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European Federation
For Immunogenetics
NEWSLETTER
April 2011 - Issue 64
………… . . from the EFI President
Dear EFI members,
Time flies! One address to the members is published and the next is
already scheduled with very little time
in-between! Indeed our Annual EFI
Conference in Prague is just around
the corner. Since my last address the
world has made some unexpected
turns. The voice of the people is heard
and in some instances this voice has
succeeded to change systems. I have
great respect for this. At the very
moment I write this address the news
of the earthquake in Japan with the
resulting destructive tsunami and fire
at a nuclear energy plant are coming
in. In the name of EFI and its membership I extend our deepest sympathy to
the victims and express our hope that
events can be settled as soon as possible. For the histocompatibility laboratories in the region I would like to offer,
in the name of EFI, our help.
related to these educational meetings.
EFI has had, in comparison to the
above, thankfully rather quiet days.
As mentioned already in my last
address POSEIDON is closed and we
are now finalizing the reports. We all
hope to have reached the set goals.
The summer school was very well
appreciated by the participants and
the teachers and you as EFI members
have already had the opportunity to
read the report in the previous newsletter. I would like to express my thanks
and gratitude to all the members of the
organizing committee and especially
to Catherine Stavropoulos-Giokas and
Frans Claas for their efforts to make
the event a very memorable one.
Regional meetings such as the Balkan
meeting have also taken place with
many participants (see report in this
Newsletter). It is fascinating to see
how lively our society is. In the future a
dedicated place within the EFI website
will help to disseminate information
A personal view of the topics offers me
the possibility to report on a scheduled meeting for the use of solid phase
assays in clinical organ transplantation.
A lot has been said to date. We should
neglect emptions and remain scientific.
Antibodies against cell surface antigens of the organ donor are not per se
deleterious. They are risk factors and
so they should be seen. Only if a good
communication exists between the different faculties e.g. nephrology and
immunology the outcome of the clinical intervention will succeed. In Essen,
Germany is such a meeting took place,
the results of which are presented in
the current issue of the Newsletter.
EFI, besides being exclusively scientific and very professional, is also a
model for a multinational and multicultural society despite the statements of
some politicians against multicultural-
ism. We speak many languages and
have different mentalities. Still we can
cooperate with each other and enjoy
every moment during our meetings
and conferences both professionally
and socially. This brings me to a proposal to collect and make accessible
recipes from the members from the
various countries of EFI. In my opinion
this will increase the understanding
between countries and their members.
A blast mail will reach you within the
next weeks with more details on this
endeavor.
The Executive Committee of EFI will
undergo several changes in membership which will be presented to you in
Prague. Some of members will leave
and make place for new members, who
will be elected in the next few weeks.
I am very confident that with the team
we had, and will have, we will be able
to cope with the various challenges we
will encounter.
continue on page 5
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… . from the editor’s desk
EFI website
htt://www.efiweb.org
Editor-in-chief
Frans H.J. Claas
Editorial address:
EFI Newsletter
LUMC, Dept. of Immunohematology
and Blood Transfusion, Bldg. 1, E3-Q
P.O. Box 9600
2300 RC Leiden, The Netherlands
EFI Executive Committee 2009
EFI President
I. Doxiadis (the Netherlands)
EFI Secretary:
A.M. Little (UK)
EFI Treasurer:
V. Dubois (France)
Membership Secretary:
S. Geelhoed (the Netherlands)
I. Abelman (the Netherlands)
Councillors:
D. Adorno (Italy).
M. Bengtsson (Sweden)
K. Fleischhauer (Italy)
J. Mytilineos (Germany)
A. Slavcev (Czech Republic)
C. Susal (Germany)
Past Presidents:
J.J. van Rood, B.A. Bradley, E. Albert,
J. Hors, M-M Tongio, J.G. Bodmer,
F.H.J. Claas, S. Curtoni, E. Thorsby,
F.Garrido, D. Charron, S.G.E. Marsh
Spring has started, the sun is shining, here in the Netherlands the bulbs are
flowering and the EFI community is looking forward to the upcoming EFI meeting in Prague.
Tony Slavcev and Gottfried Fischer have made a very attractive program, which
will certainly stimulate many people to come and visit the beautiful city of
Prague.
It is clear that our community is full of activities as reflected by this issue of
the Newsletter, which contains several reports on interesting local meetings,
on useful visits by EFI bursary recipients to other laboratories and announcements of future meetings.
Most of the current discussions in the field of histocompatibility testing concern the role of the new and very sensitive techniques for the detection of
donor specific HLA antibodies in clinical transplantation. It is clear that the
old dogma that a donor specific antibody is a contraindication for transplantation does not exist anymore. Both at the recent transplantomics meeting in
Barcelona and the extramural Eurotransplant meeting in Essen, Germany, this
topic was discussed extensively.
It is very difficult to reach consensus on this issue as the results in different
centers, obtained with different techniques, different cut-offs of positive and
negative reactions, different titers, different antibody classes and different
clinical management can not be compared. Nevertheless, one of the work
packages for the upcoming international workshop in Liverpool (an excellent
update is given by Derek Middleton) has this topic on its agenda. This is certainly a challenge, which needs the input of H & I specialists, clinicians and
companies producing these (luminex based) assays.
From this place, I would like to congratulate Jon
van Rood, the founder of EFI, with his 85th birthday. Although retired for 20 years, Jon is still very
active in the field aiming at the implementation of
the knowledge of the fetal-maternal immune regulation in clinical transplantation and immunotherapy
for cancer and autoimmune diseases.
I hope that the information in this Newsletter is
useful to you and I am looking forward to your contributions to the next one.
See you in Prague.
Frans Claas
Deadline for contributions to EFI Newsletter 65 is August 15 , 2011.
Please send your contribution by e-mail to fhjclaas@lumc.nl
The editor and the EFI officers do not accept
responsibility for the contents of published
articles. Opinions expressed by contributors
are not necessarily those of the editorial board.
Please support the advertisers in
this issue of EFI Newsletter
ISSN 0962-9521
Contents
From the EFI President
From the editor’s desk
An EFI accredited HLA Laboratory in Novi Sad-Vojvodina, Serbia
Preliminary program of the EFI meeting in Prague
Current status of the 16th IHIW Projects
EFI Region 8 H & I Laboratories Meeting in Athens
Report on my visit to the Reference Laboratory of Eurotransplant in Leiden
Report on my visits to the Medizinische Hochschule in Hannover
Report on the extramural Eurotransplant meeting in Essen
on the relevance of pretransplant HLA specific antibodies in clinical transplantation
HLA-NET Training School – Population Analyses for HLA and other Immunogenetic Systems –
Report from an EFI Education and Training Bursary recipient
1
3
5
6
8
13
15
17
19
23
25
3
4
………… . . from the EFI President (continued)
Our next EFI Conference to be held in
Prague, hosted by Tony Slavcev and
Gottfried Fischer, will be scientifically
outstanding. Together with the Scientific Committee, chaired by Ronald Bontrop, an excellent program has been put
together. The teaching sessions cover
many important areas of Immunogenetics, clearly demonstrating HLA as a
leading immunogenetic complex, not at
its end but acting as a phoenix resurrecting from the ashes. Genome wide
analyses for virally induced diseases
show that HLA is one of the most important factors. The bottom line is that it is
still very attractive to work in this field.
This is also reflected by the efforts of
our society to offer bursaries to support members to attend our annual
conference and also to cover the fees
for needy participants from emerging
countries. There will be oral sessions,
posters and plenary sessions. We will
also have esteemed awards: the Julia
Bodmer Young Scientist Award, the Jon
van Rood award for best scoring oral
presentation and finally but not least,
the Ceppellini Lecture delivered this
year by a very well known member of
our Society, Professor Eric Thorsby.
For now I wish all of you attending the
EFI Conference a pleasant journey to
Prague and an excellent Conference. I,
and all of the members of the various
EFI Committees together with the local
organising committee look forward to
welcoming you in Prague.
An EFI accredited HLA Laboratory in
Novi Sad-Vojvodina, Serbia
The Institute for blood transfusion of
Vojvodina, is established by the Government of Autonomous Province of Vojvodina and represents the transfusion
medicine institution of regional competence and importance. The Institute for
Blood transfusion of Vojvodina in Novi
Sad is the first institution in our country
in the transfusion medicine field, which
gained the ISO 9001 certificate in 1998
and ISO14000 in 2002. The institution
provides the highest number of blood
donations per year in our country. We
are very proud that the Tissue Typing
Compartment in our Institute is the
first laboratory in Serbia that obtained
the EFI accreditation, which happened
in 2010. It is an honor that the most
important organization in the field of
H&I in Europe, EFI , approved our work.
The Organogram of the Department for
laboratory diagnostics consists of four
elements, including, the Tissue Typing
Compartment, where two physicians
serve as director and technical supervisor and two laboratory technicians
perform the assays, among which one
serves as Quality Assurance manager.
The first steps of our Compartment in
process of EFI accreditation were when
we started with participation in EPT in
2005. We obtained the first certificate
for HLA class I serological typing from
Wroclaw, in Poland. In the years later,
we gradually extended our activities
and up to now we gained the satisfactory certficates from Poland, Austria,
Italy and Bulgary. The main and crucial event for our intentions to obtain
the EFI accreditation, happened when
our Commissioner, Dr Chryssa Papasteriades made the preinspection of
our Compartment in 2008. Her visit,
was most important for us, because
she encouraged us to apply for the EFI
accreditation.
After that period and in 2009. we made
following changes before the final
application for the EFI accreditation.
We invested a lot of our efforts and
strenght
- in establishing new techniques,
- on making corrections in our routine
work to fit with EFI standards,
- in establishing documents to fit to
all elements of Quality Assurance
Control which consists of 5 elements
- and to gain satisfactory certificates
for several techniques.
Our first on site inspection was on
12th July 2010. Our inspectors were
Prof.dr Elisaveta Naumova as a representative of our region, and dr Blanka
Vidan-Jeras,. Our inspectors were very
objective, friendly and open in their
communication. All their explanations
and suggestions were clear and useful.
The inspectors showed their great
experiance, knowledge and competence in the field of H&I as well as for
Quality Assurance issues. The whole
onsite inspection passed comfortably
and in very pleasant athmosphere and
was very instructive and highly beneficial for our laboratory.
All documents necessary to obtain the
EFI accreditation, including EFI standards and accreditation program, are
very well prepared. They completely
and utterly clarify to the applicant what
he should and must do. The procedure
gave us the opportunity to make several consultations with our Commissioner, Dr Chryssa Papasteriades and
to interact on some technical items
with the Manager of the EFI Accreditation Office, Ms. Sonja Geelhoed.
The EFI accreditation is a great stimulus for our further work.
In the future, the Tissue Typing Compartment in the Institute for Blood
Transfusion of Vojvodina in Novi Sad is
planning to:
- maintain the variety and level of
all activities that were performed
during the accreditation program
- continue in monitoring and incorporating the changes in EFI standards
- establish new techniques continuing in prosperity and progress
- expand the number of EFI accredited categories
- achieve re-accreditation in 2012
- participate in activities requested
for Serbia to become a full member
of the Eurotransplant organization.
Assist.Prof.Vojvodic` Svetlana MD.PhD.
HLA Laboratory,
Tissue Typing Compart.,
Dept. for Laboratory Diagnostics
Institute for Blood Transfusion of
Vojvodina
Novi Sad-Serbia
5
25th European Immunogenetics
and Histocompatibility Conference
Prague | Czech Republic
May 4–7 | 2011
CONFIRMED SPEAKERS
ORGANIZING COMMITTEE
IMPORTANT DATES
Co-chairs:
Antonij Slavcev
Gottfried Fischer
On-line Registration Opening
November 15, 2010
Deadline for Abstract Submission
January 10, 2011
version as of October 21, 2010
Stephan Beck
Marco Colonna
Katharina Fleischhauer
Federico Garrido
Adrian Hill
Lewis Lanier
Ashley Moffett
Alessandro Sette
Jean-Paul Soulillou
Caner Susal
Peter van den Elsen
Benoit Van den Eynde
Andrea Velardi
EFI2011_inz210x225.indd 1
6
Members:
Marie Dobrovolna (Prague)
Ingrid Fae (Vienna)
Pavel Jindra (Pilsen)
Marie Kurikova (Prague)
Wolfgang Mayr (Vienna)
Martin Petrek (Olomouc)
Ilja Striz (Prague)
Ondrej Viklicky (Prague)
CONFERENCE SECRETARIAT
GUARANT International spol. s r.o.,
Opletalova 22, 110 00 Praha 1
Tel.: +420 284 001 444
Fax: +420 284 001 448
E-mail: efi2011@guarant.cz
www.efi2011.eu
9.11.10 11:46
25th Immunogenetics and Histocompatibility Conference
Prague, Czech Republic, May 4 - 7, 2011
Time
9:00 - 16:30
16:30 - 19:30
20:00 - 23:00
Hall 1
Hall 2
Hall 3
Hall 4
Tuesday, May 3, 2011
Hall 5
Standards & Quality Assurance Committee
Meeting
8.00 - 12.30
Chair: K. Poulton
EFI Inspectors Workshop
Chair: G. Fischer
EFI Accreditation Committee Meeting I
Chair: G. Fischer
EFI Inspectors Dinner
Scientific Programme
8:00 - 16:30
Postgraduate Course of The Czech
Transplantation Society
9:00 - 12:00
Chair: I. Striz, A. Slavcev, V. Holan
EFI Executive Committee Meeting
8.00 - 15.30
Chair: I. Doxiadis
Wednesday, May 4, 2011
External Proficiency Testing Committee
meeting I
EFI Accreditation Commissioners Meeting II
9.00 - 14.00
Chair: G. Fischer
8.30 - 16.00
Chair: J. Vaage
Education Committee Meeting I
15.30 - 17.30
Chair: J. Mytilineos
18:00 - 18:15
Official Opening / Welcome Addresses
Chair: I. Doxiadis, A. Slavcev, G. Fischer
18:15 - 18:40
Julia Bodmer Award / R. Bontrop - Introduction
18:40 - 19:25
Ceppellini Lecture / I. Doxiadis - Introduction
19:25 - 22:00
Welcome Cocktail, Students orchestra "Three Weeks After"
8:30 - 9:00
Plenary Session I: Genetics-Epigenetics-Gene Interactions of
The HLA complex
Chair: J-M. Tiercy, D. Charron
S. Beck: Epigenetic Modification of HLA Expression
9:00 - 9:30
P. J. van den Elsen: Transcriptional Control of HLA Expression
8:30 - 10:00
9:30 - 10:00
Coffee Break
Oral Session I: Hematopoietic Stem Cell Transplantation
Chair: P. Cetkovsky, K. Fleischhauer
10:30 - 10:50
P. Sedlacek: Cord blood transplantation in children
10:50 - 12:00
Oral presentations
12:00 - 13:00
Lucheon sessions
13:00 - 14:30
Plenary Session II: From Peptide Motifs to Vaccines
Chair: J. Bartunkova, J. McCluskey
A. Sette: Peptides: the substrate of allogenicity
13:30 - 14:00
A. Hill: New vaccine design: clues from peptide motifs and the
immunogenetics of infectious diseases
14:00 - 14:30
P. van den Eynde: Tumour control with peptide vaccinations
14:30 - 15:00
15:00 - 16:30
Coffee Break
Oral Session III: Antigen Presentation and Immunological
Tolerance
Chair: I. Striz, N.M. Lardy
Concert: chamber choir L´Asenzio, St. Salvator church
20:30 - 23:00
Ceppellini Dinner; Speakers´ Dinner
08:30 - 10:00
8:30 - 9:00
9.00 - 9.30
9:30 - 10:00
10:00 - 10:30
10:30 - 12:00
12:00 - 13:00
13:00 - 14:30
13:00 - 13:30
13:30 - 14:00
14:00 - 14:30
14:30 - 15:00
15:00 - 16:30
16:30 - 17:00
Immunogenetics - future prospects I: 16th
HLA and Immunogenetics Workshop,
Liverpool 2012
Chair: S. Marsh, D. Middleton
Teaching Session I: Allocation of kidneys
throughout Europe
Chair: S. Fuggle, F. Claas
Oral Session II: MHC-peptides - Epitopes
and Prediction
Chair: A. Sette, R. Blasczyk
EFI Executive Committee and
Coordinators Meeting
12:00 - 15:00
Chair: I. Doxiadis
Teaching Session II: Posttransplant Monitoring
Immunogenetics - future prospects II:
Population genetics in Central and Eastern in Stem Cell Transplantation
Chair: A. Toubert, C. Thiede
Europe
Chair: B. Vidan-Jeras, E. Naumova
Oral Session IV: Non-HLA polymorphism
Chair: M. Bengtsson, M. Ivanova
One Lambda party
Friday, May 6, 2011
Plenary Session III: NK Cells: First or Second Line of Defence
Chair: E. Thorsby, A. Velardi
L. Lanier: Getting a license
M. Colonna: Various degrees of polymorphism in NK cell
receptors
A. Moffet: Successful pregnancy depends on KIRs
Coffee Break
Oral Session V: Solid Organ Transplantation
Chair: C. Susal, M. Howell
Oral Session VI: Immunogenetics /
Miscellaneous
Chair: R. Duquesnoy, F. Garrido
Teaching Session III: Scientific Research:
Planning and Analyses
Chair: C. Carcassi, C. Muller
Oral Session VII: COST- Europe and beyond
Chair: A. Sanchez-Mazas, G. Fischer
Luncheon Sessions
Plenary Session IV: Antibodies revisited
Chair: I. Doxiadis, P. Dyer
C. Süsal: Antibody detection with sensitive techniques and
their interpretation
G. Böhmig: Solid phase HLA antibody detection - clinical
implications
C. Taylor: Predicting HLA alloantigen immunogenicity
Coffee Break
Best abstract session
Chair: R. Bontrop, M. Colonna
Coffee Break
17:00 - 18:30
19:00 - 20:00
20:00 - 1:00
EFI General Assembly
08:30 - 10:00
Plenary Session V: Alloreactivity and Tolerance in
Transplantation
Chair: C. Susal, J. Mytilineos
K. Fleischhauer: Detecting and missing allo
A. Velardi: GvH vs GvL – any progress?
J-P. Soulilou: New insights in spontaneous tolerance state for
allogeneic transplants
8:30 - 9:00
9:00 - 9:30
9:30 - 10:00
Chair: J. Mytilineos
External Proficiency Testing Committee
Meeting II
8:30 - 11:30
Chair: J. Vaage
Wine, beer and cheese poster viewing
16:30 -18:15
18:15-19:30
19:30 - 20:00
22:00 - 2:00
Education Committee Meeting II
7:00 - 8:30
F. Garrido: The escape of cancer from immune surveillance:
HLA expression and tumor rejection
10:00 - 10:30
10:30 - 12:00
13:00 - 13:30
Thursday, May 5, 2011
Gala Dinner
10:00 - 10:30
10:30 - 12:00
Coffee Break
Oral Session VIII: Autoimmunity, Disease Association and
Cancer
Chair: M. Petrek, A. Amoroso
12:00 - 13:00
Closing Ceremony: Awards, Future EFI Conferences
Chair: I. Doxiadis, A. Slavcev, G. Fischer, J. Mytilineos, S. Marsh
Saturday, May 7, 2011
Immunogenetics III - future prospects:
trends next generation high-resolution HLA
typing
Chair: M. Tilanus, S. Marsh
Teaching Session IV: KIR, MICA and other "nonclassical" parameters in histocompatibility
laboratories: methods and clinical value
Chair: A-M. Little, P. Jindra
7
Current status of the 16th IHIW Projects
Derek Middleton
To date the following Workshop Projects, some continuing from the 15th Workshop, have been accepted.
Anyone interested should contact the organizers of each project - details are found at www.16ihiw.org.
Other proposed Projects are welcomed and will be vetted by IHIW Councillors prior to acceptance.
SOLID ORGAN TRANSPLANTATION
Post-transplant Antibody Monitoring
and Treatment of Antibodies.
Paul I. Terasaki and Mikki Ozawa.
The study has been modified to have
the following three parts and has 39
laboratories participating as of 1st
March 2011.
Part 1. One HLA Antibody Test Predicts
Future Graft Survival
Aim is to show that, with only a single
HLA antibody testing post transplantation, the future outcome of transplant
can be predicted. Each centre will
test ALL of their transplanted patients
(screening kits provided free), follow
the patients, and correlate with clinical
status.
Part 2. Post-transplant Antibody Monitoring of High Risk Patients
Centres will track the time course of
antibody development and changes
in strength and specificities, starting
from the time of transplant, in relation to immunosuppressive regimens,
clinical events, and treatments. Focus
will be on high-risk patients: pretransplant PRA 50% or greater, and/or
patients receiving a regraft. Single antigen beads provided at half of normal
charge. The 16th WS Antibody Tracking
software also provided.
Part 3. Antibody Reduction Treatment
to Prevent Graft Loss (Optional).
Centres will remove the antibodies as
preventive therapy or as antibody-mediated rejection treatment, and monitor
antibody levels to assess the efficacy
of treatments. Individual centres can
select the method of their choice (Bortezomib, Plasmapheresis/IVIg, MPAdose increase, etc.) Single antigen
beads will be provided at half of normal
charge.
Evaluation of Antibody Frequencies and
Solid Phase Assays.
Andrea Zachary and John Hart
This project is a continuation of the
antibody frequency project conducted
in the 15th IHWS. In that workshop, we
asked participants to submit information about the patient whose serum was
being reported and the CSV file from
8
the single antigen bead panel used to
test the serum. While this approach
provided some consistency in test
format, there were some difficulties
in linking the CSV files to the patient
demographics. For the 16th workshop,
we have changed the approach to have
the participants provide their final analysis, based on all test data and to provide the patient information which will
be automatically linked to the antibody
analysis.
There are two parts to this project.
The first part is to include data for
the broadest reacting serum from a
patient. This will be used to assess the
frequencies of antibodies of different
specificities and the response to transplant mismatches.
The second part is to include data from
a serum that showed high background
(MFI >200 or >300, for the Luminex
single antigen bead and/or phenotype panels, respectively, in the negative controls) or low positive control
(MFI<10,000 for either the single antigen bead or phenotype panels). The
aim of the second part of the study is
assessment of the impact of interfering
factors on antibody characterization.
Data entry is done in an EXCEL format.
The data sheet can be accessed at:
http://www.16ihiw.org/index.html
,
under this project name. To date, 12
labs have enrolled. As we will collect
data through most of this year, there
is still plenty of time to participate. If
you have questions about data entry,
please contact John Hart at jmhart@
jhmi.edu.
If you agree to participate, please let
us know by email at either aaz@jhmi.
edu or jmhart@jhmi.edu. We look forward to your participation.
Towards Standardization of Microparticle-based, Solid Phase, HLA Antibody
Identification Assay.
Robert Bray and Howard Gebel
As of March 1st, a total of 15 laboratories, worldwide, have been enrolled. As
of now, we would consider enrollment
closed. During February, sera were
selected for the workshop and a pilot
survey was sent to 5 laboratories in the
U.S. Results from this small survey are
being tabulated. It is anticipated that
the first of at least 3 send outs will take
place by the end of March. The first
send out will be 4-6 sera with the intent
of establishing a baseline for antibody
identification for each of the participating laboratories. The workshop is
designed to address issues related to
standardized testing and resulting of
solid phase assays. While flow cytometry bead data will be included, the
main goal is to identify issues related
to Luminex-based assays.
MICA-MICB Project
Peter Stasny and Yizhou Zou
The MICA Project has two components.
The first is a serum exchange in which
active participants will be provided with
well-characterized sera containing antibodies against MICA. It is envisioned
that 4 sera will be provided in 4 successive shipments. Each laboratory will
test for antibodies against MICA antigens with available reagents. Results
will be tabulated and consensus of
antibody identification will be reached
on the basis of work performed on
each of the sera supplied.
The first shipment of 4 sera has been
sent to 10 participating laboratories in
6 countries. Two additional laboratories have requested to participate. The
active laboratories are: BRAKAL, CHIHE,
USATYA, USANEL, INDMEH, USAMOS,
UASPID, CHIYE, AUSCHR, CHICHE. One
participating laboratory has already
returned results of tests from the first
shipment of sera. All results will be collected and summarized 5 weeks after
each shipment. The combined results
will be documented and distributed to
the participating laboratories before
the next shipment of sera.
Part two of the MICA Project is to
evaluate the role of antibodies against
MICA in organ transplant rejection.
For this purpose cases of organ transplant rejection in patients who did not
develop detectable antibodies against
donor HLA antigens will be collected.
At present one of the participants has
informed that some cases will be submitted. The studies will involve testing
for antibodies against MICA, confirming
that antibodies to donor HLA are negative and typing of donor MICA by SBT.
It is anticipated that other laboratories
will also contribute transplant cases for
this study.
ANTHROPOLOGY
Population global distribution of KIR
and ligand.
Jill Hollenbach, Raja Rajalingam, Derek
Middleton
We will extend the studies of the 15th
Workshop to the 16th IHIW, in particular emphasizing investigation in populations not studied in the last workshop,
as well as further investigation of
allelic variation in the KIR. Of particular
interest are non-European populations
with limited admixture. Allelic typing
will be expanded to include KIR2DL3
and KIR3DS1, as well as KIR2DL2
and KIR3DL1. This will allow a more
detailed examination of allelic variability and haplotypic associations across
the KIR complex.
To date 6 labs have agreed to participate.Populations should consist of at
least 50 healthy, unrelated individuals. Initially labs are asked to provide
details of HLA typed populations they
would like to submit. If a lab can do
its own KIR gene typing that lab will be
asked to take part in a short QC exercise.
If a lab is not able to provide KIR
results a decision will be made as to
whether it will be possible to provide
free reagents to that lab or whether it
will be possible for that population to
be typed at a reference centre. This will
depend on how useful to the overall
aim of the project that population will
be –lack of admixture etc.
If a lab has a suitable population we
will ask them for approx. 3 DNA samples to ascertain if the quality of the
DNA is good enough
AHPD: Analysis of HLA Population Data
- to reconstruct the history of modern
humans and infer the role of natural
selection.
Alicia Sanchez-Mazas
(alicia.sanchez-mazas@unige.ch)
This project is a continuation of the
AHPD project of 15th IHIW (Brazil
2008), to which 14 laboratories participated.
Up to now, 11 additional laboratories
have declared their interest for this project and have proposed population data
for population genetics analyses. The
new data include samples from Western Europe, sub-Saharan Africa, Western Asia, Southeast Asia and Oceania,
i.e. several main geographic regions
of the world. Both ethnically-defined
samples and bone marrow donors are
included.
The next 6 months will be devoted
to collaboration with the participant
laboratories through e-mail contacts
to check their data and help them to
estimate allele and haplotype frequencies and other statistics of interest. We
will also continue to encourage other
laboratories to participate in order to
improve the dataset for further population comparisons.
In autumn 2011 we will be to start
genetic diversity analyses and population comparisons by adding the new
samples to the data analysed during
the previous workshop.
Our final goal is to prepare a common
publication of the results. We plan to
write a first draft of this publication
during the first semester 2012 to discuss it during the16th IHIW. This work
should improve our knowledge on the
evolution of the HLA polymorphism
during human peopling history.
AHPD website: http://geneva.unige.
ch/ahpd
This project also benefits from the scientific achievements of the EU-funded
network HLA-NET (http://hla-net.eu).
BIOINFORMATICS
Immunogenomic Data Management
Methods
Steve Mack and Jill Hollenbach
“The goal of the 16 IHIW Immunogenomics Data-Analysis Working Group
(IDAWG) project is to develop data-management standards, analytical methods, and tools intended to facilitate
the sharing and consistent analysis of
highly polymorphic HLA and KIR data
by the immunogenomics and genomics communities. The first phase of
this project takes the form of a survey
of current immunogenetic community
data-management and data-analysis
practices. We have developed a 32
question survey, designed to collect
basic information about common practices for generating, collecting, storing,
transmitting, and analyzing HLA and
KIR genotype data; this survey is being
made available over the internet via the
SurveyMonkey web site, thanks to the
generous support of the American Society for Histocompatibility and Immunogenetics (ASHI), and can be found at
http://www.sur veymonkey.com/s/
IDAWG. All immunogenetic laboratories
are invited to complete the survey and
participate in the project. The survey
will be open for participation through
the end of 2011. Preliminary results of
the survey will be presented at EFI.
“The second phase of this project
will use the information provided by
the survey respondents to determine
the effects of the various data-management and data-analysis practices
in currently use on common applications of HLA and KIR data (e.g. registry
searches, disease-association studies,
and population studies), using combinations of real and synthetic datasets.
This aspect of the project will be fully
underway by May of 2011, and the
participation of survey respondents
is encouraged. By March of 2012, we
anticipate being able to rank current
data-management practices in terms
of their effects on specific data-applications, and look forward to working with
project participants to develop specific
data-management recommendations
for particular applications as part of
the 16th Workshop.”
Frequencies of Rare Alleles
Faviel Gonzalez, Derek Middleton
Data is continually being collected and
added to the website www.allelefrequencies.net. To date we have received
data from 33 additional laboratories
since the 15th Workshop. We are filtering the data by geographic continent.
As we receive more data we will filter
by country so that labs in a specific
country can see which alleles are rare
in their country. Labs can send data at
any time to derek.middleton@rlbuht.
nhs.uk. A spreadsheet can be supplied
to enter the data.
IHIWS Registry Diversity Project
Martin Maiers, Carlheinz Müller,
Steven Marsh
The goal of this project is: comparison,
validation and improvement of tools for
HLA haplotype frequency analysis specifically designed to address issues of
registry datasets: large sample sets,
heterogeneous methods, resolutions,
missing data and deviation from HardyWeinberg Equilibrium.
This 16th IHIWS project builds on a
foundation developed in the two previous workshops:
Task 03: “Accuracy of haplotype frequency estimations on simulated data
sets with deviation from Hardy Weinberg”.
The simulation framework we have
developed will be extended to generate
datasets of populations with specific
deviation from Hardy-Weinberg Equilibrium. These datasets will be distributed to participants and the results
compared in order to validation implementations and confirm assumptions.
One particular topic to address in this
task is the accuracy of methods of
identifying and even correcting for specific sources of HWE deviation such as
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cryptic population structure (the Wahlund effect).
Task 04: “Accuracy of methods of estimating high-resolution haplotypes from
large datasets of mixed resolution”.
All HLA typing methods include some
level of ambiguity. DNA methods are
typically reported in terms of allelic
ambiguity but there are other forms
of ambiguity: genotypic (inability to
set phase), historical (typing to a fixed
snapshot of a growing allele list) and
ambiguity due to incomplete definition of reference alleles (missing
sequence). We plan to compare results
of haplotype frequency estimation from
datasets based on population simulations typed at a variety of resolutions
and methods.
Task 05: “Worldwide donor registry
analysis: high-resolution HLA A-B-DRB1
haplotypefrequenciesfor BMDW registries”
Haplotype frequencies will be generated for all registries of the BMDW. This
data will be applied to tools to project
registry growth and diversity and the
effects on matching.
Genetic distance analysis will be performed to determine the relatedness of
donor registries.
This data has direct practical clinical
use for donor search and informing
strategies for donor recruitment
Development of an HLA Epitope
Database
Rene Duquesnoy
This project addresses the identification of antibody-defined HLA class I
and class II epitopes and what notation system should be used to describe
such epitopes. This will be based on
amino acid residues in polymorphic
sequence positions in HLA molecular
structures.
Although the steering committee is
still working on a final design, we will
likely propose that epitope notations
will have the prefix HLE (Human Leukocyte Epitope). Since many epitopes
are shared between antigens encoded
by HLA-A, HLA-B and/or HLA-C, we will
use the following notation system: HLEI-x-y for class I epitopes whereby x represents a sequence position number
indicating the molecular location of
the epitope and y is a description of
relevant amino acid residues. The HLA
epitope database will have complete
descriptions of the configurations of
epitopes and a list of antibody-reactive
alleles with the same allele. We are
also considering listing other alleles
that have not been tested with antibody but which have the same amino
acid configurations that describe the
epitope. Needless to say, the database
will have only epitopes that have been
experimentally verified with informative
antibodies that have been tested with
proper HLA allele panels.
Since there is little epitope sharing
between class II antigens encoded by
the different HLA-DR, DQ and DP loci,
the following notation system will be
proposed: HLE-R-x-y (for DRB1/3/4/5),
HLE-Q-x-y (for DQA and DQB) and HLE-Px-y (for DPA and DPB).
The steering committee is considering minimal criteria for HLA epitope
identification. Besides the need for
monospecific antibodies there should
be information about the sensitization event (transplant, pregnancy).
HLA types of immunizer and antibody
producer may identify structural differences that could determine potential
epitopes. What methods should be
used for antibody testing? As a minimum, Luminex assays with single allele
kits from two manufacturers must be
used and their reactivity patterns must
permit a proper interpretation of antibody specificity.
At this time many informative antibodies have been tested according to the
above criteria and the corresponding
epitopes will be listed in the database
that will be maintained on the 16th
Workshop and other websites.
Laboratories can participate in this
project by submitting informative epitope specific antibody preparations
together with data about the reactivity patterns with single alleles as well
as information about the sensitization
event and its clinical effect. The committee will evaluate each submission
and may request more antibody for further testing. These efforts will focus on
less well defined and newly recognized
epitopes to be added to the database.
A worldwide collaboration will likely
generate new insights into HLA epitope
structure and clinical relevance.
NEW FRONTIERS
The analysis of HLA class I non-coding
regions
Alison Castley, Linda Smith
In this workshop component we propose to identify novel HLA class I
non-coding variations in different populations. We aim to:
Survey of the level of heterogeneity of
non-coding sequence.
Identify rare versus common non-coding polymorphisms.
Determine the frequency of currently
defined alleles characterised by polymorphisms in coding sequences
Examine common polymorphisms to
determine if they are associated with
or split conserved haplotypes.
Determine if unrelated stem transplants include class I non-coding
sequence differences.
Add novel alleles to the IMGT/HLA
Database
Participants (5 Labs have agreed to
date) are to analyse SBT results for
non-coding polymorphisms. However,
if labs are unable to analyse data the
co-ordinators will perform the analysis
in Perth. We will maintain a register of
numbers of loci, alleles and non-coding sequences analysed. And we will
catalogue the number of novel alleles
including the HLA types at other loci
for haplotype characterisation. All data
submit will be collates and present at
the 16th IHIW.
Any laboratories can participate if they
can perform SBT and/or access to
HLA class I sequence data. They also
require software capable of complete
gene analysis (if this is not available
analysis can be performed in Perth). To
date 5 labs have enrolled.
Pharmacogenomics
Clara Gorodezky and Susie Leffell
The aim of this 16th IHIWS component
is to establish an on-going registry for
compilation of data on the incidence
of associations of HLA alleles and/
or other immunogenetic factors with
adverse drug reactions in different populations. Immunogenetic factors may
contribute to the responses evoked
by certain drugs, as in the case of two
established host-drug interactions,
carbamazepine and abacavir. Serious
adverse reactions to these drugs are
known to be associated with certain
HLA alleles in some ethnic groups, but
have not been investigated in other
populations. It is also likely that additional associations will be established
for other drugs with increasing awareness of possible immunogenetic predisposition. Two potential drugs for
investigation include busulfan and
desatinib, both of which have reported
adverse effects in treatment of Philadelphia chromosome negative CML
and Philadelphia chromosome positive
ALL, respectively.
To date, three laboratories have enrolled
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in this component and, as participation
can be based on retrospective data, we
encourage participation by any other
interested parties. The guidelines for
participation are as follows:
• For carbamazepine and abacavir
study:
HLA phenotypes of 50 non-white
patients with adverse reactions and
50 ethnically matched controls.
• For busulfan and desatinib:
Any ethnicity of patients with
adverse reactions plus ethnically
matched controls – preferably at
least 10 of each.
• HLA resolution (allele level) class I
and II typing
Assistance with allele level typing
will be provided on limited basis
from central labs
• Appropriate approval by institutional
review boards. Please note: All data
will be de-identified and assigned
workshop codes. In many institutions, it is possible to obtain waivers of IRB approval for retrospective
analysis of de-identified data.
The long term goal of this IHIWS component is to initiate both a registry and
potential repository for future studies
of other possible pharmacogenetic
associations.
OTHER PROJECTS
The Role of Natural Killer Cells in Solid
Organ Transplantation
Jeroen van Bergen and Ilias Doxiadis
International Histocompatibility Working Group in Haematopoietic Cell
Transplantation
Effie Petersdorf and Mari Malkki
Next Generation HLA Sequencing
Alison Castley, Richard Allcock
EFI Region 8 H & I Laboratories Meeting
in Athens
On January 2011, the EFI region 8 H
and I Laboratories meeting took place
in Athens, Greece. It was organized by
the Immunology and Histocompatibility
Dept of “Evangelismos” Hospital under
the auspices of EFI and the Scientific
Committee of “Evangelismos” Hospital.
Participants from EFI Region 8 countries, Albania, Armenia, Cyprus, Israel,
Romania, Serbia, Turkey and Greece
and from countries outside Region 8,
Netherlands, Germany, Austria and
Switzerland met at “Doma” of “Evangelismos” Hospital, a room with a superb
view of the city of Athens, Acropolis and
the sea of Piraeus.
The meeting was opened by Dr Chryssa
Papasteriades, who welcomed the participants stressing the value and the
significance of these meetings that
open the way for scientific cooperations and contribute to the amelioration of our Laboratories, tighten bonds
between EFI accredited laboratories
and draw new labs to the EFI family.
The chairman of the EFI Accreditation
committee, Prof. G. Fischer and the EFI
President, Prof. I Doxiadis greeted the
audience and expressed their satisfaction and pleasure for this meeting. The
president of the Hellenic Transplant
Organization, Prof. I. Vlachogiannis,
welcomed the audience and underlined the necessity of accreditation
of histocompatibility laboratories for
transplantation. Dr I. Datseris, KESY
vice president and Ministry of Health
representative, congratulated Dr Papasteriades for the meeting organization
and referred to her efforts to establish and enforce EFI accreditation to
all histocompatibility labs dealing with
transplantation and to combine it with
national accreditation.
Finally Hospital Manager Mr. M. Theodorou expressed his pleasure hosting this interesting meeting, stressed
the work performed by the Immunology
– Histocompatibility Dept of Evangelismos Hospital under the direction of
Dr. C. Papasteriades and wished every
success to the meeting and a pleasant
stay of the participants in Athens.
The scientific program was opened by
Dr C. Papasteriades, who presented
EFI Region 8 accredited laboratories
and laboratories interested in EFI
accreditation. She presented subjects
of interest, EPT schemes, difficulties
per standards category, educational
efforts and perspectives. It is noteworthy that there are 21 accredited labs in
the Region while during the last 6 years
with Dr C. Papasteriades as a Commissioner 14 labs acquired EFI accreditation and two more are ready for their
first on site inspection.
Prof G. Fischer (Austria), chairman of
EFI Accreditation Committee, presented
the whole picture of EFI accredited
laboratories and stressed the value of
accreditation program in everyday life.
Prof. I. Doxiadis (The Netherlands), EFI
Board President, presented a wonderful
speech entitled: “Opening the Pandora
box using solid phase assays”, very
informative and useful in transplantation and other areas.
Prof A. Sanchez-Mazas (Switzerland),
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presented during her talk entitled
“Population genetics” very interesting results of an excellent project and
impressed everybody with the amount
of information and data analysis.
“Organising a Bone Marrow Donor Registry in a small country” was the subject of Dr Frieda Jordan’s presentation.
Program, method, efficiency, offer of
the organisers and the results of these
efforts impressed everyone in the audience. Indeed the work performed by the
Armenians for the organisation of the
Armenian Registry of volunteer HCSs
donors should serve as an example to
be imitated.
Dr R. Loewenthal’s presentation entitled “How to prepare packet A/C and
B and the on site inspection” was very
useful and Prof. Il. Constantinescu’s
speech on “Improvements in the Romanian QC program for Histocompatibility” was very interesting.
During lunch time we enjoyed a nice
buffet with Greek relishes together with
the beautiful view of the city of Athens
and we were given the opportunity to
talk with our old friends and get to
know new ones.
After lunch Prof. J. Mytilineos (Germany), chairman of EFI Educational
Committee, gave an extremely useful
presentation “The new EFI educational
Committee: New people, new tasks,
new challenges”. We understood EFI
educational program, we saw the possibilities and opportunities, updates
and further education we could exploit
for our Depts.
Presentation and evaluation of Balkan
EPT, organised by Prof M. Carin and Prof
F. Oguz in Istanbul University-Turkey, for
HLA typing and by Prof Eli Naumova
and Assoc. Prof A Mihaylova in Sofia
University-Bulgaria for HLA B27 typing
and for HLA abs screening were among
the meeting purposes. Prof F. Oguz
presented the results, problems and
perspectives of last year Balkan EPT.
A constructive discussion followed. We
thank the organisers of the two EPT
schemes for their efforts.
A very interesting part of the Meeting
was the last session, where EFI Region
8 laboratories had the chance to present their experience, their problems,
their hopes, their plans. Several Laboratories came along with subjects of
different and interesting context and a
very constructive discussion among all
participants took place.
In the evening we were all invited to
dinner in a Greek taverna in the old
part of Athens, Plaka. The food was
superb, the wine was superb, the environment was great but, most importantly, the company, the discussions
among friends created a delightful
atmosphere.
Next morning the meeting was completed with a guided tour to the New
Acropolis Museum, the jewel of the city
of Athens, an unforgettable experience.
It has been a great and fruitful meeting. Dr C. Papasteriades and Immunology – Histocompatibility Dept of
“Evangelismos” Hospital did their best
and ensured the success!
We are looking forward to the next one!
Katherina Psarra MSc,
PhD, EurClinChem
Dept. of Immunology-Histocompatibility
“Evangelismos” Hospital,
Athens-Greece
15
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Report on my visits to the Medizinische
Hochschule in Hannover
Mahendra Narain Mishra,
Chief Consultant Pathologist, Dr Lal Path Labs Pvt Ltd. Delhi
I am an Immunopathologist from India
who has been supervising tissue-typing
by SSP and serology for kidney recipients and patients being worked up for
BMT in addition to crossmatch and
Panel Reactive antibody (PRA) estimation. This was in addition to my duties
as a Pathologist and teaching undergraduates and postgraduates and all
my work was done with the help of a
single technician. So many times, I was
directly a bench worker too.
Prior to the EFI conference in Ulm, I had
visited the website of the Medizinische
Hochschule in Hannover and then got
in touch with Professor Rainer Blasczyk, Laboratory Director. Dr Blasczyk
was very kind and agreed to impart me
training in Sequencing Based typing as
well as agreed to test some difficult
samples for me. I was in his department from 12-22 May 2009. The entire
staff was extremely positive, warm
and caring. I had a problem in that the
DNA concentrations were very low and
was able to circumvent the problem
by using a greater volume i.e. 80-100
micro liters instead of 8-10 micro
liters. To our pleasant surprise, we
got the results on SBT for all samples.
SBT helped us to confirm two cases
of crossing over, one at DRB1 and
another at the A locus. Subsequently
I presented a poster in the 2009 ASEATTA meeting at New Delhi on the case
of crossing over,
I next visited the same laboratory for
the period October 4-15 2010, while
returning from the ASHI meeting last
year and spent there eleven days. This
time I got an insight into Luminex Technology – a subject on which there were
many papers in ASHI meeting. It was
an opportunity to closely study antibody
detection by Luminex including donor
specific crossmatch, single antigen
detection and also HLA typing by SSO.
I also saw the method of Complement
Dependent Cytotoxicity closely and
hope to incorporate it in my laboratory
as it is a considerable improvement on
what is being performed currently.
Hannover was like a second home to
me and my visits on both occasions
were an eye opener. The first visit
served also to validate our SSP testing as 95% of the work was correct in
spite of our constraints. The work on
Luminex will certainly help me in my
new job as it has given me a certain
amount of familiarity with the technology.
From this place I wish to express my
profound gratitude to Dr Blasczyk and
his team for facilitating my stay, testing my samples and also for training
me in Luminex. I hope to continue my
association with the Institut für Transfusionsmedizin Medizinische Hochschule
Hannover for ever.
Report on my visit to the Reference Laboratory of
Eurotransplant in Leiden, The Netherlands
Angeliki Vittoraki
MD, National Tissue Typing Centre, General Hospital of Athens ‘G. Gennimatas’, e-mail: avittor2@yahoo.com
I would like to thank the EFI committee
for the bursary that I received for my
three week visit to the Department of
Immunohaematology and Blood Transfusion of the Leiden University Medical
Center. I visited both the Diagnostic and
the Eurotransplant Reference Laboratory
(ETRL) in Leiden. Currently I am working
in the National Tissue and Typing Center
of “G. Gennimatas” Hospital in Athens.
My visit to Leiden allowed me to meet
people who are working in the same field
and to exchange ideas on different topics
concerning transplantation.
The aim of this training was to improve
my knowledge in the transplantation field
and also to learn the Acceptable Mismatch (AM) program, which is run by the
ETRL. The obtained expertise and skills
will be used in our laboratory in Greece
to ameliorate the testing and defini-
tion of the “difficult” patients which are
included in the AM. We aim to evaluate
and introduce the way of working with
these patients in the AM in Eurotransplant, learning also from the difficulties of
such a program and making use of the
expertise established in Leiden for more
than 23 years. In the long term a closer
cooperation is aimed.
During the first two weeks of the training, I learned more about the techniques
used concerning HLA typing and screening in the Diagnostic laboratory of the
section Immunogenetics and Transplantation Immunology (ITI). Specifically,
Marian Witvliet gave me information on
the definition of AM program for highly
sensitized patients awaiting kidney transplantation as well as about the HLA
Matchmaker algorithm. I became familiar with the screening for HLA antibod-
ies via different solid phase techniques
as ELISA, Dynachip and CDC as well. In
addition, I followed the crossmatch procedures both for living (un)related and
postmortal kidney transplantation. Moreover, I had the opportunity to see the and
use the line probe method for HLA class
II low resolution typing and also the low
resolution typing for HLA class I. I was
pleasantly surprised with the cost of the
former “home made” technique, which is
less than 1 euro per typing. Furthermore,
I had the opportunity to visit Eurotransplant, where the medical director Dr Axel
Rahmel and his colleagues gave me a
warm welcome and they were more than
happy to give me information on the way
of allocating organs.
During the last week, I followed the procedures of the ETRL regarding the organization and analysis of the External
17
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You already rely on our LABType® products to identify many rare
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0197
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Proficiency Testing (EPT) Exercises for
laboratories participating in solid organ
transplantation and also the Internal
Quality Control of the ITI laboratory and
the Blood Transfusion Laboratory. Furthermore, I was involved in the testing
of new methodologies for the crossmatch procedure before transplantation,
specifically ELISA crossmatch. Together
with Yvonne Zoet and Simone Brand we
tested using ELISA crossmatch 14 known
human monoclonal antibodies and 5
cells lines expressing a single HLA specificity (Single Antigen Lines). Further work
is required in order to assess the utility
of this assay.
Additionally, I had the opportunity to participate in the weekly internal educational
meetings of the department. I also followed the defense of the thesis of a PhD
student. It broadened my horizons while
at the same time gave me new ideas
for continuing my
research in transplantation.
Finally, I would like
to thank Prof Dr
Frans Claas and
Prof Dr Ilias Doxiadis for offering
me the opportunity
to visit the Department and to learn
from their experience. I would also
like to thank Dr
Dave Roelen and
the remaining lab
staff for their hospitality. It was really wonderful to get to know them all and spend
three unforgettable weeks of valuable
training in the field of transplantation and
also to share with them the traditional
‘Sinterklaas’ celebrations.
I thoroughly enjoyed my stay in beautiful
Leiden and I look forward to seeing my
new friends in our laboratory in Athens
some time soon.
Report on the extramural Eurotransplant meeting
in Essen on the relevance of pretransplant HLA
specific antibodies in clinical transplantation
On the 25th of March, 2011 more than
eighty members of the Eurotransplant
Tissue Typers Community from several
countries found their way to Essen, Germany where the Extramural meeting of
Eurotransplant took place. The meeting
took place in the localities of the Institute for Transfusion Medicine, at the
Robert Koch Building and was hosted
by Peter Horn and his group. Almost
17 years ago, the first extramural meeting of Eurotransplant took also place in
Essen and concerned the same topics
i.e. screening and crossmatching and
their relevance in clinical transplantation, although the techniques used at
that time were completely different.
The current meeting was dedicated to
the use of the modern techniques (solid
phase assays (SPA) and in particular
Luminex based single microspheres
assay (LUM-SA)) for HLA antibody detection and their role in clinical (kidney)
transplantation. It was the first of many
similar meetings which for sure will
take place in the near future. Four main
topics were selected: Donor specific
antibodies (DSA): contraindication vs.
risk factor; the relevance of repeated
HLA class I and II mismatches; how
to report screening and crossmatch
results to clinicians; the role of these
methods in external proficiency testing.
The meeting started with the welcome
address of the local organizer and continued with a state of the art lecture on
the relevance of the pretransplant HLA
antibodies defined by LUM-SA, focusing
on the item: DSA contraindication or risk
factor. This was an excellent “warmingup” for the discussion of the topics mentioned above. However, prior to report
on the further discussion it is important
to state that in an organ exchange organization there are two important levels
where the degree of sensitization the
patient plays a crucial role: the allocation and the actual transplantation.
Since almost fifteen years transplantation centers within Eurotransplant must
report which HLA antigens cannot be
accepted for a given patient: the unacceptable HLA mismatches, a procedure
in the meantime known as virtual crossmatching. These unacceptable HLA antigens play a pivotal role in the allocation
process both in influencing the number
of points a patient receives at the allocation and in preventing that a patient
gets a kidney offer from a donor, who
expresses one or more of these unacceptable HLA mismatches. During the
last years and especially since the introduction of antibody treatment regimens,
some centers do accept organs towards
which the patient is sensitized considering these DSA rather a risk factor than a
contra-indication for transplantation.
The participants of the meeting spend
a long time to define and discuss the
current contraindication parameters
for allocation and transplantation. This
discussion dominated the whole meet19
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ing. Finally, the participants decided to
consider pretransplant DSA towards
HLA-A, B and HLA-DR which can activate complement a contraindication for
transplantation. Furthermore, antibodies against HLA-DQ and HLA-C can be
reported as unacceptable mismatches
when the transplantation center does
not accept such mismatches for a given
patient. HLA specific antibodies seen in
solid phase assays only, like ELISA or
Luminex based assays, are regarded as
risk factor. The complement depended
cytotoxicity (CDC) assay remains the
method of choice for the crossmatch
requiring also a screening by this
method.
Clinicians must receive all information
regarding the results of the screening assays done. This should be done
preferably in weekly meetings but also
during duty hours the information flow
must remain. Since solid phase assay
defined antibodies are thought to be
less harmful, specific antibody reducing / removing regimens can be used
according to the local policy of the transplantation center.
A lunch was served with soup and
local and also Bavarian specialties
as sandwiches with Met, Leberkäse
or Fleischsalat (translation can be
retrieved on line)!
Interesting was the discussion on the
local definition of cut-off values for CDC
and LUM-SA! For CDC the majority of
the participants use a cut-off of score
4 (25-40% lysed cells; N=22/29 participating laboratories) and for LUM-SA the
majority 10/21 use a variable cut-off
value depending on the patient immunological set-up and history. Here, a variety of fixed values were reported from
5,000 MFI to 1500 MFI. Comparing the
results to previous reports we observe
a trend towards variable quoting of cut-
off, a step in the right
direction.
Most
participants
enter repeated HLA
mismatches as unacceptable
antigens
especially when antibodies towards them
are detected. Earlier
studies showed a differential influence of
repeated HLA class II
mismatches in clinical transplantation.
When grafts are lost
early (the first year)
HLA class II mismatches should not be
accepted for a retransplant whereas
after a longer survival of the first graft
repeated HLA class II mismatches are
no contra-indication. In the absence of
antibodies HLA class I mismatches have
no negative influence as was shown in
several international, multicentral studies. For the Eurotransplant Acceptable
Mismatch program repeated HLA mismatches are accepted if the patient
does not have formed HLA specific
antibodies in either
CDC or LUM-SA after
transplantation.
The last part of
the meeting was
dedicated to the
External Proficiency
Testing, which is
an integral part of
all HLA based testing within an organ
exchange organization. Making a long
story short, the EPT
will change because
of the needs of the
participating laboratories and especially because of the
future developments in the allocation
of organs. In addition to the currently
requested typing for HLA-A, B and HLA-DR
also HLA-C and HLA-DQ typing will be
required. Sera sent to the participants
for screening for HLA specific antibodies
must be screened by the participants by
CDC. They can in addition be screened
by solid phase assays as ELISA and
LUM, where part (but certainly not all) of
the results maybe helpful to define the
specificities of the antibodies causing a
positive CDC reaction. Furthermore, the
sera can also be screened by LUM-SA.
All three possibilities are now available
and for every one of them a certificate
will be released. The same serum will
also be used for the future crossmatch
procedure. The participating laboratories will be informed accordingly by separate mail.
Future recommendations of the Tissue
Typers Advisory Committee will arise
from the above said, heading to a new
direction in HLA related transplantation
immunology matters. In summary the
following was proposed and accepted by
the participants of the meeting:
- Typing of HLA-C and HLA-DQ should
become mandatory
- Complement activating antibodies
against HLA-A, HLA-B and HLA-DR
must be avoided for transplantation.
Complement activating antibodies
against HLA-C and HLA-DQ represent
a contraindication upon agreement
of the transplantation center. Noncomplement activating antibodies
against HLA form a risk factor.
- Repeated HLA-class I and II mismatches are a contraindication for
transplantation if antibodies against
them can be defined in either CDC or
SPA at any time after graft loss.
- Clinicians must be informed on all
donor specific antibodies a patient
has. Positive crossmatches due HLA
specific antibodies are a contraindication for transplantation while DSA
not leading to a positive crossmatch
can be accepted after information
and acceptance of the clinician.
- The External Proficiency Testing for
Typing will be extended to HLA-C and
DQ.
- For the EPT on screening for HLA specific antibodies CDC is mandatory.
Screening with solid phase assays
only and Luminex SA will become
separate types of screening.
- A serum EPT crossmatch is introduced and will be used for problematic cases.
It was a memorable meeting!
21
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HLA-NET Training School – Population Analyses
for HLA and other Immunogenetic Systems –
Porto 21st Feb-24th Feb 2011
Claire Burt, Trainee Clinical Scientist, NHS Scotland, claire.burt@nhs.net
21st Feb
The HLA-NET Training School was
held in Porto and focused on
population analyses for HLA and
other immunogenetic systems. The
training school had ~30 attendees
from different scientific backgrounds
and countries, with a shared interest
in HLA. The training school began with
an introduction by the organisers and a
representative from COST, the funding
body who allowed people to attend the
training school with the help of a grant.
José Manuel Nunes from the University
of Geneva gave the first lecture, an
introduction to formats for population
data. Here, he discussed the basic
concepts of population data, and
essential tests such as Hardy Weinberg
and linkage disequilibrium, which we
would become more familiar with as
the training school went on. Following
coffee, we began our first practical
session, led by José, which introduced
us to the statistical packages that we
were to be using over the course of
the training school. These included R,
Arlequin, Textpad and Gene[RATE]. The
first tasks involved converting data files
into formats that could be used with
View of the river in Porto
each program, analysing the data and
creating graphics based on the results.
We also found out how to deal with
ambiguities that may be present in data.
Following a lovely lunch in a local café,
the afternoon began with an excellent
presentation by Dario Ligeiro from the
Centro de Histocompatibilidade do
Sul in Lisbon. He gave an overview
of the HLA system, explained the
nomenclature and discussed the
role of HLA in transplantation. The
afternoon consisted of more practical
sessions, using Hardy Weinberg to
compare populations and estimating
frequencies.
22nd Feb
The morning began with a lecture
from Stéphane Buhler who discussed
tests that could be used to compare
populations. These include the EwensWatterson test, which analyses
the number of homozygotes and
heterozygotes in a population and uses
this to determine if the population
follows the neutrality hypothesis, and
he also discussed the calculation of
genetic distances. Following the lecture,
we moved up to the computer room to
work on our practical exercises for the
day. We were given data consisting
of HLA-DRB1 types for populations
around the Mediterranean Sea. We
used Arlequin to analyse the data
to determine if the allelic frequency
distributions in the populations were
comparable with those expected
distributions under the hypothesis
of selective neutrality. We then used
R to perform a multidimensional
scaling analysis, which showed a
graphical representation of the genetic
differentiation of these populations.
After lunch we had a very interesting
talk by Hans-Peter Eberhard from the
German National Bone Marrow Registry.
He discussed the practicalities and
problems associated with analysing
registry data, highlighting the problems
with ambiguous and missing data,
as well as describing the maximum
data required to get the best match
predictions. This was followed by
another practical session, focusing on
population genetics and analysis of
genetic variance.
23rd Feb
The morning commenced with a couple
of interesting talks by Berta Martins
da Silva (ICBA) and Denisa Mendonça
(ICBA/ISPUP). Berta discussed multiple
sclerosis disease mechanisms, and
the association of HLA-DRB1 alleles,
smoking and HLA-A*02 with the
development of disease or conferring
protection from MS. She introduced
the concept of relative risk and odds
ratio, which was discussed further by
Denisa. Denisa detailed the statistical
tests that are used when performing
association studies, for example to
determine if the presence or absence
of a particular allele is associated with
disease development or protection.
The following practical session was
based on data from a study looking
at the role of HLA-DRB1 alleles on
MS susceptibility and outcome in a
population of Portuguese patients.
The afternoon session was a talk on
databases for population genetics, by
Christelle Vangenot from the University
of Geneva. She enlightened us as
to how useful databases can be for
storage and handling of datasets,
as well as showing us how to design
a basic database. The final practical
session was a continuation of the
work in the morning, with disease
associations and HLA. For our final
23
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Join us at our HLA-SBT Teaching Sessions in 2011!
Middle East Conference, Riyadh, Saudi Arabia
Thursday 14th April 2011
25th EFI, Prague, Czech Republic
Wednesday 4th May 2011
37th ASHI, New Orleans, Louisianna, USA
Monday 17th October 2011
The ABHI accredited courses are free of charge and lunch is included so make sure you
register soon, seats are limited! For more information and registration visit
www.gendx.com and go to Education.
re
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© 2011 GenDx V110314
evening in Porto we had a lovely meal in
a Portuguese restaurant and listened
to some traditional Fado music, as well
as getting the chance to sample the
Porto nightlife!
Acknowledgments: HLA-NET is funded
by COST (Action BM0803)
http://www.cost.esf.org/domains_
actions/bmbs/Actions/BM0803-
A-European-Network-of-the-HLADiversity-for-Histocompatibility-ClinicalTransplantation-Epidemiology-andPopulation-Genetics-HLA-NET-End-dateJanuary-2013
24th Feb
The final day of the training school was
free time on the computers, allowing us
to put to use all of the techniques we
had learned and use them on our own
data. I found the course particularly
useful with respect to learning how
to format data in the correct way and
knowing which statistical tests were
appropriate. All in all the training school
was very informative, useful, and we
managed to squeeze in some time to
see Porto!
HLA-NET Training School members at a
traditional Portuguese restaurant
HLA-NET Training School members at a traditional Portuguese restaurant
Report from the
“EFI Education and Training Bursary”
Study of ligands of the adaptor molecule
3bp2 in haematopoietic cells
Anna Gieryng,
Dept. of Clinical Immunology, L. Hirszeld Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, Poland, e-mail: anna.gieryng@gamil.com
The time which I spent under the care
of Dr Margarita Martin-Andorra gave
me the new knowledge about the new
molecular biology techniques and ideas
for future. Our cooperation had an influence on the science network between
my host laboratory L. Hirszfeld Institute
of Immunology and Experimental Therapy, PAS (Wroclaw, Poland) and Department of Biochemical and Molecular
Biology, UB (Barcelona, Spain).
The main point of the present work
was to evaluate a possible interaction
between 3BP2, the molecular adaptor
and Myo1f protein.
The SH3-binding protein 2 (3BP2) is a
cytoplasmic adapter protein, originally
identified by its interaction with the
SH3 domain of the Abl protein-tyrosine
kinase [1]. It has been described that
one of the biologic functions of this
adapter is the organisation of the protein macromolecular complex in the
cytoplasm in many immune cells. The
3BP2 gene is located on chromosome
4 (4p16.3 region). The human 3BP2
transcripts are express in spleen,
peripheral blood leukocytes, testis,
and ovary. The amino acid sequence
(561aa) of the 3BP2 is composed of:
a N-terminal PH domain, a proline-rich
domain and a C-terminal SH2 domain
[2]. The 3BP2 adapter is preferentially
expressed in hematopoietic tissues
and regulates transcriptional activities
via calcineurin- and Ras-dependent
pathways in T lymphocytes [2]. The
3BP2 positively regulates B cell receptors (BCR) [3], and 3BP2-deficient mice
have defects in achieving optimal B
cell activation and thymus-independent
humoral responses [4, 5]. In NK cells
3BP2 regulates cell-mediated cytotoxicity via its PH, SH2, and proline-rich
regions. Moreover, phosphorylation of
the Tyr183 on the 3BP2 protein, which
recruits the Vav-1 and the PLC-�, is
necessary for the 3BP2 to positively
regulate NK cell-mediated killing [6].
25
Announcement
19th Annual Meeting
of the
Deutsche Gesellschaft
für Immungenetik
(DGI)
September 22nd – 24th, 2011
Charité – Universitätsmedizin Berlin
Congress presidents
Marion Nagy and Constanze Schönemann
Fon: +49 30 450 525012/553223
Fax: +49 30 450 525912/553955
E-mail: marion.nagy@charite.de, constanze.schoenemann@charite.de
For program and further details, please visit the official website of the Deutsche Gesellschaft für Immungenetik (DGI)
http://www.immungenetik.de.
26
Previous work from Dr. Martin-Andorra
group showed that 3BP2 binds to the
Y337 in the CD244 cytoplasmic tail
and increases the CD244-mediated
cytotoxicity [6].
Myosins are actin-dependent molecular motors that use the energy of ATP
hydrolysis to move along actin filaments [7]. Most of the myosin heavy
chains proteins contain the three special regions. The first region named an
NH2-terminal motor or head domain
is responsible for actin binding and
ATP hydrolysis, the neck region binds
light chains, and the COOH terminal tail which binds cargo and/or is
responsible for dimerization of heavy
chains. The Myo1f gene is located on
the chromosome 19 (19p13.3-p13.2)
[8]. The Myo1f transcript is selectively
expressed in the spleen, mesenteric
lymph nodes, thymus, lung, NK cells,
macrophages, and dendritic cells [9].
It has been demonstrated that the
unconventional Myo1f gene is fused to
the MLL in infant acute monocytic leukaemia (AMoL) with a complex translocation involving chromosome 7, 11,
19 and 22. It has been published that
ENL, ELL/MEN and EEN genes are the
fusion partners of the MLL gene. Taki T.
found that the Myo1f gene on chromosome 10p13p is a novel fusion partner
of the MLL gene in a patient with AMoL
in the 2005 [10].
The Two-Three Hybrid system and the
immunoprecipitation with anti-3BP2 Ab
have been used to identify new 3BP2
ligand, and also to evaluated if this
interaction was dependent or independent on adapter´s phosphorylation.
We found that the cytoplasmic adaptor, 3BP2 associates with the Myo1f
protein. It can be supposed that this
interaction can play a key role in the
modulation of cell adhesion and motility in the immune system. The selectively expression pattern of the Myo1f
gene has been observed in the most
important organs and components of
the immune system [9]. It has been
demonstrated that cells from the
Myo1f deficient mice exhibited abnormally increased adhesion and reduced
motility. In vivo, Myo1f-deficient mice
showed increased susceptibility to
infections and an impaired neutrophils
response. The 3BP2 protein manages
the leukocyte proliferation, T-cell activation, NK cell-mediated cytotoxicity
and cells migration. The interaction
between the Myo1f and its adaptor,
3BP2 could be the one of the important points of the motility pathway of
immune system cells.
References
[1] Ren R, Mayer BJ, Cicchetti P, Baltimore D. Identification of a ten-amino
acid proline-rich SH3 binding site. Science. 1993; 259(5098):1157-61.
[2] Deckert M, Tartare-Deckert S, Hernandez J, Rottapel R, Altman A. Adaptor
function for the Syk kinases-interacting
protein 3BP2 in IL-2 gene activation.
Immunity. 1998; 9(5):595-605.
[3] Foucault I, Liu YC, Bernard A, Deckert M. The chaperone protein 14-3-3
interacts with 3BP2/SH3BP2 and regulates its adapter function. J Biol Chem.
2003; 278(9):7146-53.
[4] Chen G, Dimitriou ID, La Rose J,
Ilangumaran S, Yeh WC, Doody G,
Turner M, Gommerman J, Rottapel R.
The 3BP2 adapter protein is required
for optimal B-cell activation and thymusindependent type 2 humoral response.
Mol Cell Biol. 2007; 27(8):3109-22.
[5] de la Fuente MA, Kumar L, Lu B,
Geha RS. 3BP2 deficiency impairs the
response of B cells, but not T cells, to
antigen receptor ligation. Mol Cell Biol.
2006; 26(14):5214-25.
[6] Saborit-Villarroya I, Del Valle JM,
Romero X, Esplugues E, Lauzurica P,
Engel P, Martín M. The adaptor protein
3BP2 binds human CD244 and links
this receptor to Vav signaling, ERK activation, and NK cell killing. J Immunol.
2005; 175(7):4226-35.
[7] Krendel M, Mooseker MS. Myosins:
tails (and heads) of functional diversity.
Physiology. 2005; 20:239-51.
[8] Hasson T, Skowron JF, Gilbert DJ,
Avraham KB, Perry WL, Bement WM,
Anderson BL, Sherr EH, Chen ZY, Greene
LA, Ward DC, Corey DP, Mooseker MS,
Copeland NG, Jenkins NA. Mapping of
unconventional myosins in mouse and
human. Genomics. 1996; 36(3):431-9.
[9] Kim SV, Mehal WZ, Dong X, Heinrich V, Pypaert M, Mellman I, Dembo M,
Mooseker MS, Wu D, Flavell RA. Modulation of cell adhesion and motility in
the immune system by Myo1f. Science.
2006; 314(5796):136-9.
[10]
Taki T, Akiyama M, Saito S, Ono
R, Taniwaki M, Kato Y, Yuza Y, Eto Y,
Hayashi Y. The MYO1F, unconventional
myosin type 1F, gene is fused to MLL
in infant acute monocytic leukemia
with a complex translocation involving
chromosomes 7, 11, 19 and 22. Oncogene. 2005; 24(33):5191-7
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