Five-Locus HLA Typing of Hematopoietic Stem Cell Donor Volunteers Using PCR Sequence Specific Primers

Abstract

We have developed a strategy for five-locus human leukocyte antigen (HLA) typing of hematopoeitic stem cell (HSC) donors using the polymerase chain reaction with sequence-specific primers (PCR-SSP). The PCR-SSP method is robust, reproducible, and accurate. New PCR-SSP mixtures can be added as required and all reactions are carried out under the same conditions, which can easily be applied to the typing of other loci, e.g., ABO blood groups. Initially, 127 PCR-SSP reactions were used to detect simultaneously HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles, differentiated generally to the level of the first two digits of the allele name, essentially equivalent to the serological split specificity. Approximately 40% of subjects were tested against a further 29 HLA-A, -B SSP mixtures to exclude rare alleles and unambiguously assign a two-digit HLA allele family. This gave an overall typing resolution equivalent to or greater than the split specificity level and covered all HLA-A, -B, -C, -DRBland DQB1 alleles listed in the WHO's Nomenclature for Factors of the HLA System, 2000. The Welsh Bone Marrow Donor Registry has used this strategy to HLA type over 35,000 HSC donors over 9 years. Comprehensive and accurate five-locus HLA typing allows confident and rapid identification of potential matched HSC donors for patients requiring stem cell transplantation generally without the need for typing additional loci. This allows resources to be focused directly on allele level typing of DRB1 and other loci. This strategy decreases overall donor work-up time, which is a major benefit to patients.

Provision of Epstein‐Barr virus‐transformed B‐cell lines in a routine tissue typing laboratory: practicalities and applications

“…BCLs from our cell bank are used routinely as a source of reference DNA for primer mixture validation and control of our ‘in‐house’ PCR‐SSP typing systems (Downing et al ., 2004). In addition, BCLs have been supplied to the University of California, Los Angeles, Immunogenetics Center for distribution in their International Cell Exchange (B‐cell Line and DNA Extract).…”

“…Epstein‐Barr virus (EBV)‐transformed B‐lymphoblastoid cell lines (BCLs) have been produced in this laboratory for over 7 years. They have provided reference DNA for PCR primer validation (Downing et al ., 2004), archival material for our collection of new and rare HLA alleles, and a vital source of cells for testing patients’ sera for HLA antibodies by flow cytometry (Walters et al ., 1999). Novel, rare and unusual HLA phenotypes, e.g.…”

A “silent” nucleotide substitution in exon 4 is responsible for the “alternative expression” of HLA-A*01:01:38L through aberrant splicing

“…Because SC appeared to be homozygous at the HLA-A locus, retyping was later performed by PCR using sequence-specific primers (SSP) for HLA-A, -B, -C, -DRB1, and -DQB1 [17]. SC was subsequently serologically typed, on 2 further blood samples drawn at different times, using a total of 480 well-documented local alloantisera and 72 monoclonal antibodies (One Lambda Monoclonal Typing Tray Set, Class I [Lot 3A, LM144A and B], One Lambda, Inc., Canoga Park, CA).…”

“…PBLs were obtained by standard density gradient centrifugation methods and BCLs were prepared using our previously published methods [20]. All control cells were HLA-A, -B, and -C typed by PCR-SSP [17] and standard serology [21]. Selected HLA-A and -B alleles were confirmed by SBT [22].…”

HLA‐B*15:33, a rare allele whose product reacts as an HLA‐B62 and ‐Cw5/Cw8 specificity

“…Using PCR with sequence‐specific primers (SSP) (Downing et al. , 2004), we identified a further example of B*15:33, in a Welsh Bone Marrow Donor Registry donor (Darke, 2000), of likely north‐western European Caucasoid ancestry.…”