Human platelet antigen genotyping by using sequence-specific primers and the particle gel agglutination assay.
OBJECTIVE
Polymerase chain reaction using sequence-specific primers (PCR-SSP) is currently the most widely used technique for human platelet antigen (HPA) genotyping. Here, we describe a novel particle gel-agglutination technique for simplified visualization of the amplified products.


METHODS
Biotinylated primers were used to amplify HPA-1, -2, -3, -4, -5, -6, and -15, and the PCR products were incubated with streptavidin particles. Fluorescein isothiocyanate (FITC)-labelled primers [amplifying a fragment of the human growth hormone (HGH) gene] and anti-FITC-coated particles were used as internal controls. Agglutination of the particles in or on top of the gel indicated specific amplification. A total of 100 samples from blood donors was tested by using this new technique and a standard PCR-SSP protocol.


RESULTS
The use of biotinylated sequence-specific primers resulted in PCR products that agglutinated streptavidin particles, and the FITC-labelled HGH primers led to agglutination of anti-FITC-coated particles. Negative reactions were clearly distinguishable from positive reactions. The results of the particle gel agglutination method were in concordance with those of the electrophoretic visualization in all cases tested.


CONCLUSIONS
The new particle agglutination method is reliable and easy to use.
Meyer
O
O
Charité, Institute of Transfusion Medicine, D-13353 Berlin, Germany.
Abou-Chaker
K
K
Heymann
G
G
Bombard
S
S
Kiesewetter
H
H
Salama
A
A
D000372:Agglutination Tests / Q000379:methods; D016824:Antigens, Human Platelet / Q000235:genetics*; D001710:Biotin; D017931:DNA Primers; D016650:Fluorescein-5-isothiocyanate; D005838:Genotype; D006801:Humans; D008722:Methods; D016133:Polymerase Chain Reaction / Q000379:methods; D019809:Streptavidin
D016824:Antigens, Human Platelet; D017931:DNA Primers; D001710:Biotin; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate