2019 research

2019 research


BT Young Scientist and Technology Exhibition 2019

Brigid Lucey

I tend to associate the RDS with BioMedica, with scientific talks, a trade exhibition and with a chance to meet longstanding friends from around the country – it’s always a great couple of days out in an air of calm friendliness. Early in every January the same space is used for theBT Young Scientist and Technology Exhibition. This includes (among other stands and activities) 550 qualifying projects from second level schools, including also a separate exhibition for primary schools, which together showcase a frenetic enthusiasm for science. Each project exhibitor spends
four days minding their project display and eagerly anticipating both the arrival of judges and the public to have a welcome chance to explain their project to them. The young exhibitors bring an entourage of schoolmates, teachers and family and the public viewing days bring thousands more people. Many BT engineers divert from their usual day job to become Redcoats every year for the exhibition; their job is to make sure that the
students are all looked after and that everything runs well – they start each day at 7am and may not finish until 10pm or later.

Overall, the model works so well that it is the biggest exhibition of its kind in Europe, we are told, and it is a model that has been adopted in many other countries worldwide. As an exhibition, it brings out the best in students and develops their skills, not only in science but in communication and it is evident that they are primarily happy just to be there and presenting. It provides students with carte blanche to follow their interest and they only need to have a project ready for the deadline in order to enter the first stage of the competition, which is the qualifying round for the
RDS – all science projects will fit somewhere into one of the four categories provided. As shown below, I chose just three of the entries to describe as examples of health-associated projects, with some apologies for the irresistible bias towards microbiology and/or County Cork!

David Grey and Cillian O’Sullivan, from Scoil Treasa in Kanturk, in Cork, had a (group) project in the Intermediate Category for Biological and Ecological Sciences entitled Reducing toxic heavy metals in the global food chain. Their mission was to reduce levels of cadmium in a wheat crop. Essentially, they explained, cadmium is increased in soils through the use of phosphate fertiliser and is easily absorbed through the root
system of the wheat plant. Chronic cadmium exposure causes adverse effects in humans and animals including their lungs, kidneys and bones. What these students discovered was that Pseudomonas fluorescens could be used to immobilise cadmium, rendering it insoluble and therefore not available to the plant as it takes up water. Their experiment involved the planting of the wheat seeds with a gel inoculum of one of two different control strains of Ps fluorescens and their results (analysis assisted by use of equipment at IT Carlow) showed 30% and 23% respectively of a reduction in cadmium uptake by the plants through the use of strains L228/L321/L111 and F113/MB004. David and Cillian concluded that their method offered a cheap solution to farmers throughout the world and they were awarded First Prize in their category for their efforts.

David Grey and Cillian O’Sullivan, from Scoil Treasa in Kanturk, in Cork, had a (group) project in the Intermediate Category for Biological and Ecological Sciences entitled Reducing toxic heavy metals in the global food chain. Their mission was to reduce levels of cadmium in a wheat crop. Essentially, they explained, cadmium is increased in soils through the use of phosphate fertiliser and is easily absorbed through the root
system of the wheat plant. Chronic cadmium exposure causes adverse effects in humans and animals including their lungs, kidneys and bones. What these students discovered was that Pseudomonas fluorescens could be used to immobilise cadmium, rendering it insoluble and therefore not available to the plant as it takes up water. Their experiment involved the planting of the wheat seeds with a gel inoculum of one of two different control strains of Ps fluorescens and their results (analysis assisted by use of equipment at IT Carlow) showed 30% and 23% respectively of a reduction in cadmium uptake by the plants through the use of strains L228/L321/L111 and F113/MB004. David and Cillian concluded that their method offered a cheap solution to farmers throughout the world and they were awarded First Prize in their category for their efforts.

 

Seoda Ní Chaoimh & Muireann Ní Shé, from Gaelcholáiste Luimnigh, had a (group) project in the Intermediate Category for Biological and Ecological Sciences entitled Anti-antibiotics: study of the use of antibiotics in Country Clare. This project examined the use of antibiotics in Co. Clare through the analysis of dispensing reports from a random sample of eight Pharmacies and 5,000 prescriptions between July and November of 2018. The top five antibiotics prescribed were co-amoxiclav, amoxicillin, flucloxacillin, nitrofurantoin and clarithromycin (78% of total prescriptions) and co-amoxiclav and amoxicillin together accounted for 56.5% of the total. There was a 20.2% increase in prescriptions in October relative to August. The students concluded that it would be wise to establish a national database to include drug, drug family, reason for prescribing, doctor identifier, personal identifier, age and allergies – they felt that this would help to prescribe appropriately. They also felt that rapid identification of pathogens would be advisable to help with appropriate prescribing. Also, among their conclusions, the students suggested that there should be national public antibiotic resistance awareness campaigns. This project earned Seoda and Muireann the Maxim Integrated Special Award.

 

Rebecca Sheehan, Caoimhe Buckley and Maria O’Connor, from Coláiste Choilm, in Ballincollig, Co. Cork, had a (group) project in the Intermediate Category for Social and Behavioural Sciences entitled Technostress: An investigation into the physiological anxiety responses in students when separated from their mobile phones. Their project focused on nomophobia, the stress that millennials experience when being separated from their phone. These students used a combination of surveyed self-reporting by their subjects and technology acquired from UCC to conduct their study. The technology used was bluetooth galvanic skin response sensors or stresstracking devices that measure the electricity passing through the skin – the basis for the analysis being that sweat produced in stress mode increases electrical flow. The students set up experiments for their subjects (teachers and students) whereby they set them tasks to do while their phones were visible to the subjects but
variously out of bounds or not. Stress was measured when Maria, Caoimhe or Rebecca phoned and texted the subjects while they were e.g. tasked with conducting word searches. Their findings showed that subjects were more stressed when they were not allowed to respond to their phones, and that teachers were less stressed than students by this. Girls were more stressed than boys. This project was highly commended at the
BTYSTE.

 

 

A 20 Year Longitudinal Study of Stem Cell Potency: The Effect of Long Term Storage on Haematopoietic Stem Cells

L. O Connor1,2, N. Gardiner2,
C. Blackwell2, K. Keane2, M. Neylon2, N. Maloney2, H. Foy-Stones2, E. Kenny2,
S. Cronnolly, G. Lee2, E Higgins, M Ni Chonghaile, P. Browne2,
E. Vandenberghe2, E. Conneally2, C. Flynn2, C. Bacon2, P. Hayden2.
1University of Ulster, Coleraine, BT52 1SA, N Ireland;
2Cryobiology Laboratory Stem Cell Facility, St.James’s

 

Hematopoietic stem cell (HSC) products for autologous transplantation are cryopreserved and stored in vapour phase liquid nitrogen (-197ºC to -150ºC) for later directed infusion. A universal expiry date is not currently agreed for clinical HSC products; generally they are stored indefinitely or for as long as the patient remains a potential candidate for transplant. The aim of this study was to assess the effect of long term storage on HSC product cell viability and functionality and thereby establish a standard expiry date for long term storage of HSC products.

43 Cryopreserved Bone Marrow (BM, n=13) and Peripheral Blood Stem Cell (PBSC, n=30) clinical products in long term storage (up to 22 years) that were approved for discard were analysed. 40 cryopreserved autologous PBSC clinical products being issued for transplantation (<10 years in storage) were also included. A post-thaw sample was taken directly from the clinical product bag for analysis. Viability testing was performed using inhouse, standardised EBAO viability, 7 AAD standalone and BD™ Stem Cell Enumeration Kit. Viable CD34+ cell count was measured
using BD™ Stem Cell Enumeration Kit and functional testing was performed using an in vitro progenitor colony cultures assay CFU-GM (Colony-Forming Unit-Granulocyte Macrophage) & BFU-E (Burst Forming Unit-Erythroid).

83 HSC products tested indicate a statistically significant decrease in percentage recovery of viable CD34+ cells, over time, particularly for HSC products in storage >10 years. percentage recovery of viable CD34+ cells decrease from a median of 87.77% (products in storage < 1 year) to 54.3% (products in storage > 10 years). A similar decrease was observed for percentage recovery of BFU-E and CFU-GM progenitors. The median percentage recovery of BFU-E was 102.2% for HSC products in storage <1 year down to 24.77% for those in storage > 10 years. The median percentage recovery of CFU-GM was 109.61% for HSC products in storage <1 year, down to 56.15% for those in storage for >10 years. Post-thaw viable CD34+ cell count correlated well with the fresh CD34+ cell count and was not a better predictor of successful neutrophil or platelet engraftment.

The results agree with published data1,2 stating that Haematopoietic Stem Cell potency is lowered by long term storage of cells. We predict the rate of decline of potency to be in the order of -2% per year of storage. This may not be of clinical significance for products that have a good clinical dose, however, for patients who have a borderline dose of cells at T0 (time of cryopreservation) then an algorithm of cell potency loss of 2% per year of storage should be considered. A product with a minimal transplant dose of 2.0 x106/kg CD34 cells at T0 could potentially have a below minimum viable count of 1.6 x106/kg CD34 cells after 10 years of storage. Therefore, each stored HSC product cell dose at T0 could be assessed using the algorithm to decide if the predicted dose post-thaw is sufficient for transplant and set an appropriate expiry or ‘best before’ flag date for the product.

1. Fernyhough LJ, Relative recovery of haematopoietic stem cell products after cryogenic storage of up to 19 years. Bone Marrow Transplantation. 2013; 48(1):32-5.
2. Spurr Elisabeth E, Cryopreserved human Hemopoietic stem cells retain engraftment potential after extended (5-14 years) cryostorage. Cryobiology

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CONGENITAL MACROTHROMBOCYTOPAENIA; DO DEVIATIONS IN THE CALCIUM BINDING AFFINITY OF ACTININ-1 MUTANTS CONTRIBUTE TO ITS PATHOGENESIS?

Aidan Murphy
Authors: Murphy A, Young P
Topic: Haematology
Institution: University College Cork
Address of First Author: Haematology Department, University
Hospital Waterford,

Biography: I am currently doing my student placement at University Hospital Waterford, having recently graduated with a
Biomedical Science degree from University College Cork/ Cork Institute of Technology. I have always expressed a keen interest in Science, as is reflected in the numerous scholarship funded research projects that I have partaken in throughout my years in college. My previous research at the APC Microbiome Institute with Dr. David Clarke and Dr. Susan Joyce was Microbiology based and involved studying bile resistance mechanisms in Bacteroides species. My Final Year Project with Dr. Paul Young was haematology based and involved identifying
pathogenic mechanisms for Congenital Macrothrombocytopaenia. I am dedicated and passionate about my work and I am enthusiastic about seeing what challenges are in store for me as a Medical Scientist in the future.

Actinin-1 is a cytoskeletal protein which crosslinks actin filaments, however recent studies have shown that mutations in actinin-1 cause a rare platelet disorder known as congenital macrothrombocytopenia (CMTP). My project attempts to identify a potential pathological mechanism for this disease by studying several disease-associated mutations localised within the calmodulin domain of actinin-1. Calcium plays a critical role in
regulating actin crosslinking by interacting with the calmodulin domain in actinin-1 (Drmota Prebil et al., 2016). This regulatory role of calcium in actin crosslinking is necessary for many platelet production and platelet activation events (Machlus, Thon and Italiano, 2014). Hence, it has been hypothesised that mutations in the calmodulin domain of actinin-1 may alter its calcium binding affinity, which may consequently interfere with normal platelet processes, thereby contributing to the pathogenesis of CMTP. To test this hypothesis wild type actinin-1 and CMTP associated
actinin-1 mutant proteins (G764S and E769K) were expressed using recombinant E.coli BL21 cells, and purified using techniques such as nickel column and GST column purification, as well as size exclusion fast protein liquid chromatography. The use of novel techniques such as microscale thermophoresis and isothermal calorimetry is then used on these purified actinin-1 proteins to provide a new insight into the binding interactions and thermodynamic events that occur between the calmodulin domain of actinin-1 CMTP associated mutants and calcium.

The results obtained showed that the G764S mutated actinin-1 had no difference in calcium binding affinity (KD) against wild type actinin-1, while the E769K mutation showed a noticeable difference in calcium binding affinity against wild type actinin-1. The results provided a new structural and mechanistic insight into the basis of actinin-1 dysfunction in CMTP and helped elude to the pathogenies of CMTP in individuals carrying the mutations
studied. The results of the project elucidate that the changes in calcium binding affinity in mutant actinin-1 proteins affect the strength in which actinin-1 can secure actin filaments, which can therefore alter normal platelet processes, thereby resulting in the occurrence of CMTP.

References:
Machlus, K., Thon, J. and Italiano, J. (2014). Interpreting the developmental dance of the megakaryocyte: a review of the cellular and molecular
processes mediating platelet formation. British Journal of Haematology, 165(2), pp.227-236. Drmota Prebil, S., Slapša, U., Pavšic, M., Ilc, G., Puž,
V., de Almeida Ribeiro, E., Anrather, D., Hartl, M., Backman, L., Plavec, J., Lenarcic, B. and Djinoviç-Carugo, K. (2016). Structure and calcium-binding studies of calmodulin-like domain of human non-muscle ·-actinin-1. Scientific Reports, 6(1).15-e518.

CARBAPENEM AND COLISTIN RESISTANCE DETECTION: A CHALLENGE FOR THE DIAGNOSTIC MICROBIOLOGY LABORATORY

Kate Byrne
Topic/Discipline: Medical Microbiology
Byrne, K.1, Drudy, D.1, Keating, D.2, Coyle, E.2, Donoghue, O.2. 1Technological University Dublin, 2Microbiology, St. Vincent’s University Hospital

Background:

The global dissemination of carbapenemase producing Enterobacterales (CPE) represents a significant public health issue. Rapid and accurate detection of CPE colonisation through screening is essential to enable effective infection control strategies. Carbapenem resistance detection remains a challenge for the diagnostic microbiology laboratory (1). The EntericBio realtime® CPE assay and the GeneXpert® Carba-R assay are molecular assays designed for the in vitro diagnostic testing and qualitative detection of Enterobacterale produced, carbapenemase genes: KPC, OXA-48, NDM, VIM and IMP . Colistin resistant Enterobacterales are being increasingly reported worldwide. Colistin susceptibility testing also represents a challenge for the diagnostic microbiology laboratory. The Rapid Polymyxin NP kit detects colistin resistance in Enterobacterales (2).
The aim of this study was to evaluate the EntericBio realtime® CPE assay for direct use, the GeneXpert® Carba-R assay and the Rapid
Polymyxin NP kit for use with enterobacterial isolates in a diagnostic microbiology laboratory.

Methods:
A total of 277 rectal swabs were tested directly using the EntericBio realtime® CPE assay. A total of ten enterobacterial isolates were processed on the GeneXpert Carba-R assay and twelve enterobacterial isolates using the Rapid Polymyxin NP kit. Results: The EntericBio realtime® CPE assay demonstrated a sensitivity, specificity, positive predicted value (PPV) and negative predicted value (NPV) of 100%, 98.95%, 95.1% and 100% respectively. The GeneXpert® Carba-R assay and the Rapid Polymyxin NP kit exhibited sensitivities of 100% and 66.7% and specificities of 80%
respectively.

Conclusion:
The EntericBio realtime® CPE assay and the GeneXpert® Carba-R assay are rapid and accurate molecular assays for the detection of CPE that facilitate the improvement of infection control strategies. The Rapid Polymyxin NP kit is a rapid and easy to use method for the detection of colistin resistance. However, further study is required to determine its utility in a diagnostic microbiology laboratory.

References:
(1) Humphries, R. M. and McKinnell, J. A. Continuing Challenge for the Clinical Laboratory for Detection of Carbapenem-Resistant Enterobacteriaceae. J Clin Microbiol. 2015; 53(12):3712-3714.
(2) Poirel, L., Jayol, A. and Nordmann, P. Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes. Clinical Microbiology Reviews. 2017;30(2): 558-570.

Biography: I graduated in 2018 with a
BSc in Biomedical Science (TUD). My
undergraduate research project involved the evaluation of molecular
assays for the detection of carbapenem resistant Enterobacterales in
screening specimens. This was followed by the evaluation of a rapid
method for the detection of colistin resistance in Enterobacterales.
I am currently working as a medical scientist in the Microbiology
department of the Mater Misericordiae University Hospital.

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RESISTANCE REVERSAL IN MULTI-DRUG RESISTANT KLEBSIELLA PNEUMONIAE: EXPLORING THE SOXS DEPENDENT REGULON

Kate Dever
Topic: Medical Microbiology
Centre for Food Safety University College Dublin,
Wyckham Way, Dundrum, Dublin
Dever, K, Srikumar, S, Anes, J, Fanning, S, Corcoran, D.

Biography: I completed my undergraduate degree in the Galway Mayo Institute of Technology. I have since been lucky enough to work in the University College Dublin Centre for Food Safety, where I completed my thesis, as a research technician in molecular microbiology. In September of 2018 I was offered a permanent position as a medical scientist in the microbiology laboratory of St. Vincent’s University Hospital Dublin, where I continue to work today.

The overuse and misuse of antibiotics has led to the emergence and dissemination of multi-drug resistance in Gram-negative pathogens, jeopardising the advances of modern medicine. The SoxRS regulon plays a key role in bacterial defence against oxidative stress (1). The aim of this project was to determine the role of the transcription factor, SoxS, as a regulator of antibiotic resistance under oxidative stress conditions (2). This was achieved by employing the multi-drug resistant (MDR) wildtype Klebsiella pneumoniae MGH 78578, the deletion mutant Klebsiella
pneumoniae MGH 78578 δsoxS (with soxS deleted from the bacterial chromosome), and the complementation Klebsiella pneumoniae MGH
78578 δsoxS pBADsoxS isolate (where soxS was over-expressed from the plasmid pBAD using an arabinose inducible promoter).

The project involved the interpretation of RNA (ribonucleic-acid)- sequencing data obtained from a broader study, conducted on MDR Klebsiella pneumoniae placed under oxidative stress conditions using paraquat, at the University College Dublin-Centre for Food Safety (UCDCFS). In order to determine if genotypic observations from this data translated into distinct phenotypes, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were performed using six antibiotics namely, colistin, gentamicin, kanamycin, cefotaxime, tetracycline and rifampicin. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was employed for the confirmation of soxS over-expression in the complemented Klebsiella  pneumoniae MGH 78578 δsoxS pBADsoxS strain. Finally, the project incorporated the objective to determine the 5’transcription-start-site (5’TSS) of soxS using random amplification of cDNA (complementarydeoxyribonucleic-acid) ends.

The RNA-sequencing data outlined that under oxidative stress conditions certain antibiotic resistance genes were down-regulated in the absence of soxS. This was reflected in the phenotypic MIC and MBC assays which showed antibiotic resistance reversal in the Klebsiella pneumoniae MGH 78578 δsoxS isolate for the antibiotics cefotaxime and tetracycline.

These results highlighted that regulatory molecules, such as soxS, may provide novel targets to mitigate multi-drug resistance in the future.

References:
(1) Seo SW, Kim D, Szubin RO, Palsson B. Genome-wide reconstruction of OxyR and SoxRS transcriptional regulatory networks under oxidative stress in Escherichia coli K-12 MG1655. Cell Reports. 2015;12(8): 1289-1299.
(2) Kohanski MA, Dwyer DJ, Collins JJ. How antibiotics kill bacteria: from targets to networks. Nature Reviews Microbiology. 2010;8(6): 423-435.

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READY-TO-EAT DAIRY FOODS: A POTENTIALLY IMPORTANT VECTOR OF ANTIBIOTIC-RESISTANT BACTERIA

Shannon Diggin
Discipline: Medical Microbiology.
Institution: Department of Biological Sciences, Cork Institute of
Technology.
Authors: Diggin, S., Sleator, RD., Culligan, EP.

Biography: Biomedical Science student from Listowel, Co. Kerry. Currently undertaking clinical laboratory placement at University Hospital Kerry.

A perceived boost in nutritional value and flavour has seen the market for minimally processed, ready-to-eat (RTE) foods grow considerably in recent years. However, mounting food safety concerns have accompanied this demand, with RTE produce being increasingly recognised as a potential vehicle to transfer not only foodborne pathogens, but also antibiotic-resistant bacteria and their associated resistance genes to the consumer (1). This study examined the microbiological quality of RTE, unpasteurised milk and cheeses and investigated the antibiotic-resistance profiles of the bacteria present. Enumeration of the levels of bacteria was determined in CFU/mL and revealed that three out of the four samples tested contained unacceptably high quantities of bacteria (2), indicating poor standards of food hygiene and safety. Five species of bacteria were isolated from the dairy products; Hafnia spp. (cheese), Klebsiella oxytoca, Kluyvera cryocrescens, Escherichia coli and Hafnia paralvei (milk). All isolates were resistant to a minimum of three antibiotics (ampicillin, cephradine and fusidic acid), when subjected to antimicrobial susceptibility test using the agar disc diffusion method. The antimicrobial resistance profile of Hafnia spp. was particularly alarming; displaying resistance
to 8 out of 11 antibiotics tested and PCR revealed positive carriage of the ampC beta-lactamase gene. Moreover, bacterial isolates were shown to be capable of surviving conditions that simulated the human gastrointestinal tract and were resistant to a number of disinfectants, following stress survival testing. These findings suggest that individuals consuming unpasteurised dairy may be routinely inoculated with resistant bacteria and further reinforces the knowledge that raw milk and cheeses may represent a significant environment for the evolution of antibiotic-resistant bacteria and their spread to humans (3). The potential health implications of consuming RTE foods, therefore, need to be carefully re-evaluated. Limiting the spread of resistant bacteria throughout the food chain remains a crucial strategy to combat the global antibiotic resistance crisis.

References:
(1) Hudson, JA., Frewer, LJ., Jones, G., Brereton, PA., Whittingham, MJ., Stewart, G. The agri-food chain and antimicrobial resistance: A review. Trends Food Sci. Technol. 2017; 69:131–147. https://doi.org/10.1016/j.tifs.2017.09.007
(2) European Commission. Regulation (EC) No. 92/46/EEC of 16 June 1992 laying down the health rules for the production and placing on the market of raw milk, heattreated milk and milk-based products. Official Journal of the European Union. 1992; 268, (14):1–32
(3) Verraes, C., Van Boxstael, S., Van Meervenne, E., Van Coillie, E., Butaye, P., Catry, B., de Schaetzen, MA., Van Huffel, X., Imberechts, H., Dierick, K., Daube, G., Saegerman, C., De Block, J., Dewulf, J., Herman, L. Antimicrobial Resistance in the Food Chain: A Review. Int. J. Environ. Res. Public. Health. 2013; 10: 2643–2669. https://doi.org/10.3390/ijerph10072643

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EQUIVALENCE STUDY OF THE LYOPHILISED ENTERICBIO® NOROVIRUS ASSAY (EBVP2) TO THE ORIGINAL CE-IVD (EUROPEAN CONFORMITY – IN VITRO DIAGNOSTICS) MARKED ENTERICBIO® NOROVIRUS ASSAY (EBNOV) ON THE MARKET

Alanna Maunsell
Topic – Medical Microbiology
Institution – Serosep Ltd., Co. Limerick
Authors – Maunsell, A., O’Leary, J., Corcoran, D.”

Introduction: Norovirus is a major cause of inflammatory gastroenteritis. It peaks during the winter months and early spring giving it the name ‘Winter Vomiting Disease’. This study was carried out to assess the equivalence of the lyophilised EntericBio® Norovirus assay (EBVP2) to the CE-IVD marked EntericBio® Norovirus assay (EBNoV). These assays detect
Norovirus genogroup I and Norovirus genogroup II directly from faecal sample. The equivalence of the assays was assessed under analytical sensitivity, clinical sensitivity and clinical specificity.

Methods: The analytical sensitivity of the EBVP2 assay was examined by testing RNA solution diluted to the concentrations relevant to the limit of detection of the EBNoV assay – 100 genome copies for Norovirus genogroup I and 50 genome copies for Norovirus genogroup II. 24 replicates of each genogroup were tested. The clinical sensitivity was assessed by testing 30 faecal samples using both assays that had previously tested positive for Norovirus. The clinical specificity was assessed by testing 80 samples using both assays that had previously tested negative.

Results: The EBVP2 assay had an analytical sensitivity of 100% at the limit of detection (LOD) previously established for the EBNoV. Both assays had a clinical sensitivity and specificity of 100%. However, there was a difference in Cp values of positive samples in the assays. Reducing the time between heat treatment and testing improved this difference.

Conclusions: These results show that the assays are equivalent but poses questions about the stability of RNA in heated stool preparation solution.

THE PRODUCTION OF FORS1-POSITIVE CELLS AND PREVALENCE OF ANTI-FORS ANTIBODIES IN A PORTUGUESE POPULATION

Alyssa Corpuz
Topic: Transfusion and Transplantation Science
Department Biomedical Laboratory Sciences, ESTESC- Coimbra
Health School, Polytechnic Institute of Coimbra, Coimbra, Portugal
Authors: Corpuz, A, Mourato, C, Galvao, S, Jesus, C, Mendes, F.

Biography: I am a 1st Class Honours Graduate in Biomedical Sciences from Dublin Institute of Technology specialising in Blood Transfusion and Medical Microbiology. I completed my research project in Coimbra Health School, Portugal in the area of Transfusion Medicine. Since the completion of my project I have been working fulltime as a medical scientist in Beaumont Hospital Blood Transfusion Department.

In 2012, the FORS system was accepted by International Society of Blood Transfusion as the 31st blood group system. Forssman (Fs) antigen (Ag) expression is rare in human red blood cells (RBCs) and most commonly found on sheep RBCs. Anti-Fs antibodies (Abs) are naturally occurring in human sera and are predominantly IgM but can also be IgG. To this day the global prevalence of the FORS system is unknown. Currently, there is a lack of natural FORS1- positive RBCs available to use for anti-Fs screening in large scale populations. This study was designed to produce FORS1-positive cells viable for use in the screening and classification of anti-Fs in a Portuguese population.
A 3-5% FORS1-positive cell suspension was produced using sheep’s blood in CellStab stabilizer solution. The quality of the FORS1-positive cells was investigated through three independent experiments of AB0 titration, osmotic fragility test and supernatant haemolysis. For each batch of FORS1-positive cells produced, an extended antibody panel was performed. Demonstrating that the FORS1-positive cells can be used for up to 40 days, anti-Fs screening and classification was carried out in a patient (1115) and donor (1400) population. Antigenic expression and membrane integrity of FORS1-positive cells remained stable for 40 days. Good Fs Ag preservation was established and minimal haemolysis was observed. With this novel reagent, a large population screen was carried out providing evidence that FORS is a rare blood group system (99.97% anti-
Fs prevalence). Classification of anti-Fs Abs verified that it is predominantly IgM but may also be IgG in class.

In conclusion, a novel and easy-to-produce reagent has been developed and submitted to patent with stable Fs Ag expression. With this FORS1-positive cell suspension, it is now possible to screen and classify anti-Fs Abs in large scale populations.

Winner

TITLE: VISUAL ANALYSIS OF HUMAN ERRORS IN TRANSFUSION PROCESS FLOWS IS A SIMPLE BUT POWERFUL TOOL TO HELP TARGET IMPROVEMENT

TOPIC: Transfusion and Transplantation Science
INSTITUTION: Blood Transfusion Laboratory, Laboratory Medicine, Cork University Hospital, Wilton, Cork.

AUTHORS: O’Sullivan, P, Sheehy, J, Gilligan, O.

Introduction/Background:
In November 2015, trend analysis in the Blood Transfusion Laboratory at CUH identified little reduction in the number of Non-Conformances (NCs) occurring in the laboratory over an 8-year period since 2008. Root cause analysis (RCA) identified that “Human Error” was persistently a significant contributory factor. Given that “continual improvement” is a key element of an ISO15189 quality management system1, our Laboratory set out to address and improve this trend.

MATERIALS AND METHODS:
Over a 3 year period, the laboratory identified and classified human error in NCs (as part of RCA) using a standardised approach (i.e. Medicines Healthcare products Regulatory Authority subcategories2). Previously during the implementation of ISO15159:2012 standards, process flow diagrams had been developed in the laboratory to enhance risk management strategy. Using these diagrams, each NC was plotted at the point where the error occurred (indicating the type of human error). The findings were repeatedly discussed at staff meetings, journal clubs, and
Management Review meetings and actions were agreed.

RESULTS:
Between 2016 and 2018, analysis showed that Human error was a factor in approximately 80% of NCs mainly caused by ‘lapses in concentration’ and/or the ‘omission of procedural step(s)’. Between 2008 and 2015, on average, 75 NCs occurred in the laboratory per year. By modifying our approach to analysing NCs to target improvements, it was noted that between 2016 and 2018, on average 42 NCs occurred per year – representing a 43% reduction.

CONCLUSIONS:
Human error is very difficult to eradicate where processes cannot be modified such that they are eliminated or substituted, or where
engineering solutions cannot be provided. Repetitive use of this simple visual tool proved very powerful in reducing laboratory NCs by raising scientists’ awareness of the occurrence of human errors in processes and to help change their behaviour. It also helped identify and prioritise points in processes where further action could have the most impact.

REFERENCES:
(1) Medical laboratories – Requirements for quality and competence (ISO 15189:2012).
(2) PHB Bolton-Maggs (Ed), D Poles, A Watt and D Thomas on behalf of the Serious Hazards of Transfusion (SHOT) Steering Group. The 2013 Annual SHOT Report (2014).
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2nd Place

TITLE: POTENTIAL ROLE OF LIQUID BIOPSIES IN MANAGEMENT OF NON- SMALL CELL LUNG CANCER PATIENTS: A PROSPECTIVE INVESTIGATION OF EGFR MUTATION STATUS WITH CORRELATION IN MATCHED TISSUE SAMPLES

Amy Connolly
Amy.connolly@hse.ie
Histopathology Laboratory, Cork University Hospital.
Supervisors: Prof Louise Burke, Réiltín Werner

Molecular diagnostics has led to revolutionary treatment options for many malignancies leading to more personalised theranostic plans for individuals. EGFR mutation detection in non-squamous NSCLC is currently carried out on tumour tissue post MDM discussion in the Cork University Hospital. Evidence supports the limitations associated with this method and this has highlighted the potential usefulness of liquid biopsies in this field.

The aim of this study is to determine the sensitivity and specificity of EGFR mutation detection in liquid biopsies and correlate results with matched tissue samples. The verification of the liquid biopsy in the clinical setting will facilitate patient management with regard to beneficial therapies and furthermore act as a dynamic monitoring method of the response to this therapy.

A total of 28 patients diagnosed with adenocarcinoma of primary lung origin were enrolled on this trial following acquisition of informed consent. EGFR mutation analysis was carried out on K2EDTA blood samples and matched tissue samples from enrolled patients.

Molecular diagnostics is fundamental in the management of lung cancer patients. Liquid biopsies may provide additional information to facilitate targeted therapy in patients unsuitable for invasive biopsy procedures. Currently reflex tissue analysis is imperative to prevent false negative reports. However the utilisation of liquid biopsy provides great hope for patients who are unable to undergo biopsy procedures in order to achieve diagnosis.
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3rd Place

LABORATORY EVALUATION OF GLENBIO CALPROTECTIN TEST IN FAECES.

DISCIPLINE: Immunology
O Dea B1, Ellis A1, Louw M1, Rochev Y2, Hutchinson K1
1. Eurofins-Biomnis Ireland, Sandyford, Dublin 18, Ireland.  2. School of Chemistry, National University of Ireland, Galway, Ireland.

BACKGROUND:
Faecal calprotectin measurement is an easy, non-invasive first line test which can differentiate inflammatory bowel diseases (IBD) from irritable bowel syndrome (IBS) and other functional disorders. Faecal calprotectin correlates with disease activity and is able to predict relapses in IBD.1 This makes faecal calprotectin useful for both diagnosis and monitoring of IBD patients. We performed laboratory evaluation of GLENBIO calprotectin test on Abbott Architect.

MATERIALS AND METHODS:
Calprotectin concentration in stool samples of 50 patients was determined using GLENBIO calprotectin latex reagent on Architect ci8200, and EliA Calprotectin method on Phadia 250. Both methods involved an extraction procedure and used the relevant manufacturer’s kits.
EP-evaluator software was employed for the statistical analysis.

RESULTS:
Within-run and between-run precision for both methods were <10%. We observed an acceptable agreement (Cohen’s Kappa = 70%) between both tests. McNemar Test for Symmetry: PASSES with p = 0.439 (ChiSq=0.600); Test < Reference 9 (18.0%) and Test > Reference 6 (12.0%). UKNEQAS showed acceptable performance for both assays.

CONCLUSION:
Our study shows that GLENBIO assays on Abbott Architect analyser can be an alternative method to our current use of EliA Calprotectin on Phadia 250. But in assaying calprotectin, we are well aware of the lack of an international standardisation, of the high biological variation, and of the fact that the results vary depending on which extraction kit is used. It is important that the results should be interpreted in the light of the patients’ overall clinical conditions.

Reference:
1. Sutherland AD et al (2008). Review of fecal biomarkers in inflammatory bowel disease. Dis Colon Rectum 51:1283–1291

Judging Posters                                                                                                                                   Dr. Katrina Hutchinson receiving her award