Tracey E Madgett

University of Plymouth, Plymouth, England, United Kingdom

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Publications (17)45.85 Total impact

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    ABSTRACT: 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. Copyright © 2015 Elsevier B.V. All rights reserved.
    Biosensors & Bioelectronics 05/2015; 72:313-319. DOI:10.1016/j.bios.2015.05.034 · 6.45 Impact Factor
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    ABSTRACT: Blood group genotyping (BGG) has been in routine clinical practice ever since the molecular determination of blood groups was achieved, during the early to mid 1990s. These early methods were dependent on allele-specific PCR to detect simple single nucleotide polymorphisms (SNPs) responsible for blood group expression. As our knowledge regarding the molecular background of blood groups advanced, so did the numbers of SNPs requiring detection which necessitated the switch from allele-specific PCR to array-based technology, notably slide and bead-based approaches. All these methods thus described are totally dependent on the predefined genetic basis of each SNP. Hybrid blood group genes (as found in RH, ABO and MNS systems) are difficult to define by all of these aforementioned techniques. With the arrival of cheap next-generation sequencing (NGS) approaches in the past 5 years, we have conducted long-range PCR (LR-PCR) coupled with NGS determination of the major blood group genes. By analysis of these data using an Ion Torrent Personal Genome Machine™ (PGM™), it is readily apparent that NGS can highly effectively be applied with high resolution to blood grouping at costs no more than current array-based platforms. However, the complexity of the data obtained need careful filtering for effective clinical utilization, but provides useful insight on the evolution of blood groups and their environmental impacts which will be of undoubted value as an academic exercise, but of minimal cost (if any) to the original testing.
    ISBT Science Series 04/2015; 10(S1). DOI:10.1111/voxs.12148
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    ABSTRACT: School of Biomedical and Healthcare Sciences, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, United Kingdom The determination of the blood groups present in an individual attains a great clini- cal importance for the purposes of blood transfusion and transplantation. Blood group genotyping (BGG) has become well established in transfusion medicine. How- ever, all current technologies are based on pre-defined knowledge of known poly- morphisms. The approach of Next Generation Sequencing (NGS) circumvents this requirement and adopts a discovery mode, which is important, as almost every new BGG project discovers new alleles. NGS is capable of producing high-throughput, rapid and accurate data that results in extensive and detailed genotyping. Also, costs have considerably reduced in the past years. In this pilot study, Ion Torrent Personal Genome Machine (PGMTM) sequencer was used to optimise and develop a reliable protocol for sequencing the entire Duffy (DARC), Kidd (SLC14A1) and ABO blood group genes including flanking regions. First, a DNA library was prepared from 12 randomly selected DNA samples. DARC, SLC14A1 and ABO genes were targeted by long-range PCR, enzymatic amplicon fragmentation before ligation with barcoded adapters and size selection. Templates were immobilised on beads, then clonally amplified using emulsion PCR. Then, up to 20 samples from FY and JK samples were selected with certain serology for sequenc- ing. The reason for this is to seek the possibility that those serologically typed as negative for a particular blood group are actually genotypically weak positive. Sequencing revealed millions of reads with great coverage depth that were then aligned to the reference gene sequences. Variants were analysed and visualised with software packages, Ion Torrent SuiteTM plugins, CLC Genomic Workbench, Integrative Genomics Viewer (IGV) and SeattleSeq Annotation 138 website. Initial bioinformat- ics analysis of samples for the DARC and SLC14A1 genes revealed various single nucleotide polymorphisms (SNPs) in exons encoding for amino acid changes, such as (Gly42Asp and Ala100Thr in DARC) and (Asp280Asn, Ala270Ala and Glu44Lys in SLC14A1). In the FYsamples sequenced, a significant number (5/12) encoded the Ala100Thr mutation. In addition, the Allele JK ⃰ 01W.01 (associated with Jka+w) was found with a frequency of 8%. A great number of polymorphisms (SNPs and Indel) were found in introns in JK samples (ranging from 52 to122) and in FY samples (ranging from 2 to 4). An exam- ple of one of these SNPs is chromosomal position 43319274 in the SLC14A1 gene, close to the splice site region of exon 8. Interestingly, sets of intronic polymor- phisms present differently among samples with same phenotype. Therefore, the in- tronic polymorphisms are possibly unique to individuals or families. The ABO sequencing is in progress. We suggest that NGS shows the capability of the comprehensive sequencing of blood group genes and will supplant other genotyping platforms in the near future, becoming the potential methodology of choice for genotyping patients and donors.
    33rd International Congress of the International Society of Blood Transfusion, Seoul, South Korea; 05/2014
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    ABSTRACT: The Rh blood group system is considered as the most polymorphic blood group sys- tem and contains many variants. These variants may cause severe complications to patients, due to alloimmunisation from mismatching products of blood transfusion. Next-generation sequencing (NGS) is an intensely powerful technique capable of sequencing huge regions of the human genome. Here we sequenced the entire RHD and RHCE genes to genotype for the RhD and RhCE antigens, in order to provide a safer transfusion practice. DNA was extracted from random blood donors with known serology. By using long-range PCR (LR-PCR), four and three primer pairs were designed (giving PCR products in the range from 8 to 24 Kb) for RHD and RHCE, respectively. The sequencing libraries were then made by fragmenting the amplicons and ligating to adaptors using Ion XpressTM Plus Fragment Library Kit. Size selection was performed by SPRIselect magnetic beads. After that, the sequenc- ing template was immobilised to beads, which possess a complementary strand to the adaptors, for clonal amplification using emulsion PCR. Finally, the sequencing template was loaded onto a 316 chip and sequenced on the Ion Torrent Personal Genome MachineTM Sequencer. Millions of reads were generated and the data were analysed with CLC Genomics Workbench (Version 6.5). Sanger sequencing will be utilised in order to validate any variants from the sequencing data. NGS using the LR-PCR approach offers a crucial method assisting users to genotype many samples in a single run for detecting the Rh variants in depth. This will be extremely worth- while to genotype the difficult alleles of Rh, especially hybrid genes, and will pave the way for the discovery of novel alleles. NGS for blood group alleles may repre- sent a viable alternative to array and bead based platforms, and it is no more expen- sive and technically challenging on a per sample basis.
    33rd International Congress of the International Society of Blood Transfusion, Seoul, South Korea; 05/2014
  • British Blood transfusion Society Annual Conference 2013, Birmingham; 10/2013
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    British Blood transfusion Society Annual Conference 2013, Birmingham; 10/2013
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    ABSTRACT: 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. We developed and evaluated a multiplex ligation-dependent probe amplification (MLPA) assay to determine clinically relevant RHD and RHCE variant alleles and RHD zygosity. Study design and methodsWe analyzed 236 cases (73 normal and 163 selected samples) with the RH-MLPA assay, which is able to determine 79 RHD and 17 RHCE variant alleles and RHD zygosity. To confirm the results, mutations were verified by RHD and/or RHCE exon-specific sequencing and RHD zygosity was verified by quantitative real-time polymerase chain reaction (PCR) for 18 cases. ResultsIn 99% of the cases, the RH-MLPA assay correctly determined whether a person carried only wild-type RHD and RHCE alleles (n=69) or (a) variant RHD allele(s) and/or (a) variant RHCE allele(s) (n=164). In only three cases, including two new RHD variant alleles, the variant allele was not identified, due to lack of detecting probes. These were RHD*DCS2, a new partial RHD allele, RHD*525T (Phe175Leu), and a new D- null allele, RHD*443G (Thr148Arg). All RHD (n=175) and RHCE variant alleles (n=79) indicated by the RH-MLPA assay were confirmed by sequencing. RHD zygosity was confirmed by quantitative PCR. Two hematopoietic chimeras were recognized. Conclusion The RH-MLPA genotyping assay is a fast, easy, and reliable method to determine almost all clinically relevant RHD and RHCE variant alleles, RHD zygosity, and RHD+/RHD- chimeras in blood donors, blood recipients, and pregnant women.
    Transfusion 10/2012; 53(7). DOI:10.1111/j.1537-2995.2012.03919.x · 3.57 Impact Factor
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    ABSTRACT: 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). This MS-based assay is a rapid 5 min test and a simple "one pot reaction," requiring only 5μl of plasma. To evaluate the potential of SRM-based quantitation in a clinical setting, SAP and C1-inhibitor were quantitated in 38 normal and Down syndrome affected pregnancies. Plasma SAP levels in the Down's group were significantly raised at 10-14 weeks (p<0.0015) and 14-20 weeks (p<0.0001). Plasma C1-inhibitor levels were also observed significantly elevated in the Down's group (10-14 weeks, p<0.0193, 14-20 weeks, p<0.0001). Analysis using the IF technique did not show any significant elevation of plasma SAP levels or C1-inhibitor levels. This rapid and sensitive assay demonstrates the potential of multiplexed tandem MS-based quantitation of proteins in chemical pathology labs and in a more cost-effective, accurate manner than conventionally used antibody methods.
    Journal of proteomics 04/2012; 75(11):3248-57. DOI:10.1016/j.jprot.2012.03.037 · 3.93 Impact Factor
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    ABSTRACT: 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 100.3 kDa that was elevated in the Down's syndrome group compared to the controls (p<0.002). This protein was further isolated using SAX Q-spin columns and identified using QTOF MS and Western blotting as being plasma protease C1-inhibitor. Analysis of the 14-20 week cohort demonstrated changes in protein expression of three additional proteins. Two of these proteins were found to be up-regulated (serum amyloid P-component, p<0.004 and transthyretin, p<0.006) and complement C3-α chain was observed to be down-regulated (p<0.0005). The identification of these biomarkers in maternal plasma and their potential to improve current Down's syndrome screening are discussed.
    Journal of proteomics 03/2012; 75(9):2621-8. DOI:10.1016/j.jprot.2012.03.007 · 3.93 Impact Factor
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    ABSTRACT: ABSTRACT: 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. We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting. Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.
    Proteome Science 09/2011; 9:56. DOI:10.1186/1477-5956-9-56 · 1.88 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Haematologica 10/2009; 94(10):1354-61. DOI:10.3324/haematol.2009.006585 · 5.87 Impact Factor
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    ABSTRACT: 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. 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. 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.
    Current opinion in obstetrics & gynecology 05/2009; 21(2):175-9. DOI:10.1097/GCO.0b013e3283294798 · 2.37 Impact Factor
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    ABSTRACT: 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 Down's 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.
    Biochemical Society Transactions 05/2009; 37(Pt 2):460-5. DOI:10.1042/BST0370460 · 3.24 Impact Factor
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    ABSTRACT: 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.
    Transfusion Medicine and Hemotherapy 01/2009; 36(3):162-167. DOI:10.1159/000218192 · 2.01 Impact Factor
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    ABSTRACT: 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.
    Seminars in Fetal and Neonatal Medicine 05/2008; 13(2):91-8. DOI:10.1016/j.siny.2007.12.011 · 3.13 Impact Factor
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    Transfusion 08/2007; 47(1 Suppl):40S-6S. DOI:10.1111/j.1537-2995.2007.01309.x · 3.57 Impact Factor
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    ABSTRACT: 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.
    Expert Reviews in Molecular Medicine 02/2006; 8(13):1-20. DOI:10.1017/S1462399406010969 · 5.91 Impact Factor

Publication Stats

193 Citations
45.85 Total Impact Points

Institutions

  • 2012–2015
    • University of Plymouth
      • • Centre for Research in Translational Biomedicine (CRTB)
      • • School of Biological Sciences
      Plymouth, England, United Kingdom
    • University of Amsterdam
      Amsterdamo, North Holland, Netherlands
  • 2006–2011
    • University of the West of England, Bristol
      • Faculty of Health and Life Sciences
      Bristol, England, United Kingdom