Československý transplantační kongres
Konferenční abstrakta 2006
Abstrakta posterů – sekce lékařů
TECHNICAL ASPECTS OF KIR-GENOTYPING IN ORGAN AND STEM-CELLS TRANSPLANTATION.
Pavlová Y., Slavčev A., Kolesár L.
Natural killers (NK-cells) are known to be one of the basic components of innate immune system. They are able to kill tumor, virus infected and allogenic target cells by cytotoxicity without any pre-activation and are regulated by complex interactions between their surface receptors and HLA class I molecules. KIR (killer immunoglobulin-like) NK-cells’ receptors, belonging to the immunoglobulin superfamily recognize various HLA class I alleles and inhibit NK-cells cytitoxicity while bound to specific ligand or activate NK-cells when the ligand is missing.
The KIR gene family consists of 15 genes and 2 pseudogenes.
KIR gene polymorphisms are clinically relevant to stem-cells transplantation outcome, while their impact on solid organ transplant success has not yet been assessed. Our aim was to estimate the advantages and disadvantages of two different method of KIR-genotyping.
KIR SSP genotyping. The presence of a KIR gene is defined with a pair of primers specific for KIR intergene sequences. The amplification products are then evaluated by agarose gel electrophoresis.
KIR SSO genotyping by the Luminex 100 system combines a flow cytometry analysing method with reverse SSO DNA typing. DNA is PCR-amplified using three separate group-specific primer sets targeting exons 3, 5, and 7-9. Each PCR product is biotinylated, denatured and hybridized to complementary DNA probes conjugated to fluorescently coded microspheres. Bound DNA from the test sample is tagged with SAPE. The fluorescent intensity is then measured on the flow cytometer. Both of these methods were performed in our laboratory for analysing of KIR haplotypes of 100 healthy unrelated individuals from the Czech population panel of the Department of Imunogenetics, IKEM.
For SSP KIR genotyping we used KIR SSP genotyping kits from Dynal.And for SSO KIR genotyping was used the kits from OneLambda. Both methods were found reliable and applicable for KIR genotyping in organ and stem-cells transplantation.