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.

Using criticisms to elicit and meet unit-based learning needs.

A strategy using staff nurses' criticisms is described, which may be a useful addition to the repertoire of strategies used by staff development educators to elicit and meet unit-based learning needs. It is also argued that this strategy has the potential to equip staff nurses to move more independently from criticism to problem setting and problem solving.

Serologic and molecular investigations of a chimera.

A chimeric individual possesses two or more genetically distinct cell populations. Although the chimerism may not be evident in all gene systems, various loci display greater numbers of alleles than genetically "normal" individuals. The proposita was referred for further laboratory investigation due to a mixed-field ABO blood group reaction following routine antenatal testing. Various molecular (HLA class II, ABO genotyping, and 10 short tandem repeat [STR] microsatellites) and serologic (HLA class I and red cell blood groups) typing techniques were employed to investigate a number of polymorphic loci located on different chromosomes. Chimerism was identified in 8 out of the 14 chromosomes tested: chromosome 1 (Duffy), 6 (HLA class I and II), 9 (ABO), 11 (HUMTH01), 12 (HUMPLA2A1), 15 (HUMFES/FPS), 18 (Kidd) and 21 (D21S11). The proposita was determined to be a probable dispermic chimera, based on the results of the serology and molecular studies.

ABO genotyping by polymerase chain reaction-restriction fragment length polymorphism.

Genotyping enables the identification of both maternally and paternally derived alleles. A number of protocols have been described for the genotyping of the ABO blood group system. Generally, these methods have a number of disadvantages including the use of hazardous reagents, being technically demanding, and the excessive use of materials. In this study, a relatively simple polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) method is described. Four different amplifications were used that were specific for nucleotides sites 261, 526, 703, and 796 to distinguish the A, B, O1 and O2 alleles. The ABO genotypes of 294 random individuals were determined and were found to completely correlate with the serologic phenotypes. The protocol is applicable for investigations of weak or nonexpression of ABO alleles, paternity determinations, and population analysis.

ABO genotyping-identification of O1, O1*, and O2 alleles using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique.

ABO polymorphism at the gene level has been investigated by molecular methods, predominantly sequencing and restriction fragment length polymorphism (RFLP). We describe the application of the polymerase chain reaction-sequence specific oligonucleotide (PCRSSO) method, which is considered to be more versatile for large sample numbers, compared with conventional ABO genotyping by PCR-RFLP. PCR-SSO, while maintaining accurate and reliable results, reduces costs and labor. A population of 155 random individuals was investigated for the three O alleles, O1, O1*, and O2. The allelic frequencies were 35 percent, 26 percent, and 5 percent, respectively. PCR-SSO results correlated completely with both serologic and PCRRFLP results.

A family exhibiting the cis-AB genotype.

Studies on blood samples from a 30-year-old woman of Korean origin, her husband and children, showed the presence of the cis-AB genotype in the mother and one child. The cis-AB red cells showed abnormal reactions with both polyclonal and monoclonal anti-B reagents and an elevated level of H antigen. The serum contained weak anti-B that reacted with normal B antigen but not with that on cis-AB cells. Normal levels of A substance but reduced levels of B and H substances were present in the saliva. Conversely, the serum showed reduced A and normal B and H blood group transferase activity.