Haemagglutination is the recognised serologic technique for common (ABO & Rh) blood group antigen phenotyping with limitations; typing multi-transfused patients and non-invasive foetal blood group determination. Molecular technological advances in characterising the 30 blood group systems have also generated PCR based direct genotyping techniques. Their utility in routine blood banking practice is a rapidly evolving field. The study aims were (1) to establish PCR-SSP assays for KEL, FY and JK blood group genotyping, (2) to evaluate HEA BeadChipTM technology for SNPs detection of RHCc, RHEe, CO, DI, DO, FY, JK, KEL, LU, LW, MNS and SC and haemoglobinopathy S, against serology considering reproducibility, reliability, sensitivity and labour saving potential (3) to evaluate the specificity and sensitivity of TaqMan Real-Time PCR for NIPD of foetal RHD7, RHC, RHc, RHE and SRY status and (4) to establish Real-Time PCR assays and MGB TaqMan probes for 8 sets of gender-independent “Bi-allelic” Short Insertion/Deletion Polymorphisms (SIDPs) as internal assay controls confirming the presence of cell-free foetal DNA (cffDNA). The PCR-SSP results for KEL, FY and JK typing results showed complete concordance with serology for all samples except 1×JKa and 7×Fyb; discrepancies resolved by subsequent DNA sequencing. The HEA BeadChipTM microarray validation on gDNA (n=224) and 22 saliva samples, giving overall allele detection (ADR) and concordance rates (CoR) of >99.8% for the 24 alleles. The Fyx allele (Fyb/Fyx: 265C>T) frequency in Scottish donors (5.4%) was much higher than expected. Saliva-derived gDNA was less sensitive than buffy coat-derived gDNA; ADR 89.9% and 100% respectively. NIPD foetal blood group genotyping by Real-Time PCR of 51 alloimmunised pregnancies (n=104 samples, 12 to ≥40 weeks) with was 100% accurate for RHD7, RHC and RHE assays; 95.7% for RHc and 99% for SRY. The utility of Real-Time TaqMan assays for 8 selected SIDPs as paternal (foetal) markers, were assessed using gDNA, cell-free DNA (cfDNA) from 61 donors and 6 extended families and finally with cffDNA from 13 pregnancies. Based on these research findings, many of the molecular assays are now established in Aberdeen.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:540441 |
| Date | January 2010 |
| Creators | Varzi, Ali Mohammad |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=165837 |
Page generated in 0.0021 seconds