2020 research


HPV Primary Cervical Screening: Making the Switch from Cytology to HPV testing

Cervical screening has employed the Papanicolaou (Pap) test for over 80 years to detect cervical pre-cancer and cancer in women. The Pap test can be considered one of the most effective disease prevention strategies, preventing countless cases and mortalities associated with cervical cancer. However, it has its limitations, even with modern improvements such as liquid based cytology, sampling, sensitivity and screening subjectivity has reported up to a 40% false negative rate globally. The Human Papillomavirus (HPV) has long been known to be the primary aetiological factor in
the pathogenesis of cervical cancer and there are 14 known high-risk HPV subtypes [16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68]. Of these HPV 16 and 18 are associated with ~70% of all cervical cancer cases.

HPV testing has been used with great success in Ireland to triage low-grade cytology screening results since 2015. It has also been used as a test of cure following treatment and in the management of uncertainty at colposcopy since 2012 and 2014. More recently, using HPV rather than Cytology as the primary screening test has been shown in numerous international randomised control trials to have a significantly higher sensitivity and negative predictive value and based on this evidence, organised screening programmes worldwide are making the switch to HPV primary screening.

Following the HIQA “Heath technology assessment of HPV testing as the primary screening method for cervical screening” in 2017, The Irish Cervical Screening Programme, CervicalCheck, had committed to making this switch. In preparation for HPV primary screening, Coombe Women and Infants University Hospital, which houses the only remaining accredited Public Hospital Cervical Cytology Screening Laboratory in the country, had begun a programme of readiness for HPV testing and recently received ISO 15189 accreditation for HPV DNA testing on the Roche Cobas 4800 platform.

The core concept of HPV primary screening is to detect all women with high-risk HPV and thus those women at risk of developing cervical pre-cancer or cancer. Essentially, HPV primary screening is a test of risk for cervical malignancy, rather than of disease. But what about the high prevalence of HPV in our cervical screening population and are all HPV positive women at equal risk? The HPV primary screening approach will require that a positive HPV result is further triaged to take that test of risk into a test of disease. While many triage options exist e.g. p16/Ki67 staining of cytology smears and host gene methylation markers, with some of them being more informative than others, it has been recommended that the Pap test which has served us well so far is used to further stratify HPV positive women. There is no doubt that the addition of further risk stratification tests or the expansion of cytology as a “test of disease” will present opportunities to enhance the specificity of HPV primary screening and further refine the definition of risk.

In the future, we may see a more tailored approach, where the triage test for a HPV positive woman at the beginning of her cervical screening journey is different to a woman near exiting the programme, with HPV vaccination status also a factor. After nearly 80 years since the introduction of the Pap test cervical screening is set to dramatically change for the better with innovation and good science leading the way forward to a better cervical screening programme.

Dr Stephen Reynolds, Dr Helen Keegan, Dr Christine White, Roisin O’Brien,
Stephen Dempsey, Dr Cara Martin, Martina Ring, Prof. John O’Leary.
– on Behalf of the Cytopathology Dept. Coombe Women and Infants University Hospital, Dublin

Scientific Background on the Nobel Prize in Chemistry 2020


The Royal Swedish Academy of Sciences has decided to award Emmanuelle Charpentier and Jennifer A. Doudna the Nobel Prize in Chemistry 2020, for the development of a method for genome editing.



In 1953, J.D. Watson and F.H.C. Crick reported the molecular structure of DNA [1]. Ever since, scientists have tried to develop technologies that can manipulate the genetic material of cells and organisms. With the discovery of the RNA-guided CRISPR-Cas9 system, an easy and effective method for genome engineering has now become a reality. The development of this technology has enabled scientists to modify DNA sequences in a wide range of cells and organisms. Genomic manipulations are no longer an experimental bottleneck. Today, CRISPR-Cas9 technology is used widely in basic science, biotechnology and in the development of future therapeutics [2].

The discovery of the CRISPR-Cas system in prokaryotes.
The work that eventually led to the discovery of the powerful CRISPR-Cas9 system for genome editing began with the identification of repeated genome structures present in bacteria and Archaea. In 1987, a report noted an unusual repeated structure in the Escherichia coli genome, which contained five highly homologous sequences of 29 base pairs (bp), including a dyad symmetry of 14 bp that were interspersed by variable spacer sequences of 32 bp [3]. Some years later, similar, repeated structures were identified in the genome of the halophilic Archaea Haloferax mediterranei, with 14 almost perfectly conserved sequences of 30 bp, repeated at regular distances [4].

Subsequent bioinformatics analyses revealed that these types of repeats were common in prokaryotes and all contained the same peculiar features: a short, partially palindromic element occurring in clusters and separated by unique intervening sequences of constant length, suggesting an ancestral origin and high biological relevance [5]. The term CRISPR was introduced, an abbreviation for clustered regularly interspaced short palindromic repeats [6].

An important step towards understanding the function of CRISPR came with the identification of CRISPR-associated (cas) genes, a group of genes only present in CRISPR-containing prokaryotes and always located adjacent to CRISPRs. The identified cas genes encoded proteins with helicase and nuclease motifs suggesting a role in DNA metabolism or gene expression [6]. The association with CRISPR was used as a defining characteristic and over the coming years a number of Cas protein subfamilies were described [7, 8].

The functional importance of the CRISPR loci remained elusive until 2005, when researchers noted that the unique CRISPR sequences were derived from transmissible genetic elements, such as bacteriophages and plasmids [9-11]. Prokaryotes carrying these specific sequences appeared protected from infection, since plasmids or viruses containing a sequence matching a spacer (named protospacers) were usually absent in the prokaryote carrying the spacer [9, 11].

These correlative findings suggested a function for CRISPRs in prokaryotic defence against invading foreign DNA and the spacer sequences were described as a ‘memory of past “genetic aggressions”’ [10]. It had already been shown that CRISPRs were transcribed into long RNA molecules (pre-crRNA), which were subsequently processed by cleavage within the repeat sequences to yield small CRISPR-RNAs (crRNAs) [4, 12]. Taken together these observations indicated that crRNA could play a role in targeting viral nucleic acids, perhaps in a manner similar to RNAi in eukaryotic cells. It was also hypothesized that the Cas proteins was involved in this process [9].

Later research has indeed demonstrated that crRNA binds to one or more Cas proteins to form an effector complex that targets invading nucleic acids. Extensive efforts during the past 25 years have identified a number of different CRISPR-Cas systems, which are now divided into two major classes [13]. In the Class 1 systems, specialised Cas proteins assemble into a large CRISPR-associated complex for antiviral defence (Cascade). The Class 2 systems are simpler and contain a single multidomain crRNA-binding protein (e.g. Cas9) that contains all the activities necessary for interference.

CRISPR-Cas functions as an adaptable defence system
The hypothesis that CRISPR-Cas systems could confer resistance to invading foreign DNA was verified in 2007 [14]. In an elegant set of experiments, scientists studied a Class 2 system in a strain of Streptococcus thermophilus, which they infected with virulent bacteriophages. Next, bacteria resistant to infection were isolated and their CRISPR loci analysed. The experiment revealed that resistant bacteria had acquired new spacer sequences, which matched sequences within the infecting phage used to select resistance. Deletion of the spacer region led to loss of resistance, and the phages that were able to grow on resistant bacteria had accumulated mutations in the protospacer sequence in the phage genome. Furthermore, inactivation of one of the cas genes (cas5) resulted in loss of phage resistance. The experiments thus demonstrated a role for cas gene products in CRISPR-Cas–mediated immunity and that the specificity of the system was dependent on the spacer sequences [14].

Further insights into the function of CRISPR-Cas came from investigations of E. coli, which contains a Class 1 CRISPR-Cas system encoding no less than eight different Cas proteins. Five of these gene products could be purified as a multiprotein complex termed Cascade (CRISPR-associated complex for antiviral defence). Cascade was shown to function in pre-crRNA processing, cleaving the long transcripts in the repeated regions and thereby producing shorter crRNA molecules containing the virus-derived sequence [15]. After cleavage, the mature crRNA molecules were retained by Cascade, and, assisted by a cas-encoded helicase, Cas3, they served as guide molecules that enabled Cascade to interfere with phage proliferation. The results thus suggested two different steps in CRISPR function: first, CRISPR expression and crRNA maturation, and second, an interference step that required the Cas3 protein. The results also provided evidence suggesting that the E. coli CRISPR-Cas system targets phage DNA and not RNA, inasmuch as crRNA with complementarity to either of the two DNA strands could interfere with phage proliferation [15].

Conclusive evidence for DNA being the target of CRISPR-Cas interference came from elegant experiments using a strain of Staphylococci epidermidis that contained a CRISPR array with a spacer sequence homologous to a gene present in a conjugative plasmid [16]. Transfer of the plasmid into the strain occurred only if the spacer sequence was mutated or deleted. A self-splicing intron was inserted into the target sequence on the plasmid. In this way, the CRISPR spacer would be complementary not to the DNA, as it is disrupted by an intron, but to the RNA, which would be spliced, reconstituting the sensitive target. Indeed, insertion of the self-splicing intron was sufficient to overcome CRISPR-Cas inhibition of plasmid transfer, strongly implicating DNA as the primary target [16]. This conclusion was further supported from studies of S. thermophilus, in which the CRISPR-Cas system was shown to cleave both bacteriophage and plasmid DNA in vivo [17].

Protospacer adjacent motifs distinguish CRISPR from invading DNA.
If spacers lead to cleavage of DNA with matching sequences, how do they avoid cleaving their own CRISPR spacers? The answer to this question came from studies of sequences around protospacers, i.e. the sequences in the phage genomes that had given rise to spacers. Short sequence motifs were noted just a couple of nucleotides away from protospacer sequences [11, 18]. These motifs were later labelled protospacer adjacent motifs or PAMs [19].

The functional importance of PAMs became clear from work studying the phage response to CRISPR-encoded resistance in S. thermophilus. In these studies, phages that had overcome bacterial resistance were isolated and analysed. These studies revealed that a number of those resistant to CRISPR immunity had acquired mutations in the PAMs, implicating these short sequences as important for targeting [20]. Later studies have demonstrated that the PAM sequences are required both for target interference and for uptake of new spacer sequences into CRISPRs [21, 22].

Discovery of the CRISPR-Cas9 system
By 2011, it was clear that CRISPR-Cas systems were widespread in prokaryotes and functioned as adaptive immune systems to combat invading bacteriophages and plasmids (Figure 1). Studies had also established that the Cas proteins functioned at three different levels: (i) integration of new spacer DNA sequences into CRISPR loci, (ii) biogenesis of crRNAs, and (3) silencing of the invading nucleic acid [23, 24].

The identification of CRISPR-Cas9 as a tool for genomic editing came from studies of the Class-2, Type-II CRISPR-Cas system in S. thermophilus and the related human pathogen Streptococcus pyogenes. This system contains four cas genes, three of which (cas1, cas2, csn2) are involved in spacer acquisition, whereas the fourth, cas9 (formerly named cas5 and csn1), is needed for interference [14]. In support of this notion, inactivation of the cas9 gene prevented cleavage of target DNA [17]. To further define the elements required for immunity, the S. thermophilus CRISPR-Cas system was introduced into E. coli, where it provided heterologous protection against infection with phages and plasmids [25]. Using this experimental model, parts of the system were inactivated to define the components required for protection. The work clearly demonstrated that the Cas9 protein alone was sufficient for the CRISPR-encoded interference step, and that two nuclease domains present in the protein, HNH and RuvC, were both required for this effect [25].

Figure 1. A general scheme for the function of the CRISPR-Cas adaptive immune system as presented in [26]. Three stages are identified. Adaptation: Short fragments of double-stranded DNA from a virus or plasmid are incorporated into the CRISPR array on host DNA. crRNA Maturation: Pre-crRNA are produced by transcription and then further processed into smaller crRNAs, each containing a single spacer and a partial repeat. Interference: Cleavage is initiated when crRNA recognize and specifically base-pair with a region on incoming plasmid or virus DNA. Interference can be separated both mechanistically and temporally from CRISPR acquisition and expression.

Discovery of tracrRNA and its role in crRNA maturation
In 2011, Emmanuelle Charpentier and colleagues reported on the mechanisms of crRNA maturation in S. pyogenes [27]. Using differential RNA sequencing to characterize small, non-coding RNA molecules, they identified an active CRISPR locus, based on expression of pre-crRNA and mature crRNA molecules. Unexpectedly, the sequencing efforts also identified an abundant RNA species transcribed from a region 210 bp upstream of the CRISPR locus, on the opposite strand of the CRISPR array (Figure 2a).

Figure 2. Identification of tracrRNA in S. pyogenes as reported in [27]. a. Differential RNA sequencing (dRNA-seq) reveals expression of tracrRNA and crRNAs. Sequence reads of cDNA libraries of RNA are shown on top. Below is the genomic organisation of tracrRNA and CRISPR01/Cas loci. Red bar: tracrRNA is encoded on the minus strand and detected as 171-, 89- and ~75-nt tracrRNA species. Black rectangle inside the red bar: 36-nt sequence stretch complementary to CRISPR01 repeat. The pre-crRNA is encoded on the plus strand. Black rectangles: CRISPR01 repeats; green diamonds: CRISPR01 spacers; 511, 66 and 39-42 nt: pre-crRNA and processed crRNAs. b. Base-pairing of tracrRNA with a CRISPR01 repeat is represented. Cleavages observed by dRNA-seq and leading to the formation of short overhangs at the 3′ ends of the processed RNAs are indicated by two black triangles.

The transcript was denoted trans-encoded small RNA (tracrRNA) and contained a stretch of 25 nucleotides (nt) with almost perfect complementarity (1-nt mismatch) to the repeat regions of the CRISPR locus, thus predicting base pairing with pre-crRNA [27]. The RNA duplex region that would form included processing sites for both pre-crRNA and tracrRNA, which immediately suggested that the two RNAs could be co-processed upon pairing (Figure 2b).

In support of the proposed idea, deletion of the tracrRNA locus prevented pre-crRNA processing and vice versa. Charpentier and colleagues also noted that a co-processed duplex involving tracrRNA and pre-crRNA would have short 3′ overhangs, similar to those produced by the endoribonuclease RNase III, and they went on to demonstrate that this enzyme could process a heteroduplex formed between tracrRNA and pre-crRNA in vitro and was required for tracrRNA and pre-crRNA processing in vivo. Finally, the researchers found that processing also involved the Cas9 protein, since deletion of the cas9 gene in bacteria impaired both tracrRNA and pre-crRNA processing. Based on their findings, Charpentier and coworkers suggested that the Cas9 protein acts as a molecular anchor that facilitates base pairing between tracrRNA and pre-crRNA, which in turn allows recognition and cleavage by the host RNase III protein [27].

Previous reports had revealed the importance of Cas9 for interference. Charpentier and Jennifer A. Doudna initiated a collaboration to investigate if crRNA could be used to direct the sequence specificity of the nuclease. In contrast to what had been hypothesised in Charpentier’s report a year earlier, addition of crRNA to purified Cas9 could not stimulate Cas9-catalysed target DNA cleavage [27, 28].

At this point, the two scientists made a crucial discovery. Addition of tracrRNA to the in vitro reaction triggered Cas9 to cleave the target DNA molecule. The tracrRNA thus had two critical functions: triggering pre-crRNA processing by the enzyme RNase III and subsequently activating crRNA-guided DNA cleavage by Cas9.

In a series of in vitro biochemistry experiments, the researchers investigated the biochemical mechanisms of the reaction [28]. The two nuclease domains in Cas9, HNH and RuvC, were each shown to cleave one strand of target DNA. Cleavage occurred 3 bp upstream of the PAM sequence, which in S. pyogenes has the sequence 5′-NGG-3′, with N corresponding to any of the four DNA bases. Furthermore, as predicted from previous reports, target recognition and cleavage were inhibited by mutations in the PAM sequence [20].

A peculiar aspect of PAM sequence dependence was that cleavage of double-stranded DNA was sensitive to mutations in both the complementary and non-complementary strand whereas cleavage of single-stranded DNA targets was unaffected by mutations in the PAM motif. These observations led the authors to conclude that PAM motifs may be required to allow duplex unwinding [28].

Similar findings were also published in another report using the related CRISPR-Cas system in Streptococcus thermophilus. As in Charpentier and Doudna’s work, this report also demonstrated that Cas9 cleaves within the protospacer, that cleavage specificity is directed by the crRNA sequence, and that the two nuclease domains within Cas9, each cleave one strand. However, the researchers did not notice the crucial importance of tracrRNA for sequence-specific cleavage of target DNA [29].

In their study, Charpentier, Doudna and colleagues also worked to delineate the regions of tracrRNA and crRNA that are absolutely required for Cas9-catalysed cleavage of target DNA. This led to the identification of an activating domain in tracrRNA and the realisation that a “seed region” of ∼10 nt in the PAM-proximal region of the target strand was especially important for target recognition.

Based on their in vitro biochemical analysis, the authors hypothesized that the structural features in the two RNA molecules required for Cas9-catalysed DNA cleavage could be captured in a single RNA molecule. In a crucial experiment, they demonstrated that this was indeed possible: the RNA components (crRNA and tracrRNA) of the Cas9 complex could be fused together to form an active, chimeric single-guide RNA molecule (sgRNA).
Furthermore, Charpentier and Doudna demonstrated that the sequence of the chimeric sgRNA could be changed so that CRISPR-Cas9 would target DNA sequences of interest, with the only constraint being the presence of a PAM sequence adjacent to the targeted DNA. They had thus created a simple two-component endonuclease, containing sgRNA and Cas9, that could be programmed to cleave DNA sequences at will.

The importance of this finding was not lost on them. In the abstract of the paper reporting their findings, the authors wrote: “Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA programmable genome editing” [28].

A molecular understanding of the CRISPR mechanism
Today, there is a detailed structural understanding of how the Cas9-gRNA complex recognizes its target and mediates cleavage. This information has been important for efforts to engineer new versions of the system, with altered PAM specificity and reduced off-target activities [30].

The structure of Cas9 in free form revealed two distinct lobes, the recognition (REC) lobe and the nuclease (NUC) lobe, with the latter containing the HNH and RuvC nuclease domains. When Cas9 binds to sgRNA, it undergoes a structural rearrangement, with the REC lobe moving towards the HNH domain (Figure 3).


Figure 3. A schematic representation of the mechanism by which CRISPR-Cas9 recognizes and targets DNA for cleavage as presented in [30]. Binding of sgRNA leads to a large conformational change in Cas9. In this activated conformation, the PAM-interacting cleft (dotted circle), becomes pre-structured for PAM sampling, and the seed sequence of sgRNA is positioned to interrogate adjacent DNA for complementarity to sgRNA. The process starts with PAM recognition, which in the next step leads to local DNA melting and RNA strand invasion. There is a step-wise elongation of the R-loop formation and a conformational change in the HNH domain to ensure concerted DNA cleavage. Abbreviations: bp, base pair; NUC, nuclease lobe; PAM, protospacer adjacent motif; REC, recognition lobe; sgRNA, single-guide RNA.

For target recognition, the 20-nt spacer sequence must form complementary base pairs with the protospacer sequence. In the structure of Cas9 in complex with sgRNA, the 10-nt seed sequence in the spacer adopts an A-form conformation and is positioned to engage with the target sequence in DNA [31, 32]. The seed sequence is located in the 3′ end of the 20-nt spacer sequence and is essential for target recognition [25, 28, 33]. In genome editing, similarities between the seed sequence and genome sequences can cause off-target effects, even if there are many mismatches elsewhere in the spacer region of sgRNA [34].

As noted, a PAM sequence must also be present next to the target site, and mutations in this motif prevent Cas9-dependent cleavage at the target sequence. The Cas9 protein first searches for the PAM sequence, and once found, probes the flanking DNA for complementarity to the sgRNA. The GG dinucleotides in PAM are recognized by base-specific hydrogen-bonding interactions with two arginine residues in a PAM interacting site, which is disordered in the apo-form of Cas9, but becomes ordered after sgRNA binding. The interactions between PAM and Cas9/sgRNA lead to destabilization of the adjacent double-stranded DNA, which in turn facilitates for sgRNA to invade the double-stranded DNA. The destabilization is in part explained by a kink in the target DNA strand, which is caused by Cas9 interactions with the phosphate group immediately upstream of the PAM in the same strand [22].

Once a stable RNA–DNA duplex, an R-loop, has been formed, Cas9 is activated for DNA cleavage. Each of the two nuclease domains cleaves one strand of the target double-stranded DNA at a specific site 3 bp from the 5′-NGG-3′ PAM sequence, and in most cases, the ends that are formed are blunt. By inactivating one of the two domains, a nickase can be formed, i.e. an enzyme that cleaves only one strand of a DNA duplex [28, 29]. Nickases are very useful for practical applications of CRISPR-Cas systems, since they can be programmed to target opposite strands and thus make staggered cuts within the target DNA. In this way, a Cas9 nickase mutant, combined with a pair of sgRNA molecules, can introduce targeted double-strand breaks with very high sequence specificity [35].

The application of the CRISPR-Cas9 technology in higher cells
Genome editing relies on the existence of natural pathways for DNA repair and recombination. Double-stranded breaks typically lead to either non-homologous end joining (NHEJ) repair or homology-directed repair (HDR). In the case of NHEJ, the ends are directly ligated back together and the process usually results in a small insertion or deletion of DNA at the break, frequently causing frame shifts in coding sequences and loss of protein expression. The HDR pathway instead uses a homologous DNA sequence as a template to repair the break. By introducing modified genetic sequences as templates for the HDR, it is thus possible to introduce defined genomic changes such as base substitutions or insertions.

DNA can be introduced into mice embryonic stem cells and recombine there with the matching sequence within the host genome to produce gene-modified animals. This method is powerful but labour-intensive, since recombination events are rare and require a selectable marker, such as an antibiotic resistance gene, to be identified. Recombination efficiency is enhanced if a double-stranded break is introduced at the site of the desired recombination event, which led to a search for endonucleases that can be programmed to cleave DNA at locations of interest.

An important earlier step in the engineering of sequence-specific nucleases came with the development of zinc finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs). When linked to a nuclease domain, zinc finger proteins can function as site-specific nucleases that can cleave genomic DNA in a sequence-specific manner and stimulate site-specific recombination [36, 37]. TALENs provide yet another DNA-binding modality that recognizes DNA in a modular fashion and that can be fused to nuclease domain [38]. Both ZFNs and TALENs are powerful tools for genome editing. However, their widespread use has been limited by the inherent difficulties of protein design, synthesis and validation.

In their work, Charpentier and Doudna defined a simple two-component system that could rapidly be programmed for sequence-specific cleavage of target DNA and thereby sparked a revolution in genome editing. The first experimental demonstration that CRISPR-Cas9 could indeed be harnessed for genome editing in human and mouse cells came in early 2013 [39, 40]. These influential studies demonstrated that Cas9 nucleases could be directed by crRNA of a defined sequence to induce precise cleavage at endogenous genomic loci in mouse and human cells. For the reaction to occur, tracrRNA, crRNA, and Cas9 were all required, whereas RNase III was replaced by endogenous enzyme activities.

Just as observed by Charpentier and Doudna in vitro, the system could be further simplified in vivo, and a chimeric sgRNA molecule together with Cas9 was sufficient to cleave target DNA. The system has also been used to introduce genome modification in a number of other eukaryotic systems [41], including Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Danio rerio and Arabidopsis thaliana [42-46], demonstrating its broad applicability.

In ongoing work, scientists are trying to expand the usefulness of the CRISPR-Cas system for genome editing. In addition to Cas9 from S. pyogenes, a number of other Cas homologues are used today for genome editing and related purposes. Naturally occurring CRISPR systems have other PAM requirements, and in addition, new Cas9 variants are continually engineered to have altered PAM compatibilities. CRISPR-Cas systems can also be used to target RNA. Studies of Pyrococcus furiosus demonstrated that in this species, the system encodes for a crRNA-guided Cas complex, which targets foreign mRNA [47].

Efforts are also under way to develop evermore precise CRISPR-Cas–based genome editing strategies [48]. These efforts include strategies for base editing at specific sites in eukaryotic genomes (Figure 4). As an example, a cytidine deaminase enzyme has been fused to a mutant form of Cas9 that cleaves only one strand – a nickase. When programmed with sgRNA for the desired sequence, this system can be targeted to a specific genomic location, induce a nick in the DNA there, and mediate the direct conversion of cytidine to uridine, which after replication results in a cytosine-to-thymine conversion [49].

Another elegant example is a method called prime editing, in which a Cas9 nickase is fused to a reverse transcriptase enzyme [50]. In this approach, the sgRNA contains an additional piece of RNA, creating a “prime editing guide RNA” that both specifies the target site and encodes the desired edit. Once produced by the reverse transcriptase, the DNA synthesized can be installed at the nick, replacing one of the original DNA sequences.

Figure 4. Genome editing with Cas9 as presented in [48]. a. The Cas9 enzyme is directed to target DNA by a guide RNA and produces a double-stranded break. A piece of DNA can be used as a template for homology-directed repair (HDR). b. Cas9 can be fused to a deaminase enzyme. The mutant Cas9 produces a nick, which stimulates deaminase activity. The deaminase converts a cytidine base (C) to uracil (U). DNA repair then repairs the nick and converts a guanine–uracil (G–U) intermediate to an adenine–thymine (A–T) base pair. c. Prime editing. A nick-producing Cas9 and a reverse transcriptase enzyme produce nicked DNA, into which sequences corresponding to the guide RNA have been incorporated. The original DNA sequence is cut off, and DNA repair then fixes the nicked strand to produce a fully edited duplex.

Concluding remarks
In 2012, Charpentier and Doudna reported “that the Cas9 endonuclease can be programmed with guide RNA engineered as a single transcript to cleave any double-stranded DNA sequence” [28]. Their discovery has led to widespread applications of the CRISPR-Cas9 system as a powerful and versatile tool in genome editing.

By introducing a vector encoding the Cas9 nuclease and an engineered sgRNA, scientists are now able to make precise single-base-pair changes or larger insertions. Coupled with the availability of genome sequences for a growing number of organisms, the technology allows researchers to explore these genomes to find out what genes do, move mutations that are identified as associated with disease into systems where they can be studied and tested for treatment, or where they can be tested in combinations with other mutations. The technology has enabled efficient targeted modification of crops and is currently being developed to treat and cure genetic diseases, for instance by modifying hematopoietic stem cells to treat sickle cell disease and β-thalassemia.

Finally, it should be emphasised that the power of the CRISPR-Cas9 technology also raises serious ethical and societal issues. It is of utmost importance that the technology is carefully regulated and used in responsible manner. To this end, the World Health Organization has recently established a global multi-disciplinary expert panel to examine the scientific, ethical, social and legal challenges associated with human genome editing, with the aim to develop a global governance framework for human genome editing.

Claes Gustafsson
Professor of Medical Chemistry
Member of the Royal Swedish Academy of Sciences
Member of the Nobel Committee for Chemistry


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An evaluation of the clinical effectiveness of the EUCAST Rapid Antimicrobial Susceptibility Testing (RAST) method for positive blood cultures
at Letterkenny University Hospital.

Julie McGee

Sepsis is a medical emergency; the mortality rate of sepsis is greatly increased with each hour treatment is delayed. This lifethreatening condition is as common as heart attacks and kills more people than any cancer. Septic shock is the most severe form of sepsis and is seen in 10-20% of intensive care unit admissions. Traditional laboratory methods for identification and susceptibility testing of positive blood cultures are labour
intensive with long turnaround times. The new standardised EUCAST rapid antimicrobial susceptibility testing disk diffusion method directly from positive blood cultures gives the opportunity to improve current identification and antimicrobial susceptibility testing turnaround times. Susceptibility breakpoints for 8 of the most common blood stream pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis can be accurately
determined within 4-8 hours of a blood culture signalling positive.

This study aimed to evaluate the EUCAST RAST disk diffusion method at Letterkenny University Hospital. Timely microbiology analysis in bacteremia or sepsis is critical for improving patient outcome. Bacteria were identified using MALDI-TOF MS. Results were interpreted using EUCAST rapid disk diffusion method. The EUCAST RAST recommended reading times at 4,6- and 8-hours incubation were compared to the standard Vitek 2 susceptibility results. Additionally, investigation of acceleration of Vitek 2 susceptibilities at 8 hours incubation to provide a final susceptibility report 24hrs earlier was carried out. RAST and accelerated Vitek 2 susceptibilities was performed on a total of 146 positive blood cultures at 8 hours incubation.

The optimal time for zone reading was found to be 8 hours incubation. During this study there were six Extended Spectrum ßeta Lactamase producing E. coli isolates and four Vancomycin Resistant Enterococci isolates detected from positive blood cultures. Six of these patients benefited with escalation of appropriate antimicrobial treatment at 8 hours after their blood culture signaled positive. There was strong agreement in this comparison study with a low number of errors recorded. Accelerated Vitek 2 susceptibilities delivered excellent agreement when compared to overnight incubation. RAST combined with accelerated Vitek 2 analysis is an enormous quality improvement in Blood culture processing at Letterkenny University Hospital providing a complete AST report 24hrs earlier than using the standard method.

Julie Mc Gee has worked in the microbiology laboratory of Letterkenny University Hospital for 15 years and has recently
completed a Biomedical science MSc with Ulster University. This has unlocked an interest in quality improvement in clinical
microbiology. There are always ways to do things better.



An evaluation of the operational, clinical and economic impacts of a rapid molecular influenza and Respiratory Syncytial Virus diagnostic testing service at Letterkenny University Hospital.

Laura Wilson,1 Dr. Jacqui Clarke1 and Judith Rodgers1.
1 Pathology Department, Letterkenny University Hospital, Kilmacrennan Road, Letterkenny, Co. Donegal F92 AE81.

Influenza and Respiratory Syncytial Virus (RSV) outbreaks are burdensome on hospital resources and are associated with increased emergency department presentations and hospital admissions each winter season. The early, rapid and accurate laboratory diagnosis of influenza and RSV is necessary to facilitate prompt clinical decision making while also enabling the initiation of infection control measures in reducing the risk of nosocomial spread. To determine if onsite rapid molecular diagnostic testing for influenza and RSV has any impacts on patient care and treatment, accurate impact evaluations are required.

This study aimed to assess the operational, clinical and economic impacts following the implementation of an onsite rapid molecular diagnostic test for influenza and RSV at Letterkenny University Hospital. A large retrospective data analysis including 2,391 patients tested over two time periods (referral testing v’s onsite testing) was conducted to evaluate the impacts that a newly implemented onsite rapid testing service had on inpatient length of stays, testing turnaround times, the prescribing rates of Oseltamivir and influenza vaccine uptake rates among healthcare workers. In addition, an economic evaluation was carried out to evaluate the cost-effectiveness and feasibility of the onsite testing service.
An instrument evaluation comparing two commercially available assays, the Cepheid® GeneXpert Xpress Flu/RSV and Roche® Cobas® LIAT Flu/RSV, was conducted to determine performance characteristics and assay suitability when compared to a gold standard Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) comparator test. Both the GeneXpert and Roche Cobas LIAT are rapid, automated, in-vitro diagnostic tests employed for the qualitative detection and differentiation of influenza A/B and RSV RNA using RT-PCR in the nasopharyngeal swabs of patients presenting with influenza-like-illness.

Results The mean inpatient length of stay during the onsite testing period was significantly reduced by three days (P <0.001) for influenza positive patients when compared to the previous referral testing period (5 days vs 2 days). A significant reduction in turnaround times was observed (7 days vs 1 hour, P <0.001) between the two testing periods and an increase in antiviral prescribing rates and influenza vaccine uptake rates by healthcare workers was observed. A saving of almost €2400 per patient positive for influenza/RSV was determined due to reductions in length of stays during the onsite testing period. Both the Cepheid® GeneXpert and Roche® Cobas® LIAT performed equally with respect to high sensitivity (100%), specificity (100%) and accuracy (100%) in the detection of influenza A/B and RSV.

It is concluded from this study that the rapid onsite testing of influenza/RSV is associated with significant clinical improvements in patient care pathways. The findings in this study also supports and influences the importance and significant benefits that the provision of a highly sensitive, specific and accurate onsite influenza/RSV testing service has on patient care, hospital management and healthcare expenditures.

Laura Wilson is a medical scientist at Letterkenny University
Hospital and has just completed a Masters in Biomedical
Science at Ulster University. She carried out this research
project as part of her MSc in the Microbiology Laboratory
at LUH.


Performance Verification of the Sysmex UF-5000 for Urinalysis with a Comparison to Manual Microscopy.

Clodagh Beatty, BSc. (Hons) Medical Science, Galway Mayo Institute of
Supervisors: Ultan Smith, Senior Medical Scientist, Microbiology Laboratory, Midlands Regional Hospital Tullamore and Dr Sharon Duffy,
Lecturer, GMIT.

The Sysmex UF-5000 is a new third generation automated flow cytometry analyser for urinalysis. This study aimed to verify its performance in relation to specifications provided by Sysmex. A comparison study to manual microscopy using 216 samples from hospital and primary care settings was additionally investigated.

The precision, limit of detection (LOD), linearity and carryover were assessed for white blood cells (WBCs), red blood cells (RBCs), bacteria (BACT), and epithelial cells (ECs) on the Sysmex UF-5000. A total of 13 patient samples were used in the verification process. Manual microscopy was carried out for WBCs and RBCs using KOVA chambers for 216 samples. Bland Altman plots assessed agreement and correlation was assessed using Spearman’s rank correlation.

Sysmex UF-5000 showed excellent precision, LOD and linearity results within the provided specifications. Carryover was high at 0.44%, 2.30%, 0.40% and 2.04% for WBCs, RBCs, BACT and ECs respectively. Good agreement was observed between the methods with a bias observed of 5.637 (WBCs) for 135 samples and -9.003 (RBCs) for 194 samples. Correlation ranged between 0.961 for WBCs and 0.779 for RBCs. Diagnostic performance of the Sysmex UF-5000 at a threshold of =/>10cells/μL yielded a sensitivity of 90.9%, 88%, a negative predictive value (NPV) of 73.2%, 89.2% and a diagnostic accuracy of 88%, 74% for WBCs, RBCs when compared to manual microscopy.
The Sysmex UF-5000 showed an overall good performance with further investigations needed to assess sample carryover. Moreover, it exhibits excellent characteristics for urinalysis in a diagnostic laboratory.

Clodagh graduated as a medical
scientist from GMIT thisyear.
Currently, she is working in the
Microbiology Laboratory
in the Galway Clinic.


A Real-Time Polymerase Chain Reaction (qPCR) Validation of an Amoebic Gill Disease RNA Sequencing data set

Nicole Curley, BSc. (Hons) in Medical Science, Galway-Mayo Institute of Technology
Supervisors: Dr. Joan O’Keeffe, Lecturer, GMIT and Dr. Anita Talbot, Marine and Freshwater Research Centre (MFRC), GMIT

The aim of this research project was to validate an RNA Sequencing (RNA-Seq) data set which analysed the expression of genes in the liver of Atlantic salmon following an Amoebic Gill Disease challenge study, previously conducted in the Marine and Freshwater Research Centre in Galway-Mayo Institute of Technology. AGD is caused by the parasite Neoparameoba perurans which affects Atlantic salmon aquaculture in Ireland. The RNA-Seq data set was generated to study the host-pathogen interactions and to identify potential biomarkers of disease. This data set must first be validated and proven true by comparing it with another method of gene expression analysis, Real-Time quantitative Polymerase Chain Reaction (qPCR). Ten liver genes differentially expressed using RNA-Seq were analysed using the Biomark Microfluidics qPCR system.

The objectives of this project were achieved through the data analysis of the qPCR data and the correlation with the RNA-Seq data set. The qPCR raw data was analysed to quantify the relative gene expression of the ten liver genes. A housekeeping gene was chosen to normalise the qPCR raw
data. The relative expression of the genes was calculated using the 2-ΔΔCt method. The data analysis determined there is strong positive correlation between the qPCR and RNA-Seq data sets (ρ = 0.83).

The RNA-Seq data set, which has an excess of 2,000 genes was successfully validated using the qPCR method which can now be analysed further by members of the ADIOS project team.

Nicole is a graduate of the Medical Science
Class of 2020 at GMIT and is currently working
as a Medical Scientist in the Blood and Tissue
Establishment in University Hospital


Multi-triazole-resistant Aspergillus fumigatus and SARS-CoV-2 co-infection: A lethal combination research

Aia Mohamed(a), Tidi Hassan(b), Marta Trzos-Grzybowska(a), Jubil Thomas(c), Aidan Quinn(d), Maire O’Sullivan(f,g),
Auveen Griffin(f), Thomas R. Rogers(f,g), Alida Fe Talento(a,e,*)

a Department of Microbiology, Our Lady of Lourdes Hospital Drogheda, Co. Louth, Ireland
b Department of Medicine, Our Lady of Lourdes Hospital Drogheda, Co. Louth, Ireland
c Department of Anaesthesia and Intensive Care, Our Lady of Lourdes Hospital Drogheda, Co. Louth, Ireland
d Department of Radiology, Our Lady of Lourdes Hospital Drogheda, Co. Louth, Ireland
e Department of Microbiology, Royal College of Surgeons, Ireland, Dublin, Ireland
f Department of Microbiology, St. James’s Hospital, Dublin, Ireland
g Deparment of Clinical Microbiology, Trinity College Dublin, Dublin, Ireland

We report a case of severe COVID-19 pneumonia complicated by fatal co-infection with a multi-triazole resistant Aspergillus fumigatus and highlight the importance of recognising the significance of Aspergillus sp. isolation from respiratory samples. Early diagnosis and detection of triazole resistance are essential for appropriate antifungal therapy to improve outcome in patients with coronavirus associated invasive aspergillosis.

1. Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing COVID-19 infection, is a newly recognised pathogen that has been traced to Wuhan (Hubei province) in China [1]. The clinical spectrum of COVID-19 varies from asymptomatic infection to severe pneumonia requiring mechanical ventilation. The overall case fatality rate is estimated to be 2.7%, which increases to 4.5% in those ≥ 60 years old [2]. Secondary
infections with bacterial and fungal pathogens have been reported in patients with COVID-19 pneumonia which may be due to virus induced mucosal damage and/or the dysregulated immune response seen in patients with acute respiratory distress syndrome [3,4].
We report severe COVID-19 pneumonia with multi-triazoleresistant Aspergillus fumigatus co-infection in a patient not known to be immunocompromised to highlight the importance of early diagnosis and detection of triazole resistance.

2. Case
A 66-year-old male presented to the emergency department with a history of progressive shortness of breath (SOB), myalgia, headaches,non-productive cough and fever.
Two days previously, he had contacted his general practitioner (GP) describing a seven-day history of fever and cough. He had returned from the United Kingdom eight days earlier after visiting a relative who was subsequently diagnosed with COVID-19. He was advised to self isolate and monitor his symptoms. Due to progressive SOB, he re-presented to his GP who referred him to the hospital for further management.
The patient had Type 2 diabetes mellitus, hypertension, hyperlipidaemia and obesity with a body mass index of 35.5 (weight 90kg) with no hospital admissions in the past eight years. His medications included metformin, aspirin, simvastatin and ramipril. He had no history of prior triazole antifungal therapy. He was an ex-smoker but had not been previously diagnosed with chronic lung disease. He works in ground maintenance where he is exposed to fungicides daily.
On presentation, a portable chest radiograph was performed which showed unilateral peripheral left basal airspace shadowing (Fig. 1A).

Fig. 1. A. Portable chest radiograph taken on day of admission
showing unilateral peripheral left basal airspace shadowing. B.
Portable chest radiograph taken on Day 12 of hospitalisation (Day 20
of COVID-19 infection). The endotracheal tube and bilateral central
lines are in satisfactory position. There has been interval progression
of the left peripheral airspace shadowing with additional right upper
and lower zone peripheral airspace shadowing with no evidence of

In view of the clinical presentation, radiographic findings and the recent exposure history, the patient was admitted under contact and droplet precautions.
On admission (Day 7 of COVID-19 illness), the patient was noted to be pyrexial (temperature of 38.6 °C), tachycardic (pulse rate of 174 beats per minute) with new onset atrial fibrillation, normotensive (BP 117/69 mmHg), tachypnic (24 breaths per minute) with oxygen saturation of 84% on room air. He was commenced on a trial of continuous positive airway pressure with oxygen therapy. Preliminary laboratory results revealed
white cell count of 4.7 × 109/L (reference range 4.0–11.0 × 109/L), neutrophil count 3.0 × 109/L (reference range 2.0–7.0 × 109/L), lymphocyte count 0.8 × 109/L (reference range 1.0–3.0 × 109/L) and C-reactive protein of 118 mg/L (reference range < 5.0 mg/L). Nasopharyngeal and oropharyngeal swabs detected SARS-CoV-2 using real-time reverse transcriptase polymerase chain reaction on day 3. He was commenced on azithromycin (500 mg on day 1 and 250 once daily on days 2 and 3) and hydroxychloroquine (200 mg twice daily) orally as per our local
protocol at that time.
On day 4 (Day 11 of COVID-19 illness) he developed worsening hypoxic respiratory failure with a Sequential Organ Failure Assessment (SOFA) score of 6 necessitating intensive care unit (ICU) admission for ventilatory support. The patient was proned initially for 15 hours which did not improve his oxygenation. This, combined with significant hemodynamic instability, were contraindications for further proning. His condition continued to deteriorate progressing to multi-organ failure on day 7 (Day 14 of COVID-19 illness) which included acute respiratory failure, acute kidney injury requiring continuous renal replacement therapy and vasopressor support due to septic shock. His SOFA score increased to 12. He had continuing pyrexia and copious amounts of purulent respiratory secretions prompting a repeat septic screen which included
culture of blood and endotracheal aspirate (ETA).
Empiric antimicrobial therapy for hospital-acquired pneumonia with intravenous (IV) piperacillin-tazobactam (4.5g three times a day) was started. Subsequently, ETA culture grew Klebsiella varicola (susceptible to piperacillin-tazobactam), Aspergillus sp. and Candida albicans. Antifungal therapy with IV liposomal amphotericin B (3 mg per kg once daily) was commenced due to the patients rapidly deteriorating status and uncertainty of the full identification of the mould isolate which may be resistant to triazoles. The Aspergillus sp. was referred for further identification and antifungal susceptibility testing, as were serum samples for detection of 1–3, ß-d-glucan (BDG) antigen and galactomannan
(GM) antigen and ETA for GM antigen.
His SOFA score on day 11 (Day 18 of COVID-19 illness) increased to 16. Antibiotic therapy was escalated to IV meropenem (1g twice daily) and vancomycin, with dosing based on renal function, due to ongoing pyrexia and deteriorating status. He was not prescribed systemic steroids at any time during his critical illness.
A repeat portable chest radiograph on day 12 (Day 20 of COVID-19 illness) showed progression to additional right upper and lower zone peripheral airspace shadowing with no evidence of cavitation (Fig. 1B). He remained haemodynamically unstable which precluded further radiological investigations. In view of his non-responsive multi-organ failure, and after discussion with his family, the patient’s life-support was withdrawn. He died on day 14 of hospitalisation (Day 22 of COVID-19 illness).
The Aspergillus sp. isolated from the ETA was confirmed to be A. fumigatus. Phenotypic testing utilising a 4-well triazole resistance screen (VIP check™, Mediaproducts BV, The Netherlands) (Fig. 2A and B) as well as determination of minimum inhibitory concentrations (MIC) utilising gradient strips (Liofilchem, Waltham MA, USA) were suggestive of triazole resistance (Table 1) [5]. The MIC to amphotericin was 0.125 mg/L (susceptible ≤ 1 mg/L) [5]. Genotypic testing utilising a commercial assay (Aspergenius™, Pathonostics B.V. The Netherlands) on the ETA detected A. fumigati complex with the TR34L98H resistance mutation in the cyp51A gene, the most common mutation in triazoleresistant A. fumigatus originating from an environmental source. The serum and ETA GM optical density index (ODI) were 1.1 and 5.5 respectively (Platelia™
Aspergillus Ag EIA, WA, USA). Serum BDG was 202 pg per milliliter (Fungitell™ Assay, Associates of Cape Cod, USA). These results (Table 1) were consistent with severe COVID-19 pneumonia and triazole-resistant invasive pulmonary aspergillus co-infection.



3. Discussion

Previously known to cause infections in severely immunocompromised patients, A. fumigatus is now recognised as an emerging pathogen in critical care patients suffering chronic respiratory disorders and as a complication of severe influenza infection [6]. Aspergillus colonisation can rapidly lead to invasive aspergillosis following severe influenza infection due to multiple pathways including structural lung damage coupled
with disruption of mucociliary clearance, leukopenia, Th1/Th2 imbalance and diffuse damage to the respiratory mucosa [7]. In addition, severe respiratory viral infections such as influenza have been identified as an independent risk factor for IPA with high mortality [6]. Although our patient did not receive steroids, their use in critical care patients is linked to increased risk of invasive fungal infections [6]. Like other causes of viral pneumonias, SARSCoV-2 may impair local mucosal and systemic immune defenses [3].
Thirty-four cases of COVID-19 associated invasive aspergillosis (CAPA) have been reported to date [8–17]. These were all patients with severe pneumonia with adult respiratory distress symdome, most of whom had no known history of immunocompromise. These has led to challenges in early diagnosis since most of them do not have the classical risk factors for invasive aspergillosis.
Histopathology and culture from sterile site samples and biopsy remain the gold standard for proven IPA however the definition of probable or “putative” IPA has expanded but remains a composite of the host factors, clinical features and mycological evidence [6,18]. Although severe viral pneumonia is not considered a risk factor for IPA or other invasive mould disease according to the European Organisation for Research and Treatment of Cancer/Mycoses Study Group definitions, the structural damage to the lung parenchyma, in addition to the dysregulated immune response, can lead to IPA [6]. Recently, a panel of experts proposed a case definition for influenza associated invasive aspergillosis (IAPA) which may be useful to classify COVID-19 patients with invasive aspergillosis. Following this case definition a patient with COVID-19 detected
in respiratory sample by PCR, pulmonary infiltrates and a positive serum or bronchoalveolar lavage galactomannan has criteria consistent with probable CAPA [19]. Our patient had radiographic findings consistent with COVID-19 pneumonia. Unfortunately, his clinical status did not allow for computed tomography (CT) to be performed. Other mycological evidence of IPA include culture of Aspergillus sp. from non-sterile sites or detection of fungal antigens such as serum BDG. Our patient had COVID-19 pneumonia, A. fumigatus from a tracheal aspirate with elevated
serum BDG and GM and elevated ETA GM, findings consistent with COVID-19 and probable IPA co-infection.
Multi-triazole resistance in A. fumigatus has recently emerged and is linked to the use of triazole-containing compounds as agricultural fungicides or less commonly prolonged triazole use [20]. The former mechanism of resistance typically affects azole näive patients and is characterised by elevated MICs to itraconazole, voriconazole and posaconazole as was found in our patient. This is of serious concern since triazoles are
recommended as the first-line and most effective treatment for IPA [21]. Of the 34 cases reported so far, only 7 cases had reported susceptibility results of which one was triazole resistant A. fumigatus [16] similar to our case. Our patient’s exposure to fungicides daily in his work has most likely led to exposure and colonisation with triazoleresistant A. fumigatus which was further supported by detection of cyp51A TR34L98H mutation from our patient’s ETA, the most prevalent triazole resistance mutation from environmental source [20].
This case highlights the importance of early evaluation of patients with COVID-19 pneumonia because of the risk of secondary or co-infection with fungal pathogens. Case definitions have been proposed for IAPA [6,19] which can be modified for early recognition of CAPA. The occurrence of multi-triazole resistance in this case emphasises the urgent need for antifungal drug susceptibility testing of Aspergillus isolates using a rapid and
simple phenotypic method and/or by detection of Cyp51 gene associated triazole resistance mutations directly on respiratory samples.

Funding sources
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interestCOI
T.R.Rogers has received grants and personal fees from Gilead Sciences, personal fees from Pfizer Healthcare Ireland, personal fees from Menarini Pharma outside the submitted work, A.F.Talento has received grant and personal fees from Gilead Sciences and personal fees from Pfizer Healthcare Ireland outside the submitted work. The other authors have no conflict of interests

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[21] T.F. Patterson, G.R. Thompson, D.W. Denning, J.A. Fishman, S. Hadley, R. Herbrecht, et al., Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the infectious diseases society of America, Clin. Infect. Dis. 63 (4) (2016) e1–60.

Point of care testing in General Haematology

Ciarán Mooney,1 Mary Byrne,2 Patience Kapuya,3 and Lorna Pentony,4 Barbara De la Salle,5Tony Cambridge,6 DavidFoley,7 on behalf of the British Society for Haematology Guideline

1Rotunda Hospital, 2 St James Hospital, Dublin, Ireland, 3 St. Mary’s Hospital Newport, Isle of Wight, Newport, UK, 4Rotunda Hospital, Dublin, Ireland, 5UK NEQAS Haematology, Watford, 6University Hospitals Plymouth NHS Trust, Plymouth, UK and 7MaterHospital, Dublin, Ireland
First published: 02 October 2019 https://doi.org/10.1111/bjh.16208
This guideline is an update of the BSH 2008 Guideline for point of care testing: haematology (Briggs et al, 2008).

This guideline was compiled according to the BSH process at https://b-s-h.org.uk/guidelines/proposing-and-writing-a-newbsh-guideline/.

The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate levels of evidence and to assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org.

Point of care testing (POCT) refers to any testing performed outside the hospital laboratory, near or at the site of the patient where the result influences patient management
(ISO 15189:2012; ISO 22870:2016). There has been a major expansion in POCT since the publication of the 2008 BSH guideline (Briggs et al, 2008). In the intervening period, the range and complexity of POCT technologies and the repertoire of POCT assays has grown, both in primary care (pharmacies, general practice surgeries and community clinics) and hospitalbased settings. A major driver of this growth has been the
development of relatively simple testing methods, which allow the generation of results close to the patient for both therapeutic monitoring and diagnostic purposes. At the same time, the accreditation requirements for clinical laboratories, specified in the International Organization for Standardization (ISO) 15189:2012 and ISO 22870:2016 standards (ISO, 2012, 2016), have driven the development of comprehensive clinical, financial and quality governance.

The aim of this guideline is to provide an overview of point of care (POC) assays available, and a framework for implementing and maintaining a POCT service compliant with international standards (ISO, 2012, 2016). This guideline does not specifically encompass POCT systems in a primary/community setting; however, the same principles may be applied. This guideline does not apply to general medical devices in a ‘near patient’ setting, such as blood pressure monitors, pulse oximetry and thermometers. (https://ec.europa.eu/growth/single-market/european-standards/harmonised-standards/iv-diagnosticmedical-devices_en, last accessed 8 June 2018)



Validation of extended reporting times for APTT and D-dimer tests in Sligo University Hospital

Chloe Maguire, BSc. (Hons) Medical Science, Galway Mayo Institute of Technology

Supervisors: Sonia Gilmartin, Chief Medical Scientist in Haematology/Blood Transfusion in Sligo University Hospital, Dr. Eleanor
Rainsford, Lecturer, GMIT.

Activated Partial Thromboplastin Time (APTT) testing and D-dimer testing are commonly requested in the Haematology/Coagulation laboratory. However, sodium citrate samples for APTT and D-dimer analysis must be tested within four hours of collection (Rimac and Coen Herak, 2017). This restricts the use of add-on tests after initial testing has been performed, and results in the rejection of some GP samples if not received in time. Studies in recent years have validated extended stability and reporting times for APTT and D-dimer samples. This study aims to determine if the stability time for APTT samples can be extended to 8 hours post-collection, and for D-dimer samples to 12 hours post-collection. Samples were collected and run at set intervals postcollection.

Samples for APTT analysis were re-tested at 4, 6 and 8 hours post-collection. Samples for D-dimer analysis were re-tested at 4, 8 and 12 hours post-collection. The baseline results were then compared to the extended results and the data was analysed to determine if a statistically or clinically significant difference existed.

The results found that there was a statistically significant difference in APTT results even at four hours of storage, however no statistically significant difference in D-dimer results was found up to eight hours post-collection. There was no clinically significant difference in APTT results after eight hours of storage, and no clinically significant difference in D-dimer results after 12 hours of storage. In conclusion, APTT testing can be performed up to eight hours post-collection and D-dimer testing can be performed up to 12 hours post-collection.

Chloe graduated from GMIT this year
and is currently working in the
Histology/Cytology Department
in Sligo University Hospital.



Bioinformatics Analysis of Bioactive Peptides from Seaweed Proteins.

Aoife Mc Enery, BSC. (Hons) in Medical Science, Galway-Mayo Institute of Technology
Supervisors: Dr. Joan O’Keefe, Dr. Orla Slattery, Dr. Sharon Duffy Lecturers, GMIT

Seaweeds are a potential source of bioactive peptides that could potentially be utilised in the treatment of conditions like diabetes mellitus. Bioinformatics is a novel approach used to investigate potential bioactive peptides in seaweeds. This study aimed to investigate using bioinformatics tools antidiabetic and antimicrobial bioactivities in five seaweed species namely Palmaria palmata, Chondrus crispus, Gracilariopsis lemaneiformis, Ectocarpus siliculosus and Saccharina japonica.

The positive datasets (15 antidiabetic peptides) and (3 human-defensins) were extracted from Antimicrobial Peptide Database and UniProt respectively. BLASTp and T-Coffee software were used to identify sequence similarities between a particular seaweed and a chosen positive datasets with putative functions. The biological activities of identified peptides sequences were confirmed in silico using the predictive algorithms BIOPEP-UWN and APD3

Peptides sequences LPKVLNYESLFKNSFTY and FLPVLAGAASF (found in Phenylalanine tRNA synthetase beta subunit and Phosphate transporter of G. lemaneiformis), KLLSTASKLGLLSKL (found in Expressed unknown protein of E. siliculosus) and FLPLLGSLVAGF (found in NADH dehydrogenase subunit 5 of S. japonica) were selected as being the most likely antidiabetic peptides based on lowest E-value (< 10-5). These peptides sequences confirmed Dipeptidyl Peptidase 4 inhibitor activity. This is a novel finding. No antimicrobial peptides were confirmed. Through the use of bioinformatics tools, antidiabetic bioactivities were successfully confirmed in specific seaweeds proteins. Further studies are warranted to test the efficiency of these antidiabetic bioactivities in vitro. Bioinformatics tools are an innovative approach in identifying potential bioactive peptides in seaweed.

Aoife is currently working
as a Medical Scientist in
Microbiology at Childrens’
Health Ireland in Crumlin.

Introduction of a multi tissue control block for Immunohistochemistry in the Histology Laboratory at Midlands Regional Hospital Tullamore

Meghan Farrell, BSc. (Hons) in Medical Science, Galway-Mayo Institute of Technology

Supervisors: Naomi Cronin, Chief Medical Scientist in Histology the Midlands Regional Hospital, Tullamore, Terri Muldoon, Chief Medical Scientist in Histology Galway University Hospital & Lecturer, GMIT

The aim of this study was to introduce a multi-tissue control block (MTCB) that would cover a wide range of antibodies and improve the daily internal quality control (IQC) procedures in Midlands Regional Hospital Tullamore (MRHT). This MTCB will reduce the risk of false positive
and false negative staining and reduce the number of blocks in the current immunohistochemistry (IHC) control library. The MTCBs include tonsil, appendix, normal and malignant colon. The tissues were retrieved from samples that were due for disposal or archived samples. The Leica
ASP 300S tissue processor and Leica EG1150H were used to process the tissues and embed the MTCBs respectively. A section from each MTCB was stained with haematoxylin and eosin (H&E), and the MTCBs with sufficient tissue structures were used for antibody staining. The VENTANA
Benchmark ULTRA was used to stain the MTCBs for 48 antibodies, which were assessed using microscopy.

Overall, the MTCB has the potential to provide strong positive, weak positive, and negative staining for 62% of the 55 suitable antibodies. Thus, improving the daily IQC procedures and reducing the risk of false negative reactions. The MTCB will also introduce a negative tissue control into daily IQC procedures (for all 55 antibodies), which will reduce the risk of false positive reactions. Currently, a total of nine control tissues are used for the 55 antibodies tested. If this MTCB is put into routine practise, it will also condense the current control library from nine to four tissues for these antibodies.

Meghan worked as an intern medical scientist in the Blood Transfusion Laboratory
at Midlands Regional Hospital Tullamore. She is now working in the Haematology
Laboratory there.

Evaluation of an Electrochemiluminescent Immunoassay for measurement of Adrenocorticotropic Hormone in EDTA plasma

Sinead Corry, BSc. (Hons) in Medical Science, Galway-Mayo Institute of Technology
Supervisors: Nicole Walsh, Martina Doheny, Dr. Paula O’Shea, Department of Clinical Biochemistry, Galway University Hospital and Dr. Brian Moran, Lecturer, GMIT

ACTH is a main constituent of the HPA axis which allows the body to respond to homeostatic changes or ‘stressors’. Inappropriate levels of ACTH are seen in a variety of conditions such as adrenal insufficiency and glucocorticoid excess and therefore measurement is useful in their investigation and in monitoring treatment. Before introducing a new method to a clinical laboratory, a verification study should be carried out to ensure it is performing to the standard intended by the manufacturer when used under new conditions.

In UHG, their local procedure suggests carrying out five different experiments as part of this study: a precision study, assessment of bias, a method comparison, assessment of linearity and determination of the functional sensitivity of the assay. Once these performance characteristics are defined under the conditions of the laboratory in UHG, they are compared to the performance claims stated by the manufacturer. The precision study concluded that the assay was performing more precisely than the manufacturer claimed, the method comparison indicated good agreement between the current and proposed method although there were limitations in this study and the functional sensitivity or limit of quantitation was found to be slightly lower than the manufacturer’s claims. However, it was not possible to complete the bias study or assess linearity due to COVID-19 restrictions. Before the assay can be introduced into the laboratory further research will have to be conducted to verify the performance of the assay in UHG.

Sinéad completed her project in the
Biochemistry Laboratory in University
Hospital Galway and since then
has joined the biochemistry team in the
National Maternity Hospital as a medical scientist.

Comparison of three Kleihauer-Betke Acid Elution methods for Fetomaternal Haemorrhage estimation at Portiuncula University Hospital

Siobhán Reynolds, BSc. (Hons) in Medical Science, Galway-Mayo Institute of Technology
Supervisors: Paula McMorrow, Chief Medical Scientist in Haematology & Blood Transfusion, Portiuncula University Hospital and Helen
Cregg, Lecturer, GMIT

Fetomaternal haemorrhage in Rh D negative women carrying a Rh D positive foetus can lead to maternal alloimmunisation and consequently, haemolytic disease of the foetus and newborn. Upon administration of an appropriate dose of anti-D immunoglobulin within 72 hours of a sensitising event, alloimmunisation can be prevented. The Kleihauer-Betke acid elution test is the most common technique for initial fetomaternal haemorrhage screening. The Immucor Kleihauer-Betke kit is currently in use at Portiuncula University Hospital, however, it is being discontinued. Therefore, two new kits, Guest Medical and Shepard’s Kleihauer-Betke kit will be compared to determine which kit produces the
best results. A series of ten artificial samples were prepared consisting of adult blood spiked with varying amounts of cord blood to imitate FMH volumes ranging from 1ml to 60ml. These sample were tested in replicates of five for each FMH volume per kit. This study showed no statistical differences observed between the means of the new kits and the Immucor kit (P > 0.05). A Pearson’s correlation coefficient, r, value of 1 was obtained for each kit in comparison to expected values, indicating strong positive correlation. All three kits were inaccurate and showed bias. A coefficient of variation value of less than 20 was considered acceptable. All FMH volumes per kit satisfied these criteria, except the 1ml FMH volumes. Each kit had a sensitivity and specificity of 100%. A comparison of staining was performed between Guest Medical and Shepard’s. Results show that the Guest Medical kit yielded better results.

In conclusion, the Guest Medical kit was the superior kit. Although it suffers from sediment issues, it has a longer sample stability and superior
staining quality. Accuracy and precision results of this kit were the most comparable to the Immucor kit. This kit is currently in use at Portiuncula.

Siobhán worked as an Intern Medical Science between the
Microbiology and Biochemistry laboratories at Portiuncula
University Hospital to help out during the COVID-19 crisis.
Currently, she is working in the Blood Transfusion Laboratory
at the Beacon Hospital.


Blood Transfusion: Evaluation of Electronic Issue in the Republic of Ireland – Over 30 Years in Existence and Still Underutilised.

Blood Transfusion (BT) became an area of great interest to me during my clinical placement at Regional Hospital Mullingar (RHM). I was very fortunate that the BT Department of RHM were able to facilitate my GMIT undergraduate research project. My project involved the successful validation of their Laboratory Information Management System (LIMS) (NetAcquire Version 7.5) for the purpose of Electronic Issue (EI) with an evaluation of EI in the Republic of Ireland (ROI). The evaluation of EI in the ROI was by means of a national survey. The current use, advantages,
drawbacks and future use of EI in the ROI became a part of my research project. However, the survey was sent out in the height of the COVID-19 pandemic. It was purposely condensed in the hope of a 100% response rate. For the comprehensive completion of a national report, a second survey was required. The second survey targeted the BT laboratories that currently perform EI. It identified when and where EI was first introduced in the ROI and whether the majority of RCC units issued by the Irish Blood Transfusion Service (IBTS) in the ROI were crossmatched
and transfused in the hospitals that have EI. Both distributed surveys had a 100% response rate (n=47 and n=14).

The crossmatch is one of the elements of pre-transfusion compatibility testing. The crossmatch was first performed in 1908. At that time, the crossmatch was solely serological and consisted of the immediate spin. The serological crossmatch developed to further include the Indirect Antiglobulin Test (IAT) in the 1950s [1]. By the 1980s, a need for urgent blood transfusions during surgery and the reduction to the overall
cost to a laboratory arose. EI was developed to fulfil this need [2]. Unlike the serological crossmatch which physically involves testing a potential recipient’s plasma with a suspension of donor red cells, EI uses a computer algorithm to ascertain ABO compatibility between donor and recipient. Despite internationally recognised guidelines being readily available, EI is generally underutilised [3].

At present, only 30% (n=14/47) of ROI BT laboratories are performing EI. EI was first introduced to the ROI in Beaumont Hospital in 1993. This was just one year after the first widescale implementation of EI at the University of Michigan in 1992 [1]. The progressive introduction of EI in the ROI over the last 27 years is illustrated as follows:

It wasn’t until after 2015 that the majority (n=8/14) of BT
laboratories that currently perform EI implemented it. This
highlights the fact that despite being in existence for over 30
years, EI is relatively underutilised in the ROI.
EI has many advantages. All 30% of BT laboratories stated at
least three advantages from its introduction. Advantages stated
are illustrated as follows:

While an improved TAT for red cell issue, improved bloodstock inventory management, optimisation of staff time and reduced work life stress were predictable advantages associated with the introduction of EI based on other publications, an interesting unexpected advantage stated by one BT laboratory was the transfusion of DAT positive RCC units. Previous studies have shown the transfusion of DAT positive RCC units are not directly associated with any adverse transfusion reaction [4]. Currently ROI BT laboratories
performing the IAT crossmatch will return these RCC units to the IBTS for refund. The return of these DAT positive RCC units increases the overall workload to a scientist and potentially causes a delay in the provision of a RCC unit to a patient. With EI, the status of a potential DAT positive RCC unit will remain unknown (provided that RCC unit is never crossmatched using the IAT phase of testing) and therefore, can safely be transfused to a patient. Blood donation is on the basis of altruism. Every donor assumes their blood will be used to save a life. There are often shortages with regards to blood supplies and therefore to
not use a donated RCC unit is less than ideal. While predominately advantageous, EI can be associated with some drawbacks. Of the 30% performing EI, 50% (n=7/14) stated at least one drawback from its introduction while 50% (n=7/14) stated none. Drawbacks stated were predominantly associated with:
1. LIMS packages for EI that result in the permanent exclusion of patients from EI if the patient has ever had a positive antibody screen
2. The requirement in the BSH guidelines to exclude patients with modified results from EI who would otherwise be eligible

Where a BT laboratory did not currently perform EI, they were asked if they planned to introduce it in the future. Of the 70% (n=33/47) of ROI BT laboratories who are not currently performing EI, 61% (n=20/33) plan on introducing it in the future with 20% (n=4/20) stating they are in the process of implementing EI in their laboratory. If all 20 BT laboratories who stated they plan to introduce EI in the future implement EI, then 72% (n=34/47) of BT laboratories in the ROI will perform EI. This is encouraging and will without a doubt have many benefits for these laboratories. Maximum Surgical Blood Ordering Schedules (MSBOS) can be revised and updated for patients that are eligible for EI. A revision to the MSBOS will mean that less RCC units will be held for patients that do not necessarily require them. Therefore, outdating of RCC units will be less likely to occur with the potential to reduce the overall number of expired units. Although time expiry of RCC units is very low in the ROI, (<1% in HSE hospitals) this number could potentially be even further reduced by the implementation of EI to these 20 BT laboratories in the future. EI facilitates blood ‘on-demand’ to patients suitable for EI, should the need to transfuse arise.
While only 30% of hospitals utilise EI, these hospitals account for 58% of RCC units that are transfused in Irish Hospitals. The IBTS issued a total 122,933 RCC units to Irish hospitals in the year 2019. An approximate total of 71,000 of these RCC units were transfused in the Irish hospitals with EI. These hospitals have the biggest impact with regards to the national management of blood supplies. EI allows these laboratories to individually improve their management of RCC units which overall improves the national management of blood supplies. Ten BT laboratories were able to provide figures with regards to the total RCC units crossmatched and total RCC units electronically issued for the year 2019. Fifty seven percent (n=55,616/97,572) of RCC units crossmatched were electronically issued. This highlights that the majority of Irish patients are eligible for EI and will benefit from its advantages.
Thirty nine percent (n=13/33) of ROI BT laboratories do not plan on introducing EI in the future. With respect to that 39%, 46% (n=6/13) of respondents provided a reason. The reasons from these respondents for not planning on introducing EI in the future are displayed as follows:

The reasons for not introducing EI in the future (where given) are related to an absence of adequate IT and/or automation. When the national LIMS (MedLIS) is introduced, the BT laboratories which stated that their barrier to introducing EI was inadequate IT, should then be able to
consider EI.
While it may not be possible for EI to be implemented in all BT laboratories in the ROI, I am hopeful that after highlighting its use and associated outcomes in the ROI that EI may become more widely implemented.

I would like to thank Carol Cantwell, John Quigley, Eunan Connolly, Frances Walsh and all the team in Mullingar for facilitating and helping me with both my project and the subsequent survey that allowed for this evaluation. Additionally, I would like to thank all the BT laboratories for their willing participation and to Tony Finch and Barry Doyle for providing the IBTS 2019 figures.


1. Chapman, J.F., Milkins, C. and Voak, D. (2000). The computer crossmatch: a safe alternative to the serological crossmatch. Transfusion Medicine, 10(4), 251-256.
2. Marzepa, M.A. and Raval, J.S. (2014). Pathology Consultation on Electronic Crossmatch. American Journal of Clinical Pathology, 141(5), 618–624.
3. Reesink, H.W., Davis, K., Wong, J., Schwartz, D.W.M., Mayr, W.R., Devine, D.V., Georgsen, J., Chiaroni, J., Ferrera, V., Roubinet, F., Lin, C.K., O’Donovan, B., Fitzgerald, J.M., Raspollini, E., Villa, S., Rebulla, P., Makino, S., Gounder, D., Säfwenberg, J., Murphy, M.F., Staves, J., Milkins, C., Mercado, T.C., Illoh, O.C. and Panzer, S. (2013). The use of the electronic (computer) cross-match. VoxSanguins, 104(4), 350-364.
4. Steciuk, M.R., Brown, M.R, Freeman, J.B. and Huang, S.T. (2007). Evaluation of a Possible Transfusion Reaction With a Positive Direct Antiglobulin Test in a 29-Year-Old Male. LABMEDICINE, 38(12), 719-720.

Rebecca Salmon was
recently awarded a first
class honours degree
in Medical Science
(BSc) from GMIT. She
is currently working as
a Medical Scientist in
the Blood and Tissue
Establishment at UHG.



Improving Blood Stock Management: A collaborative approach

Helena Begley, Senior Medical Science, Naas General Hospital

Blood stock management is a balancing act. Blood transfusion scientists must ensure they have enough blood on site to meet clinical demands at all times. However, if hospital blood transfusion laboratories hold excess stock it increases the risk of expiry and may also impact on blood stock levels elsewhere in the country.

In May 2018, a blood stock review meeting was held between the heads of transfusion at Tallaght University
Hospital (TUH), The Coombe Women and Infants University Hospital (CWIUH), Naas General Hospital (NGH), the IBTS and the HSE. We needed to discuss our blood ordering and in particular the high numbers of O RhD Negative units we were keeping in stock at all 3 sites. It is recommended that approximately 12% of hospital blood stock should be O Negative. Our stock levels of O Negative were much higher than this. While very few O Negative units had expired, it was certainly true that many of them were being used inappropriately i.e. remaining in stock until close to expiry and then either being transfused to a non O Negative patient or re-routed. O Negative blood is often in short supply as only 8% of the population is O Negative and donors are therefore scarce. Blood transfusion medical scientists are all too familiar with IBTS notifications informing us that O Negative stock levels are well below the 7 day target. We needed to improve our blood stock management to lessen the demand we were placing on the IBTS for O Negative blood and ensure it was used appropriately as far as possible.

We were all reluctant to drastically reduce the numbers of O Negative units we held in stock as we were fearful that it might be unsafe. So we decided to begin a weekly blood exchange programme. Every Wednesday morning, blood with 2 weeks to expiry is sent from Naas hospital and the
Coombe hospital to TUH and exchanged for units with 3-4 weeks left till expiry using First Direct Medical vans. We decided to exchange all blood groups, not just O Negative. Our blood exchange initiative started in June 2018. At the end of 2019 we reviewed our blood stock ordering and
usage statistics and were very impressed by our findings across all 3 sites.

Improvements we noted were as follows:
• A reduction in blood ordering, especially O Negative units, therefore less demand was being placed on the IBTS to supply them.
• Safe numbers of O Negative units on site without placing extra orders with IBTS.
• Appropriate use of O Negative improved i.e. O Negative units being transfused to O Negative patients, in emergencies, or to patients with sickle cell disease went from 65.2% to 82.8%.
• Transfusion of short-dated O Negative units to patients of other blood groups was essentially halved.
• Marked reduction in re-routing of short dated units to TUH from NGH and CWIUH.
• Less expired red cell units at all 3 sites.
• Reduction in patients being transfused alternative suitable blood groups to use up expiring blood.
• Less pressure on medical scientists if ordering additional stock during an emergency as it can be exchanged.
• Can exchange a non-suitable unit in stock for a suitably antigen typed unit for patients with red cell antibodies.
• NGH and CWIUH can arrange to get groups they don’t stock from TUH rather than the IBTS e.g. B-, AB+, AB for a particular patient.
• Can exchange units for alternative blood groups if there is a large number of one particular blood group on site.
• Conversely, NGH and CWIUH can take units from TUH if they have a large number expiring within a short timeframe.
• Savings in excess of €30,000 were achieved in 2019 due to the blood exchange programme.

Challenges of blood exchange:
While the benefits are very much evident, blood exchange does not come without some challenges. Regular communication between hospitals is crucial to re-assess current stocks and arrange exchange as stocks are ever changing. It is time consuming. Blood transfusion laboratories must maintain records of all blood exchanged which adds to the vast quantity of records which must be stored to comply with ISO15189. Laboratories must use an alternative source for exchanged blood when entering blood units into their LIS to ensure accurate blood stock statistics.
Occasionally, blood exchange is not possible due to stock levels and expiry dates of blood at TUH.

Blood is precious
One in four of us will need a transfusion in our lifetime. In 2008, my own daughter Chloe had a bleedduring the pregnancy and lost approximately 75% of her total blood volume. Her haemoglobin was 3.9g/dl at birth. She needed immediate emergency transfusion when she was born or she would not have survived. Donated blood is a very precious resource. It is so important that we do all we can to use and manage blood stocks efficiently so that blood is available for all patients whenever and wherever they need it.

At present we are the only group exchanging blood but this exchange programme could easily be rolled out nationwide. We have now also started to exchange blood products other than red cells e.g. LG-Octaplas with a year left to expiry. Approved First Direct Medical temperature controlled vans are already contracted by the HSE to deliver blood to every hospital in the country. With the addition of perhaps one more van per region, exchange could easily start between every hospital group. This would hopefully result in a further reduction in expiry rates and improved blood stock management, particularly with regard to O Negative units, nationwide. In addition, we predict that the HSE could save in excess of €370,000 annually if this program was implemented nationally.

This initiative was short-listed for the 2020 HSE Excellence awards and won the team category for the Dublin region at this year’s Spark Ignite competition. Sincere thanks to Alison Harper, Fergus Guilfoyle, the medical scientists at NGH, TUH, CWIUH, Tony Finch (HSE), Marian Barry (IBTS) and First Direct Medical for all their help in delivering this very successful project to date.