WINNER

 

Investigation of the antimicrobial effects of both aerial and root parts of selected plants against Staphylococcus aureus

Simon Meehan, Coláiste Choilm, Ballincollig, Co. Cork

My grandfather has had a lifelong interest in medicinal herbs, and because of this I was taught from a young age how to identify, gather and grow plants. My grandfather prescribed personal remedies for people suffering from various illnesses. I am attempting to do the same by searching for a new antibiotic. My own feelings about the shortage of antibiotics are that this is an urgent matter that needs more attention. We need to obtain new information about plant extracts and how they function in order to create antibiotics to target infection. The idea that invasive plants may contain an antimicrobial constituent excites me and the phrase “killing two birds with one stone” would seem to apply. There is also an opportunity to make use of invasive plants (weeds) that prevent the healthy growth of crops. In my project, the crops tested were Asparagus, Elecampane (previously grown by my grandfather), Horseradish, Physalis and Lemon balm, and the weeds tested were Nettle, Mare’s tail, Bramble (including fruit in this case), Dandelion and Sea beet.
This project incorporated botany, microbiology and analytical chemistry.

I began by gathering, cleaning, drying and grinding the roots of the plants in winter, 2015. Each root was suspended in an equal ratio of 40% ethanol w/v (vodka). In June 2016, the aerial parts were gathered and placed in airtight jars, also at an equal ratio in ethanol. Both sets of extracts were left for >30 days in a dark area. After 30 days, the herbs were strained from the liquid. This liquid obtained was tested for its effect on Staphylococcus aureus. In August, 2016, the aerial part of the bramble was harvested a second time, and this, along with Bramble fruit, was suspended in ethanol, and prepared as described above for aerial preparations. After risk assessment and demonstration, antimicrobial
susceptibility testing was carried out in Cork Institute of Technology using Mueller Hinton Agar and an agar well diffusion assay. The agar was swabbed with Staph aureus after which wells were created in the agar. The extracts were introduced by pipette, and agar plates were placed in an incubator at 37∞C for 24 hours. Elecampane root extract was used as a positive control and 40% ethanol was used as a negative control. After incubation, the results were noted. An inhibition zone was observed around plant extracts that were inhibitory to Staph aureus. For the root extracts the tests were conducted nine times, and the reproducibility of the assay was assessed by calculation of mean and standard deviation.
Using the same method, all possible paired combinations were tested of the set of root extracts. All possible paired combinations were tested of the set of aerial extracts. For each of the ten plants the two extracts for each plant were tested in combination. Where noteworthy inhibition of Staph aureus was recorded, further testing was conducted on a variety of bacteria, both Gram-positive and Gram-negative, i.e. Mare’s tail and Bramble were also tested against Pseudomonas aeruginosa, Enterococcus faecalis, Enterobacter spp. and Escherichia coli. The extracts of Mare’s tail root and Bramble fruit were tested against MRSA in Cork University Hospital by Medical Scientists.

In an effort to understand the basis of my findings, I investigated possible techniques, and discovered that High Performance Liquid Chromatography was useful. I emailed UCC independently, stating that I was at a crucial point in my project and that I would like, if possible, to access specialised equipment i.e. HPLC. I received a positive reply and was granted not only access but also freedom to test my own plant extracts. I was trained in the use of HPLC and was able to operate the instrument independently, fulltime, for six days.

The following are all conclusions drawn from my results:
• It can be concluded from this project that some of the plant extracts contained antimicrobial constituents and this project has shown novel findings not reported elsewhere
• Bramble leaf collected in June and Bramble fruit collected in August demonstrated strong anti-staphylococcal effects, while Bramble root
demonstrated little activity. The Bramble fruit extract was also tested against MRSA by staff in a hospital laboratory and proved effective
against it
• Bramble fruit extract was equally effective against Pseudomonas aeruginosa as Enterobacter spp. but generated a reduced effect against Enterococcus faecalis and partial inhibition of Escherichia coli. Bramble fruit extract may show moderately broad-spectrum activity against Gram-positive and Gram-negative bacteria
• Bramble leaf collected in August at the same time as the fruit did not show anti-staphylococcal properties, which demonstrates that harvesting time is critical for Bramble leaf
• Mare’s tail root was inhibitory to methicillin sensitive Staph aureus, which has not been reported previously. Mare’s tail root was not as strongly effective against MRSA, however
• Based on testing combinations of extracts for antimicrobial effects in this project, it can be concluded that if a medicinally based drug/ treatment were to be manufactured from plant extracts comprising more than one active ingredient, the effect might not generate the desired result due to new bonds being formed with different properties to the original compounds; this study showed that some extracts inhibited the effects of others
• HPLC analysis of the four extracts of Bramble produced chromatograms with differing peaks, and three of the peaks appeared to be common to Bramble fruit and Bramble leaf extracted in June, but not to the other Bramble extracts, so these peaks may represent antimicrobial agents
Since the 18th Century, organisms have been classified according to the method of Linnaeus i.e. their biological format including genus,
species and variety. All humans have their individual identity which is universally recognised. However, we classify plants as something far
less individual than ourselves. I feel that plants should be classified at a greater depth by a more specific system i.e. chemical composition.
During library searches of Rubus fruticosus (Bramble), there was no direct correlation between my own results and others. In my own study, the extracts were not concentrated. The antimicrobial effects of Bramble were demonstrated by 50mL volumes (in 40% vodka), showing effective concentrations for treatment of Staph aureus (or MRSA) infection combined with non-toxicity.

The long term aim of this project is to work in a field that allows me to create a freely-available database of plant constituents with inhibitory effects against pathogens. What would this new classification provide in the long run? It could prevent constant gathering, preparing, extracting, testing, reproducing, failing, understanding and retesting. It will allow the prediction of the antimicrobial efficacy of any plant once the constituents are known, even in advance of testing them against pathogens. If a plant outside my back door has the capability to inhibit Staph
aureus, MRSA and Pseudomonas aeruginosa to such a degree that it can be classified as being an antibiotic:
1. Has the concept of science led people to view science in a sophisticated way and to ignore the simpler solutions?
2. It may be that people do not view wild plants as relevant to themselves. The extraction method for Rubus fruticosus as an antibacterial
agent used in this project is patent pending.

Acknowledgements
Firstly, I wish to thank my teacher, Ms Karina Lyne, for her fantastic mentoring and support of myself during this project. I also thank Ms Breda O’Callaghan and Mr Michael O’Donoghue at the Dept. of Microbiology at Cork University Hospital for testing two of the extracts against MRSA. I am very grateful to Dr Eric Moore and Dr Anna Hogan at the School of Chemistry, University College Cork, for providing me with access and training in HPLC. My thanks also to my mother, Dr Brigid Lucey for providing me with technical training in the lab and for education in how to read scientific papers. Finally, I wish to thank the Dept. of Biological Sciences at CIT, for laboratory access and for the use of equipment.

WINNER, Simon Meehan with his teacher, Karina Lyne and Richard Bruton, Minister for Education and Skills.

Conference Bursary

Mark Wright M.Sc. Candidate, Medical Scientist, Histopathology Department, St. James’s Hospital, Dublin

I was honoured to be the recipient of the Academy Conference Bursary Award recently. This will provide an invaluable opportunity to disseminate my research to a large clinical and scientific audience. It will also lead to further networking, collaborative and grant opportunities for our research group as well as developing my knowledge base as a Medical Scientist and an allied health care professional. My research is based on immuno-oncology and relates to the development of a novel companion diagnostic test for programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC). Globally, lung cancer is diagnosed in approximately 1.8 million people each year, with 1.59 million dying from the disease. NSCLC is the most common histological subtype,
with 70% – 80% of patients presenting with metastatic disease at diagnosis. Despite significant progress in molecular diagnostics and targeted therapies over the past decade, the prognosis for patients with advanced NSCLC remains poor. Currently, platinum based combination chemotherapy
   is the mainstay of palliation, however, a number of immunotherapies have now been approved by the  US FDA in the first and second line setting in NSCLC. These drugs are based on a humanised antibody that targets programmed cell death protein 1 (PD-1), which is a cell surface receptor expressed on T cells and pro-B cells. Immunohistochemistry (IHC) is the current gold standard method for detecting PD-L1 protein expression on formalin fixed paraffin embedded (FFPE) tissue. Scoring IHC stained sections for PD-L1 is very difficult and has proven to be subjective. A positive score of 50% or greater must be given for a patient to be considered a candidate for at least one major anti-PD-1 immunotherapy, and in addition several other cut-off points have been defined depending on the drug, tumour type and indication. Furthermore, several studies have shown that a percentage of PD-L1 negative patients (via IHC) actually responded to anti-PD-1 immunotherapy. Therefore, this raises questions relating to PD-L1 IHC scoring. My study seeks to address these issues using RNA in-situ hybridisation (RISH) as a companion biomarker for immune checkpoint inhibitors in NSCLC in conjunction with image analysis scoring technologies.

Writing –
tips and trip ups

Allison Cartwright looks at the challenges of a writing
assignment and shares some tips that have worked for her…

 

Writing can be one of the most difficult tasks. It’s hindered me over the past 8 years at university. On hearing the word ‘essay’, I prove the ‘fight or flight’ principle by feeling instant panic. I don’t mind talking, but writing is different.
Or is it? Recently (and I wish I’d found this out sooner) it became clear that it’s acceptable to write in a similar way to how I talk. My role as web publication officer for the ECS has given me the chance to test this. Chatting is fun and so I figured it would make writing less laborious.

Gift of the gab
So why didn’t I write like this sooner? Part of the problem was a fear people would be judgemental. Most people tell me I talk too much. Many question my work ethic, highlighting my endless chatter. With this kind of feedback, I was afraid to write in a conversational manner. There was also a concern that this style wouldn’t be suitable for examination. It took some persuasion before I would write this way for an academic or peer audience. Since employing this tactic, readers of my work seem happier, as the text has passion and narrative. Submit work that’s easier to read and there’s a greater chance an reviewer will view it with a kinder eye.

On your marks
What are my tips for penning an assignment? Start by looking at the marking scheme. The area with the highest scores should demand the most time and effort. This may not mean they are the largest sections though. For example, you may spend a lot of time doing data analysis for the results section, but only present two graphs. This may get you a lot of marks, even though it is one or two pages.

Keep it simple
Next up – content. Think of the reader. It’s one thing to write everything you know on a topic, but would anyone want to read it? Keep it simple by making the key points. It may help to list the main points before starting to write. It’s easier to work backwards and give the reader the information they need to understand your main findings. Be careful with your use of acronyms and jargon. It may make the work seem ‘smart’ but if the reader can’t easily understand it, you may suffer as a result.

Mapping the route
Planning is key. You need to have researched the topic and understand how you got to the final points. Think about your own journey to finding that information. I often like to clarify the basic. For example, I worked with two bacteria species: Escherichia coli and Enterococci faecalis. Even in that one line, there’s a clarification that the presented
species names are indeed bacteria. Many of you will know that, but I only knew of E. coli before my PhD, E. faecalis
could have been anything, maybe a little dung beetle?

Build the scaffolding
Once you know the key points, it’s an idea to create a document with the points as titles. These can be removed later but it solves the problem of staring at a blank page. You can then start to fill in info under each title and reference later by inserting (ref) throughout the work. It can help to jump between sections. Bored or uninspired? Start adding info under a different title and return later. You just need to sit down and write. This was something I found hard, so ask a friend to help. Alternatively, why not set a reward or punishment to ensure you meet your writing target. My reward was an ice cream after dinner if a target was reached. If not, I made myself write at the weekend or miss swimming to make up the time. Forgoing swimming was probably the most severe punishment I could give myself, so I only failed the writing target once.

Express yourself
Final point – don’t be afraid to express your opinions. As scientists, we’re meant to give a balanced view of contrasting literature, particularly in discussion chapters. This may highlight the level of research you have done, but it can help to have a stance, or even a final note on how you think the research field will progress. It shows you’re fully engaged with your work. After editing, get feedback. Don’t worry if you get drafts that look like a blood bath. This just means someone has taken an interest in helping your work have maximum impact. Writing is a craft, it takes practice.
One of the best compliments I’ve received relating to my writing is, “I can hear you talking to me through the piece.” Focus on the compliments, as it is easy to give too much weight to criticism. So all I can do now is wish you good luck with your writing!!!

Allison Cartwright is the Publications Officer for the Early Career Scientists group of the Society of Applied Microbiology. She is currently awaiting examination of her PhD studying the microbial ecology of freshwater sponges in Ireland. Allison is a regular contributor to microbeblog.org
The Early Career Scientists (ECS) group is part of SfAM

 

The Association of The Human Papillomavirus (HPV) With P16ink4a In Head and Neck Cancer

Foy-Stones, H., Nor, N., Donegan, L., Murtagh, F., Muldoon, T.
Department of Biopharmaceutical and Medical Science, GMIT. Department of Histopathology, MRHT.

Introduction:
The Human Papillomavirus (HPV) has emerged from anonymity as a carcinogenic agent in head and neck squamous cell carcinomas (HNSCCs). This sexually transmitted virus principally targets the oropharynx, which has recently led Ireland’s department of health to consider extending the HPV vaccine to Irish men.(1) There is no gold standard for HPV testing, but HPV in situ hybridization (ISH) is employed to directly detect HPV
DNA.(2) The over expression of the tumour suppressor protein p16INK4a is detected using p16INK4a immunohistochemistry (IHC), which indirectly represents the presence of HPV.(3)

Materials and Methods:
In total, thirty-five HNSCC samples from the pharynx (n=2), oropharynx (n=18), larynx (n=14) and salivary gland (n=1) were retrospectively selected and tested using the Ventana BenchMark Ultra staining system. The detection of p16INK4a involved using the CINtec p16INK4aantibody for IHC staining and the high-risk HPVs were detected using the Ventana HPV III Family 16 Probe for ISH staining.

Results:
Analysis of the thirty-five cases demonstrated that p16INK4a tested both negative (n=17/23) and positive (n=6/23). The HPV ISH and p16 INK4a IHC correlated in 82.9% of cases. A statistical relationship between HPV and p16INK4a in the oropharynx was observed (p<0.05) with 61.1% of the selected oropharyngeal samples exhibiting positivity (n=11/18). The HPV/p16INK4a positive cases were prominent in males aged 60-69 but no statistical relationship was observed (p>0.05).

Conclusion:
The correlation between HPV and p16INK4a in the thirty-five HNSCC cases highlights that p16INK4a IHC shows potential as a screening tool that would prompt HPV ISH testing in oropharyngeal cancers cases. The link between HPV and p16INK4a in cancers of the oropharynx should be highlighted to all health care systems throughout Ireland to promote routine histopathology testing. This would allow prevalence statistics to be generated and reinforce the importance of vaccinating both males and females to reduce HPV-associated oropharyngeal cancers for future generations.

References:
1. Cancer Trends 33 – HPV-associated cancers | National Cancer Registry Ireland [Internet]. Ncri.ie. 2018 [cited 7 May 2017]. Available from:
https://www.ncri.ie/publications/cancer-trends-andprojections/cancer-trends-33-hpv-associated-cancers
2. Ventana, INFORM HPV III Family 16 Probe (B). [Pamphlet]. Arizona USA: Ventana. 2017.
3. Ventana, CINtec p16 Histology. [Pamphlet]. Arizona USA: Ventana. 2016. 34.3% tested positive for HPV and p16INK4a(n=12/35). HPV
tested negative in 65.7% of cases (n=23/35) and of these,

Persister Formation in Escherichia coli.

Dunlea, AD, School of Microbiology and APC Microbiome Ireland, UCC.

In bacteria, persister cells represent a subset of cells within a population that have the ability to survive potentially lethal stresses including antibiotic treatment. Current dogma suggests that the slow growing or dormant nature of persister cells enables them to avoid antibiotic killing while the majority of the cell population is killed (1). The clinical relevance of bacterial persistence lies with the contribution of these cells to antibiotic failure and the recurrence of infections. The aim of this study was to investigate the relationship between metabolism and persister
cell formation in Escherichia coli HM605, a colonic mucosal isolate associated with Crohn’s disease. During antibiotic stress, metabolism plays a central role in establishing and maintaining a persister state (2). The level of persister formation in E. coli HM605 was assessed using a gentamicin persistence assay (3).

Persistence of HM605 was found to be enhanced in the presence of glucose. High performance liquid chromatography analysis revealed the production of various organic acids by persister cells under antibiotic stress. Using mutants defective in the production of specific organic acids it was shown that the production of acetate, a byproduct of glucose metabolism, was not required for persister formation in E. coli HM605.

However, there was a strong correlation between HM605 persister formation and the production of lactate. These findings suggest that persister cells exist in a metabolically-active state, contradicting previous beliefs that persister cells are in a metabolically inactive or dormant state.
Moreover, these studies indicate that a better understanding of metabolic flux during antibiotic stress and persister cell formation could determine how wild type HM605 persister cells adapt to favor survival during antibiotic exposure. With this knowledge, the discovery of additional persister elimination strategies could be achieved so that the problem of antibiotic failure caused by persister cells could potentially be solved.

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Assessment of the Comparability Of CLSI, EUCAST and Stokes Antimicrobial Susceptibility Test Methods for Escherichia Coli uropathogenic isolates

O’Halloran. Cork Institute of Technology; Pathology Department, University Hospital Limerick.

Nowadays, as many clinical laboratories transition between Stokes, CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) methods for susceptibility testing of uropathogens, the problem of comparing differently-derived sets of antimicrobial susceptibility testing data with each other arises, owing to a scarcity of knowledge of inter-method comparability.

The purpose of the current study was to establish the potential degree of discrepancy associated with each of these three methods and their respective interpretive breakpoint guidelines, for six oral antimicrobial agents commonly used to treat urinary tract infection, namely
ampicillin, amoxicillin-clavulanate, trimethoprim, ciprofloxacin, nitrofurantoin and cephradine/cephalexin.

A total of 100 uropathogenic Escherichia coli isolates obtained from boric acid-treated urine samples of GP patients were investigated. For the EUCAST and CLSI testing, the Kirby-Bauer method of disk diffusion and associated interpretive guidelines was employed. For the Stokes method, direct susceptibility testing was performed on the urine samples, and results were interpreted using the Stokes guidelines.

When comparing the interpretive results, only 45% of isolates showed accordance among all three methods across the 6 antibiotics tested. Certain antibiotics had high levels of accordance (trimethoprim – 2% discrepancy), while other antibiotics had large discrepancy levels, particularly amoxicillin-clavulanate (40% discrepancy rate) and ciprofloxacin (11% discrepancy rate). The annual adjustments to EUCAST and CLSI breakpoint guidelines (from 2011-2017) also affected the comparability of the 3 methods.

Our novel data indicate that the discrepancies generated through using different AST methods and different interpretive guidelines may result in confusion and inaccuracies when prescribing treatment for urinary tract infection. These results also show that care should be taken to compare susceptibility test data between studies only when the same method has been used in each case.

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Confirmation of Clostridium difficile Toxin Expression in tcdB Gene Positive Stool Samples using an Enzyme Immunoassay and Antimicrobial Susceptibility Testing of Clostridium difficile Isolates in an Irish Hospital

P. Ryan, Galway Mayo Institute of Technology

Clostridium difficile infection (CDI) is the leading cause of infectious nosocomial diarrhoea. The organism’s virulence is related to its toxin production. The laboratory must differentiate between non-toxigenic and toxigenic strains of C. difficile (Polage et al, 2016). C. difficile AST is essential for the monitoring of emerging resistance. C. difficile AST provides invaluable epidemiological data relating to ribotype prevalence (Solomon et al, 2011). A total of 26 tcdB positive stools were tested for the presence of detectable toxin using an EIA.

Patients were divided into EIA toxin positive and EIA toxin negative groups. The white cell count (WCC), CRP value and length of stay (LOS) were recorded for each patient. The Bristol Stool Chart (BSC) score was also used to describe stools submitted in both groups. Antimicrobial susceptibility testing (AST) was performed on 27 isolates in accordance with bioMérioux Etest® guidelines. Ribotype data was available for 25 of these isolates. CLSI breakpoints were followed for the antibiotics used in the study (metronidazole, clindamycin and moxifloxacin).

Study findings, showed that the WCC (U=11.5), Ct value(U=36) and BSC score (U=44) were statistically higher in the EIA toxin positive group. The CRP (U=48.5) and LOS (34.5) were not statistically higher in the EIA toxin positive group. AST results demonstrated all isolates were susceptible to metronidazole. Clindamycin resistance appeared to be associated with ribotype prevalence.

In conclusion, the introduction of a second-step EIA test for tcdB positive stools was useful for identifying those with CDI. The reliability of metronidazole as the drug of choice for CDI was reaffirmed. The study provided further insight into the relationship between broad-spectrum antibiotic susceptibility and ribotype prevalence.

Optimisation of Recombinant GP41 Protein For Use In HIV Diagnostic Assay

Gilligan M, School of Biological Sciences, Dublin Institute of Technology

Human Immunodeficiency Virus (HIV) has been long established as the causative agent for Acquired Immune Deficiency Syndrome (AIDS). Recommendations are that initial testing for HIV should be carried out using an antigen/antibody combination immunoassay, such as an
enzyme-linked immunosorbent assay (ELISA) (1). The project objective was to assess the functionality of two variants of the reference recombinant HIV protein Gp41 used in a commercial ELISA kit; a wild-type variant with a poly-His tag fused to its C-terminus and a mutant variant without cysteines produced using splicing by overlap extension PCR. High-throughput cloning and transformation methods were used to establish the optimal expression vectors for both constructs via expression screening (2). The best expression vectors, determined by SDS-PAGE and Western Blot analysis, were selected for large-scale expression testing. The protein was purified by Immobilised Metal Affinity
Chromatography (IMAC) (3). The wild-type and mutant protein products were biotinylated and their reactivity assessed in ELISA.

Although both constructs were observed to be functional in ELISA, neither the wild-type nor the mutant construct performed as well as the reference construct. The mutant construct performed comparatively well with an average reactivity of 54% of the reference standard. The wild-type protein product had an average reactivity determined to be less than 30% of the reference standard. However after use of automated purification techniques and optimisation of the biotinylation process, the mutant product was observed to be more reactive in ELISA than the current reference standard.

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CIRP As A Therapeutic Vaccination Platform Against Cancer

Keane, E, Centre for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain; DIT

The approval of immune checkpoint inhibitors (ICIs) was a major development in the treatment of certain cancers. However, response to ICI treatment is linked to high numbers of tumour infiltrating T-cells which are not always present(1). Therefore, it is of interest to produce a therapeutic vaccination capable of stimulating tumour infiltrating T-cells in order to maximise effectiveness of ICIs.
This study explored the production of a therapeutic vaccination platform based on cold-inducible RNA binding protein (CIRP), an endogenous TLR4 ligand capable of eliciting a targeted immune response when fused to tumour antigen. CIRP-linked constructs were cloned in bacterial vectors. The protein constructs were then produced and purified via affinity chromatography. Immunogenicity was tested in vivo by Enzyme-Linked ImmunoSpot (ELISPOT).
Five separate CIRP-linked constructs were successfully cloned. Two of these constructs (OVA-CIRP and Spy-CIRP) were selected for protein production and purification. The immunogenicity of the OVA-CIRP product was tested in vivo, however, no statistically significant response was observed when compared to the OVA antigen alone. Overcoming limitations in purity in future studies may improve immunogenicity of OVA-CIRP.
Additionally, the effectiveness of a previously established construct SIIN-CIRP was tested in combination with ICIs (anti-CTLA4 and anti-PD1) against tumours in murine subjects. Tumour volume and survival rates were measured over 56 days.
The treatment group receiving SIIN-CIRP and ICIs in combination resulted in delayed tumour growth and improved survival rates overall, although results failed to reach statistical significance when compared with ICI treatment alone. Repeating the study with improved homogeneity of tumour size at treatment initiation and increased treatment group size may yield significant results.
Further study is warranted to fully appreciate the potential of CIRP in combination therapy. However, its ability to be combined with tumour antigen and elicit an immune response remains promising.

Poster Competition BioMedica 2018

This year I had the great privilege of being the chairperson of the Poster competition at Biomedica. There were over thirty abstracts accepted for the post graduate competition, all of which reflected the high standard of research being carried out nationally in both our hospitals and research
centres. I was also delighted to be involved in the introduction of the under-graduate poster competition section to the conference this year. This is a fantastic addition to the competition as it facilitates the show-casing of the tremendous research that is being carried out by our under-graduate
medical science students from the three accredited colleges in the Republic of Ireland. We hope that this section will continue to grow over the next few years and welcome the submission of under-gradate research.

I, together with a panel of judges, had the difficult task of judging both the undergraduate and postgraduate posters. Each poster was reviewed and judged independently by each member of the panel. Overall everyone agreed that the standard was high and it was difficult to choose but a
consensus was reached for both categories and awards given at the closing ceremony to very deserving winners. It was fantastic to see the variety and high quality of research that is being carried by Medical Scientists, both at an undergraduate and post graduate level, being displayed over
the two days. I would encourage anyone who is involved in research to showcase it at every given opportunity. Remember you have put in the hours of hard work, make sure to let others see the results which are very often not only invaluable to your laboratory but also to others.
Dr Debbie Corcoran, Academy Council

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First Prize Winner
NOVEL GENOTYPING OF CRYPTOSPORIDIUM PARVUM VIA HIGH RESOLUTION MELT ANALYSIS OF TANDEM REPEAT LOCI.

Topic: Medical Microbiology
Authors: O’ Leary, JK(1); Blake L(2), Corcoran D(2), Sleator, RD(1, 3); Lucey, B(1)

1 Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland
2 Department of Medical Microbiology, Cork University Hospital, Wilton, Cork, Ireland
3 APC Microbiome Institute, University College Cork, Cork, Ireland

Abstract:
Microscopy still provides the gold standard means by which to diagnose clinical Cryptosporidium infection. However, differentiation between species and sub-species for epidemiological purposes requires the use of molecular techniques, including real time PCR. There is currently no
consensus on the most suitable method for sub-speciation of Cryptosporidium spp1.

High resolution melt (HRM) analysis has been used successfully with pathogens other than Cryptosporidium for subtyping purposes2, while within Ireland, approximately 80% of clinical cases of Cryptosporidium infection are attributable to Cryptosporidium parvum3. The purpose of the current study was to develop a method capable of differentiating between C. parvum sub-species.

This was approached by first identifying a panel of established variable number tandem repeat (VNTR) loci known to vary between C. parvum sub-species. These VNTR loci were targeted via nested real time PCR and HRM analysis. HRM
analysis is based upon the detection of small genetic variations via minute differences in melting temperature determined when amplified double-stranded DNA is subjected to denaturation and reannealing. The generated HRM data was subsequently analysed via melting curves and melting peaks, allowing for differentiation between sub-species. Multi-locus sequence typing (MLST) analysis was employed in conjunction with HRM
in order to definitively identify the sub-type of each clinical sample and verify HRM results.

This application was employed to discriminate between allelic differences within the selected loci, based on the variable number and sequence of the tandem repeats within these regions. Our newly-developed method was tested using a set of 12 sequenced reference samples from the Cryptosporidium Reference Laboratory (CRU), Cardiff, Wales, and a bank of clinical Cryptosporidium-containing samples (2015-2018) from the department of Clinical Microbiology, Cork University Hospital. Our study reports on the analysis of these samples, and provides a novel method for the subtyping of C. parvum.

References
1. Chalmers RM, Robinson G, Hotchkiss E, et al. Suitability of loci for multiple-locus variable-number of tandem-repeats analysis of Cryptosporidium parvum for inter-laboratory surveillance and outbreak investigations. Parasitology. 2017;144(1):37-47.doi:10.1017/S0031182015001766
2. Keeratipibul S, Silamat P, Phraephaisarn C, et al. Genotyping of Salmonella enterica Serovar Typhimurium Isolates by Multilocus Variable Number of Tandem Repeat High-Resolution Melting Analysis (MLV-HRMA). Foodborne Pathog Dis. 2015;12(1):8-20. doi:10.1089/fpd.2014.1761
3. Zintl A, Proctor A., Read C, et al. The prevalence of Cryptosporidium species and subtypes in human faecal samples in Ireland. Epidemiol Infect. 2009;137(2):270-277. doi:10.1017/S0950268808000769

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2nd Prize
TARGETING THE DNA DAMAGE RESPONSE IN CANCER: FUNCTIONAL CHARACTERISATION OF NOVEL RAD9-INTERACTING PROTEINS

Kiely, AK(1), O’Halloran, FO’H(1), Grenon, MG (2,3), Lowndes, NL (2,3) and Finn, KF(1).

1. Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Co. Cork
2. Department of Biochemistry, School of Natural Sciences, National University of Ireland, Galway
3. Centre for Chromosome Biology, Biomedical Sciences Building, National University of Ireland, Galway

“By 2020 it is estimated that 1 in 2 adults will get a cancer diagnosis in their lifetime”. Cancer is a disease caused largely by changes to genes that control cell growth and proliferation. These genetic changes can be inherited and/or acquired by direct damage to DNA via exposure to DNA damaging agents, such as ultraviolet (UV) radiation and carcinogenic agents, as well as by-products of normal cellular metabolism. Cells have
evolved a complex system, termed the DNA damage response (DDR), which detects DNA damage and promotes DNA repair(1).

Rad9 was the first DNA damage checkpoint protein identified in budding yeast. Loss of Rad9 function results in significant sensitivity to DNA damaging agents due to defects in the DDR (2). In undamaged cells, Rad9 exists as a large complex (>850kDa) and is remodelled following DNA damage (3). A preliminary proteomic study has identified 55 putative Rad9-interacting proteins.

This project aims to phenotypically characterise these putative interactors to determine whether these proteins play an important role in Rad9-dependent DNA damage checkpoint activation and/or DNA repair. To test this hypothesis deletion strains will firstly be analysed via droptest to determine DNA damage sensitivity to a variety of DNA damaging agents.

To date several deletion strains screened have exhibited sensitivity to (UV) radiation (80Jm-2) and/or the double-stand break-inducing antibiotic, Zeocin (4Ìg/ml). Sensitivity to Hydroxyurea (HU)-mediated DNA damage is currently ongoing. Sensitive strains identified in the screen will be further characterised to determine whether the protein of interest plays a role in DNA damage checkpoint activation (G1, intra-S, G2/M) and/or DNA repair.

As cellular events governing the DDR are highly conserved from yeast to mammals, novel findings in budding yeast may be extrapolated to humans. Thus, data from this project could significantly impact cancer research and influence the development of novel cancer therapeutics.

References
1. Jackson SP. UKPMC Funders Group The DNA-damage response_: new molecular insights and new approaches to cancer therapy. Tennis.
2010;37(Pt 3):483–94.
2. Weinert TA, Hartwell LH. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science .1988 Jul 15 [cited 2018 Mar 1];241(4863):317–22.
3. Gilbert CS, Green CM, Lowndes NF. Budding yeast Rad9 is an ATPdependent Rad53 activating machine. Mol Cell. 2001 Jul [cited 2018 Mar 1];8(1):129–36.

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3rd Prize
SUCCESSFUL IMPLEMENTATION OF DEMAND MANAGEMENT OF VITAMIN B12 AND FOLATE REQUESTS IN THE PATHOLOGY LABORATORY IN NAAS GENERAL HOSPITAL

Topic: Haematology
Claire Burke, Chief Medical Scientist, Haematology Department, Naas General Hospital.

Authors: Claire Burke, Johnny McHugh, Ronan Desmond.

In the current economic climate it is vital that Laboratories play an increasing role in ensuring that testing is evidence based.

With this in mind, we at Naas General Hospital looked at B12 and Folate requesting patterns in our catchment area. In 2016, our laboratory received 55,077 requests for B12, Folate and Ferritin. This was an increase of 44% since 2011. Naas General Hospital provides B12, Folate and Ferritin testing for a population of 180,011, which is an increase of 6.9% since 2011(HSE health atlas). The increase in numbers doesn’t correlate with the population increase.

In May 2016 the National Laboratory Handbook Volume 1 was published and together with a GP survey that was sent to our GP’s, we decided to implement demand management for B12 and Folate test requests. The demand management process was implemented in June 2017.This process involved rejection of tests based on inappropriate clinical details and assessment of the patients full blood count. The
average monthly referral decreased by 48% in 2017 in the post implementation period. To determine whether the reductions in the number of total B12 samples processed and low B12 results were statistically significant we performed and independent- samples t-test.

By the end of 2017 there was a cost saving of at least €87,000 euro.

The issuing and enforcement of guidelines produced a dramatic reduction in testing without affecting the ability of Clinicians to detect clinically relevant B12 deficiency.

References:
1. Freyer AA, Hanna FW. Managing demand for pathology tests: financial imperative or duty of care? Ann Clin Biochem 2009;200946:435-7
2. National Laboratory Handbook Volume 1

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Undergraduate Poster Winner
INVESTIGATION OF THE OVERESTIMATION OF PARAPROTEINS IN SERUM BY IMMUNOTURBIDIMETRIC METHOD: TIME FOR A
CHANGE OF PROTOCOL IN SLIGO UNIVERSITY
HOSPITAL?

Location: Sligo University Hospital & Galway-Mayo Institute of Technology
Topic: Immunology

Authors: Roe, M., Mahon, C., Montgomery, N., McGrath, M.

Introduction/ Background:
A key aspect of the treatment and monitoring of patients with monoclonal gammopathies relies on obtaining reproducible and accurate quantitation of paraproteins. Discrepancies between quantitation of paraproteins by Serum Protein Electrophoresis (SPE) and Immunoturbidimetry has been well documented [1, 2]. In Sligo University Hospital (SUH), all immunoglobulin results are assayed and reported on a daily basis, independently of SPE results which may not be reported for some days later. The aim of this research was to: • Examine the extent of discrepancies that sometimes occur in the quantitation of a paraprotein band by SPE and the result of the immunoglobulin isotype by
Immunoturbidimetry
• Evaluate the current practices in the reporting of SPE and immunoglobulin results particularly in patients with a paraprotein band
• Suggest recommendations (if required) for a change in the reporting protocol

Method:
A retrospective search was carried out of 240 serum samples that had paraproteins to examine the relationship between SPE and immunoturbidimetric analysis. Due to noted discrepancies, a selection of samples (n=75) were analysed for Immunoglobulins by Immunoturbidimetry and diluted if outside the reference range and reanalysed. From these samples, SPE (n=24) were performed. Identification of paraprotein bands was carried out and paraprotein concentrations quantified. Comparisons were made with the relevant immunoglobulin
concentration.

Results:
Significant discrepancies between Immunoturbidimetry and SPE were evident, especially at higher paraprotein concentrations. Dilution of the samples did not offer an improvement in the overestimation of paraproteins by immunoturbidimetric analysis.

Conclusions:
A consistent approach is required by laboratories and clinicians so that the paraprotein concentration is monitored on an individual basis using the same method. Based on the findings of this research project many recommendations were suggested. At a minimum, if immunoglobulin results are to be reported independently of SPE results, a disclaimer highlighting the possibilities of inaccuracies should be appended to the
result. Ideally, all immunoglobulin results should be withheld and reported only when SPE results are available. In cases where a discrepancy is evident, overestimated values by immunoturbidimetry should not be reported and only the non-paraprotein serum immunoglobulins reported to
demonstrate immunoparesis [3]. In these cases the paraprotein concentration quantified by SPE should be given reported solely.
Update: These recommendations were implemented in SUH in January 2018. All immunoglobulin results (IgG, IgA, and IgM) outside the reference range are placed in a hold file and are validated in conjunction with SPE results.

References:
[1] Jelinek, A.G. and Bachmann, L.M. (2014). Unexpected test results in a patient with multiple myeloma. Clinical chemistry, 60 (11), 1375-1378.
[2] Keren, D.F. and Schroeder, L. (2016). Challenges of measuring monoclonal proteins in serum. Clin Chem Lab Med, 54 (6), 947-61.
[3] Tate, J., Caldwell, G., Daly, J., Gillis, D., Jenkins, M., Jovanovich, S., Martin, H., Steele, R., Wienholt, L., Mollee, P., (2012). Recommendations for standardized reporting of protein electrophoresis in Australia and New Zealand. Annals of Clinical Biochemistry, 49 (3), 242-256.

 

RESEARCH

 

 

Academy Conference Bursary Award

Carol Troy, Surveillance Scientist, Microbiology Laboratory, University Hospital Waterford

Iwas honoured to receive the Academy Conference Bursary Award this year. This award provided me with an
opportunity to present the findings of my Master’s thesis as a poster at the recent European Congress of Clinical
Microbiology and Infectious Diseases (ECCMID) conference in Madrid. The title of my thesis was “A Retrospective Review of Community Associated and Healthcare Associated ESBL Producing E. coli and K.
pneumoniae in the South East of Ireland 2015-2016” and was undertaken as part of a Masters in Healthcare Infection Management in Trinity College, Dublin. This review demonstrated a significant increase in ESBL producers over a 24 month period. High levels of resistance were observed among both healthcare associated and community associated isolates. The majority of isolates were found to be community associated indicating a reservoir of ESBL in the community.

ECCMID has been an annual event since 1999. It is the annual conference of the world’s largest society European Society for Clinical Microbiology and Infectious Diseases, (ESCMID) covering microbiology and infectious diseases. This year’s conference was held in Madrid from the 21st to the 24th of April. The programme of events over the four days included educational workshops, oral sessions, keynote lectures, meet-theexpert sessions and poster presentations. In addition to the extensive educational opportunities, a large arena was also dedicated to displays by leading diagnostic and pharmaceutical companies on new and emerging trends in laboratory diagnostics and drug therapies.

Being able to present my poster at the conference allowed discussion of my findings with fellow attendees and also to explore the numerous other areas of research and study currently being undertaken on ESBL producing organisms.

With a vast array of topics on offer over the course of the four days in the various lecture halls and presentation rooms, trying to get to see all my chosen lectures was a challenge!

As my job is primarily focused on surveillance my choice of lectures were primarily in this area. Some of the more interesting lectures I was able to see included Surveillance and Prevention of Surgical Site Infection and The Burden of Healthcare Associated Infection(HAI). The implementation of a SSI programme results in positive patient outcomes and can be achieved even in the resource limited environment as was demonstrated
by a study at a Tanzanian hospital. The speakers on HAI covered a variety of topics including ESBL HAI in ICU adding to the costs of isolation, but the overwhelming message was that HAI is a worldwide problem that is being fought in every healthcare environment.

Among the diagnostic companies’ stands that I visited, the ever growing emphasis on rapid identification and antimicrobial susceptibility testing was evident. In particular the area of bacterial identification where molecular testing is providing diagnostic answers at an ever increasing pace. This can only impact positively on both the patient and the healthcare environment allowing earlier treatment of patients, and identification of patients who are infected or colonised with MDROs.

I am extremely grateful to the Academy for awarding me this bursary that allowed me to bring the findings of my study to a wider audience and also to engage in the educational experience of ECCMID.

A Retrospective Review of Community Associated and Healthcare Associated ESßL Producing E.coli and K.pneumoniae in South East Ireland 2015-2016

Background: Extended-spectrum beta-lactamases (ESBL) are a major public health problem. A retrospective study was undertaken of ESBL-producing E. coli and K. pneumoniae isolated from clinical specimens in the regional diagnostic microbiology laboratory in the South East of
Ireland during the period January 2015 to December 2016. The aim of this study was to review the patient demographics, geographical data, primary acquisition site and antimicrobial susceptibility data.

Materials/methods: Data on all ESBL-producing E. coli and K. pneumoniae for 2015 and 2016 was extracted from the laboratory and patient information management systems. This included antimicrobial susceptibilities and patient demographics. Isolates were designated healthcareassociated, community-associated, or long-term care facility associated. Under these designations, data was analysed under the following headings: age and gender distribution, length of stay for patients admitted to an acute healthcare facility, specimen types, and antimicrobial susceptibilities. In total 75 isolates from the study were examined for the presence of blaCTX-M by real time PCR.

Results: A total of 896 ESBL-producing isolates met the study criteria; 822 E. coli and 74 K. pneumoniae. SBLproducing E. coli isolates increased from 255 in 2015 to 567 in 2016 and ESBL-producing K. pneumoniae isolates increased from 27 in 2015 to 47 in 2016. This represents an overall increase of 118% in ESBL-producing isolates. Community-associated isolates increased from 57% in 2015 to 67% in 2016, healthcare-associated isolates also increased from 15% in 2015 to 20% in 2016. Long-term care facility associated isolates decreased from 28% in 2015 to 13% in 2016. Female patients represented 73% of isolates and 71% of patients >65 years of age. Urine accounted for 730 of the 896 specimens. The proportion of isolates that were multidrug-resistant rose from 74% in 2015 to 82% in 2016. In total 70/75 (90%) harboured a blaCTX-M gene (63 = blaCTX-M group 1, 7 = blaCTX-M group 9)

Conclusions: Community-associated ESBL producing E. coli and K. pneumoniae represent an increasing threat to public health. Failure to control the spread of these isolates through prudent antimicrobial use and screening may potentially result in an increase in the use of carbapenems and the spread of carbapenem resistance.

Survey of British Society of Haematology Antenatal Transfusion Guidelines in Irish Maternity Units

Eamon Clavin(1), Fergus Guilfoyle, Fabian McGrath, Catherine Flynn The Coombe Women and Infants University Hospital, Dublin Institute of Technology(1); Corresponding Author: Eamon Clavin, Blood Transfusion Laboratory, Tallaght University Hospital, eamon.clavin@amnch.ie

Abstract
Background: The British Society of Haematology (BSH) has published several guidelines regarding transfusion testing of pregnant patients. Compliance with these guidelines is not mandatory. There is likely to be variation in the extent of implementation of these guidelines in the policies of Irish maternity units. Objectives: This investigation was designed to assess the implementation of selected recommendations outlined in the BSH guideline for the estimation of fetomaternal haemorrhage, guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the foetus and newborn and the guideline for blood grouping and red cell antibody testing in pregnancy in Irish maternity units.
Materials/Methods: A survey of 10 questions was electronically generated and distributed to senior scientific staff in the transfusion laboratories of the 19 maternity units in the Republic of Ireland.
Results: A wide variation was observed in the guidelines adopted by each hospital. No single guideline was implemented in the same way in all of the maternity units surveyed. Certain hospitals reported combinations of BSH compliant practices that may be incompatible.
Conclusion: Performance of similar audits to the present study at an individual hospital level may be useful for the identification of minimally beneficial combinations of practices.

Introduction
Many of the guidelines issued by the British Society for Haematology (BSH) pertain to the prediction, prevention, monitoring and treatment of haemolytic disease of the foetus and new-born (HDFN) in transfusion laboratories of maternity units. The BSH Guidelines for the Estimation of Fetomaternal Haemorrhage (FMH) provide recommendations regarding the quantification of FMH, and the labelling of samples for such
analyses. These guidelines advise that FMH quantitation and ABO and RhD grouping should not be performed on the same maternal EDTA sample at delivery (Austin et al. 2009). The BSH has published a guideline regarding the use of anti-D immunoglobulin for the prevention of HDFN (Qureshi et al. 2014). The offering of routine antenatal anti-D prophylaxis (RAADP) to RhD negative women between weeks 28 and 34 of
pregnancy is recommended by this guideline and by the Health Service Executive (HSE) in the Republic of Ireland (ROI) (The Irish Haematology Society et al. 2012). It is recommended that cord blood samples or neonatal venous samples should be obtained for ABO and RhD grouping of neonates born to RhD negative women. This guideline advises against the routine performance of a direct antiglobulin test (DAT) on all neonatal
samples, due to the risk of positive DAT findings in the absence of HDFN. The BSH Guideline for Blood grouping and Red cell Antibody Testing in Pregnancy advises that all pregnant women should undergo ABO and RhD grouping and be screened for erythrocyte alloantibodies at booking and at 28 weeks gestation (White et al. 2016). Where antibodies with clinical significance in HDFN are not detected at week 28, no further antibody
screening is required for the remainder of the pregnancy. The BSH and The National Institute for Care and Health Excellence (NICE) have both published guidelines that recommend that anti-D, anti-c and antibodies of the Kell blood group system be quantified every 4 weeks until the 28th week of gestation and every 2 weeks thereafter until delivery. Other clinically significant antibodies detected at booking should be quantified through titration at booking and at week 28 (White et al. 2016; Royal College of Obstetricians & Gynaecologists 2014). Continuous flow analysis (CFA) is regarded as the method of choice for the quantitation of anti-D and anti-c during pregnancy. Where anti-D is detected, the BSH recommends that samples be obtained for CFA quantitation, except where the antibody is detected directly prior to delivery. Such investigations are advised regardless of whether or not the antibody is suspected to be attributable to prophylactic anti-D administration (White et al. 2016).
While BSH guidelines are evidence based and derived from literature reviews, they are not standards and therefore compliance with these recommendations is not mandatory. The guidelines are commonly adopted into clinical and laboratory policies at an individual hospital level, though the extent of their implementation in each maternity unit and laboratory in the ROI are not known.
Objectives
The aim of this study was to assess the extent and variation of implementation of selected guidelines issued by the BSH in the transfusion laboratories of all maternity units in the ROI.
Materials and Methods
A survey containing 10 questions was constructed using the QuestionPro® online survey generation resource and a link to the survey was issued via e-mail to senior or chief medical scientists in the transfusion laboratories of the 19 maternity units in the ROI in February 2017. The questions addressed specific recommendations outlined in:
• The BSH guideline for the estimation of fetomaternal haemorrhage (Austin et al. 2009),
• The BSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn (Qureshi et al. 2014), and
• The guideline for blood grouping and red cell antibody testing in pregnancy (White et al. 2016). The questions were based on observations made regarding the BSH guidelines implemented in the Coombe Women and Infants University Hospital (CWIUH). Where response options such as “other” were selected by responding hospitals, respondents were asked to describe their relevant policies.

1. What is the name of your hospital?
2. Following delivery, is the same maternal sample used for both FMH testing and group and screen analysis?
3. Are all samples for FMH testing labelled as per BSH Transfusion Guidelines? (I.E. using Handwritten or Bloodtrack®-generated labels)
4. Does your laboratory policy currently state that a DAT must be performed on all neonatal samples?
5. Is your laboratory planning on changing its policy in relation to the DAT analysis of neonatal samples? Please state the change if any.
6. Does your hospital offer RAADP to all RhD negative patients?
7. On what category of patients does your hospital currently carry out grouping and screening at approximately 28 weeks of gestation?
8. In the event of detection of an antibody in the sample of a pregnant woman, does your transfusion laboratory inform clinicians as to the clinical significance of the antibody in HDFN and potential complications of transfusion?
9. What antibody specificities are titrated or quantitated on your patients and what is the frequency of titration or quantitation?
10. In the event of detection of anti-D in the sample of a pregnant woman, where the antibody may be immune or due to prophylaxis, does your hospital send samples to the Irish Blood Transfusion Service blood establishment (National Blood Centre) for quantitation of anti-D via continuous flow analysis?

Results
Of the 19 Irish maternity hospitals to which the survey was distributed, 17 hospitals responded.

Figure 1: Responses of 17 Irish maternity units to
questions 2 (Figure 1A) and question 3 (Figure 1B)
regarding sample requirements for group and screen
(G&S) and labelling of samples for FMH testing.

 

 

 

 

 

 

 

 

 

Question 2: Following delivery, is the same maternal sample used for both FMH testing and Group and Screen analysis?
Approximately one quarter (n=4) of the responding hospitals described local FMH sampling requirements that differed from the answer options presented in this question (Fig 1A). One of these centres stated that the same sample was acceptable for group and screen (G&S) and FMH testing, provided that an
aliquot was removed for FMH testing prior to centrifugation of the sample for G&S analysis. Three transfusion laboratories stated that they did not perform FMH testing.

Question 3: Are all samples for FMH testing labelled as per BSH transfusion guidelines?
(I.E. Using handwritten or Bloodtrack®-generated labels?) Samples for FMH testing were described as being labelled according to standards other than those described by the BSH in six hospitals (35%). One of the hospitals stated that samples were labelled as per blood science guidelines before being sent to an external laboratory for analysis (Fig 1B). Another hospital stated that samples were labelled at the patient’s bedside using maternal-neonatal clinical management system
(MN-CMS)-generated labels. The remaining four hospitals described alternative policies and stated that samples for FMH testing were labelled as per local haematology guidelines.

Question 4:
Does your laboratory policy currently state that a DAT must be performed on all neonatal samples?
Four hospitals (Fig 2A) stated that their DAT policies differed from the outlined answer options. Two of these maternity units stated that DATs were performed on neonatal samples when clinically indicated by neonatal jaundice or in cases where maternal antibodies of immune origin were detected during pregnancy. Another unit stated that DATs were performed on cord blood samples following the detection of maternal antibodies during pregnancy. This unit stated that DATs were performed on neonatal venous samples, except in cases where the mother is RhD negative. The remaining maternity unit did not provide adequate information to answer this question.

Figure 2: Responses to questions 4(A), 5(B) and 6(C)
regarding current and future policies of performance of a
DAT on neonatal samples and the offering of RAADP to
RhD negative patients.

Question 5:
Is your hospital planning on changing its policy in relation to DAT analysis of neonatal blood samples?
Of the three centres (18%) that stated their intention to change their neonatal DAT policy, one specified that its policy was scheduled to change to the performance of DATs as requested or as indicated by clinical details (Fig. 2B). Another respondent indicated that their policy was scheduled to change from the performance of a DAT on all neonatal samples to the performance of these investigations only when requested. The remaining maternity unit stated that its policy of routine DAT testing of all neonatal samples was scheduled to change pending available technology but did not state the nature of the change.

Question 6:
Does your hospital offer RAADP to all RhD negative patients?
The most common policy regarding RAADP, as described by 65% of respondents (n=11) was the provision of RAADP to all RhD negative patients, regardless of the RhD phenotype of the foetus (Fig 2C). In only one maternity hospital, the policy of RAADP administration was found to consider foetal RhD type.

Question 7:
On what Category of patients does your hospital routinely perform grouping and screening at approximately 28 weeks of gestation?
Policies regarding 28 week screening of patients that differed from the presented answer options were described by two maternity units. The first of these stated that 28 week screening was performed on all RhD negative patients and only RhD positive women with a previous positive antibody screen, but certain departments within that hospital had commenced 28 week screening of all patients. Another hospital stated that alloimmunised patients were monitored, but not necessarily at the 28 week period.

Figure 3: Responses of hospitals to questions 7 (3A) and
8 (3B) regarding 28 week screening of patients and
communication of clinical significance of antibodies to
clinicians.

 

 

 

Question 8: In the event of detection of an antibody in the sample of a pregnant woman, does your transfusion laboratory inform clinicians as to the clinical significance of the antibody in HDFN and potential complications of transfusion?
Of all participating maternity units, four (23.5%) described alternative practices regarding the communication of results to clinicians. A single unit stated that clinicians were informed of the specificity, titre and clinical significance of detected antibodies via a comment in the laboratory report and were provided with a copy of reports from the referral centre where such investigations were performed. Where obtained, critical findings would be communicated to clinicians via telephone. Another of these respondents stated that the specificity and titre were routinely issued via report while quantitation results were issued as separate findings. This centre also stated that clinicians were informed of potential delays in the
crossmatching of erythrocytes arising from alloantibody detection and the unlikelihood of antibodies of the Lewis blood grouping system to cause HDFN. A single centre stated that clinicians were informed of the quantitation result and specificity of detected antibodies via laboratory report but were directed to the BSH guidelines for information on the clinical significance of the detected antibody. Another centre stated that clinicians
were informed of critical findings via telephone.

Question 9: What antibody specificities are titrated or quantified on your patients and what is the frequency of titration or quantitation?
Six of the responding hospitals stated that titrations were not performed by their transfusion laboratories and that samples requiring such investigations were referred to other centres. A policy of titration or quantitation of all clinically significant antibodies every four weeks until week 28 of gestation and every two weeks thereafter was described by three maternity units. A single maternity unit specified that titration and
quantitation of antibodies were performed at the request of clinicians. The policy of quantitation of anti-D, anti-c and titration of anti-K every four weeks until week 28 of gestation and every 2 weeks thereafter until delivery was stated to be in operation in six maternity units. Another unit described a similar policy of quantitation of anti-D and anti-c, which involved the titration of anti-K and other clinically significant antibodies at booking and week 28. Titration of other clinically significant antibodies at booking and at 28 weeks gestation was stated to be performed in these six hospitals. The remaining centre stated that samples were referred for quantitation at the request of clinicians.

Question 10: In the event of detection of anti-D in the sample of a pregnant woman, where the antibody may be immune or due to prophylaxis, does your hospital send samples to the Irish Blood transfusion Service blood establishment (National Blood Centre) for quantitation of anti-D via continuous flow analysis?
All 17 participating maternity units stated that samples for anti-D quantitation were sent to the National Blood Centre for continuous flow analysis (CFA). Almost all respondents (88%) stated that samples were sent for CFA where anti-D was known to be immune in origin, or where it was uncertain whether the antibody arose due to immunoprophylaxis or the maternal immune response. Two centres (12%) indicated that samples
were sent for anti-D quantitation regardless of history of immunoprophylaxis. No hospital indicated any other approach with regard to CFA quantitation of anti-D.

Discussion
Prior to this investigation, no published data regarding the implementation of BSH guidelines in Ireland were available. Seventeen of nineteen hospitals who received the questionnaire responded (89% response rate).

At the time of completion of this survey, the majority of maternity units (n=10) reported compliance with the BSH recommendation for separate EDTA samples for FMH and G&S testing (n=9), or allowed a single sample to be used for both investigations, provided that an aliquot was removed for FMH quantification prior to centrifugation of the sample for G&S (n=1). The compliance with this guideline of the four hospitals that use the same EDTA sample for both investigations is uncertain. The BSH states that if the same sample is used for both investigations, FMH estimation should be performed first. As these hospitals were not asked to outline the order in which these two investigations were performed, the actual level of compliance with this guideline could not be assessed by asking this question.

Compliance with the BSH recommendation for labelling of FMH samples was reported by 65% of maternity units. These units require such samples to be labelled with the name, date of birth and unique hospital number of the patient, as well as the time and date of phlebotomy and the physical signature of the individual taking the sample or electronic signature generated using a secure labelling system. A single hospital
requires samples to be labelled using an MN-CMS, though it is unclear if this system of sample labelling is compliant with BSH recommendations.

The most common practice relating to DAT analysis of neonates in the ROI was the routine performance of a DAT on all neonatal blood samples (n=11). This practice differs from BSH recommendations. Only the hospitals that stated that DATs were performed at the request of clinicians or based on clinical symptoms suggestive of HDFN (n=5) complied with this guideline. Once the scheduled changes to DAT policy outlined by two hospitals have been implemented, compliance would be expected to be observed in 41% of Irish maternity units.

When this study was conducted, the offering of RAADP to all RhD negative patients had been introduced into the policies of the majority of hospitals in the ROI (Fig. 2C). While this practice is known to significantly lower the degree of anti-D alloimmunisation and severe anti-D mediated HDFN (Pilgrim et al. 2009), provision of RAADP to RhD negative women carrying RhD negative foetuses has no benefit. The use of procedures such as cell free foetal DNA analysis may be feasible for targeting of RAADP only to women carrying RhD positive foetuses (Tiblad et al. 2013).

Six hospitals (Fig 2) were found to offer RAADP to all RhD negative mothers and routinely perform a DAT on all neonatal samples. The introduction of RAADP into systems where routine DAT analysis of all neonatal samples is performed has been shown to lead to a significant increase in the rate of positive DATs observed (Dillon et al. 2011) and may lead to unnecessary clinical and laboratory monitoring of the neonate, including increased blood sampling, which may contribute to iatrogenic anaemia.

While a considerable proportion of maternity units (n=8) complied with the BSH recommendation for 28 week antibody screening of all patients, these units were in the minority (Fig.3A). Different policies for screening of patients at 28 weeks were followed by ten other maternity units.

Clinicians in all surveyed maternity units (n=17) were reported to be informed of the titre or quantitation result and specificity of antibodies detected during antenatal screening. While eleven were found to have a policy of also informing clinicians of the clinical significance of detected antibodies, only those that communicated the clinical significance of antibodies through the laboratory report (n=9) complied with the BSH.

Six hospitals, (including the largest specialist maternity hospitals), reported that their local policy of antenatal antibody titration and quantitation complied with recommendations outlined by the BSH. The policies of these hospitals involved
referring samples for quantitation of anti-D, anti-c and performance of anti-K titration every four weeks until week 28 and every two weeks thereafter until delivery. These hospitals also reported a policy of titration of all other clinically significant antibodies at booking and at week 28.

Only a minority of maternity units (n=2) in the ROI adhere to the BSH recommendation that all samples containing anti-D, regardless of suspected origin, should be referred for CFA quantitation. The policy of referring samples containing immune anti-D or anti-D of uncertain origin, as was implemented in 15 maternity units, may be effective in cases where the history of prophylactic anti-D administration is suggestive that the anti-D was passively acquired.

While an accurate assessment of adherence to certain guidelines was not successfully obtained, this study revealed
variation in the implementation of BSH recommendations in Irish maternity units. Certain hospitals appear to have adopted incompatible combinations of BSH compliant and local policies such as the offering of RAADP to all RhD negative patients and the performance of a DAT on all neonatal samples. Further audits of practice similar to the present study conducted at an individual hospital level may be useful for identifying minimally beneficial combinations of policies.

References

Austin, E., Bates, S., Silva, M., Howarth, D., Lubenko, A., Rowley. M., Scott M., Thomas, E., White, J. & Williams, M. (2009) Guidelines for the Estimation of Fetomaternal Haemorrhage. BCSH Guidelines, 1–23.

Dillon, A., Chaudhari, T., Crispin, P., Shadbolt. & B. Kent, A. (2011) Has anti-D prophylaxis increased the rate of positive direct antiglobulin test results and can the direct antiglobulin test predict need for phototherapy in Rh/ABO incompatibility? Journal of Paediatrics and Child Health, 47, 40–43.

Pilgrim, H., Lloyd-Jones, M. & Rees, A. (2009) Routine antenatal anti-D prophylaxis for RhD-negative women: a systematic review and economic evaluation. Health Technology Assessment, 13, 1-103.

Qureshi, H., Massey E., Kirwan, D., Davies, T., Robson, S., White, J., Jones, J. & Allard, S. (2014) BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn. Transfusion Medicine, 24, 8–20.

Royal College of Obstetricians & Gynaecologists, 2014. The Management of Women with Red Cell Antibodies during Pregnancy. Green-Top Guideline 65.

The Irish Haematology Society, Institute of Obstericians and Gyanecologists, Royle College of Physicians of Ireland & Obstetrics and Gynaecology Programme HSE Directorate for Quality and Clinical Strategy. 2012. Clinical Practice Guideline: The Use of Anti-D Immunoglobulin for the Prevention of RhD Haemolytic Disease of the Newborn. Clinical Practice Guideline 13.

Tiblad, E., Taune Wikman, A., Ajne, G., Blanck, A., Jansson, Y., Karlson, A., Nordlander, E., Holländer, B. S. & Westgren, M. 2013. Targeted Routine Antenatal Anti-D Prophylaxis in the Prevention of RhD Immunisation – Outcome of a New Antenatal Screening and Prevention Program C. Thorne, ed. PLoS ONE, 8 p.e70984.

White, J., Qureshi, H., Massey, E., Needs, M., Byrne, G., Daniels, G. & Allard, S. 2016. Guideline for blood grouping and red cell antibody testing in pregnancy. Transfusion Medicine, 26, 246–263.