The Anthony Nolan Trust Histocompatibility Laboratories & Operations Department Service Provision

Transcription

1 The Anthony Nolan Trust Histocompatibility Laboratories & Operations Department Registered Charity /SC038827

2 Contents 1. Introduction Summary Procedures Undertaken for Haematopoietic Stem Cell Transplantation Identification of a Related Donor for a Patient HLA Typing Requirements of Patients Undergoing an Unrelated Donor Search, when typing is performed externally from the Anthony Nolan Trust Performing Searches for Unrelated Donors The Initial Unrelated Donor Search Request The Search Report Extending the Search to other Volunteer Bone Marrow Donor Registers Confirmatory Typing of Potential Matched Donors Identified Post-transplant Chimerism Monitoring Target Turnaround Times Additional Services Provided by The Anthony Nolan Trust Histocompatibility Laboratories 4.1 Solid Organ Transplantation Renal Transplantation Liver Transplantation Disease Associations Target turnaround times Haemochromatosis Mutation Detection Contract HLA typing Histocompatibility and Immunogenetics Procedures Utilised by the Anthony Nolan Trust Histocompatibility Laboratories Introduction Serological Typing DNA Based Typing Techniques PCR-SSOP testing PCR-SSP testing PCR-Sequencing testing Virology and Blood Group Typing Antibody screening Cross-matching Preparation of HLA Typing Reports Participation in Quality Assurance Schemes Summary of Current Services Provided by the Histocompatibility Laboratories Histocompatibility Laboratories Supervisory Staff Request forms and simple type Histocompatibility laboratory address and opening hours Time limits for requesting additional examinations Histocompatibility Laboratories contact details Histocompatibility Laboratories senior staff 7. Operations Department and Register Staff Aims for the Future Complaints procedure... Page N o

3 1. Introduction The Anthony Nolan Trust takes back lives from leukaemia by managing and recruiting new donors to the UK s most successful bone marrow register. This independent charity also conducts pioneering research into the treatment of bone marrow disorders and improving the effectiveness of bone marrow transplants. Currently, The Anthony Nolan Trust Register contains more than 400,000 potential haematopoietic stem cell (HSC) donors and has provided over 5,000 HSC donors for transplantation. This prospectus is aimed at the following individuals and organisations to ensure that all those affected by the services provided by The Anthony Nolan Trust are informed of the processes undertaken: Transplant centres and associated laboratory staff in the UK and overseas General practitioners Patients in need of a HSC transplant and their families in the UK and overseas Potential HSC donors Government agencies The wider national and international scientific and medical community The Anthony Nolan Trust s supporters and fundraisers International registries Local hospital haematology units The aim of The Anthony Nolan Trust remains to provide a lifesaving HSC transplant for those patients in need. This aim is achieved by maintaining an active volunteer donor register, through focused donor recruitment, retention and tracing strategies. Comprehensive HLA typing of donors and patients is undertaken by The Anthony Nolan Trust Histocompatibility Laboratories employing the most up to date methodologies. The Laboratories also provide a Histocompatibility and Immunogenetics service to the liver and renal transplant units of the Royal Free Hospital. Disease association testing is also undertaken. 2 Authorised By: Kieran Herrity

4 2. Summary The Anthony Nolan Trust The Anthony Nolan Trust is comprised of several departments all working together to ensure the charity s aims are achieved. This document presents the services offered by the Operations Department and the Histocompatibility Laboratories for donor identification and HLA typing. Other departments within The Anthony Nolan Trust also have an important role to play in the realisation of our goals, such as scientific research, information technology and fundraising. For further details please visit our website: The Anthony Nolan Trust acts as the UK national hub for volunteer unrelated HSC transplantation. A hub is defined in the World Marrow Donor Association (WMDA) guidelines as: a national organisation responsible for the processing of incoming requests from other countries and outgoing requests to other countries, to co-ordinate within each country the activities of donor, harvest and transplant centres, and to supervise a system of accreditation for such centres. As the UK hub, The Anthony Nolan Trust coordinates all aspects of extending a donor search internationally, from foreign donor and cord blood sample requests through to donor work-up for donation and import of a haematopoietic stem cell product, and to underwrite the funding of such operations to hubs in other countries. Authorised By: Kieran Herrity 3

5 3. Procedures undertaken for haematopoietic stem cell transplantation 3.1 Identification of a related donor for a patient The first procedure usually undertaken when a patient is identified as potentially requiring a HSC transplant is to perform HLA typing on the patient and any available, suitable and willing relations, usually siblings. CMV screening and blood group typing is also performed. There is a 25% chance that a HLA matched sibling will be identified for a patient in need of a haematopoietic stem cell transplant. Whenever possible HLA typing of parents should be performed to confirm the patient s type and to accurately determine haplotypes within a family. For final selection of a related donor, HLA typing of both donor and recipient must be repeated using a freshly drawn typing sample from each, such that each individual s typing and Identity are confirmed. HLA typing of patients is performed by The Anthony Nolan Trust Histocompatibility Laboratories. Request forms are available by contacting labs@anthonynolan.org.uk or are downloadable from The Anthony Nolan Trust Search Website for those individuals with appropriate access. (See section 5 for description of HLA typing technologies utilised). Patients are screened for certain viruses and are A,B,O, RhD blood group typed (see Section 5.4). HLA typing of potential related donors is undertaken to identify a histocompatible match. For those patients where a related matched donor is not identified, upon request from the referring centre, the next step is to identify a matched volunteer unrelated donor (section 3.3). In some circumstances an extended family search may be performed (see box 1, Page 5). Advice on matching donors and patients can be provided by The Anthony Nolan Trust. However, the final decision on donor suitability is always the responsibility of the transplant centre. 4 Authorised By: Kieran Herrity

6 Box 1 If requested, extended family searches may be performed if a sibling match or unrelated donor match is not available. Relatives, such as grandparents, aunts, uncles or cousins, may share one of the patient s haplotypes and it may be possible that the patient s second haplotype (or a closely matched second haplotype) is introduced into the family via spouses. Even a child of the patient could be a match. Extended family searches are advisable if the patient possesses at least one haplotype that is considered common within the general population. The extended family search should focus on analysis of the side of the family from which the that the frequent haplotype has been introduced through marriage. Extended family searches may also be undertaken if the patient is from an ethnicity not widely represented on donor registers. In the diagram below, the patient has inherited a rare haplotype from his father and a common haplotype from his mother. His cousin also shares the same phenotype, with the same rare haplotype inherited from the cousin s father and the second haplotype inherited from the cousin s mother, who is unrelated to the patient. least common haplotype is inherited, in the hope grandfather grandmother mother father patient cousin Common haplotype in population Rare haplotype in population }Other haplotypes in population Authorised By: Kieran Herrity 5

7 3.2 HLA typing requirements of patients undergoing an unrelated donor search, when typing is performed externally from The Anthony Nolan Trust. The first step in identification of an unrelated donor for a patient is to submit a Donor Search Request form to The Anthony Nolan Trust Operations Department. Please also attach a copy of the HLA typing report provided for the patient. It is essential to ensure that the HLA typing has been performed to high resolution, preferably to allele level for HLA-A, B and DRB1 and to first field (two digit) 3.3 Performing searches for unrelated donors The initial unrelated donor search request. Within the UK, transplant physicians or their representatives, or haematologists at referring hospitals may request unrelated donor searches. Transplant centres must be JACIE (Joint Accreditation Committee EBMT-Euro/ISHAGE) accredited for unrelated donor transplants. As such, there are no restrictions on patient age or diagnosis, although certain unusual requests may require approval from The Anthony Nolan Trust Medical Advisory Committee ( Searches will not be performed at the request of patients, their family or friends. To collect all the necessary information to initiate an unrelated donor search, a standard Donor Search Request form is used. Completed forms should be faxed or allele level for other loci or serological split level, whichever gives the higher resolution. Where a string of alleles is given as a type, it is preferential to group alleles into serological equivalent clusters e.g. for the HLA-B*15 (B62, B63, B70, etc) and HLA-B*40 (B60, B61 and B4005) families. This level of typing is mandatory to expedite the search process. Low resolution HLA types will slow down the search procedure by identifying donors who on subsequent analysis may prove to be mismatches. As some international registries require high resolution HLA typing of patients prior to provison of donor, we recommend high resolution patient typing at the beginning of the unrelated search process. ed, rather than posted to, The Anthony Nolan Trust to expedite the search process. When the HLA typing has been performed by The Anthony Nolan Trust Histocompatibility Laboratories, the referring centre will be asked if a search should be performed at the time of issue of the HLA typing report. High resolution HLA typing will be performed on all patients requiring an unrelated donor search The Search Report. The Search Report will comprise a front page identifying the patient and recommending an appropriate course of action based on the accompanying donor listings (see figure 1, page 7). 6 Authorised By: Kieran Herrity

8 The Anthony Nolan Trust Search Report Figure 1 Patient: Not Applicable HLA Search Details: A*0201,2402;B*0702,4402;Cw*0501,0702;DRB1,0401;DQB1*0602,0301 A-B DR Typed Donors - Exact Matches: 0 Potential Matches: 121 Minor Mismatches: 30 (Limited to 13) Donor Ref/ s-stored PM AN S PM AN S PM AN S Sex/ Age Male 23 Male 38 Male 26 PM AN Male 31 AN Male PM 36 AN Male PM 39 PM AN Male 40 AN Male PM 40 AN Male PM 41 PM AN Male 43 AN Male PM 44 AN Male PM 44 AN Male PM 44 CMV/ Date Positive 08/11/2004 Negative 27/02/2001 Positive 17/09/2004 Positive 02/03/1999 Negative 02/03/1999 Negative 11/03/1996 Blood Group/ Ethnic O Positive N European B Positive N European A Negative N European O Positive N European O Positive N European O Negative N European Wgt (kgs) N European 80 O Positive N European 64 N European 83 N European 92 A Positive N European A Positive N European N European State A B DRB1 DRB3 DRB4 DRB5 DQA1 DQB1 Cw DPA1 DPB1 *02ATEZ *24AMSK *02XX *24GMW *02JJJ *24KVX 2 24 *02XX *24XX *02XX *24XX *07XV *4402 *07BD *4402 *07JKH *44JYR 7 44 *07BD *44XX *07BD *44XX *01TMB *01YXC *01DBH *01BFK *01TMB *01YXC *0103 *0101 *0103 *0101 *0103 *0101 *06BNK *03AF *06WG *0301 *06GPK *03NX *0602 *0301 *0602 *0301 *0602 *0301 *0602 *0302 *0602 *0301 *05ANJN *0702 *07CZA *05MN *05UA *07DNF *0501 *07BC *0501 *07BC *0501 *07BC Printed on 13 September 2005 at 17:47 KEY Exact Matches: EM Potential Matches: PM Minor Mismatches: x Mismatches: X Note: Alphabetical characters in the HLA fields are NMDP codes, denoting intermediate level resolution typing. Donors with intermediate level typing are classified as potential matches denoted by the symbol`?. As some HLA antigens have more allelic variations than others and frequencies of these in the target population differ, it is important that search reports and donor selections are reviewed by someone with extensive experience in Histocompatibility and Immunogenetics. Authorised By: Kieran Herrity 7

9 The donor listing is divided into those donors matching with HLA-A, -B and -DRB1 data and those with only HLA-A and -B data. The matching program developed at The Anthony Nolan Trust includes the HLA A, B, and DRB1 molecular typing data in the sorting algorithm. Where ambiguities exist, resulting in intermediate resolution results, the allele codes developed by the National Marrow Donor Program, USA ( are utilised to enter data onto the computer. Searches will be extended to look for donors with a single mismatch at the HLA-A, -B or DRBI locus if deemed appropriate. The standard sort format for donor listings is male donors before female (based on transplant centre preference) and younger before older, within degree of HLA match. Additional information provided on matching donors, if known, includes blood group, cytomegalovirus (CMV) status, ethnic group and weight. Most of these factors may be applied as optional filters to reduce a long list of matching donors e.g. a list of CMV negative matching donors only may be requested. Donors who are known to be temporarily unavailable are marked `Unavailable with a release date, and those in test for another patient are marked `In Test. The higher the HLA typing resolution provided on the patient, the more informative the donor listing will be. The minimum level of typing required on the patient is that recommended in The Anthony Nolan Trust Patient HLA Typing Guidelines ( The Search Report and recommendations will be issued within two working days of receipt of the Donor Search Request form Extending the search to other volunteer bone marrow donor registers In the UK there are three HSC panels that function independently but co-operate over exchange of search requests. If the initial request from a UK transplant centre is received by The Anthony Nolan Trust, it will be faxed or electronically transmitted on to the British Bone Marrow Registry (BBMR) and the Welsh Bone Marrow Donor Registry (WBMDR) on day of receipt. A separate donor listing should be expected from each panel, the formats of which may differ. Simultaneously with The Anthony Nolan Trust search, for each UK referred patient, we will conduct an extended search of the international panels through `Bone Marrow Donors Worldwide (BMDW). BMDW comprises the HLA types of all the donors on panels around the world, collated in Leiden, the Netherlands. Cord blood units are also included. By this process the requesting physician has a global picture regarding all possible matches at the outset of the search. Advice on matching donors and patients can be provided by The Anthony Nolan Trust. However, the final decision on donor suitability is always the responsibility of the transplant centre. 8 Authorised By: Kieran Herrity

10 3.4 Confirmatory typing of potential matched donors identified Once potential matched unrelated donors are identified, their HLA type must be confirmed. Blood samples are requested from the unrelated donors (on The Anthony Nolan Trust register or any other register where a potential matched donor has been identified). It is necessary to request a fresh blood sample from prospective donors to ensure they are still willing and available to donate, and still fulfil the medical requirements for donation. Virology screening also needs to be performed on a recent blood sample. HLA typing of matched unrelated donors can be performed by the transplant centre s designated laboratory or by The Anthony Nolan Trust Histocompatibility Laboratories. HLA typing at The Anthony Nolan Trust Histocompatibility Laboratories is performed by the same procedures as described for the patient, including high resolution typing. Virology and blood group typing are also included as before. Results will not be issued until all tests including virology and blood group testing are complete. The results of donor HLA typing performed outside The Anthony Nolan Trust must be sent to The Anthony Nolan Trust Operations Department, as it is necessary to know whether the donor is required for transplant; if the donor is to be put on hold (maximum 90 days) or if the donor can be released and made available for other patient searches. This information is crucial not only for The Anthony Nolan Trust donors but also donors provided by international registries. At this time it is necessary to perform confirmatory typing on a second blood sample from the patient, if this has not been performed. This step eliminates any remote possibility of sample error at the time of initial bleed and typing. 3.5 Post-transplant chimerism monitoring Please contact laboratories for further details: labs@anthonynolan.org.uk 3.6 Target turnaround times We aim to process all patients, potential related and unrelated donor samples received for testing within an average of seven working days. Authorised By: Kieran Herrity 9

11 4. Additional services provided by The Anthony Nolan Trust Histocompatibility Laboratories 4.1 Solid organ transplantation The Histocompatibility Laboratories, Solid Organ Division, provides a service to support the requirements of the renal and liver transplant units of the Royal Free Hospital, London. This service is funded by the Royal Free Hospital NHS Trust Renal Transplantation For kidney transplantation, potential recipients are HLA typed by serological (HLA-A, B) and DNA based methods: HLA-A, B, C, DRB1,3,4,5, DQB1. HLA- DPB1 testing is performed where appropriate. Two independent samples are tested for each patient. Patients are also screened for HLA alloantibodies and if present the specificity of such antibodies is defined. Patients are screened at three monthly intervals in accordance with British Transplantation Society standards ( Confirmatory blood group typing is also performed on all patients. Family members may also be HLA typed to resolve patient s haplotypes and to aid definition of antibodies arising from sensitisation through pregnancy. The cadaveric transplant (UK Transplant, UKT; now part of NHS Blood and Transplant) is maintained up-to-date with patient details, and monthly updates are issued to all patient centres. A 24 hour, 7 days per week, on-call service is available to provide tissue-typing and cross-matching tests at any time. The North Thames transplant coordinators inform laboratory staff of potential cadaver organ donors in local hospitals and arrange for EDTA blood to be collected and sent to the laboratory for HLA typing. All local cadaver donors are HLA typed by serology (HLA-A, B, DR, DQ) and by DNA methods (HLA-A, B, C, DRB1,3,4,5, DQB1). HLA-DPB1 testing is performed when appropriate. Non-local donors also have their HLA type confirmed by serology during on-call hours and subsequently by DNA methods. Crossmatching between donor and potential recipient is performed using both Complement Dependent Cytotoxicity (CDC) and Flow Cytometry methods. This enables detection of the presence of donor specific antibodies in the patient serum, which if present are a contraindication for transplantation. Results are reported to physicians and surgeons on duty. UKT are notified of donor HLA type by fax. Where a living related or unrelated donor transplant is being considered, all appropriate and willing potential donors are blood group typed to determine compatibility. HLA typing (HLA-A, B, C, DRB1,3,4,5 and DQB1) is performed to determine compatibility. Crossmatching between patient and potential donor is performed using both CDC and Flow Cytometry methods at the initial time of donor selection and again within 7-14 days of the proposed transplant. All results are communicated in writing to the transplant nurse and clinicians. Serum samples from all recipients of a transplant are received and screened at intervals in accordance to British Transplant Society guidelines. 10 Authorised By: Kieran Herrity

12 4.1.2 Liver Transplantation All potential liver recipients are HLA typed by DNA (HLA-A, B, C, DRB1,3,4,5, DQB1) methods. HLA types of cadaver donors are obtained from UK Transplant. Retrospective crossmatching between liver transplant recipients and donors is performed by CDC. All results are communicated in writing to the liver transplant coordinators Target turnaround times Registration of new patients on national renal transplant list, tests include: HLA typing, blood group testing and HLA antibody screen: 10 working days. Routine HLA antibody screen by ELISA or luminex technologies: 10 working days. Routine HLA antibody screen by CDC: 3 months. 4.2 Disease Associations HLA typing will be undertaken to provide support in the diagnosis of the following associated diseases: Ankylosing Spondylitis HLA-B typing is performed by serology, and where appropriate also by DNA methods including HLA- B27 allele typing. Cadaver renal crossmatch (CDC and flow cytometry): 7 hours. This does not include HLA typing time, which will differ depending on whether the donor is local or not. Narcolepsy HLA-DRB1 and DQB1 typing is performed by DNA methods and if positive for DRB1*15 and DQB1*06, further allele level typing is undertaken. Cadaver liver donor crossmatch (CDC only): 4 hours. Live renal donor crossmatch (CDC and flow cytometry, including HLA typing and blood group testing: 5 working days. Behçet s Disease HLA-B typing is performed by serology, and where appropriate also by DNA methods including HLA- B51 allele typing. Abacavir Hypersensitivity HLA-B*57 testing is performed by DNA methods. The presence of HLA-B*5701 is performed by allele level typing. For more details on disease association tests and costs incurred please contact the laboratory: labs@anthonynolan.org.uk Target turnaround time Our target turnaround time for reporting disease association requests is 5 working days. Authorised By: Kieran Herrity 11

13 4.3 Haemochromatosis Mutation Detection Haemochromatosis is an autosomal recessive disorder which causes an increase in iron absorption leading to iron overload. Two mutations within the HFE gene (found telomeric to the HLA genes on chromosome 6) have been found to be commonly associated with the disorder. These mutations which alter amino-acid 63 (histidine to aspartate) and amino-acid 282 (cysteine to tyrosine) are detected by the PCR-SSP technique. All reports are issued via the Centre for Hepatology, Royal Free Hospital, London. Please contact the laboratory for a request form labs@anthonynolan.org.uk. 4.4 Contract HLA typing The Histocompatibility Laboratories are able to undertake low and high throughput contract typing when related to the improvement of medical sciences. For more details contact the laboratory: labs@anthonynolan.org.uk 12 Authorised By: Kieran Herrity

14 5. Histocompatibility and Immunogenetics procedures utilised by The Anthony Nolan Trust Histocompatibility Laboratories 5.1HLA typing Introduction HLA typing is performed to define compatibility between donor and patient. An individual s HLA type is determined by a group of genes which produce proteins expressed on virtually all tissues in the human body. These genes are extremely variable (or polymorphic), making it unlikely that any two random individuals would have an identical HLA type. There are different laboratory methods which are utilised to determine HLA type, and these methods can be divided into two groups. The first, called serology, targets the definition of the HLA protein at the cell surface where many different antibodies with specificity against different HLA molecules are used to determine HLA type. The second group of methods targets the DNA molecules directly, usually using a large number of reagents which can detect the many different polymorphisms in the population. There are currently over 3,700 different HLA alleles (or variants) ( therefore the typing methods and interpretation of results can appear complex. Although a combination of typing tests is generally performed on each sample, it is not always possible to define the type to a single allele, and frequently results will appear as allele strings. For example in certain allele combinations for heterozygous loci (majority of individuals are heterozygous at HLA loci) we may not be able to differentiate between the common HLA DRB1 and the rare HLA- DRB1*0426, and would therefore report the allele string HLA- DRB1/26. A full list of HLA alleles currently defined by the WHO Nomenclature Committee for Factors of the HLA System can be found at: and can also be accessed on our website: Serological typing HLA typing can be performed by the complement dependent lymphocytotoxicity reaction (serology). Live peripheral blood mononuclear cells are required for this assay. CD8 + T-cells and/or CD19 + B-cells are purified from whole blood and incubated against a panel of antibodies with specificity against polymorphic epitopes expressed on HLA-A and -B proteins (CD8 + T-cells or HLA-DR and DQ proteins (CD19 + B-cells). In the presence of complement cells expressing HLA proteins which react with a particular antibody are lysed, allowing these damaged cells to uptake a stain which is detected by fluorescent microscopy. The pattern of negative and positive reactions is scored and interpreted to give an HLA serological type. Authorised By: Kieran Herrity 13

15 Box 2 The HLA-DR gene family The HLA-DR gene family consists of a single HLA-DRA gene encoding the α-chain of the DR molecule and a single DRB1 gene encoding the β-chain of the DR molecule. Other DR molecules can be encoded by the same α-chain and a β- chain from a DRB3, DRB4 or DRB5 gene. The β- chain from the DRB3, DRB4 and DRB5 genes encode the serological determinants DR52, DR53 and DR51 respectively. All individuals possess a DRB1 gene. However the presence of DRB3, DRB4 and DRB5 genes is haplotype dependent. DRB3 is found on haplotypes possessing DRB1*03, *11, *12, *13 and *14. DRB4 is found on haplotypes possessing DRB1*04, *07 and *09 and DRB5 is found on haplotypes possessing DRB1*15 and * DNA based typing techniques PCR-SSOP testing All patients, matched related donors and unrelated donors are typed for HLA-A, B, C, DRB1, DRB3,4,5, DQB1 by PCR-SSOP methods. All newly recruited donors to The Anthony Nolan Trust Register are also tested for HLA-A, B, C and DRB1 by PCR-SSOP methods. The SSOP assay involves the incubation of locus specific PCR products (e.g. HLA-A) with a panel of immobilised oligonucleotide probes with sequences complementary to polymorphic sequences within the HLA alleles. Currently we are using two technologies. The first is called Luminex xmap in which the oligonucleotide probes are immobilised on polystyrene microspheres (or beads). Reactivity between PCR product and beads is detected by measurement At present all patient and potential matching donor samples (HSC and renal transplant) are typed by serology, with additional DNA based typing performed. Serological typing is the quickest method to establish identity within a family, and it also allows the identification of null alleles (HLA alleles detected by DNA techniques, which do not result in protein expression) when further DNA based typing is performed. of fluorescence at two wavelengths, the first to identify the specificity of the bead and the second to determine if PCR product has bound. The second method used has the oligonucleotides immobilised on a nitrocellulose membrane ( strip ). Detection of bound PCR products causes the appearance of a coloured band at a particular oligonucleotide probe specific location on the strip. 14 Authorised By: Kieran Herrity

16 PCR-SSP testing PCR-SSP typing utilises a panel of PCR primer pairs which target known polymorphic nucleotide motifs within HLA alleles. The presence or absence of a PCR product (in the presence of a positive internal control PCR product) determines the presence or absence of a particular nucleotide sequence. This pattern is interpreted to give the HLA type. PCR-SSP is used to achieve HLA-DRB1 allele level typing on haematopoietic stem cell patients and their selected donors. PCR-SSP is also used to provide a fast on-call HLA type of cadaveric solid organ donors. It is also used to resolve ambiguities arising from other methodologies and to confirm unusual associations between alleles in an individual (e.g. HLA-B-C associations) PCR-sequencing testing Direct sequencing of PCR products is performed on an automated DNA sequencer using dyeterminator chemistry. Sequencing is used mostly to determine HLA-A, B, C, DRB1 and DPB1 high resolution type for haematopoietic stem cell patients and their selected unrelated donors. Sequencing is also used to resolve any novel alleles detected by other methods. Any unacceptable ambiguities arising from sequencing are resolved by PCR-SSP. Virology, Royal Free Hospital, Pond Street, London, NW3 2QG. Blood group typing is performed by haemagglutination reaction to detect red blood cell antigens. Any ambiguous/equivocal results are confirmed by referral to the CPA accredited Blood Transfusion, Royal Free Hospital, Pond Street, London NW3 2QG. 5.3 Antibody screening Three methods are used in rotation to establish the presence and specificity of HLA class I and II reactive antibodies in both pre- and posttransplant patients. These include: Complement-dependent Iymphocytotoxicity (CDC) This method is used to determine the presence of IgM and IgG complement fixing HLA-specific antibodies. Test patient sera are reacted with panels of T-cells (expressing known HLA class I antigens) and B-cells (expressing known HLA class I and II antigens), that have been specifically selected on the basis of their HLA types. Complement mediated lysis of T and B-cell panels, is recorded using microscopy. Enzyme-linked immunosorbent assay (ELISA) Method discontinued 5.2 Virology and blood group typing Human sera are screened for the presence of CMV (antibody detected), HIV1/2 (antibody/antigen), hepatitis B (antigen detected) and C (antibody detected) infection by ELISA. Any ambiguous/equivocal results are confirmed by referral to the CPA accredited Department of Authorised By: Kieran Herrity 15

17 Luminex Luminex technology uses fluorescent microbeads coated with purified HLA class I and II proteins. The beads are incubated with patient sera and bound antibodies detected after reaction with a fluorescently labelled IgGspecific secondary. Test sera are screened to initially determine the presence of HLA class I or II specific antibodies. Sera scoring positive for HLA specific antibodies, are further analysed with luminex kits designed to define HLA specificities. Highly sensitised patients with IgG antibodies reactive with multiple HLA class I or II antigens are further characterised with luminex beads possessing immobilised recombinant single class I or II molecules, which provides the highest resolution of HLA-specific antibody analysis. Currently the laboratory uses in alternative cycles products from two different manufacturers. 5.4 Cross-matching The purpose of the crossmatch (XM) test is to determine the presence of pre-formed antibodies in a patient that are reactive with HLA antigens on donor cells and could therefore cause hyperacute rejection of a renal graft. Both CDC and flow-cytometric (FC) tests are performed by reacting both fresh and selected historic recipient sera with separated donor T and B-cells (allogeneic XM), and recipient T and B-cells (autologous XM). The results of these tests, in conjunction with the known immunological and clinical parameters of a potential renal transplant recipient, are used to advise renal transplant surgeons on the likely risk (high, intermediate of low) of graft rejection occurring, should a given renal transplant be performed. The FC XM is recognized to be more sensitive and objective than the CDC XM and detects both complement fixing and non-complement fixing IgG antibodies. The CDC XM although less sensitive, detects both IgG and IgM complement fixing antibodies. Both CDC and FC XM can be performed simultaneously. 5.5 Preparation of HLA typing reports All HLA typing data collected are interpreted independently by two different experienced laboratory staff. Data from individual tests are submitted to SOLAR, The Anthony Nolan Trust central database, and patient and donor reports are prepared after all tests requested are completed. Reports related to patients that have been typed by The Anthony Nolan Trust Histocompatibility Laboratories (related or registry donors) are prepared by a Laboratory/Clinical Scientist and checked by either the Laboratory Head, Laboratory Manager, or Senior Supervisor. Other potential donor reports may be issued directly from the SOLAR database via the Operations Department. All HLA typing data is checked for appropriate haplotype associations based on published and in-house data. Any unusual results will be checked further and sent for DNA sequence analysis if deemed appropriate. 16 Authorised By: Kieran Herrity

18 5.6 Participation in quality assurance schemes The Histocompatibility Laboratories take part in the following external quality assurance programmes and consistently achieve satisfactory results. United Kingdom National External Quality Assurance Schemes (UK NEQAS) for Histocompatibility and Immunogenetics; Scheme 1A HLA Phenotyping Scheme 1B HLA-B27 Testing Scheme 2A Cytotoxic Crossmatch Scheme 2B Crossmatching by Flow Cytometry Scheme 3 Scheme 4 Scheme 5 Scheme 6 Scheme 7 HLA Antibody Specificity Analysis HLA DNA Typing Haemochromatosis Candidate Gene Mutations Antibody Detection Educational Scheme HLA-B*57 UK NEQAS for Microbiology Immunity Screen (for CMV testing) Blood Borne Virus Scheme (for Hepatitis B, Hepatitis C and HIV Serology) Diagnostic Serology (for EBV, Syphilis and Toxoplasma testing) United Kingdom NEQAS for Blood Transfusion Laboratory Practice ABO RhD Blood Group Typing University of California at Los Angeles (UCLA) International Cell and DNA Extract Exchange Authorised By: Kieran Herrity 17

19 5.7 Summary of current services provided by the Histocompatibility Laboratories The Histocompatibility Laboratories of The Anthony Nolan Trust are able to provide the following genetic testing and typing services to the defined users. Service Haematopoietic stem cell transplantation HLA-A, B and DRB1 typing of potential unrelated HSC donors and cord bloods HLA-A, B, C, DRB, DQB1 and DPB1 confirmatory typing of potential unrelated HSC donors User The Anthony Nolan Trust register ANCTC The Anthony Nolan Trust register External registers Transplant centres 1 Transplant centres 1 HLA typing on stored frozen potential unrelated donor DNA HLA-A, B, C, DRB, DQB1 and DPB1 typing of patients Transplant centres 1 and potential related HSC donors HLA-A, B, C, DRB, DQB1 and DPB1 typing of non-ant Transplant centres 1 HSC donors for patients within the U.K. ABO RhD blood group typing of HSC donors, Transplant centres 1 patients and cord blood ANCTC Virology screening of potential HSC donors and Transplant centres 1 patients Crossmatching of patients with potential HSC donor Transplant centres 1 Chimerism detection in post transplant patients Transplant centres 1 Solid organ transplantation HLA-A, B, C, DRB, DQB1, DPB1 typing of liver and renal potential transplant recipients HLA-A, B, C, DRB, DQB1 typing of cadaver liver and kidney donors HLA-A, B, C, DRB, DQB1 typing of living kidney donors HLA alloantibody screening and definition for patients pre and post-renal transplant Crossmatching of liver and renal transplant recipients and donors 24 hour, 7 day / week on-call HLA typing and crossmatching service for liver and renal transplantation Non-transplantation services HLA disease associations Surgical and Associated Services Division, Royal Free Hospital Surgical and Associated Services Division, Royal Free Hospital, United Kingdom Transplant UK Transplant Surgical and Associated Services Division, Royal Free Hospital Surgical and Associated Services Division, Royal Free Hospital Surgical and Associated Services Division, Royal Free Hospital Surgical and Associated Services Division, Royal Free Hospital Royal Free Hospital and other related organisations HLA-A, B, C, DRB, DQB1 and DPB1 typing for nontransplantation projects organisations 1 Commercial and non-commercial Haemochromatosis gene (HFE) mutation typings Centre for Hepatology, Royal Free Hospital, and other requesting centres. 1 Currently within the United Kingdom and overseas 18 Authorised By: Kieran Herrity

20 6. Histocompatibility Laboratories Information 6.1 Request forms and sample type Request forms for the following tests, can be obtained from the laboratories (see contact details below) Patient histocompatibility testing (Haematopoietic stem cell transplantation) Related donor histocompatibility testing (Haematopoietic stem cell transplantation) Renal and liver transplant histocompatibility testing (patient and/or donor tests) Disease association studies Haemochromatosis mutation detection It is the requesting centre s responsibility to ensure appropriate patient and donor consent as stipulated by the Human Tissue Act 2004 is obtained for the requested tests. All details required for sample collection are on the request forms (please use Block capitals). HLA typing for transplantation or disease association: 4ml EDTA (never heparin). Virology: 4ml clotted sample Blood grouping: 4ml clotted sample Antibody screening: 10ml clotted sample Crossmatch: Patient: 20ml EDTA and 10ml clotted sample Donor: 40ml EDTA All samples should be stored at room temperature and can be shipped at room temperature. Please ensure all mailing containers are compliant with UN3373 and IATA 650 transport regulations. All pathological specimens should be packaged by a recognised laboratory or institution, a qualified medical or dental practitioner or a veterinary surgeon, who then posts the package. The package should show the name and address of the sender and must be handled and transported with the understanding biological samples could transmit infectious agents. 6.2 Histocompatibility laboratory address and opening hours Postal address of Histocompatibility Laboratories: Histocompatibility Laboratories The Anthony Nolan Trust Royal Free Hospital Pond Street London NW3 2QG Courier address for Histocompatibility Laboratories: The Round Table Laboratories The Anthony Nolan Trust Royal Free Hospital Fleet Road NW3 2QG (Entrance behind doctors surgery at no. 75) The laboratories are open Monday to Friday, excluding bank holidays from 8am to 5pm. 6.3 Time limits for requesting additional examinations Samples from all patients and donors (related and unrelated) who have undergone a transplant are securely stored frozen in the laboratories as either DNA or whole blood for a minimum period of 11 years 1. Authorised By: Kieran Herrity 19

21 Samples from patients and potential matching donors (related and unrelated) who may not have undergone a transplant are securely stored frozen in the laboratories as either DNA or whole blood for a minimum period of 11 years 1 Serum samples from renal and liver transplant patients (pre and posttransplant) are securely stored frozen in the laboratories as sera for a minimum period of 11 years 1 Samples from patients tested for disease associations (not haemochromatosis) are securely stored frozen in the laboratories as either DNA or whole blood for a minimum period of 5 years 1 Samples from patients tested for HFE are securely stored frozen in the laboratories as either DNA or whole blood for a minimum period of 10 years 1 1 Space permitting 6.4 Histocompatibility Laboratories contact details Laboratory reception telephone number: Laboratory fax number: Laboratory address (general enquiries): labs@anthonynolan.org.uk Director of Operations: Ailsa.Ogilvie@anthonynolan.org.uk Laboratory Manager: Richard.Holman@anthonynolan.org.uk 20 Authorised By: Kieran Herrity

22 7. Histocompatibility Laboratories and Operations Department Senior Staff. Histocompatibility Laboratories Research & Scientific Director: (Co-Director of H&I): Laboratory Manager: Consultant Clinical Scientist: Senior Supervisors: Professor Alejandro Madrigal, MD, PhD, FRCP, FRCPath, DSc Professor Steven Marsh, BSc, PhD, ARCS Richard Holman, BSc, SRCS Henry Stephens, BSc, PhD, SRCS Finnuala A Fowles, BSc, SRCS (Confirmatory Testing) Joyce Grant, BSc, MSc, SRBS (Solid Organ Group) Helen Ogilvie, BSc, SRCS (Clinical Services) Anita Shah, BSc, SRCS (New Donor Testing) Franco Tavarozzi, BSc, SRCS (High Resolution Typing) Operations Department Director of Operations: Head of Donor Recruitment: Search Manager: Pre-Harvest Manager: Harvest and Welfare Manager: Ailsa Ogilvie TBA Irina Evseeva, MD, PhD Edel Harkin Rochelle Roest Operations Department Please contact: Ailsa.Ogilvie@anthonynolan.org.uk ANT Quality Manager: Kieran Herrity, BSc (Hons), MSc, FIBMS, SRBS Authorised By: Kieran Herrity 21

23 the future 1. To continue to provide an excellent Histocompatibility and Immunogenetics service for patients in need of a stem cell transplant. 2. To continue to increase the number of donors recruited and HLA typed on The Anthony Nolan Trust Register. 3. To increase laboratory automation. 4. To increase the level of resolution performed for register HLA typings, thus minimising time required to identify and perform confirmatory typing. 5. To increase the resolution of the HLA typing performed on patients and potential matched donors whilst maintaining desired sample turnaround times. 6. To promote closer collaboration with transplant centres in order to assess their needs and to maintain an effective and efficient service. 7. To establish additional genetic testing services as the need becomes apparent for the improvement of donor and patient matching in haematopoietic stem cell transplantation and other clinical procedures. 8. To continue our research and analysis of The Anthony Nolan Trust donor database such that we can improve our understanding of the diversity of donor phenotypes which will enable us to specifically select donors at an early stage for whom further typing will be useful. 9. To increase the ethnic diversity of the Register through black and minority ethnic recruitment initiatives. 10. To maintain and improve the quality of Histocompatibility and Immunogenetics service provided to support the solid organ transplant programme of the Royal Free Hospital, London. 11. To maintain laboratory accreditation via Clinical Pathology Accreditation (UK) Ltd. and European Federation for Immunogenetics accreditation. 12. To maintain World Marrow Donor Association (WMDA) Registry accreditation, and HTA license status. 9. Complaints procedure If you wish to make a formal complaint regarding the services provided by The Anthony Nolan Trust Histocompatibility Laboratories or Operations Department. Or report a Serious Adverse Event or Reaction (SAER) please send details / contact the Trust Quality Manager: Mr Kieran Herrity, BSc (Hons), MSc, FIBMS, SRBS Kieran.Herrity@anthonynolan.org.uk 22 Authorised By: Kieran Herrity