cytomegalovirus infections, because such infections may in-
pothesis, as does the recent report that leukodepletion does duce alterations in immune regulation in the hostaswell
not remove membrane microparticles bearing HLA anti- as provoke modifications on cell surfaces, increasing the
gen~.~~." susceptibility of host target cells to attack by effector
Besides the critical role in alloimmunization, the presence An excellent article describing the molecular basis
of leukocytes in platelet concentrates during storage has been of the cytokine dysregulation that occurs in acute GVHD
reported to be associated with changes in platelet glycopro- has recently been published.'5
EIOLOGICEFFECTS OF LEUKOCYTES
The occurrence of a TA-GVHD in a blood transfusion
ecipient depends on the immunocompetence of the host,
he genetic similarity between donor and recipient, and the
rumber of viable donor T lymphocytes present in the trans-
h s e d blood product.w,lOO The precise number of transfused
lymphocytes needed to induce TA-GVHD is not known.
Data from animal studies suggest that lo7 lymphocytes per
lilogram of body weight may be required:' In humans, as
few as lo4lymphocytes administered to children with severe
combined immunodeficiency has resulted in TA-GVHD."'
Typically, TA-GVHD has been reported to occur in immu-
nosuppressed patients whose immune systems are impaired
is a result of prematurity, a congenital immunodeficiency
state, a hematologic malignancy, a solid tumor, or associated
vith bone marrow transplantati~n.~~*'~ Accumulated evi-
aence indicates that TA-GVHD can also affect immunocom-
ptent individuals.'"" The risk of the development of TA-
GVHD in nonimmunocompromised individuals appears to
he greater if there is some HLA-identity between donor and
recipient such as occurs with transfusions from first-degree
The likelihood that blood from unrelated ho-
nozygous donors will be transfused into heterozygous pa-
Cents is greater in a monoracial population and has been
tstimated to be 1 in 16,835 in France,Iog l in 660 in Ja-
Pan,'03.104*"0 and 1 in 7,174 in American whites.Iog
The prevention of TA-GVHD can be accomplished by
dtering either the viability or the total number of T lympho-
cytes in a cellular blood product before transfusion. y-Irradi-
ition is the procedure generally used to prevent TA-GVHD.
"he recommended minimum dose of 15 Gy has been shown
b decrease the lymphocyte mitogen response by 90% with-
Put compromising the function of the other blood cell^.^'
Although higher doses of y-irradiation, such as 500 Gy, may
Sfect granulocyte chemotaxis and bactericidal activity,"'
granulocyte function remains essentially normal after y-irra-
tiation ranging from 0 to 175 Gy."I*'Iz The y-irradiation of
blood products does not appear to cause significant adverse
tffect on stored blood cells; nevertheless, some investigators
ecommend that y-irradiated RBC concentrates, used for
iltrauterine or pediatric transfusions, be washed to reduce
ne elevated plasma potassium levels often seenin such
Inits.113.114 Furthermore, irradiated RBC concentrates have
tmeen shown to have minor changes in ATP, pH, lactate
tehydrogenase (LDH), and plasma-free hemoglobin levels
&er 42 days of storage when compared with non-y-irradi-
aed RBC ~ n i t s . "Nonetheless,
~ the routine washing of irradi-
aed RBC before transfusion does not appear to be war-
nnted.'I6
A survey conducted in 1989 showed that various institu-
tons associated with the American Association of Blood
Banks used the following y-irradiation doses: 15 Gy
(42.3%),15 to 25 Gy (31.8%), 25 to 35 Gy (22.8%),and
3 to 50 Gy (3.1%).'17 Because a small percentage of lym-
$ocytesmay survive 15 Gy irradiation, it has been sug-
gested that doses of 25 to 30 Gy of y-irradiation may be
rppropriate to prevent TA-GVHD."','19 The FDA in the
United States has recently established regulations regarding
7-irradiation doses of blood products intended for clinical
me. These guidelines require 2,500 cGy (25 Gy) to the cen-
1707
tral part of the container, with 1,500 cGy (15 Gy) as the
minimum dose to any other point."'
Alternative approaches to prevent TA-GVHD have been
reported. In animal studies, the UV-irradiation of transfused
leukocytes has been shown to abolish TA-GVDH in recipient
dogs."' Another approach has been to reduce the number of
donor lymphocytes using leukocyte filters that provide at
least 3 log,, removal. In vitro studies indicate that leukode-
pleted cellular blood components may be associated with
decreased risk for TA-GVHD"'; however, TA-GVHD has
been reported to occur even with the transfusion of leukode-
pletedblood components (99% to 99.6% leukocyte re-
m~val).''~
Imrnunornodulation
Bloodtransfusionand allograji transplantation. The
beneficial effect of allogeneic blood transfusions (ABT) in
renal transplantation was described morethan 20 years
ag0.Iz4A highly significant correlation was reported between
increased numbers of pretransplant blood transfusions and
improved allograft survival in aprospective analysis of 1,360
kidney transplant^."^ Over the past 20 years, these results
have been replicated in animal experiments and confirmed
clinically in many transplant centers. Overall, patients trans-
fused with allogeneic blood products have a significantly
better renal allograft survival rate than nontransfused pa-
tients, regardless of the number of HLA-A locus and HLA-
B locus mismatches. Similar data are also available for
HLA-DR mismatching. Even with HLA-identical sibling al-
lografts, it has been reported that 33% of allogeneically
transfused recipients experience graft rejection, compared
with 75% of untransfused recipients.Iz6Thus, although data
exist to indicate that the beneficial effect of ABT occurs
even in HLA-identical recipients, the ABT effect is most
evident when the degree of HLA mismatching is greate~t.''~
Although it is generally accepted that ABT can improve
renal allograft survival, the influence of such transfusions
has declined significantly over the last decade, probably
caused by the advent of very effective immunosuppressive
drugs and improved patient management, particularly the
treatment of rejection episodes.'28.'29However, a recent
multicenter study reporting on 58,036 renal transplants since
the advent of the use of cyclosporine indicates that patients
receiving ABT are still more likely to have successful renal
allografts than those that did not.'" Similar results have been
observed in patients receiving living-related-donor kidney
tran~plants.'~'The mechanism involved in producing the
ABT-associated prolonged allograft survival has not yet been
completely elucidated. In addition, there are manyunan-
swered questions about the use of ABTin such patients,
such as the optimal number of transfusions, the component
of blood required to produce the effect, the volume of blood
required with each transfusion, the optimal timing of the
transfusions to produce the effect, the concurrent hazards of
the blood transfusion induced immunosuppressive effect,
and whether ABT are still n e c e s ~ a r y . ' ~ ~ ~ ~ ~ '
It has been proposed that the ABT effect may occur be-
cause of the selection of nonresponders, the development of
a suppressor cell network, the inactivation of alloreactive
1708
clones by immunosuppression, and/or the induction of anti-
idiotypic and anticlonotypic a n t i b o d i e ~ . ~The
~ . ' ~exclusion
~ stomach, cervix, vulva, head and neck, larynx, soft tissue,
of responders in the cross-match test cannot account for the
beneficial effect, because ABT have been shown to improve
renal allograft survival even in HLA-identical donor-recipi-
ent combination^.'^' Similarly, the clonal deletion theory
proposed by Terasaki is no longer ~ u p p o r t e d . ' ~ ~There ."~
is considerable evidence to suggest that ABT induce the
production of suppressor T cells; however, this effect does
not seem to persi~t.'~' Anti-idiotypic antibodies, which are
predominantly of the V,6 gene family, have been shown to
be present in serum of ABT recipients and in patients with
long-term functioning allografts."' Furthermore, the pres-
ence of an anti-Fc receptor antibody in the serum of kidney
recipients has been associated with an allograft survival rate
of 93% at 1 year and 71% at 3 years, compared with 29%
at 1 year and 25% at 3 years in patients lacking such antibod-
ie~.'~~
It has been suggested that the beneficial effect of ABT on
renal allograft survival increases with the number of transfu-
sions. Thus, patients who received 6 to 10 transfusions have
been reported to have higher l-year graft survival rates than
patients who had received fewer allogeneic transfusions. In-
terestingly, patients who receive more than 10 transfusions
appear to have lower overall graft survival rates.'35 This
may be caused by the greater likelihood that multitransfused
patients will develop cyotoxic antibodies and, thus, are at
greater risk for earlier and more severe allograft rejection
episodes.'3o Relevantly, recipients of whole blood or RBC
concentrates have been shown to have better l-year cadav-
eric graft survival than individuals receiving leukocyte-poor
blood components such as washed RBC concentrates or fro-
zen-deglycerolized RBCs. Such data indicate that allogeneic
donor leukocytes somehow participate in the production of
the beneficial ABT effect.'36Thus, pretransplant ABT remain
an intervention that can be useful in the management of
selected patients scheduled for kidney transplantation. Pa-
tients transfused during surgery had no impact on subsequent
renal allograft survival.125Nonetheless, additional data are
required to understand how ABT induce their beneficial ef-
fect in renal transplantation. In addition, the potential hazards
of the ABT should be considered before being undertaken
in a particular patient.
Blood transfusion and tumor growth. The possible asso-
ciation between ABT and cancer recurrence was first sug-
gested more than 10 years ago, when concern was raised for
patients undergoing curative surgery for a malignant disorder
who might be adversely affected by the immunomodulatory
effects of ABT administered perioperati~e1y.I~~ No definitive
data proving this effect in humans have been published as
yet. What have been reported are mainly nonrandomized
retrospective clinical data. Many reports have appeared eval-
uating the effect of perioperative ABT on tumor recurrence
and/or overall prognosis in patients with a malignancy under-
going curative cancer surgery. A considerable amount of
such data has been obtained from studies of patients with
colorectal carcinoma (summarized in Table 3).138"67Evi-
dence for a deleterious ABT effect has also been provided
by more than 40 clinical studies in patients with a variety of
BORDIN,HEDDLE, AND BLAJCHMAN
other malignancies, including breast, lung, kidney, prostate,
bone, and liver metastases. The adverse effect of ABT has
thus been reported in approximately 50% of such studies,
with the remaining 50% reporting no adverse effect.'"
The most relevant clinical studies evaluating the effect of
perioperative ABT on theoutcome in patients with colorectal
carcinoma have been subjected recently to meta-analysis by
two groups of investigator^.'^^^'^^ One such analysis indicates
that the cumulative odds ratio of colorectal carcinoma recur-
rence, cancer-associated death, and death from any cause in
allogeneically transfused patients are 1.80,1.76,and1.63,
re~pective1y.l~~ These investigators concluded that their anal-
ysis supports the hypothesis that perioperative ABT are asso-
ciated with an increased risk of colorectal carcinoma recur-
rence and death from this disease.'69 The second group
concluded that the adverse effect of ABT might increase the
relative risk of cancer recurrence by 37% (95% confidence
index [CI], 20% to 56%)."" Interestingly, a recentlypub-
lished prospective randomized study in colorectal cancer pa-
tients concluded that the risk of cancer recurrence was in-
creased in patients transfused with either allogeneic or
syngeneic blood compared with untransfused In
contrast, a similar prospective randomized study concluded
that ABT in colorectal carcinoma patients were an indepen-
dent prognostic factor on the cancer recurrence rate.'63Thus,
whether ABT affect tumor growth, in humans, is still an
open issue.
The tumor growth-promoting effect of ABT has been
shown to occur in experimental ani mal^.'^'^''^ Data from
bothinbredand outbred animal models indicate that the
tumor growth-promoting effect of ABT is mediated immuno-
logically; as the ABT tumor growth-promoting effect can be
adoptively transferred, using spleen cells, to naive animals.173
These studies indicate that the ABT effect on the growth of
animal tumors is related to the presence of donor leukocytes
in the allogeneic blood, and that this deleterious effect can
be ameliorated by the prestorage leukodepletion of the allo-
geneic b l ~ o d . ' ~ ' .Moreover,
'~~ the data provide evidence for
thelack of efficacy of poststorage leukodepletion in pre-
venting the ABT tumorgrowth promotion effect.175Although
results from experimental animals cannot necessarily be ex-
trapolated to the clinical situation, the results obtained indi-
cate that the bedside (poststorage) leukodepletion of alloge-
neic blood products may not be effective in preventing the
tumor growth-promoting effect of ABT, if this effect indeed
occurs in humans.
Blood transfision and infection. ABT are administered
frequently to patients who are at risk for infections. The
association between perioperative ABT andthe increased
incidence of bacterial infections after surgery has been exam-
ined in both clinical and experimental animal studies. The
available clinical data are mainly from uncontrolled studies.
The results of 9 relevant studies are summarized in Table
4.176-184 The only prospective trial conducted in colorectal
cancer patients, in which subjects were randomly assigned
syngeneic or allogeneic blood, reported a significant higher
postoperative infection rate in patients transfused with allo-
geneic blood compared with patients who received syngeneic
BIOLOGIC 1709

Table 3. Summary of 30 Clinical Studies That Analyzed the Effect of Perioperative Allogeneic Blood Transfusions
on Clinical Outcome
(Recurrence Rate andlor Overall Prognosis) in Patients With Colorectal Carcinoma
No. of Patients Adverse Effect of
ABT on Outcome
Investigators Study Design Untransfused Transfused
Total IPvalue)

Burrows andTamer'" Retrospective 123 58 65 1.005

Nathason et allm Retrospective 366 199 167 <.005
Blumberg et allw Retrospective 197 129 68 <.0001
Foster et all" Retrospective 146 65 81 .03
Frankish et all'' Prospective, 174 103 71 NS
not randomized
Ota et Retrospective 207 162 45 NS
Parrot et all4' Retrospective 517 373 144 <.005
Burrows et Retrospective 295 177 118 <.005
Francis and Judson'" Retrospective 87 53 34 NS
ROSS"~ Retrospective 159 95 64 NS
Voogt et all" Retrospective 113 86 27 <.01
Weiden et all'' Retrospective 171 103 68 .Ol*
Corman et allso Retrospective 400 343 57 1.001
van Lawick van Pabst et all5' Retrospective 164 117 47 ,039
Beyon et all5' Retrospective 519 385 134 <.001
Crowson et all5' Retrospective 525 373 152 NS
Mecklin et a P 4 Retrospective 520 355 165 <.001
Vente et all" Retrospective 212 158 54 NS
Waymack et allw Retrospective 155 101 54 1.0001
Wobbes et a P 7 Retrospective 270 184 86 <.001
Cheslyn-Curtis et allw Retrospective 961 591 370 NS
Jakobsen et allSB Retrospective 315 268 47 NS
Liewald et allrn Retrospective 439 304 135 .02
Marsh et allB1 Retrospective 132 62 70 <.005
Ouintiliani et al'" Prospective, 58 35 23 NS
not randomized
Heiss et allm Prospective 120 NA NA .008
Randomized
Tamer"' Prospective, 339 110 229 <.0001
not randomized
Sene et a P 5 Prospective, 379 221 158 NS
not randomized
Tang et allw Retrospective 725 472 253 NS
Busch et all" Prospective, 475 236 2391 .01
randomized
Total 3,225 5,918 9,263
Abbreviations: NS, not significant; NA, not available.
*Transfused patients had a lower tumor recurrence rate than untransfused patients.
t Patients in the control group received syngeneic (autologous) blood transfusions.

blood transfusions (27% v 12%,P = .036). Using multivari-
ate regression analysis to adjust for other factors, the odds
ratio for risk of postoperative infections in the allogeneic
versus syngeneic blood transfusion group was 2.84 (95%
CI, 1.02 to 7.98). The observed number of noninfectious
complications was similar in the two groups of patients.ls4
The dose-response relationship between the number of
allogeneic blood units transfused and the risk of infection
has also been examined. Thus, in a study of 137 patients
who underwent surgery for a penetrating colonic injury, the
risk of an infection was 7.5% for untransfused patients, and
25%, 37%, and 57% for patients transfused with 1 to 5
units, 6 to 9 units, or 10 or more units, respe~tively.'~~In a
retrospective study of patients who underwent surgery for
gastric carcinoma, patients who developed postoperative in-
fections were transfused with a higher number of allogeneic
units compared with those who did not. However, infected
patients were found to have been subjected to longer surgery,
more frequent use of urinary tract catheters and drains, and
had an operation involving a resection.Is6
The role of allogeneic leukocytes in the development of
postoperative infections has also been investigated. A pro-
spective randomized trial involving patients who underwent
colorectal surgery has shown that patients transfused with
allogeneic whole blood had a significantly higher frequency
of postoperative infections than patients who received
99.98% leukodepleted allogeneic blood. This study also pro-
vided evidence that NK cell function remained significantly
impaired for up to 30 days after surgery in those patients
transfused with nonleukodepleted allogeneic wholeblood
 1710 BLAJCHMAN BORDIN, HEDDLE, AND

Table 4. The Effect of Allogeneic Blood Transfusions on the Incidence of Postoperative Infectious Complications
Infection Rate

Allogeneic Statistical
Type of No. of Transfusion Control Significance
Investigators Study Design Surgery Patients
Group Group (Pvalue)

NORGAS”‘ Prospective, Abdominal 1,537 221357 16/1,180* <.0001

not randomized (1.4%) (6.2%)
Tartter et all7’ Prospective, Abdominal 343 331134 9/209* <.0001
not randomized (4.3%) (24.6%)
Jensen et al”’ Prospective, Abdominal 31 1 571202 2/109* <.0001
not randomized (28.2%) ( 1.8%)
Murphy et all7’ Retrospective Orthopedic 84 16/50 1/34t ,0029
(2.9%) (32.0%)
Mezrow et al1’’ Retrospective Gynecologic 100 8/50 2/50t 1.05
(4.0%) (16.0%)
Triulzi et alle1 Prospective, Orthopedic 109* 512 4 2/60t ,0185
randomized(3.3%) (20.8%)
Fernandez et all8’ Retrospective Orthopedic 3761 5/72 7/140t .oo 1
(5.0%) (6.9%)
Jensen et alls3 Prospective, Abdominal I 971 13/56 1/@/1 <.Ol
randomized (23.2%) (2.0%)
et Heiss alla4 Prospective, ,036 Abdominal17/62
7/58t 120
randomized (12.1%) (27.4%)
Total -
4711,888 -
176/1,007 3,177 -
(2.5%) (17.5%)
*The control group did not receive any blood transfusions.
t The control group received syngeneic blood.
* Of 109 patients evaluated, 24 received allogeneic blood, 60 received syngeneic blood, and 25 did not receive any blood transfusions.
§ Of 376 patients evaluated, 122 did not receive any blood, 140 received syngeneic blood, 72 were transfused with allogeneic blood, and 42
received both syngeneic and allogeneic blood. There are no differences across the four groups with regard to the infection rate; however,
patients transfused with allogeneic whole blood had a higher relative risk of infection ( P = ,001).
1Of 197 patients evaluated, 104 received allogeneic blood transfusions. Forty-eight were randomized to receive leukodepleted (99.98%
leukocyte removal) allogeneic blood products, and 56 to receive nonleukodepleted allogeneic whole blood.
11 This group of 48 patients received leukodepleted allogeneic blood products, not syngeneic blood products.

compared with patients who received leukodepleted blood.’83
Transfused allogeneic leukocytes have also been identified
as the blood component responsible for the gut-derived in-
fection in a murine model.L87 In contrast, in a bacterial perito-
nitis animal model that compared syngeneic blood transfu-
sions to ABT, the latter were not shown to influence survival
of mice.’88
Based on the published clinical studies, it appears that
susceptibility to postoperative bacterial infection is associ-
ated with the transfusion of nonleukodepleted ABT. The
reported bacterial infection rate in allogeneically transfused
patients ranges between 20% and 30% compared with be-
tween 5% and 10% in either untransfused or syngeneically
transfused subjects. Nevertheless, the relationship between
ABT and bacterial infection has not been proved. This is
partially because of the problem of defining the term “infec-
tion” in such patients. Limiting the definition of infection
to positive cultures underestimates prevalence, whereas ex-
tending the definition to include fever overestimates preva-
lence. It is our opinion, therefore, that a large prospective
controlled randomized clinical trial is required to validate
definitively the association between ABT and increased risk
of infection.
Blood transfusion and spontaneous recurrent abortions.
Because the fetus represents a semiallogeneic graft to its
mother, the maintenance of a pregnancy is dependent on
immunologic equilibrium between the implantated fetus and
the maternal immune response to the fetus. It has been pro-
posed that an idiotypic-anti-idiotypic network downregu-
lates maternal rejection during p r e g n a n ~ y . ’ ~This
~ * ’delicate
~
balance may be altered in situations in which the spouses
share HLA antigens. In such situations, maternal blocking
antibodies may not form and the implanted fetus is rejected.
Based on such an hypothesis and also on evidence of pro-
longed graft survival seen in allogeneically transfused kidney
transplant recipients, leukocyte transfusions have been used
as a form of immunotherapy to treat women with spontane-
ous recurrent abortions.19’ Different allogeneic leukocyte
transfusion protocols have been used with leukocytes from
either sexual partners or third-party donors. Such allogeneic
leukocytes have been transfused as pooled buffy-coats; as
single donor buffy-coats; as RBC suspensions containing
leukocytes; or intravenously, intracutaneously, or as an intra-
dermal injection of mononuclear cells obtained by gradient
~eparation.’~’
The success rate of approximately 75% obtained with the
transfusion of either paternal or third-party leukocytes is
considerably better than that observed with maternal cells
of approximately 50% (Table 5).’9”202The latter figure is
derived from three prospective nonrandornized studies that
 BIOLOGIC EFFECTSOF LEUKOCYTES 1711

Table 5. Summary of the Clinical Outcomeof Patients With a periods of considerable disease activity, patients with
History of Recurrent Spontaneous Abortions who Received Crohn’s disease require surgical treatment because of a
Leukocyte Transfusions bowel obstruction or perforation. The recurrence rate after
~ ~~~

Success Rate (no. of live births or pregnancies surgical resection is approximately 50% at 10 years.’” Al-
beyond 20 wklno. of pregnant women) though the etiology of the Crohn’s disease is stili uncertain,
According to Treatment
it has been suggested that immunologic mechanisms are in-
Third-party
Paternal volved in its pathogenesis. In 1986, Tamer et a1’” reported
Investigators
Lymphocytes
Maternal
Donors Cells that patients with Crohn’s disease had a lower number of
Faulklg’
and
Taylor - 3/4 (75.0) - circulating total lymphocytes and T lymphocytes compared
Mowbray
et (77.3)
17/22 - (37.0)10127 with normalcontrols, and that multiple ABT were associated
all” et
Mclntyre - 23/26 (88.5) - with a significantly lower peripheral total lymphocyte and
Mowbray
et allg5 (80.0)
24/30 - - T-cell counts after surgery. Postulating that the immunosup-
Reznikoff-Etievant et al’” 29/34 (85.3) - -
- - pressive effects of allogeneic blood might be beneficial to
Smithand C o ~ c h o c k ’ ~ ~43/74 (58.1)
Takakuwa et aI1” 28/35 (80.0) - - patients with Crohn’s disease, several studies examined
Carp et a P (71.9) 64/89 - - whether the postoperative recurrence rate is affected by the
et Cauchi aI2Oo 13/21 (61.9) - - perioperative use of ABT. The pooled data from 5 available
Ho et al”” 31/39 (79.5) 8/11 (72.7) 32/49
(65.3) studies suggest that the recurrence rate in the two groups is
Unander’” -(92.4) 243/263 - similar (Table 6).212~2’6 However, the studies summarized in
Gatenby
et alzo2 (68.4)
13/19 - 9/19 (47.4) Table 6 are difficult to compare because they have different
Total 262/363 (72.2)(53.7)
51/95
277/304
(91.1) follow-up periods and use different surgical treatments. Most
Percentages are in parentheses. importantly, they are all retrospective.
The effect of ABT on the number of postoperative septic
complications in patients with Crohn’s disease has also been
compared patients who received either paternal or maternal analyzed. In 1988, Tartter,’I7 using multivariate analysis,
leukocytes.193,201.202
The number of allogeneic mononuclear implicated ABT as a major factor in the development of
cells inoculated may also influence the outcome. The infu- infectious complications in Crohn’s disease patients sub-
sion of less than 60 million mononuclear cells may result in jected to surgical procedures. Such an association was not
a suboptimal effect, whereas the inoculation of excessive shown to reach statistical significance in another similar
cells may not be e f f e c t i ~ e . ~The ’ ~ of allogeneic leuko-
~ ~ .use study.’I3 Because many factors might affect recurrent rates
cytes in the treatment of patients with spontaneous recurrent of Crohn’s disease, as well as the rate of septic complications
abortions is of course associated with the same risks associ- after resection, it will be necessary to conduct well-designed
ated with anyother leukocyte transfusion, including neonatal prospective trials to clarify whether ABT have an impact in
GVHD. Moreover, alloimmunization to leukocyte antigens the disease activity of patients with Crohn’s disease.
may cause fetal growth retardation, congenital anomalies,
and alioimmune neonatal neutropenia.L92~2032”5 THE MECHANISM OF THE ABT-INDUCED
The mechanism of the beneficial action of the allogeneic IMMUNOMODULATION
leukocyte infusions in patients with spontaneous recurrent From the available evidence, it appears that ABT-induced
abortions is unknown. However, it has been suggested that immunomodulation is caused by the presence of contaminat-
the beneficial clinical response observed may be associated ing leukocytes in the transfused cellular blood prod-
UCtS~125,127.173.175 The mechanism of the immunornodulation
with the production of auto-antidiotypic antibodies?% re-
lease of IL-lor decrease in maternal IL-2 receptor has not yet been precisely elucidated; however, it has been
leve1.208 postulated that the immunosuppressive effects are immuno-
The efficacy of leukocyte transfusions in patients with logically mediated.53.127.2L8
Allogeneic leukocytes present in
spontaneous recurrent abortions remains to be established cellular blood products bear class I1 major histocompatibility
by proper prospective randomized studies. Limitations to complex (MHC) antigens andit has been suggested that
data currently available include small numbers of women in donor APCs present these allogeneic MHC antigens to the
some studies; heterogeneity of the study populations; great T lymphocytes of the re~ipient.’~ This MHC antigen-T-
diversity of the therapy protocol used; and possible cointer- lymphocyte interaction provides the first signal that culmi-
vention effects derived from placebo treatments used in some nates in the expression of the IL-2 receptor. This is insuffi-
of the control groups of patient^.^^^.'^ Only well-designed cient to elicit an immune response, because a second signal
randomized cooperative studies will clarify the possible true is required to induce the secretion of the various cytokines,
benefit, risks, and clinical indications for allogeneic leuko- which, in turn, cause the proliferation and differentiation of
cyte transfusions in the treatment of women with spontane- alloantigen-specific T lymph~cytes.’’~
ous recurrent abortions. The immunogenicity of MHC antigens present on alloge-
Blood transfusion and injammatory bowel disease. neic blood products thus depends on the ability of donor
Crohn’s disease is a chronic inflammatory condition that APCs to stimulate recipient T cells by delivering two differ-
can affect both the small and large bowel to cause clinical ent signals. The absence of the second signal has been shown
symptoms of diarrhea and crampy abdominal pain. Typi- to induce T-cell anergy.220In this context, it has been sug-
cally, after many years of quiescent periods alternating with gested recently that, during storage, leukocytes lose their
1712 HEDDLE, BLAJCHMAN BORDIN. AND

Table 6. Effect of Allogeneic Blood Transfusions on the Recurrence Rate of Crohn’s Disease

Recurrence Rate Statistical

Study Design No. of Significance
Investigators Transfused (time period) Patients Not Transfused ( P value)

Williams and Hughes’” Retrospective 60’ 4/28 15/32 ,006

(1973-1986) ( 14.3%) (46.9%)
Peters et a P 3 Retrospective 79t 10145 15/34 ,035
(1976-1985) (22.2%) (44.1%)
Sutherland et aI’l4 Retrospective 68 12/26 13/42 >.05
(not cited) (46.2%) (31.O%)
Scott et Retrospective 197$ 381102 35/95 2.05
(1947-1988) (37.3%) (36.8%)
Steup et a P 6 Retrospective 1042 36/65 20139 ,165
(1980-1985) (55.4%) (51.3%)
Total - 508 100/266 981242 -
(37.6%) (40.5%)
* Both groups were similar in age, duration of Crohn‘s disease, and serum albumin level. They had different hemoglobin levels, length of
bowel resected, and location of small bowel involvement.
t Both groups were similar in age, sex, race, and duration of disease. The multitransfused group had significantly lower hematocrit and
albumin levels on admission. The untransfused group included patients receiving 1 unit of blood.
*There were significant differences in the hemoglobin and albumin levels between the two groups. The distribution of AB0 and Rhesus
blood groups was similar in the two groups.
§ The transfused group had significantly lower hemoglobin levels and longer bowel segments resected.

i m m ~ n o g e n i c i t yThus,
. ~ ~ ~the transfusion of allogeneic cellu-
lar blood products stored for prolonged periods of time might
be associated with the induction of T-cell anergy and thus
to ABT-induced immunosuppression. Relevantly, ABT re-
cipients have been shown to have various alterations in im-
munocyte function. These include decreased helper-suppres-
sor lymphocyte ratio; decreased NK cell function; decreased
antigen presentation; suppression of lymphocyte blastogene-
sis; and reduction in delayed hyper~ensitivity.‘~~~’~~~’’~ Thus,
although the precise mechanism of ABT-induced immuno-
modulation has not been elucidated, there is a considerable
body of information indicating that allogeneic blood transfu-
sions are associated with alterations in immune function and
that this effect is transferable to naive animals by immuno-
~ytes.’~~
LEUKOCYTESASTHEVECTOR FOR TRANSFUSION-
ASSOCIATEDINFECTIOUSDISEASES
Cytomegalovirus (CMV} Infections
Transfusion-associated CMV (TA-CMV) infections have
been recognized as a significant cause of morbidity and mor-
tality in blood product recipients at risk. These include
CMV-seronegative pregnant women, premature infants born
to CMV-seronegative mothers, CMV-seronegative alloge-
neic bone marrow transplant recipients, and CMV-seronega-
tive patients with acquired immunodeficiency syndrome
(AIDS).”’ CMV antibody prevalence rates in North America
range from 30% to 80%.222 Whether all seropositive blood
donors can transmit CMV i s unknown, and recipient factors
may be critical in determining the development of TA-
CMV.223Thus, it might be possible to reduce the risk of TA-
CMV infection from asymptomatic seropositive donors by
limiting the number of blood units transfused to subgroups
of patients at high risk, and/or improving the ability to detect
IgM anti-CMV toidentify acutely infected blood donors who
may be more infectious than chronic carriers.223
CMV has been isolated from the peripheral blood leuko-
cytes of many infected individuals.2?”1.225
Moreover, individu-
als transfused with granulocyte concentrates have been
shown to be at increased risk for CMV infection.”’ Thus,
it has been suggested that the removal of leukocytes from
allogeneic donor blood may prevent TA-CMV.226,227 this In
context, the use of frozen-deglycerolized lU3C has been re-
ported to prevent the transmission of CMVZ2*and the transfu-
sion of washed RBC (87% leukocyte removal) from CMV-
seropositive donors to neonates is associated with a lower
frequency of anti-CMV seroconversion (1.3%) than that seen
in recipients of unwashed RBC (13% to 35%).229
The maintenance of a satisfactory inventory of CMV-
seronegative blood donors may be difficult for some centers.
The use of leukocyte filters in preventing TA-CMV infection
has therefore been the subject of several studies. In a
multicenter controlled trial, 21% of newborn infants trans-
fused with unfiltered blood products developed CMV infec-
tion, whereas no TA-CMV was observed in the group of
infants transfused with leukodepleted blood products ( P =
.005).230In one retrospective study, none of 59 CMV-sero-
negative patients with acute leukemia or non-Hodgkin’s
lymphoma had evidence of seroconversion 2 months after
transfusion with leukodepleted blood prod~cts.’~’Another
retrospective study reported that only 2 of 13 CMV-seroneg-
ative patients who were transfused with lymphocyte depleted
allogeneic bone marrow from CMV-positive donors, and
who were transfused with leukodepleted CMV-unscreened
blood products, developed clinical manifestations of CMV
infe~tion.’~’It has been reported that leukocyte filters are
also capable of removing CMV-DNA from CMV-infected
donor blood.233The data from a prospective randomized
study in 487 patients undergoing bone marrow transplanta-
BIOLOGIC EFFECTS OF LEUKOCYTES
tion indicate that the use of leukodepleted blood products is
equivalent to the use of CMV-seronegative blood products
in preventing TA-CMV infection.z34
Other Infections
Leukocytes, particularly B and T lymphocytes, are also the
reservoir of other infectious agents that can be transmitted by
blood transfusion. These include the human immunodefi-
ciency viruses (HIV-1 and HIV-2), the human T-cell leuke-
mia viruses (HTLV-I and HTLV-11), the Epstein-Barr virus
(EBV), as well as the hepatitis C virus (HCV).873z35 However,
the minimum number of leukocytes capable of transmitting
such infections has not yet been determined. In this context,
it has been shown that the leukocyte filtration process can
remove intact HIV-l-infected cells and infectious particu-
late debris but not free virus.z36.z37 Interestingly, allogeneic
peripheral blood mononuclear cells have recently been
shown to induce the activation of HIV-l-infected cells and
the subsequent dissemination of HIV-1 to uninfected cells.
This in vitro effect was not observed with leukodepleted
allogeneic blood.z38Additionally, a recently published retro-
spective study has shown an increased incidence of opportu-
nistic infections in previously transfused HIV- 1-infected pa-
t i e n t ~Such
. ~ ~ findings
~ could have important implications in
clinical decision-making regarding the use of leukodepleted
blood products in HIV-infected individuals.
Although leukodepletion is not sufficient to completely
remove HTLV-I from infected blood, it has been suggested
that the transmission of HTLV-I can be prevented, at least
partially, by donor blood l e ~ k o d e p l e t i o n . ~Additionally,
~”~~
it has been shown that the infectivity of HTLV-positive
blood products decreases with storage, probably because of
the death of infected lymphocytes at refrigerator tempera-
t ~ r e . Experimental
’~ data indicate that only 10%of HTLV-I-
positive units stored for 20 days were virus culture-positive,
whereas all units stored for less than 7 days were still posi-
Although EBV has not often been reported as a transfu-
sion-associated infection, this B-cell-associated virus can
be transmitted by ABT causing an infectious mononucleosis
type of ~ y n d r o r n e EBV . ~ ~ can also be transmitted to trans-
plant recipients by transfused allogeneic leukocytes, thus
exposing them to the potential risk of a lymphoproliferative
d i s ~ r d e r . ~ The
” . ~ ~removal
~ of leukocytes from blood prod-
ucts thus might prevent EBV transmission; however, no evi-
dence has yet been provided to support this hypothesis.
The bacterial contamination of the blood products remains
a significant problem in transfusion m e d i ~ i n e . ’ ~ ~For- ’ ~the
~ provides accurate detection of 0.5 to 1 leukocytes per micro-
past 13 years, the annual incidence of positive bacterial cul-
tures in blood components tested by the Canadian Red Cross
Society has been approximately 0.3%.z47.z49 Moreover, post-
transfusion septic reactions account for approximately 10%
of the transfusion-associated deaths reported in the United
States.z46Possible measures to minimize transfusion-associ-
ated sepsis include improved donor screening, reduction in
blood product storage periods, improved techniques for
blood collecting and processing, quality control programs,
bacteriologic screening, and the removal of contaminating
b a ~ t e r i a . ’ ~Recently,
~ - ~ ~ the removal of bacteria contaminat-
1713
ing blood products has been demonstrated by leukodepletion.
It appears that removal of leukocytes from blood products
that have phagocytosed bacteria lowers the risk of transfu-
sion-associated septic r e a ~ t i o n s . ~However,
~ ~ - ” ~ the removal
of leukocytes should be performed only after an appropriate
incubation period to enable the phagocytosis of the bacteria
p r e ~ e n t .Leukocyte
~ ~ ~ . ~ ~depletion
~ filters have also been re-
ported to be able to remove contaminating uningested bacte-
ria that may be present in blood components d i r e ~ t l y . ~ ~ ~ ~ * ~
The relative contribution of the phagocytic mechanism ver-
sus the direct mechanism in the removal of bacteria from
blood products has not yet been delineated.
THE LEUKOCYTE DEPLETION OF BLOOD PRODUCTS
Leukocyte Depletion of Blood Products
The removal of leukocytes from blood products can be
accomplished by various techniques including sedimenta-
tion, centrifugation, washing, freezing-thawing, and leuko-
filtration.z56Recently, a number of articles have provided
information about the filtration of blood products including
leukocyte depletion.’~z34~53~z57~259
First-generation blood filters
(170 to 240 pm) are intended to prevent the infusion of
large clots and large cellular aggregates to blood product
recipients; second-generation filters (40 pm), also called mi-
croaggregate filters, remove cellular microaggregates that
often form during blood storage; and third-generation blood
filters can retain not only microaggregates but also cells and
are generally referred to as leukocyte depletion filters.z8
Significant progress has been made over the past decade in
the biotechnology of leukofiltration. Thus, donor leukocytes
can be removed by both physical (barrier retention, surface
phenomena, and charge density) and biologic (cell adhesion
and cell-cell interaction) mechanisms. For an excellent dis-
cussion about what is known currently about leukofiltration,
the interested reader is referred to the report by D ~ i k . ’ ~ ~
Currently available leukocyte filters provide depletion
rates that exceed 3 log,, (99.9%), resulting in less than 3 X
lo6 leukocytes in a 300-mL unit of a blood component, ie,
approximately 10 residual leukocytes per microliter. Various
techniques have been devised to count accurately such low
levels of residual leukocytes in leukodepleted blood prod-
ucts, because traditional cell counting techniques are inaccu-
rate at this low number.260The most widely accepted current
methoduses a counting chamber known as the Nageotte
chamber. The Nageotte chamber is a large-volume hemocy-
tometer that uses a sample volume of 50 pL. This approach
liter. This corresponds to approximately 3 X lo5 residual
leukocytes in a 300-mL unit of RBCs.z61,z6z Flow cytometry
techniques have been reported to have a detection sensitivity
to approximately 0.1 l e ~ k o ~ y t e s / p and
L , ~to~ have
~ ~ ~ less
~~
variability than the Nageotte chambe?60.z6z;however, flow
cytometry requires expensive equipment and reagents, and
may not be available in many centers requiring such technol-
ogy.
The leukodepletion of the blood products can be per-
formed shortly after collection (prestorage leukodepletion)
or after storage, just before transfusion (poststorage leukode-
1714
pletion). The prestorage leukodepletion of blood products
has been shown to prevent some of the adverse effects caused
by metabolites released from leukocytes during blood prod-
Moreover, the prestorage leukodepletion of
uct storage.2M’26s
blood products has been shown to be more effective than
poststorage leukodepletion in preventing platelet alloimmu-
nization and the tumor-growth promoting effect associated
with ABT.173.’7sThe latter observations are from studies in
experimental animals and need to be confirmed in humans.
Clinical Indications for Using Leukodepleted Blood
Products
The proposed clinical indications for the use of leukode-
pleted blood products have been reviewed in several recent
publication^."^*^^* These include the prevention of FNHTR,
GVHD, the transmission of disease, refractoriness to trans-
fused platelets, and immunomodulation. However, in most of
these, the effectiveness of leukodepletion remains unproven.
Moreover, the exact degree of leukodepletion required for
the various proposed clinical situations remains to be de-
fined. Thus, it has been suggested that l log,, leukocyte
reduction prevents FNHTR; that a 2 log,, reduction may
prevent the transmission of viruses; and that a 2 3 log,,
reduction may be necessary to prevent platelet alloimmuni-
~ a t i o n . ”However,
~ because there are no data available as
guidelines for the use of leukodepleted blood products for
most clinical indications, the use of leukocyte filters should
be restricted to selected patients for whom such data exist.
Despite the latter, it has been suggested recently that the use
of leukodepleted blood components might be a very cost-
effective strategy to prevent platelet alloimmunization in
adult patients with acute leukemia.’% In our opinion, further
data are required before the routine implementation of leuko-
depletion for all blood products to prevent platelet alloimmu-
nization as well as other indications.
SUMMARY
A considerable literature has accumulated over the past
decade indicating that leukocytes present in allogeneic cellu-
lar blood components, intended for transfusion, are associ-
ated with adverse effects to the recipient. These include the
development of febrile transfusion reactions, graft-versus-
host disease, alloimmunization to leukocyte antigens, and
the imunomodulatory effects that might influence the prog-
nosis of patients with a malignancy. Moreover, it has become
evident that such leukocytes may be the vector of infectious
agents such as CMV, HTLV-MI, and EBV as well as other
viruses. An interesting development that has occurred coinci-
dentally in transfusion medicine is that agencies responsible
for the provision of blood products are being designated
manufacturers of biologicals. The trend among manufactur-
ers of biologicals is to try to produce pure products to provide
for the specific therapeutic need of patients. Thus, with the
realization that allogeneic leukocytes and their products may
have adverse biologic activities, there is increasing pressure
from various sources for the reduction of the leukocyte con-
tent in allogeneic blood components to minimize the occur-
rence of their adverse effects. Although the threshold leuko-
BORDIN,HEDDLE, AND BLAJCHMAN
cyte number below whichthese effects might nolonger occur
is still to be determined, a 2 to 3 log,, leukocyte reduction,
provided by the currently available commercial leukocyte
filters, has been shown to reduce the frequency of many of
such reactions.
On the other hand, the immunosuppressive effects pro-
duced by the infusion of allogeneic leukocytes might be
beneficial for some patients, ie, for the maintenance of kid-
ney allografts, in possibly reducing the relapse rate in pa-
tients with inflammatory boweldiseases, and in ameliorating
recurrent spontaneous abortion. Moreover, therapeutic gran-
ulocyte transfusions may be ofbenefit in certain well-defined
categories of patients infected with bacteria, yeast, andor
fungi. These include neonates with bacterial sepsis associ-
ated with bone marrow failure as well as severely neutro-
penic leukemic patients withan infection unresponsive to
appropriate and specific antibiotic therapy.267,268
Manyof the results obtained with the useof leukocyte
depletion filters are tantalizing, but the actual clinical benefit
of leukodepletion has not been established in most instances,
because much of the available data are retrospective or from
uncontrolled clinical trials. Moreover, issues of cost-effec-
tiveness and quality control have not been considered ade-
quately. Properly designed prospective clinical trials are es-
sential to provide data with which to answer such questions
and also to help define the optimal conditions required for
the preparation of blood components ultimately destined for
clinical use. Only with the availability of such data can sound
decisions be made about the clinical value of leukodepletion.
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