Diverse and novel RHD variants in Australian blood donors with a weak D phenotype: implication for transfusion management.

BACKGROUND AND OBJECTIVES: Variant RHD genes associated with the weak D phenotype can result in complete or partial D-epitope expression on the red cell. This study examines the genetic classification in Australian blood donors with a weak D phenotype and correlates RHD variants associated with the weak D phenotype against D-epitope profile. MATERIALS AND METHODS: Following automated and manual serology, blood samples from donors reported as 'weak D' (n = 100) were RHD genotyped by a commercial SNP-typing platform and Sanger sequencing. Two commercial anti-D antibody kits were used for extended serological testing for D-epitope profiles. RESULTS: Three samples had wild-type RHD exonic sequences, and 97 samples had RHD variants. RHD*weak D type 1, RHD*weak D type 2 or RHD*weak D type 3 was detected in 75 donors. The remaining 22 samples exhibited 17 different RHD variants. One donor exhibited a novel RHD*c.939+3A>C lacking one D-epitope. Weak D types 1·1, 5, 15, 17 and 90 showed a partial D-epitope profile. CONCLUSION: The array of RHD variants detected in this study indicated diversity in the Australian donor population that needs to be accommodated for in future genotyping strategies.

Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms.

BACKGROUND AND OBJECTIVES: Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fy(a) /Fy(b) polymorphism, for weak Fy(b) antigen, and for the red cell null Fy(a-b-) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. MATERIALS AND METHODS: Samples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a-b-) n = 47 (one with anti-Fy3 antibody). RESULTS: Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fy(b) expression discrepant with Fy(b-) (Group 1 and 3). For three, SNP genotyping predicted Fy(a) , discrepant with Fy(a-b-) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null. CONCLUSION: Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status.

A novel FY*A allele with the 265T and 298A SNPs formerly associated exclusively with the FY*B allele and weak Fy(b) antigen expression: implication for genotyping interpretative algorithms.

BACKGROUND AND OBJECTIVES: An Australian Caucasian blood donor consistently presented a serology profile for the Duffy blood group as Fy(a+b+) with Fy(a) antigen expression weaker than other examples of Fy(a+b+) red cells. Molecular typing studies were performed to investigate the reason for the observed serology profile. MATERIAL AND METHODS: Blood group genotyping was performed using a commercial SNP microarray platform. Sanger sequencing was performed using primer sets to amplify across exons 1 and 2 of the FY gene and using allele-specific primers. RESULTS: The propositus was genotyped as FY*A/B, FY*X heterozygote that predicted the Fy(a+b+(w) ) phenotype. Sequencing identified the 265T and 298A variants on the FY*A allele. This link between FY*A allele and 265T was confirmed by allele-specific PCR. CONCLUSION: The reduced Fy(a) antigen reactivity is attributed to a FY*A allele-carrying 265T and 298A variants previously defined in combination only with the FY*B allele and associated with weak Fy(b) antigen expression. This novel allele should be considered in genotyping interpretative algorithms for generating a predicted phenotype.

A standardized immunofluorescence test method with human neutrophil antigen-expressing cell lines to enhance antibody detection.

There is an international need for a large-scale human neutrophils antigen (HNA) antibody screening platform to minimize the risk of antibody-mediated transfusion-related acute lung injury. However, sourcing a substantial, reliable source of HNA, as well as the scarcity of well-characterized HNA antisera for validating new screening platforms, remain as major obstacles. This short communication presents an improved protocol for the effective use of HNA-expressing KY cells as a screening platform using eight well-characterized HNA antisera of a single defined specificity.

Differential incorporation of precursor moieties into cerebral cortex and cerebellum glycerophospholipids during aging.

The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [3H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [3H]choline was inhibited. However, the incorporation of [14C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([3H]palmitic) and polyunsaturated ([3H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [3H]palmitic, [3H]oleic and [3H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 month old) and adult (3.5 month old) rats. [3H]oleic acid incorporation into neutral lipids (NL) and [3H]arachidonic acid incorporation into PC, PE and phosphatidylinositol (PI) were increased in aged rats with respect to adult rats. Present results show the ability and avidity of aged brain tissue in vitro to incorporate unsaturated fatty acids when they are supplied exogenously. They also suggest a different handling of choline and serine by base exchange enzyme activities to synthesize PC and PS during aging.

PCR monitoring for tetracycline resistance genes in subgingival plaque following site-specific periodontal therapy. A preliminary report.

BACKGROUND: The selection of antibiotic resistance genes during antibiotic therapy is a critical problem complicated by the transmission of resistance genes to previously sensitive strains via conjugative plasmids and transposons and by the transfer of resistance genes between gram-positive and gram-negative bacteria. The purpose of this investigation was to monitor the presence of selected tetracycline resistance genes in subgingival plaque during site specific tetracycline fiber therapy in 10 patients with adult periodontitis. METHOD: The polymerase chain reaction (PCR) was used in separate tests for the presence of 3 tetracycline resistance genes (tetM, tetO and tetQ) in DNA purified from subgingival plaque samples. Samples were collected at baseline, i.e., immediately prior to treatment, and at 2 weeks, and 1, 3, and 6 months post-fiber placement. The baseline and 6-month samples were also subjected to DNA hybridization tests for the presence of 8 putative periodontal pathogenic bacteria. RESULTS: PCR analysis for the tetM resistance gene showed little or no change in 5 patients and a decrease in detectability in the remaining 5 patients over the 6 months following tetracycline fiber placement. The results for tetO and tetQ were variable showing either no change in detectability from baseline through the 6-month sampling interval or a slight increase in detectability over time in 4 of the 10 patients. DNA hybridization analysis showed reductions to unmeasurable levels of the putative periodontal pathogenic bacteria in all but 2 of the 10 patients. CONCLUSIONS: These results complement earlier studies of tet resistance and demonstrate the efficacy of PCR monitoring for the appearance of specific resistance genes during and after antibiotic therapy.

The effect of using different levels of shrimp meal in broiler diets.

The objective of this study was to measure the effect of substituting different levels of shrimp meal (SM) for soybean meal (SBM) in broiler diets. In Experiment 1, 0, 10, 20, 30, and 40% of the crude protein contributed by the SBM in broiler diets was substituted by crude protein from SM. In Experiment 2, 0, 60, 80, and 100% of the crude protein contributed by SBM in broiler diets was replaced by crude protein from SM. Body weight, cumulative feed consumption, and feed conversion (feed:gain) were determined on a weekly basis for 49 d in Experiment 1 and 42 d in Experiment 2. Mortality was reported daily. Carcass weight and percentage yield were determined on a prechilled basis. In Experiment 1, no significant differences were found for body weight, feed consumption, feed conversion, mortality, carcass weight, or yield. In the second experiment, body weight was found to be significantly higher (P < 0.01) at 21, 28, 35, and 42 d of age in treatments in which SM was introduced at a 100% substitution for SBM. Growth responses to SM were also seen at lower levels of substitution at 21, 28, and 35 d. No significant differences were observed for feed consumption, feed conversion, mortality, or carcass yield for any of the treatments. Carcass weight increased significantly by 12.1% when SM was substituted 100% for SBM. Results of the present study show that the particular SM used in this study could partially or totally replace SBM in broiler diets without negatively affecting performance or carcass quality.

Age-associated changes in the content and fatty acid composition of brain glycerophospholipids.

The fatty acid composition of total lipids and individual phospholipids and the ratio of cholesterol-to-phospholipid content were studied in cerebral cortex, subcortical white matter, cerebellum and medulla oblongata/pons in 4-, 21.5- and 28-month-old rats. The cholesterol-to-phospholipid molar ratio in subcortical white matter, medulla oblongata/pons and cerebellum in 28-month-old rats was found to be 17, 17 and 16% higher, respectively, than in adult rats. These alterations in the molar ratio were produced as a result of a net increase in cholesterol content rather than by changes in the total phospholipid content. The content of alkenylacylglycerophosphoethanolamine (alkenylacyl GPE) increased in 28-month-old rats with respect to 4-month-old rats, following the order cerebral cortex > cerebellum > medulla oblongata/pons > subcortical white matter. The fatty acid composition of total lipids showed an increase in monounsaturated fatty acids (MUFA) and a decrease in polyunsaturated fatty acid (PUFA) content with increasing age in all regions studied, such changes being more marked in cerebellum, medulla oblongata/pons and subcortical white matter than in cerebral cortex. The proportion of 22:6(n-3) in cholineglycerophospholipids in the different brain regions of 28-month-old rats showed a slight decrease with respect to that in adult rats following the order cerebellum > medulla oblongata/pons > subcortical white matter, whereas that of 20:4(n-6) decreased only in cerebellum. Ethanolamineglycerophospholipid fatty acid composition was modified in 28-month-old rats through a marked increase in monounsaturated fatty acids (18:1(n-9) and 20:1(n-9) specifically) in all brain areas. The MUFA content of alkenylacyl GPE increased with increasing age in all the regions studied, in the order cerebral cortex > medulla oblongata/pons > subcortical white matter > cerebellum. An increase in the MUFA content of diacylglycerophosphoethanolamine was observed in medulla oblongata/pons and cerebellum of aged rats with respect to control rats. PUFA content of alkenylacyl GPE decreased mainly in medulla oblongata/pons, cerebral cortex and subcortical white matter of aged rats. The PUFA content of serineglycerophospholipids was the most affected by aging. Changes occurred mainly in the content of 22:6(n-3), 22:4(n-6) and 20:4(n-6) in cerebellum and of 22:6(n-3) and 22:4(n-6) in subcortical white matter. Changes in the inositolglycerophospholipid fatty acid content of 28-month-old rats were observed mainly in medulla oblongata/pons, which showed a decrease in PUFA (22:4(n-6) and 22:6(n-3)) content and an increase in MUFA (18:1 and 20:1).