4.75
#54
OVERRATING OR UNDERESTIMATION AMBIGUITIES?.
Erik H. Rozemuller 1, Anette Dijk van 1, Petra Weide van der 1, Martin Maas van der 1 and Marcel G.J. Tilanus 1. 1 Department of Pathology, H04.312, Heidelberglaan 100, University Medical Centre Utrecht, Utrecht, Netherlands .
HLA typing approaches are dependent on available allele sequence information, and the technological design of the typing technique All typing techniques show ambiguous typing results, in which no unique identification of the alleles can be determined. Three major groups of ambiguities occur: 1. Incomplete sequences cause ambiguities in combination with known alleles. 2. Allele ambiguities refer to alleles which differ only outside the determined regions 3. Genotype ambiguities are (heterozygous) allele combinations with identical patterns or sequences, and thus can not be discriminated. All three kinds of ambiguities can be resolved. The genotype ambiguities are usually resolved by separating the alleles, either at the amplification step or by specific sequencing the generic template. Among all techniques, SBT shows the lowest number of ambiguities. We evaluated the ambiguities encountered during routing typing by SBT of patient and (potential) donors for unrelated stem cell transplantation. Our approach is based upon exons 2, 3 and 4 sequencing for HLA-A and HLA-B, and exons 2 and 3 and, when necessary exons 1,4,5,6 and/or 7 for HLA-C, minimizing the number of allele ambiguities encountered. In HLA-A genotype ambiguities (40%) of predominantly frequent alleles are encountered compared to about 10% for HLA-B and HLA-C. All ambiguities are resolved by group specific sequence primers (GSSP). GSSP primers are designed to cover many different genotype ambiguities. The nomenclature indicates the specificity. GSSP-A362GG-F discriminates between A*010101,02010101 and A*0236,3604 at position 362. The ease of design and applicability makes the GSSP approach favourable compared to prior separation of alleles.