| Donor Registry Typing |
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Overview. A primary goal of the International Histocompatibility Working Group (IHWG) is the development of genomic methods for analysis of the human MHC and the application of this technology to clinical typing. The clinical project selected as a component of the IHWG to accomplish this goal is HLA typing for volunteer marrow and umbilical cord blood donors. The aims of this project are to: 1) coordinate an international strategy for HLA typing of volunteer marrow and umbilical cord blood donors; 2) implement a common DNA-based typing method and standard set of reagents; 3) establish quality control procedures to verify data; 4) build a database consisting of sequence polymorphisms; and 5) facilitate transfer of typing data to local and international donor registries. Background. Since 1964 International Histocompatibility Workshop and Conferences have played a major role in defining HLA polymorphism and facilitating the development of standards for HLA typing. Over the last 30 years the characterization of reagents and the definition of new HLA antigens has been made possible largely through international collaboration. Standardization of typing methods initially depended on the exchange of selected antisera and typing trays. Recent advances in genomic analysis, however, have virtually eliminated the need to exchange reagents for the purpose of standardizing specificity and defining new antigens. Primers and probes used for DNA typing can be easily characterized and manufactured according to uniform specifications. Nevertheless, the assignment of an HLA genotype can be difficult because of the complex polymorphisms that define individual HLA genes, and the relatively large number of primers and probes sometimes required to fully characterize each allele. Most of the methods currently used for typing HLA-DR and DQ, and more recently HLA-A and B, employ a limited number of primers and probes resulting in an assignment of genotypes variably defined at a low-to-intermediate level of resolution. Specific Aims. The HLA Typing for Marrow Donor Registries component of the IHWG will focus on five specific aims: 1) the coordination of an international strategy for HLA typing of volunteer marrow and umbilical cord blood donors; 2) the implementation of a common DNA-based typing method and standard set of reagents; 3) the establishment of quality control procedures to verify data; 4) the building of a database consisting of sequence polymorphisms; and 5) the facilitation of the transfer of typing data to local and international donor registries. Achievement of these goals will be facilitated by the international collaborative effort that will characterize the scientific projects and core components comprising the IHWG. Donor Typing and Matching. The problem posed by the extreme complexity of the HLA system is highly germane to marrow donor and cord blood registries. Cost considerations tend to limit the number of different primers and probes used for registry typing, thereby constraining typing to an intermediate level. Invariably, the assigned genotype based on the available data must include two or more alleles. Examples now exist where the assigned specificity can be encoded by one of 20 different possible alleles. The number of different specificity groups assigned has grown dramatically, and many of these groups consist of overlapping clusters of alleles. The assignment of specificity becomes even more complex when donor types are deduced by various typing methods using different primers and probes. Over time, the assigned specificity becomes more ambiguous as typing reagents change and new alleles are discovered. The solution to this problem is to recognize that typing with either sequence specific primers (SSP) or sequence specific oligonucleotide probes (SSOP) identifies sequence polymorphisms (1). Rather than relying on a database of assigned HLA types, donor registries should retain the actual primer reactions and hybridization patterns. The donor HLA data saved by the registry should consist of the actual sequence polymorphisms identified. New donor search algorithms should be developed that identify potential matches according to the sharing of these common motifs. It is now feasible with the aid of Informatics systems to match according to virtual sequences (2). Genomic Analysis of HLA. The development of genomic typing for HLA is a major goal of the IHWG. DNA-based typing for HLA-DR and DQ was introduced during the 11th workshop, with the distribution of a common protocol and a standard set of reagents consisting of locus specific primers and sequence specific oligonucleotide probes (SSOP) (3). DNA-based typing for class I genes was initially undertaken during the 12th workshop with the introduction of protocols and reagents for HLA-A (SSOP) and HLA-C (SSP) at a level of resolution equivalent to serology (4). The further improvement and application of DNA-based typing for HLA-A, B and C and the need for establishing a common method and nomenclature for donor registry typing will be addressed during the 13th workshop. Standardization of Typing Methods and Reagents in the IHWG. The IHWG has chosen hybridization of PCR amplified DNA with SSOP as the core technology for HLA typing. The SSOP method is robust, defines individual sequence polymorphisms, and can be adapted relatively easily to high volume typing. An SSOP advisory committee chaired by Dr. Carolyn Hurley has updated the primers and probes used for the typing of HLA-DR, DQ and DP in the 11th and 12th workshops, and has selected primers and probes for the typing of HLA-A, B and C. The sequences of these primers and probes will be posted on the International Histocompatibility Working Group website (5). Two sets of SSOP reagents have been selected: one for intermediate resolution and one for high resolution typing. The intermediate reagents, which will type at a level equivalent to serology, will be appropriate for donor registry typing. An RFP was issued and the SSOP Advisory Committee evaluated product samples prior to designating the official vendor. A purchase agreement is being negotiated to assure competitive pricing. The goal of this agreement is to make quality reagents conveniently available to investigators and laboratories around the world in a cost-effective manner. Quality Control. The SSOP Advisory Committee will supervise a pretest administered to all IHWG participants. A standard panel of DNA will be distributed by the SSOP Quality Control (QC) Laboratory supported by The CW Bill Young Marrow Donor Recruitment and Research Program, Georgetown University. Participating laboratories will be asked to type these QC samples with IHWG SSOP reagents. Results of the pretest including the scores for each probe will be submitted to the QC Laboratory for evaluation. Laboratories that achieve a typing accuracy of at least 90% per locus will be allowed to submit data to the IHWG. The performance of participating laboratories will be monitored during the course of the workshop by periodic testing of blind control samples. A panel of coded cells will be distributed, typed along with workshop samples, and results reported to the SSOP Quality Control Laboratory. A reference cell panel provided by the IHWG Cell and Gene Bank Core Laboratory (Fred Hutchinson Cancer Research Center, Seattle) will be used to monitor the specificity of primer and probe hybridizations. Most of the cells in this panel will be homozygous for specific HLA alleles to provide optimal quality control of typing reagents. The panel is being selected by the SSOP Advisory Committee. Marrow Donor and Cord Blood Registry Typing. Participating HLA laboratories will arrange sample acquisition with a local donor center, donor registry or cord blood bank. Informed consent and confidentiality will be the responsibility of the donor center. All samples will be coded and no donor-specific information will be transmitted to the typing laboratory or the IHWG. Data Reporting and Analysis. A data report form will be developed that recognizes the IHWG SSOP primers and probes and captures the positive and negative hybridization reaction of each probe. Donor identity will be confidential and only coded data will be submitted via a secure file-transfer format by e-mail or through the Internet to a central database. An interactive Informatics system will be developed to verify the accuracy of data entry and provide the user a preliminary genotype assignment (allele or allele-group). Unexpected hybridization patterns and new variants will be reported. With prior consent of the typing laboratory, verified coded results will be reported directly to the designated donor registry and if requested this data can also be forwarded directly to Bone Marrow Donors Worldwide (BMDW) (6-7). In addition to providing common reagents and facilitating the standardization of donor registry and cord blood HLA data, this international effort will be helpful in guiding future strategies for donor recruitment aimed at broadening the HLA diversity of the virtual global donor registry. Summary International Histocompatibility Workshops have played a critical role in disseminating technical advances and establishing standards for clinical typing. A primary goal of the IHWG is to facilitate the further development of genomic methods for HLA analysis and the translation of new technology to relevant clinical applications. The need for high volume HLA typing has increased dramatically the last few years with the remarkable growth of unrelated marrow donor registries. The superior accuracy achievable with DNA-based typing can improve the efficiency and reliability of the unrelated donor search by decreasing the errors that can occur when searching for HLA phenotypes defined by serological methods. Various options currently exist for DNA-based typing but the use of different methods and reagents has resulted in significant heterogeneity in the data reported and this is complicating donor searches. The situation will become more complex as new alleles are discovered. Agreement among marrow donor registries around the world to use a common method and set of reagents for genomic typing will greatly simplify creation of a high quality database and permit retention of the actual sequence polymorphisms of each donor, thereby facilitating the unrelated donor search and matching process. The ultimate goal is to provide more efficient and timely access to HLA matched hematopoietic stem cells for patients in urgent need of a transplant. References 1. Hurley CK. Acquisition and use of DNA-based HLA typing data in bone marrow registries. Tissue Antigens 49: 323-328, 1997. 2. Helmberg W, Lanzer G, Zahn R, Weinmayr B, Wagner T, Albert E. Virtual DNA analysis - a new tool for combination and standardised evaluation of SSO, SSP and sequencing-based typing results. Tissue Antigens 51: 587-592, 1998. 3. Kimura A, Sasazuki T. Eleventh International Histocompatibility Workshop reference protocol for the HLA DNA-typing technique. In: K Tsuji, M Aizawa, T Sasazuki (eds): HLA 1991. Oxford: Oxford University Press 1992: 397-419. 4. Tonks S, Marsh SGE, Bunce M, Moses JH, Drausa P, Sadler AM, Petronzelli F, Bodmer JG. HLA Class I DNA typing study. In: D Charron (ed): Genetic diversity of HLA. Paris: EDK 1997: 199-215. 5. International Histocompatibility Working Group website: http://www.IHWG.org/ 6. Oudshoorn M, Leeuwen A, Zanden HGMv, Rood JJv. Bone marrow donors worldwide: a successful exercise in international cooperation. Bone Marrow Transplant 1994:14:3-8. 7. BMDW program information and statistics are available through BMDW online, URL: http://bmdw.leidenuniv.nl/ |
