HUMAN PLATELET ANTIGEN GENOTYPING USING A FLUORESCENT SSCP TECHNIQUE WITH AN AUTOMATIC SEQUENCER

The typing of human platelet antigene (HPA) can be useful in many clin ical situations such as neonatal alloimmune thrombocytopenia, post-tra nsfusion purpura, and platelet transfusion refractoriness. The fluores cent-based single-strand conformation polymorphism (F-SSCP) technique is a fast and convenient way to perform HPA genotyping, Universal sequ ences from phage M13 were introduced at both ends of specific PCR-prod ucts by using 5'-tailed primers, A short second round of PCR with univ ersal primers coupled to Cy-5 enabled the PCR-products to be fluoresce ntly labelled. F-SSCP was performed by gel electrophoresis on an autom ated fluorescent DNA analyser. Genotyping of the three major HPA syste ms carried by the GP IIb-IIIa complex; showed the F-SSCP technique to be accurate and reliable. A single gel procedure has been sufficient t o detect HPA genetic polymorphisms tested to date. Neither restriction enzyme, radioactive material, nor any other hazardous chemicals such as ethidium bromide were required. This technique enabled us to genoty pe HPa-1, -3 and -4 alleles easily and to diagnose materno-fetal incom patibility in a rare alloantigenic system. F-SSCP is a promising techn ique for the detection of new mutations and/or DNA polymorphisms on a large scale.