BACKGROUND: 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. STUDY DESIGN AND METHODS: Donor samples(n 5 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. RESULTS:The average sequencing depth per target region was 66.2 6 39.8. Each sample harbored on average 43 6 9 variants, of which 10 6 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. CONCLUSION:The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting.H uman blood group antigens are of significance in transfusion medicine, because patients who have made antibodies to red blood cell antigens are at risk of being affected by hemolytic transfusion reactions after the transfusion of incompatible blood. The International Society of Blood Transfusion has defined 36 blood group systems and over 350 blood group antigens.1 Different blood group systems exhibit varying degrees of antigen polymorphism, and the clinical significance of red blood cell antibodies also varies. [2][3][4] As a minimum requirement in blood transfusion safety, all blood donors are screened for ABO and the D antigen as well as for blood group antibodies known to be clinically significant. 5The majority of antigens are missense mutations and a consequence of single nucleotide variants (SNVs); however, genetic variations, such as insertions/deletions and splice-site variants, have a qualitative and/or quantitative impact on antigen expression. Blood group systems, such as RH and MNS, exhibit an additional layer of genetic variation. These arise because each system comprises homologous genes in which gene crossover or g...

Targeted exome sequencing resolved complex serology problems and defined both novel blood group alleles (CD55:c.203G>A, ABCB6:c.1118_1124delCGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) and rare variants on blood group alleles associated with altered phenotypes. This study illustrates the utility of exome sequencing, in conjunction with serology, as an alternative approach to resolve complex cases.

ABSTRACT. In this communication we describe for the first time the isolation of 3 iridoviruses from the toad Bufo marinus and an unknown species of frog Leptodactylus in Venezuela, South America. The viruses are icosahedral with electron-dense cores, each of which IS surrounded by an inner membrane, capsid and a cell-derived envelope. The virus(es) have an average vertex to vertex diameter of 160 nm and replicate in the cytoplasm of a range of cell lines. Wlthin the cytoplasm of infected cells, ra~ified areas could be observed; structures lacked cellular organelles dnd contained complete, empty and developing viruses. Results from antigen-capture enzyme-linked immunosorbent assays (ELISA) with polyclonal antibody raised against epizootic haematopoietic necrosis virus (EHNV) indicated crossreactivity between these isolates, Bohle iridovirus (BIV) and frog virus 3 (FV3). Comparison of polypeptide and genomic profiles indicated that the Venezuelan viruses shared many polypeptides of equivalent molecular weight with type species FV3. There were, however, differences between the group of Venezuelan viruses and FV3 and BIV The viruses belongs to the family Iridoviridae and the genus Ranavirus.

Dose-intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34+ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum-free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800-fold expansion in cell number was achieved for UCB, and up to 4,000-fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10-fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave-type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients.

A serological survey was conducted for antibodies against 'ranavlruses' in the giant toad Bufo marinus in Australia and Venezuela. Sera contaming antibodies against 'ranaviruses' were found in both countries. In Australia positive antibodies were identified in populations throughout most of the known range of B. rnarinus. Results were confirmed by immunofluorescence and immunoelectron microscopy where a characteristic stalning pattern of 'ranaviruses' in infected cells was observed. Whilst a 'ranavirus(es)' has been isolated from populations of B. rnarinus in Venezuela, no virus has been isolated from Australian B. marinus populations. The significance of 'ranavirus' sero-positive B. marinus in Australia is discussed. KEY WORDS. Giant toads .

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The genetic basis for five GP(B-A-B) MNS system hybrid glycophorin blood group antigens results from rearrangement between the homologous GYPA and GYPB genes. Each hybrid glycophorin displays a characteristic profile of antigens. Currently, no commercial serological reagents are currently available to serologically type for these antigens. The aim of this study was to develop a single nucleotide polymorphism (SNP) mapping genotyping technique to allow characterisation of various GYP(B-A-B) hybrid alleles. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) assays were designed to genotype five GYP(B-A-B) hybrid alleles. Eight nucleotide positions were targeted and incorporated into the SNP mapping protocol. The allelic frequencies were calculated using peak areas. Sanger sequencing was performed to resolve a GYP*Hop 3' breakpoint. Observed allelic peak area ratios either coincided with the expected ratio or were skewed (above or below) from the expected ratio with switching occurring at and after the expected break point to generate characteristic mass spectral plots for each hybrid. Sequencing showed that the GYP*Hop crossover in the intron 3 region, for this example, was identical to that for GYP*Bun reference sequence. An analytical algorithm using MALDI-TOF MS genotyping platform defined GYPA inserts for five GYP(B-A-B) hybrids. The SNP mapping technique described here demonstrates proof of concept that this technology is viable for genotyping hybrid glycophorins, GYP(A-B-A), GYP(A-B) and GYP(B-A), and addresses the gap in current typing technologies.

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.