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EUROPEAN FEDERATION FOR IMMUNOGENETICS

NEWSLETTER JANUARI 2008 - ISSUE 55

…………..FROM THE EFI PRESIDENT

Toulouse, oh Toulouse ! La « ville rose » will welcome the 2008 EFI annual con-ference.The scientifi c program is as attractive as is exciting the cultural city and I encourage all of you to join and enjoy.Also, 2008 will be the year of the 15th International Histocompatibility and Immunogenetics Workshop and Confer-ence in Brazil. I sincerely hope that 2008 will make progress towards a structured inter-national organization in which EFI will have an essential and proactive role.On the professional side of our Soci-

ety, 2008 will be a landmark. Over 200 laboratories are accredited by EFI. The challenge is for us to be ready for the next 200 !!!

EFI is engaging a refl ection on how to best contribute to the International development of accreditation schemes in all the Eastern European states, in the Mediterranean area, in the Middle East and also in Asia, and in South America in collaboration with ASHI, ASE-ATTA and other national and regional organizations that share our goals, values and projects. I have secured

DEAR EFI MEMBERS:

the participation of EFI to AHCTA and Poseidon which are instrumental for this development and we will all work together toward this important goal.

I wish every EFI member and one’s family a New Year 2008 full of success, joy and serenity.

Dominique CHARRONEFI President

MESSAGE FROM THE PRESIDENT

....FROM THE EDITOR’S DESK

IMPORTANT ANNOUNCEMENT TO EFI MEMBERSFor security reasons we need to change the username password for the EFI web-site on a regular basis. Changes to the password will be announced in the EFINewsletter and will apply from the publication date of the Newsletter in which thechange is announced.The new username and password are:

Username: efi userPassword: BarcelonaURL: http://www.efi web.org/members/

CONTENTSFrom the EFI President 1From the editor’s desk 3New EFI President-Elect 5Summary of the EFI executive committee meeting 5Election of new members of the EFI Executive Committee 2008 6New horizons in Immunogenetics and Histocompatibility 8Satellite Session at EFI Meeting 9Proposal of candidates for the Julia Bodmer young scientist award 2008 11Future EFI conferences 11Update on EFI ept issues 13Proposals for EFI Standards version 5.6 132007 European Clinical Histocompatibility Workshop, Rome, Italy. 21Conference on Quality Assessment 25Annual meeting of the Croatian immunological society 2007 26Report of the French 11th educational meeting 27Report on the 4th EFI Summer School 28Reports of EFI bursary recipients on the meeting in Barcelona. 29

Also from the editors desk a happy New Year. Hopefully 2008 will bring you all happiness and good health. First of all I would like to congratulate Steve Marsh with his appointment as president-elect. I am sure that he will be an excellent president for our federation.In this issue, the members are invited to vote for new councilors. It is good to see that several excellent candidates are available.The EFI meeting in Toulouse is coming near and the very attractive program is certainly for many of you a good reason to visit this beautiful city in the south of France.The deadline for applications for bursaries has been extended to February 15 and , please, do not forget to nominate candidates for the Julia Bodmer young scientist award!In this Newsletter you will also fi nd reports from the different committees including some proposals for adaptations of the standards.The International Summer school on Immunogenetics in Pecs was again a great success as described by one of the participants. Next year the summer school will be organized in Brazil just before the International Workshop. Furthermore, several reports of local meetings are included. Please note the extremely beautiful places, where these meetings have been organized.Hopefully, you all enjoy reading this Newsletter and I am looking forward to your contributions to the next one.

Frans Claas

The deadline for contributions to EFI newsletter no 56 is April 24, 2008. Please send your contribution to Frans Claas, Leiden, the Netherlands by e-mail: fhjclaas@lumc.nl

EFI websitehtt://www.efi web.org

Editor-in-chiefFrans 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 2007

EFI President:D. Charron (France)

President-elect: S.G.E. Marsh (UK)

EFI Secretary:M. Tilanus (Holland)

EFI Treasurer:C. Raffoux (France)

EFI deputy Treasurer:A. Moine (France)

Membership Secretary:S. Mesander (Holland)

Councillors:K. Fleischhauer (Italy)

E. Naumova (Bulgary)

I. Doxiadis (Holland)

A.M Little (UK)

C. Navarrete (UK)

R. Blasczyk (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

The editor and the EFI offi cers 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

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We just received the information that Prof. G.B. Ferrara has passed away. A great loss for the EFI community. An obituary will appear the next issue of the Newsletter

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NEW EFI PRESIDENT-ELECTNominations were sought for the position of EFI President-Elect. Dr Steven Marsh was nominated for this position, and no further nominations were received. Due to the delay in seeking nominations for this position he will take offi ce immedi-ately. His CV is given below.

Dominique Charron

Dr Steven GE Marsh, London UK

I am the Deputy Director of Immunoge-netics at the Anthony Nolan Research Institute (ANRI) in London. After obtain-ing my fi rst degree in Biochemistry from Imperial College, University of London, in 1981, I spent three years at the Blood Transfusion Centre in Oxford, where I was fi rst introduced to HLA. In 1985 I moved to the Tissue Antigen Laboratory at the Imperial Cancer Research Fund where I worked with Julia Bodmer for eleven years and was awarded a PhD for my work on Epitope Mapping in Major His-tocompatibility Systems in 1996. I Chair the WHO Nomenclature Committee for Factors of the HLA System having been active on this committee since 1989, and more recently the KIR Nomenclature Committee. I have been responsible for the maintenance of a database of HLA sequences since 1989, and since 1998 the IMGT/HLA Sequence Database. I currently head a research group at the

ANRI where much of my work has con-centrated on the understanding of HLA polymorphism in terms of the serological epitopes and also in understanding the role of HLA matching in haematopoietic stem cell transplantation. I have over 240 publications in the HLA fi eld, and serve on the editorial boards of Tissue Antigens, Immunogenetics, Human Immunology and the International Journal of Immunogenetics. I have participated in the last fi ve International Histocompat-ibility Workshops, and was elected as an International Histocompatibilty Councillor following the 13IHWC. I was elected as an EFI councillor in 1999 and have been EFI Deputy Secretary since 2002. I look forward to continuing to serve EFI as its next President and building on the work of previous presidents in maintaining EFI as a high profi le society providing expertise within the clinical and academic world of immunogenetics.

SUMMARY OF THE EFI EXECUTIVE COMMITTEE (EC) MEETING

Saturday 10 November 2007; Castle Vaeshartelt, Maastricht

Annual EFI meetingsThe EFI meeting in Barcelona was evalu-ated and the board thanks and congratu-lates Jordi Vives and Antonio Nunez-Roldan for the excellent organisation of the EFI 2007 annual meeting. The annual EFI meeting in Toulouse is approaching rap-idly with early registration available until February 1st, 2008. Mogens Thomson with his local organising committee are working with various EFI committees and the meeting liaison, Ralf Wassmuth, to make sure that the Toulouse meeting is unforgettable scientifi cally, educationally and socially. In addition to the selection of the best oral abstracts, three best posters will be selected and awarded. Subsequent EFI meetings will be held in ULM, Ger-many 2009, May 9-12, chaired by Joan-nis Mytilineos; Firenze, Italy 2010,May 15-18, chaired by Katharina Fleischhauer and for 2011 the board selected Prague, Czech Republic which will be chaired by Toni Slavcev (May 4-7).

The new EFI website is currently being developed and it is the intention to launche this website in 2008. The new website will facilitate an easier exchange of documents and improve the stream of information to the mem-

bers. EFI membership cards will also be provided via the web. Several new features will be included such as indi-vidual member access; accreditation forms and information will be made available electronically.

The Chair of the education committee, Paulo Santos, is unable to continue as chairman for personal reasons. As there are a number of EFI Education issues that need immediate address-ing, the EFI board is pleased to appoint Cristina Navarrete as the new Chair of the education committee. We would like to thank Paulo for all his efforts under-taken whilst Chair of this committee and wish him all the best for the future.

The EFI booth was visited frequently during the Barcelona meeting and many members paid their fees at the booth, either by cash or were helped through the online payment system using their credit cards. Suggestions and compliments can be put forward to the booth. The board thanks Sonja Mesander, James Robin-son, Colette Raffoux and Agnes Moine for occupying the booth. At the Toulouse meeting the booth will have a central position at the registration area.

The board is proud to announce that Professor Dr Hans-Georg Rammensee

accepted the invitation as Ceppellini Lecturer at the annual meeting in Tou-louse, 2008.

Elections: No other nominations for the vacancies of president elect and deputy secretary were received. We are pleased to announce that Steven Marsh is appointed president elect and wish him success for realising his ideas and contri-bution to the EFI community. Ann-Marga-ret Little was nominated for the position of deputy secretary, she will take up her position following the EFI meeting in Tou-louse. Four nominations were received for the three positions of councillor, the CVs of the candidates and the voting form is included in this newsletter.

Poseidon; Ilias Doxiadis and Elisavetta Naumova are currently interacting with Ann Cambon-Thomson on how the Poseidon projects will be organised in relation to the EFI -education, accredi-tation and EPT committees.

Marcel G. Tilanus, EFI secretary

THE EFI EXECUTIVE COMMITTEE WISHES ALL THE EFI MEMBERS

A HAPPY AND SUCCESSFUL 2008

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ELECTION OF NEW MEMBERS OF THE EFI EXECUTIVE COMMITTEE 2008Nominations were sought for the functions of Deputy Secretary and three Councillors.

Ann-Margaret Little was nominated as Deputy Secretary

No further nominations for this positions were received. If the General Assembly during the 22nd European Immunogenet-ics and Histocompatibility Conference in Toulouse approves of this nomination, Ann-Margaret Little will take offi ce after the General Assembly

We have four nominations for the three positions of councillor:

Domenico AdornoCiaran DunneAntonij SlavcevCaner Süsal

If you are paid-up EFI member you should use your right to vote. So please, fi ll in the ballot form, which is enclosed with this Newsletter, and send it to the EFI deputy secretary as soon as possible, following the instructions as indicated. To assist you with your decision, a short presentation of all candidates is included below.

Dominique CharronPresident

European Federation for Immunogenetics

Ballot form for EFI Councillor 2008

Candidates:

1. Domenico Adorno

2. Ciaran Dunne

3. Antonij Slavcev

4. Caner Süsal

How to Vote:1. Select three candidates and give a mark beside their name. You must vote for three candidates. Ballot forms with only

one or two votes will not be counted.2. Enclose the ballot sheet in a blank and anonymous envelope and seal.3. Enclose this envelope into another envelope on which you should put your name, address and EFI membership number

(if known). If you fail to do this your vote will not be counted!4. Return this envelope before March 7, 2008 to:

Prof. dr. M. G. J. TilanusTissue Typing LaboratoryUniversity Hospital MaastrichtPO Box 95006202 AZ MaastrichtThe Netherlands

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Nomination for EFI Deputy Secretary

Ann-Margaret LittleMy experience within the Histocompat-ibility and Immuno-genetics fi eld began 21 years ago when I embarked on my PhD training in Manchester with Dr. Phil Dyer. This was followed by post-doctoral studies at Stanford, USA with Professor Peter Parham. My interests have predominantly been to understand the structure and function of HLA mol-ecules, particularly their role in clinical medicine. My experience in research and diagnostics has lead to my current position as Operations Director of the Anthony Nolan Trust in London. Our lab-oratories provide a service not only to the Anthony Nolan Trust volunteer hae-matopoietic stem cell donor register but also to many transplant units within the UK. We also support the renal and liver transplant programmes within our hos-pital and perform a range of research activities covering various aspects of health and disease. Over the years, I have also been actively involved in various committees of the British Soci-ety for Histocompatibility and Immu-nogenetics. I have been a member of EFI for 11 years, and an accredita-tion inspector for the past fi ve. I have enjoyed the opportunities to meet and interact with fellow EFI inspectors and laboratory staff, and from this I have gained much knowledge that has had a positive impact on the way in which our laboratory operates. I was elected as an EFI councillor in 2007 and now look forward to serving EFI as Deputy Secretary.

Nominations for EFI Councillor

Domenico AdornoI graduated from Medi-cal School at the Uni-versity “La Sapienza” in Rome in 1971. While completing my board in Pneumology and Clinical Immunology, I started to work in the Tissue Typing Laboratory of the city of L’Aquila, which I directed from 1978 to 1983. In 1983, I was appointed Associated Professor for Surgical Physiopathology at the University “Tor Vergata” in Rome, and became Director of the Laboratory of Tissue Typing and Transplantation Immunology at the same University in 1991. This laboratory in 1998 received the governmental appointment of Coor-

dinating Transplant Center for the Lazio Region. In 1986, I was appointed Direc-tor of the Institute for Organ Transplan-tation at the National Research Council (CNR) in L’Aquila. The two laboratories directed by me were among the fi rst in Italy to receive accreditation by ASHI in 1994 and by EFI in 1998, the year in which I was appointed inspector for EFI accreditation.

I am convinced that a strong col-laboration between histocompatibility laboratories and transplant centres is crucial for optimising the results of tissue grafting. As Councillor to the EFI Board, I intend to put my experience at the service of promoting international exchange programs for quality control, educational and research purposes.

Ciaran DunneI was born in 1964 in Dublin, Ireland. I began my career in the fi eld of H&I in 1993 at the National Histocompat-ibility and Immunogenetics Reference Laboratory (NHIRL), Irish Blood Trans-fusion Service and completed my doc-toral thesis at the University of Ulster in 2006. In 1997 I was appointed Chief Medical Scientist of the NHIRL where I have continued to be actively involved in research, teaching and the clinical application of immunogenetics par-ticularly in the fi elds of allogeneic hae-matopoietic stem cell transplantation, blood transfusion and disease asso-ciation. The laboratory was accredited by EFI in 2001 and the Irish Unrelated Bone Marrow Registry, to which we are affi liated, was awarded WMDA accredi-tation earlier this year.

I have been an active member of EFI for 10 years. I previously served as a Councillor to the EFI Board between 2001 and 2004. I have been Chairper-son of the EFI External Profi ciency Test-ing Committee since 2004 and have been a member of the EFI Standards Committee since 2005. I became an EFI Inspector in 2003. I have also been a member of ASHI and BSHI since 2001 and 1996 respectively. I was local co-organiser of the 15th Annual BSHI Conference in Dublin, 2004. I have been very fortunate to have had the opportunity to meet and cooper-ate with many colleagues from the EFI community and from other parts of the world. I would feel that the extensive experience that I have gained both per-sonally and professionally should help

me to further contribute to the educa-tional and scientifi c objectives of our wonderful society.

Antonji SlavcevI was born in 1960 in Sofi a, Bulgaria. I com-pleted my medical edu-cation at the Medical Academy in Sofi a in 1986. In 1987 I received a scholar-ship and became a PhD student in the Department of Immunology of the Institute for Clinical and Experimental Medicine (IKEM), Prague, Czechoslo-vakia. After defending my PhD thesis in immunology 1992, I spent one and a half years on a postdoctoral stay at the Department of Transplantation Immunology at the CLB, Amsterdam. Back to the HLA laboratory in Prague, I was engaged in research and coor-dinated various national and collab-orative projects with laboratories in Holland, France and Bulgaria. In 2002 I became head of the Department of Immunogenetics and an EFI inspector. My current research interests include HLA and non-HLA antibodies in trans-plant patients and the role of cytokine and KIR gene polymorphisms for trans-plant outcome. In the recent years, I was involved in the organization of various educational workshops for HLA laboratories in the Czech Republic and the neighboring countries. In collabo-ration with Gottfried Fischer and col-leagues from the region, we organized two East-West Immunogenetics Confer-ences in Prague (2006 and 2007) with the objective to support education and EFI accreditation in Central and East-ern Europe.

Caner SüsalI studied medicine at the University of Heidelberg between 1980 and 1986. Already during that time I was fascinated by the international nature of research carried out at the transplantation immunology department of our university. In 1984, I asked Prof. Gerhard Opelz, head of the department, for a thesis in his labora-tory. He accepted my wish, and since then, I have been working at the same institution where I am currently super-vising the Antibody Laboratory. My scientifi c interest is focused on the role of HLA and non-HLA immunity in organ transplantation, pre- and post-transplant risk estimation, and mark-ers of immune tolerance. I authored more than 90 publications and received

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NEW HORIZONS IN IMMUNOGENETICS AND HISTOCOM-PATIBILITY The 22nd conference of the European Federation for Immunogenetics, EFI, will take place in Toulouse from the 2nd to the 5th of April 2008. From the site of the last EFI conference (Bar-celona, 2007), you just have to look across the Pyrenean Mountains to fi nd Toulouse, and that was one of the rea-sons for naming the conference “New horizons in Immunogenetics and Histo-compatibility”. Toulouse is situated at the border of the Garonne River which takes its sources from the Spanish side of the mountains. Toulouse is a cross-road for trading since Antiquity and it is a town with a rich cultural heritage of more than 2000 years. It is thus not surprising that Toulouse is a candidate for being named the “European cultural city” in 2013. Toulouse is called the pink city (“La ville rose”), because most traditional as well as modern buildings are constructed with red bricks and tiles. Toulouse was a fl ourishing city of pastel trade in the Renaissance but is nowadays an active part of the techno-logical revolution through new high-tech industries. The technologies necessary to build airplanes as the giant airbus 380 are present in Toulouse and the various satellites sent up by the Ariane rockets in Guyana are not only con-ceived and constructed in Toulouse but are also tracked after the launch-ing by teams in Toulouse National Centre for Space Studies. In the bio-medical area, Toulouse is an important centre in France, both in fundamental and clinical research. A recent survey shows that the university hospitals of Toulouse are among the best in France. The Universities of Toulouse make it the second biggest university town in France and out of a population of about half a million, more than 20 % are stu-dents. The “Pierre Baudis” congress centre is located very centrally in the town, 10 minutes walk from the town hall “Le Capitole”. We have booked the whole congress centre so there will be ample space for all the activities in con-nection with the congress. The newly opened metro line has a stop next

to the congress centre and the con-nection to the main railway station is a question of minutes. The airport is served by a direct shuttle, only 15 min-utes away.

The name of the conference “New hori-zons in Immunogenetics and Histocom-patibility” is full of promises. Together with the scientifi c committee a very exciting plenary programme has been constructed. The fi rst plenary session is dedicated to Immunogenetics of diseases where Rikard Holmdahl from Sweden will reveal the latest develop-ments in the fi eld of rheumatoid arthri-tis; Ashley Moffett from UK will tell us about Immunogenetics of Preeclamp-sia and fi nally Federico Garrido from Spain will review the important fi eld of HLA expression on tumour cells and escape mechanisms. The second plenary session is devoted to hema-topoietic stem cell transplantation. Effi e Petersdorf from USA, (President elect of WMDA, World Marrow Donor Association) will give an overview of clinical results in this area, Carlheinz Muller from Germany will present data from donor registries and Alois Grat-wohl from Switzerland will tell us about alternative donor sources. In the third plenary session on Friday, we have succeeded in gathering three very well known researchers in the fi eld of trans-plantation tolerance, namely Michel Goldman (Belgium), Kathryn Wood (UK) and Robert Lechler ((UK). They all par-ticipate in the European project RISET (Reprogramming the Immune System for the Establishment of Tolerance) coordinated by Michel Goldman and they will give new information about their experience in clinical and experi-mental transplantation and in vitro studies. In the fourth plenary session the area of markers in disease and population studies will be covered by Steve Mack (USA), Xavier Estivill (Spain) and Benedicte Lie (Norway). Worldwide immunogenomics will be reviewed, the important question of copy number variants will be dealt with and new

autoimmune disease markers in vari-ous populations will be discussed. Finally, non-classical MHC genes and their function will be the subject of the fi fth plenary session. The important work on soluble HLA-G and NK activa-tion by the group of Eric Long (USA) will be presented by his close collaborator Sumi Rajagoplan, viral evasion will be summarised by Emmanuel Wiertz (the Netherlands) and HFE and its role in haemochromatosis will be reviewed by Marie-Paule Roth (France).

In addition to this ambitious pro-gramme, we will have an opening ses-sion in the afternoon of the 2nd of April with distribution of the Julia Bodmer award to a young researcher and the Ceppellini lecture, which this year will be given by Hans Georg Rammensee from Germany. On the website of the Conference www.efi 2008.eu you will fi nd more details about the programme. Among the more than 350 abstracts received, 56 will be chosen for oral pre-sentations and there will be a special best abstracts session where the pre-senters will receive an award. The other accepted abstracts will be displayed as posters and on the 3rd of April a spe-cial poster session with wine tasting and regional cheese specialties will take place at the end of the afternoon. The 5 best posters will be rewarded by a special scientifi c committee and the authors will receive their award during the closing session, Saturday.

multiple awards for my research in the fi eld. In 2005, I became Professor of Immunology. Currently, I am Secretary of the German Society for Immunogenetics, and during the last 23 years, I was fortunate to

have the opportunity to cooperate with colleagues from all over the world and to host scientists from Europe and other continents in my lab. I would be delighted if I could share the expertise that I gained over the years with the

members of the European Federation for Immunogenetics and serve the Fed-eration as a Councillor.

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Educational sessions are integral part of the scientifi c programme. Four such sessions have been planned by M Abbal and C Navarrete from the educa-tion committee:

1. Screening for HLA antibodies: rel-evance of the different antibody types

I Doxiadis, JD Bignon, F Claas 2. Platelet and granulocyte allo-

immunisation M Abbal and C Navarrete 3. Bioinformatics in immunogenetics

P-A Gourraud and S Marsh4. European regulatory framework for

organ and cell transplantation and clinical assays

(Poseidon-RISET EU projects train-ing sessions)

E Rial-Sebbag and L Lwoff

There will of course also be special sessions, and these are coordinated by Anne Cambon-Thomsen. (see web-site for details)

1. 15th International Histocom-patibility workshop in BrazilMaria Gerbase-DeLima and Maria Elisa Moraes Chairs of the 15th International Histocompatibility and Immunogenetics Workshop. This session will allow updating the work already done and planning the remaining tasks for the various axes of the workshop, in particular those including European laboratories.

2. POSEIDON A Cambon-Thomsen, D Charron,

I Doxiadis, E Naumova The project POSEIDON for Promot-

ing Optimisation, Safety, Experience sharing and quality Implementa-tion for Donation Organisation and Networking in Unrelated Haemato-poietic Stem Cell Transplantation in Europe, is project N° 2006210 co-funded by the Public Health Programme of the European Com-mission (http://ec.europa.eu/phea/documents/2006_Health_Threats.pdf) where EFI is a partner.

3. Special Eurovillage poster session A. Cambon-Thomsen Numerous EU supported projects

have some components related to immunogenetics, but it is not easy to trace them in the cohort of EU projects. A special call for contribu-tions is therefore done for this ses-sion where posters will have the aim to inform about the existence, the aims and the structure of EU con-sortia and projects where immuno-genetics is involved. Such posters will be grouped in a specifi c area in the poster session where a board for announcing positions opened, especially for young investigators will also be available. Interested project coordinators or participants should contact A. Cambon-Thomsen (cambon@cict.fr) for further infor-mation.

Several Satellite symposia are being organised, one is already posted on the website:NON-HUMAN PRIMATE IMMUNOGENETICSThis symposium is organised by

Antoine Blancher (Immunology Labo-ratory of the University Hospital of Toulouse) and it will take place on April 2nd from 9 a.m. to 3.30 p.m.If you wish to present data at this satellite symposium, please contact: blancher.sec@chu-toulouse.fr. In addition to the scientifi c programme, there will be an interesting social pro-gram, with reception at the town hall, an organ recital at the old roman church St Sernin, a gala dinner at the new mediathèque and various mani-festations organised by the sponsors. Also interesting tourist programmes are proposed as for instance a visit to the giant airbus 380 construction site or maybe a visit to a rugby match; in fact, Toulouse has recently qualifi ed for the quarter fi nals of the European cup that will be played the same week-end as the congress ends. As usual there will be an attractive commercial exhibition close to the poster area, where coffee will be served during coffee breaks. Special sponsored lunch sessions will also take place Thursday and Friday, where refi ned sandwich lunches will be avail-able to all registered participants. On behalf of the local organising com-mittee and the EFI executive commit-tee we look forward to seeing you in Toulouse in April

Anne Cambon-Thomsen and Mogens Thomsen.

Tuesday April 2, 2008 at 1:30 - 3:30 PM

“Structurally Based HLA Matching: Are We Ready for Its Application in the Clinical Setting?”

Moderators: Rene J. Duquesnoy, University of Pittsburgh Medical CenterFrans Claas, Leiden Uni-versity Medical Center

Ever since the beginning of clinical trans-plantation, HLA compatibility has been determined by counting the number of mismatched (or matched) HLA-A, HLA-B and HLA-DR antigens. Although the so-called zero-A, B, DR mismatched transplants have the best outcome, we do not understand why many mis-

matches do so well. The antibody and cellular immune responses to HLA are directed against epitopes that can now be defi ned at the structural level. Accordingly, each HLA antigen repre-sents a set of structural epitopes and histocompatibility can be assessed by determining which donor epitopes are different from the epitope repertoire of the recipient. A recent EFI newsletter has an update about structurally based matching.This session addresses the question whether the structural epitope concept can be applied to determine mismatch acceptability for sensitized patients and mismatch permissibility for non-sensitized patients considered for an organ transplant, stem cell transplanta-tion or platelet transfusion. There will

SATELLITE SESSION AT EFI MEETINGbe lots of discussions and participants are invited to describe their experience and views. For more information contact: Duques-noyr@upmc.edu and F.H.J.Claas@lumc.nl

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The next EFI Conference, to be held in Toulouse, France 2. -5. April 2008, is quickly approaching. One of the highlights of any EFI Conference is the presentation of the Julia Bodmer Young Scientist Award. As you know this award is given as an outstanding recognition of the scientifi c work of a young scientist. It goes without saying that the award also represents a rec-ognition of the laboratory in which the award winner has performed his or her work. Last year’s winner was Blanca

Rueda from Granada (see p. 18 in the last issue of the EFI Newsletter).

Any EFI member can propose candi-dates for this award, preferably sup-ported by the head of the laboratory where the candidate is working. The candidate needs to be an EFI member and under the age of 35. The winner will give the Julia Bodmer Young Scientist Award lecture at the Opening session of the EFI Conference in Toulouse. Fur-ther, he or she will receive 1000 euro

PROPOSAL OF CANDIDATES FOR THE JULIA BODMER YOUNG SCIENTIST AWARD 2008

to cover travel and hotel expenses and free registration at the conference.

Send proposals of candidates by e mail to the chairman of the EFI Scientifi c Committee, Erik Thorsby, erik.thorsby@medisin.uio.no, before 20. February 2008. The proposal must be accom-panied with the CV of the candidate, including his or her list of publications.

EFI conference 2008 in Toulouse,France

By the time you read this EFI Newslet-ter the EFI conference in Toulouse will be soon upon us. I am sure that Anne Cambon-Thomsen and Mogens Thom-sen are in the midst of the preparation of the 22nd EFI conference to be held in Toulouse on April 2nd to 5th next year. Under the motto “New horizons in Immunogenetics and Histocompatibil-ity”, you shall fi nd an exciting program presented in pleasant springtime in the attractive city of Toulouse. Detailed information can be found at the confer-ence web site ( www.efi 2008.eu ) and the EFI homepage ( www.efi web.org ).

EFI conference 2009 in Ulm, Germany

In 2009, the EFI conference will be held in Ulm, Germany, hosted by Joannis Mytilineos, Carl-Heinz Müller and Rainer Blasczyk from May 9th to 12th, 2009. It is planned that this 23rd EFI confer-ence will held in conjunction with 17th Annual Meeting of the German Society for Immunogenetics (Deutsche Gesell-schaft für Immungenetik). An update indicating what can be expected in Ulm will be given during the EFI meeting in Toulouse and detailed information will be available after the Toulouse meeting on the congress web site www.efi 2009.eu and/or www.efi 2009.de.

EFI conference 2010 in Florence,Italy

The EFI conference in 2010 will be held in Florence, Italy, from May 15th to 18th. Katharina Fleischhauer as Chair and Mario Savi for the local organis-ing committee will be in charge for this conference which is also planned as a joint event bringing EFI together with the Italian National Society of Immuno-genetics, AIBT (Associazione Italiana di Immunogenetica e Biologia dei Tra-pianti).

EFI conference 2011 in Prague, Czech Republic

As announced in the last EFI newslet-ter, applications for hosting the con-ference in 2011 were sought. The Executive Committee received appli-cations for hosting the conference in Bucharest (Romania), Maastricht (The Netherlands), Prague (Czech Republic) and Vienna (Austria). Given the high quality of the applications and the attractiveness of proposed venues, the Executive Committee had a very diffi cult task to select the venue for 2011. The selection was based on the credentials of the applicant (relation-ship to EFI and the fi eld; organisations skills), suitability, attractiveness and accessibility of the proposed venue, together with EFI’s interest to support regional development and while main-taining balance, as well as economic

FUTURE EFI CONFERENCES

aspects. After much discussion the EFI Executive Committee choose Prague, Czech Republic, to hose the 2011 conference. Antonij Slavcev will be in charge of the organisation of the meet-ing. More details will be given in a later issue of the EFI Newsletter.

Ralf Wassmuth

12

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Ciaran Dunne

The EFI EPT Committee met in Maas-tricht on 9th November 2007.

Approval of External Profi ciency Test-ing (EPT) schemesThe objective of EFI working towards standardisation between H&I EPT pro-grammes, provided to laboratories affi liated to EFI, as set out in Rules for EPT Organisers, was the primary topic for discussion. This was originally requested by the EFI Accreditation Com-mittee in Leiden 2006 and endorsed by the EFI Board in Barcelona 2007. It was acknowledged that working towards standardisation of regional, national, multinational, voluntary and commer-cial EPT programmes across Europe will be diffi cult. In comparison, the USA has one national EPT programme.

The EFI EPT Committee agreed to “approve” EPT Organisers and their individual schemes. This approval programme refers specifi cally to EPTs (assessed and scored) rather than to cell/DNA exchange schemes.

Proposed timeframes1st January 2008 – implementation of version 4 of EFI Rules for EPT Organisers

1st January 2009 – start date for submission of applications from EPT Organisers for approval by EFI EPT Committee. Implementation of version 5 Standards for EPT Organisers’ will coincide with this date.

1st January 2011 – deadline for EFI approval of EPTs (3 years from present).

1st January 2012 – deadline for labora-tory participation in EFI approved EPT schemes (4 years from present).

EPT Approval application EPT Organisers to submit applications titled numerically i.e. Applications 1 or 2 or 3 as distinct from EFI Accredita-tion Packets A or B or C. The Application shall include CVs of the Director and/or local manager, overview of the duties of the personnel involved in providing the scheme(s), organogram outlining lines of responsibility, history of EPT pro-grammes, list of EPT schemes provided, quantitative information on activities, copy of prospectus/customer handbook/manual, scoring systems employed to assess schemes, completed standards checklist based on version 5.

EPT Approval processTo be modelled on current EFI laboratory accreditation process. Applications to be reviewed by a committee appointed from the EFI EPT Committee. EPT Committee member can not partake in the review of their own submission. Applications in Eng-lish. EFI EPT Chairperson to function as EPT Commissioner. Applications to be received by June for fi nal assessment at EPT autumn meeting. EPT Commissioner to make deci-sion on application – issue recommenda-tions in a report or recommend approval.

Certifi cate for EPT schemesTo be awarded for analytes covered by EFI Standards – Human Histocompatibility Test-ing at present – certifi cate to refl ect this:1. Class I typing – serology or low reso-

lution DNA – based typing2. Class II typing – serology or low res-

olution DNA – based typing3. Class I typing – high resolution4. Class II typing – high resolution5. Class I HLA antibody detection6. Class II HLA antibody detection7. Class I HLA antibody identifi cation8. Class II HLA antibody identifi cation9. Crossmatching (reference to meth-

ods for decision in Toulouse)

UPDATE ON EFI EPT ISSUESDuration of EPT Approval for 3 years before next review - as per laboratory accreditation. EPT Organisers shall notify all participants of their approval status for each scheme. “Approved” EPT Organisers can use EFI logo irre-spective of the number of approved schemes provided.

EFI EPT Committee to upgrade EPT Rules version 4 to EPT Standards ver-sion 5 in Toulouse. Go live date from 1st January 2009. Applications to be inspected by EPT Committee over the following 2 year period.

Any Other Businessi. Agreed that EPT Organisers must ensure that samples circulated for serological typing are DNA typed by the Organiser or another EFI accredited laboratory.ii. Agreed not to approve EPT schemes for B*27 typing as this could also be extended to other alleles or allele groups. Some of this testing may not be performed in H&I laboratories accredited by EFI. Approval of EPT schemes for HLA class I/II typing should suffi ce initially.iii. Recommended that the number of participating laboratories in a scheme should be suffi cient for valid analysis. A minimum number of 15 was consid-ered appropriate.iv. Reaffi rmed that French EPT is under contract for 3 years with AFSSAPS and cannot change the requirement for HLA typing from 8 samples to 10 before 2009. v. The scoring, assessment, consensus level and determination of successful annual EPT performance for HLA anti-body identifi cation were discussed in detail. Eurotransplant, Instand, NEQAS and AFSSAPS – LNH to review their data and report back in Toulouse.

PROPOSALS FOR EFI STANDARDS VERSION 5.6Here is a proposal for changes to be included in the new version 5.6 of the EFI Standards. These modifi cations have been prepared and accepted by the Standards Committee following the Barcelona (May 2007) and the Maas-tricht (November 2007) meetings. The changes are based on feed-back from the Inspectors Workshop, and on cross-checking EFI and ASHI standards, with a major commitment to replace stan-dards with “shoulds” by “must stan-

dards” and to delete standards which are simple statements or recommen-dations. Standards on HLA-C,-DQ.-DP serology have thus been deleted. The use of NMDP codes is allowed for reg-istry typing only. Also the subsection on Complement has been revised, as well as the entire section J on Trans-fusion and section K on HLA-disease. The latter now includes DNA typing for single allele(s) or allele-group(s).This new version is now submitted for

public comment. The EFI members are invited to send their proposals to the chaiman of the Standards Committee BEFORE March 31, 2008. All proposals will be discussed during the next Com-mittee meeting in Toulouse.

PD Dr. J.-M. TiercyChairman of the Standards and QA Com-mittee

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C - QUALITY ASSURANCE

C1.700 The laboratory must establish and employ policies and procedures for the proper maintenance of equipment, instruments and test systems by 1) defi ning its preventive maintenance programme for each instrument and piece of equipment at least once a year, and by 2) performing and documenting function checks on equipment with at least the frequency specifi ed by the manufacturer.

C2.210 All blood and tissueC1.710 Assays must be performed with calibrated dispensing instruments (e.g. pipettes, etc.). Calibration must be performed at least once a year and must be documented.

(= former N1.200)C2.210 All biological samples shouldmust be handled and transported in accordance with the understanding that they

could transmit infectious agents.C4.100 External Profi ciency Testing (EPT) and Competency Evaluation.C4.140 EPT samples must be tested and interpreted in a manner identicalcomparable to that for routine testing of clinical

samples.C4.145 (NEW) Participating laboratories must ensure that all EPT related documents including submitted worksheets, EPT

summary/scheme reports, annual performance and participation certifi cates, outcomes of investigations of any unsatisfactory results, corrective or preventive actions are maintained and are made available to EFI inspectors for assessment.

C4.150 The laboratory must, at least once a year, give each individual performing tests, and not involved in daily routine testing in the Histocompatibility and Immunogenetics laboratory (e.g on call personnel only), a characterised spec-imen as an unknown to verify his or her ability to reproduce test results. This standard must be applied to all tests regularly performed by an individual. The laboratory must maintain records of these results for each individual.

C4.440 The laboratory must maintain permanent fi les of all internal and external quality control tests according to any regulation to which the laboratory is obliged to abide, but for a minimum of four years.

C5.140 Reports or records, as appropriate, shouldmust include a brief description of the specimen (blood, lymph node, spleen, bone marrow, etc.) used for testing.

C5.150 Molecular typing: a record must be kept which is appropriate to the technique used, such as a photographic record of a gel, a membrane, an autoradiograph, an electronic fi le, or the read out from a sequencer. The record must be kept according to any regulation to which the laboratory is obliged to abide, but for a minimum of four years.

C5.200 For marrow transplantation, donor records should be maintained so that donors can be rapidly retrieved according to HLA type.

D - HLA ALLELES AND ANTIGENS

D1.250 Broad antigens and the epitopes Bw4 and Bw6 may be reported in brackets.D1.300 If only aD1.110 (NEW) Use of NMDP codes is only allowed for recording donors or cord blood unit typings into

databases or for communication of the donor, cord blood unit or recipient typing with the registries.D1.300 If no more than one single antigen or allele is found at a locus by serological typing or DNA typing, the phenotype

may include it twice only if homozygosity is proven by family studies, Conversely, a “blank antigen or allele” can only be assigned if proven by family studies.

D1.310 If2-digit DNA typing unequivocally demonstrates the presence of heterozygosity for two different alleles from the same specifi city(e.g. DRB1*1301/1359, DRB1*1303/1333), the report may include it twice (e.g. DRB1*13,*13) even in the absence of family studies. . Conversely, a “blank antigen or allele” can only be assigned if proven by family studies.

D2.000 Haplotype assignment.D2.100 Determination of haplotypes can onlymust be done by typing immediate family members including parents, sib-

lings and/or children of the patient.D2.110 Typing for HLA-A, B and DR locus alleles or antigens is mandatory.

E - SEROLOGICAL HLA CLASS I AND CLASS II TYPING

E1.000 HLA-A, -B, -DR locus antigens.E1.100 The laboratory must be able to type for the HLA-A, -B, and-B/ or -DR specifi cities, which are offi cially recognised by

the WHO and for those deemed relevant by EFI.E1.200 Typing for HLA-C locus antigens is not mandatory.E2.000 HLA-DR, -DQ antigens.E2.100 The laboratory must be able to type for the HLA-DR specifi cities offi cially recognised by the WHO and for those

deemed relevant by EFI.E2.200 Typing for HLA-DQ antigens is not mandatory.E3.220 Typing results may be invalid if the positive control fails to react as expected.E3.240 Cell viability in the negative control well at the end of incubation must be suffi cient to permit accurate interpreta-tion of results. For most techniques, viability should exceed 80%.E3.610 It is recommended that the specifi city of typing sera obtained locally be confi rmed in at least one other HLA laboratory.

15

E3.220 If the positive control fails to react as expected, there must be a procedure in place as whether to accept or reject the test.

E3.240 Procedures regarding the minimal viability of the cells and the reactivity of the control sera, for the validation of a serological typing must be described in the laboratory manual.

E3.640 Each lot of newtyping trays must be evaluated by testing either with at least fi ve different cells of known phenotype representing major specifi cities or in parallel with previously evaluated trays. Each new shipment of previously evaluated typing trays must be verifi ed with at least one cell of known phenotype.

E3.700 Complement.E3.710 Each batchlot of complement must be tested to determine that it mediates cytotoxicity in the presence of specifi c

HLA antibody but is not cytotoxic in the absence of HLA specifi c antibody. The complement must be kept at an appropriatethe recommended temperature.

E3.720 The test shouldmust employ multiple dilutions of complement to ensure that it is maximally active at least one dilution beyond that intended for use.

E3.730 The test should be carried out with at least two antibodies, which should react with at least two different test cells and at least one cell, which should not react. A strong and a weak antibody should be selected for the test, or serial dilutions of a single serum may be used.

E3.E3.730 Each lot and shipment of complement must be evaluated by either i) testing with at least 3 previously evaluated

trays for every application for which it is intended for or ii) testing a combination of at least 3 sera and 2 cells selected to include negative, weak positive and strong positive reactions.

E3.740 Complement shouldmust be tested separately for use with each type of target cell, since a different dilution or preparation may be required for optimal performance.

F - ANTIBODY SCREENING AND CROSSMATCHING

F1.230 Positive control sera shouldmust be from highly alloimmunised individuals and documented to react with HLA antigens. The antibodies must be of the appropriate isotype for each assay. Each assay must include positive control(s).

F1.320 To detect HLA class II specifi c antibodies, B lymphocytes, chronic lymphocyte leukaemia cells or B cell lymphoblas-toid lines may be used.

F1.350 To identify the specifi city of an antibody with certainty, the laboratory should test the serum with additional cells expressing and lacking the candidate antigen and cross-reacting antigens.

F2.200 If sera are screened after treatment with dithiothreitol, then IgG and IgM HLA-specifi c positive controls must be included.

F5.100 Laboratories performing assays using fl uorescent microbead arrays in conjunction with a dedicated cytometer-like instrument must additionally conform to relevant parts of section M4.000.

F6.120 For lymphocytotoxic crossmatching each serum must be tested undiluted and in duplicate and undiluted.F6.150 If crossmatches are performed after treatment of the patient sera with dithiothreitol, then IgG and IgM positive

and negative HLA specifi c controls must be included.

G - RENAL and/or PANCREAS TRANSPLANTATION

G3.120 If prospective crossmatch is not systematically performed, there must be evidence that the laboratory maintains a record of potentially sensitising events for each patient.Otherwise such a record should be maintained.

G3.220 Final crossmatches performed prior to transplantation shouldmust utilise a recipient serum sample collected within the previous 48 hours before transplant if the recipient has HLA antibodies or has had a recent sensitising event. Otherwise, the most recent available serum collected as defi ned in G2.110 must be used.

G3.240 Serum samples usedstored for crossmatching shouldmust be retained in the frozen state for future use.

I - HAEMATOPOIETIC STEM CELL TRANSPLANTATION

I1.110 HLA typing for HLA phenotypically identical siblings must include adequate testing to defi nitively establish HLA identity by descent (D2.120 applies), or use high resolution Class I and/or Class II typing (4-digit allele assign-ment) by DNA methods to determine the degree of HLA matching as appropriate fordocumented in the transplant protocol.

I1.120 HLA typing for recipient and potential intra-familial donors who are not HLA identical siblings must include high resolution Class I and Class II typing (4-digit allele assignment) by DNA methods as required bydocumented in the transplant protocol.

I2.260 For unrelated donors HLA-A,-B,-DR concordant results are required on two separate samples. Registry data are typing is acceptable foras one of these samplesthe two required results.

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J – HLA AND TRANSFUSION

J1.000 HLA typingJ1.100 Platetelet refractoriness.J1.100 The patient shouldJ1.110 Platelet refractory patients who require HLA matched platelets must be typed for HLA-A

and HLA-B.J1.200 The donors should be selected according to national regulation/legislation.J2.000 Cross-matching. J2.100 Lymphocyte cross-matches are optional.J2.200 Cross-matching by techniques which utilise donor platelets or granulocytes as the target cells are preferred.J1.120 For the laboratory investigation of suspected alloimmune platelet refractoriness the patient must be tested for

HLA class I antibodies .J1.130 The specifi city of detected HLA antibodies must be defi ned and recorded or crossmatching must be performed to

provide compatible platelets. For crossmatching using lymphocytes standards in section F6.000 apply.J1.140 All selected plateletpheresis donors used for the provision of HLA matched platelets must be typed for HLA-A and HLA-B.J1.200 Transfusion Related Acute Lung Injury (TRALI).J1.210 For the laboratory investigation of Transfusion Related Acute Lung Injury (TRALI) the sera from implicated donors

must be tested for both HLA class I and class II antibodies.J1.220 The specifi city of detected HLA antibodies must be defi ned and recorded.J1.230 The patient and the donor must be typed for HLA-A, HLA-B, and HLA-DR.

K - DISEASE ASSOCIATION

K1.000 CompleteIf complete HLA typing is an appropriate option.K1.100 Typing may alsoperformed by serology standards in section E must be limited to all products of a single or limited

number of HLA loci.followed.K3.000 If HLA typing is performed by DNA techniques standards in section L must be followed.K3.100 Typing for a single allele-group by molecular techniques (e.g. HLA-B*27).K3.110 Where typing for a single allele-group is performed a positive control DNA known to encode the allele-group of

interest must be included in each test.K3.120 Where typing for a single allele-group is performed a negative control DNA known not to encode an allele belonging

to the allele-group of interest must be included in each test.

L – NUCLEIC ACID ANALYSIS

The nucleic acid analysis standards apply to histocompatibility testing.L1.2100 Contamination must be monitored for all amplifi cation products that are produced in the laboratory.L1.4200 Laboratories must have a policy for quality control of each lot or shipment of primers. The specifi city and quantity

of amplifi ed product must be confi rmed with reference material. For commercial kits, each newlot or shipment must be tested against at least one DNA sample of known type. The signal intensity must be defi ned, monitored and fall within an acceptable range.

L1.4300 Primers must be utilized under empirically determined conditions that achieve the defi ned specifi city for templates used in routine testing. Each new setlot of local primers must be tested for amplifi cation specifi city and quantity using reference material whenever available.

L1.4400 Local primer pairs Each lot of local primers must be monitoredtested with reference DNA for appropriate sensitiv-ity and specifi city at regular intervals. The frequency between intervals should not exceed six months.

L2.1100 Nucleic acids must be extracted and purifi ed by a standardpublished method that is documented and has been validated in the laboratory.

L3.2130 MethodsValidation of the methods for template preparation must ensure that the accuracy of the fi nal typing is not altered (e.g. mutations during cloning, preferential amplifi cation).

L3.2560 The laboratory must document the sequence database utilized to interpret the primary data. Records of the data-bases used must be maintained according any regulation the laboratory is obliged to abide, but for a minimum of four years.

L3.3130 Laboratories must have a policy in place for quality control of each lot and shipment of probes. The specifi city of hybridization must be confi rmed with reference material. For home made kits, each new lot shouldmust be tested with reference DNA so that each probe is tested for specifi city and signal intensity at least once. For commercial kits each newlot and shipment must be tested in parallel against at least one DNA sample of known type. The specifi city and signal intensity for each probe must be defi ned and monitored.

L3.3230 Each assay must include appropriate controls to validate the hybridisationhybridization and the revelation (detec-tion) step of the assay.

L3.3240 Each amplifi cation assay must include a negative (no DNA) control. In forward SSOP this negative control must be included in the hybridization and revelation steps of the assay. For reverse SSOP the negative control must either be included in the hybridization and revelationdetection step of the assay whenever false positive hybridization in any of the amplicons is observed.or monitored by gel electrophoresis.

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L3.3271 Where a scanner is used for acquisition of the raw data, a second visual reading shouldmust be performed to confi rm data.

M - FLOW CYTOMETRY

M1.1400 The fl ow cytometer shouldmust be cleaned regularly in accordance with the manufacturer’s instructions.M1.2100 This standard may be incorporated in the beads or other particles used for optical standardisation or may be a

separate bead or fi xed cell preparation.M1.2300 The results of fl uorescent standardisation must be recorded in a daily quality control log.M1.2300 In the event that acceptable fl uorescence separation cannot be attained, a written protocol for instituting cor-

rective action must be available. This protocol shouldmust include appropriate corrective action including clear guidelines describing when a service call is warranted.

M1.3100 Compensation must be carried out using peripheral blood lymphocytes or microparticles individually stained with suitableeach of the fl uorochromes or with other dual stained particles, which have been shown to be suitable by the laboratory. in use.

M1.4000 For laser based instruments, the current input (amps) and laser light output (milliwatts), at the normal operating wavelength measured after the laser is peaked and normal operating power set, shouldmust be recorded as part of a daily quality control record.

M2.0000 Flow cytometric crossmatch technique/Antibody screening.M2.1000 A multi-colour technique is recommended. However, if a single colour technique is used, the purity of the isolated

cell population must be documented and shouldmust be suffi cient to defi ne the population for analysis. M2.1200 In order to assess binding of human immunoglobulin to cell population(s), the sub population(s) shouldmust be

identifi ed by two or three colour staining with differently labelled monoclonal antibodies to the appropriate CD marker(s) (e.g. phycoerythrin conjugated CD3 monoclonal antibody to identify T cells).

M2.1300 Two or three colour staining of other immunoglobulin classes may also be justifi ed.M2.2000 Controls.M2.2100 Control sera must be tested at the same time and under the same conditions as the sera under test. Tests must

be done in duplicate as a minimum requirement. Each assay must include positive and negative controls.M2.2200 The normal human serum (negative) control must be from non-immunised and otherwise healthy individuals and

may be a pool of several donors. It must be screened by fl ow cytometry to ensure lack of reactivity against human leukocytes.

M2.2300 The positive control shouldmust be a human serum specifi c for HLA antigens and of the appropriate isotype. Additionally, other positive controls for other alloantigens deemed to be important for detection in the crossmatch shouldmust be used.

M2.2400 The secondary antibody reagent shouldmust be titered to determine the dilution with optimal activity (signal to noise ratio). If a multicolour technique is employed, the reagent must not demonstrate crossreactivity with the other immunoglobulin reagents used to mark the cells.

M2.3000 Reagents.M2.3200 ReagentsMonoclonal antibodies which have been reconstituted from lyophilised powder form for storage at 4°C

must be storedcentrifuged according to the manufacturer’s instructions or accordinglocally documented proce-dures to remove micro aggregates prior to maintain stability by documented local tests. use in preparation of working stains.

M3.2000 Cell preparation.M3.2100 The method used for cell preparation shouldmust be documented to yield appropriate preparations of viable

cells.M3.2200 The viability of cell preparations shouldmust be recorded and shouldmust exceed the laboratory’s established

minimum standards for each procedure used.M3.3400 Reagents must be stored according to manufacturer’s instructions or according to conditions verifi ed to maintain

stability by documented local tests.M4.000 Bead array techniques.M4.400 For antibody screening, and identifi cation and HLA typing the following standards in section M also apply :

M1.1100, M1.1200, M1.1310, M1.1400, M1.1500.M4.500 For antibody screening and identifi cation the following standards in section N also apply : N1.200, N2.100,

N2.200, N2.210, N2.220, N2.400, N2.500, N2.700.M4.600 For HLA typing using bead array techniques the standards in L3.3200300 also apply.

N - ENZYME-LINKED IMMUNO SORBENT ASSAY (ELISA)

N1.200 AssaysN2.220 The positive control must be performed with calibrated dispensing instruments (pipettes). Calibration must be

performed routinely and must be documentedN2.220 The positive control should be a human serum specifi c for HLA antigens and of the appropriate isotype. N2.700 New lotsLots of reagents must be validated by side-by-side testing with a lot known to give acceptable perfor-

mance or by testing with test specimens of known reactivity.

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Anz_A4_hoch_AbCross_020707.indd 1 03.07.2007 9:48:16 Uhr8:16 Uhr

The venue for this meeting could not have been in a more beautiful location than the spa town of Fuiggi, south of the Italian capital, Rome. I was invited by VHBio (on behalf of One Lambda) to give a ‘short informal talk’ but until the meeting began with little idea of what to expect.!

As it turned out the educational content of the meeting was excellent. There were speakers from all over Europe and the US, who updated us with the latest work on MICA and other antibod-ies, problematic renal transplantation, stem cell and liver transplantation. The hospitality provided by One Lambda was exceptional in line with the meet-ing and we could only wonder at the organisation skills of Ricardo Ordonez. In this article I have presented merely a selection of the talks and results that the group shared with the delegates. I apologise to anyone whose work I’ve omitted but all the data will be pub-lished by One Lambda.

The fi rst session of the ten minute pre-sentations opened with the Major His-tocompatibility Complex Class I related chain-A (MICA). These molecules have been proposed as targets for antibody recognition during kidney allograft rejec-tion.

The session began with an excellent presentation from Dr. Antonina Piazza (Institute of Organ Transplantation, Rome). Their study analysed the pres-ence and specifi city of MICA antibod-ies in kidney transplant patients and looked for a role for MICA antibod-ies on graft outcome in 421 cadaver donor kidney transplant recipients. All patients were negative for donor spe-cifi c HLA antibodies (HLA-DSA) before the transplant. Post transplant, the patients were tested for DSA specifi c antibodies using FLOW PRA, Class I & II screen and Luminex single antigen beads. The patients had been divided into three clinical groups: those with a functioning graft (FG): those with chronic allograft dysfunction (CAD) and those with graft failure (GF) based on their serum creatinine levels. The results showed that MICA anti-bodies were absent in the majority of patients (n=360) but positive in 29 patients pre- transplantation and in 32 patients post transplantation. MICA

antibodies alone were signifi cantly asso-ciated with CAD whereas HLA antibod-ies alone were signifi cantly associated with GF but there was no association with either HLA/MICA antibodies in the FG group. This study also confi rmed a strong association between the devel-opment of post transplant HLA –DSA and kidney graft failure.

Dr. Bignon ( Nantes, France) pre-sented the observation that there was low graft survival in patients who had received a third transplant. One year graft survival was 92.4% vs. 86.8% but 10 year graft survival 69.1% vs. 37.5% for fi rst and third grafts respectively. However, the rate of rejection was simi-lar regardless of whether the patient had received a fi rst or third graft (22% versus 17% respectively). 41% of patients waiting for a third transplant had HLA specifi c antibodies greater than 80% PRA, but had been grafted with well HLA matched grafts: 21% being fully matched for HLA-A,-B,-DR and 59% having less than two HLA mis-matches. The 47 donor/recipient pairs receiving a third renal transplant were MICA typed. The group investigated the genetic linkage between MICA and HLA and whether MICA mismatching affected graft survival. Typing was performed using MICA kits LABTYPE RSSOP (One Lambda). The IMGT data-base lists 61 MICA alleles 001-0053 and this kit recognised 55/61 alleles with 6 ambiguities using 45 beads. In this population the frequency distri-bution of MICA alleles was similar to that published for the European Ameri-can population (Goa et. al.) excepting MICA *008, *009 and *0010 frequen-cies. They found a strong genetic link-age between a number of HLA-B alleles but demonstrated that poor third graft survival could not be explained by MICA mismatching. Dr. Arnold, (Erlangen–Nurnberg) addressed the question of whether MICA antibodies contain non-comple-ment binding (NCB) isotopes IgG2/IgG4 and IgA1/IgA2. Using a modifi ed Labscreen assay, which substituted the IgG conjugate with IgG2/IgG4 and IgA1/IgA2, they tested patients for both HLA and MICA specifi c antibod-ies.19% of patients with 0 % PRA for HLA had MICA antibodies present: in patients with >5% PRA , 35% had MICA antibodies.16 patients had only class I

2007 EUROPEAN CLINICAL HISTOCOMPATIBILITY WORK-SHOP, ROME, ITALY.

HLA antibodies and 25% of these sera also contained MICA specifi c antibod-ies and of those with only HLA Class II 29% had these. In serum containing both HLA class I + II specifi c antibod-ies 40% had MICA antibodies. In trans-planted patients with no HLA specifi c antibody, 28% had MICA antibodies. In pretransplant patients with no HLA antibody, 14% were MICA antibody positive. Patients who had a previous transplant had higher risk of developing MICA compared with patients receiving a fi rst transplant (39% versus 11%). In the study 14% of males who were non transplanted and non transfused were positive for MICA specifi c antibod-ies! The modifi ed Luminex allowed the detection of non complement binding IgG subclasses among the anti HLA and anti MICA specifi c antibodies. IgA1 anti-bodies were signifi cantly higher against MICA rather than HLA (65% versus 15% (p=0.01)).The session ended with a presenta-tion by Professor Paul Terasaki who enthralled the audience with some updated ‘old fashioned’ serology. The professor presented data on eluted antibodies from specifi c HLA antigens (i.e. B44) where his team tested the eluted antibody against panels of single antigen beads and observed their reac-tivity. In the example cited antibody activity was observed with HLA speci-fi cities B13, B*4005, B41, B44, B45, B47, B49, B50, B60 and B61 where he was able to map the epitope to three amino acid positions aa35 ( R ), 41( T ), 44 ( R ) with position 41 conferring the unique shared epitope. He went on to defi ne the epitopes of antibodies eluted from HLA-A24, -A25,-A80 and defi ne multiple epitopes eluted from A1 antigens. This was old fashioned serology with currently available tools; a pleasure to listen to.

Prof. Ella Van den Berg-Loonen from Leiden was the guest lunch time lec-ture on the fi rst day and she presented an excellent study on ’de novo’ HLA Class I and II antibodies before and after transplantectomy. 63 fi rst renal transplants in patients who were HLA antibody negative prior to transplant were studied.The majority of patients received calcineurin inhibitors for immu-nosuppression. Post transplant serum was collected weekly during hospitali-sation, then monthly for one year fol-

22

lowed by annually for life (a huge task in itself!). Samples were screened using Flow PRA and antibodies identifi ed by ELISA and CDC. The results were fasci-nating: 62% (39) of patients developed HLA antibodies post transplant; 82% of these were directed against HLA Class I and 59% to Class II. The median time to antibody production was 4 months. The majority (90%) of these antibodies were produced post kidney removal whereas only 10% of patients devel-oped antibodies prior to this point. In addition HLA class I antibodies were signifi cantly associated with increased donor age and with a reduced age of recipient. HLA Class II antibodies were associated with Class II mismatches and acute rejection.

Dr. Illias Doxiadis (Leiden, Nether-lands) presented the Eurotransplant (ET) scheme for non-acceptable HLA mismatched donors avoiding HLA specifi cities for which the patient has preformed cognate antibodies. This scheme is aimed at highly sensitised patients defi ned as having antibodies with > 85% PRA.

It is well known that the higher the number of HLA mismatches, at the fi rst transplant, the greater the chances of sensitisation and this factor should be heavily weighted for in paediatric trans-plants. The historical DSA is the most signifi cant factor affecting graft survival in the fi rst 6 months post transplant.The current approach at Eurotrans-plant is to list negative panel donors on antibody screening, followed by a cross match using a patient specifi c panel of sera against single antigen beads or single antigen expressing cell lines (SALs), and use the HLA match-maker programme to defi ne acceptable mismatches (AM). Following the AM procedure every potential donor HLA type is submitted to ET. The recipient is selected on the basis of matching of the donor with the patient HLA-A,-B -DR antigens in combination with compat-ibility with AM. Selected donor kidneys are shipped to the recipient centre without any cross match at the donor centre.

In 1985 the fi rst of 470 transplants that have taken place in Leiden between 1996-2006 was performed. Although only 200 patients are now on the AM-waiting list, potentially there should be 345. This means that not all centres in Holland participate in the scheme. 60% of the patients on the waiting list were transplanted within 2 years. As a

result of this scheme the mean wait-ing time rose from 10 months in 2002 to 21months in 2006 but this increase was due to the better defi nition of the DSA and some patients had previously waited for 10 years! Preliminary data to be published soon shows that from 248 transplants performed between 1989 and 2006 there was a 65-69% graft survival. Professor Doxiadis con-cluded that the AM scheme of ET helps sensitised patients receive a kidney with a signifi cantly reduced waiting time and with excellent graft survival. Both historical and current sensitisa-tion is regarded as important. In con-trast to desensitisation strategies the costs are minimal. A useful computer programme can be found on www.etrl.org and helps in the defi nition of the v(virtual)-PRA enhancing the chances of fi nding a suitable donor for a given patient.

Dr. Taupin presented data on the impact of a virtual cross match strat-egy on kidney transplantation in Bor-deaux, France. The allocation kidneys for France Transplant involves retain-ing one kidney locally and the second is allocated the national pool where priority is give to patients with a PRA >80% with HLA compatible recipients and to patients who have waited a long time. If there are no suitable can-didates, kidneys are allocated to the larger Southwest area of France in a reciprocal arrangement. For two and a half years Bordeaux have changed their cross matching strategy in the light of better solid phase assays for HLA anti-body identifi cation. For HLA antibody ELISA has been replaced by Luminex although CDC for PRA is a mandatory requirement so this is still performed. Antibody identifi cation by CDC for class I & II is performed by Luminex id and single antigen beads.

Results of antibody defi nition could predict a B cell positive cross match for sensitised patients and so the prelimi-nary cross match (CDC) was dropped completely and the prospective fi nal cross match remained unchanged with fl ow and CDC. Results vindicated this strategy as the number of sensitised patients receiving transplants has increased since the Luminex antibody analysis was introduced, the number of transplants performed despite a posi-tive cross match has increased and the total number of cross matches per-formed has fallen.

Dr. Taupin also described a patient with

HLA-DP antibodies following a failed transplant giving positive B cell positive cross match, but was also was compli-cated by occasional T cell positives for cells that were HLA-B18 and some -B44 positive reactions. The patient was HLA B44 positive himself! Dr Taupin asked the delegates what strategy they would recommend for this patient. Answers ranged from allelic typing of the recipi-ent and positive X-match cells to see if differences at the allelic level could explain the B18 ‘short’ B44 positive reactions to simply avoiding B44 anti-gens in spite of the patient being B44. There was no consensus on the sig-nifi cance of the HLA – DP antibodies in this case.

Dr. VItkisiuo, (Dept Immunolgy-His-tocompatibilty, Greece) analysed NK alloreactive effects in graft outcomes for human renal transplantation. They analysed KIR ligand incompatibility on renal graft survival line 83 donor/recip-ient pairs. These pairs were fully HLA typed and designated as KIR incompat-ible or compatible according to their Cw and Bw status. The former group had 17.5% early graft loss compared with 9.2% in the latter group. Those with potential alloreactivity had no late graft loss compared with 14.5% in the compatible group. They concluded the early graft loss could be related to KIR related alloreactivity. This group then presented data on comparative analy-sis for Luminex, ELISA and CDC to determine %PRA. They concluded that both Luminex and CDC yield false posi-tive results and ELISA produces false negatives.

Mr. Tim Key (Cambridge, UK) compared acceptable matches (AM) identifi ed using single antigen beads and HLA Matchmaker in 24 patients who had a PRA IgG > 85%. The presence or absence of antibody correlated with the number of amino acid triplet matches present for each HLA specifi city. When combining these two methods they identifi ed an average of 19 AM for each patient.

Dr. Ann Green (NBS, Bristol, UK) ques-tioned the role for HLA specifi c donor derived HLA antibodies in HSCT The published literature in this area is scant. A recently published paper in Blood suggests that in mice, sera rather than primed T cells could cause non-engraftment when adoptively trans-ferred. Dr.Green used several recent HLA mismatched stem cell transplant to outline the current understanding

23

of HLA antibodies in HSCT. Recipient derived donor HLA specifi c antibodies lead to graft failure in the mismatched transplants. In two cases antibody removal strategies were used success-fully to allow a second transplant to engraft There was a suggestion from the cases that class II antibodies may be potentially of less clinical signifi -cance. However it was emphasized that this was only preliminary data.

Miss Katherine Sills from Sheffi eld outlined a methodology for molecular typing the minor histocompatibilty anti-gens, HA1 and HA2. In this study there was no signifi cant effect of mismatch-ing for these antigens and graft versus host disease. However, the numbers were small and the study is ongoing.

DrJ.M.Tiercy gave a very useful talk (published recently BMT 40, 515-522.) indicating potential improvements to the search algorithm for the selection of unrelated donors for HSCT. At the start of the search the aim was to provide the clinician with an early estimated probability of fi nding an HLA-A,-B,Cw,-DRB1, -DQB1(10/10) matched donor. Patients who had a >95% chance of a donor were in the high probability group. In these cases clinicians could concentrate on the non-HLA criteria for selection of a donor e.g. young male, CMV status. The intermediate group were the most problematic group as they had a roughly 50% chance of a 10/10 match after requesting 3-5 con-fi rmatory typing samples. If there was no clinical emergency then the typing could continue to search for donors. The low probability group was indicated by a <5% chance of fi nding a donor. The group assigned related to the hap-lotype of the patient, the ethnic group, the presence of one or more ‘rare’ alleles and having less than 3 potential donors on Bone Marrow Donors World-wide search. The group studied 350 searches retrospectively; results show that estimates turned out to be accu-rate for 96%, 88% and 56% of patients in the high, intermediate and low prob-ability groups respectively. Interest-ingly there was an increased survival in patients in the high probability group with a three year survival of 75% vs. 51% in the low/intermediate probabil-ity group. It could be argued that this refl ected HLA matching and indeed the 10/10 group had a signifi cantly better three year survival than the 6-9/10 mismatched transplants 63% vs. 46% (p=0.02)When they analysed all of the patients

who received a 10/10 matched graft regardless of their group, those in the high probability group had a signifi cantly better survival and this was believed to be linked with the presence of highly conserved haplotypes (p= 0.00014). Over 80% of these patients had one or more highly conserved haplotypes as determined using micro satellite analysis. It was found that there was > 95% matching of these in patients with conserved haplotypes in the high prob-ability group for whom 10/10 matches were found. In light of this data we can start to apply some fi ne tuning to donor selection.

Dr. J Mytilineos presented data on using haplotype frequency as predic-tive criteria for fi nding a HSCT donor. A 1162 UD HSCT transplants and 740 related transplants have been performed in Germany since 1998. A hierarchy of searches was described : for patients classifi ed as ‘green ‘ searches, three confi rmatory (CT) samples were ordered because there was a 99% concordance with expected haplotypes, ‘yellow’ searches required four CT samples to be ordered as they were likely be mismatched and ‘red’ searches required 6 CT samples. They looked at all searches performed in 2005, divided these according to the colour coded groups, looked at search duration, success rate, the number of identifi ed HLA DRB1 and DQB1 identi-cal donors. The questions asked were; is there a chance to optimise search outcome and to reduce its duration? What is the optimal number of donors to be ordered?

Mr. Cierran Dunne (Dublin, Eire) reviewed the chances of fi nding an HLA matched donor from the geneti-cally homogenous unrelated stem cell donors on the IBMDR for Irish Cauca-sian patients. He presented interesting data on the probability of fi nding full matches for Irish patients on worldwide registries and for international patients looking for donors on the Irish registry (IBMDR). The aim of this work was to assess whether the search algorithm could be improved by assessing the frequency of patients HLA haplotypes (HLA-A,-B,Cw, DRB1 and DQB1). 250 registry searches were used to pre-dict the likelihood of identifying allele matched unrelated donors. Probability of fi nding a 10/10 allele match was considered ‘high’ if >90%, ‘intermedi-ate’ 50-80% and ‘low’ <50%. In an earlier study from 2004 when patients were typed HLA-A and–B at low resolution and -DRB1 at the allele level: 94% of patients had at least one matched donor on the Irish registry. In 2003 allele typing was introduced and 73% had at least one 10/10 matched donor, this increased to 82.7% for a 9/10 mismatched donor. In 1998-2006, of 137 unrelated trans-plants performed for Irish recipients 28% received a graft from Irish donors and the remainder (72%) used interna-tional donors. Ciarran showed some data (Carl Heinz –Muller, ZKRD) in which the registry size was compared with the chances of fi nding one or more HLA matched donors in all donor regis-tries. Japanese patients have the high-est chance of fi nding a matched donor in their local registry!

24

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25

The frequency with which Irish patients found donors in other registries were 40% in UK and Australia but 30% chance of identity in Northern Europe and US registries. In ‘oriental’ registries there was a poor chance of an Irish patient fi nding a donor. However, patients from the UK, Sweden, or Holland have only a 30% chance of a matched donor (cf 40% equivalent patients Irish) on the Irish registry! This was explained by the being limited haplotypes diversity in the Irish registry.

Native Irish patients retain a high prob-ability of fi nding a donor despite the introduction of allelic HLA typing and extending matching to additional loci.

Dr. Robert Bray presented an excel-lent review of the methodologies used for antibody specifi cation with particu-lar emphasis on single antigen bead technology, demonstrating how this

technology can further be developed to even higher levels of sensitivity using tertiary biotinylated antibodies. He also demonstrated that several allele specifi c antibodies were identifi ed in patients with an alternate allele of the same antigen grouping. These antibod-ies were found for alleles in HLA- A, -B, ---DRB1, -DRB3, -DQB1 and -DQA1 loci. He posed the question - what is the clini-cal signifi cance of higher sensitivity and specifi city in HLA antibody detection. In answering this he cited work by Warner and Nelson showing a decreased graft survival in patients with these previ-ously undetected antibodies. Dr. Bray went on to review transplantation for highly sensitised patients looking at MICA and endothelial antibodies as well as HLA. He left us enthused that there was still much work to be done in determining the best strategy for trans-planting diffi cult cases with high levels of sensitisation.

There were a number of very interest-ing talks that we have left out of our report. One Lambda will publish all the abstract of this meeting.

We would like to thank both One Lambda and VHBio (who invited the UK delegates) for sucha wonderful meet-ing where even us ‘old timers’ learnt a great deal more about HLA!

The meeting ended with a superb dinner in the conference hotel. We even man-aged to get our photos taken with the great man himself!

Dr Ann Green and Nr John Harvey, His-tocompatibility and Immunogenetic, National Blood Service, Bristol, UK.

CONFERENCE ON QUALITY ASSESSMENT Conference on Quality Assessment in Immunology, Immunogenetic and Haematopoietic Stem Cell Transplantation. (Borovets, Bulgaria)

This conference was organized in the Borovets resort in the heart of Rila mountain on December 1-2, 2007 by Elissaveta Naumova and coworkers under the patronage of the Minister of Health in Bulgaria. The meeting was attended by more than 110 people – mainly immunologists, hematologists and some other specialists from Bul-garia, Romania, Greece, Serbia, Arme-nia, Czech Republic, Spain, Ireland, Austria, Turkey and France.The conference was run in two days in Serdika Hall at Samokov Hotel and the scientifi c program was scheduled in four sessions.The fi rst session was focused on the National EPT System in Immunology and there was made an analysis of EPT schemes in fl owcytometric immu-nophenotyping, immunoglobulins and complement, autoantibodies by the lecturers: Maria Nikolova and Anas-tassia Myhailova. Daniel Bitoun (Bel-gium) in his talk has considered some interesting points concerning Evolution of QC in immunology from a medical manufacturing perspective. During the second session Kamen Tzachev and Momchil Enchev (Bulgaria) made clear to the audience some important issues related to the Accreditation of medical laboratories and EPT schemes as well

as some Legal aspects of stem cell, tissue and organ transplantation.Session 3 was opened by Dominique Charron (France) who presented his lecture on Immunogenetics in motion. The Serdika Hall was full up with par-ticipants willing to listen to his inter-esting talk on the latest developments in immunogenetics. Another presen-tation, given by Catherine Stavropou-los-Giokas (Greece), was dedicated to

Cord blood mesenhymal cells – from banking to therapy and has been fol-lowed by interesting discussion. Ver-ginia Lepage (France) has drawn the attention of the participants to her talk on HLA and non HLA associations with bone marrow transplantation outcome. Another interesting part of the Session 3 was the lecture presented by Elissa-veta Naumova who introduced to the attendants some aspects of National

26

ANNUAL MEETING OF THE CROATIAN IMMUNOLOGICAL SOCIETY 2007

Katarina Stingl, Zagreb, CroatiaThe Croatian Immunological Society, founded in 1968, is one of the oldest immunological societies in Europe. Its continuing purpose is to contribute to the advancement of immunology in all its aspects and to improve the commu-nication between immunologists work-ing in different fi elds in Croatia. These aims are in large part achieved through the annual meetings of the Society. This year, the Annual Meeting of the Croatian Immunological Society was held in October, at the beautiful island

Crveni Otok near Rovinj. The program of the meeting usually includes plenary lectures from prominent scientists, short oral communications selected by the scientifi c committee and poster section. One of the sessions was dedi-cated to transplantation. The fi rst lec-ture in this session was given by Prof. Boris Labar (Zagreb, Croatia), with the title: “Graft versus tumor effect in allo-geneic transplantation”. It is of interest to the EFI community that the second invited speaker in transplantation ses-sion was Prof. Ilias IN Doxiadis (Leiden,

The Netherlands), one of the leading fi g-ures in the HLA worldwide, who gave a lecture entitled: “Transplantation as a model for autoimmunity or vice versa”. In his talk Prof. Doxiadis compared the mechanisms underlying the etiology of autoimmune diseases and transplanta-tion related immunological processes, gave an overview of the current views about the role of T and B cell mediated reactions involved in the organ rejection and addressed the questions regarding the possibilities of tolerance induction. This lecture was one of the meeting’s highlights, and especially appreciated by the participants from the Tissue Typing Centre Zagreb. We would like to use this opportunity to thank Prof. Doxiadis for fi nding the time to attend our meeting.

and EU Projects refl ecting HSCT. Spe-cial attention was paid to the aims and importance of Poseidon project which is coordinated by Anne Cambon-Thomsen(France). During session 4, which was chaired and moderated by Antonio Nunez-Roldan (Spain) and Chryssa Papasteriades (Greece), con-cerned the EFI accreditation program: Staying competent in a complex fi eld given by Gottfried Fischer (Austria) and EPT organization within EFI, accredi-tation of EPT Schemes presented by Ciaran Dunne (Ireland). Also there have been reported results on EFI Region 8 EPT schemes in HLA typing, alloantibodies and HLA-B27 testing by the organizers Mahmut Carin (Turkey) and Elissaveta Naumova (Bulgaria). Chryssa Papasteriades in her talk described a present status and future directions in EFI Region 8. The Borovets EPT conference turned out an excellent example to get together renowned lecturers, participants from EFI Region 8 and people from National immunological society giving to all of

them unique possibilities for future col-laborations and partnerships.Furthermore, the attendants had great opportunities for discussions and infor-mal talks in a nice and friendly atmo-

sphere. Also they had a rare chance to enjoy a winter beauty of Rila mountain, social events full of fun as well as the incredible Bulgarian hospitality.

27

Report of the French 11th educational meeting: the lesson of the foetus to organ transplantation? Mireille Drouet, representative of the French EFI educational committee

The 11th French educational meet-ing took place the 27th and 28th of September at Limoges, capital of the Limousin region, famous for its refi ned “Porcelaine” and the quality of its beef meat.

First of all, I like to thank all the mem-bers of my staff for their help in the organisation, a heavy job for a small lab.

Dominique Charron, president of the EFI, MJ Cantournet, representative of the direction of Limoges hospital wel-comed the two hundreds participants, including representatives of Algeria, Morocco, and Portugal.

The theme of the fi rst session was dedicated to the lesson brought by the maternal-foetal immunology. G Chaouat (Insern Clamart) told us the complexe story of the immunology of pregnancy… a story which had changed in the last decade. The classical scheme which considered the foetus as a tolerated allograft was reviewed : “The concept has evolved from the maternofoetal tolerance to a dialogue between the embryo and the mother, which involve specifi c regulations mechanism at the gestation phase”. The materno foetal immunity involves specifi c uterine NK cells (not T cells) and their KIR recep-tors as well as various cytokines. Pregnancy is preceded by the implan-tation phase which requests an infl am-matory reaction. The comprehension of these mechanisms contributes to understand the process involved in spontaneous abortion and preeclamp-sia. HLA molecules expression has its own characteristics. In particular HLA-G molecules expression on the tropho-blast play a role in the tolerance of the

foetus. A Naji (Paris) demonstrated the implication of HLA G in transplanta-tion. In heart transplant patients who expressed HLAG on their graft do not develop chronic rejection. A high quan-tity of HLA-G molecules in the plasma appears to correlate with a decrease of acute rejection and an increase of graft survival. We all know that moth-ers can develop HLA antibodies against the paternal HLA inherited antigens. Mothers can develop antibodies again others polymorphic proteins. Those alloantibodies might be deleterious to the foetus. V Guigonis (Limoges) report cases of glomerolopathies occurring in newborn babies due to an alloimmune reponse to CD10 molecules. A refi ned analysis of these antibodies can lead to the identifi cation of non HLA antibod-ies involved in organ rejection. The development of cord blood stem cell transplantation is a new link in the relation between foetus and trans-plantation. E Marry, director of France Greffe de Moelle, describe the French cord blood bank whose development started in year 1999. The collection of new units allows enriching the bank with more than 85% of new HLA geno-types each year. The increase of HLA-C typing becomes an important criterion of selection, even if only 40% of the patients are transplanted with a 6/6 or 5/6 HLA matched units. The risk of loosing the units during the process must lead to have a security process such as localization of another donors or collection of autologous stem cells. The increased of cord blood transplan-tation is due to its feasibility in adult patient with a milder conditioning. The advantage and disadvantage of cords blood versus unrelated haematopoietic stem cell was developed by S Furst (Marseille). In France, when an unre-lated bone marrow transplantation is considered, the current strategy is to interrogate simultaneously unrelated bone marrow panel and cord blood bank to search for a 10/10 or 9/10 matched alive donors. In absence of donors, one or two cord blood units are searched.

The third session was dedicated to the transplantation of highly sensitized patients. A Parissiadis (Strasbourg) reviewed the recommendations edited by the French expert committee to per-form the follow up of HLA antibodies

for those patients. The experiences of acceptable missmatch programs of the Agence de Biomédecine (C Antoine, ABM)) and Eurotranplants (I Doxiadis, Leiden) were compared and allowed an animated debate. The good news is that both programs are useful to patients. The high number of positive cross match in France could be due to HLA class II antibodies which are not currently taken into count. On the side of acceptable miss match program, Y Le Meur (Limoges) reviewed others strategies to give access for such patients to the transplantation.

Plenary session were completed by four teaching sessions : Quality session (A Cesbron, P Perrier, C Gautereau, B Mercier, P Tracco), Platelet transfu-sions (J Chabod, V Dubois), Beginning in lymphocytotoxicity (L Absi, P Perrier), the 24 hours duty in organ transplanta-tion (I Jollet, JL Taupin).

Just as well we had a pleasant meal in an old Limousine farm, to warm up from an unusual bitterly cold weather !!!

You can already note in your agenda next year educational meeting which will take place at Besançon on the 9-10 October.

REPORT OF THE FRENCH 11TH EDUCATIONAL MEETING

28

REPORT ON THE 4TH EFI SUMMER SCHOOL KATARINA LUDAJIC

Pecs is a quaint little town in the middle of the Mecsekalja wine region in the south of Hungary. All the chances are you’ve never heard about it before, but be prepared to hear plenty, as the city is selected the European Capital of Culture in 2010. It was there that the 4th Summer School of Immunogenetics was held, organized by local coordinator Dr. Rajczy and EFI representative Prof. Claas with friends. The fi rst introductions of school participants, or in some cases reunions, took place over the dinner featuring delicious Hungarian specialty of goulash soup. During following four days many of immunogenetic topics were covered, experiences were exchanged and some new co-operations plotted - all in the same friendly and relaxed manner.

The school was opened with the population genetics and HLA and disease association lectures (Cristina Navarrete and Sophie Caillat-Zucman), followed by HLA typing (Deborah Crowe), histocompatibility and organ transplantation (Karen Nelson and Frans Claas), antigen processing and peptide presentation (Jim McCluskey) and lastly the lecture on KIR genes (Frank Christiansen) that closed the meeting. Each of the lectures offered both general overview as well as detailed approach to corresponding topics, depending on the lecturer’s exper-tise. The school attendants also used the opportunity to present their work, mostly related to the theme of the day, and get the advice from all present.One of the lectures that resisted classifi cation due to its all-encompass-ing nature was the one on immunogenomics, given by Andras Falus from Semmelweis University, Hungary. I believe I was not the only one thankful for the in-depth explanation of very resonant and profound, but somewhat nontransparent terms like glyomics, immunomics (and other omics), immunogrid, intelligent vaccines, virtual immune system etc. The cross-section through various knowledge engines, OMIM, Gene-Spring and other databases, Filter Genome and similar tools was an overwhelming undertaking, and extremely useful one, no need empha-sizing. Another special of this year’s School was the opportunity to attend the Congress of the Hungarian Nephrologists, where Frans Claas gave the lecture on Eurotransplant experience concerning kidney waitlist and

transplantation. The school attendants could, in addition, benefi t from Karen Nelson’s talk, who briefed us on the current situation of organ transplanta-tion in the USA. This short congress visit underlined an important difference between the two events: unlike the congress were one has to choose the session, run from one lecture to the other, say one’s polite hellos to company representa-tives or greet the cooperation partners - time in the school was ample, effi -ciently used and enjoyable, evenings included. Whether a guided tour around the city, visit to a local craftsmen, wine tasting in a family cellar or a dinner, conversa-tions never run out of topics. Consider-ing properly mixed company (Aseatta, EFI and ASHI) it was no surprise. Our different professional backgrounds (tissue typers, medical doctors, basic research biologists) and variable national origins provided the constant inspiration for lively conversations. I would like to thank all the tutors and organizers for making this success-ful and rewarding event possible and as enjoyable as it was. To the future attendants of 2008 Summer School I wish many good experiences in Brasil and I’m looking forward to reading here about their impressions after the 5th school is over.

29

REPORTS OF EFI BURSARY RECIPIENTS ON THE MEETING IN BARCELONA.

The annual meeting in Barcelona was also attended by a number of recipi-ents of an EFI bursary. Receiving a bursary implies writing a report on one of the sessions. A summary of these contributions is given in this paper. All recipients of a bursary were grateful to EFI for receiv-ing the support, which enabled them to attend the meeting.

The relevance of genetic polymor-phisms on HSC transplantation

David S. DeLuca , Hannover,Germay

Genetic variation is the major compli-cating factor following a hematopoietic stem cell transplantation (HSCT), with HLA matching representing the state of the art solution. However, non-HLA variation also affects HSCT outcome via various avenues, including minor histocompatibility antigen presenta-tion, SNPs in genes associated with immune response, as well as SNPs in genes associated with drug metabo-lism, response to infection, etc. Dr. Dickinson began her presentation with an example involving polymorphisms in proteins in disrupting a metabolic path-way. Methylenetetrahydrofolate reduc-tase (MTHFR) is a major enzyme in the folate metabolism pathway, a pathway which is also adversely affected by the pharmaceutical methotrexate (MTX). Non-synonymous SNPs in the gene coding for this protein were shown to associate with increased GvHD and decreased overall survival in MTX and non-MTX sibling cohorts. These results contribute to a series of studies which have similarly demonstrated decreased survival in non-wild type carriers. For example, Murphy et al 2006 demon-strated that MTHFR variant carrying donors associate with GvHD in HSCT patients treated with MTX. Robien et

all 2006 similarly found an association between an increased risk of GvHD and MTHFR polymorphism in a 350 pair cohort.Furthermore, studying SNPs can help to answer questions like: Why do some patients develop more severe com-plications, infections, GvHD (acute vs. chronic)? Why do some patients respond better to therapy than others? What factors affect the extent of the “cytokine storm” after transplanta-tion? A variety of cytokine gene poly-morphisms have been associated with GvHD and infection, including TNF-�, IL-10, IL-5, IFN-�, and IL-Ira. These con-cepts could be brought to the clinic by defi ning hi- and low-risk patient groups, allowing for the reduction in the inten-sity of conditioning in certain groups. Dr. Dickinson concludes that the evalu-ation of new genetic risk factors beyond HLA should contribute to individualized patient and donor selection as well as selection of prophylactic and therapeu-tic strategies.

Dr. Guillermo F. Sanz gave an overview of the advantages and disadvantages of umbilical cord blood transplanta-tion (CBT) over HSCT, particularly for children without an available HLA iden-tical family donor. The problem of the long wait associated with the search for a matched unrelated HSC donor is overcome by the approximately 14-day cord blood search. This is particularly critical for patients requiring urgent transplantation. Furthermore, one or two HLA mismatches are tolerated for a CBT, increasing the chance of fi nding a “matched” donor. On the other hand, engraftment is slowed by the low levels of hematopoietic stem cells in the cord blood. However, the effect on survival can be counterbalanced by a lower inci-dence of severe graft-versus-host dis-ease. Dr. Fanz suggests that the low HSC level in cold blood could be com-pensated with a double dosage.

Antoine Toubert’s talk focused on immune reconstitution in HSCT which is necessary for avoiding relapse via a GvL effect and to achieve a broad T cell response against pathogens. As summarized nicely in Barrett J., Curr Opin Immunol 2006, recipient lympho-cytes drop drastically in number within the fi rst week after transplantation,

after which time the donor NK cells quickly proliferate. Recipient and donor antigen presenting cells on the other hand decline and increase slowly over months respectively. Donated T-cell precursors can mature in the recipient thymus, allowing for a broad repertoire of long-lived naïve T cells, leading to full immune recovery. In parallel, donated memory T cells expand in the periphery, leading to a skewed memory T cell rep-ertoire, but with the positive effect of providing early response to pathogens. A delay in the T-cell repertoire reconsti-tution can occur especially in the case of T-cell deplete CD34+ grafts. Further-more there is a correlation between TCR repertoire disturbance and the GvHD/infection status of the patient. While thymic-derived naive T cells from donor lymphoid precursors can pro-vide a fully functional immune system, the thymus itself may be a target of GvHD. This can furthermore inhibit thymic negative selection, resulting in the escape of autoreactive T cells. To measure thymic function Dr Toubert’s group quantifi es T-cell receptor exci-sions circles (TRECs) using real-time PCR. TRECs are DNA fragments which result from TCR locus recombination. TREC levels have been previously asso-ciated with GvHD, opportunistic infec-tions and leukemia relapse. The group demonstrates dramatically that high levels are a clear predictor of cumula-tive survival (~90%), and conversely, low TREC levels predict very poor survival (<50%) for patients receiving transplants from HLA identical siblings. They furthermore demonstrate that low TREC levels lead to increased GvHD as well as bacterial and CMV infections.

Federico Sizzano, Turin, Italy

Matching of donor and recipient for the HLA antigens and alleles improved strongly the outcome of transplanta-tion, especially in kidney allografts. Moreover, the use of unrelated donors matched with recipients for the HLA loci, ameliorated the outcome of hema-topoietic stem cell transplants (HSCT) in terms of acute and chronic graft-versus-host disease (GvHD). Never-theless, in solid organ transplantation episodes of acute or chronic rejec-tion could lead to the organ failure

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whereas the development of severe GvHD remains a cause of morbidity and mortality after HSCT. This could be due to differences between donor and recipients for genes that not belong to the HLA complex. In recent years, immunogenetic research was focused on the identifi cation of genes and their polymorphic variants involved in the immune response after a solid organ transplantation or HSCT. For example, many investigators reported associa-tions between immunological adverse reactions (rejection, GvHD) and the presence of a particular cytokine gene polymorphism in the donor or recipient. Several investigators proposed not only to defi ne a “structural matching”, i.e. to characterize allelic variants of genes involved in the immune response, but to assess also a “functional matching”. The use of in vitro methods measur-ing the strength of immune response between responder and stimulator cells of donor/host origin could give informa-tion about a general activation of the immune system, taking into account all the genetic differences among the individuals. Furthermore, the analysis of the humoral response against HLA alloantigens before and after trans-plantation, as well as the detection of serological markers, could give pre-dictive information about solid organ rejection. Dr. Fuggle presented data about the Living Donor Kidney Transplantation Program in the UK. The study assessed the association between the degree of HLA matching in living related (LRD) and unrelated donor (LURD) allografts and the transplantation outcome. The level of HLA matching was signifi cantly poorer in LURD transplants compared to the LRD (29% LURD allograft had 4

HLA-B+DR mismatches compared to 2% of LRD). Data showed that three-year graft survival was greater in LURD allografts, whereas the 3-year patient survival was poorer in this allograft setting. Prof. Doxiadis described the use of a new tool to identify a cross-match negative donor: the virtual Panel Reactive Antibody (PRA). The presence of donor-reactive antibodies against HLA alloantigens is unfavorable for the outcome of transplants. The PRA as a parameter for organ allocation is informative for the probability of a posi-tive crossmatch with a potential donor. Some problems occur in this proce-dure, mainly due to the heterogeneity of the methods used and the compo-sition of the panel. The “Virtual PRA” program of Eurotransplant calculates a virtual PRA value based on the HLA typing of 4000 organ donors from the Eurotransplant. The advantage of this method is that results from different techniques used to determine anti-bodies specifi cities can be combined, giving more reliable data on the chance to receive a crossmatch-negative donor in the specifi c donor population. Dr. Galvani showed the importance of allo-antibodies against HLA-class I antigens in a murine model of chronic vascular rejection. Human mesenteric arteries from organ donors were grafted into immunodefi cient SCID/BEIGE mice, that received weekly i.v. injections of monoclonal antibody anti HLA-class I, whereas control mice, grafted with the same arteries, received i.v. injec-tions of an irrelevant isotype-matched antibody. Mice receiving anti-HLA class I antibody developed neointimal thick-ening, whereas control mice had little or no thickening. The pathogenesis of chronic rejection could be due to the proliferative effect of anti HLA-antibody on the smooth muscle cells. Dr. Vago described the lack of Graft-versus-leu-kemia (GvL) effect after haploidentical HSCT (in terms of relapse incidence) despite the predicted donor NK allo-reactivity on the basis of KIR ligand incompatibility. NK cells in the fi rst month after allografting were NKG2A positive and KIR negative (in 13% of cases), similarly to NK CD56bright from healthy donors. However, NK cells from the patients had heterogeneous inten-sities of CD56 fl uorescence, expressed high levels of CD16 and were partially positive for c-kit and CD25. This phe-notype is common to NK cells differ-entiated from CD34+ progenitors after autologous and matched unrelated HSCT. Furthermore, these cells had an impaired capability to secrete IFN- in

response to leukemic blasts and had an impaired cytotoxicity. The authors suggested that the NK cell mediated-GvL effect could be obtained mainly when donor mature NK cells are allo-grafted. Dr. Figueiredo presented data concerning the ex-vivo modulation of the alloreactivity in primary NK cells. Incubation of NK with IL-1 , IL-2, IL-4, IL-12, IL-15 increased the expression of the activating natural cytotoxicity recep-tors, whereas IL-1 and IL-2 decreased the expression of inhibitory receptors KIR2DL1 and 2DL2. The NK cytotoxicity after the treatment with this panel of cytokines was increased by up to 20%. When NK cells were incubated with HLA-C1 or HLA-C2-positive B-LCLs, the subsets expressing KIR 2DL1 and 2DL2 increased by up to 30%, respectively. The subset KIR3DL1-positive, in pres-ence of Bw4-positive-B-LCLs, increased by up to 17%. Cytotoxicity was found augmented when these cells were incubated with target cells not express-ing the corresponding ligands. These results suggested that the ex vivo manipulation of the NK cell subsets could increase the alloreactivity against leukemic blasts. In the oral session 7, I was particularly interested in the talk about the immunomodulatory effects of human mesenchymal stem cells (by Dr. Naji, O-72). Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types and possess immunomodulatory properties. Currently, there’s a great interest in the use of MSCs in HSCT fi eld, especially for the GvHD treatment (see Le Blanc K et al. Lancet, 2004). In the presentation at EFI congress, the Authors showed that adult MSCs express membrane bound HLA-G1 mol-ecules and secrete soluble HLA-G5 iso-forms. These factors are very important in the maintenance of the immune privi-lege. Moreover, the blockade of HLA-G molecules decreased the capability of MSCs to induce the expansion of Treg, inhibit T cell proliferation and NK-medi-ated cytolysis.

Antibody removal in solid organ trans-plantation

Maja Puc, Zagreb, Croatia.

The shortage of donor organs, espe-cially in renal transplantation, leads to an increasing discrepancy between the number of end-stage renal disease patients on waiting lists and the number of available deceased donor kidneys. Expansion of the donor pool can be

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y

f f

y f

o f

y

f

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achieved by increasing the numbers of living kidney transplantation and by overcoming the immunological barriers of ABO-incompatibility and HLA-sensiti-zation. Despite a substantial increase in the number of patients, receiving living kidney transplant, otherwise suitable donors have to be rejected due to pre-existing human leukocyte antigen (HLA) antibodies or ABO-incompatibility. As blood group antigens are expressed by the endothelium of solid organs including the kidney, transplantation across the blood group barrier can result in hyperacute antibody mediated allograft rejection. So removal of those is necessary prior to ABO-incompatible grafting for kidney recipients with high anti-A/B antibody titres. There are several protocols for ABO incompatible kidney transplanta-tion, all based on some principle-to remove existing antibodies and pre-vent rebound of antibodies in the kidney recipient after transplantation. Antibodies can be removed by plasma exchange, unspecifi c adsorption and specifi c adsorption. To prevent rebound, removing antibodies is combined with immunosuppressive therapy, including oral immunosuppression, intravenous immunoglobulin and antibody induc-tion. The donor specifi c antibody levels are monitored and transplantation is performed when the antibodies are suffi ciently eliminated.

Dr. Rydberg presented protocol and results of his team which included patients pretreatment with 3-6 immuno-adsorptions using sepharose columns with synthetic A- or B carbohydrates attached (Glycosorb®A/B). The aim was to reduce the titre of anti-A/B IgM/IgG antibodies, as measured by stan-dard blood banking techniques, to 8 or below. The immunosuppressive treat-ment included anti-CD20 (rituximab), mycophenolic acid, tacrolimus and ste-roids. Kidneys from ABO incompatible living donors can successfully be used after reduction of recipient’s ABO anti-body titres and with a carefully moni-tored immunosuppressive protocol.A positive cross-match is generally an absolute contraindication to transplan-tation. To overcome this problem, some centres have developed desensitisation protocols very similar to those used for ABO incompatible transplantation. The removal of HLA antibodies is carried out either by plasmapheresis or by pro-tein A immunoadsorption. In contrast to kidney transplantation a positive crossmatch is no contraindication for liver transplantation. In liver transplan-tation, antibody mediated rejections are rarely reported and a liver graft is suspected to have protective effects for kidney grafts when transplanted simultaneously. So, a simultaneous transplantation of kidney and a par-tial auxiliary liver graft from the same

donor, with the sole purpose of protect-ing the kidney from harmful lymphocy-totoxic antibodies, can be performed successfully despite a positive cross-match and may thus be a new option of treatment for highly sensitized patients waiting for a kidney transplant.Dr. Briggs presented HLA incompatible kidney transplantation and guidelines that are used in UK for antibody incom-patible transplantation (AiT) since September 2006, which are based on British Transplantation Society. A laboratory supporting a programme of AiT have robust methods to distinguish between total HLA antibodies, often reported as ‘panel reactive antibod-ies’ (PRA), and HLA antibodies directed against the donor. DSA levels may change independently of PRA, espe-cially in the post-operative period. The introduction of more sensitive methods using purifi ed HLA antigens either in ELISA formats or attached to beads are a major advance in monitoring DSA before and after transplantation. While the newer techniques allow for rapid and sensitive monitoring of DSA levels, CDC and FC XM are still performed in all individuals. If the CDC XM indicates reactivity, serum is titred in order to measure the highest dilution at which the CDC XM is positive. The titre of the CDC XM is regarded as an effec-tive measure of the amount of antibody present, and is correlated with the

32

amount of plasmapheresis needed pre-transplant. It is also associated with the risk of antibody-mediated rejection post-transplant. However, there are cir-cumstances where a transplant may be performed with a current positive CDC XM.The level of HLA incompatibility (HLAi) that causes hyperacute rejection depends on factors including the anti-gen type, antigen density, and the antibody level. The CDC XM was once thought to represent an adequate in vitro evaluation of the risk of hyper-acute rejection, as antigen and anti-body-dependent factors both infl uence the test result. However, it has recently become clear that for some types of HLAi, a positive CDC XM does not nec-essarily indicate that hyperacute rejec-tion will occur. This is most apparent for class II HLA antibodies, especially the products of non DRB1 genes, ie HLA-DR51/52/53. Transplants with positive CDC XM titres of up to 1/16 have been performed in such cases with good immediate graft function. The relatively low level of expression of HLA-DR 51/52/53 is thought to be the main factor that reduces the risk of hyperacute rejection. The use of LDT may also facilitate transplantation in the presence of class II donor specifi c antibodies (DSA), as the relevant anti-gens are expressed to a rather lower extent than on deceased donor trans-plants. Although low titre positive CDC class II DSA may be tolerated by the

graft at the time of transplantation this has only been shown to be safe in the context of live donor transplants.The rationale for performing pre-trans-plant plasmapheresis in subjects with low levels of HLAi, for example negative CDC/positive FC XM, is empirical. Plas-mapheresis-based protocols have been used to treat patients with a negative CDC/positive FC XM, with good clini-cal outcomes. Although it may appear to make sense to remove all DSA prior to a transplant, this is not necessarily the case. In vitro data indicate that low levels of DSA may upregulate defen-sive mechanisms in vascular endothe-lium, perhaps enhancing the ability of a kidney to resist antibody-mediated rejection at a later stage. The optimal treatment protocol for subjects with HLAi that is negative CDC/positive FC XM remains uncertain.The likelihood of re-synthesis of DSA after the transplantation cannot be predicted accurately in advance. It is associated with the amount of antibody present before treatment was started, but this is not always the case. When re-synthesis does occur, it may be rapid, leading to rejection with oliguria over a period of less than 24 hours. In other cases little or no change in graft function may occur. Therefore careful clinical and laboratory monitoring of all patients seems sensible, regardless of the pre-treatment levels of antibody.The clinical signifi cance of very low levels of antibody (for example nega-

tive CDC/negative FC XM but positive using purifi ed antigen methods, eg anti-gen coated beads) remains to be eluci-dated. It is probably low risk, certainly at the time of surgery, though there may be an increased risk of antibody-medi-ated rejection subsequently. It would be reasonable to treat such patients without pre-transplant plasmapheresis, but to monitor them very carefully post transplantation.

Chronic transplant rejection.

Sylvain Galvani, Toulouse

As I’m doing my PhD about chronic vascular rejection, I’ll focus this short report on the important role played by HLA antibodies in chronic transplant rejection.Even if great progress have been done to avoid acute and hyperacute rejection by a better cross-matching between donor and recipient and a better moni-toring of recipient immunosuppression by the use of new drug associations, long term transplant survival is still a problem and chronic rejection is the main cause of organ loss and patient death. there is increasing evidence that the development of chronic vascu-lar rejection is a determinant factor of graft survival.After a slow deterioration of the organ function, the main feature of chronic vascular rejection (CVR) also known as graft arteriosclerosis, is a specifi c histopathological process in the arter-ies of the graft, characterized by a con-centric proliferation of smooth muscle cells (SMC) in the intima of the arter-ies of transplanted organs. This pathol-ogy leads to a progressive occlusion of the blood vessels and ischemia of the transplant organ. The physiopatho-logical mechanisms of CVR are still poorly defi ned and there is currently no effi cient therapy to avoid this develop-ment.It is now clear that immunonological and non immunological factors can act in the development of the lesion, but the role of the humoral alloreactivity is still an open question.In our lab, we want to describe the important role played by these antibod-ies in the lesion development.Our study is based on in vitro studies on human SMC but also on in vivo stud-ies using a promising animal model. We have grafted human mesenteric arter-ies into SCID/beige mice (which are immunodefi cient mice), and we have injected intravenously HLA antibodies

33

(w6/32 clone) weekly. Six weeks after grafting, arteries were removed to study arteriosclerotic lesions. Using differ-ent patients with different haplotypes, age, sex… we have shown that HLA antibodies can provoke a strong neo-intimal proliferation. The time course of this proliferation is quite short (in fi ve weeks we have observed a mean thick-ening of 40% of the intima). Moreover, we have shown that HLA antibodies can provoke human SMC proliferation, in vitro, by the activation of 2 signalling pathways, the PI3K pathway (results also found by the team of E. Reed), and the MAPK pathways.

During the congress, one of the teaching lectures drew particularly my attention.Pr Terasaki who is one of the great researchers on HLA implication in transplantation had presented us results obtained from many centres on sera from transplant patients. The aim of the project was to correlate appear-ance of HLA antibodies in serum, and rejection episodes. Do these antibod-ies appear before rejection and if so are they actors of this rejection, or do these antibodies appear after rejec-tion, playing a minor role in chronic rejection.To do this study, HLA antibodies were screened in sera from patients, dif-ferent time post-transplantation. One screening was made directly after kidney transplantation; the other was done some time after. These results were correlated with the rise of serum creatinin level which is a good indica-tor of kidney function degradation. The multi-centre bank of sera was a mine of information (the “goldmines”) and an important point to validate his hypoth-esis. The result is really relevant because, most of the transplants were lost after production of HLA antibodies. The presence of MICA antibodies was also checked. MICA antibodies (MHC class I chain-related protein A) are antibodies which appear after transplantation. The precise role of these antibodies is not well known but they might play a crucial role in transplant rejection. The study of Pr Terasaki highlighted the fact that when no HLA antibodies were found in the sera of patient in chronic rejection, MICA antibodies were present. The co-presence of MICA antibodies and HLA antibodies in patient serum was asso-ciated with an earlier and rapid trans-plant loss. The multi-centre bank of sera is a mine of information (the “gold-mines”). Professor Teraski pushed his results by showing us different patient

sera dosing, showing in parallel serum creatinin level, HLA antibodies levels, MICA antibodies level, and graft sur-vival. In most case, serum creatinin increase after antibodies apparition in the serum. But graft loss is delayed in the time (more than 2 years).During his talk, he also presented us new results on antibodies against graft dosing in case of other transplanta-tion, like lung, heart and the survival of graft. In all case antibodies apparition was correlated with a poor outcome of graft.This study appears really innovative in the fi eld of the comprehension of the cause of chronic rejection, because all centres made the same conclusion that HLA antibodies but also non-HLA antibodies against the graft (donor specifi c of not) play a crucial role in the development of chronic rejection. These results pushed by the results found by our studies can lead to a new approach of the support of post transplant patient, in which strong monitoring of HLA antibodies and other antibodies against the graft seems to be crucial for graft survival.These works lead to a strong important conclusion: Antibodies play a role in the development of lesions in chronic rejection; they really are responsible for chronic rejection.

Meeting the Challenge in Sequencing-Based Typing

Helder Spinola, Madiera, Portugal

Verboom and Blasczyk presented three different challenges in Sequencing-Based Typing (SBT): Genetic; Techni-cal; and Financial. Genetic challenges are specially associated to ambigui-ties, null alleles and new alleles. Two kinds of ambiguities are present in HLA SBT typing: the ones caused by poly-morphisms in undefi ned regions out-side exon 2 in class II or exon 2 and exon 3 in class I loci; and heterozygous ambiguities caused by undefi ned cis-linkages. Null alleles are mostly solved looking exon 2 and exon 3 of HLA class I loci. From 84 HLA class I null-alleles known, 54 are excluded in exon 2 and exon 3 and 9 requires exclusion in pro-moter or introns. Nine of the 11 HLA class II null alleles known could be excluded in exon 2 and only 2 require exclusion in intron 1 or exon 3. Null alleles are extremely rare and most are covered by sequencing exon 2 + 3 for class I, and exon 2 for class II genes. Pretending solve all null alleles could

be considered a waste of resources.On the contrary, new alleles are a fre-quent phenomenon and are a source of mistyping that could be minimized solving cis-linkages through SBT and group-specifi c amplifi cation.Most technical challenges relays on allele assignment, data management, and throughput. Allele assignment and data management are candidates for bioinformatics solutions with the advantage that sequencing is the typing technique with the highest potential for automation. Otherwise, high through-put is only possible with a good data management, which means that these technical challenges on SBT are inter-related. Financial challenges on SBT are related to the expenses in employee training and to the equipment invest-ments. Training for technicians is the elementary basis for a good running sequencing process, which is the most economical typing technique in a high throughput procedure with the advan-tage that have no need for another supplement technologies.To meet this SBT challenges it is important to maximize allele separa-tion, minimize sample mix up, and maximize sample throughput. Group-specifi c amplifi cation is one of the available techniques to maximize allele separation reducing ambiguities. Soft-ware documentation is strongly rec-ommended to avoid sample mix up, and automation and homogenization of processes is essential to maximize sample throughput.

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> W O R L D M A R R O W D O N O R A S S O C I A T I O N

7th InternationalDonor Registry Conference and

Working Group MeetingsSecond announcement

16 to 19 april 2008 in Bern / Switzerland

35

I am pleased to be presenting you with the 2nd announce-

ment for the 7th International Donor Registry Conference

and Working Group Meetings in Switzerland, to which

you are warmly invited.

I am sure that we can offer you an exciting programme

for this congress. Please consult the following pages for

more detailed programme information. The organising

committee in collaboration with the board of WMDA has

made every effort to provide you with a conference

structure which will give you new information as well as a

challenge to prepare the future work within this world

wide so important network.

In addition to the hard work you will be asked for, I

would like to point out our welcome reception to be held

on Thursday, 17 April 2008. Our Swiss registry will be

celebrating its 20th anniversary next year. This is a moment

for reflexions, a time to thank all those who contributed

to this achievement and an occasion to celebrate the

evening – with you – as a sort of «birthday party». Come

along and see what we have in store !

I am looking forward to as many registrations as possible,

enabling me to welcome you in Bern in April 2008.

Yours sincerely,

Prof. Alois Gratwohl, MD

President, Swiss Blood stem cells

Dear Colleagues

> I N V I T A T I O N

2007 © Invitrogen Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen catalog or our website, www.invitrogen.com).

LESS A BIGUI Y

MOR ACCUR C

.

.Invitrogen’s Transplant Diagnostics team offers proven, innovative technologies to help make a difference in HLA analysis –

and patients’ lives. Our products are designed to improve the quality, reliability, and speed of transplant diagnostics. To meet your resolution

and throughput needs, we deliver a range of methodologies, including SBT, SSO, SSP, and Antibody Analysis. Our Transplant Diagnostics team

also offers years of HLA expertise and involvement in the community by sponsoring the key transplant organizations. We’re here to support your

efforts at making a difference. See how at www.invitrogen.com/transplantdiagnostics.