| dc.description.abstract | Traditional antibody-based serologic typing methods are widely used to identify red blood cell (RBC) antigens. However, serology has limitations which might be circumvented by red cell genotyping. Currently, RBC genotyping is commonly practiced in specialized immunohematology reference laboratories. However, in-house RBC genotyping implementation at local transfusion services might have several advantages, such as reduced turnover time and establishment of an antigen-negative red cell unit supply.
Part one:
In the present study, the commercial and certified Ery Q® red cell genotyping kits were tested and evaluated to be used in-house at a local blood bank, supplementing traditional serology technique. A cost analysis was conducted to compare the expenses of sending blood samples requiring RBC genotyping to the Norwegian National Advisory Unit on Immunohematology (NAU-IH) at Oslo University Hospital (OUH) versus performing RBC genotyping by use of Ery Q® kits in-house. In addition, a questionary survey was performed to clarify the needs and wishes of various blood banks regarding RBC genotyping. Blood samples from 34 blood donors were collected during regular blood donation at Innlandet Hospital Trust (IHF) and conducted for RBC genotyping by use of quantitative PCR (qPCR) instrumentation and Ery Q® kits, comprising allele variants within the Rh, Kell, Kidd, Duffy, and MNS blood group systems. The Ery Q® kits were found to be highly accurate in comparison with serology findings and user-friendly with low hands-on time and software displaying detected allele variants and molecular predicted phenotypes. This ready-to-use kit might be suitable for in-house implementation. The questionnaire revealed that none of the responders have RBC genotyping established in their laboratory inventory, however, several consider implementation in future time.
Part Two:
The second part of this study aimed to develop the novel and multiple 25-plex LAD genotyping platform as RBC genotyping assays and benchmark them against the Ery Q® KKD/MNS kit. Primers and probes were designed to detect allele variants included in the Ery Q® kit, comprising KEL*02, KEL*01.01, JK*01N.06, JK*01, JK*02, JK*02N.06, FY*02N.01, FY*01, FY*02, FY*02W.01, GYPA*01, GYPA*02, GYPB*03, GYPB*04, GYPB*03N.01, and GYPB*03N.04 allele variants within the Kell, Kidd, Duffy, and MNS human blood group systems. Sequence-specific PCR primers were designed with 5’ universal A- and B primers to increase and equalize PCR amplification yield. Five fluorescence channels of detection with a maximum of five melting temperatures in each channel were applied. LAD showed detection of ten out of 22 allele variants in four multiplex reactions, comprising KEL*01.01, KEL*02, FY*01, FY*02, JK*01, JK*02, JK*01N.06 wild type, JK*02N.06 wild type, FY*02N.01 wild type, and FY*02W.01 wild type. In addition to weak signals, false positive quenching was observed in several, both sample and “no- template” control, reactions. In conclusion, the LAD technology showed to be a promising tool for RBC genotyping. Further bioinformatic investigation is needed to elucidate false positive quenching and detection of remaining allele variants. | |