Genghis H. Lopez

Evaluation of targeted exome sequencing for 28 protein‐based blood group systems, including the homologous gene systems, for blood group genotyping

Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next‐generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS.

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

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.

Molecular typing for the Indian blood group associated 252G>C single nucleotide polymorphism in a selected cohort of Australian blood donors.

BACKGROUND The Indian blood group antigens, In(a) and In(b), are clinically significant in transfusion medicine. However, antisera to type these antigens are difficult to obtain. The In(b) antigen is a high frequency antigen present in all populations, while the frequency of the antithetical In(a) ranges from 0.1% in Caucasians up to 11% in Middle Eastern groups. This antigen polymorphism is encoded by the single nucleotide polymorphism (SNP) 252G>C in CD44. The aim of this study was to establish and compare two genotyping methods to measure the frequency of the IN*A and IN*B alleles in a blood donor cohort. MATERIALS AND METHODS Donor blood samples (n=151) were genotyped by a novel real-time polymerase chain reaction (PCR) high-resolution meltcurve (HRM) analysis and a custom matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) assay. Samples with the rare IN*A allele were further investigated by nucleotide sequencing, red cell agglutination, and flow cytometry techniques. RESULTS In this study group, 149 IN*B homozygous and 2 IN*A/B heterozygous samples were detected with 100% concordance between HRM and MALDI-TOF MS methods. For PCR HRM, amplicon melting alone did not differentiate IN*A and IN*B alleles (class 3 SNP), however, the introduction of an unlabelled probe (UP) increased the resolution of the assay. Sequencing confirmed that the two non-homozygous samples were IN*A/B heterozygous and phenotyping by red cell agglutination, and flow cytometry confirmed both In(a) and In(b) antigens were present as predicted. DISCUSSION Genotyping permits conservation of rare antisera to predict blood group antigen phenotype. In PCR UP-HRM the IN*A and IN*B alleles were discriminated on the basis of their melting properties. The In(a) frequency in this selected donor population was 1.3%. Application of genotyping methods such as these assists in identifying donors with rare blood group phenotypes of potential clinical significance.

A D+ blood donor with a novel RHD*D‐CE(5‐6)‐D gene variant exhibits the low‐frequency antigen RH23 (DW) characteristic of the partial DVa phenotype

Blood donors whose red blood cells (RBCs) exhibit a partial RhD phenotype, lacking some D epitopes, present as D+ in routine screening. Such phenotypes can exhibit low‐frequency antigens (LFAs) of clinical significance. The aim of this study was to describe the serologic and genetic profile for a blood donor with an apparent D+ phenotype carrying a variant RHD gene where D Exons 5 and 6 are replaced by RHCE Exon (5‐6).

Targeted exome sequencing defines novel and rare variants in complex blood group serology cases for a red blood cell reference laboratory setting

We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single‐nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP‐typing cases.

GYP*Kip, a novel GYP(B‐A‐B) hybrid allele, encoding the MNS48 (KIPP) antigen

G P(B-A-B) hybrid glycophorins, designated GP.HF, GP.Mur, GP.Bun, and GP.Hop, arise from insertion of a homologous segment from the Exon 3 region of the GYPA gene into the GYPB gene, including the 50 end of Intron 3. This insertion repairs the inactive splice site (that makes the Exon 3 region of the GYPB gene a pseudoexon) and leads to the generation and expression of characteristic profiles of novel antigens. A novel red blood cell serologic profile for a GP(B-AB) hybrid (positive with anti-Mur, -Hil, -MINY, -MUT, and -Hop1Nob) was reported by the International Blood Group Reference Laboratory (IBGRL). It was postulated that this was a new GP(B-A-B) hybrid with a proposed designation GP.Kip after the German propositus. In 1992, the IBGRL reported detection of an identical serologic pattern for an Australian blood donor. The genetic bases of four GP(B-A-B) MNS system hybrid glycophorins (GP.HF, GP.Mur, GP.Hop, and GP.Bun) that result from rearrangement between the homologous GYPA and GYPB genes are well characterized. This report describes a new GYP(B-A-B) hybrid allele from the Australian blood donor reported with the GP.Kip antigen profile.

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

Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fya/Fyb polymorphism, for weak Fyb 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.

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

An Australian Caucasian blood donor consistently presented a serology profile for the Duffy blood group as Fy(a+b+) with Fya 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.

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.

An alloantibody in a homozygous GYP*Mur individual defines JENU (MNS49), a new high-frequency antigen on glycophorin B.

R ed blood cell (RBC) antigens present in more than 90% of a population are classified as highfrequency antigens. Nine of these are in the MNS blood group system with two antigens, ‘N’ (MNS30) and U (MNS5), on glycophorin B (GPB). Hybrid glycophorins such as GYP(B-A-B) are formed by gene conversion events between homologous genes GYPA and GYPB. Gene conversion resulting in GYPA Exon 3 insertion into the 30 end of GYPB Pseudoexon 3 forms a composite exon with a repaired splice donor site (that is inactive in GYPB) and, depending on the crossover sites, encodes various GP(B-A-B) hybrid glycophorins: GP.HF, GP.Mur, GP.Bun, GP.Hop, and GP.Kip. A patient with thalassemia of Thai ethnicity was transfused with 2 RBC units to correct anemia. Posttransfusion, the patient developed multiple alloantibodies including a pan-reactive alloantibody of unknown specificity. A total of 3600 E–c–Jk–S– RBC units were screened and 2 compatible units were found. We present typing data for the patient and these compatible donors as well as epitope mapping defining this pan-reactive antibody.