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

Authors

  • Ole Olerup,

    Corresponding author
    1. Center for Bio Technology, Karolinska Institute, NOVUM, Huddinge, Department of Clinical Immunology. Karolinska Institute at Huddinge Hospital, Huddinge. Sweden.
      Address: Olle Olerup, M.D., Ph.D. Center for Bio Technology Karolinska Institute NOVUM S-141 57 Huddinge Sweden Tel: +46–8–60891 19 Fax: +46–8–774 55 38
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  • Henrik Zetterquist

    1. Center for Bio Technology, Karolinska Institute, NOVUM, Huddinge, Department of Clinical Immunology. Karolinska Institute at Huddinge Hospital, Huddinge. Sweden.
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Address: Olle Olerup, M.D., Ph.D. Center for Bio Technology Karolinska Institute NOVUM S-141 57 Huddinge Sweden Tel: +46–8–60891 19 Fax: +46–8–774 55 38

Abstract

Abstract: In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR “low-resolution” typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR “low-resolution” PCR-SSP technique, TagI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing and RFLP analysis was 100% The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR “low-resolution” typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.

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