Methods for Diagnostic HLA Typing in Disease Association and Drug Hypersensitivity

  • Michael D. Varney
  • Alison S. L. Castley
  • Katri Haimila
  • Päivi Saavalainen
Part of the Methods in Molecular Biology book series (MIMB, volume 882)


This chapter describes the application of diagnostic HLA typing for disease association and five methods used for specific HLA genotypes. The methods utilise a combination of polymerase chain reaction (PCR) amplification to detect sequence polymorphism by the presence or absence of amplification, nucleotide sequencing of the PCR product, and hybridisation of the PCR product with labelled probes. The probes are specific for sequence polymorphism associated with the genotype and are attached to either a Micro Bead or a Solid Phase. In addition, the detection of single nucleotide polymorphism(s) which “tag” for the genotype using a real-time PCR is described.

Key words

HLA Disease Association Hypersensitivity Methods B*27 B*57 Coeliac B*1502 Narcolepsy DQ 



The DR/DQ SSP, SBT, and micro bead hybridisation methods are a culmination of input by the molecular typing staff of the Transplantation and Immunogenetics Service of the Australian Red Cross Blood Service.


  1. 1.
    Warrens AN (2000) Statistical considerations in analyzing HLA and disease associations. In: Lechler R, Warrens A (eds) HLA in health and disease, 2nd edn. Academic, LondonGoogle Scholar
  2. 2.
  3. 3.
    Brown MA (2009) Genetics and the pathogenesis of ankylosing spondylitis. Curr Opin Rheumatol 21:318–323PubMedCrossRefGoogle Scholar
  4. 4.
    Wu DY, Ugozzoli L, Pal BK, Wallace RB (1989) Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci USA 86:2757–2760PubMedCrossRefGoogle Scholar
  5. 5.
    Olerup O, Zetterquist H (1992) HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 39:225–235PubMedCrossRefGoogle Scholar
  6. 6.
    Sayer DC, Whidborne R, De Santis D, Rozemuller EH, Christiansen FT, Tilanus MG (2004) A multicenter international evaluation of single-tube amplification protocols for sequencing-based typing of HLA-DRB1 and HLA-DRB3,4,5. Tissue Antigens 63:412–423PubMedCrossRefGoogle Scholar
  7. 7.
    Lind C, Ferriola D, Mackiewicz K, Heron S, Rogers M, Slavich L, Walker R, Hsiao T, McLaughlin L, D’Arcy M, Gai X, Goodridge D, Sayer D, Monos D (2010) Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing. Hum Immunol 71:1033–1042PubMedCrossRefGoogle Scholar
  8. 8.
    Monsuur AJ, de Bakker PI, Zhernakova A, Pinto D, Verduijn W, Romanos J, Auricchio R, Lopez A, van Heel DA, Crusius JB, Wijmenga C (2008) Effective detection of human leukocyte antigen risk alleles in celiac disease using tag single nucleotide polymorphisms. PLoS One 3:e2270PubMedCrossRefGoogle Scholar
  9. 9.
    Koskinen L, Romanos J, Kaukinen K, Mustalahti K, Korponay-Szabo I, Barisani D, Bardella MT, Ziberna F, Vatta S, Széles G, Pocsai Z, Karell K, Haimila K, Adány R, Not T, Ventura A, Mäki M, Partanen J, Wijmenga C, Saavalainen P (2009) Cost-effective HLA typing with tagging SNPs predicts celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations. Immunogenetics 61:247–256PubMedCrossRefGoogle Scholar
  10. 10.
    Helmberg W, Lanzer G, Zahn R, Weinmayr B, Wagner T, Albert E (1998) Virtual DNA analysis—a new tool for combination and standardised evaluation of SSO, SSP and sequencing-based typing results. Tissue Antigens 51:587–592PubMedCrossRefGoogle Scholar
  11. 11.
    Sayer DC, Goodridge DM, Christiansen FT (2004) Assign 2.0: software for the analysis of Phred quality values for quality control of HLA sequencing-based typing. Tissue Antigens 64:556–565PubMedCrossRefGoogle Scholar
  12. 12.
    Rozemuller EH, Ploeger LS, Mulder W (2008) SBTengine; the ultimate solution for high resolution HLA typing. Hum Immunol 69:131Google Scholar
  13. 13.
    Olerup O, Aldener A, Fogdell A (1993) HLA-DQB1 and -DQA1 typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours. Tissue Antigens 41:119–134PubMedCrossRefGoogle Scholar
  14. 14.
    Olerup O (1994) HLA-B*27 typing by group specific PCR amplification. Tissue Antigens 43:253–256PubMedCrossRefGoogle Scholar
  15. 15.
    Sayer DC, Cassell HS, Christiansen FT (1999) HLA-B*27 typing by sequence specific amplification without DNA extraction. Mol Pathol 52(5):300–301PubMedCrossRefGoogle Scholar
  16. 16.
    Martin AM, Nolan D, Mallal S (2005) HLA-B*5701 typing by sequence-specific amplification: validation and comparison with sequence-based typing. Tissue Antigens 65:571–574PubMedCrossRefGoogle Scholar
  17. 17.
    Man CB, Kwan P, Baum L, Yu E, Lau KM, Cheng AS, Ng MH (2007) Association between HLA-B*1502 allele and antiepileptic drug-induced cutaneous reactions in Han Chinese [Erratum in: epilepsia 2008]. Epilepsia 48:1015–1018PubMedCrossRefGoogle Scholar
  18. 18.
    Kotsch K, Wehling J, Blasczyk R (1999) Sequencing of HLA class II genes based on the conserved diversity of the non-coding regions: sequencing based typing of HLA-DRB genes. Tissue Antigens 53:486–497PubMedCrossRefGoogle Scholar
  19. 19.
    Voorter CE, Kik MC, van den Berg-Loonen EM (1998) High-resolution HLA typing for the DQB1 gene by sequence-based typing. Tissue Antigens 51:80–87PubMedCrossRefGoogle Scholar
  20. 20.
    Van Dijk A, Melchers R, Tilanus M, Rozemuller E (2007) HLA-DQB1 sequencing-based typing updated. Tissue Antigens 69(Suppl 1):64–65PubMedCrossRefGoogle Scholar
  21. 21.
    Dunn PP, Day S, Williams S, Bendukidze N (2005) HLA-DQB1 sequencing-based typing using newly identified conserved nucleotide sequences in introns 1 and 2. Tissue Antigens 66:99–106PubMedCrossRefGoogle Scholar
  22. 22.
    Tilanus MGJ (ed) (2000) IHWG technical manual. Genomic analysis of the human MHC. DNA based typing for HLA Alleles & linked polymorphisms. Fred Hutchinson Cancer Research Centre, Seattle, USAGoogle Scholar
  23. 23.
    Cereb N, Yang SY (1997) Dimorphic primers derived from intron 1 for use in the molecular typing of HLA-B alleles. Tissue Antigens 50:74–76PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Michael D. Varney
    • 1
  • Alison S. L. Castley
    • 2
  • Katri Haimila
    • 3
  • Päivi Saavalainen
    • 4
  1. 1.Victorian Transplantation and Immunogenetics Service, Australian Red Cross Blood ServiceMelbourneAustralia
  2. 2.Department of Clinical Immunology, PathWest Laboratory MedicineRoyal Perth HospitalPerthAustralia
  3. 3.Clinical LaboratoryFinnish Red Cross Blood ServiceHelsinkiFinland
  4. 4.Research Program for Molecular Medicine, Department of Medical Genetics, Haartman Institute, Biomedicum HelsinkiUniversity of HelsinkiHelsinkiFinland

Personalised recommendations