No description available.

No description available.

No description available.

No description available.

No description available.

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.

Previously it has been shown that sequences containing both Trp²⁵ and Leu³³ are the most effective at inducing Th cell proliferation in HLA-DRB3*0101 positive women, alloimmunised with anti-HPA-1a.  The Leu³³/Pro³³ polymorphism is embedded in the N-terminal plexin/semaphorin/integrin (PSI) domain of GPIIIa.  In the present study, amino acids 1-62 of the GPIIIa (Leu³³ or Pro³³) PSI domain were cloned into the vector pGEX-6p-1.  The recombinant proteins were expressed and tested by ELISA, Luminex and Absorption Assays.  The presence of the HPA-1a/-1b epitope was confirmed by the ability of PSI-Leu³³/-Pro³³ recombinant fragments to specifically capture its corresponding HPA-1 antibody.  Cells from a human B cell line (HHKB), homozygous for HLA-DRB3*0101, were pulsed with the recombinant PSI domain fragment of GPIIIa expressing the HPA-1a antigen.  MHC class II/peptide complexes were isolated from the pulsed cells using an immunoaffinity column.  A nested set of naturally processed and presented HPA-1a derived peptides, each containing the residues Trp²⁵ – Leu³³ core epitope was identified.  For the first time a naturally processed and presented HPA-1a peptide that spans the HPA-1a polymorphism has been identified, bound to the class II molecule encoded by HLA-DRB3*0101.  The efficient processing and presentation of this peptide, which includes the putative dominant Th epitope, is likely to be an important contributory factor in the immunogenicity of HPA-1a.  Such peptides may also provide the basis for novel treatments to tolerise the corresponding Th response in HPA-1b1b women at risk of NAIT with an HPA-1a-positive fetus.

Thrombin activatable fibrinolysis inhibitor inhibits fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. It is expressed by the liver and circulates in plasma as a zymogen TAFI, which can be activated to an active carboxypeptidase, TAFIa, by plasmin or thrombin. Thrombomodulin in complex with thrombin increased the activation 1250-fold. The active carboxypeptidase TAFIa is unstable with a half-life of about 10 min at 37 °C and is inactivated to TAFIai by conformational change.

The induction of phenylketonuria in mice through the use of excess dietary phenylalanine is an area in which limited research has been done. This study intends to pursue further work in this area, more specifically, to study the effects of excess dietary phenylalanine and the phenylalanine analogue A.P.B.A. (2-amino-3-phenyl butanoic acid) (7) on brain serotonin and brain norepinephrine. In addition, the effects of these two compounds on the incidence of audiogenic seizures will be explored.

Platelets of divers undergo changes following exposure to high pressures and decompression. Since the latter is associated with formation of intravascular bubbles, platelet-rich plasma (PRP) was treated to reproducible doses of bubbles of air, with and without enrichment by 5% Co2 (to maintain physiological pH). PRP was also subjected to stirring and a 'rocking' procedure. Bubbles caused significant aggregation, measured turbidimetrically, of human (but not bovine) platelets, particularly without pH control, whereas little occurred with stirring and rocking. Bubbling inhibited subsequent ADP-induced aggregation of human platelets, more markedly without pH control. Bovine cells were unaffected, thus apparently less sensitive to bubbles. Since rocking was shown to be equiconvective to bubbling, aggregation associated with bubbling is not due simply to exposure of the gas/liquid interface, shear stress exerted by bubbles possibly being culpable. The importance of pH control in in vitro work was also demonstrated. PRP was next separately equilibrated (by rocking) with 5 atmospheres absolute (ata) pN2O, with 101 ata pN2 (these being approximately narcotically equipotent) and with 100 ata pHe (extremely weakly narcotic). 5 ata pN2O caused least inhibition of ADP-induced aggregation, and hydrostatic pressure (applied independently, using He and a floating plastic barrier which reduced dissolving of gas) was shown to swamp narcotic effects. It was suggested that inhibition by high pressure was due to blocking of prostaglandin synthesis. Finally, the effect of hydrostatic pressures of up to 51 ata on platelet ATP release was examined using a luciferin-luciferase assay. ADP-induced secretion was either completely blocked or partially reduced by high pressure. These effects could be important to divers suffering haemorrhages at great depths. Furthermore, in vitro hyperbaric studies are of potential value in elucidating activation pathways in platelets and other excitatory cells.