ASSIGNEMENT OF Lub-NEGATIVITY FOR RARE DONOR SELECTION REQUIRES SOPHISTICATED TECHNIQUES BASED ON MOLECULAR GENOTYPING
Christian Rüegg, Adriana Mendez, Edi Matheis, Annelies Böhlen and Beat M. Frey Stiftung Zürcher Blutspendedienst SRK, CH-8001 Zürich (www.zhbsd.ch)
Clinically, the most important antithetical antigens of Lutheran (Lu) blood group system are Lua and Lub (frequency 7.6% and 99.8% resp) which is characterized by single amino acid exchange at position 77 (His77Arg) resulting from point mutation in exon 3 (229A>G). Three types of Lunull phenotypes have been recognized: 1. autosomal-recessive type: amorph allele at LU locus, 2. autosomal-dominant type: inhibitor gene In(Lu) unlinked to LU locus and 3. x-chromosomal inhibitor gene (XS2). The autosomal-recessive type is the only real Lunull as weak expression of Lu antigens is detectable in the two other types by adsorption/elution techniques (AET) but not by indirect agglutination test (IAT). Two mutations creating amorph LU gene (deletion of exon 3/4 and a stop mutation of exon 6 (733C>A) have been described. We observed a patient with anti-Lub alloantibodies requiring Lub-negative red blood cells, which were provided by Swiss Red Cross Rare Donor Registry (RDR) and unexpectedly showed positive cross-matches, rendering the unit not suitable for transfusion. Therefore, pheno- and genotyping of Lub was evaluated to appropriately select rare blood donors (BD) of Lub negative phenotype.
Samples from 12 Lub negative BD as by IAT were re-examined. In order to define LUA/LUB genotype and to exclude missense mutation 733C>A, protocols to study Restriction Fragment Length Polymorphism of PCR products (PCR-RFLP) by AciI and MwoI restriction resp. were developed. Results of genotyping were compared with those of serological typings by IAT, AET and flowcytometry (FC).
8/12 BD (67%) disclosed homozygous for LUA. 4/12 BD (33%) were positive for LUB. Two of them, which were homozygous for LUB by genotyping were positive for Lub by AET. They represent incomplete suppression of Lub by inhibitory mechanisms. The other two BDs with Lu(a+b-) phenotype were heterozygous for LUA and LUB by genotyping. One of them showed weak expression of Lub by AET. The other one was negative for Lub by AET but was found positive for Lub by FC. None of the samples were positive for the missense mutation 733C>A.
- Lub phenotyping by IAT requires confirmation by molecular genotyping in combination with more sensitive assessment of phenotype by AET and/or FC.
- PCR-RFLP provides a reliable method to distinguish between Lub negativity and In(Lub) status.
- Mechanisms of differential inhibition of Lua/b expression by inhibitory loci remains to be elucidated.