HSL Molecular
Pathology
Foreword
As for everyone, 2020 has been a year
like no other for HSL Molecular Pathology.
At the beginning of the year, our molecular scientists were working across many different specialist disciplines; a few months later, everyone had converged on one assay – the COVID-19 PCR test. Now, with a team that has more than doubled in size, we are again running all of our normal services alongside the coronavirus testing.
When we set up HSL Molecular Pathology, our vision was that, by combining molecular testing across the biological disciplines, we could learn from each other, share ideas, and enhance our services across the group. For example, scientists working on cancer genetics and haematology have come together to develop NGS panels that test an array of targets in an efficient and cost-effective manner. We have staff with genetics backgrounds training with virologists and senior BMSs, and our clinical scientists are sharing ideas to improve our repertoire and workflows. Before COVID, we were processing the largest number of molecular tests in one laboratory in the UK (which we were very proud of).
Today, the number of tests we are processing has increased nearly five-fold, and it is testament to the structure and dedication of the team that we have been able to scale up so effectively in just a few months. During the lockdown period in the spring and early summer of 2020, the number of samples from other areas dwindled as patients stayed away from hospitals – so spare staff, already used to working together across the disciplines, helped to ramp up the COVID-19 testing. As lockdown eased, the routine work returned and we continued to see rising numbers of COVID-19 samples. New IT solutions were put in place, processes were streamlined, and more staff were hired to support the workload. As we settle into the ‘new normal’, we are getting back to projects that will improve our routine and specialist repertoire beyond COVID-19.
Although the last six months have been extremely challenging and demanding, the team has come together in a remarkable way. We have supported our existing staff, and the new staff have joined a busy but satisfying, productive atmosphere, where we all know we are making a difference.
Dr Lisa Levett
Director of Genetics and Molecular Pathology
Virology
COVID-19
HSL has been at the forefront of
COVID-19 testing for the UK since the
disease first emerged. The team has
developed new testing methods, worked
closely with hospitals, helped others
open their own labs, and by the end of
September 2020 was processing more
than 10,000 PCR tests a day.
When the outbreak of COVID-19 occurred in Wuhan City, China, in December 2019, our consultant virologist Dr Gee Yen Shin warned us of the potential for this to spread. We were already running a respiratory panel that included seasonal coronavirus and a separate assay for MERS CoV, and our scientists were on high alert. When the sequence of SARS-CoV-2 (as the virus is now known) was published, the team’s Scientific Lead, Dr Paul Grant, designed an assay to detect it by PCR and integrated it with the usual respiratory testing workflow.
As the weeks went by and more positive cases began to emerge in the hospitals, however, more testing was required. The UK, as one of the later countries to develop positive cases, was at a distinct disadvantage for reagents – most of the supplies for the PCR test had already been used by our neighbouring European counterparts. Panther Fusion reagents were in limited supply and the chemicals needed to extract the RNA from the virus were non-existent. Even though we had two separate assays to detect COVID-19 by PCR, both assays had limited supplies.
After encouragement from our chairman Lord Carter and a visit from NHSE’s chairman, Lord Prior, we decided that it could be possible to tweak the assay to enable us to run it without the extraction reagents that were in short supply. Dr Grant developed it within days and refined the assay to improve sensitivity. This resulted in a preprint publication in the online journal BioRxiv, which enabled us to share our findings with other NHSE laboratories and with colleagues within our global Sonic network.
At the same time, the team helped The Francis Crick Institute convert their research facility into a COVID-PCR testing laboratory. And subsequently the team set up another COVID-19 PCR lab at University College London for frontline NHS staff testing; this lab went live in August 2020.
The team have worked closely with London hospitals, supporting the Royal Free Hospital Group’s High Consequence Infectious Diseases Unit, where the initial index patients with Covid-19 in the UK were treated at the outset, and other hospitals that were worst affected by the peak of Covid-19. TDL also supported the London Nightingale Hospital with clinical leadership and pathology transport, and is contributing to research projects investigating vaccine production, use of convalescent plasma and analysis of prevalence in care homes throughout England. In September, TDL provided the COVID-19 testing for the ‘cataract drive’ at Moorfields Eye Hospital. The project aimed to carry out 1000 cataract operations in a week – four times the cataract procedures that they usually perform.
By the end of September 2020, the Halo laboratory was processing more than 10,000 samples a day, and over the next few months, the capacity will grow even further. In partnership with UCL and MedCity, the team is setting up a winter COVID surge laboratory at the Halo – a major project where we are providing all the expertise and intensive management input for this national Government project. This new COVID lab on level 9 of the Halo will allow a further 10,000 people to be tested a day in addition to our current capacity.
Random, rapid access
HSL uses a high-throughput platform called the Panther Fusion for many of its large-scale tests. With many other platforms, samples have to be analysed in batches (often of 96 samples), so turnaround times are constrained by sufficient samples for each batch. However, one of the Fusion’s features is its ‘random access channel’, a completely automated system that enables samples to be loaded on individually and analysed as soon as they arrive at the laboratory. The results are then transmitted automatically to the laboratory IT systems.
The team has now moved all of its respiratory and gastrointestinal virology testing to the random access channel, improving turnaround times by 48%. These tests are often the most urgent for hospitals, needing fast turnaround times for infection control.
MERS
HSL has introduced a test for another zoonotic virus – MERS-CoV (Middle-East Respiratory Syndrome Coronavirus). This first emerged in 2012, causing severe illness in humans. Up to the end of August 2019, 2468 confirmed cases and 850 MERS-CoV-associated deaths, mainly in the Arabic peninsula, were reported.
The World Health Organization recommends the use of two independent PCR assays for confirmation of MERS-CoV cases, so one test targets a region upstream of the E gene (upE) and the other targets open reading frame 1a (orf1a) of the MERS-CoV genome.
“This is clearly an extraordinary year for HSL virology and virology labs everywhere, with new COVID-19 challenges appearing every week, if not every day. HSL has met these challenges with flexibility, adaptability and resilience, introducing, after appropriate verification, no less than 4 new molecular SARS-CoV 2 (COVID-19 virus) assays since March 2020. This unusually diverse portfolio of assays has been driven by well-publicised constraints in the global supply chain. It has ensured that every patient who has needed a COVID-19 test in the HSL network has had one.
The other challenge has been one of scale. Throughout this pandemic, HSL has been asked by others, such as NHS England, to support multiple other London NHS Trusts, nursing homes etc. At the time of writing, HSL’s Halo lab is testing around 10,000 COVID-19 PCR samples per day. HSL is certainly playing its part in the UK’s response to the pandemic. Dr Gee Yen Shin
HSL Clinical Specialty Lead for Virology Consultant Virologist, UCLH
Microorganisms
Rapid detection
of enteric organisms
A new test for gastrointestinal
infections has integrated targets across
disciplines – microbiology, parasitology
and virology – and enabled expert
consultants, biomedical scientists
and BMSs and clinical scientists
to discuss cases together.
Acute gastrointestinal infections can be caused by a variety of pathogens, including viruses, bacteria and parasites, but they generally present with nearly indistinguishable clinical signs and symptoms. The new DiagCORE® Gastrointestinal Panel,
introduced by HSL’s Molecular Pathology team, detects DNA from the most common viral, bacterial and parasitic pathogens that cause gastrointestinal infections. Rapidly determining the cause helps with timely decisions on treatment, hospital admission, infection control, and return of the patient to work and family. It also supports improved antimicrobial stewardship and other important public health initiatives.
VIRUSES (6)
Clostridium difficile toxin A/B
Enteroaggregative E.coli (EAEC)
Enteroinvasive E.coli (EIEC)/Shigella
Enteropathogenic E.coli (EPEC)
Enterotoxigenic E.coli (ETEC) lt/st
Campylobacter spp.
(C.jejuni, C.upsaliensis, C.coli)
Plesiomonas shigelloides
Salmonella
Shiga-like toxin producing
E.coli (STEC) stx1/stx2
Shiga-like toxin producing
E.coli (STEC) O157:H7
Vibrio cholerae Vibrio parahaemolyticus Vibrio vulnificus Yersinia enterocolitica PARASITES (4) Cyclospora cayetanensis Cryptosporidium spp. Entamoeba histolytica Giardia lamblia
BACTERIA (14)
Clostridium difficile toxin A/B
Enteroaggregative E.coli (EAEC)
Enteroinvasive E.coli (EIEC)/Shigella
Enteropathogenic E.coli (EPEC)
Enterotoxigenic E.coli (ETEC) lt/st
Campylobacter spp.
(C.jejuni, C.upsaliensis, C.coli)
Plesiomonas shigelloides
Salmonella
Shiga-like toxin producing
E.coli (STEC) stx1/stx2
Shiga-like toxin producing
E.coli (STEC) O157:H7
Vibrio cholerae Vibrio parahaemolyticus Vibrio vulnificus Yersinia enterocolitica
Cervical Screening London
The NHS has commissioned HSL to provide a comprehensive, single-site HPV primary screening service for London. The service operates within the NHS Cervical Screening Programme, and is a partnership with London North West University Hospital NHS Trust under the new banner of Cervical Screening London (CSL). Unlike previous cytology-based screening, the new programme tests for high-risk subtypes of human papilloma virus (hrHPV) as a first step in the screening process. These hrHPV subtypes are known to be linked to the development of abnormal changes in the cervix which, in some cases, can lead to cervical cancer. Early identification of women with hrHPV means they can be more closely monitored and treated.
From December 2019, all women across London are being offered a safe HPV primary screening test, with samples collected by TDL Collect couriers and brought to the central laboratory at the Halo building. The first test performed on each sample will be the HPV test. If hrHPV is not detected, there is a very low risk that the woman will develop a significant abnormality before her next test. She will be asked to return in 3 or 5 years depending on her age. If hrHPV is detected, a cytology slide will be prepared from the same sample and examined under a microscope. If there are no abnormal cell changes, the woman will be asked to come back in 12 months for another HPV test. If abnormal cells are detected, the woman will be referred for colposcopy.
Sexual health
Many bacteria that cause sexual health diseases – such as those that cause Chlamydia or Gonorrhea – do not grow well in culture. This makes identification difficult and so patients have traditionally been given broad-spectrum treatments.
HSL has introduced a portfolio of sexual health screening tests using molecular techniques. These tests are more accurate and targeted and can identify organisms that would often have been missed before. This not only provides new pathways for treatment, but also allows population-level screening at an affordable cost.
Mycoplasma genitalium, for example, has a
prevalence of 1-2% in the general population, and has been implicated as a cause of acute and chronic non-chlamydial non-gonococcal urethritis in males and post coital bleeding, cervicitis, endometritis and pelvic inflammatory disease in females. It is extremely difficult to culture, and nucleic acid amplification testing (NAAT, from urine or a swab) is the only method for routine
detection. It also develops resistance to antimicrobials spontaneously, and resistance to macrolides,
generally considered the first-line treatment, seems to be increasing worldwide typically – exceeding more than 40% in male patients who are detected positive for Mycoplasma genitalium at screening. Macrolide resistance testing can therefore be undertaken from the same sample if Mycoplasma genitalium is detected.
Culture techniques still provide useful information for clinicians. They can be used to determine sensitivities, or to test for antibiotic resistance. An example of a molecular profile test:
7 STI PROFILE BY PCR – 7 TESTS FROM 1 SAMPLE (Urine, Swab, Thin Prep or Semen)
Chlamydia trachomatis Neisseria gonorrhoea Mycoplasma genitalium
Macrolide Resistance Test (M. gen)* Ureaplasma Trichomonas vaginalis Gardnerella vaginalis
Herpes simplex I/II
*included if Positive M. gen is detected from the same sample.
Genetics
Haemophilia and
Thrombosis Genetics
Our Genetics teams are using
high-throughput sequencing to
combine the testing for rare bleeding,
platelet and thrombosis disorders
into a single workflow.
The Haemophilia and Thrombosis Genetics team currently offer full gene Sanger sequencing and variant analysis for the F7, F8, F9, F10, F11,
SERPINC1 (Antithrombin) and PROC genes.
Targeted Sanger sequencing and variant analysis of von Willebrand Factor (VWF) is carried out. To help inform the understanding of patients’ clinical and laboratory phenotypes, and of their disorder in general, two custom gene panels have been created for sequencing by synthesis on a high-throughput platform – an 87 gene panel for bleeding and platelet disorders, and a 15 gene panel for thrombosis disorders. The genes in the panels were selected in conjunction with HSL consulting clinicians and Genomics England. Genetic variants are interpreted and reported in line with ACGS/ACMG guidelines, and reports are issued following discussion and authorisation by Clinical Scientists and Consultant Haematologists.
Non-invasive prenatal testing
TDL Genetics were the first to offer
large-scale non-invasive prenatal
testing (NIPT) in the UK, and since
then the service has expanded to
help thousands of pregnant women
every month.
The Harmony® test is a well-proven, cell-free
DNA-based prenatal blood screen that tests for Down syndrome, Edwards syndrome and Patau syndrome. It can be used in singleton, twin and egg-donor pregnancies, and has been validated for use in pregnant women aged 18 to 48. Clinicians have used Harmony to screen over one million pregnancies in more than 100 countries around the world.
The most recent development with the service is 22q11.2 deletion screening as an additional option in the Harmony prenatal test menu. 22q11.2 deletion is the underlying cause of conditions described as DiGeorge syndrome and velocardiofacial syndrome (VCFS) and is the most common chromosomal microdeletion, occurring in up to 1 in 1000 pregnancies.
The test is aimed at a screening population, as it has been shown to identify 75% of pregnancies with a 22q11.2 deletion. It therefore requires careful genetic counselling, and pregnancies with a known higher risk of 22q11.2 deletion, whether ascertained through ultrasound scan or family history should consider invasive diagnostic testing as 1 in 4 (25%) cases will not be identified. If a high-risk pregnancy for 22q11.2 deletion is identified, a confirmatory aCGH (microarray) on the CVS or amnio is offered.
Cancer genetics
Rapid testing for leukaemia
HSL’s Haem-Oncology team is
introducing rapid genetic testing
to help with the treatment
management of leukaemia patients.
To help with the treatment management of patients with acute myeloid leukaemia and acute lymphoblastic leukaemia, HSL has introduced rapid testing for FLT3 and NPM1 gene mutations and for common translocations. Two new treatments for acute myeloid leukaemia were licenced by NICE in 2018, so it is particularly important to determine patients’ FLT3 status three days after diagnosis, to decide which drug they get.
The team have reduced this to a 48-hour turnaround, and soon, using a newly available assay in conjunction with an automated set-up and real-time PCR, this will be same-day testing.
For patients with chronic lymphocytic leukaemia, the team is working on rapid TP53/IgVH mutation testing. Patients with TP53 mutations do not respond to chemotherapy – they need targeted therapies. If this testing identifies such mutations, they qualify for ibrutinib and do not need to come to hospital to receive chemotherapy.
At present, TP53 testing is carried out as part of a next-generation sequencing panel and IgVH mutation status is obtained using Sanger sequencing. These two tests currently have 2- and 4-week turnarounds respectively. The new test will combine the two tests together in a multiplex and will use nanopore sequencing to interrogate the TP53 gene for mutations and gross deletions and to assess IgVH mutation
The team is also exploring the use of nanopore sequencing in conjunction with existing variant and fusion panels to give full genomic data on all newly diagnosed acute leukaemia patients within 72–96 hours. This has the potential to be of enormous clinical benefit; initial exploratory work will commence shortly. Other under development are the identification of KRAS/NRAS mutations and of IDH1/2 mutations by rapid PCR. It is important to identify KRAS/ NRAS mutations in patients with relapsed acute lymphoblastic leukaemia as the presence of certain KRAS/NRAS mutations opens up the possibility of MAP kinase inhibitors as a form of targeted therapy. Similarly it is also important to identify IDH1/2
mutations in patients with acute myeloid leukaemia as targeted therapy is available for patients with mutations in these genes. Rapidly identifying mutations in these genes means that treatment can potentially be given much more quickly.
For patients with a new diagnosis of B-Lymphoblastic Leukaemia, the team is working-up a new PCR-based multiplex assay that will allow the rapid detection and identification of fusion genes associated with B-ALL – including the rare ETV6-ABL1, NUP214-ABL1, RCSD1-ABL1, RCSD1-ABL2, PAD1-ABL2, SSBP2-CSF1R, SSBP2-PDGFRB, PAX5-JAK2, TERF-JAK2 and BCR-JAK2 fusions. This will help to identify patients who may benefit from tyrosine kinase inhibitors. It is anticipated that this will be available in early 2021.
Staff training and development
ELAINE HOLGADO
Elaine joined the team in 2001, when molecular genetics was still in its infancy as a discipline. She became a clinical scientist in 2005, gained her FRCPath Part 1 in 2008, and Part 2 in 2011. Later, she became a UKAS Assessor, a position that also enables her to build connections with other labs, and to share ideas and practice. As the first to follow this pathway, and now as training officer, she is helping others – as a mentor and by sharing her experience on how to apply this pathway to TDL as a lab and an institution.
“The support of the department and TDL has been invaluable throughout my career.”
DANIEL ODUKOYA
Daniel joined the team in 2013, initially working in sample reception in genetics. With a background in biomedical science, he soon began working in the laboratory on DNA extractions and PCR. He expanded and developed his experience, and with the move to the Halo he was promoted in 2018 to Lab Supervisor – one of two such positions who oversee the genetics and virology work.
He became registered as a Biomedical Scientist in 2019, choosing this route as the qualification gives versatility to his career at this stage and fits with the breadth of activities within Molecular Pathology at HSL.
“I love helping people in the lab, hands-on”
HSL is committed to helping staff with their development,
encouraging them to reach their full potential.
For the members of the Molecular Pathology team, the broad range of disciplines, specialties and technology that make up the division means that there are many opportunities to build their expertise and develop their careers.
Health Services Laboratories The Halo Building
1 Mabledon Place London WC1H 9AX T +44 (0)20 7307 9400 E [email protected] www.hslpathology.com
