Blood Reviews (2000) 14, 130144

2000 Harcourt Publishers Ltd

doi: 10.1054/ blre.2000.0131, available online at http://www.idealibrary.com on

Transfusion medicine

Immunological aspects of blood transfusions

A. Brand
Donor selection based on blood group phenotypes, and blood processing such as leukocytedepletion, gamma-irradiation or washing to remove plasma, are approaches for therapeutic or
preventive use to manage the immunological complications of transfusion. Indications for specific
components are prescribed in guidelines provided by (inter)national Transfusion Societies. Although
the use of guidelines and protocols is in line with modern medicine, these can create a state of tension
with the political sense of values to improve the viral safety of blood products and with the
commercial exploitation of pooled plasma-products.
A century of blood transfusion therapy has facilitated cancer treatment and advanced surgical
interventions. The transfusion product has improved progressively, although mostly in response to
disasters such as wars and AIDS. Every blood transfusion interacts with the immune system of the
recipient. There are, however, very few quantitative figures to estimate the consequences. This review
is based on the available literature on the clinical consequences of transfusion induced immunization
and modulation. To a large degree the clinical consequences of transfusion induced immune effects
are still a mystery.
A blood transfusion is a medical intervention, which in many cases remains experimental with
respect to the benefit/risk ratio. Ideally, this uncertainty should be communicated to patients and
every transfusion included in a study. Such studies preferentially should be randomized because the
perceived need for transfusion is associated with clinical conditions with a worse prognosis than those
that do not receive transfusion. This difference may mask the interpretation of the transfusion effect.
Since the blood supply services in almost all Western countries have been reorganized and
nationalized, or at least operate to national quality standards, the measurement of risk: benefit of
transfusion, whether political or evidence-based, needs to be reconsidered. Differences in emphasis
and responsibilities between transfusion providers and transfusion prescribers will drive the providers
to political and liability criteria ever safer products that will increase hospital costs with
undetermined clinical benefits. 2000 Harcourt Publishers Ltd
expected that in the new millennium state of art blood
products will be depleted of the majority of leukocytes, which generally serve no transfusion purpose
and can cause unwanted side-effects. Despite leukoreduction, blood components remain impure, unique
batches, containing hundreds of donor specific antigens including many viruses that are hosted by
donors. The collection and processing of blood
exposes soluble proteins and cells to artificial surfaces. Storage not only leads to apoptotic cells, but

INTRODUCTION
Some 60 years ago, speeded up by wars, banking of
donor blood started. Gradually, whole blood was
replaced by more or less purified components. It is
A.Brand MD, PhD, FRC Path, Sanquin Blood Supply
Foundation, Bloodbank Leiden-Haaglanden, P.O. Box 2184, 2301
CD Leiden, The Netherlands. Tel.: +31 71 526 38 30; Fax: +31 72
524 82 52; E-mail: abrand@sanquinbblh.nl

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Immunological aspects of blood transfusions

viable cells produce biologically active factors and

chemicals derived from the container accumulate.
Pooled blood plasma from more than 12 donors must
be treated as a pharmaceutical product and measurements to inactivate the lipid-enveloped viruses HIV,
HTLV, HBV and HCV are mandatory for such derivatives. Virus and bacterial inactivation of single
donor cellular components may become possible in
the next few years.

TRANSFUSION-INDUCED IMMUNITY
When a laboratory animal receives two antigenic stimuli simultaneously, the response against one of the
two is generally decreased. Blood transfusions introduce a multitude of foreign antigens and indeed
immunization and immunosuppression are the consequences.
Almost all acute and/or severe immunological
transfusion reactions are caused by alloantibodies.
The clinical consequences of transfusion-induced
changes in cellular immunity are virtually unknown.
When obvious, such as the improvement of outcome
of organ transplantation after pre-transplantation
blood transfusion, in vitro assays to explain or monitor this effect are lacking. In this review immunodominant alloantigens relevant for immunization and
silent transfusion side-effects, and their possible relevance for immune suppression, will be discussed.

MAJOR HISTOCOMPATIBILITY (MHC)

ANTIGENS
The immune response towards alloantigens needs processing and presentation by the recipients antigen
presenting cells (APCs). An exception is donor cells
bearing major histocompatibility complex (MHC)
antigens, which can directly stimulate recipient cells.
In the circulating blood there is a large amount of
HLA Class I antigens. About 70% is present on
platelets, followed by 1520% as soluble HLA in
plasma, while red cells and leukocytes express comparable amounts.1 Soluble HLA-Class I and II antigens
are present as shed intact antigens but also as cleaved
heavy chains and smaller fragments. Part of the HLA
on platelets and red cells is adsorbed from the plasma.
The HLA-class I antigens (HLA-A, B and C) are,
with a variable degree of expression, present on all
cells, bound by a membrane-anchored heavy (44 kd)
chain, which has three extracellular domains. The
3 domain binds the 12 KD non-polymorphic 2microglobulin (B2m) light chain. Polymorphism
within the MHC is caused by unique amino-acid

131

sequences in the groove constructed by the 1 and 2

domains. In this groove peptides of 811 amino acids
are bound at both ends. Most of these peptides are
derived from endogenous proteins and can be unique
for that particular cell or for viruses infecting the cell.
The peptide/Class I complex is recognized by CD8+
(cytotoxic) T cells.
HLA-Class II molecules have two anchored chains
which pass through the cell membrane (the 33 kd
chain and the 28 kd chain), and each form two
extracellular domains: 1 and 2 and 1 and 2.
The most external 1 and 1 domains form a groove
open at both ends and which binds peptides of 1228
amino acids.
HLA class II antigens are constitutively expressed
on APCs, such as dendritic cells and monocytes, but
also on non-antigen presenting responder cells, B lymphocytes and activated T cells. APCs take up (exogenous) antigen, degrade it and bring peptides to the cell
surface in the groove shaped by their class II antigens.
CD4+ helper T cells recognize this MHC class II/peptide complex through their T cell receptor (TCR). The
initial contact between T cells and APCs, often
referred to as signal 1, is facilitated by adhesion
molecules and their ligands. Activated T cells need
secondary, co-stimulatory, signals to proceed with
proliferation and differentiation. If not received, these
T cells die, which can result in anergy towards the
antigenic stimulus.
In contrast to this indirect antigen presentation,
allogeneic APCs (dendritic cells and activated monocytes) can directly activate recipient T cells. Because at
least 100 times more T cells have receptors for alloMHC antigens, for HLA immunization this direct
pathway is far more efficient than the indirect pathway.
Both indirect and direct T cell activation need second signals provided by interaction between co-stimulatory molecules on both cells. Co-stimulatory
molecules on APCs are the CD40 and B-7 molecules
(CD80 and CD86). Their ligands on T cells and activated B cells are CD40L (CD 154) and CD28 respectively. Dendritic cells can activate naive T cells as they
carry sufficient adhesion and co-stimulatory molecules, together with a very high expression of HLAclass II antigens. Monocytes need activation for
effective antigen presentation.
After antigen recognition in the presence of second
signals, T cells differentiate, to the Th1 or Th2 subset
dependent on the type of intracellular antigen processing. Other T cell differentiation pathways, such as
those which lead to regulatory cells, are currently
being unravelled.
Cytokines associated with the induction of Th-1
cells are interleukin (IL)-12 and IFN-. Th1 cytokines

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activate macrophages and stimulate B cells to produce

potent antibodies. For the Th2 pathway IL-4,5 and 10
are characteristic cytokines. Type Th2 activated B
cells produce IgE and non-complement fixing IgG
antibodies.
The role of soluble HLA antigens is more likely to
be immunosuppressive than stimulatory. In-vitro,
sHLA antigens compete with their ligand expressed
on responder cells and downregulate proliferative
CD4 and cytotoxic CD8 responses.2

MHC ANTIGENS AND BLOOD

TRANSFUSIONS
Pregnancy, blood transfusion and transplantation
introduce allogeneic APCs, which can directly activate
recipient responder cells. A difference between blood
transfusions and pregnancy/transplantation model is
that transfusions contain processed blood with, dependent on storage condition and interval, a variable quality
of APCs. The induction of HLA antibodies, indicative
of a Th1 type immune response, is the result of donor,
patient and product characteristics (Table 1).

Number of white blo\od cells

In inbred mice, approximately 104105 purified white
blood cells (WBCs) are needed for lymphocytotoxic
antibody formation. In the presence of platelets, which

carry abundant HLA-class I antigens, much lower

numbers of 102103 WBC can evoke antibodies. Purified
platelets alone are not immunogenic.3,4 Extrapolating
mice to men this would mean that 106 107 WBC in the
presence of platelets induce HLA-antibodies.
HLA-immunization by platelet transfusions has been
studied extensively. Based on 33 studies, published
between 1973 and 1992, involving almost 3000 haematooncological patients, the HLA-immunization rate was
39%, with a range between 21% and 71%.5 The clinical
observation that leukoreduced platelet concentrates
cause less platelet transfusion refractoriness indicates
that donor APCs are the major inducers of an immunological cascade leading to HLA class I antibodies.6 A
reduction of HLA immunization by more than 70%
after leukoreduced platelet concentrates was found in
three meta-analyses of partly overlapping controlled
studies.5,7,8 In a large prospective randomized study, the
TRAP study (Trial to Reduce Alloimmunization by
Platelets), the incidence of HLA immunization by four
different platelet products was compared in 530 evaluable patients. Forty-five per cent of the patients receiving
standard non-leukoreduced platelet transfusions developed lymphocytotoxic antibodies compared to 17% of
the patients after leukoreduced platelet transfusions.9 A
similar lower HLA-alloimmunization rate was observed
with platelets treated with UVB-light, inactivating costimulatory molecules. There was no reduced immunogenicity of apheresis platelets from single donors.
The threshold dose of WBC in platelet transfusions is well defined. In a study to evaluate an

Table 1 Factors determining transfusion-induced HLA antibodies

Properties of APCs

Characteristics for immunization

Weakly immunogenic or
immunosuppressive

Number of WBC

> 5.106 in platelet concentrates

>108 in RBC?

<5.106 WBC in 3.1011 platelets

Nature of WBC

Dendritic cells/monocytes

Granulocytes, B cells, platelets

Viability of WBC

Viable WBCs are necessary for

immunization by direct presentation

Non-viable WBC, membranes, elicit

after repeated stimulation weak
antibodies by indirect presentation

Storage of product

WBC must be stored <72 h at 20C

and <13 days at 4C

Beyond this intervals loss of

co-stimulatory capacity
and release of downregulatory sHLA and FasLigand

HLA (class II) differences

between donor and recipient

Two class II mismatches evoke more

frequent and broader HLA-antibodies

Sharing of one HLA class II antigen

reduces formation of antibodies

Immunocompetent recipient

Sensitized patients with previous

pregnancy produce antibodies
upon lower numbers APC
*Lymphocytic leukemia patients may be
lower responders

WBC: White blood cells.

Immunological aspects of blood transfusions

immunosuppressive role of platelets in prospective

kidney transplant patients, Fisher et al. compared two
groups of 12 patients. After three platelet transfusions
with less than 5.106 WBC none of 12 patients became
immunized. Five of 12 patients developed HLAantibodies after platelet transfusions with 15.106
WBC. Three of these 5 patients experienced a primary
immunization in the absence of a transfusion/pregnancy history.10 Comparing two leukoreduction techniques for platelet transfusions, Marwijk Kooy et al.
observed an 8% incidence of HLA antibodies by
products with <50.106 WBC compared to 59% antibody responders by leukoreduced platelet products
still contaminated with >50.106 WBC.11
The immunogenicity of WBC in whole blood or
red cell concentrates is less extensively studied. In
human volunteers administration of 25 ml fresh
whole blood (12. 108 WBC) causes accelerated
rejection of a subsequent skin graft from this donor.
Repeated injections of 20 ml of whole blood for 1684
weeks evoked stable HLA-antibodies in 80% of 56
mismatched donor-recipient combinations.12
After one to three transfusions from prospective
live kidney donors, 1045% of dialysis patients
develop HLA-antibodies.13,14 Immunogenic threshold
numbers of WBC in red blood cell concentrates are
not known. Buffy-coat removal results in ill defined
products with WBC, ranging in number between 2.108
to 1,2.109. The technique of buffy-coat depletion
determines the composition of residual WBC varying
from mainly granulocytes to predominantly mononuclear cells.15 A few studies evaluated HLA antibodies
after buffy-coat depleted RBC and observed a low
(<15%) incidence of antibody responders.16
Nature and viability of WBC
For in vitro stimulation of allogenic T cells, viable APCs
are needed. Cells fixed with paraformaldehyde, cell fragments, purified membranes and soluble antigens do not
stimulate allogenic lymphocytes in vitro and hardly elicit
by indirect stimulationa lymphocytotoxic antibody
response in animals. Allogeneic B cells do not activate
T cells.17 More likely, B cells are immunosuppressive as
they lack co-stimulatory molecules. Experiments in
mice, evaluating the nature of immunogenic WBC in
purified platelets, suggested that removal of B cells
enhanced lymphocytotoxic antibody formation and the
adding-back of B cells decreased immunity.18
Storage of WBC
During storage at 2024C, such as is routine for
platelet concentrates, class II antigen expression
decreases on mononuclear cells and the stimulatory

133

potential is lost after 3 days.19 At 26C, such as in

stored whole blood or RBCs, the co-stimulatory activity of mononuclear cells is abolished after 12 days.17
Responder T cell functions are much more resistant
and alloproliferative T cells are present in red cell
components at the end of shelf-life. During storage,
cytokines are produced by leukocytes and, in particular, granulocytes release soluble class I antigens and
Fas-ligand, both of which downregulate in-vitro CD4
alloproliferative T cells and cytotoxic CD8 cells.20 The
reduced incidence of HLA immunization by stored
red cell and platelet transfusions may thus be the
result of lack of co-stimulatory molecules and immune suppression by soluble factors.
HLA-(in)compatibility between donor and recipient
HLA class II differences between donor and recipient
determine stimulation of the Th1 response. When cells
from two individuals, which share an HLA haplotype or
one HLA-DR antigen, are co-cultured in vitro, more
IL-10 favoring a Th2 type response is produced
compared to cultures between completely HLA-mismatched individuals.21 Lagaay et al. evaluated the clinical effect of HLA-DR sharing on the development of
lymphocytotoxic antibodies. Seven out of 10 dialysis
patients formed strong HLA-antibodies after 1 to 3
RBC transfusions containing (viable) leukocytes from
completely HLA-DR mismatched donors. In comparison, only three out of 10 weak antibody responders
were observed after a similar number of transfusions
from donors sharing one HLA-DR antigen.22
Immunecompetence of the recipient
Patients with previous pregnancies or prior transfusions
produce HLA antibodies more often than immunologically virgin patients.9,23,24 In certain patients leukoreduced blood still provides sufficient stimulatory capacity
to activate (memory) antibody responses.
The ability to form HLA-antibodies may furthermore be influenced by disease and treatment. Patients
with acute lymphoblastic leukemia (ALL) develop
HLA antibodies less often than patients with AML.
The highest antibody responder rates have been
observed in aplastic anemia patients. It is unknown
whether diagnosis, treatment, or both causes leads to
reduced immune responses.25
TRANSFUSION-INDUCED HLA AND
NON-HLA ANTIBODIES: TRANSFUSION
REACTIONS
Transfusion induced immunity often does not cause
clinical symptoms, but reactions sometimes occur and

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Blood Reviews

are almost always the result of antibodies. Such transfusion reactions are classified into three groups: febrile
non-hemolytic, hemolytic and allergic (see Table 2). This
classification, however, is based on a mixture of cause
and effect and is often not very helpful at the bedside.
Febrile non-hemolytic transfusion reactions to whole
blood or RBC transfusions
It is unknown whether febrile non-hemolytic transfusion reactions (FNHTR) cause long-term damage,
but people feel unwell and diagnostic tests must
exclude infections and hemolytic reactions. The incidence and severity of FNHTR is dose dependent
associated with the number of leukocytes in blood
products, either in the transfusate or during storage.
Before 1970 febrile reactions were reported in >16%
per unit transfused.26 After 1970 using less fresh whole
blood this declined to between 0.210.73% per unit.27
FNHTR upon transfusion of buffy-coat depleted
RBC is estimated to occur in 0.1%/unit.28 Sera from
patients with FNHTR show in 25100% either HLAantibodies only or mixtures of HLA, platelet- and
granulocyte reactive antibodies. Unfortunately, differ-

ent criteria for FNHTR have been used in the literature, but after exclusion of patients with infections
the percentage of leukocyte antibodies as the explanation for FNHTR increased to >75%.29 Reactions due
to pro-inflammatory cytokines produced in stored
blood cause less prominent fever and often chills are
the only symptom.
FNHTR and platelet transfusion refractoriness
Antibodies account for less than 50% of platelet
transfusion failures and a shortened platelet life-span
is more often the result of infection, malignancy or
endothelial damage.30 HLA, ABO and antibodies
against platelet specific (HPA) antigens can destroy
transfused random donor platelets.
Despite a strict leuko-reduced transfusion policy,
HLA-antibodies are still the major cause of immunological platelet refractoriness. Not all patients with
HLA-antibodies are refractory. Lack of transfusion
increment is often restricted to patients with multispecific antibodies reactive with > 60% of the donor population.23 Moreover, HLA-antibodies can be transient

Table 2 Signs and symptoms of frequent acute/severe immunological transfusion reactions

Classification

Symptoms

Cause and mechanism

Non-hemolytic
febrile reactions

Fever >1.5C or chills within 4 h

after RBC or platelet
transfusion, sometimes associated
with dyspnoea.
Fever, chills, agitation, chest and
low back pain, hypotension

*HLA/HGA antibodies destroying donor leukocytes

leading to pro-inflammatory cytokines
*Passively transfused cytokines, produced during
storage of leukocyte-containing blood components
*Antibodies against RBC antigens induce TNF-,
activate complement and release free hemoglobin and
reactive oxygen radicals
*IgE antibodies against plasma proteins/haptens cause
histamine release from mast cells
*Antibodies against IgA in IgA-deficient individuals
*Antibodies against IgG allotypes in polytransfused
patients
*Antibodies against Chido/Rodgers, soluble plasmaantigens also expressed on red cells
*Contact activation through negatively charged filters
induce bradykinins, which have shown catabolism in
patients on ACE-inhibitors
*Donor leukocyte antibodies cause pulmonary
leukostasis and local complement activation

Acute and delayed

hemolytic reactions
Allergic reactions

Quinkes edema, urticaria

Erythroderma
Hypotension, bronchospasm

TRALI

Dyspnoea, cyanosis, pulmonary

infiltrates within 6 h after
transfusion
Non-cardiogenic lung-oedema up
to 2 days after (multiple)
transfusions
Bleeding

ARDS

Unclassified

*Micro-aggregates and bioreactive lipids in stored

blood
*Hemolytic reactions leading to DIC
*Post-Transfusion Purpura

HGA: human granulocyte antigens; TRALI: Transfusion related acute lung injury; RBC: red blood cell antigens; ARDS: Acute respiratory
distress syndrome; ACE: angiotensin converting enzyme; DIC: diffuse intravascular coagulation.
Fever is caused by pro-inflammatory cytokines, of which pyrogens such as TNF- act upon the temperature center in the hypothalamus.
All types of transfusion reactions can cause fever, but most often fever and chills result from destruction of donor leukocytes by recipient
antibodies or from passively administered cytokines, produced in vitro during storage of blood containing leukocytes, in particular in
platelet concentrates.

Immunological aspects of blood transfusions

and disappear in 1050% of the patients, despite continuation of transfusions.

ABO-antibodies reduce the platelet survival, but in
practice this is only relevant if they limit the number
of available HLA-matched donors.23,31
Platelet-specific (HPA) antigens need processing by
recipient APCs. The clinically most important, antibodies HPA-1a and-5b, show strong, but not absolute,
MHC-restrictions.32 After repeated platelet transfusions, HPA-antibodies are found in <10% of the
patients,9,23 but in high responder patients with multispecific HLA-antibodies an incidence over 20% was
observed.33 For patients with a combination of HLA,
HPA and ABO-antibodies great difficulties arise in
finding compatible donors.
Destruction of transfused platelets is not usually
accompanied by clinical symptoms. Contaminating
leukocytes or cytokines produced by leukocytes during storage are more often responsible for FNHTR.
This is exemplified by the following studies.
Muylle et al. transfused 570 platelet concentrates,
containing 2108 leukocytes per unit to 74 patients.
Concentrates stored for longer than 3 days caused
FNHTR in 17.6% of the recipients in contrast to 8.7%
reactions when transfusions had been stored for less
than 3 days.34 Anderson35 found 17% febrile reactions in
13 patients after 117 platelet transfusions contaminated
with 4108 WBC. In comparison, 158 transfusions to
17 patients caused 3.8% reactions if platelets contained
less than 107 WBC. Further reduction of WBC below
106 did not result in less FNHTR. Heddle36 separated, at
the end of their shelf-life, platelets from the supernatant
plasma and showed that the cytokine containing
plasma and not the platelets were the major inducers
of FNHTR. Indirect further evidence that cytokines
produced by leukocytes during storage caused FNHTR
is given by Williamson who observed no difference
in FNHTR in a randomized study comparing standard
non-leukoreduced and leukoreduced platelets, which
after storage were filtered at the bedside.37
Hemolytic transfusion reactions
Acute hemolytic transfusion reactions (HTR) cause
agitation, low back and substernal pain as well as
fever, chills, hypotension and dyspnoea. Antibody
mediated haemolysis causes a massive cytokine storm
with complement activation, neopterin production,
and may lead to DIC.38,39 The incidence of antibodies
other than ABO-RhD is dependent on the number of
transfusion episodes rather than on the number of
transfusions. After one transfusion episode, RBCantibodies are, depending on the technique used,
detected in 1/50 to 1/700 RBC units.40,41 The titer is
maximal after 3 weeks and often decreases rapidly

135

to undetectable levels.41 Two to 15 days after a

subsequent transfusion, acting as booster, a delayed
hemolytic reaction can be very severe. In order to prevent these delayed hemolytic transfusion reactions
life-long matching is indicated for patients who ever
developed potentially dangerous irregular red cell
antibodies.
The occurrence of irregular antibodies increases
to 10% of the patients after 1020 transfusion
episodes42,43 and reaches a plateau to over 30% in
recipients of chronic transfusion regimens such as in
hemoglobinopathy.44
Immunodominant red cell antigens are Rh and
Kell, followed by Duffy and Kidd antigens. The preventive use of Rh and K matched donors for patients
dependent on chronic red cell substitution is an effective prophylaxis for the prevention of broad red cell
alloimmunization.44
The patients immune status plays a role in red cell
allo-antibody formation. Neonates rarely develop red
cell antibodies and neonatal transfusions even seem to
tolerize for red cell antigens. Neonatally transfused
thalassaemia patients develop red cell antibodies 50%
less often compared to patients who start transfusion
therapy later in life.44
Patients with lymphocytic malignancy also produce RBC-antibodies less often compared to patients
with other hematological malignancies or myelodysplastic syndromes.43 High responders are Rh-D immunized women.45 Incidently, HLA-antibodies can cause
hemolysis by destruction of erythrocytes strongly
expressing HLA-antigens.46
Allergic reactions
Allergic reactions with erythema, urticaria and sometimes bronchospasm, suggestive for IgE antibodies and
mast cell activation, are common and estimated to occur
in 13% of all transfusions. These reactions can show
upon the first transfusion and are not related to donor
allergy but rather to allergic diathesis of the patient.47
Severe hypotension with or without respiratory
distress can result from bradykinins. These can be
passively administered, for instance after contact-activation of plasma on negatively charged bedside white
cell reduction filters. ACE-inhibiting drugs slow the
inactivation of bradykinins and predispose patients to
these reactions.48 For severe allergic reactions with
hypotension with or without respiratory symptoms
often no explanation can be found. Antibodies to class
specific IgA in IgA-deficient individuals (1: 800 of the
population) explain only a minority of severe allergic
reactions. Evaluation of 359 sera referred to the
American Red Cross Laboratory because of severe
allergic reactions, suspected to be due to anti-IgA

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antibodies, revealed such antibodies in only 17.5% of

the cases.49
Chido (Ch) and Rodger (Rg) blood groups are
located on the complement factor C4d, and are passively adsorbed from the plasma onto red cells.
Antibodies do not cause hemolysis but can cause
severe hypotensive plasma reactions.50,51 Anti-IgA and
Ch/Rg antibodies are of the IgG-class. Also, IgE
antibodies against undefined proteins have been
demonstrated in severe allergic reactions to platelet
transfusions.52
After multiple transfusions, more than 40% of the
patients produce antibodies against various plasma
proteins. Upon subsequent transfusions these antibodies can form immune-complexes (with albumin,
complement-components or fibrinogen). The clinical
consequences of such immune-complexes have not
been extensively studied but were associated with
reduced survival of transfused platelets by platelet Fcreceptor adherence.53
Because often no explanation can be found for
(severe) allergic reactions, empirical washing of cellular blood components is used to prevent them
Transfusion-related lung injury and respiratory distress
Transfusion-related lung injury (TRALI), a non-cardiogenic lung edema is, after hemolytic reactions, the
second most common of severe acute transfusion
complications.54 The estimated incidence is 1:5000 and
the mortality 515%. In over 90% of the published
cases, donor derived IgG antibodies capable of leukoagglutination or cytotoxic activity against HLA
and/or granulocyte/monocyte antigens of the recipient have been identified. In lethal cases leukostasis in
the pulmonary circulation was found. Dyspneoa
occurs 15 min < 6 h after products, which may contain as few as 30 ml plasma. Interdonor incompatibility in pooled platelet products has also been identified
as a cause of TRALI.55
Approximately 30% of female blood donors who
have had multiple pregnancies, have leuko-agglutinating and/or cytotoxic antibodies in their plasma.
Considering this percentage, TRALI should occur
more often. It has been postulated that susceptibility
for TRALI requires at least two factors. Besides antibodies in donor plasma, critical illness and/or additional transfusions which activate granulocytes, by
micro-aggregates or granulocyte activating lipids
which are both present in stored blood, can act as cofactors.54,56
Another type of non-cardiogenic lung edema may
be difficult to distinguish from TRALI, but tends to
have a longer interval from transfusion to onset of up
to 3 days. This type cannot be distinguished from acute

respiratory distress syndrome associated with sepsis,

micro-emboli induced by multiple trauma or as the presenting sign for multi-organ failure after major surgery.
Due to these multifactorial circumstances a causal relationship with micro-aggregates in transfused blood was
extensively debated but no consensus of opinion was
reached.57 As a final possibility, volume overload may
contribute to post transfusion pulmonary symptoms.
Bleeding symptoms
A rare, but serious, transfusion complication caused
by HPA-antibodies is post-transfusion purpura,
occurring almost exclusively in older females with previous pregnancies. Thrombocytopenia and bleeding
presents 715 days after transfusion of whole blood,
RBC or platelets. The HPA antibodies directed
against the donor transiently destroy autologous
platelets, possibly due to passive adsorption of HPA1a antigen or of an antigen-antibody complex onto
the patients platelets. In one case report a fatal thrombocytopenia was described after transfusion of
plasma from a donor with HPA-1a antibodies.
In patients with marginal platelet counts, such as in
myelodysplasia, micro-aggregates in transfused blood
have been shown to be a cause for transient platelet
sequestration, preventable by the use of leukoreduced
transfusions.58

CELLULAR IMMUNITY
Cellular changes after blood transfusions rarely cause
symptoms and are only detected if they are sought. In
man, two types of studies are available: in vitro evaluation of post transfusion cellular immunity; and
clinical epidemiological inventories. Unfortunately,
a relation between specific changes in cellular immunity in vitro and clinical sequelae has yet to be
established.
Mixed lymphocyte reactivity
The fate of donor lymphocytes after transfusion is not
well-known. Three to 5 days after transfusion atypical
lymphoid blast cells, incorporating H3-Thymidine for
DNA-synthesis, appear in the circulation of the recipient.60 Fresh whole blood and packed RBC, but not
frozen-thawed leukocyte-depleted RBC, cause such
atypical lymphocytosis. The occurrence of lymphocytic blasts was found to be associated with the subsequent production of lymphocytotoxic antibodies.61 It
is likely that this blast-formation represents an in vivo
mixed lymphocyte reaction as recipient and donor
cells proliferate.62

Immunological aspects of blood transfusions

Chimerism
5

Using sensitive techniques detecting less than 1/10

cells, prolonged persistence of allogeneic cells, often
referred to as chimerism, is found after pregnancy and
organ transplantation. After a neonatal exchange
transfusion donor cell concentrations up to 50% of
the white blood cells can be detected for several
years.63 When investigated by genomic amplification
of the male Y chromosome-DNA or probes against
HLA-DRB1, it was observed that the majority of
transfused nucleated donor cells rapidly disappear
from the circulation within days but that minor populations can persist for many weeks, in particular after
transfusion from donors sharing an (HLA-B/DR)
haplotype with the recipient.6466 Female trauma
patients transfused with multiple units of blood
showed male DNA within the B, T, NK and myeloid
cells even after 1.5 years.67 It is speculated that these
cells are derived from viable hematopoietic progenitor
cells. The immunological and clinical consequences of
chimerism are unknown. An association with immunological tolerance, but also with chronic antigenic
stimulation relevant for the development of autoimmune diseases in females in relation to postpregnancy microchimerism, have both been postulated.68
Transfusion-associated graft versus host disease
Transfusion-associated graft versus host disease (TAGVHD) is caused by immune competent donor T
cells and is, in contrast to bone-marrow transplantation associated GVHD, almost invariably lethal.
Delayed diagnosis and pancytopenia, because the
hematopoietic cells are targets for GVHD effector
cells, contribute to the high mortality.
TA-GVHD occurs in the immunocompromised
and is rare in immunocompetent patients, except in
Japan. Guidelines for prevention of TA-GVHD by
irradiation of cellular blood products with at least 25
Gy are widely available. Risk factors for TA-GVHD
in presumed immunocompetent patients are HLAhomozygous donors, haplo-identical HLA-type with
the patient, the use of relatives as donors and transfusion of fresh blood containing viable lymphocytes.
Homozygosity for HLA occurs in 2% of the population and the estimated chance of transfusion from a
haplo-identical donor to a patient is 1:800, a small
fraction of these being associated with GVHD.73 This
suggests a protective mechanism against TA-GVHD.
Indeed it has been shown that transfusions induce T
cells in the host able to down regulate donor cell proliferation against the recipient HLA type. This may
explain why TA-GVHD is a rare event while persistence of donor cells seems quite common.74 In line
with this hypothesis is the observation that among

137

1000 bone marrow transplant patients the frequency

of GVHD was significantly reduced in recently transfused compared to non-transfused patients.75
As few as 8 104 stored leukocytes/kg body
weight of the recipient can cause GVHD.76 Probably
due to technical failures, TA-GVHD has been occasionally observed after irradiated blood. In this
respect it may be relevant that after irradiation with
20 Gy, 3 out of 65 donors still showed a mixed lymphocyte reactivity of 1014%, compared to their
non-irradiated cells.77

CLINICAL IMPLICATIONS OF TRANSFUSIONINDUCED ALTERED CELLULAR IMMUNITY

Several comprehensive reviews appeared on transfusion-induced immune modulation in animal models
and clinical settings. A summary of clinical studies
will be given.
Allograft tolerance
Transplantation tolerance is a clinical phenomenon
that was revealed by multivariate analysis of factors
contributing to graft rejection. Two types of blood
products, presumably acting by different mechanisms,
have been shown to be associated with allograft tolerance. Improved kidney graft survival is demonstrated
after multiple transfusions from random donors,
dose-dependent with the number of transfusions
received.70 Immune suppression by stored random
transfusions can be the result of lack of co-stimulatory signals on leukocytes and/or high concentrates of
soluble HLA and Fas Ligand.71
Improved graft survival was also shown after a single transfusion with non stored blood.69 Later studies
showed that this effect is produced by viable leukocytes from a donor sharing one HLA-DR and who is
mismatched for the other haplotype.21,22 The major
drawback of pre-transplantion blood transfusions is
HLA alloimmunization. After multiple transfusions
up to 60% of the patients develop antibodies. In comparison, after a single transfusion from a donor sharing an HLA-DR antigen only about 10% of the
recipients become immunized. HLA antibodies on
the other hand may be responsible for (part of) the
improved transplantation outcome by selection of a
crossmatch negative graft.
The use of pre-transplantation blood transfusions is
no longer applied on a large scale since potent
immunosuppressive drugs improved kidney graft survival to >80%. However, chronic immunological rejection still occurs in 50% of kidney transplant recipients
and profound immunosuppression is associated with

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Blood Reviews

an increased incidence of post-transplant cancers. In a

retrospective study in cardiac surgery patients, none of
45 recipients of HLA-DR matched transfusions, who
received less rejection treatment, developed cancer
compared to 6/55 patients transfused with HLA-mismatched blood.72 In transplantation settings with a less
favourable outcome such as pancreas or heart transplantation, conditioning transfusions may still be an
attractive immunomodulatory approach and relatively
safe compared to drug induced immune suppression.

surgery and precise location of the tumour. These

factors are generally not equally distributed between
transfused and non-transfused colorectal cancer
patients and transfusion may act as a surrogate
marker for these prognostic signs. Three randomized trials,8486 all from Europe, found no differences
in cancer recurrence between standard (buffy coat
depleted in all three studies) red cell transfusions
and alternative products. These alternative products
were autologous red cells in two studies84,85 and
leukoreduced RBC in one study.86 From a meta
analysis of these three prospective studies it was
concluded that if a deleterious effect is present it is
smaller than a 1.3 increase in relative risk of standard buffy coat depleted RBC compared to autologous or leukoreduced RBC.83

Cancer immunosurveillance
The beneficial effect of blood transfusions on kidney
graft survival resulted in the classical question raised
by Gantt in 1981:
is it possible that patients with malignant tumours who
receive transfusions of whole blood are suppressed to the
point where the malignant tumour has a better chance to
survive?78

Other types of cancer

Meta-analysis of univariate results of the studies are
shown in Fig. 1 and Table 3.87 When not corrected for
confounding factors, such as in Fig. 1, there is a significant association between cancer recurrence and
blood transfusions in all cancers except in cervical
carcinoma. After correction for potential confounding factors by multivariate regression analysis the
deleterious role of blood transfusions on cancer prognosis is less obvious (Table 3).
In head and neck cancer the majority of the patients
were treated between 1955 and 1990 and had received
whole blood or red cells containing buffy-coat. Ten
studies performed multivariate regression analysis. In
four of these 10 studies, transfusion was significantly
associated with worse cancer prognosis. A deleterious
role of blood transfusions on head and neck cancer
recurrence can thus not be excluded.
The role of blood transfusions on breast cancer
was evaluated in 10 retrospective studies, all applying

Since this question was asked, hundreds of publications, mainly retrospective, reviews and meta-analyses about this subject have been published. Table 3
summarizes all studies up to 1999 comparing transfused and non-transfused patients for an adverse
effect on cancer outcome. Most studies, including the
only three prospective randomized studies, evaluated
outcome of colon cancer.
Colorectal cancer
Five meta-analyses7983 concluded that blood transfusions were significantly associated with decreased colorectal cancer prognosis with an increased relative
risk of cancer recurrence between 1.3382 and 1.8.83
The authors, however, could not correct for potential confounding factors, such as complexity of
Table 3 Association between blood transfusions and cancer prognosis
Tumor type

Colorectal
Head/neck
Breast
Gastric cancer
Lung
Cervix
Prostate
Total

Significant negative association

Number of studies (patients)

Lack of association
Number of studies (patients)

Univariate

Multivariate*

Univariate

2 (920)
3 (301)

5 (1546)
4 (511)
3 (1662)**
2 (329)
5 (898)

7 (1541)
1 (240)

2 (449)
1 (168)
8 (1938)

2 (335)
21 (5281)

1 (283)
5 (734)***
14 (2798)

Multivariate*
13 (6985)
6 (920)
7 (3742)
4 (2198)
2 (447)
1 (302)
3 (2203)
36 (16.797)

* If univariate and multivariate analyses were performed, only the multivariate analysis is shown.
** In two of the three studies significance was only present in subgroups.
*** In a small subgroup of 42 patients there was a deleterious transfusion effect.

Total number of
studies (patients)

27 (10.992)
14 (1972)
10 (5405)
8 (2976)
8 (1658)
7 (1162)
5 (2538)
69 (26.703)

Immunological aspects of blood transfusions

multivariate analysis. In nine studies, patients were

treated before 1980 and the majority of the patients
were in more advanced stage II cancer. A deleterious
transfusion effect, if present, is very small and this
possible risk must be weighed against indicated transfusions.
In gastric cancer the indication for blood transfusions is highly associated with cancer stage and size of
the tumor. In six investigations multivariate analysis
was performed, and in only two studies, representing a
minority of the patients, transfusion remained an
independent risk factor for cancer recurrence. In one
of these publications, a significant deleterious effect of
blood transfusions was only present in patients with
occult bone marrow metastasis present at surgery.
In lung cancer, five of seven studies that applied
multivariate analysis found a small but significant
negative effect of blood transfusions on cancer recurrence, suggesting that a deleterious effect of transfusions may indeed exist
In cervix carcinoma a role for blood transfusions on
cancer recurrence is not suggested. This is interesting
because cervical carcinoma is a papilloma virus
(HPV) associated tumor and relevant HPV proteins
have been shown to elicit a cytotoxic T cell response
that may be susceptible for the putative immunosuppressive transfusion effects.
In prostate cancer, two of five studies found a negative transfusion effect, but mainly on non-cancer
death. In one of the retrospective studies there was no
difference in outcome when autologous blood instead
of allogeneic transfusions had been administered.
In major cancer surgery, such as in renal cell cancer,88,89 pancreatic head cancer,90 esophageal cancer91
and liver carcinoma,92 one or two retrospective studies
for each indication have been published. In none of
these was transfusion associated with recurrence, but
massive blood transfusions were a significant prognostic factor for postoperative complications leading
to increased mortality.
In contrast to presumed deleterious immunosuppressive effects of transfusions on solid tumors, in
acute leukemia, the opposite, a beneficial transfusion
effect, was presumed. Ford observed a possible prolongation of remission in AML patients who received
granulocyte transfusions.93 Tucker et al. reported in
1994 that 23 AML patients who had received leukoreduced transfusions presented with an increased
relapse rate of 56% compared to 13% in patients after
standard non-leukoreduced blood.94 Four subsequent
retrospective analysis could not confirm this observation.9598 In contrast, Oksanen et al.98 found a lower
relapse rate after leukoreduced transfusions, but this
difference disappeared when HLA-alloimmunized
patients, refractory for random platelet transfusions,

139

were excluded. HLA-immunized patients were assumed to have received less optimal chemotherapy.
The initial reports of Ford and Tucker differed from
the other studies by the huge amounts of leukocytes,
either given with the granulocyte-transfusions or in
the platelet transfusions. It is therefore possible that
large doses of allogeneic leukocytes contribute to
graft versus leukemia.
Prior history of blood transfusion
The putative role of blood transfusion as a risk factor
for enhanced cancer growth, or for the transmission
of oncogenic viruses, and so by these mechanisms
leading to cancer, has been addressed in six observational studies.99104 In particular the relation between
non-Hodgkin lymphoma (NHL) and previous transfusions was studied extensively.
Greenwald et al.99 followed, for an average period
of 7 years, 105 recipients of blood from donors who,
after donation, developed haemato-oncological cancers. None of the recipients developed cancer. This is
despite the fact that it was recently shown that inadvertently transfused BCR-ABL positive chronic
myeloid leukaemia (CML) cells were not rejected
immediately and survived for many months, until the
recipient died of an unrelated disease. However, such
recipients did not develop CML.
In an early Swedish study,100 3177 individuals
transfused between 19811982 were compared with
29,910 hospital patients. Both groups were followed
for 39 years and compared with data from the
regional cancer registry. There were 134 cancer diagnoses in the transfused group observed versus 113
expected, the increase being caused by renal cancer,
squamous skin cancer and NHL.
In Iowa, USA, the transfusion history was
obtained from 37,337 women aged between 55 and 59
years of age. During a 5 year follow-up period
an increased incidence of lymphoma (RR 2.2;CI
1.355.38) and renal cancer (RR 2.53;CI 1.344.78)
was observed in the transfused group.101 From
England, Wales and Scotland 21,329 babies transfused perinatally between 19421970 were evaluated
for cancer in 1992 and compared with figures from the
national cancer registers. Transfused patients had no
increased incidence of cancer except four cases of
NHL instead of 1.85 expected cases, which was a nonsignificant increase.102
A second Swedish study was based on questioning
all newly diagnosed NHL patients about a history of
blood transfusions of whom 298 (70%) responded.
The odds ratio for having received a blood transfusion
was 1.74(CI 1.242.44), with the highest association in
patients transfused 615 years before diagnosis.103

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Blood Reviews

The pattern of malignancies observed in these four

studies suggest an immunosuppressive role of blood
transfusions, leading to similar types of tumors as
observed in transplant patients and in patients with
congenital or acquired immunodeficiency.
Surprisingly a third, nationwide, Swedish study104
found the risk for developing lymphoma after transfusions to be 0.93 (CI 0.711.23). The authors explain
the discrepancy with previous studies because buffycoat was routinely removed since the mid-1970s and
the older studies included more patients transfused
with whole blood or RBC with buffy-coat.
A small increase of malignancies after transfusions
containing allogeneic leukocytes cannot be excluded,
and the type of these cancers show a striking similarity with tumors arising in immunosuppressed patients.

Blood transfusions and susceptibility for postoperative

infections
In many observational studies in hip replacement
surgery, trauma, abdominal and coronary bypass
surgery, the receipt of allogeneic blood transfusions
was found to be associated with a higher incidence of
postoperative infections compared to non-transfused
patients Even though in many of these studies multivariate analysis could exclude identified confounders,
unknown factors cannot be excluded and a causal
relationship remains unproven. Randomized prospective studies can only compare different blood products, and not the need for transfusion or not. Seven
randomized studies have been performed, six of these
in abdominal and one in cardiac surgery (Table 4).
In colorectal cancer surgery, two randomized controlled trials (RCTs) compared allogeneic with autologous red cell transfusions.84,85 Both studies used
buffy-coat depleted RBCs in the control arm as well

as in the study arm. In the single center study84 a

difference in postoperative infections between the two
groups was found, whereas in the multicenter study
with 14 participating hospitals85 no difference was
observed. Four RCTs evaluated filtered leukoreduced
red cells in abdominal surgery, two of these compared
to whole blood105 or RBCs with buffy-coat107 and
in two studies the control arms received buffy-coat
depleted RBCs.85,106 The three single center studies
found a significant reduction of postoperative infections in the patients allocated to receive filtered RBCs.
In the multicenter study86, applying stratification in 15
hospitals, no difference was found. The possibility
that the pooling of hospital data, with a huge difference in infection rates between hospitals, might be
responsible for masking an eventual true deleterious
transfusion effect was discussed extensively in order to
explain the difference between the single center and
multicenter studies.
One further study compared buffy-coat depleted
RBC with filtered RBC in cardiac surgery patients108
and found a reduction of postoperative infections
from 23% to 17% with filtered RBC. In this study a
significant difference (31% versus 22.5%; P 0.04)
between trial arms was only present in the subgroup
of transfused patients who received four or more units
of RBC.
There is little doubt that transfusions are associated with postoperative infections. The RCTs suggest
a causal role for allogeneic leukocytes but the magnitude of the benefits of alternative products (leukoreduced by buffy coat depletion or by filtration or
autologous RBC) varies widely. Differences in incidence as well as in the reporting/recording of infection may account for this. Also, differences in
leukocyte load and the nature of residual WBC in the
products used in the controls arms may have played a
role.

Table 4 Randomized trials comparing different perioperative RBC products and postoperative infections
Ref
n patients
Surgery type
Study design
% transfused
Control group
Study group
Infections*
Controls
Study patients
% difference
Significance

84
120
Colorectal Ca
Single center
80
Buffy-coat
depleted
Autologous
20%
16%
7%
9
P0.042

85
470
Colorectal Ca
Multicenter
69
Buffy-coat
depleted
Autologous
26%
25%
27%
ns
P0.6

105
197
Abdominal
Single center
51
Whole blood
Filtered WB
10%
23%**
2%
21
P0.0033

86
697
Colorectal Ca
Multicenter
64
Buffy-coat
depleted
Filtered RBC
33%
32%
36%
ns
P0.42

* Overall incidence including non-transfused patients.

** Comparison was only performed in the subgroups of transfused patients.

106
586
Abdominal
Single center
44
Buffy-coat
depleted
Filtered RBC
17.5%
17%**
3,5%
18
P<0.0001

107
221
Abdominal
Single center
27
RBC

108
861
Cardiac surgery
Single center
92
Buffy-coat
depleted
Filtered RBC Filtered RBC
16%
20%
44%**
23%
16%
17%
28
6%
P0.063
P0.13

Immunological aspects of blood transfusions

BLOOD TRANSFUSION, CRITICALLY ILL

PATIENTS AND MORTALITY
Trauma and surgery lead to profound alterations in
cellular immunity. Decreased natural killer cell functions, reduced delayed type hypersensitivity reactions
and altered cytokine responses have been demonstrated after various surgical interventions. Decreased
IL-12, a factor crucial for dendritic cells to induce
naive T cells to Th1 responses,109 and necessary for the
upregulation of immune functions against infectious
pathogens, was found to be reduced after surgery and
in critically ill patients.110 In joint replacement surgery
patients it was shown that IL-12 production was further decreased in those case where allogeneic perioperative transfusions had been administered.111
This implies that blood transfusions further skew
the immune response towards a helper type 2 reactivity with the release of suppressive factors as demonstrated by reduced LPS responses and generation of
suppressive cytokines like IL-4 and IL-10, increased
levels of which have been found to be associated with
the length of hospital stay.111 Matzinger et al.116 proposed that a Th2 type response is a normal stress reaction in the absence of a specific antigen stimulus in
order to protect an individual against a systemic
inflammatory response (SIRS). Early de-activation of
mononuclear cells by suppressive peptides, endogenously released and/or exogenously administered in
the transfused blood, could play a role in down regulation of any Th1 response.112 Shortly after blood
transfusions to critically ill patients a transient reactive leukocytosis has been observed.113115
A few recent observational and randomized studies
have addressed the question of the role of blood
transfusions on morbidity and mortality in critical illness and major surgery. Van de Watering et al108
observed in cardiac surgery a 7.8% mortality after
standard buffy-coat depleted RBC compared to 3.4%
in patients who received filtered components.
Increased death in the standard transfusion arm
occurred due to multi organ failure in patients who
received more than 4 units of RBC.
Hebert et al. randomized 838 intensive care
patients for two transfusion triggers.117 A liberal transfusion strategy aimed to maintain hemoglobin
between 100120 g/l and in a restrictive arm, the
transfusion level was between 7090 g/l. Patients randomized in the liberal arm received on average 5.6
units compared to 2.5 units in the restricted arm.
Hospital mortality was 23.3 versus 18.7% in the liberal and restricted groups respectively. This difference
was significant only in subgroups of patients younger
than 55 years of age and less acutely ill. The authors
hypothesized that the dominant explanation for worse

141

outcome may be the shelf life of the blood. The shelf

life of red cells was subsequently retrospectively investigated in cardiac surgery patients by Vamvakas. He
found an increase in post operative pneumonia after
bypass surgery related to the storage time of the RBC
transfused.118

CONCLUSIONS
The immunological side effects of blood transfusions
can be related to immunization and immunomodulation. Immunization is the result of antigen specific
indirect or direct recognition, the latter restricted to
MHC-alloantigens. Clinically manifested transfusion
reactions are almost always mediated by allo-antibodies, the production of which can be considered as a T
helper type 1 (Th1) response. Blood transfusions can
also induce a helper type 2 (Th2) response, which synergizes with the decreased type 1 immunity associated
with trauma, surgery and critical illness and may lead
to reduced clearance of postoperative infections and
increased susceptibility to the development of multi
organ failure. The balance between a Th1 and Th2
predominance may be influenced by donor selection
(matching or mismatching), product processing, storage, transfusion dosage and the clinical condition of
the patient. Knowledge about these mechanisms
should improve blood safety by suggesting more precise indications for particular products and components. Hemovigilance, even though it should lead
to improvements in safety where no or inadequate
surveillance systems were in place shall not, because
of its observational nature, answer most of the questions about the cellular side effects of transfusions,
which will require further scientific study and RCTs in
order to elucidate the full effects.
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