Tracey E. Madgett

The BloodGen project: toward mass-scale comprehensive genotyping of blood donors in the European Union and beyond.

Neil D. Avent, Antonio Martinez, Willy A. Flegel, Martin L. Olsson, Marion L. Scott, Núria Nogués, Martin Písǎcka, Geoff Daniels, Ellen van der Schoot, Eduardo Muñiz-Diaz, Tracey E. Madgett, Jill R. Storry, Sigrid H. Beiboer, Petra A. Maaskant-van Wijk, Inge von Zabern, Elisa Jiménez, Diego Tejedor, Mónica López, Emma Camacho, Goedele Cheroutre, Anita Hacker, Pavel Jinoch, Irena Svobodova, and Masja de Haas

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 36.7% increase, respectively, in comparison with that of a bare printed graphene or reduced graphene oxide electrode. The good electron transfer activity is attributed to a combination of the large number of electroactive sites in reduced graphene oxide and the high conductivity nature of graphene. The probe ssDNA was further immobilized onto the surface of the reduced graphene oxide-graphene double-layer electrode via π-π bonds and then hybridized with its target cDNA. The change of peak current due to the hybridized dsDNA could be used for quantitative sensing of DNA concentration. It has been demonstrated that a linear range from 10(-7)M to 10(-12)M is achievable for the detection of human immunodeficiency virus 1 gene with a detection limit of 1.58 × 10(-13)M as determined by three times standard deviation of zero DNA concentration.

The Bloodgen Project of the European Union, 2003-2009

The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism.

Molecular biology of Rh proteins and relevance to molecular medicine

The Rhesus (Rh) blood group system is expressed by a pair of 12-transmembrane-domain-containing proteins, the RhCcEe and RhD proteins. RhCcEe and RhD associate as a Rh core complex that comprises one RhD/CcEe protein and most likely two Rh-associated glycoproteins (RhAG) as a trimer. All these Rh proteins are homologous and share this homology with two human non-erythroid proteins, RhBG and RhCG. All Rh protein superfamily members share homology and function in a similar manner to the Mep/Amt ammonium transporters, which are highly conserved in bacteria, plants and invertebrates. Significant advances have been made in our understanding of the structure and function of Rh proteins, as well as in the clinical management of Rh haemolytic disease. This review summarises our current knowledge concerning the molecular biology of Rh proteins and their role in transfusion and pregnancy incompatibility.

The SAFE project: towards non-invasive prenatal diagnosis.

After the revolutionary detection of ffDNA (free fetal DNA) in maternal circulation by real-time PCR in 1997 and advances in molecular techniques, NIPD (non-invasive prenatal diagnosis) is now a clinical reality. Non-invasive diagnosis using ffDNA has been implemented, allowing the detection of paternally inherited alleles, sex-linked conditions and some single-gene disorders and is a viable indicator of predisposition to certain obstetric complications [e.g. PET (pre-eclampsia)]. To date, the major use of ffDNA genotyping in the clinic has been for the non-invasive detection of the pregnancies that are at risk of HDFN (haemolytic disease of the fetus and newborn). This has seen numerous clinical services arising across Europe and many large-scale NIPD genotyping studies taking place using maternal plasma. Because of the interest in performing NIPD and the speed at which the research in this area was developing, the SAFE (Special Non-Invasive Advances in Fetal and Neonatal Evaluation) NoE (Network of Excellence) was founded. The SAFE project was set up to implement routine, cost-effective NIPD and neonatal screening through the creation of long-term partnerships within and beyond the European Community and has played a major role in the standardization of non-invasive RHD genotyping. Other research using ffDNA has focused on the amount of ffDNA present in the maternal circulation, with a view to pre-empting various complications of pregnancy. One of the key areas of interest in the non-invasive arena is the prenatal detection of aneuploid pregnancies, particularly Downs syndrome. Owing to the high maternal DNA background, detection of ffDNA from maternal plasma is very difficult; consequently, research in this area is now more focused on ffRNA to produce new biomarkers.

Post-genomics studies and their application to non-invasive prenatal diagnosis.

Non-invasive prenatal diagnosis (NIPD) offers the opportunity to eliminate completely the risky procedures of amniocentesis and chorionic villus sampling. The development of NIPD tests has largely centred around the isolation and analysis of fetal cells in the maternal circulation and the analysis of free fetal DNA in maternal plasma. Both of these techniques offer difficult technical challenges, and at the current moment in time the use of free fetal DNA is the simplest and most effective method of defining paternally inherited fetal genes for diagnosis. Post-genomics technologies that explore the proteins (proteomics) and transcripts (transcriptomics) released by the placenta into the maternal circulation offer new opportunities to identify genes and their protein products that are key diagnostic markers of disease (in particular Down syndrome), and might replace the current screening markers in use for prediction of risk of Down syndrome. In the ideal situation, these markers are sufficiently diagnostic not to require invasive sampling of fetal genetic material. Post-genomics techniques might also offer better opportunities for defining fetal cell-specific markers that might enhance their isolation from maternal blood samples. This review describes progress in these studies, particularly those funded by the Special Non-invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence.

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

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. We developed and evaluated a multiplex ligation–dependent probe amplification (MLPA) assay to determine clinically relevant RHD and RHCE variant alleles and RHD zygosity.

Cell-free fetal DNA in the maternal serum and plasma: current and evolving applications

Purpose of review Free fetal nucleic acids, found in the plasma of every pregnant woman, have made a substantial impact on prenatal diagnosis. The past decade has seen the introduction of routine noninvasive prenatal diagnosis (NIPD) using DNA extracted from maternal plasma for a number of clinical complications of pregnancy, notably feto-maternal blood group incompatibility, fetal sexing and exclusion/detection of single-gene disorders. It appears that mass-scale analysis of all RhD-negative pregnant women will be adopted to conserve stocks of prophylactic anti-D and avoid the administration of a blood product unnecessarily. For the majority of prenatal diagnostic procedures, the assessment of trisomy, particularly trisomy 21, is the highest priority. Because RHD genotyping, fetal sexing and analysis of single-gene disorders all depend on the detection of paternally inherited alleles, they were relatively simple to adapt on the basis of PCR analysis of DNA obtained from maternal plasma. However, for assessment of chromosome copy number, this is not so straightforward. Recent findings The assessment of polymorphisms among placentally expressed mRNAs found in maternal plasma has enabled the detection of trisomy 21 fetuses using a combination of reverse transcriptase PCR and mass spectrometry to define allelic ratios of maternally and paternally inherited single nucleotide polymorphisms. Interesting recent developments also include the finding that direct sequence analysis of maternal plasma extracted DNA using ‘next-generation’ DNA sequencers can differentiate between normal and trisomy fetuses. Summary NIPD using nucleic acids obtained from maternal plasma and serum is now a clinical reality, particularly in the management of hemolytic disease of the fetus and newborn. Recent advances signal that NIPD for common aneuploidies will soon be possible.

4.1R-deficient human red blood cells have altered phosphatidylserine exposure pathways and are deficient in CD44 and CD47 glycoproteins

Phosphatidylserine exposure on the surface of the red cell membrane initiates the process of eryptosis, the red cell death program. The 4.1R protein is a phosphatidylserine binding protein. In this article, the authors demonstrate that erythrocytes from two patients with 4.1R deficiency show alterations of other proteins of the 4.1 multicomplex such as CD44 and CD47 and significantly increased phosphatidylserine exposure, suggesting a role for 4.1 protein in a signaling pathway relevant for red cell turnover. Background Protein 4.1R is an important component of the red cell membrane skeleton. It imparts structural integrity and has transmembrane signaling roles by direct interactions with transmembrane proteins and other membrane skeletal components, notably p55 and calmodulin. Design and Methods Spontaneous and ligation-induced phosphatidylserine exposure on erythrocytes from two patients with 4.1R deficiency were studied, using CD47 glycoprotein and glycophorin C as ligands. We also looked for protein abnormalities in the 4.1R - based multiprotein complex. Results Phosphatidylserine exposure was significantly increased in 4.1R-deficient erythrocytes obtained from the two different individuals when ligands to CD47 glycoprotein were bound. Spontaneous phosphatidylserine exposure was normal. 4.1R, glycophorin C and p55 were missing or sharply reduced. Furthermore there was an alteration or deficiency of CD47 glycoprotein and a lack of CD44 glycoprotein. Based on a recent study in 4.1R-deficient mice, we found that there are clear functional differences between interactions of human red cell 4.1R and its murine counterpart. Conclusions Glycophorin C is known to bind 4.1R, and we have defined previously that it also binds CD47. From our evidence, we suggest that 4.1R plays a role in the phosphatidylserine exposure signaling pathway that is of fundamental importance in red cell turnover. The linkage of CD44 to 4.1R may be relevant to this process.

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). Using Y-specific and RHD-specific targets, we investigated variation in the cell-free fetal DNA (cffDNA) fraction and determined the sensitivity achieved for optimal and suboptimal samples with a novel Droplet Digital™ PCR (ddPCR) platform compared with real-time quantitative PCR (qPCR). RESULTS The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes (P < 0.001). In samples expressing optimal cffDNA fractions (≥4%), both qPCR and digital PCR (dPCR) showed 100% sensitivity for the TSPY1 (testis-specific protein, Y-linked 1) and RHD7 (RHD exon 7) assays. Although dPCR also had 100% sensitivity for RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions (<2%), dPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 (multicopy target) assay. CONCLUSIONS qPCR was not found to be an effective tool for RHD genotyping in suboptimal samples (<2% cffDNA). However, when testing the same suboptimal samples on the same day by dPCR, 100% sensitivity was achieved for both fetal sex determination and RHD genotyping. Use of dPCR for identification of fetal specific markers can reduce the occurrence of false-negative and inconclusive results, particularly when samples express high levels of background maternal cell-free DNA.