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EUROPEAN FEDERATION FOR IMMUNOGENETICS

NEWSLETTER MAY 2008 - ISSUE 56

…………..FROM THE EFI PRESIDENT

It gives me great pleasure, and it is indeed a great honour, to address you for the fi rst time as President of EFI. I am not new to the EFI Board, I was fi rst elected onto the EFI Executive Board as a Councillor in 1999. Since that time I have served two terms as Deputy Secretary and for six years chaired the Web Committee. As such I have worked with the past fi ve EFI presidents, and have for some time found myself in the unenviable position of being the longest serving member (but not the oldest!) of the Executive Board.

Communication between members of EFI is key to the successes of the society and in particular the achievements of the committees that serve the EFI member-ship. During my time on the Executive Board, in my role as Deputy Secretary, I have been responsible for accurately remembering details of the many dis-cussions and decisions made that have taken place at Executive Board meetings, all of which are summarised in our news-letter and presented to the membership at the annual general meeting. However, I feel that with the technology available to

DEAR EFI MEMBERS:us we ought to be able to make greater use of our website to store and dissemi-nate more fully the details of the deci-sions taken by the board, the minutes of meetings, the operating structures of our various committees and their activi-ties. These can all be housed within the members section that has existed on the website for some time now. We will soon be seeing the new updated version of the website with a much enhanced members section. This is the result of the hard work undertaken by Ralf Wassmuth and the Web Committee. Once launched it is planned to introduce new sections of the web site as they become available. Ultimately the new site will give us much more functionality than the existing one.

I would like to see electronic communi-cation via both the web site and through email become more usual than it is at present. Not only will this help EFI save money on postage but also prevent the unnecessary use of paper and save a few trees along the way. It is perhaps surprising that some 20% EFI members do not have an email address listed in the membership database and a further 40-50 have supplied old or incorrect addresses. I would ask all members to check their details online and send an email address to the membership sec-retary <membershipsecretary@efi web.org> if one is not listed.

The fi rst EFI meeting that I attended was EFI’s third, which took place in Strasbourg in 1988. Since then I have attended a further 18 meetings, and have always been impressed by the standard that we achieve. This year’s meeting in Toulouse was no exception; Ann Cambon and Mogens Thomsen, together with their local organising com-mittee, are to be congratulated on their success. As indeed are the Scientifi c committee with Erik Thorsby as chair,

our new EFI president

for putting together such a stimulating and high calibre scientifi c program.

I would like to take this opportunity to wish a belated happy birthday to Jon van Rood, EFI’s fi rst president, who cel-ebrated his eightieth birthday recently. This year also marks the 50th anniver-sary of his seminal publication in Nature on June 21st 1958, on the identifi cation of “Leucocyte antibodies in sera from pregnant women”. This publication with George Eernisse and Aad van Leeuwen as co-authors, opened the doors to the generation of a new medical technology of tissue-typing with the tissue typers screening the sera from women immun-ised against paternal HLA antigens, and then using these as reagents for HLA typing. Some of us were able to share in this celebration recently at the World Marrow Donor Association meeting in Bern. Jon’s continued energy and enthu-siasm never ceases to amaze me, I am sure he will continue to play an important role in our fi eld for many years to come.

Lastly the 15th International Histocom-patibilty Workshop and Conference will take place in Brazil in September. Many of you will have seen the presentation by Maria Gerbase-DeLima. The venues in Buzios and Rio de Janeiro look fan-tastic as do the scientifi c and social programs. I am sure that EFI will be well represented, and I look forward to seeing you there.

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....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: UlmURL: http://www.efi web.org/members/

CONTENTSFrom the EFI President 1From the editor’s desk 3Giovanni Battista (GB) Ferrara (1932-2008) 5Report of the General Assembly, 62007 balance General Offi ce 82008 budget General Offi ce 9Balance and budget of the accreditation offi ce 11Report of the Standards and Quality 11EFI EPT Committee Meeting 13Launch of the new EFI website 13Future EFI conferences 14Reports of EFI bursary recipients 15Reliability of chimerism testing 26A Collection of EBV-Transformed B-cell Lines 28Welcome to the 5th International Summer School on Immunogenetics 29Italian Workshop on EFI Accreditation in Bari 30

The EFI meeting in Toulouse was an enormous success both scientifi cally and socially. Not only an all time high record with respect to the number of participants was reached but the quality and variety of the scientifi c presen-tations was great. On behalf of the EFI community, I would like to congratulate Anne and Mogens with this success, also because the social program ( for many of us an (or the) important parameter to validate a meeting) was so excellent. I wish the local organizers of the meeting in Ulm good luck in their efforts to reach a similar level next year.This Newsletter refl ects many aspects of the Toulouse meeting including reports on the general assembly, of several committees and by recipients of a travel award.Hopefully, these reports and the pictures will stimulate the people, who did not attend the meeting, to join us next year in Germany.For our new president, Steve Marsh, who actually did not include a picture of his car in the issue, the challenge to guide EFI the coming years to an ever brighter future.Another successful concept is the international summer school on Immuno-genetics. This year the meeting, organized by ASHI, will take place in Sep-tember in Buzios just before the International Workshop. More information for possible applicants is given in this Newsletter.Furthermore, important information on the new website and several reports on national meetings are included in this issue.Hopefully, you all enjoy reading this Newsletter and I am looking forward to your contribution to the next one.

Frans Claas

The deadline for contribution to EFI Newsletter no 57 is October 15, 2008. Please send your contribution by e-mail to : 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 2008

EFI President:S.G.E Marsh (UK)

EFI Secretary:M. Tilanus (Holland)

EFI deputy secretary:A.M. Little (UK)

EFI Treasurer:C. Raffoux (France)

EFI deputy Treasurer:A. Moine (France)

Membership Secretary:S. Mesander (Holland)

Councillors:R. Blasczyk (Germany)

C. Dunne (Ireland)

E. Naumova (Bulgary)

C. Navarrete (UK)

A. Slavcev (Czech Republic)

C. Susal (Germany)

Past Presidents:J.J. van Rood, B.A. Bradley, E. Albert,

J. Hors, M-M Tongio, J.G. Bodmer,

F.H.J. Claas, S. Curtoni, E. Thorsby,

F.Garrido, D. Charron

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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GIOVANNI BATTISTA (GB) FERRARA (1932-2008)

On January 4th of this year our dear friend and colleague Giovan Battista Ferrara passed away. Ferrara, known to his friends simply as “GB”, was an International Councillor for the International HLA and Immunogenetics Workshop and the recipient of the 2005 EFI medal. He had recently retired from his post as Professor of Molecular Biology at University of Genova, Italy.

After taking a degree in biology at University of Milano, GB pursued his early interests in Immunogenetics, and joined the laboratory of Ruggero Ceppellini in Torino. Here he initiated his career in Histocompatibility and also met Anna Longo, a key-fi gure in Ceppellini’s HLA typing team, who became GB’s life and science companion until his death. In 1965, he left Torino to start his independent laboratory of Immunogenetics in the Cliniche Gavazzeni of Bergamo and then in General Hospital of Massa, Italy. At that time, he established a fruitful collaboration with the AVIS Research Center in Bergamo, which led to the generation of an impressive panel of human anti-HLA alloantisera. This collection was an extremely valuable resource in the early days of HLA typing and was also widely used for studying HLA polymorphisms and biochemical characterization of HLA, particularly the class II proteins. GB generously made this resource available not only to well-established HLA laboratories, but also to HLA typing laboratories in developing countries that had little access to any source of alloantisera. Through this activity GB demonstrated one of his strengths, the ability to establish an interna-tional network of collaborations to exchange scientifi c information and reagents.

In 1982 he became the head of the Laboratory of Immunogenetics at the National Institute for Cancer Research in Genova and, later on, Professor in the Department of Molecular Biology at University of Genova. While in Genova he demonstrated his inclination and passion for technological advancement, fi rst by estab-lishing anti-HLA human monoclonal antibodies, then shifting gears towards emerging DNA typing techniques, from DNA blot analysis, to oligonucleotide typing and DNA sequencing. His fi nal 2005 paper on the use of DNA microchip arrays for HLA typing epitomizes his constant endeavor to be on the cutting edge of the fi eld. Although Ceppellini initially resisted GB’s independence, he eventually recognized the success of his pupil and re-established a warm relationship with GB that lasted for rest of Ceppellini’s life.

GB’s innate enthusiasm for HLA was his driving force. However, he was also interested in applying his expertise in genetic polymorphisms to other problems, particularly cancer and forensic medicine. Given the broadness of his research interests and commitment to emerging technologies as well as access to an inter-national network of outstanding collaborators, GB’s laboratory attracted many young investigators not only from Italy, but also from many other countries in Europe as well as Latin America and the United States. In the late eighties, when I was there, the laboratory was at times an organized chaos, yet it became the launching pad for an extraordinary number of students and post-docs who have gone on to become independent inves-tigators in academia and industry in the areas of HLA, Immunology and Cancer.

GB’s approach with students and post-doc as well as his colleagues was direct, passionate, sometimes emo-tional but always wholehearted and this was his defi ning trait. His comradeship, spontaneity, and friendship will be deeply missed.

Marco Colonna Washington University School of Medicine in St Louis, USA

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REPORT OF THE GENERAL ASSEMBLY, HELD FRIDAY APRIL 4, 200817:30-19:00 PM DURING THE EFI ANNUAL MEETING IN TOULOUSE, FRANCE

1. Opening The president, Dominique Charron,

opens the General Assembly meet-ing at 17:45 pm and welcomes all members.

2. Minutes General Assembly 2007 The minutes of the General Assem-

bly, Monday May 7, 2007 held during the EFI annual meeting in Barce-lona, Spain that were published in the EFI Newsletter Issue 54, 2007 were approved.

3. Report of the Secretary - Deputy Secretary

Dr Steven Marsh was nominated as President Elect and Ann-Mar-garet Little as deputy secretary; both were elected unopposed. Four nominations were received for three positions of councillor; Domenico Adorno, Ciaran Dunne, Anthonij Slavcev, and Caner Susal. A total of 181 members voted, 15 votes were excluded due to the absence of a name or EFI number. Ciaran Dunne, Anthonij Slavcev, and Caner Susal were elected by majority votes.

Since more and more communi-cation will be done electronically please provide an updated email address to the membership secre-tary.

4. Report of the EFI executive Com-mittee meeting.

Extension of the Central Offi ce func-tion is currently being worked out including consideration of require-ment of the secretaries capabili-ties for the website management. The newsletter remains an excel-lent way of providing information to the members. All can make use of this possibility. Frans Claas is acknowledged for his continuing outstanding contribution in produc-ing the newsletter. Ralf Wassmuth, meeting liasion, coordinated the selection for the annual EFI 2011 meeting. Prague, Czech Republic was selected and will be chaired by Dr. Antonij Slavcev. The excellent collaboration with Tissue Antigens as offi cial EFI journal was evaluated and will be continued.

5. Report of the EFI treasurer More than 135 members paid their

annual fee at the EFI booth during the congress and 8 new members were registered. Sonja Mesander

(membership secretary), Colette Raffoux and Agnes Moine manned the booth. The budgets presented were approved (see tables).

6. Report of the EFI committees Standards and Quality Assurance,

Chairman Jean-Marie Tiercy Comments and suggestions to the

second draft of standards version 5.6 published in the newsletter (Jan 2008) has been discussed by the committee and adapted accord-ingly. The new standards regard-ing internal profi ciency testing was reformulated. The revised subsec-tion on complement and new stan-dards for platelet refractoriness and transfusion related acute lung injury have also been addressed. In the coming year new standards on cord blood and HPA/HNA standards will be worked out. Next challenge may include standards for chimerism analysis.

Andrea Hammer and Jean-Marie Tiercy are leaving the committee. Two new members are welcomed: Susan Fuggle and Christien Voorter. Kay Poulton has accepted the chair of the standards and QA committee. In 2009 there will be one vacancy for this committee. Jean-Marie thanks the pioneer members of the committee with whom he has had the priviledge to collaborate during his time as chairman (2000-2008): E. Albert, J-D Bignon, L. Gebuhrer, C. Loliger, S. Martin, P. Reekers, E. van den Berg-Loonen, A. van Leeu-wen and all the members of his committee for the excellent collabo-ration and efforts in this productive committee. Jean-Marie on his turn is thanked for the excellent man-agement of the committee and the continuous progression of the stan-dards.

Education, chairman Cristina Nava-rette

The current activities include the planning and co-ordination of Educa-tion Sessions at Toulouse meeting in close collaboration with the local organizer, Michel Abbal).

Two bursaries for educational activities (visit laboratories) were awarded.

Ongoing and planned activities include

a) Discuss/review the structure, composition and future activi-ties of the Education Commit-tee including a proposal to have Education representatives by region (accreditation committee regions)

b) Bursary applications: rewrite the application procedure and review the application form to promote the use of these bursa-ries. Announce the availability of such bursaries in the EFI news-letter, EFI Web etc

c) Identify the needs for Education and Training in each country through a Questionnaire distrib-uted through the regional repre-sentatives

d) Defi ning the role of Education Committee in the Poseidon proj-ect (WP6 and WP9)

e) Plan Education sessions for EFI meeting 2009 In Ulm, Germany (local representative Carl-Heinz Muller )

External Profi ciency Testing, acting chairman Ilias Doxiadis

Personal circumstances hindered Ciaran to chair the meeting. It is very much appreciated that Ilias Doxiadis was willing to chair the EPT meeting in Toulouse. Major issues that were discussed included 1. the defi nition and evaluation of HLA specifi cities and 2. the approval of the Eurotransplant EPT schemes. A summary of their meeting is reported elsewhere in this newslet-ter. John Vaage will take over the chair during the absence of Ciaran Dunne.

Accreditation, Chairman Gottfried Fisscher

As of March 2008, 205 labora-tories have been accredited. The strategic goals of the committee include accessibility, consistency and cooperation. For consistency the inspectors workshop was orga-nized and 64 inspectors attended. Marilyn Pollack (ASHI) attended the accreditation committee meeting. New activities that are addresses include

• Establish a new database for the accreditation offi ce

• Evaluate customer satisfaction through post inspection ques-

7

tionnaire and a web based ques-tionnaire.

• Joint inspection procedures together with national accredita-tion bodies

• Promotion of the EFI accredita-tion programme beyond Europe. Proposed to the board is an EFI Middle-East workshop early 2009.

Web committee, chairman Ralf Wassmuth The EFI website is currently imple-

mented and can be accessed through the new web address: at: www.efi web.eu. & www.efi web.org. A major function that is created is the introduction of a fi le system to share documents for the secre-tariat, the committees and special interest groups. The system is cur-rently online and adjustments to user specifi cations implemented.

The transfer of the information of the “old” website is transferred and new functions stepwise included. The fi rst available new function is a Personalised Member Access:

• Individual member login, data maintainance, platform for mem-bership card, etc.

Membership Fees: • Payment function imple-

mentedwith online registra-tion of credit card and cash payment, generation of receipts

Detailed information on the new website is published elsewhere in this issue.

Scientifi c Committee, chairman Erik Thorsby

A major task of the committee is to establish a very close collaboration with the local organizing committee of the annual EFI meeting, maintain-ing several degrees of freedom to the LOC. For the EFI annual meeting in Toulouse a total of 358 abstracts were submitted and anonymously (no author names or institutions) scored (0-10) by each of the mem-bers of the scientifi c committee independently. Associated mem-bers were not allowed to score. All abstracts were accepted. The Julia Bodmer awardee was selected and Ceppillini lecturer proposed to the board. The close collaboration with the local organizing commit-tee of the EFI meeting organizers in Toulouse resulted in an excellent scientifi c program including basic-, applied and clinical research. Erik Thorsby thanks Mogens Thomsen

and the LOC for their collaboration and excellent program.

An alternative procedure for applica-tion for the Julia Bodmer award has been proposed to the board.

The members appointed in the sci-entifi c committee 2007-2008 are: Antonio Amoroso, Ronald Bontrop, Marco Colonna, Els Goulmy, John Trowsdale and Erik Thorsby (chair) Ex offi cio: EFI President, LOC chair-man of last and next conference; i.e. Dominique Charron, Antonio Nunez-Roldan and Mogens Thomsen. Erik Thorsby will step down from the committee and Ronald Bontrop accepted the chair. Els Goulmy will also step down. Erik expresses many thanks for a very fruitful collabora-tion - a real teamwork. Erik Thorsby on his turn is acknowledged for his important contribution and chairing the scientifi c committee.

7. Report of the EFI President The president commentated on the

growth of the EFI community, with over 800 members and 205 accred-ited laboratories. The Toulouse meeting was a huge success with over 850 attendees of which more were from a younger age group.

The society consists of two inte-grated pillars: professional and scientifi c with education crossing both pillars. The next challenge is to integrate the activities of the accreditation and central offi ce with a more powerful website – this will require increased personnel for its successful management.

In the future there would appear to be a requirement for a single budget, this is an EU legal require-ment that one organisation has a single consolidated budget.

The treasurer was thanked for the improvements to the current accountancy system.

During the presidents term of offi ce, the relationship between ASHI and EFI have improved and the role of EFI has been recognised at the Euro-pean Council by having the award of the POSEIDON grant.

The interactions with the EFI’s named journal, Tissue Antigens was proving to be very positive and Tissue Antigens have agreed to pub-lish a manuscript from the current and future Ceppellini Lecturer.

The board members were thanked for their work during the Presidents term and the new members to the board were welcomed.

Dominique Charron was thanked by the new president, Steven Marsh, and given a gift of English Cham-pagne. Steve Marsh described his aim to build on the achievements made by the previous presidents, to increase the international profi le of EFI, all of which would be depen-dent on the cohesive support of the board and the chairs of the various committees

8. Future EFI conferences – Ulm, Germany 9-12 May 2009 – Firenze, Italy, 15-18 May 2010 – Prague, Czech Republic, 4-7 May 2011 Instructions for the application for

the 2012 meeting will be announced in the EFI newsletter.

9. Other Topics Katharina Fleischhauer led a

moments silence in remembrance of the sad loss of two Italian col-leagues, Professor G.B. Ferrara and Dr. Pier Luigi Mattiuz.

10. Installation of new offi cers and Councillors

Ann-Margaret, Ilias Doxiadis and Katharina Fleischhauer resigned as councillor. All are thanked for their contribution to the executive com-mittee. Ann-Margaret is welcomed as deputy secretary. Ciaran Dunne, Anthonij Slavcev, and Caner Susal. are welcomed to the board. Domi-nique thanks Colette Raffoux and Marcel Tilanus for their support during his presidency. Steve Marsh takes over the presidency of Domi-nique Charron.

11. EFI medal: Prof. Cathy Stavropoulos-Giokas

received the EFI medal for her long-standing major contribution to EFI. Dominique Charron presented a summary of many of her contribu-tions.

Marcel G.J. Tilanus, EFI secretaryAnn-Margaret Little, EFI deputy secretary21-05-2008

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2007 BALANCE GENERAL OFFICE

Provisional Budget Profi ts and loss Income Membership Fees 25.028 22.122Advertisements Newsletter 14.237 15.153Benefi ts Oslo Conference 35.542Benefi ts Barcelona Conference 36.957 34.154JB YS Award Stock and shares 155 238Interests (Saving account) 26 32Plus Value on Stocks 3.107 6.580

Total 79.510 113.821

Expenses Website 763 4.334Auditor fee 2.000 2.000Newsletters 8.193 14.619Education Bursaries 2.550 610Board Meetings 11.015 19.031Committee Meetings 3.094 4.942Bursaries Congress 8.000 5.980Treasurer Offi ce 2.260 1.664EFI Offi ce Leiden 13.800 13.800Bank Charge 1.189 1.264Contribution IMGT/HLA Database 4.600 4.600Summerschool 9.027 4.800JB YS award 1.000 1.000International affairs 1.000 14JB award /Ceppellini Plaques 468 391Banner 3.000 1.215

Total 71.958 80.264 Net Result 7.552 33.557

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2008 BUDGET GENERAL OFFICE

2007 BALANCE 2008 PROVISIONAL BUDGET IncomeMembership Fees 22.122 22.000Advertisements Newsletter 15.153 15.000Conference Benefi ts 35.542 Oslo 60.000 Toulouse 34.154 Barcelona JB YS Award Stock and shares 238 238Interests (Saving account) 32 32Plus Value on Stocks 6.580 6.500

Total 113.821 103.770Expenses Website 4.334 4.300Auditor fee 2.000 1.000Newsletters 14.619 16.260Education Bursaries 610 6.000Board Meetings Organisation 19.031 10.000Board Travel and accomodation 10.000Committee Meetings Organisation 4.942 1.000Committee Travel 4.000Bursaries Congress 5.980 6.000Treasurer Offi ce 1.664 2.000EFI Offi ce Leiden 13.800 13.800Bank Charge 1.264 1.300Contribution IMGT/HLA Database 4.600 4.600Summerschool 9.027 5.000JB YS award 1.000 1.000International affairs 14 500JB award /Ceppellini Plaques 391 400Banner 1.215 0Advance Ulm Conference 10.000

Total 84.491 97.160 Net Result 29.330 6.610

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ACCREDITATION OFFICE:PROFIT AND LOSS ACCOUNT 2007

2007 2006Operating income 130,250 118,799Travel costs received 52,622 63,008Financial income and expense 4,266 3,589

Total income 187,138 185,396

Travel expenses 87,268 101,720Operational fee offi ce 5,445 5,445Secretarial fee 42,457 38,448Bank charges 697 689Offi ce expenses 20,879 25,607Other operational expenses 33,895 18,886

Total expenses 190,641 190,795Result (3,503) (5,399)

ACCREDITATION OFFICE:EXPECTATIONS 2008 2007 2008Operating income 130,250 150,000Travel costs received 52,622 65,000Financial income and expense 4,266 4,000 Total income 187,138 219,000

Travel expenses 87,268 105,000Operational fee offi ce 5,445 5,445Secretarial fee 42,457 45,000Bank charges 697 700Offi ce expenses 20,879 25,000Other operational expenses 33,895 33,000Web 4000

Total expenses 190,641 218,445Result (3,503) 555

The members of the standards and QA Committee met on April 1st 2008. A major topic on the agenda was the revision of version 5.6 (as published in the January 2008 Newsletter), based on the comments/proposals that were received from G. Fischer, D. Hanau, J.-L. Taupin, I. Doxiadis, L. Pecoroni, E. Albert, J. Merenmies (on behalf of Scandiatransplant), and M.-P. Edmonds. Most of proposed standards have been validated and accepted. A few changes have been included, in particular standard C4.150 on internal

EPT, and will be ultimately published in a forthcoming EFI Newsletter. The com-mittee has discussed the framework for new standards on HLA testing for cord blood samples, based on a pre-liminary draft prepared by T. Gervais, focusing on minimal HLA typing require-ments and confi rmatory typing on a second sample. The standards on cord blood will be submitted to Netcord for comments. Also the section on platelet transfusion will be implemented with a new subsection on HPA/HNA testing (A. Harmer, J. Mytilineos, C. Navarrete).

REPORT OF THE STANDARDS AND QUALITY ASSURANCE COMMITTEE

Concerning the rotations in the commit-tee, two members, A. Harmer and J.-M. Tiercy have terminated their mandate. The new chair is K. Poulton who has been appointed by the EFI Executive Board. The committee is welcoming the collaboration of two new members, S. Fuggle and C. Voorter. In 2009 there will be one vacancy, candidates should send their application form to K. Poul-ton.

J.-M. Tiercy, former chair of the committeeK. Poulton, new chair

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TOULOUSE 2ND APRIL 2008

Present : Ilias Doxiadis (ID acting chair-person), Guadelupe Ercilla (GE), John T. Vaage (JTV), Chantal Gautreau (CG), Francesca Quintieri (FQ), Martin Petrek (MP), Susan Corbin (SC), Falko Heine-mann (FH), Guest and observer ASHI: Marilyn Pollack

1. Announcements ID chaired the EPT Committee (EPT-

C) meeting in the absence of CD. ID pointed the need for a co-chairper-son or deputy chairperson.

2. Minutes of 9th November meeting in Maastricht and matters arising

The minutes were accepted. The UCLA schemes on high resolu-

tion DNA typing may be acceptable as it is assessed , provided the organiser seeks and is granted approval by the EPT-C.

3. 75% consensus for serological typing tests

The current rules state that results are assessed using a 75% consen-sus, which is diffi cult to ascertain if the reported results are obtained by serological or DNA-based methods. The EPT-C will have to discuss this point further.

4. Defi ning and evaluating results of HLA specifi cities in antibody screening

The committee then discussed appropriate levels of consensus for assessment of results for both “false negative” and “false posi-tive” reports.

It was agreed that the 75% consen-sus for antibody assessment be changed to:

“The Organiser must establish a system to (score or assess) the results. The Organiser will decide if the results will be analysed by technique.”

5. Trial approval of Eurotransplant EPT schemes

The documentation provided by ID was discussed. ID reported that the amount of work required to produce this application was considerable and much of the information was duplicated if the EPT host labora-tory is EFI accredited (e.g. CVs). ID recommended that all EPT approval submissions be made on CD or other electronic means.

It was decided that version 5 of the EPT Standards will be re-written by ID and SC by the end of June. CG and GE will draw up a ‘checklist’ of required EPT approval documenta-tion by October.

6. Contact with other Committees

7. Future of the EPT Committee The size of the EPT committee and

possible expansion of the commit-tee was discussed. It was felt that a large committee was not desir-able but new members from other EPT schemes should be accepted, as appropriate.

EFI EPT COMMITTEE MEETING –

JTV will be the new Chair by Novem-ber, A co-Chair will be proposed in the November meeting. ID will step down by November from both the interim Chair and as a member of the Committee.

8. Any other business ID reported that a request to inves-

tigate platelet typing EPTs had been received from the Accreditation Committee. SC commented that a platelet typing EPT is already in exis-tence in the UK but is run by another UK NEQA Scheme.

ID requested that information on new EPT schemes is added to the website.

ID reported that B-cell crossmatch-ing will be accredited and consider-ation must be given to expand EPT schemes to encompass this testing.

The Committee agreed that Scheme Organisers are required to produce the performance certifi cates by 6 weeks from the start of the calen-dar year.

9. Date of next meeting: Will be set when the date of the EC and the Accreditation Meeting are known.

LAUNCH OF THE NEW EFI WEBSITE

Dear EFI Members,

The work of the EFI Web Committee has had two major objectives recently:

– Enhanced support for the profes-sional work of the EFI Central offi ce and the EFI committees

– Improved service for the EFI mem-bers and the EFI community.

In order to provide the necessary infra-structure for support and service, an EFI fi le serving system was imple-mented and the EFI website has been technologically updated.

EFI fi le serving system

Similar to your fi le system on your local computer, the EFI fi le serving system allows to deposit electronic docu-ments, however, on a web-based plat-form. It serves the purpose of providing access to documents to be shared among members of committees or spe-cial interest groups in a securely pro-tected environment. This platform has been used since last autumn by the Executive Committee. Beginning this year, the EFI fi le serving system was made available to all EFI committees

and special interest groups, for e.g. EFI conference organisation or working parties such as POSEIDON.

New EFI website

The current EFI website was developed and fostered mainly by Steven Marsh and James Robinson under the aus-pices of the EFI Web Committee. As technology progresses and needs and expectations on the functionality of the website have changed, an overhaul of technology and content of the EFI web-site became necessary. In cooperation with I S D - Internet Systems GmbH

14

Dresden – the EFI website reengineer-ing carried out over the last months and is now ready for takeoff and will become live at the beginning of June 2008. In a fi rst step, the new website has received a revision of its content struc-ture, however, provides the same functionality as before with a general access for members (User: user; Pass-word: efi 2008). The new EFI homepage can be reached under www.efi web.org as previously as well as under www.efi web.eu.

In a second step, new functions, such as a personalised member access including improvements for the profes-sional work of the EFI Central Offi ce and the EFI Treasurer will be implemented until the autumn 2008. Subsequently, the support for the organisation of EFI conferences and electronic commu-nication with the EFI members will be enhanced.Your ideas, comments and wishes on the new website and on what you would like to see and have as an EFI member is very welcome.

At last, I would like to thank James Rob-inson, who has now stepped-down from his role as webmaster, for his many achievements and support of the EFI web activities.

Ralf WassmuthEFI Web CommitteeCorrespondence to: ralf.wassmuth@uniklinikum-dresden.de

FUTURE EFI CONFERENCES

First of all, I would to thank and congrat-ulate Anne Cambon-Thomsen, Mogens Thomsen and their team for organis-ing such a splendid and successful EFI conference in Toulouse. Looking ahead, for the following EFI conferences preparations are well under way:

EFI conference 2009 in Ulm, GermanyIn 2009, the EFI conference will be held in Ulm, Germany, hosted by Joannis Mytilineos, Carlheinz Müller and Rainer Blasczyk from May 9th to 12th, 2009. This 23rd EFI conference will held in conjunction with 17th Annual Meeting of the German Society for Immunogenet-ics (Deutsche Gesellschaft für Immun-genetik). The theme of the conference will be “Genetics of Allorecognition and Immune Tolerance”. Detailed informa-tion will be available on the conference web site www.efi 2009.net.

EFI conference 2010 in Florence,ItalyThe 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 RepublicThereafter, in 2011, we will all convene in Prague, Czech Republic, for the 25th EFI conference. The tentative dates, May 4th to 7th, 2011, have been pro-posed. Antonij Slavcev will be in charge of the organisation of the meeting. It is planned to cooperate in the organisa-tion of the conference with the Czech Transplant Society. More details are to come in the near future.

EFI conference 2012For 2012, the decision will be taken this year by the EFI Executive Committee. To ensure we continue to have excellent annual conferences and to provide more assistance to the candidates for future meetings, the Executive Committee has instituted the following procedure:

In a fi rst step, beginning with this announcement, all parties or individu-als interested in being considered as hosts for the next annual EFI confer-ences are invited to express their inter-est by sending an informal letter of intent by email to the EFI Meeting Liai-son Offi cer. Your letter of intent should be received by the 31st July 2008.

After review by the EFI Executive Com-mittee, candidates will be selected and invited in August 2008 to submit a complete formal application. This appli-cation should be submitted by 31st October 2008. It will be reviewed at the EFI Committee meeting in mid-Novem-ber 2008 and decision taken at this time. This formal application process will be overseen by the EFI Meeting Liaison Offi cer to ensure consistency and standardisation of the applications to a high level.

The selection of the future host will based on the credentials of the appli-cant (relationship to EFI and the fi eld; organisations skills), suitability, attractiveness and accessibility of the proposed venue, together with EFI’s interest to support regional develop-ment and while maintaining balance, as well as economic aspects.

Ralf WassmuthMeeting Liaison Offi cerCorrespondence to: ralf.wassmuth@uniklinikum-dresden.de

15

The annual meeting in Toulouse was also attended by several recipients of an EFI bursary. Receiving a bursary implies writing a report for the EFI Newsletter on one of the scientifi c or teaching sessions. Herewith you fi nd some of these contributions.

Elizabeth Bulpitt, Southampton, UK.

Toulouse or la ville rose as it is known is a charming city with a large student population and a laid back atmosphere. Spring had come early with clear blue skies and temperatures in the twen-ties, which was a welcome break from the grey skies of England at the time!

Eight hundred like-minded delegates converged at the Centre de Congres Pierre Baudis, an imposing glass building set in pretty grounds, for the opening session of the 22nd EFI Con-ference. With over three fl oors of lecture halls and poster stands the sheer size of this meeting place was awe-inspiring.

With 358 submitted abstracts I was pleased to be among the 302 invited poster exhibitors (P-286). I arrived early, taking the opportunity to mount the poster and explore the conference centre before attending the molecular chimerism quantitation satellite sym-posium for which we (Molecular Pathol-ogy, Southampton General Hospital) had submitted data.

The symposium organised by M.Alizadeh was very well attended with standing room only! The aim was to compare different technologies for quantifying known serial dilutions in a simulation of a mixed chimeric recipient. STR anal-ysis has long been considered the gold standard for post-transplant monitoring however the results from the recipient

laboratories revealed that the analysis was very variable and the sensitivity of the assay questionable. It appears from the collated data (results being prepared for publication) and the fi nd-ings of others that quantitative PCR is outperforming STR for chimerism anal-ysis both in sensitivity and its ability to predict relapse earlier (Koldehoff, M et al. Am J Hematol. 2006 Oct; 81(10): 735-46).

In the opening session of the confer-ence the invited Ceppellini lecture was given by Hans Georg Rammensee from Tubingen on his work to sequence, all peptides presented by all HLA mol-ecules from all proteins or the ‘HLA ligandome’ and it’s application to immunotherapy. This captivating talk presented the idea that antigen pre-sentation is the ‘real time analysis of the functional genome’ and that by sequencing peptides presented at the cell surface of normal versus tumour cells you could identify tumour-asso-ciated peptides that may prove to be good targets for immunotherapy. This was being done in renal cell carcinoma where vaccination with a cocktail of these peptides presented by HLA-A*02 had been successful in producing anti-bodies stabilising or decreasing tumour loads in responders.

The Julia Bodmer award winner Roberta Rizzo from Italy presented her work on the role of soluble HLA-G (sHLA-G) mol-ecules in pregnancy. By analysing the supernatants surrounding IVF embryos for the presence of sHLA-G they found that pregnancy only occurred when sHLA-G was detected. sHLA-G is detect-able within 48 hours of fertilisation but the human embryonic genome is not activated until 72 hours post-fertilisa-tion. Could the mother’s genome play a role in sHLA-G activation? Further work is required to assess whether sHLA-G could be used as a marker of oocyte and embryonic competence.

This was followed by an informative talk by Arnd Hoeveler on the health col-laborative research framework 7 and the innovative medicines initiative in Europe, which highlighted the opportu-nity for researchers at all career stages to apply for EU grants.

Following the lectures there was an opportunity to attend an organ recital

REPORTS OF EFI BURSARY RECIPIENTSat the Saint Sernin Basilica, a beauti-ful Romanesque style church dating back in parts to the 11th century. The talented organist Jean-Baptiste Dupont played well-known pieces by Bach and other composers before playing some of his own interesting compositions.

The welcome reception, which followed, was set in the Town Hall ‘Capitole’ in an ornate room known as the ‘Salle des Illustres’ with fl oor to ceiling paint-ings dating from the 19th century. This seat of local government was the set-ting for our welcome by the Mayor of Toulouse and the local organisers.

The quality of lectures and entertain-ment organised over the following three days of the conference did not disap-point after this very promising start. There was something for everyone regardless of scientifi c background. I would like to take this opportunity to thank the local organisers for their hos-pitality, EFI for the bursary, Emma for the photographs and everyone in Mol.Path, SGH for their support and contri-butions to the poster and the chime-rism workshop.

Katharina Stingl, Zagreb, Croatia

For the past several EFI meetings, special sessions, with the topics that cannot be easily integrated into one of the traditional plenary or educational session, but are none the less of great importance to the EFI community, are

Centre de Congres Pierre Baudis. Photograph by E.Lougee, Guy’s Hospital, UK

Salle des Illustres Photograph by Emma Lougee.

16

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organized. This year’s meeting in Tou-louse was not an exception in this regard, and three special sessions were included in the scientifi c program. The aim of the fi rst session was to introduce the Poseidon project to the participants of the EFI conference. This project, as can also be seen from its acronym, is dedicated to Promoting Optimization, Safety, Experience shar-ing and quality Implementation for Donation Organization and Networking in unrelated hematopoietic stem cell transplantation (HSCT) in Europe. The importance of this particular fi eld in the work of every institution involved in the tissue typing and transplantation program strongly justifi es the organiz-ers’ decision to add this presentation to the conference schedule.

The Poseidon is a three year long project funded by the Public Health Programme of the European Commis-sion. Therefore, the fi rst talk of this session was given by a representative of the European Commission, Dr. Arnd Hoeveler who is the Head of Unit Bio-technology for Health. Dr. Hoeveler has highlighted the role of EU in this proj-ect. Following, Dr. Anne Cambon-Thom-sen, the project’s coordinator, gave an overview of the organization, aims, expected results and participating institutions. The project is divided into nine work packages (WP), with each work group dealing with a specifi c fi eld and aiming to achieve clearly defi ned objectives. The fi rst WP, „Coordina-tion“ is led by the Institute National de la Santé et de la Recherche Médicale (INSERM, France), which is also the initiator of the project. The remaining WPs are dealing with the dissemina-tion of the results, evaluation of the

project, legal issues in HSC donation, donation promotion and quality issues, quality and safety aspects of immuno-genetics for HSCT in Europe, economic and statistical tools for optimal imple-mentation of effi cient HSC registries, synthesis and interdisciplinary inte-gration and fi nally education and train-ing. The institutions which are leading these subprojects are: Agence de la biomédecine (France), Universidad del Paìs Vasco/Euskal Herriko Unibertsi-tatea (Spain), Oslo University - Faculty of medicine, section of Medical Ethics (UIO, Norway), Tudástársadalom Alapít-vány (TSA, Hungary), European Federa-tion for Immunogenetics (EFI), Centre National de la Recherche Scientifi que (CNRS, France), and Országos Gyógyin-tézeti Központ (Hungary). The fi nal goal

of this project is to generate two sets of recommendations for recruitment strat-egies and typing. One will be directed to the health professionals and institu-tions which are involved in processes taking place prior to the donation, while the second will be forwarded to the policy makers at national and EU level. As a result of these activities, an improvement in the safety of HSCT as well as the equalization of access to this treatment throughout Europe can be realistically expected. Next talk concentrated on the role of a national public health agency and was given by the representative of the Agence de la biomédecine, the institu-tion in charge of the essential parts of the Poseidon project, namely the distri-bution of the results to the interested parties and public and integration of the results achieved by all work groups. The mathematical and economical approaches in building and evaluating

recruitment strategies were presented by Prof. Jean-Pierre Florens from the Toulouse school of economics. Finally, the role of EFI in the Poseidon project was addressed by Dr. Elissaveta Nau-mova, co-chair of the EFI Poseidon Com-mittee. Dr. Naumova has presented the preliminary results obtained by the analysis of questionnaires sent to various tissue typing centers regarding the accreditation status of laboratories participating in the transplantation pro-gram. The importance of this project was once more proven during the round table, led by Prof. Dominique Charron, which took place at the end of the ses-sion. Namely, most of the questions from the auditory were inquiries about the possibility of registries from coun-tries which are not part of the EU to join or in some other way participate in the project. The fi nal conclusion of the session was that the results of the Poseidon project will be of great ben-efi t to the entire HLA community, espe-cially professionals actively involved in the HSCT program. This was also my personal impression, as well as, I strongly believe, the impression from all the attendants to this session. For that reason, I would like to thank the organizers for the opportunity to learn more about this project and to express my hope to hear about the results in the near future.

Jeroen Blokhuis, Rijswijk, the Netherlands

The EFI congress started with an excel-lent satellite meeting on nonhuman primate immunogenetics organized by Prof. Dr. Antoine Blancher. Topics were diverse and included MHC gene evo-lution, microsatellite typing of DRB in humans and rhesus macaques, high resolution genome analysis of primate MHC regions, innate peptidoglycan recognition proteins, humans with chimpanzee-like MHC-specifi cities who control HIV-1 infection, immunogenet-

18

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ics of the histo-blood group ABO, and concluded with an interesting talk on primate glycomes. This meeting was relevant for a fundamental understand-ing of the evolution of immune related genes and selective pressures on them. I enjoyed this satellite meeting, and the warm welcome of our host. From the main congress I have selected two ple-nary sessions that I found particularly interesting.

Dr. Ashley Moffett discussed interac-tion of killer cell immunoglobulin-like receptors (KIR) expressed by uterine NK cells with trophoblast HLA-C molecules for successful human reproduction. As Sir Peter Medawar already noted in 1953, there is an immunological prob-lem with pregnancy. Because the fetus is part foreign to the mother, one would expect an immune response to start unless their are factors involved that modulate this response. It seems that one cell population, notably the natu-ral killer (NK) cells are very abundant in the female uteris during pregnancy and seem to play important roles in blood vesicle formation and immuno-modulation. A hypothesis was put forth that the activation potential of these NK cells can have an infl uence on the succes of pregnancy. Pre-eclampsia and fetal growth restriction are condi-tions were trophoblast invasion of the myometrium is inadequate, resulting in less transformation of the spiral arteries and ultimately poor blood fl ow to the fetus. A cohort was screened consisting of (i) control women with a normal fi rst pregnancy (ii) women with low birth weight babies (iii) those who

had three or more spontaneous mis-carriages (iv) a new large cohort of over 700 women with pre-eclampsia. Because extravillous trophoblast cells express a combination of HLA-C, -E and -G, and HLA-A and -B are not expressed, it makes sense to look specifi cally at certain combination of HLA-C and their ligands. The role of HLA-G, its different isoforms and affi nity to KIR2DL4 was briefl y mentioned and the observation that HLA-G1 can bind CD160 on endo-

thelial cells was noted, but in this pre-sentation emphasis was placed on the association studies. Indeed a strong association in all women with repro-ductive failure was presented with certain maternal KIR and fetal HLA-C genotypes. It seems that women that are homozygous for the KIR A haplo-type (which lacks activating genes) and homozygous for HLA-C2 (which is the ligand of the activating KIR2DS1) had the highest frequency of reproduc-tive conditions. Being AA for KIR and heterozygous for C1C2 was also a risk factor. But having at least one KIR B haplotype protects agains pre-eclamp-sia. It seems in humans there is a fi ne balance to be kept between extreme invasion of the trophoblast cells which can ultimately lead to the death of the mother, and restriction of fetal growth which may lead to miscarriage.

The presentation I would also like to discuss was Dr. Sumi Rajagopalan who showed data about how KIR2DL4 inter-acts with endocytosed soluble HLA-G molecules. KIR2DL4 is a conserved member of the KIR protein family and found in humans and nonhuman pri-mates. It is unique in that it has a charged amino acid in its transmem-brane domain, and an immunorecep-tor tyrosine-based inhibitory motif (ITIM) located on its cytoplasmatic tail. These characteristics predict the protein to have both inhibitory and acti-vating potential, although the former role was only postulated. HLA-G can stimulate uterine NK cells to prolifer-ate and release cytokines. When look-ing at the cell surface expression of KIR2DL4 it seems low expressed, but Dr. Rajagopalan showed that KIR2DL4 actually localizes to a subset of endo-somes containing Rab5. The role of

HLA-G was further shown by an in-vitro assay where membrane shed HLA-G was tested on T cells and NK cells. It seems there was a differential effect on both cell populations. While in T cells anti-apoptotic pathways were being expressed, NK cells were stimu-lated to proliferate and secrete IFN-gamma. Using an anti-2DL4 antibody at fi rst glance it seemed that this has a pro-infl ammatory and pro-angiogenic effect on NK cells. Expression of TNF-alfa, VEGF, IFN-gamma, IL-1beta, IL-6, IL-8 and IL-23 was presented. In 293 T cells anti-2DL4 induces IL-8 secre-tion. Cells transformed with KIR2DL4 that lacks its charged amino acid in the transmembrane domain could still produce IL-8 upon 2DL4 crosslink-ing. These mutagenesis experiments showed that yet unknown motifs in the cytoplasmic tail were responsible for this production. Selective deletions of the extracellular domains showed that they were essential for transport of the protein to the endocytic vesicles. The presentation ended with a discussion whether or not dimers or monomers of HLA-G had differential effects, and although it seemed that dimers might be functional more important, empha-sis was placed that one possible role of soluble HLA-G is to bypass membrane bound receptor ligand interaction, acti-vating different cells in this manner.

Chrissy Roberts, London, UK

Plenary Session 1 : Immunogenetics of Disease

The fi rst speaker of the day was Profes-sor Rikard Holmdahl, from the section for medical infl ammation research at Lund University, Sweden. The lecture

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concerned the immunogenetics and possible triggering factors of Rheuma-toid Arthritis (RA), a chronic imfl am-matory autoimmune disease that is currently incurable but which is respon-sive to a number of medical interven-tions such as treatment with anti-TNFa. We learned that disease diagnosis tends to follow a number of years in which pre-clinical levels of auto-immu-nity take place and during which time the full-blown disease may be predicted through testing for rheumatoid factors and genetic markers.RA is partly genetically regulated and the predisposing polygenic system apparently requires environmental trig-gers in order to initiate the disease phe-notype. Such triggers may not only be pathological factors such as infectious disease, and physiological factors such as sex hormone levels, but may also include other ‘social’ factors such as alcohol consumption and smoking.Prof. Holmdahl showed how genome-wide association (GWA) studies had identifi ed a role for the Major Histo-compatibility Complex (MHC), along with other more specifi c markers such as PTPN22, PADI4 and the TRAF1-C5 haplotype, in the genetic predisposi-tion towards RA. The MHC association seemed to be focussed upon the Class II region although the specifi c gene involved remained elusive. Similarly hard to identify was the autoantigen, which may have required specifi c modi-fi cation, perhaps in the form of citrul-lination or glycosylation, prior to the initiation of the auto-response through this pathway.Type II collagen (CII) has been identi-fi ed as an autoantigen in RA and mice immunised with CII have been shown to suffer from collagen induced arthri-tis (CIA). The responses to CII appear to be MHC Class II dependent and spe-cifi cally require that the CII be glycosyl-ated rather than hydroxylated in order to allow the manifestation of the CIA phenotype. Transgenic murine lines expressing the human HLA-DRB1*0401 allele were shown to suffer from CIA as a result of the same glycosylation event.In the last part of the lecture, we learned how the severity of responses that are mediated by the autoreactive T-cells might be moderated by polymorphism in the Ncf1 gene, a component of the NADPH oxidation pathway. The oxida-tive or respiratory burst is a component of innate immunity that is delivered by the MHC Class II expressing Antigen Presenting Cells (APC), in which reac-

tive oxygen species are released into the environment of pathogens; causing free-radical damage to the invader and presumably triggering infl ammation. Contrary to this latter presumption, Professor Holmdahl showed how the disease susceptibility allele of Ncf1 was in fact the allele that induced a reduced oxidative burst and he went on to hypothesise that the failure of the reactive oxygen species to oxidise the membranes of and thereby to inhibit the action of the autoreactive T-cells, was in fact the root cause of the differ-ential disease severities in the allelo-morphs.

The second speaker of the session was Dr. Ashley Moffett of the Cambridge University Department of Pathology. Dr. Moffett’s lecture emphasised the central role of the placenta, rather than of the developing foetus as the allo-genic entity during human pregnancy. The focus of Dr. Moffet’s research has been on the immunogenetic factors involved in the interplay between the maternal tissues and the trophoblast, that layer of cells that forms the ulti-mate boundary between mother and her child and which provides nourish-ment to the developing embryo. The immunogenetics of pregnancy is quite unique and cells of the villous tropho-blast are wholly MHC negative, probably in order to prevent recognition of the placental tissue as an allograft, which would ordinarily lead to its rejection as part of a ‘non-self’ targeted immune response. A proportion of trophoblast cells can be seen to migrate into the uterine decidua and these extravillous

trophoblast cells start to express HLA-C, HLA-E and HLA-G. As the extravillous trophoblast cells invade the uterine decidua, they interact with the decidual leucocytes and it appears to be through these interactions that the extent or ‘depth’ of placentation is determined. Increased placentation increases the degree of vascular interaction between the maternal and foetal systems and is linked with both increased birth-weight and reduced incidence of pre-eclamp-sia.Pre-eclampsia is a hypertensive condi-tion that affects around 10% of pregant women and which puts both mother and child at risk in the later stages of preg-nancy. The incidence of pre-eclampsia is partly characterised by a differential risk of the disease manifestation in individual mothers when they change sexual partner. Because foetal geno-types are an indirect representation of the paternal genotype, the disease pattern suggests a role for a mater-nal immune response to ‘dangerous’ foetal genotypes that may in a propor-tion of cases be contributed solely by the father.The population of decidual leucocytes consists of more than sixty percent Natural Killer (NK) cells and we learned that Killer cell Immunoglobulin-like Receptors (KIR) are highly expressed on these cells during early pregnancy. This upregulation refers in particular to the 2 domain receptors, some of which have been shown to interact with HLA-C. A role in pregancy for HLA-G inter-actions with the LILRB1 receptor has been proposed, in that these interac-tions move APC function away from

22

alloreactivity and towards foetal toler-ance as part of some kind of toleris-ing ‘pregnancy signal’. Dr. Moffett focussed the remainder of her talk on the importance of the HLA-C receptor. Arguing that alone amongst the HLA molecules expressed by the extravillous trophoblast, HLA-C is polymorphic, the logic applied was that allelic variation in HLA-C may be suffi cient to explain the various successes or failures of pregnancy in relation to pre-eclampsia. More specifi cally it seemed possible that a combinatorial genotype of mater-nal KIR and foetal HLA-C could be to blame for the differential manifestation of pre-eclampsia and indeed, it became clear that this was exactly the case. Dr. Moffett showed how mothers who were homozygous for the KIR ‘A’ hap-lotype were at risk of pre-eclampsia, but only when the foetal HLA-C geno-type included alleles of the HLA-C2 epi-tope group. This model can explain the observation that certain fathers tend to produce pre-eclamptic pregnancies with certain mothers. The data goes further to show that the maternal ‘AA’ KIR genotype and paternal HLA-C2 are both present at increased levels in cou-ples who have suffered from recurrent miscarriages.Collectively this data implies that a component of the KIR ‘B’ haplotype is protective and Dr. Moffett has inferred that the activating KIR2DS1 gene, which interacts with HLA-C2, is this factor. In summary, some consideration was given to the need for balance in the con-trolling factors of placentation. Whilst we generally consider pre-eclampsia to be a poor outcome of pregnancy, we must also consider that extremely high birthweight and massive intrusion of the trophoblast into the decidua may be similarly damaging to the mother and her child.

In the fi nal lecture of the session, Pro-fessor Federico Garrido, of the Hospital Virgen de las Nieves, Granada, spoke on the topic of HLA class I expression and tumour immune escape. In brief

summary, Professor Garrido showed how tumour cells may be detected by T-cell recognition that is based upon the identifi cation of specifi c tumour anti-gens, or through the downregulation of all HLA class I molecules, which is detected by NK cells according to the ‘missing self’ hypothesis. Further to this, the downregulation or loss of indi-vidual Class I antigens is a means of T-cell escape, but that downregulation of all Class I antigens enables the NK cell responses. The take-home message in this is that tumour cell immune-escape is most successful when antigenic Class I molecules are downregulated to a degree that is suffi cient to escape both the T-cell and NK cell mediated responses.HLA downregulation in this manner is associated with a failure of metastatic tumourous tissue to respond to cancer immunotherapy based upon the use of peptides delivered as adjuvants to T-cell responses. Professor Garrido explained how additional mutational events in different populations of metastatic lesion could make them either susceptible (restored MHC class I expression) or resistant (continued downregulation) to such immunothera-pies and suggested that by screening for such abberant regulation one could pre-emptively seek alternative thera-pies, such as direct gene-therapy, for those ‘hard’ lesions that were resistant to the immunotherapeutic autologous vaccination approach.

Alina Lemnrau, London, UK

I am studying for a PhD in Immunoge-netics and my interest is in MHC hap-lotypes and tagSNPs. For that reason, I will focus this report on the very inter-esting talk of Dr Effi Petersdorf on MHC haplotype matching for unrelated hae-matopoietic cell transplantation.

Haematopoietic stem cell transplan-tation (HCT) represents the standard method of care for various types of haematological cancers. However, post-transplant complications including acute and chronic graft-versus-host dis-ease (GVHD), are still the main barriers to successful HCT. GVHD can affect all parts of the body and in severe forms (grade III to IV) can lead to the death of the patients. GVHD occurs when there are differences in the HLA anti-gens involved in the immune response between the donor and the patient who receive the transplant. Most often, GVHD occurs in patients who receive

transplants from unrelated donors, even though the donor-recipient pairs are matched for the most HLA anti-gens known to be involved in immune rejection. A current criterion for selec-tion prior transplantation for unrelated donors involves matching of classical HLA-A, -B, -C, -DR and DQ alleles and antigens. However, the human genome is organized into segments or blocks of closely linked genetic variants that are inherited as haplotypes on the same DNA strand of a chromosome. The HLA genes are highly polymorphic and tightly linked on the chromosome, with low recombination frequencies. The genes are inherited as genetic units known as haplotypes, one from each parent. The hypothesis is that haplotypes that share the same HLA alleles may also share discrete segments or blocks of highly conserved sequences in strong positive linkage disequilibrium (LD) with these HLA alleles. In addition, major efforts have been directed at catalogu-ing the genes and variation content of the entire MHC region. Recently, much interest has been directed at document-ing the extent of genomic variations, predominantly in the form of single nucleotide polymorphism (SNPs).

The degree of HLA matching between donor and patient is a critical param-eter for the successful of haematopoi-etic stem cell transplantation (HSCT). Although current PCR-based and sequencing methods provide high reso-lution typing for alleles at individual HLA loci, it is increasingly evident that the haplotype in which they reside is also important. Recent fi ne mapping stud-ies of the MHC demonstrated that hap-lotypes could be defi ned by a limited number of tagSNPs. The major histo-compatibility complex (MHC) is recog-nised as one of the most important regions in relation to human common diseases. The MHC containing the human leukocyte antigen (HLA) loci on the short arm of human chromosome 6p21.31 is a gene dense region span-ning nearly 4 Mb. The complete map of the MHC resulted from the HapMap project gave a better insight into the fi eld of human leukocyte antigen. The proteins encoded by the classical HLA genes in the MHC are highly polymor-phic and play a vital role in self versus non-self immune recognition.

During the congress, one of the teach-ing lectures drew my attention. Dr Effi Petersdorf from Seattle, USA, who is one of the key researchers in haema-

23

topoietic stem cell transplantation, had presented us with the results of a study of MHC haplotype matching for unrelated haematopoietic stem cell transplantation. In her presentation, Dr Petersdorf talked about the current criteria for the selection of unrelated donors for haematopoietic stem cell transplantation (HCT), complications regarding the development of GVHD and also possible solutions to identify the genes involved in the transplanta-tion outcome.

The aim of their study was to defi ne extended MHC haplotyes in unrelated donor-recipients pairs to see if using this technique might provide a way to better assess the outcome of GVHD than matching for alleles individualy. As part of their study they developed a method of defi ning HLA-A, -B and -DR haplotypes in recipients an their HLA-matched unrelated haematopoietic donors (URD) using DNA spanning 2 mil-lion base pairs across the MHC region. The method used for haplotype sepa-ration is described in Guo et al paper published in 2006. Family studies are not available for unrelated transplant donors, so the group hypothesized that some donor-recipient pairs have identi-cal MHC haplotypes of physically linked HLA alleles, while others have different haplotypes. Dr Petersdorf and her team studied 248 haematopoietic stem cell transplantation recipients and their cor-responding donors, previously matched for HLA-A, -B, -C, -DRB1, -DQB1 by cur-rent techniques. Using their new tech-nique, they found out that more than 20% of the donor-patients pairs were haplotype mismatched. After transplan-tation, they assessed the outcome of GVHD and they observed that indeed haplotype mismatching was associated with an approximately four time greater risk severe acute GVHD and transplant-related mortality. Since the patients and donors were identical regarding HLA-A, -B, -C, -DRB1 and –DQB1 alleles, GVHD could not have been caused by disparity of these HLA alleles. These results suggest that the MHC region contains additional genes that encode

transplantation antigens. Therefore, the severity of GVHD after HCT in unre-lated individuals could be lowered not only by matching HLA alleles individu-ally but also matching their extended haplotypes. Dr Petersdorf also talked about using a panel of 1228 exon-cen-tric tagSNPs to defi ne functional genes within the MHC. Their hypothesis was that MHC haplotypes share blocks of conserved sequences depending on their HLA alleles which can be tagged with a panel of informative tagSNPs. Dr Petersdorf concluded that, in the future, SNPs haplotypes could be used in association studies to defi ne genes involved in transplantation outcome.

This study appears really important in the fi eld of haematopoietic stem cell transplantation because it could provide a way to reduce the risk of GVHD from unrelated donors, and in the future could be considered as a technique to match donors and recipi-ents. Moreover, this method could be explored further to assess the clinical importance of haplotypes in solid organ transplantation and to discover genetic determinants associated with autoim-munity, infection and cancer. To summarize, these studies have a great signifi cance for the fi eld of transplantation and immunogenetics because it could provide with new alter-native ways to match donor and recipi-ents.

Anna Gieryng, Wroclaw, Poland

The session on population genetics and evolution was dedicated to the relation-ship between the major histocompati-bilty complex (MHC) and the evolution, detection of the source population, and the convergence between the species. The HLA typing of the recipients and donors is the fi rst step in the hema-topoietic stem cell transplantation (HSCT). Hirv presented data about the relationship between the incorrect typing of HLA null alleles and higher risk for graft-versus-host disease (GvHD) or graft failure. The HLA null alleles can be caused by one of the underlining mechanisms: SNP related stop codon generation, insertion or deletion muta-tions associated with the change of a frame shift, mutations within the pro-moter regions, and the mutations in the splice sites. The HLA null alleles appear with the frequency of 0.002-0.5%. Hirv studied 10 690 donors from the BMDR Ulm using HLA-A and -B typing by serology followed by the

PCR-SSO for homozygous (potential null) individuals detected in serological typing. The samples with different sero-logical and DNA typing results were analyzed one more time by the serology and confi rmatory typing of null alleles was performed by SBT. The serologi-cal typing data showed that: 1560 samples were homozygous for HLA-A (14.6%) and 784 samples were HLA-B (7.3%) in donors cohort. The discrep-ancies between serology and molecu-lar typing PCR-SSO were associated with: HLA-A 69/1554 (4.4%) and HLA-B 46/780 (5.5%) (molecular typing of 8 samples was not possible). The forty HLA-A typing results (2.6% of all 1560 homozygous samples) were discrep-ant as a consequence of the serologic technical restriction. The differences between data of HLA-B typing in donors were found in 43 samples (5.5% of 784 homozygous samples). The PCR-SSO typing showed the following frequencies of the HLA-A null alleles 4-7/10 690 (0.04 - 0.07%), the HLA–B null alleles 2/10 690 (0.02%) donors, giving 6-9/10 690 (0.06 - 0.08%) HLA-A, -B null alleles. However, some differences appeared also between the results of PCR-SSO and SBT. This fi rst method could not detect the low expression alleles/null alleles the HLA-A*6818N, HLA-A*74 variant (stop codon in 2 exon), and A*2439. The SBT confi rma-tory typing also identifi ed 10 HLA low expression alleles (0.09% of all sam-ples), including: HLA-A*24020102L. In addition, HLA-A*6818N, HLA-A*74var, HLA-A*2439, and HLA-B*140N and B*5111N null alleles were found. As the HLA-A*2439 and one of the HLA-A*03010101’s were not detected by serology, Hirv concluded that the further analysis (mRNA analysis, fl ow cytometry detection) is needed to assess the characteristics (N or L) of those alleles. The knowledge about the HLA can be used in clinical immunology and trans-plantation. The analysis of the HLA within bone marrow donor registries show that the population background determines HLA of the individual and screening of the registries can be used for founding the donor for recipient – both belonging to the some parental population. Klitz presented data about the identifi cation of source popula-tions using HLA and the impact of the migration and ancestry on the compo-sition of the registry. The difference between populations is extinction, and admixture among populations is nearly popular. For identifi cation of the paren-tal population and the source analysis

24

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25

of: SNPs, tandem repeats, indels, Y chromosome haplotypes, and mito-chondrial haplotypes can be used. As 80-95% of genetic variations in humans are shared, the remaining (5-20%) of DNA sequence differences are used to assess ancestry informative markers (AIMs). The HLA belong to this group, because: a lot of variations are pres-ent within several linked loci, there are millions of individual types, and very old allelic lineages. Klitz used the HLA variations of the A, B and DRB1 loci as the AIMs to fi nd the differences / simi-larities between the USA donors from the National Marrow Donor Program. The model of the derivation of founder population showed that the European Americans contributed to both the Afri-can Americans and Hispanics. HLA hap-lotypes demonstrated nearly competes separation according to continental source (African, American and Europe). Klitz analyzed the samples sizes of two digit HLA – A, B and DR haplotypes from NMDP Registry, and assessed the group European Americans includ-ing Ashkenazi Jews and Native Europe-ans. The next speaker Gragert presented data about the using of HLA to detect the way of separation and migration from the single ancestral Jewish popu-lation. The samples from 32 countries (n= 23 856 individuals) were analyzed at 2 digit HLA-A and HLA-B and 4 digit DRB1 with some high-resolution data also available to identify haplotypes at the 4 digit level. The Nei’s genetic dis-tance on the HLA-A, -B and -DR in the population dendrogram and correlation of haplotypes frequency between coun-tries showed that the Ashkenazi popula-tions appears relatively homogeneous in opposition to the non-Ashkenazi pop-ulations. Pairwise test for homogene-ity showed the statistically signifi cant difference between most populations, ever between Poland and Russia. The data presented that the Jewish people who emigrated from Europe and the Americas are draw from the same Ashkenazi source population. Only one haplotype: HLA-A*2402-B*3502-DRB1*1104 named as the ‘Abraham Haplotype’ was found in each Jewish regional population, and they have one origin from the single paternal popula-tion. The most common Jewish haplo-type designated as ‘Moses Haplotype’ (HLA-A*2061-B*3801-DRB1*0402) is either not found or present at a very low level in Asia, India, Middle East, and Yemen, indicating an early diver-gence of these Jewish populations with those west of the Levatan.

The HLA typing in human and analy-sis of the highly conserved and place accumulated mutations in MHC encod-ing genes, can be useful for typing the evolution and homologues between the species. That is why the part of the ses-sion was dedicated to the knowledge about the polymorphisms in various species - in the MHC-DR region in the primate species. These included: Yama-moto who described the immunogenet-ics of the histo-blood ABO system, van der Wiel who concentred on the func-tion of classical and non-classical MHC class I, Shiina, who compared the MHC regions of shark, rainbow trout, quail, chicken, rat, whale, pig, marmoset, rhesus macaque, chimpanzee etc. The primate species constitute an impor-tant model of preclinical transplanta-tion and for disease association study, when comparison of gene sequences could give us the knowledge about his-tory of the evolution of the MHC encod-ing genes. One of the most interesting presentations in this part of the session was given by de Groot, who compared the sequences of DRA and DRB genes of rhesus macaques, chimpanzees and human to show the evolutionary history of the various HLA-DR regions. The data showed that the sequence of the exon-intron of the DRA gene, accumulates a low level of the gene polymorphism and has been highly conserved during the evolution. The second gene - DRB is not so consecrated in the evolution and accumulated new polymorphisms.

Yarúa Jaimes, Hannover. Germany.

In the session on Immunogenetics and transplantation tolerance, professor Lechler started his presentation with a Cosmos picture showing a leg trans-plantation. He pointed out the optimism in the transplantation fi eld illustrated on the picture and compared it with the actual expectations regarding the achievement of tolerance. This lecture gave us an overview about the recognition pathways in allogeneic transplantation and their clinical impli-cations.There are three major problems in allo-geneic transplantation: the shortage of organs, the morbidity and/or mor-tality that arises as side effect of long term immunosuppression and chronic rejection. Therefore, a major goal in transplantation is the induction of a drug-free permanent state of immuno-logical tolerance to donor alloantigens. Allorecognition is mediated by the Major Histocompatibility Complex (MHC). In

the fi rst part of this presentation the direct and the indirect pathway of rec-ognition were described. In the direct pathway T cells recognize determinants on the intact donor MHC molecules dis-played on the surface of transplanted cells. In the indirect pathway donor derived proteins are processed and pre-sented as peptides on recipient MHC class II molecules to CD4+ T cells. Transplantation studies in rodents have shown that the tolerance is more easily achieved when MHC incompatibility is absent or limited. In 1999, Turka and coworkers showed that alloreactive T cell death might be required to achieve tolerance. Furthermore, in 2006 Strom and colleagues demonstrated that tol-erance is favored by T cell deliberate deletion. Thus, in the context of MHC incompatibility the deletion of T cells may be required.In mice models it was found that periph-eral T cell tolerance can be achieved by adoptive transfer of CD4+ T cells into a naive host. This suggests several inter-actions between different cell types, which diffi cult the draw of a model for T cell mediated regulation in tolerance. A high frequency of Treg cells with direct allospecifi city for a particular allopeptide was detected in long-term transplanted patients. However, in mice models transferable tolerance appeared to be controlled by CD4+ T cells with indirect anti donor allospecifi city. These cells were induced by stimulation with autologous bone marrow derived dendritic cells (DCs) pulsed with an immunodominant pep-tide of the MHC class I. Also, several studies proposed that the direct and the indirect pathway of allorecognition are connected through a cross- talk. A four- cell, unlinked, model for inter-actions between direct and indirect pathway T cells in alloimmunity was

26

purposed. In this model, helper or suppressor CD4+ T cells with indirect specifi city are activated by recipient DCs that reside in secondary lymphoid organs, whereas in the direct pathway, effectors CD8+ T cells must recognize determinants expressed on the grafted cells. However, this hypothesis does not explain how CD4+ T cells regulate CD8+ T cell activity. This effect may be mediated by soluble factors (IL-10, TGF-�) acting on APC or the neighboring cells, or if it is a consequence of cell to cell contact, involving other molecules. It was demonstrated that proliferation of CD25- line was suppressed in vitro only when co-cultured with CD25+ line, but not when both lines were separated by a semi-permeable membrane. This indicates that the suppression effect was due to cell contact and not to dif-fusible products. In the co-cultures, the presence of regulatory cytokines (IL- 10 and TGF- �) was not detected by ELISA. Several results suggested the exis-tence of a new allorecognition pathway, the semi-direct pathway. This was fi rst revealed by MHC class I and class II molecules exchange during co-culture of mismatched DCs. The transferred MHC complexes could be recognized

by T cells. Also, DCs can acquire MHC class I molecules derived from epithelial cells upon treatment with IFN-�. In vivo murine models showed that peptide- MHC class I complex could be trans-ferred to recipient APCs and effi ciently presented peptides to recipient CD8+ T cells. After those fi ndings, the aim was the integration of new knowledge in ther-apeutic strategies that could promote tolerance in allogeneic transplantation. One approach could be the adoptive therapy with “customised” regulatory cells, selected and expanded ex vivo. The two main questions concerning this therapy were: if the cells with allospeci-fi city for donor antigens were superior to polyclonal “auto-reactive” cells and if the cells with direct or indirect allospeci-fi city were more effective. In a mouse system, Treg cells with indirect allospecifi city for a MHC class I alloantigen can be selected from the pool of naturally occurring Treg cells and can be expanded in vitro in pres-ence of IL-2, without affecting the initial phenotype and the suppressive proper-ties. In addition, those donor- specifi c Treg cells are able of regulating the alloresponse of the effectors CD4+ T cells in vivo and this was revealed by adoptive transfer of Treg cells to skin transplanted animals after thymec-tomy. Furthermore, this CD4+ CD25+ cell line showed more potent suppres-sive capacities than the freshly isolated CD4+ CD25+ T cells. It was also shown that adoptive transferred Treg cells are able to proliferate, migrate and accu-mulate in vivo, preferentially in the allograft. This was demonstrated by injecting intravenously mice with CFSE- labeled CD4+ CD25+ T cell line. In T cell depleted recipients, CD4+ CD25+

T cell line effi ciently improved skin graft survival, but could not prevent 3rd party skin graft rejection. To evaluate whether indirect allospeci-fi city can be conferred on Treg cells by gene transfer, a CD4+ CD25+ T cell population was transduced with spe-cifi c TCR- gene delivering constructs. This TCR- transduced Treg cells were selectively expanded by repeated stim-ulation in vitro, and were able to prolif-erate in response to allopeptide pulsed DCs. In addition, it was demonstrated the homing properties of the trans-duced cells to the site of the antigenic challenge, which lead to an increase in the survival rate of heart transplanted mice. It was also documented the fea-sibility to isolate antigen specifi c CD4+ CD25+ cells following tetramer sorting. These T cell regulators can suppress the indirect alloresponse by controlling the IL-2 secretion of CD4+ CD25- T cells in a dose dependent manner. In conclusion, this interesting lecture provided us an insight in the different presentation pathways after allogeneic transplantation and an overview of T cell regulation mechanisms contribut-ing to the achievement of tolerance.

Unless stated otherwise, pictures included are made by studio Jean Cousin, Toulouse.

FROM DECIMALS TO DYNAMICS: MAKING CHIMERISM TESTING MORE INFORMATIVE AND RELIABLE

Chimerism testing has proven its util-ity for managing patients following allogeneic hematopoietic stem cell transplantation (HSCT) (1). The dynamic “chimera“, created by the HSCT, is defi ned by the ratio of donor (D) cells to recipient (R) cells, in the recipient. Since this D-R relationship is intrinsi-cally quantitative, chimeric status also should be evaluated quantitatively. That goal can now be implemented using an STR-PCR/DNA-based assay. This is an important advance because it enables us to compare sequential values lon-gitudinally in a single patient (2). That

capability is important because it allows us to detect, and report, the dynamic aspects of the D-R relationship in the patient, which changes over time. For this reason, a longitudinal approach uniquely reveals the progressive kinet-ics of each patient’s mixed chimeric state. It, thereby, allows us to provide individualized monitoring that facilitates anticipatory therapeutic responses, fi ne-tuning of a patient’s chimeric state, and consequently, disease status. In contrast, isolated, STR-based assess-ments of % chimerism (%Chm) offer little value over qualitative assays. Addi-

tionally, the biological implications of individual, absolute values of mixed chi-merism are unknown; the signifi cance of 40% vs. 50% Chm remains obscure. In short, the novel dimension of modern chimerism testing is the assessment of changes in chimeric status, i.e. focus-ing on the patient’s engraftment dynam-ics, not on static percentages or mere decimals. Practically, longitudinal testing need not be labor intensive or expensive. One feasible, basic schedule could be 3, 6, 12 weeks, 6 mos, 1 year and then yearly. This framework can be easily

27

adjusted to the actual events in the patient’s course. There are times when closer monitoring may be critical, such as following a therapeutic maneuver, or a change in chimeric level greater than 5%. The purpose of this short note is to review several recent enhancements in the processes of chimerism monitor-ing that have relevance to laboratories providing this critical service. Although our experience is primarily based on the commonly used STR/DNA-PCR assay (2,3), the following comments are meant to have more general application, since they address fundamental aspects of monitoring.

What’s New?Two recent developments in the area of chimerism testing bear on the useful-ness and reliability of this test for clini-cal decision making. These advances address the problem of recognizing the occurrence of both technical and biological sources of error. Such errors can lead to a misinterpretation of the results, providing a basis for an inap-propriate therapeutic response or a critical delay in initiating therapy. Two areas particularly deserve our atten-tion. First, the assessment of technical reliability of our estimates of chimeric level. Second, the detection of biologi-cal sources of error using multi-lineage chimerism analysis (MLCA) integrated with longitudinal testing.

Are your results reliable?STR is a complex technology platform for indirect DNA measurement (3). It is associated with inherent variability origi-nating during preparation, amplifi cation, and uncalibrated product detection. Nonetheless, these semi-quantitative measurements of DNA quantity are used to estimate percent chimerism (%Chm). Multiplex PCR partially overcomes this limitation by using a set of simul-taneously amplifi ed STR markers that enables statistical assessment of the variance among loci of a sample. One can then compare the variance with allowable levels published previously (2). In this regard, when three informa-tive loci are evaluated, we fi nd that the locus error (difference to the sample mean) and coeffi cient of variation (cv) to be most useful (2) in providing a general assessment of platform performance on a individual sample. But this statistical evaluation is still indi-rect, and limited to multiplex assays. A more direct, and universal, basis to assess reliability requires determining whether one can depend on the mea-

sured values for allelic DNA for each locus in a sample. For this, we utilize operational parameters. Although the detailed formulation for each of these parameters is available from previous work (2,3), two examples are worth dis-cussing. The DNA Measurement Error, ME, represents the most direct indi-cation of platform performance. This parameter is based on determining whether the STR platform is perform-ing optimally at a locus in regards to the measurements for all of its alleles. For instance, the allelic measurements should be equal in a locus with two normal heterozygous alleles. In that instance, ME = 0. But if the measure-ments are not equal, then the differ-ence in measurements between the two alleles indicates the measurement error at the locus, and ME > 0. Obvi-ously, an error in measurements will affect the computation of %Chm: 10% ME is associated with a 6-7% error in chimerism estimation and a 20% ME, with a 12% error. Another critical aspect of reliability assessment is the sensitivity of the system to detect a minor component in a chimeric locus, which may either rep-resent DNA from D or R. Although in principle, this is 2-5%, the performance of the platform for a specifi c locus in a particular sample can be highly variable (3). Reporting results based on mea-surements from a locus with sub-stan-dard detection sensitivity for the STR markers can be misleading to the clini-cians. For instance, 100% Chm could be erroneously reported, if the minor R component was not detected because of poor performance. This can result in missing an early relapse or graft failure. Sensitivity can be gauged by examining the measurement of the major peak(s) in a locus, and determining whether their measurements are suffi ciently large to ensure that a 2-5% minor peak would have been detected if present. A sum of the major peak heights of approximately 2000 RFU indicates suffi cient sensitiv-ity at a locus for a reliable diagnosis of 100% or 0%. However, lower values may be adequate if precise estimates of sen-sitivity, on a specifi c locus, were made by computing the sensitivity parameter based on the platform’s actual settings for detection threshold, as described previously (2). Incorporating assessment of these two parameters into a testing program can help insure that the study was reliable and that a report can be issued. On the other hand, this assessment may indicate that the study was sub-optimal and needs to be repeated, either by re-

running the sample, or performing the analysis on a new sample.

Importance of Multi-lineage Chimerism AnalysisThe foregoing has focused on technical sources of error in chimerism monitor-ing. However, purely biological sources also exist that may likewise confound the interpretation of the results. In the latter case, the cause is usually com-plete reliance on non-fractionated periph-eral blood or bone marrow samples. An important approach in overcoming this particular limitation to chimerism test-ing is to assess %Chm for a number of key subpopulations in the sample. Multi-lineage (lineage specifi c) Chm anal-ysis, naturally, provides more informa-tion for assessing a patient’s prognosis, and characterizing the events of his clini-cal course (1, 2). But more importantly, examining sub-fractions is often critical for the correct interpretation of the labo-ratory results on chimeric level. Omit-ting MLCA – and relying exclusively on non-fractionated samples -- may result in being misled by biological factors occur-ring in the patient in response to the graft. For instance, myeloid chimerism is often dominant and stable prompt-ing us to view the patient’s engraftment status as stable, when actually incipi-ent failure of the T cell population was being masked. With MLCA data you can avoid this pitfall. On the other hand, patients with a hematopoietic malig-nancy, who show a progressive drop in the level of donor chimerism, would raise the concern of relapse or incipient graft failure. Yet, some of these cases are actually undergoing reactive recipi-ent hematopoiesis involving a non-T cell subpopulation. MLCA can clarify this biological conundrum by demonstrating the cell group responsible for the drop in donor chimerism. The latter conclusion, of course, should be confi rmed by an examination of specifi c tumor markers. In short, multi-lineage chimerism analy-sis is important for correctly interpreting the biology of the graft-host relationship that may be obscured by exclusive use of non-fractionated samples. Since highly enriched fractions are studied, the sen-sitivity of the assay also is improved at least 10-fold. MLCA can be performed cost-effectively if an immuno-magnetic bead approach is used, which is also much less labor intensive than FACS (4). Also, it is not necessary to examine every sample for MLCA. We recommend establishing a baseline early in most cases, but par-ticularly mixed chimerism, and then fol-lowing-up with MLCA periodically during

28

A COLLECTION OF EBV-TRANSFORMED B-CELL LINES

J. Street, H. Bass and C. Darke

Welsh Transplantation and Immunoge-netics Laboratory, Welsh Blood Ser-vice, Wales, United Kingdom

IntroductionWe have established a collection of locally identifi ed and transformed B-cell lines (BCLs) at the Welsh Transplanta-tion and Immunogenetics Laboratory (WTAIL). These are used as reference material for DNA-based typing, cells for antibody screening by fl ow cytometry and archival material for our growing collection of new and uncommon HLA alleles. Details of the initiation, meth-odology and application of this collec-tion have been previously reported (1). Here we give a brief account of the collection’s current status after some 10 years of development.

Donor consentInformed written consent is obtained before HBsAg, HIV, HCV screening and subsequent B-lymphocyte transforma-tion.

BCL collectionThe collection is currently divided into two groups. Both sets are typed for the ABO blood group alleles A1, A2, O1, O2 and B by PCR-SSP (2) and ABO grouped by conventional serology.

1. Selected CellsPotential novel, rare and ‘unusual’ HLA types are identifi ed during routine PCR-SSP (3) /serology-based (4) typing of patients and Welsh Bone Marrow Donor Registry (5) donors. These are comprehensively investigated and selected subjects form the basis of the Collection’s ‘Selected Cells’.

12

34

56

78

%DL

%LE

%ME

0%

10%

20%

30%

Percent

Informative

Loci

Performance

Parameters

Platform Performance for Sample

Graph 1

Graphic Displays for

Tracking Chimerism and

its Reliability

D

2

D

1

R

1 R

2

T-Cell

Gran

NK-Cell

B-Cell

PBC0%

20%

40%

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%

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CellSubsets

Multi-Lineage

0%

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513724232151TX2434025221282

Weeks Post SCT

TC PBLongitudinal Course

There are currently (March 2008) 306 BCLs in this group. These include 28 that provide the fi rst reported examples of 24 new alleles (8 HLA-A, 11 -B, 1 -C, 3 -DRB1 and 1 -DRB3), including, e.g., the ‘null’ alleles A*0116N, A*03010102N, A*0311N, A*2448N, B*0819N and B*4423N; 40 BCLs that afford 37 dif-ferent confi rmatory sequences (8 HLA-A, 17 -B, 3 -C, and 9-DRB1). For two alleles (B*2723, B*040302) sequenced mate-rial is not catalogued but relatives’ cells with these alleles (PCR-SSP defi ned) are available. In addition, examples of cells with uncommon associations including: B*14 Cw*02; B*27 Cw*03; B*40(B60) Cw*01; B*07 Cw*12; B*07 Cw*04; DRB1*11 DQB1*06; DRB1*1303 DQB1*02. A total of 33.5% (95) of the ‘selected cells’ are DPB1 typed, most of these (97.9%) have a DPA1 type.

the initial post-transplantation period (ca. 6 mos). Obviously, we would do MLCA more frequently in the face of a discrepancy between our impressions based on the longitudinal PBC analy-sis and a patient’s clinical status. Our standard panel includes: PBC, T cells, B-cells, NK cells and granulocytes. The T cell fraction should be considered the most critical (1,2). Although the information from a single sample that is fractionated is greater than if it were not fractionated, this should not preclude serial sampling; it is still fundamentally critical to integrate MLCA into a framework for longitudinal tracking. Indeed, the rich interplay of dynamics between the different subpop-ulations strongly reinforces the impor-tance of doing MLCA longitudinally. Figure 1 illustrates the feasibility of inte-grating MLCA and longitudinal monitor-ing into a single Excel-type spreadsheet,

Figure 1. Graphic displays from ChimerTrack©, a software application for tracking multi-lineage chimerism analysis and its reliability. This Excel-based tool uses the same data to simultaneously perform the computation for chimerism and for fi ve reliability parameters. Graphic displays for the longitudinal results are automati-cally generated on the report page for all the fractions. The reliability assessments appear simultaneously on the laboratory section of the spreadsheet. The bottom panels show two different graphic formats for reporting MLCA, which are comple-mentary since each emphasizes a different aspect of the chimerism dynamics. Top left – Electropherogram (from Genescan®) showing a chimeric locus; Top right – Graph showing results of reliability assessment for four loci in a sample. DL = sen-sitivity; LE = locus error; ME = measurement error. Bottom left – Three dimensional bar graph of longitudinal MLCA showing the course for four sub-populations com-pared to PBC; Bottom right – Line graph comparing the longitudinal behavior of the T-cell fraction with parallel non-fractionated PBC samples from the same patient.

along with a parametric assessment of reliability for each locus in a sample.

Concluding Comment:Inherent in modern STR-based chime-rism testing are both technical and biological sources of error. Recent developments have shown that these limitations can be identifi ed and over-come in a cost-effective manner. This is accomplished by performing longitu-dinal MLCA integrated with concomitant reliability assessment. With this focus on a reliable characterization of engraft-ment dynamics – not decimals – chime-rism monitoring can be optimized and individualized.

Don Kristt and Tirza KleinLaboratory of Histocompatibility and ImmunogeneticsRabin Medical CenterPetach Tikvah, Israel

References1. Baron F, Sandmaier BM: Chimerism

and outcomes after allogeneic hema-topoietic cell transplantation follow-ing nonmyeloablative conditioning. Leukemia 2006; 20: 1690-1700.

2. Kristt D, Stein J, Yaniv I, and Klein T. Assessing quantitative chimerism lon-gitudinally: technical considerations, clinical applications and routine fea-sibility. Bone Marrow Transplantation 2007; 39: 255-268.

3. Kristt D, Klein T: Reliability of quan-titative chimerism results: assess-ment of sample performance using novel parameters. Leukemia 2006; 20, 1169-1172.

4. J. Stein J, Kristt D, Klein T, Kodman Y, Yaniv I. Lineage specifi c chime-rism analysis using immunomagnetic cell separation in children following haematopoietic stem cell transplan-tation. EBMT Congress, Florence, 2008, Abstract 1140.

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WELCOME TO THE 5TH INTERNATIONAL SUMMER SCHOOL ON IMMUNOGENETICS Dear Colleagues, Dear Friends, The American Society for Histocom-patibility and Immunogenetics (ASHI) together with the other international societies for histocompatibility and immunogenetics, The European Fed-eration for Immunogenetics (EFI) and The Australasian and South East Asian Tissue Typing Association (ASEATTA), would like to cordially invite you to participate in the 5th International Summer School on Immunogenetics. It will be held from September 9-13, 2008 in the beautiful seaside city of Buzios, Brazil immediately preceed-ing the 15th International workshop in Buzios from September 13 and the International Conference from Septem-ber 17-20, in Rio de Janeiro. The aim of the summer school is to promote the fi eld of immunogenetics through an intensive 4-day course cov-ering a broad range of related topics in immunology, genetics, histocompat-ibility and transplantation. The program will be in English and will emphasize interaction between participants and faculty. Introductory lectures will be fol-lowed by discussions of recent devel-opments and student presentations of their own research. The faculty includes: Jim McCluskey (ASEATTA), Campbell Witt (ASEATTA),

Deborah Crowe (ASHI), Carol Pancoska (ASHI), Andrea Zachary (ASHI), Sophie Caillat-Zucman (EFI) and Frans Claas (EFI). Topics to be covered include immuno-genetics of HLA and non-HLA genes, molecular typing strategies, structural and functional aspects of the MHC, population genetics, antigen presenta-tion, NK cells and KIR genes, autoim-munity, host pathogen response, solid organ and bone marrow transplanta-tion, HLA disease associations. Par-ticipants will have the opportunity to present their own research data. The summer school will accept 25-30 graduate students and postdoctoral fel-lows. Applications: applications must include a current C.V., statement of motivation for attending the summer school, a letter of recommendation and a sum-mary of current research project.

Applications should be sent to: Katherine Miranda Association Headquarters 15000 Commerce Pkwy Mt. Laurel, NJ 08054 kmiranda@ahint.com

The deadline for applications is July 15, 2008.

A committee consisting of representa-tives from EFI, ASHI and ASEATTA will review the applications and select the candidates. Applicants will be notifi ed of their acceptance by August 1, 2008. After selection, successful candidates will receive registration information.

Registration fee: 150 USD which includes registration, lodging and meals

Bursaries: Participants are expected to pay their own travel expenses to Rio de Janeiro; a few travel stipends will be available for participants who provide reasonable justifi cation at the time of registration. Contact for the Summer School:

Maria Gerbase-DeLima, MD, PhDImmunogenetics Division, Federal University of São Paulo, Rua Loefgreen, 1235 04040-031 São Paulo, SPBrazil e-mail: gerbase@igen.epm.br Further information about the Summer School may be found on the websites of ASHI (www.ashi-hla.org) EFI (http://www.efi web.org), ASEATTA (http://www.aseatta.org.au/) and 15th International Histocompatibility and Immunogenetics Workshop (www.15ihiws.org).

2. Random CellsThis group currently consists of 98 BCLs (will rise to 166) from a contigu-ous ‘random’ group of 166 local blood donors, used as a local reference DNA set. These are typed for 11 HLA loci (DRB1, DPB1, at the 4-digit level as a minimum, remainder at 2-digits mini-mum) and C4A, C4B, Bf, MICA and MICB (6), HFE (7), HPA (8) and HA-1, HA-2.

Antibody screening by fl ow cytometryA set of 22 BCLs, covering 78% of all offi cial HLA-A, B, C and 90% of DR, DQ specifi cities, are identifi ed for use in anti-body screening by fl ow cytometry (2).

Availability and provision of BCLsThis resource is available as frozen BCLs or genomic DNA to bona-fi de labo-ratories for use as ‘research reagents’ on a strictly non-commercial basis.

They are provided free of charge but a handling fee is levied that covers: cell culture, microbiological quality control, authentication, processing, packag-ing and postage/transportation costs. Procedures are provided for the resus-citation of frozen BCLs, their routine culture, cryopreservation and process-ing for use in antibody screening by fl ow cytometry.

Material has been provided to 15 institutions in the UK and overseas; including 77 BCLs for the UCLA Immu-nogenetics Center’s ‘exchanges’.

Our complete BCL collection, user responsibilities and conditions of supply are detailed on our Laboratory’s website at: www.wtail.org.uk or for further information regarding supply contact Dr. Helen Bass (Telephone: +

44 (0) 1443 622169; Fax: + 44 (0) 1443 622176; e-mail: Helen.Bass@wbs.wales.nhs.uk; Welsh Transplanta-tion and Immunogenetics Laboratory, Welsh Blood Service, Pontyclun CF72 9WB, Wales, United Kingdom).

WTAIL References

1. Eur J Immunogenet 2004, 31, 87. 2. Eur J Immunogenet 2003, 30, 295. 3. Genet Test 2004, 8, 301. 4. Histocompatibility Testing: A Practical Approach. IRL Press Practical Approach Series, Oxford, pp 51-80. serology5. Bone Marrow Transplant 2000, 25, 771. 6. Genet Test 2005, 9, 93. 7. Genet Test 2000, 4, 111. 8. Eur J Immunogenet 1999, 26, 393.

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ITALIAN WORKSHOP ON EFI ACCREDITATION IN BARI

Rising demand for EFI accreditation from laboratories in Italy, fostered by recent national legislation requiring EFI accreditation for all Italian laboratories typing for hemtaologic stem cell trans-plantation as of January 2009, has prompted the Italian National Immuno-genetics Society AIBT to coordinate a dedicated workshop on accreditation. This event was organized in Bari from January 24 to 25 by Marco Andre-ani (Rome), Biagio Favoino (Bari) and Mario Savi (Parma). It was aimed at 1) elucidating the theoretical and practi-cal aspects of accreditation, and 2) discussing diffi cult standards and how to interpret them.For logistic reasons, admission had to be limited to a maximum of 30 par-ticipants, with one member per labora-tory. Acceptance was on a fi rst come fi rst serve basis, with priority given to representatives of as yet not accred-ited laboratories. In the end, of 48 applications received, the 30 accepted participants all represented as yet not accredited labs.The course was divided into presenta-tions by invited speakers on Thursday and Friday morning, and round table discussions between all participants and the eight Italian EFI inspectors on Thursday afternoon and Friday morn-ing. Invited speakers included the past (Francesca Poli, Milan) and present (Katharina Fleischhauer, Milan and Carlo Carcassi, Cagliari) EFI commissioners for Region 07, the Italian EPT coordina-tor (Francesca Quintieri, Rome), and, the EFI Accreditation Committee Chairman (Gottfried Fischer, Vienna), who gave an interesting overview about the past, present and future of the EFI Accredita-tion Program in his presentation “Staying competent in a complex fi eld”, on Friday morning. The other speakers gave each 30 minutes lectures on Friday morning, respectively on general aspects of EFI accreditation, guidelines for completing the A packet, a simulation of an on-site EFI inspection, and an overview over the EPT schemes.

For the round table discussions, par-ticipants were divided into four groups that rotated around four different tables, each dedicated to discussion of different sections of the standards and chaired by two EFI inspectors. Table 1, chaired by Francesca Poli (Milan) and Benedetta Mazzi (Milan), discussed standard sections A, B and C on General Policies, Personnel Qualifi ca-tions and Quality Assurance, respec-tively. Table 2, chaired by Domenico Adorno (L’Aquila) and Luca Mascaretti (Monza), discussed standard sections E and F on Serological Typing and Anti-body Testing. Table 3, chaired by Maria Edvige Fasano (Turin) and Carlo Car-cassi (Cagliari) discussed standard sec-tion L on Nucelic Acid Analysis. Table 4, chaired by Marco Andreani (Rome) and Katharina Fleischhauer (Milan), discussed standard sections D and I on HLA nomenclature and hematopoi-etic stem cell transplantation. The two-hours round table discussion with the different groups gave suffi cient time for lively interaction between inspec-tors and participants, making it pos-sible to discuss numerous questions concerning interpretation of “diffi cult” standards and ways for implementa-tion. Gottfried Fischer rotated between tables and thus, along with the inspec-tors, got an interesting overview about the most frequently asked questions and practical issues, being able also to give helpful advice in different mat-ters. The emerging picture confi rmed

the evidence obtained from previous EFI inspector workshops that the most “tricky” standards regard Quality Assur-ance (Section C), Nucleic Acid Analysis (Section L), and HLA Nomenclature (Section D). An important issue that became evident was the importance of consistency between inspectors and commissioners. Discussions were very lively and it became evident that Italy, which has the largest number of HLA laboratories pro capite in Europe, has a pool of competent specialists work-ing in these laboratories that may, in the future, become useful beyond the boarders as the EFI accreditation scheme widens. The friendly and collegial atmosphere of this successful meeting was com-pleted by Bari hospitality, greatly appre-ciated by all attendants who on Friday evening enjoyed the beautiful old city center of Bari and, as a special high-light, a remarkable seafood dinner in the famous restaurant “Ai 2 Ghiottoni”. We hope this initiative may soon be fol-lowed by similar ones in Italy and/or abroad, possibly under the umbrella of the Poseidon EC project whose precise aim is the promotion of EFI accredita-tion throughout Europe.

Mario Savi (Parma)Marco Andreani (Rome) Biagio Favoino (Bari) Katharina Fleischhauer (Milan) Gottfried Fischer (Vienna)

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