RHCE genotyping using next generation sequencing: Allele specific reference sequences.

Background: The Rh blood group system (ISBT004) is encoded by two homologous genes, RHD and RHCE. Polymorphism in these two genes gives rise to 56 antigens, which are highly immunogenic and clinically significant. This study extended previous work on the establishment of RHD allele specific reference sequences using next generation sequencing (NGS) with the Ion Personal Genome Machine (Ion PGM) to sequence the complete RHCE gene.

Clinical Validation of a Prenatal Cell-Free DNA Screening Test for Fetal RHD in a Large U.S. Cohort.

Objective: To present a large U.S. clinical validation of a next-generation sequencing-based, noninvasive prenatal cell-free DNA test for fetal RHD .

Rh Blood Group D Antigen Genotyping Using a Portable Nanopore-based Sequencing Device: Proof of Principle.

Background: Nanopore sequencing is direct sequencing of a single-stranded DNA molecule using biological pores. A portable nanopore-based sequencing device from Oxford Nanopore Technologies (MinION) depends on driving a DNA molecule through nanopores embedded in a membrane using a voltage. Changes in current are then measured by a sensor, thousands of times per second and translated to nucleobases.

Next-generation sequencing of 35 RHD variants in 16 253 serologically D- pregnant women in the Finnish population.

Fetal RHD screening for targeted routine antenatal anti-D prophylaxis has been implemented in many countries, including Finland, since the 2010s. Comprehensive knowledge of the RHD polymorphism in the population is essential for the performance and safety of the anti-D prophylaxis program. During the first 3 years of the national screening program in Finland, over 16 000 samples from RhD- women were screened for fetal RHD; among them, 79 samples (0.

Complete next-generation sequencing: establishment of reference alleles.

The Rh blood group system (ISBT004) is the second most important blood group after ABO and is the most polymorphic one, with 55 antigens encoded by 2 genes, and This research uses next-generation sequencing (NGS) to sequence the complete gene by amplifying the whole gene using overlapping long-range polymerase chain reaction (LR-PCR) amplicons. The aim was to study different alleles present in the population to establish reference allele sequences by using the analysis of intronic single-nucleotide polymorphisms (SNPs) and their correlation to a specific Rh haplotype. Genomic DNA samples (n = 69) from blood donors of different serologically predicted genotypes including RR (DCe/DCe), RR (DcE/DcE), RR (DCe/DcE), RR (DcE/DCE), Rr (DCe/dce), Rr (DcE/dce), and Rr (Dce/dce) were sequenced and data were then mapped to the human genome reference sequence hg38.

Red cell genotyping precision medicine: a conference summary.

This review summarizes the salient points of the symposium 'Red Cell Genotyping 2015: Precision Medicine' held on 10 September 2015 in the Masur Auditorium of the National Institutes of Health. The specific aims of this 6th annual symposium were to: (1) discuss how advances in molecular immunohematology are changing patient care; (2) exemplify patient care strategies by case reports (clinical vignettes); (3) review the basic molecular studies and their current implications in clinical practice; (4) identify red cell genotyping strategies to prevent alloimmunization; and (5) compare and contrast future options of red cell genotyping in precision transfusion medicine. This symposium summary captured the state of the art of red cell genotyping and its contribution to the practice of precision medicine.

Rapid Zygosity Determination Using Digital PCR.

Background: Paternal zygosity testing is used for determining homo- or hemizygosity of in pregnancies that are at a risk of hemolytic disease of the fetus and newborn. At present, this is achieved by using real-time PCR or the PCR, which can be difficult to interpret and unreliable, particularly for black African populations.

Methods: DNA samples extracted from 53 blood donors were analyzed using 2 multiplex reactions for -specific targets against a reference () to determine gene dosage by digital PCR.

Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR.

Background: Noninvasive genotyping of fetal RHD (Rh blood group, D antigen) can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. We evaluated laboratory methods for such genotyping.

Methods: Blood samples were collected in EDTA tubes and Streck® Cell-Free DNA™ blood collection tubes (Streck BCTs) from RHD-negative women (n = 46).

Graphene electrode modified with electrochemically reduced graphene oxide for label-free DNA detection.

A novel printed graphene electrode modified with electrochemically reduced graphene oxide was developed for the detection of a specific oligonucleotide sequence. The graphene oxide was immobilized onto the surface of a graphene electrode via π-π bonds and electrochemical reduction of graphene oxide was achieved by cyclic voltammetry. A much higher redox current was observed from the reduced graphene oxide-graphene double-layer electrode, a 42% and 36.

Prenatal testing for hemolytic disease of the newborn and fetal neonatal alloimmune thrombocytopenia - current status.

Incompatibility of red cell and platelet antigens can lead to maternal alloimmunization causing hemolytic disease of the fetus & newborn and fetal neonatal alloimmune thrombocytopenia respectively. As the molecular background of these polymorphisms emerged, prenatal testing using initially fetal DNA obtained from invasively obtained amniotic fluid or chorionic villus was implemented. This evolved into testing using maternal plasma as source of fetal DNA, and this is in routine use as a safe non-invasive diagnostic that has no risk to the fetus of alloimmunization or spontaneous miscarriage.

Non invasive prenatal diagnosis of aneuploidy: next generation sequencing or fetal DNA enrichment?

Current invasive procedures [amniocentesis and chorionic villus sampling (CVS)] pose a risk to mother and fetus and such diagnostic procedures are available only to high risk pregnancies limiting aneuploidy detection rate. This review seeks to highlight the necessity of investing in non invasive prenatal diagnosis (NIPD) and how NIPD would improve patient safety and detection rate as well as allowing detection earlier in pregnancy. Non invasive prenatal diagnosis can take either a proteomics approach or nucleic acid-based approach; this review focuses on the latter.

Maternal plasma biomarkers for down syndrome: present and future.

Down syndrome is the most common cause of mental retardation and has an incidence of between 1:600 and 1:800 pregnancies. It is the condition for which prenatal diagnosis is requested the most and most developed countries have adopted a screening program based around maternal plasma/serum testing and ultrasound. Advances have been made recently to eliminate invasive testing for genetic diagnosis of this condition based on the analysis of free fetal DNA in maternal plasma.

Non-Invasive Screening Tools for Down's Syndrome: A Review.

Down's syndrome (DS) is the most common genetic cause of developmental delay with an incidence of 1 in 800 live births, and is the predominant reason why women choose to undergo invasive prenatal diagnosis. However, as invasive tests are associated with around a 1% risk of miscarriage new non-invasive tests have been long sought after. Recently, the most promising approach for non-invasive prenatal diagnosis (NIPD) has been provided by the introduction of next generation sequencing (NGS) technologies.

RHD and RHCE variant and zygosity genotyping via multiplex ligation-dependent probe amplification.

Background: The presence of a D variant may hamper correct serologic D typing, which may result in D immunization. D variants can be determined via RHD genotyping. However, a convenient single assay to identify D variants is still lacking.

The development of a peptide SRM-based tandem mass spectrometry assay for prenatal screening of Down syndrome.

Two new biomarkers, serum amyloid-P (SAP) and plasma C1-inhibitor protein are elevated in the maternal circulation of mothers carrying Down syndrome foetuses. Much emphasis of late\ has been put on the lack of translational tests being developed following the identification of new biomarkers. We have created a single-reaction-monitoring (SRM) tandem mass spectrometry-based assay for the quantitation of these biomarkers and compared these results with an in-house developed immunofluorescence-based technique (IF).

Identification of new biomarkers for Down's syndrome in maternal plasma.

Using ProteinChip Technology (SELDI TOF MS), the maternal plasma of 53 chromosomally-normal control and 28 Down's syndrome affected pregnancies was profiled between 10 and 20 weeks' gestation. Preliminary studies demonstrated two distinct phases of changes in protein expression, the first at 10-14 weeks and second at 14-20 weeks. Using this data, analysis of the 10-14 weeks' plasma samples (Down's syndrome n=13, control n=20) showed the presence of a protein of mass 100.

2D DIGE analysis of maternal plasma for potential biomarkers of Down Syndrome.

Background: Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results: We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers.

Quantification of free fetal DNA in multiple pregnancies and relationship with chorionicity.

Objective: Free fetal DNA (ffDNA) in the maternal plasma appears to originate mainly from the trophoblast. We tested the hypothesis that ffDNA concentration is increased in multiple pregnancies where trophoblastic mass has been shown to be increased.

Methods: Quantitative real-time PCR was used to measure the plasma concentration of DYS14 in singleton and twin pregnancies with one or two male fetuses.

Transfusion medicine education session.

The European Haematology Association annual meeting is the second largest conference organized specifically for hematologists. This meeting report gives an overview of one particular session within this meeting, that of the transfusion medicine subject speciality, which included two speakers from the USA and one from the UK. The following lectures will be reviewed here: Jo-Ann Moulds--Transfusion policies and practices in alloimmunized sickle cell patients; Derwood Pamphilon--Transfusion problems in stem cell transplant patients; and Naomi Luban--Transfusion issues in neonatal and pediatric hematology.

Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells.

Aims/hypothesis: This study used proteomics and biochemical approaches to identify novel glucose-regulated proteins and to unveil their role in pancreatic beta cell function. Translationally controlled tumour protein (TCTP) was identified to be one such protein, and further investigations into its function and regulation were carried out.

Methods: Global protein profiling of beta cell homogenates following glucose stimulation was performed using two-dimensional gel electrophoresis.

Toxicity testing: the search for an in vitro alternative to animal testing.

Prior to introduction to the clinic, pharmaceuticals must undergo rigorous toxicity testing to ensure their safety. Traditionally, this has been achieved using in vivo animal models. However, besides ethical reasons, there is a continual drive to reduce the number of animals used for this purpose due to concerns such as the lack of concordance seen between animal models and toxic effects in humans.