Agreement among HLA antibody detection assays is higher in ever-pregnant donors and improved using a consensus cutoff.

Background: HLA antibodies might contribute to the pathogenesis of transfusion-related acute lung injury (TRALI). HLA antibody detection methods include ELISA, flow cytometry, and multiplex bead-based assays, as well as the older lymphocytotoxicity assay, and it is not obvious how to compare results across platforms.

Study Design And Methods: Five hundred twenty-five serum samples were selected from 7841 donors in the Leukocyte Antibody Prevalence Study (LAPS) repository based on risk for the development of HLA antibodies, using the number of pregnancies as the risk factor.

Multiplex single nucleotide extension: a robust and high throughput method for HLA-A locus typing.

This report describes a typing method that can identify all known human leukocyte antigen A (HLA-A) alleles by determining nucleotides present at polymorphic sites using single nucleotide extension. Allele specific primers are bound to capture oligonucleotides which allows for a multiplex approach during single nucleotide extension (SNE) reactions. Eleven group-specific polymerase chain reaction amplifications were performed to obtain the templates to be analyzed with sets of primers designed to investigate the polymorphisms.

Study of MICA alleles in 201 African Americans by multiplexed single nucleotide extension (MSNE) typing.

We have developed a method for major histocompatibility complex class I chain-related gene A (MICA) genotyping using multiplexed single nucleotide extension (MSNE) and flow cytometric analysis of an array of fluorescent microspheres. This technique employs a polymerase chain reaction-derived target DNA containing all the polymorphic sites of MICA, synthetic complementary primers, biotinylated dideoxynucleotide triphosphate, fluorescent reporter molecules (streptavidin-phycoerythrin), and thermophilic DNA polymerase. Genomic DNA was amplified by MICA locus-specific primers and the MSNE reactions were carried out in the presence of 30 MSNE primers used to assay polymorphisms in exons 2, 3, and 4 of the MICA genes.

Characterization of a novel MICA allele, MICA*047.

We report the identification of a novel MICA allele, MICA*047. It was initially detected because of an unusual hybridization pattern with sequence-specific oligonucleotides (SSOP) in a normal subject of Caucasian origin. Cloning and sequencing of both strands, and comparison of the sequence with previously defined MICA alleles, revealed that the new allele is similar to MICA*041 except for one nucleotide substitution at position 811 (C-->G).