Vox Sanguinis (2009) 96, 266–269

© 2008 The Author(s)

REPORT Journal compilation © 2008 International Society of Blood Transfusion
DOI: 10.1111/j.1423-0410.2008.01144.x

Recommendations of the ISBT Working Party on Granulocyte

Blackwell Publishing Ltd

Immunobiology for leucocyte antibody screening in the

investigation and prevention of antibody-mediated
transfusion-related acute lung injury
ISBT Working Party on Granulocyte Immunobiology; (P. Bierling,1 J. Bux,2 B. Curtis,3 B. Flesch,4 l. Fung,5 G. Lucas,6
M. Macek,7 E. Muniz-Diaz,8 l. Porcelijn,9 A. Reil,2 U. Sachs,10 R. Schuller,11 N. Tsuno,12 M. Uhrynowska,13 S. Urbaniak,14
N. Valentin,15 A. Wikman16 & B. Zupanska13)
1
Platelet and Leucocyte Immunology Laboratory, EFS Ile de France, Hopital Henri Mondor, Creteil, France
2
Leucocyte and Platelet Immunology Laboratory, Blood Service West of the German Red Cross, Hagen, Germany
3
The Blood Center of Southeastern Wisconsin, Platelet and Neutrophil Immunology Laboratory, Milwaukee, USA
4
Institute of Transfusion Medicine, University of Schleswig-Holstein, Kiel, Germany
5
Australian Red Cross Blood Service-Queensland, Brisbane, Australia
6
Platelet and Granulocyte Immunology Laboratory, National Blood Service, Bristol, UK
7
Blood Transfusion Center of Slovenia, Ljubljana, Slovenia
8
Centre de Transfusio I Banc de Teixitis, Hospital Vall d'Hebron, P. Vall d'Hebron, Barcelona, Spain
9
Sanquin Diagnostics, Immunohaematology Diagnostic Department, Amsterdam, The Netherlands
10
Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig University, Gießen, Germany
11
American Red Cross North Central Blood Services, Neutrophil Serology Laboratory, St. Paul, USA
12
Department of Transfusion Medicine, Graduate School of Medical Sciences, University of Tokyo, Tokyo, Japan
13
Department of Immunohaematology and Transfusion Medicine, Institute of Haematology and Blood Transfusion, Warsaw, Poland
14
Aberdeen and North East Scotland Blood Centre, Scottish National Blood Transfusion Service, Aberdeen, UK
15
Platelet and Leucocyte Immunology Laboratory, Institute of Biology, Nantes, France
16
Department of Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden

Background Transfusion-related acute lung injury (TRALI) is currently one of the

most common causes of transfusion-related major morbidity and death. Among the
many TRALI mediators, leucocyte antibodies have been identified as important
triggers of severe TRALI.
Study Design and Methods These recommendations were compiled by experts of the
ISBT Working Party on Granulocyte Immunobiology, based on the results obtained in
eight international granulocyte immunology workshops, their personal experiences
and on published study results.
Results Leucocyte antibody screening has to include the detection of human leucocyte
antigen (HLA) class I, class II and human neutrophil alloantigen antibodies using
established and validated techniques. HLA class I antibody detection should be
restricted to antibodies clinically relevant for TRALI. To avoid unnecessary workload,
TRALI diagnosis should be assessed by consultation with the reporting clinician and
thorough exclusion of transfusion-associated circulatory overload/cardiac insufficiency.
In patients diagnosed with TRALI having donors with detectable leucocyte antibodies,
evidence of leucocyte incompatibility should be provided by either cross-matching or
typing of patient for cognate antigen.

Correspondence: Dr Jürgen Bux, Blood Service West of the German Red Cross, Feithstrasse 182, D-58097 Hagen, Germany
E-mail: j.bux@bsdwest.de

266
 Leucocyte antibody detection in TRALI 267

Conclusion Leucocyte antibody screening for the immunological clarification of

Received: 17 October 2008,
revised 21 November 2008,
accepted 22 November 2008,
published online 29 December 2008
TRALI cases as well as for identification of potentially alloimmunized blood donors
is feasible and can be performed in a reasonable and quality assured manner. This
practice can contribute to the prevention of antibody-mediated TRALI.
Key words: TRALI, HNA, leucocyte antibody detection

Workshops established in 1989. The combination of GIFT and

Introduction
Transfusion-related acute lung injury (TRALI) is a clinical
diagnosis that is the symptomatic expression of a non-
cardiogenic lung oedema induced by blood transfusion [1,2]
in which transfusion overload is excluded. Soon after the first
descriptions, TRALI has been associated with leucocyte antibodies
in donor and on rare occasions in recipient blood [3,4]. After
publication of an abundance of clinical and experimental
data in the last decade, it is generally accepted that TRALI can
be triggered by leucocyte antibodies [2]. These antibody-mediated
cases are often classified as ‘immune’ TRALI that can be distin-
guished from ‘non-immune’ TRALI reactions that can be induced,
for example, by cytokines or soluble neutrophil-priming lipids
[5]. Therefore, identification of alloimmunized donors and
recipients in TRALI cases and screening donors at risk for the
presence of leucocyte antibodies have been considered as a
preventive measure for antibody-mediated TRALI. The
following recommendations are made to provide a basis for
appropriate and qualified leucocyte antibody detection.
Leucocyte antibodies, antigens and techniques
for antibody detection
Leucocyte antibodies to be detected: (i) human neutrophil
alloantigen (HNA) antibodies; (ii) human leucocyte antigen
(HLA) class I antibodies; and (iii) HLA class II antibodies.
Leucocyte antigens at least to be covered by panel: (i)
HNA-1a, -1b, -2 and -3a; (ii) HLA class II; and (iii) HLA-A2
and other frequent HLA class I antigens.
Techniques for the detection of HNA antibodies: (i) granulo-
cyte immunofluorescence test (GIFT); (ii) granulocyte
agglutination test (GAT); (iii) granulocyte chemiluminescence
test; and (iv) any other validated test.
Techniques for the detection of HLA class I antibodies:
(i) enzyme immunoassay; (ii) flow cytometry with microbeads;
(iii) lymphocytotoxicity; (iv) lymphocyte immunofluorescence;
and (iv) any other validated test.
Techniques for the detection of HLA class II antibodies:
(i) enzyme immunoassay; (ii) flow cytometry with microbeads;
and (iii) any other validated test.
Human neutrophil alloantigen antibody detection
Recommendations for HNA antibody detection are based on
the experience of the International Granulocyte Immunology
GAT remains the best means of detection and has become the
standard approach in most of the laboratories participating
in the international workshops [6,7]. GIFT is widely considered
to be the more sensitive technique; however, GAT has an
enhanced ability to detect granulocyte agglutinins (leucoag-
glutinins), such as antibody to HNA-3a (former 5b).
Granulocyte agglutinins have been associated with severe
(i.e. mechanical ventilation required) and fatal cases of TRALI
[4,8]. Not only does GAT offer the advantage of assessing the
antibody’s clinical significance, but it also has the ability to
detect other agglutinins that have not been classified.
Alternative methods should be validated using neutrophil-
specific sera, which have been well-characterized by
participating International Granulocyte Immunology
Workshops laboratories. At a minimum, any other validated
method should have equivalent sensitivity and specificity to
the combination of both GIFT and GAT.
If possible, the HNA specificity of detected antibodies
should be confirmed in an antigen-specific assay, such as the
monoclonal antibody-specific immobilization of granulocyte
antigens assay.
Human leucocyte antigen antibody detection
The recommendations for HLA antibody detection techniques
are based on their current use. Some investigators prefer
anti-globulin tests for HLA antibody detection as they also
detect non-complement activating antibodies. The majority
of currently used HLA class I antibody screening tests are
designed for transplantation needs and these are therefore
both very sensitive and also designed to detect antibodies to
low frequency antigens.
In the May 2008 granulocyte workshop discussion, this
working party raised the question regarding the clinical
relevance of detecting weak and low frequency HLA antibodies
in TRALI for the following reasons: (i) transfused HLA class
I antibodies are primarily adsorbed to platelets and soluble
HLA class I molecules that represent 90% of the HLA class I
antigens in blood [9]; (ii) it is unlikely that an antibody
directed against a low frequency HLA class I antigen will be
transfused into a patient who carries the cognate antigen and
who is susceptible to TRALI.
Reports demonstrate that only few transfused HLA class I
antibodies will actually induce TRALI [10–12]. These findings
are in accordance with the questionable clinical relevance of

© 2008 The Author(s)

Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 266–269
268 ISBT Working Party on Granulocyte Immunobiology
weakly reactive HLA antibodies detected in assays with
increased sensitivity.
Since sera from alloimmunized individuals usually contain
a mixture of different HLA class I antibodies resulting in a
more or less pronounced broad reactivity [13,14], most
alloimmunized donors will be identified even if a less
comprehensive panel of HLA class I antigens is used for HLA
antibody screening.
Clinical cases with apparent pulmonary
transfusion reactions
Diagnosis of transfusion-related acute lung injury
Diagnosis of transfusion-related acute lung injury [1]: (i) acute
respiratory distress; (ii) temporal association with blood
transfusion, that is, occurrence within 6 h of transfusion
(typically within 2 h); (iii) new bilateral infiltrations in the
chest radiogram; and (iv) careful exclusion of (the more
frequent) transfusion-associated circulatory overload (hyper-
tension, pre-to-post transfusion elevation of B-type natriuretic
peptide (BNP) levels, history of and/or radiographic signs of
heart failure).
Since TRALI investigation can be complex, it is important
to assess the case with the reporting clinician, document the
clinical symptoms and to confirm clinical diagnosis [15].
Samples to be tested
Leucocyte antibody screening for HLA class I, class II and
HNA antibodies using the techniques listed above should be
performed on blood donations/donors that were transfused
within 6 h of the suspected TRALI reaction. Donor investigation
can be prioritized on the likelihood of the donor having
leucocyte antibodies such as parous female donors and trans-
fused donors. It should be noted that male donors have been
implicated in TRALI and that inappropriately stored residual
specimens from blood bags as well serum samples can cause
false-positive test results.
If non-leucocyte-reduced platelets or red blood cells were
transfused, the recipient’s blood should be also investigated
for leucocyte antibodies.
Provision of evidence of leucocyte incompatibility
When a donor leucocyte antibody is detected, a cross-match
between donor serum/plasma and recipients leucocytes is
recommended. When a recipient leucocyte antibody is
detected, the cross-match is between recipient serum/plasma
and donor leucocytes. Note that in the case of HNA antibodies
a granulocyte cross-match must be performed.
In cases where suitable cells for cross-matching are not
available, the recipient, and when necessary the donor,
Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 266–269
should be typed for the cognate antigens recognized by the
detected leucocyte antibodies. In cases where the cognate
antigens are present, the leucocyte antibodies likely caused
or contributed to the TRALI reaction.
Leucocyte antibody screening in blood donors
Screening blood donors for leucocyte antibodies is a possible
strategy to reduce the risk of antibody-mediated TRALI.
Transplanted donors, parous female donors, and, to a lesser
extent depending on the custom to transfuse leucocyte-reduced
blood components, transfused donors are most likely to have
leucocyte alloantibodies. Leucocyte antibody screening, that
is, HLA class I, class II, and HNA, should be performed using
the techniques listed above. Blood components with a high
plasma fraction (typically fresh-frozen plasma, apheresis
platelets and whole blood) should not be prepared from blood
donated by donors with leucocyte antibodies. In the case of
HNA-3a antibodies, deferral of the donor from blood
donation should be considered, as these antibodies have been
frequently implicated in fatal TRALI reactions [12,16] and
one fatal TRALI reaction occurred even after transfusion of
red blood cells [12]. Donors with negative test results for
leucocyte antibodies should be retested only after re-exposure
to leucocyte antigens.
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