A rapid and simple method for reversing the inhibitory effect of heparin on PCR for HLA class II typing.

Centro Trasfusionale e di Immunologia dei Trapianti, Ospedale Maggiore Policlinico, 20122 Milan, Italy Blood samples collected with heparin as anticoagulant have been shown to yield decreased quantities of DNA, and an inhibitory effect of this anticoagulant on PCR has been demonstrated. Furthermore, in our experience, the amplification by PCR of HLA-DRB1 second exon was unsuccessful when the DNA template was obtained from heparinized blood. In this paper a simple method for e l iminat ing the inhibi tory effect of heparin on Taq polymerase is described. It employs a chelating ion resin, and DNA obtained can be used successfully for PCR and for HLA class II sequence-specific oligonucleotide (SSO) typing. This method, validated on >600 typings, is a useful alternative when no other source of DNA, except for heparinized blood, is available. Since 1989 we have been storing peripheral blood from donors and graft recipients in liquid nitrogen at -40~ to perform retrospective studies. With DNA technology, we began to re-evaluate the role of HLA class II matching by sequence-specific oligonucleotide typing (SSO typing) after DNA amplification by PCR. Most of the samples stored consisted of heparinized blood, as we were unaware of the inhibitory effect of this anticoagulant on several DNA polymerases/1'2) As a consequence, a significant number of samples could not be amplified by PCR and typed at the gene level. To overcome this problem, the use of heparinase has been suggested (z'3), but it is costly and sometimes fails. (z) To reverse the inhibi tory effect of heparin on Taq polymerase we used Chelex 100, an ion-exchange resin (Bio-Rad Laboratories, Richmond, CA). This agent is usually employed in our laboratory to improve the quality of DNA for PCR (4) and by others in a variety of PCR situations. (s) Here, we report our experience with the use of this resin both for "cleaning" DNA extracted from heparinzed blood, which previously failed to amplify by PCR, as well as for DNA extraction. The amplified DNA was employed for genomic HLA class II typing. The interaction mechanism between Chelex 100 and heparin has also been investigated.