Academy of Clinical Science and Laboratory Medicine President’s Prize – 2020

President’s Prize
Although Biomedica 2020 has been postponed to November 30th, the Academy continues with it’s award scheme for graduates in the degree courses in Biomedical Science from TU Dublin, GMIT and CIT/UCC. Each year the colleges are requested to nominate their two students who have achieved the highest marks in the project component of their final exams. The nominated students are invited to our annual conference to give a 15 minute presentation. The President’s Prize is awarded to the student who presents a project judged to be the winner by a panel of selected members and other professionals. The winner goes forward to compete for the Martin Nicholson Award at the EPBS (European Association for Professions in Biomedical Sciences) annual conference.
Helen Barry

The President’s Prize 2020 Competition entrants presented their research at Biomedica – A Virtual Festival of Medical Science, 3rd December 2020. Congratulations to Cliona O’Shea on winning the 2020 President’s Prize with her work ‘Investigation of the ability of the bacteriocin nisin to inhibit coagulase negative Staphylococci’.

Improving the Process of Mitigating Daratumumab Interference in Pre-transfusion Compatibility Testing

Liptrot, C., Cregg, H., Scally, E. Galway-Mayo Institute of Technology, Old Dublin Rd, Galway
Irish Blood Transfusion Services, National Blood Centre, St. James’s Street, Dublin

Multiple myeloma (MM) is a malignant plasma cell disorder. Daratumumab (DARA), a monoclonal antibody directed against CD38, may be used for the treatment of MM as CD38 is over expressed on the malignant plasma cells¹. Treatment with DARA, however, interferes with pre-transfusion compatibility testing as DARA binds CD38 on reagent red cell. Several methods may be utilised in order to mitigate DARA interference including DTT treatment of reagent cells and masking CD38 present on reagent-cells. The aim of this research was to improve the current process of mitigating interference due to DARA in pre-transfusion compatibility testing.

The utility of extending the shelf life of DTT-treated reagent red cells was assessed.   Batch DTT-treated reagent red cells remained stable for up to 28 days post treatment. However, based on this study, due to haemolysis of cells and a decrease in reaction strength of some red cell antigens by day 28, the shelf-life of DTT-treated cells should be extended no longer than 14 days.

The validity of a room temperature crossmatch for patients who have been administered DARA and have a negative antibody screen was also assessed. Using NaCl gel cards, 22 patient plasma samples containing DARA were crossmatched against group O Rh D negative donor cells with compatible reactions observed.  The implementation of a room temperature crossmatch would eliminate the need to DTT-treat donor red cells for compatibility testing and thus reducing laboratory turnaround-times.

The efficacy of DaraEx, an anti-CD38 neutralizing agent was evaluated. The ability of DaraEx to inhibit DARA interference was found to be dependent on the amount of DARA circulating in patient plasma and possibly the number of CD38 antigenic sites on reagent cells. An advantage of the use of DaraEx-treated reagent-cells, in comparison to the DTT based method of pre-transfusion compatibility testing for this patient group, is that DaraEx-treated cells may be used for the detection of anti-K.

Reference

1. Chapuy, C., Aguad, M., Nicholson, R., AuBuchon, J., Cohn, C., Delaney, M., Fung, M., Unger, M., Doshi, P., Murphy, M., Dumont, L. and Kaufman, R. (2016). International validation of a dithiothreitol (DTT)-based method to resolve the daratumumab interference with blood compatibility testing. Transfusion, 56(12), pp.2964-2972.

Áine O’Connor graduated with a first class degree honours in Biomedical
Science (BSc) from TU Dublin- City Campus.
She completed a translational research project in Karolinska
Institutet Sweden for her undergraduate thesis, analysing the
regenerative potential of human placental amnion epithelial cells.
She is currently working in Tallaght University Hospital.

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In Vitro Expansion of Human Placental Amnion Epithelial Cells

Karolinska Institutet Flemingsberg, Trevet Grange Dunshaughlin Co. Meath.  O’ Connor, AOC., Brankin, BB., Gramignoli, RG. 

Liver failure represents a major public health concern worldwide. According to the World Health Organisation, the total deaths caused by cirrhosis and liver cancer have increased by 50 million/ year since 1990 (1). Until recently orthotopic liver transplantation (OLT) has remained the only established curative therapy for treating acute, chronic liver disease or metabolic defects in liver function. However, organ donor availability remains one of the main constraints on health services.  Over the past 3 decades, cell-based therapies have been gaining importance since they can contribute to regeneration of failing organs or damaged tissues by either direct replacement of the lost cells or by facilitating the body’s natural regenerative processes. Hepatocyte transplants (HT) have been gaining recognition as an alternative therapeutic option, although it is limited by the need for strict immunosuppressive therapy in support of long-term engraftment. Furthermore, allograft rejection remains a primary issue that prevents the long-term sustained therapeutic benefits of HT (2)

The placenta, or specifically the amniotic epithelial (AE) cells are a promising non-controversial source of potential stem cells. Their constitutive ability to mature into different cell types and correct life threatening liver disease in preclinical models have highlighted them as a potential candidate for liver-based therapy. Ex vivo expansion of AE cells may be important to generate an adequate yield for transplantation, in addition to reducing the number of allogenic donor AE cells per recipient. We isolated AE cells from six gestational full term placentas which generated high amounts of cells (up to 240 million of AE cells per placenta) characterised by high viability (91.5 ± 3.4 %). Whilst preserving several of their native characteristic features, we successfully expanded AE cells in a xenobiotic-free culture medium (PneumaCult™) supplemented with human platelet lysate in comparison to the current gold standard medium formulation. Our results showed AE cells proliferated robustly in PneumaCult™. Gene expression analysis by qRT-PCR showed expanded AE cells maintained their native epithelial marker expression (CD326 and cytokeratin 18). Using albumin ELISA quantitation AE cells secreted albumin upon expansion, albeit at levels equal to half of what is observed in healthy hepatocytes. Focusing on the wider clinical application of AE cells, it could be envisaged from these results that AE cells may aid in expanding the donor hepatocyte pool, generating functionally improved hepatocytes without the need to increase hepatocyte donor source.

References:

1. Murray, C. and Lopez, A. (2013). Measuring the Global Burden of Disease. New England Journal of Medicine, 369(5), pp.448-457.
2. Iansante, V., Mitry, R., Filippi, C., Fitzpatrick, E. and Dhawan, A. (2017). Human hepatocyte transplantation for liver disease: current status and future perspectives. Pediatric Research, 83(1-2), pp.232-240

Áine O’Connor graduated with a
first class degree honours in Biomedical
Science (BSc) from TU Dublin- City Campus.
She completed a translational research
project in Karolinska Institutet Sweden for
her undergraduate thesis, analysing the
regenerative potential of human placental
amnion epithelial cells. She is currently working
in Tallaght University Hospital.

Investigation of the ability of the bacteriocin nisin to inhibit coagulase negative Staphylococci

 O’ Shea, C., Twomey, E., Field, D., Begley, M. Department of Biological Sciences, Cork Institute of Technology

Introduction: Coagulase negative Staphylococci (CoNS) are ubiquitous colonizers of human skin ⁽¹⁾. While previously seen as harmless commensals, CoNS are now viewed as opportunistic pathogens and are reported to be the leading cause of infections related to implanted medical devices ⁽²⁾. Of particular concern, is the increasing development of resistance to conventional antibiotics. Furthermore, CoNS can form biofilms that aid adherence to biotic or abiotic surfaces, and biofilms are inherently refractory to treatment with antibiotics ⁽³⁾. As the number of medical device implantation surgeries is projected to increase, owing to the ageing population and improving healthcare in developing countries, alternative approaches to control CoNS are therefore urgently required. The present study investigates the ability of the natural microbially-produced peptide bacteriocin nisin to inhibit CoNS.

Materials and Methods: A bank of commensal and clinical CoNS was assembled, and strains were characterized using a variety of standard microbiological and biochemical tests. The ability of the strains to form biofilm was examined using a standard plastic microtiter plate-based assay. Agar-based deferred antagonism assays were employed to assess whether CoNS were inhibited by a nisin producing bacterium. Nisin peptide was purified from the nisin producer by reversed-phase high performance liquid chromatography (RP-HPLC), and minimum inhibitory concentration assays were carried out. The microtiter plate-based assay was employed again to assess how nisin affected biofilm formation on plastic surfaces. Subsequent experiments of this study focused on optimizing a method to examine biofilm formation by CoNS on a medical device substrate i.e. stainless steel.

Results: All of the CoNS strains in this study were shown to form biofilm, however the extent of biofilm formation varied between strains, as determined by final optical densities of crystal violet stained biofilms. It was observed using the agar-based deferred antagonism assays, that a nisin producing bacterium had the ability to inhibit the growth of all CoNS. The minimum inhibitory concentration of nisin against the tested CoNS strains was shown to be in the micromolar (μM) range. The addition of nisin during microtiter plate-based assays resulted in a significant reduction of biofilm formation. Furthermore, biofilm formation on stainless steel was also shown to be significantly reduced in the presence of nisin.

Conclusion: In summary, this study reports the ability of nisin to inhibit a bank of CoNS and their ability to form biofilms. Nisin may ultimately be used to develop novel coatings for medical devices, or be impregnated into them, with the aim of reducing the incidence of or preventing, medical device related infections caused by CoNS.

 References:

1. Otto, M. 2009. Staphylococcus epidermidis – the “accidental” pathogen. Nat. Rev. Microbiol. 7:555-67.
2. Campoccia D., Montanaro L., and Arciola C.R. The significance of infection related to orthopaedic devices and issues of antibiotic resistance. Biomaterials 27:2331-9.
3. McCann M., Gilmore B.F. and Gorman S.P. Staphylococcus epidermidis device-related infections: pathogenesis and clinical management. J. Pharm. Pharmacol. 60:1551-71.

Clíona O’ Shea graduated in 2019 with a first-class
honours joint Biomedical Science degree from Cork
Institute of Technology and University College
Cork (CIT/UCC). She is currently completing
her 5th year clinical placement in the
Mercy University Hospital (MUH) in
Cork City. She is investigating Uncertainty
of Measurement for antibiotic susceptibility
testing using the disc diffusion method.

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CHARACTERIZATION OF A LARGE GROUP OF LISTERIA MONOCYTOGENES USING WHOLE GENOME SEQUENCING: ASSESSING THEIR VIRULENCE POTENTIAL.

UCD-Centre for Food Safety, School of Public Health, Physiotherapy and Sports Science, Science Centre South, University College Dublin, Belfield, Dublin D04 N2E5.

Unrath, N., McCabe, E., Hurley, D., Herra, C., Fanning, S.

Listeria monocytogenes is the etiological agent of listeriosis, a foodborne illness associated with high hospitalization and mortality rates.⁽¹⁾ L. monocytogenes may be classified as hypovirulent, intermediate and hypervirulent, based on the presence of Listeria pathogenicity islands (LIPIs).⁽²⁾ This bacterium can exist in food associated environments for years with persistent strains being linked to outbreaks of foodborne disease.⁽³⁾ This study aimed to improve food-borne subtyping and identification of virulent and persistent L. monocytogenes in food associated environments through the application of whole genome sequencing (WGS).

WGS was applied to a collection of 143 L. monocytogenes from a longitudinal study of a large food production facility, collected between 2014 and 2017. Sequence data was analysed using bioinformatic tools. Virulence was assessed using the Virulence Factor Database (VFDB). Persistent strains were detected using multilocus sequence typing (MLST) in silico, single nucleotide polymorphism (SNP) analysis and the determination of biocide resistance using the BacMet database.

The assessment of virulence profiles revealed that only one isolate was hypovirulent and two others were hypervirulent, with the majority (98%) being intermediate. In silico MLST and SNP analysis showed that 77% of the isolates are potential persisters and harbor resistance genes to biocides commonly used in food environments. These findings suggest that virulent L. monocytogenes are present and persisting within Irish food environments which can have adverse effects on both the food producer and public health.

WGS can achieve more than just discrimination between unrelated isolates. An additional benefit of WGS is the opportunity to extract specific information, such as, the determination of virulence, antibiotic or biocide resistance status, as well as the assignment of serotypes. WGS is the ultimate tool for characterization of bacterial isolates as it provides the highest possible resolution in strain typing and represents a paradigm shift for outbreak investigation and contamination-source tracking.

References:

• EFSA and ECDC. The European Union One Health 2018 Zoonoses Report. EFSA Journal. 2019;17(12): 5926.
• Maury MM, Tsai YH, Charlier C, Touchon M, Chenal-Francisque V, Leclercq A, Criscuolo A, Gaultier C, Roussel S, Brisabois A, Disson O, Rocha EPC, Brisse S, Lecuit M. Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity. Nature Genetics. 2016;48(3): 308-313.
• Ferreira V, Wiedmann M, Teixeira P, Stasiewicz MJ. Listeria monocytogenes persistence in food-associated environments: epidemiology, strain characteristics, and implications for public health. Journal of Food Protection. 2014;77(1): 150-170.

Natalia Unrath graduated from Technological University
Dublin in 2019 with a BSc in Biomedical Science. She
started her career in the microbiology department in Connolly
Hospital. She is currently pursuing a PhD in the Centre for
Food Safety, University College Dublin specialising
in the use of Whole Genome Sequencing to assess the virulence and
persistence potential of Listeria monocytogenes under the principal
supervision of Professor Séamus Fanning.

Angiotensin-Converting Enzyme 2 ACE2) Is A Novel Substrate For γSecretase-Dependent Intramembrane Proteolysis

 AUTHORS: Harte, J.V.^1,2, McCarthy J.V^.3
1 Department of Biological Sciences, Cork Institute of Technology, Rossa Avenue, Bishopstown, Cork, Ireland. ² School of Biochemistry and Cell Biology, University College Cork, College Road, Cork, Ireland. ³ Signal Transduction Laboratory, School of Biochemistry and Cell Biology, ABCRF, 3.41 Western Gateway Building, Western Road, University College Cork, Cork, Ireland.

Introduction: The renin-angiotensin system (RAS) is a critical component of the response to hypovolaemia and hypotension; however, RAS dysfunction is an appreciable mechanism of disease1. The recently discovered angiotensin-converting enzyme 2 (ACE2) is known to counterregulate the potentially destructive effects of uncontrolled angiotensinergic activity2. Subversion of ACE2 by pathophysiological conditions, including infection by zoonotic coronaviruses, can contribute to inappropriate activation of the RAS and accentuate inflammatory and fibrotic disease. Although ACE2 is known to undergo ectodomain shedding, a feature shared by many substrates of γ-secretase-dependent regulated intramembrane proteolysis3, no studies have investigated the subsequent cleavage of the ACE2 carboxyl-terminal fragment (CTF).

Materials and Methods: The molecular mechanism of ACE2 cleavage following ectodomain shedding was investigated by Western blot analysis in cultured cells expressing exogenous or endogenous ACE2 using pharmacological inhibitors and genetic deficiency of presenilin. The subcellular localization of γ-secretase-dependent proteolysis was also investigated using inhibitors of clathrin-dependent endocytosis.

Results: In this study, we show that following ectodomain shedding, the membrane-bound ACE2 CTF is subsequently cleaved by γ-secretase, and that specific biochemical and genetic loss of γsecretase activity independently compromise sequential cleavage of ACE2. Pharmacological inhibition of proteasomal degradation also revealed a putative γ-secretase generated ACE2 intracellular domain (ICD). Moreover, we show that clathrin-mediated internalization of ACE2 is necessary for γ-secretase-dependent proteolysis.

Conclusions: These observations demonstrate that ACE2 is a novel γ-secretase substrate and the formation of a potentially biologically active intracellular domain may be partly responsible for the counter-regulation of the “classical” RAS. Internalization-dependent cleavage may also partly explain the uptake of zoonotic coronaviruses. Thus, the identification of ACE2 as a γ-secretase substrate may facilitate translational approaches for manipulating RAS activity in vivo, with potential biomedical significance in terms of health and disease.

 REFERENCES:
1. Forrester S, Booz G, Sigmund C, Coffman T, Kawai T, Rizzo V, et al. Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology. Physiological Reviews. 2018;98(3):1627-1738.
2. Paz Ocaranza, M, Riquelme, J, García, L, et al. Counter-regulatory renin–angiotensin system in cardiovascular disease. Nature Reviews Cardiology. 2020;17(2):116-129.
3. McCarthy, A, Coleman-Vaughan, C, and McCarthy, J. Regulated intramembrane proteolysis: emergent role in cell signalling pathways. Biochemical Society Transactions. 2017; 45(6); 1185– 1202.

James Harte is a graduate of the BSc (Honours) in Biomedical
Science from Cork Institute of Technology and University
College Cork, and is currently completing his Diploma in
Clinical Laboratory Practice in Cork University Hospital.

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EVALUATION OF A FAECAL CALPROTECTIN ASSAY FOR INTRODUCTION INTO ROUTINE TESTING IN BIOCHEMISTRY LABORATORY, UHL

 Authors: ⁽¹⁾ Lalor C, McGrath M, Conmy B. ⁽¹⁾ Riversdale, North Circular Road, Limerick.

Institution: Dept. of Biochemistry, University Hospital Limerick in collaboration with Dept. of Biopharmaceutical and Medical Science, Galway-Mayo Institute of Technology.

Introduction: Faecal calprotectin (FC) is a routinely used, non-invasive biomarker for the diagnosis and monitoring of Inflammatory Bowel Disease (IBD) [1]. The primary aim of this study is to evaluate the Bühlmann fCAL faecal calprotectin turbo assay in order to bring FC testing into routine testing on site in University Hospital Limerick (UHL).

Material and Methods: The performance characteristics of fCAL turbo assay were verified on the Abbott Architect C16000 analyser. Precision was carried out by analysing two levels (low and high) of Internal Quality Control (IQC) in three replicates, daily for five days. Accuracy was measured by analysing nine EQA samples. Additionally, a comparison study was performed which involved the measurement of 20 patient samples on both the Abbott Architect C16000 and Phadia 250 instrument.

Results: The precision study confirmed consistency with the manufacturer’s claims and revealed a total coefficient of variation (CV) of 1.53% and 3.8%, respectively. A high percentage inaccuracy of 21.83% and significant negative bias was observed for accuracy studies. A percentage bias of 88.9% and Passing-Bablok analysis revealed poor agreement between the two methods, further emphasised by a p value of 0.007 (α=0.05). The null hypothesis was thereby rejected.

Conclusion: The fCAL turbo assay performed in accordance with the manufacturer’s specifications of precision. However, accuracy and method comparison results were unsatisfactory for the verification of the assay. Further analysis is required before introduction of the assay into routine testing. Once the analytical performance is evaluated successfully, the assay will be implemented on site, drastically reducing turnaround times, decreasing referral costs and improving service to users.

References:  [1] Seenan JP, Thomson F, Rankin K, Smith K, Gaya DR. Are we exposing patients with a mildly elevated FC to unnecessary investigations? Frontline Gastroenterology. 6^th ed. 2015; 156-160.

Clodagh Lalor graduated in 2019 with a BSc. in Medical
Science from GMIT. She completed her clinical
laboratory placement in University Hospital Limerick
(UHL). Following this, She majored in Clinical
Chemistry and Medical Microbiology and is now
working in the Biochemistry Laboratory, UHL.