by Dr Carsten Lange Flow cytometry is a powerful technique for the detailed analysis of complex populations which, over the last two decades, has evolved from a staple technique of the research laboratory into an essential part of the modern clinical laboratory.
Some of the current ‘norms’ for clinical flow cytometry include its critical use for phenotyping hematological malignancies, as well as playing a vital role – along with other testing methods – in diagnosing disease, informing treatment plans and monitoring patients. Only time can tell how this powerful analytical technology will contribute to the clinical lab of the future. We can, however, anticipate that it will only continue to increase in importance, based on technical innovations that have driven the evolution of flow cytometry over the last decade.
In addition to today’s applications in disease diagnostics, the power of this technology continues to be used in cell biology research and pharmaceutical discovery. This evolution has been made possible by a higher number of analytical parameters to measure cells in suspension. The first cytometers were systems capable of merely three or four parameters, using a single laser and four detectors, and were the size of a small car. Today, however, flow cytometers (including cell sorters) can analyse more than 30 parameters, and new technology in benchtop analysers can deliver exponentially better performance in a smaller footprint. Shifting paradigms This paradigm shift, toward higher performance in a small instrument, is driven by clinical laboratories that want to capture the power of flow cytometric analysis, but don’t want to invest a significant amount of time in learning the instrumentation. The democratization of flow cytometry is enabled by key advances in technology. Advantage is being taken of prominent concepts in other scientific fields, such as the telecommunications industry, to allow the subsystems to be miniaturized while at the same time providing even better performance. These compact high-performance systems not only deliver better performance than historically expensive systems, but they are also easy to set up, operate and maintain, enabling a greater number of clinical laboratories to maximize the power of flow cytometry. The power to see more Performance of flow cytometers is typically measured by their capacity to resolve and their sensitivity to detect dim and/or rare populations. In this regard, efficient light management for optimal excitation and emission of fluorochrome-tagged cells is critical to performance.
With conventional flow cytometers, laser excitation sources are optimized by shaping and focusing light through a series of lenses and filters onto a flow cell where cells are hydrodynamically focused. However, newer flow cytometers use unique laser designs that are focused onto a flow cell with integrated optics. These systems can ensure increased excitation of the dyes not only on (and within) cells, but also increased collection of the emitted light for integration and measurement. When designing a compact clinical cytometer, the use of fibre optics to carry light is an efficient way of transmission, providing flexibility in laying out system components. These cables capture emitted light to deliver it onto a unique detector array, reducing crosstalk between channels, which improves performance.
Another recent development is a key concept borrowed from the telecommunications industry, the wavelength division multiplexer (WDM), which is used for light detection and measurement. Wavelength division multiplexing is a method used to deconstruct and measure multiple wavelengths of light as signals that relate to analytical parameters. The detectors used to measure each parameter are avalanche photodiodes (APDs), which are highly sensitive semiconductor devices. By contrast, conventional clinical cytometers to date have (and continue to use) photomultiplier tubes (PMTs). The major advantages of using APDs over PMTs include but are not limited to:
enhanced linearity;
4–5 times the quantum efficiency;
higher dynamic range, 106 versus 103;
smaller size and about one-tenth the cost.
Shows the WDM of the first commercially available clinical cytometer to use compact APDs which reduce the overall instrument footprint (DxFLEX, Beckman Coulter). Each WDM contains optical and detector components to selectively measure specific wavelengths. This improves light collection for higher sensitivity to detect dim populations.
The WDM’s innovative and simple design uses a single bandpass filter to select the various colours of light. This contrasts with traditional clinical cytometers, which use a series of dichroic steering filters and bandpass filters that bounce the light along an array, leading to successively less available light, resulting in diminishing light collection efficiency, and ultimately compromising fluorescence sensitivity and resolution. Simplifying high complexity Leveraging the linearity of detection systems that use APDs in the operation of the cytometer can be dramatically simplified owing to the predictability of the signals. The linear gain and the normalization performed during the daily quality control routine takes care of the relative variations during instrument set-up commonly seen in instruments. Further, setting up a highcomplexity assay is simplified by using a software gain-only adjustment. The linearity of gain adjustment also simplifies the typically arduous task of spectral compensation which has been the barrier for many to push to a higher number of colours/ parameters. To maximize the benefit of the APD linearity, new software algorithms have been developed that facilitate set-up and analysis of high-complexity experiments by simplifying compensation.
It is now possible to create a compensation library that stores the APD gain settings and spectral spill-over coefficients for every parameter and multicolour combination. This allows users to make a virtual spectral compensation matrix selecting various single colours from the library. In addition, the library can intelligently adjust the compensation values when gains are adjusted owing to the predictive responses of linear APDs. The result is a dramatically simplified and intuitive method of setting up high-complexity applications. The size factor For most cytometers, measuring size of particles less than 300|nm is difficult because they deliver relative sizing information using forward scattered light from the 488|nm blue laser. For these systems, particles of less than 1|mm (1000|nm) usually fall below the noise threshold of the laser and detector subsystems. In contrast, newer systems use principles of Mie scattering, which predicts that with lower wavelengths of excitation there will be an increased amount of scattered light and improved resolution.
Therefore, measuring scattered light from a shorter-wavelength 405|nm violet laser versus a longer-wavelength 488|nm blue laser will allow the system to resolve smaller particles. The use of the violet side scatter parameter enables systems to detect particles of less than 0.2|mm (200|nm) in size, enabling excellent resolution of microparticles. The future is now Combining powerful performance and innovative design and technology, it is possible to deliver a compact, easy-to-use flow cytometer. Pushing the ‘norms’ of conventional flow cytometry, today’s – and tomorrow’s – cytometers simplify high-complexity applications in the clinical laboratory, as well as a deeper understanding in the frontier applications of hematopoietic cancers. Flow cytometry remains a powerful tool for interrogating complex questions. Today’s clinical laboratories want to harness that power and are demanding smaller and more powerful flow cytometers that are more affordable and easier to use. Using innovation, engineers can deliver solutions to meet the challenge. The author Carsten Lange PhD Beckman Coulter GmbH, 47807 Krefeld, Germany
by Peter Murphy It was first noticed that the rate of erythrocyte sedimentation changed owing to illness in the 1700s. The use of this attribute as a measure of inflammatory activity due to underlying disease was formalized into a test in the early 1900s and what has become known as the Westergren test has again recently been proposed to be the reference method for measuring erythrocyte sedimentation rate, which is still a commonly used hematology test today. This article allows you to understand why it is used, how the results are affected by physiological factors and how to perform it to obtain useful and reliable results. Using erythrocyte sedimentation rate measurement to indicate inflammation Explaining erythrocyte sedimentation rate measurement The erythrocyte sedimentation rate (ESR) is a general condition indicator and serves as a guide to determine diagnosis and treatment follow-up of different autoimmune diseases, acute and chronic infections and tumours. ESR is the speed at which erythrocytes settle in a tube and provides medical practitioners with valuable information for the diagnosis of their patients. Normal-sized erythrocytes are negatively charged and repel each other, which limits their sedimentation rate. Erythrocytes that form clumps fall faster than small ones, so factors that increase aggregation will increase sedimentation. This increased sedimentation indicates health problems, resulting in a need for additional tests. Applications of ESR measurement There’s a long list of conditions for which ESR can be used to assist in making a correct diagnosis or managing the care of a patient: autoimmune diseases such as rheumatoid arthritis, temporal arteritis and polymyalgia rheumatica are well known examples, as is multiple myeloma. When the presence of inflammation is suspected, ESR is a simple and cost-effective way of confirming this. Moreover, for patients with a known condition, the ESR test can provide useful information into the overall effectiveness of their treatment. The Westergren method The discovery of the ESR dates back to 1794, but in the 1920s, pathologist Robert Fåhraeus and Alf Westergren developed ESR measurement as we know it. To this day, the so-called Westergren method is recognized as the gold standard, among others by the Erythrocyte sedimentation rate: getting the most out of this test by Peter Murphy It was first noticed that the rate of erythrocyte sedimentation changed owing to illness in the 1700s. The use of this attribute as a measure of inflammatory activity due to underlying disease was formalized into a test in the early 1900s and what has become known as the Westergren test has again recently been proposed to be the reference method for measuring erythrocyte sedimentation rate, which is still a commonly used hematology test today. This article allows you to understand why it is used, how the results are affected by physiological factors and how to perform it to obtain useful and reliable results. Hematology and Flow Cytometry June 2020 13 | Clinical and Laboratory Standards Institute (CLSI). In 2017, the International Council for Standardization in Hematology (ICSH) reconfirmed the Westergren method as the reference method for ESR measurement. The Westergren method owes its popularity to the fact that it’s a simple and inexpensive first-line test, providing valuable information to GPs in the investigation of inflammation after only 60 (or even 30) minutes. Critical factors of a reliable ESR test Although the Westergren method may be the gold standard, many factors can meddle with its reliability. Therefore, always keep in mind the following requirements:
non-hemolysed blood anti-coagulated with EDTA at collection;
blood sample is thoroughly mixed and diluted 4|:|1 using a sodium citrate solution;
the tube is held in vertical position at a constant temperature (±1|°C) between 18|°C and 25|°C in an area free from vibrations, drafts and direct sunlight; and
results are interpreted after at least 30|minutes.
Can we speed up ESR measurement? In the original Westergren method, the ESR is read after 60|minutes. You can imagine this puts practical limitations on the workflow in clinical laboratories. A laboratory investigation, however, showed that 30-minute ESR readings correlate highly with the corresponding 60-minute ESR readings, which is why today most laboratories perform 30-minute ESR readings and then extrapolate them to derive the 60-minute ESR result. There are Westergren alternatives that claim to measure ESR after only 20|seconds, but as it takes at least 10|minutes before sedimentation starts at a constant rate, these tests risk leading to a number of false negatives. Why speeding up ESR measurement is not a good idea The Westergren method and faster alternatives As mentioned above, the 30-minute version of the Westergren test has become the standard in most hospitals and laboratories. However, even though 30|minutes can be regarded as a short time frame, some companies have worked on Westergren alternatives that can be read after mere minutes or even seconds. A major step forward, or so it seems. What’s the deal with fast ESR measurement methods? There are several conditions that ESR methods should comply with in order for them to be reliable. For example, test tubes must be held in vertical position, and the blood must be thoroughly mixed and diluted. Still the most important condition of all doesn’t revolve around equipment; it revolves around time. It takes approximately 10|minutes before red blood cell sedimentation starts at a constant rate. This means that ESR readings after 20|seconds do not actually measure sedimentation but calculate a mathematically derived ESR. This, in turn, leads to ESR readings that don’t correlate with the Westergren standard, leading to a number of false negatives. So, in their attempt to speed up the diagnosis of patients, laboratories that use Westergren alternatives risk overlooking important signs of disease. Speed or reliability? Healthcare and in vitro diagnostics are being improved daily and theories are constantly evolving. This makes it hard to determine which ESR method is the right one to choose. The choice is even harder when you consider that ESR alternatives are comparable to the Westergren method, as long as you treat healthy people under Erythrocyte sedimentation rate test normal circumstances. It’s when people are ill that the results start to deviate. This is why our advice is to always choose a method that adheres closely to the Westergren method [such as automated ESR analysers Starrsed (RR Mechatronics), MixRate and Excyte (ELITech)]. Westergren has always been the method of choice in fundamental studies, meaning that ESR is essentially based on this procedure. Moreover, the Westergren method is recommended by the CLSI and reconfirmed as the gold standard by ICSH, two organizations that inform healthcare professionals on state of the art technologies for in vitro diagnostic testing. Not everything can be rushed Moving forward is part of human nature; it’s why we’re always so busy making things better, faster and more comfortable. But in the case of ESR measurement, we simply have to face the fact that not everything can be rushed. We may be able to speed up the way we live, work and travel; we cannot force red blood cells to settle faster than they do. What we can do, is make ESR measurement tests as reliable as possible and have them help us improve diagnostics and save lives. Physiological and clinical factors that influence ESR values
In the investigation of inflammation, ESR measurement is often the first-line test of choice as it’s simple, inexpensive and – if based on the Westergren method – reliable, reproducible and sensitive. But as is the case with every test, there are physiological and clinical factors that may influence ESR results. In this section, we’ll tell you more about them. However, when reading about factors that influence ESR results, please keep in mind that much, if not all of this information, is based on studies undertaken with the Westergren gold standard ESR method only. This is mainly due to the fact that the Westergren ESR method has been almost universally used to investigate the clinical utility of the test in a range of disease states, with much of this work published in peer reviewed journals. As a result, there’s a deep body of knowledge that describes the impact of disease, the limitations and sources of interference with the Westergren ESR. As the Westergren method for ESR measures a physical process under a defined set of conditions, this expansive body of knowledge cannot simply be ‘transferred’ to estimations of ESR by methods that use centrifugation or optical rheology. What’s normal in ESR? Before discussing the factors that influence ESR results, first we should answer the question: what is normal? When patients suffer from a condition that causes inflammation, their erythrocytes form clumps which makes them settle faster than they would in the absence of an inflammatory response. However, ‘faster’ is a relative term, and what’s ‘normal’ changes based on sex and age category. Physiological and clinical factors that increase ESR The most obvious explanation for increased ESR is inflammation. During acute phase reactions, macromolecular plasma proteins, particularly fibrinogen, are produced that decrease the negative charges between erythrocytes and thereby encourage the formation of cell clumps. And as cell clumps settle faster, this increases ESR. Inflammation indicates a physical problem, meaning additional tests and follow-up are needed. However, there are other factors that increase ESR but don’t necessarily come with inflammation. For example, ESR values are higher for women than for men and increase progressively with age. Pregnancy also increases ESR, which means you’ll be dealing with ESR results above average. In anemia, the number of red blood cells is reduced, which increases so-called rouleaux formation so that the cells fall faster. This effect is strengthened by the reduced hematocrit, which affects the speed of the upward plasma current. Another factor that increases ESR revolves around high protein concentrations. And in macrocytosis, erythrocytes have a shape with a small surface-to-volume ratio, which leads to a higher sedimentation rate. Physiological and clinical factors that decrease ESR Apart from factors that increase ESR, medical practitioners and laboratory scientists should also consider the factors that decrease ESR. This is especially important as decreased ESR results may lead to missed diagnoses, whereas increased ESR results either lead to the right follow-up or false positives. Polycythemia, caused by increased numbers of red blood cells or by a decrease in plasma volume, artificially lowers ESR. Red blood cell abnormalities also affect aggregation, rouleaux formation and therefore sedimentation rate. Another cause of a low ESR is a decrease in plasma proteins, especially of fibrinogen and paraproteins. The four factors that determine ESR reliability (dos and don’ts)
As with any test, the reliability of ESR measurements stands or falls with proper implementation. When not reliably performed, the nonspecific indicator for inflammation may point in the wrong direction, and result in either a false positive or a false negative. This may lead to the initiation of unnecessary investigations or worse: the overlooking of serious problems that actually needed follow-up. In this section, we discuss some do’s and don’ts when performing ESR measurement, to guarantee ESR reliability. Factor 1: blood collection Do: make sure you mix and dilute the sample 4:1 using a sodium citrate solution. If you adhere to these practices, you standardize the way you handle the blood samples, and therefore their suitability for ESR. Don’t: leave the sample for too long before testing. We can imagine you’re pretty busy, and that you can’t do everything at the same time. However, when it comes to blood collection for ESR tests, some speed is required. After four hours, the results won’t be as accurate as before, which may negatively impact the reliability of the result. We therefore recommend performing the test within these four hours. If you really can’t make it in time, 24|hours is the max, but only if the sample is stored at 4|°C. Factor 2: tube handling Do: hold the tube vertically. A tube that is not held completely vertical can lead to increased sedimentation rates and is one of the technical factors that can affect ESR readings. And as we discussed in the previous paragraph, temperature is a factor too. Therefore, always place the tube in a stable and vertical position and at a constant temperature. Don’t: expose the sample to vibrations, draft and sunlight, as all of these factors can have a strong influence on the final result obtained. Factor 3: result reading Do: wait 30|minutes. This is a very important one. Before reading ESR results, you should always wait 30|minutes. There are ESR testing methods that claim to show reliable results within only 20|seconds, but as it takes 10|minutes before sedimentation starts at a constant rate, these tests do not actually measure sedimentation. In fact, they calculate a mathematically derived ESR, leading to a number of false negatives. Don’t: include the buffy coat (which is made up of leukocytes) in the erythrocyte column. Factor 4: test quality Do: go with an automated ESR test. They provide you with more reliable results, not least because they can correct hazy results. Moreover, automated ESR tests have a higher throughput compared to manual tests and minimize human contact with the tubes, which helps you reduce operations costs and minimize occupational health and safety risks. Don’t: choose an ESR test that deviates from the Westergren standard. This method has always been the method of choice in fundamental studies, meaning that ESR is essentially based on this procedure. ESR tests that deviate from the Westergren will logically provide you with different ESR values, meaning they can lead you in the wrong direction. This is why the Westergren method is recom-mended by the CLSI and reconfirmed as the gold standard by ICSH. ESR test as a reliable tool
If you keep these dos and don’ts in mind, you’re well on your way to making the ESR test a reliable tool that’s going to help you diagnose patients fast and error-free. The author Peter Murphy MBA(TechMgt), MAACB, BSc, GradDipEd ELITech Group, Braeside, Victoria 3195, Australia E-mail: p.murphy@elitechgroup.com
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by Professor Paul Kaye Leishmaniasis is classified as a neglected tropical disease. It is the cause of a huge health burden and is common in Asia, Africa, South and Central America, and even southern Europe. This article discusses how flow cytometry can help to evaluate diagnosis, monitor the effects of therapy and help in the creation of a vaccine.
Background
The leishmaniases are a family of devastating diseases, affecting a great many people across the globe and presenting a significant risk to both public health and socioeconomic development. The leishmaniases are vector-borne diseases, caused by infection with one of 20 species of the parasitic protozoan Leishmania (Fig. 1), transmitted through the bite of the infected female phlebotomine sand fly.
They can be broadly classified as tegumentary leishmaniases (TLs), affecting the skin and mucosa, and visceral leishmaniasis (VL), affecting internal organs. Whereas VL is responsible for over 20¦000 deaths per year, TL are non-life-threatening, chronic and potentially disfiguring, and account for around two-thirds of the global disease burden.
Within TL, there are three subtypes: self-healing lesions at the location of sand fly bite (cutaneous leishmaniasis; CL), lesions that spread from the original skin lesion to the mucosae (mucosal leishmaniasis; ML), and those which spread uncontrolled across the body (disseminated or diffuse cutaneous leishmaniasis; DCL). VL, also known as kala azar, involves major organs including the spleen, liver and bone marrow. In addition, patients recovering from VL after drug treatment often develop post kala-azar dermal leishmaniasis (PKDL), a chronic skin condition, characterized by nodular or macular lesions beginning on the face and spreading to the trunk and arms. As it may develop in up to half of patients previously treated and apparently cured from VL, it is thought that PKDL plays a central role in community transmission of VL.
The World Health Organization designates leishmaniasis as a neglected tropical disease (NTD), which together affect more than one|billion people across 149 countries worldwide; true prevalence may be even higher. Disproportionately, NTDs affect the poorest, malnourished individuals, and contribute to a vicious circle of poverty and disease. The significant physical marks, including ulcers, often left in the wake of the TLs may have an impact on mental health and perpetuate social stigma associated with the diseases [5]. There are over 1|million new cases of TL and 0.5|million new cases of VL each year, which together account for the loss of approximately 2.4|million disability-adjusted life years.
Treatment challenges
Leishmaniasis treatment can be quite difficult since at-risk populations may lack access to healthcare, and the limited battery of drugs has been increasingly compromised by resistance. Additionally, because the parasites in question are eukaryotic, they are not dissimilar from human cells, so the medication is also liable to be harmful – even fatal – to host as well as to pathogen.
Although the burden of VL in South Asia has been reduced with single-dose liposomal amphotericin B, the drug is less effective in other geographic locations, namely East Africa. Various drug combinations have been tested, unsuccessfully, and new chemical entities and immune-modulators are in early stages of development and as yet untested in the field. Unfortunately, little has changed in the treatment for CL for the past 50|years.
No vaccines are currently approved for any form of human leishmaniasis, although vaccines for canine VL have reached the market. Barriers to vaccine development include the limited investment in leishmaniases R&D and the high costs involved in bringing new products to those that need them.
Current work
My work on leishmaniasis has taken a holistic view, rooted in the immunology of the host-parasite interaction, but employing tools and approaches that span many disciplines: mathematics, ecology, vector biology and most recently neuroscience. Thirty years of discovery science has led to the development of a candidate for a therapeutic vaccine for PKDL, the mysterious sequela to VL [6]. ‘Therapeutic’ vaccines are given after an individual is infected with a pathogen and are designed to enhance our immune system and help eliminate the infection.
With colleagues from Sudan, we are in the midst of a phase IIb clinical trial funded by the Wellcome Trust, evaluating the efficacy of this therapeutic vaccine in Sudanese patients with persistent PKDL.
However, the research has been a long time in the making and has a long way to go. To continue to make progress, we linked with colleagues in Ethiopia, Kenya and Uganda and at the European Vaccine Initiative (http://www.euvaccine.eu/) in Germany, to develop a new research consortium to evaluate the immune status of people suffering from leishmaniasis. For example, using flow cytometry for blood and multiplexed immunohistochemistry for tissue biopsies, we can enumerate the proportions of lymphocytes, monocytes and neutrophils based on surface marker expression (e.g. CD3, CD19, CD14, CD16), and characterize their function, for instance by expression of cytokines (e.g. interferon-gamma) or other cell surface proteins that define function state. To support this endeavour, we recently received a grant from the European & Developing Countries Clinical Trials Partnership (EDCTP) that will allow us to not only extend our vaccine programme in Sudan [9] but also to address other important research challenges.
To develop vaccines and indeed new drugs, we often need tools capable of performing in-depth comparisons of how the body’s immune system is coping with the infection when a patient is first admitted to hospital and how it changes as the patient undergoes treatment and is hopefully cured. For example, recent evidence suggests that during infection, T lymphocytes may become ‘exhausted’ and unable to fight infection and the exhausted state can be identified by expression of surface molecules such as programmed cell death protein|1 (PD-1) and lymphocyte activation gene 3 protein (LAG-3). It is important to know if exhaustion can be reversed following treatment or whether we need to stimulate new populations of T lymphocytes. By understanding these nuanced changes in immune cells in our blood, we can design ways to improve how vaccines and drugs work in concert with immune cells, and understand why some patients might relapse from their disease or develop PKDL. Flow cytometry is a central tool for immunologists and plays a critical role in uncovering mechanisms of immunity and in assessing how well vaccines work and biomarkers of drug response. It uses antibodies that recognize specific molecules or markers on the surface or inside immune cells, such as those mentioned above, that help us predict their function. These antibodies are fluorescently labelled and the fluorescent signal can be detected by exposing each cell individually to laser light as they pass through a small aperture, the essence of flow cytometry.
For flow cytometry to be beneficial in this project, we needed to purchase five new flow cytometers that could meet exacting standards. They needed to be sufficiently sensitive to identify rare cell populations, often with low levels of surface marker expression. For multicentre research projects, reproducibility of data between sites is essential. Hence, we needed excellent inter-machine reproducibility and the Figure 2. Initial training course with recently appointed flow managers (Credit: Dr Karen Hogg, University of York) | 10 manufacturer had to be able to provide service support across the region. In our search for the right flow cytometer to support the consortium, we settled upon the CytoFLEX, Beckman Coulter Life Sciences’ research flow cytometer, which uses avalanche photodiode detection to arrive at the required level of sensitivity. With assistance from Beckman Coulter, we devised and have run initial training courses with a group of recently appointed flow managers from each partner country, to share standard operating procedures, develop high-level data analysis strategies as well as to provide instruction in routine instrument maintenance.
Beckman Coulter also provides another important aid to reducing errors in flow cytometry for multisite projects such as this, namely freeze-dried antibody cocktails (DURAClone panels) [10], that allow highly multiplexed phenotyping of small volumes of blood added directly to a single tube. Particularly for investigators in remote locations, the use of dry, preformulated reagents, rather than liquid (‘wet’) antibodies, removes the need for a cold chain. Equally importantly, staining of cells when manual mixing of 15 or 16 antibodies is required can introduce data inconsistencies when conducted by different individuals and at different locations.
Together, these innovations have allowed us to establish a new network for flow cytometry in East Africa that will allow us to identify and functionally characterize and identify the types of immune cells present in the blood during these devastating diseases. We will match this data with similar multiplexed techniques in pathology to compare blood immune cell profiles with those of cells found in the skin, to give a more complete picture of the host response to infection before and after treatment or vaccination.
Future Directions
As mentioned, we are currently in the midst of an efficacy trial of our therapeutic vaccine, ChAd63-KH. The technology we are using is similar to that being used by researchers at the university of Oxford to develop a coronavirus vaccine. In short, we introduce two genes from Leishmania parasites (KMP-11 and HASPB1) into a well-studied chimpanzee adenovirus (ChAd63 viral vector). After vaccination with this vaccine, host cells become infected with the virus and express the Leishmania proteins in a way that can be recognized efficiently by the immune system. We are particularly interested in how well this vaccine can generate T|cells to fight the infection.
With the first of our clinical objectives now well underway – the ongoing therapeutic clinical trial in patients with PKDL will be completed in mid-2021 – we have two additional goals. The next, funded by EDCTP, is to start a new clinical trial to determine whether the vaccine can prevent progression from VL to PKDL. And finally, we hope to develop a human challenge model of leishmaniasis to test the vaccine for its ability to protect against infection by different forms of parasite. This would open the way to the development of a cost-effective prophylactic vaccine to prevent these diseases occurring in vulnerable populations across the world.
Our research also has larger ambitions for the long term. Our East African partners are also linked together through their work on leishmaniasis in drug development, as members of the Leishmaniasis East Africa Platform group, established to help coordinate drug development activities in the region by the Drugs for Neglected Diseases Partnership. Central questions about why the disease varies between countries are being addressed, and the increased capacity for flow cytometry will additionally support patient monitoring during drug trials conducted by DNDi or other groups. Indeed, through the capacity building this project provides, we hope this project will extend its reach beyond leishmaniasis, providing muchneeded support for research on other neglected diseases of poverty that affect people in the region, including bacterial, fungal, other parasitic and viral diseases. By continuing to demonstrate the analytical power of flow cytometry and its role in helping design much-needed therapies, we hope to open up additional discovery research possibilities for colleagues in Africa and around the world.
The research described in this article is part of the EDCTP2 programme supported by the European Union (grant number RIA2016V-1640; PREV_PKDL; https://www.prevpkdl.eu). The author Paul Kaye PhD, FRCPath, FMedSci Hull York Medical School, University of York, York, UK E-mail: paul.kaye@york.ac.uk
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Expert opinions from Dr Heidi Mendoza There are many assessments to make when adding a new test to a lab’s collection. Dr Heidi Mendoza, acting consultant clinical biochemist at Raigmore Hospital, Inverness, UK, shares her experiences and observations of doing exactly that in both ordinary circumstances and during a pandemic, as well as having to contend with the geographic challenges imposed by the nature of life in the Scottish Highlands. Can you provide a little background about yourself and where you work, please?
I am a clinical biochemist based in Raigmore Hospital, which is a small hospital in the Scottish Highlands. In my current role I provide clinical advice and interpretation for biochemistry tests for general practitioner (GP) practices and three hospitals across the Highlands. Working in the Highlands is incredibly rewarding, but also very challenging! It can take between 2 and 6|hours to travel between hospitals and our patients may have to travel by plane or boat to be seen, with journey times of +12|hours depending on where they live. It really puts the laboratories under pressure to get it right for the patient. Repeat testing isn’t as simple or straightforward as it would be in a city and we have to have excellent systems in place for reporting critical results and getting patients into hospital or transferring them between hospitals. Getting the right test, in the right place, with the right turnaround time is really important for our patients and for our clinicians. What are the usual circumstances in which you would think about bringing a new test into the lab’s repertoire?
Any new test is a cost pressure on our National Health Service (NHS) and can only be brought in when it demonstrates clear benefits for patients. We have brought in two new tests in the last 12|months that are good examples of the different ways we can bring in new tests to our laboratory.
The first test is the NT-proB-type natriuretic peptide (NTproBNP) test. NTproBNP is used to investigate patients with suspected heart failure and the results can be used to determine whether a patient needs an echocardiogram (ECHO) or not. If they do need an ECHO the NTproBNP result can be used to split patients into those who need urgent ECHO (2|weeks) or routine ECHO (6|weeks). In theory this is a perfect test to implement as it will benefit patients and is cost-effective with respect to the more expensive ECHO investigation. However, NTproBNP has been implemented in other hospitals without reducing ECHO waiting times or the number of ECHOs performed! To ensure that this didn’t happen in our service, I spent 6|months before implementation of the test liaising with cardiologists and GP representatives from across the Highland region. We changed the ECHO referral pathway to include NTproBNP and created useful guidance for GPs on when to, and importantly when not to, request NTproBNP. We implemented the test just under 1|year ago and have seen a positive effect on ECHO referrals. We will still have to attend a 1|year post-implementation review with the Hospital Board to present our audit data and show that investment in the service by introducing a new test has benefited patients and other areas of the service.
Procalcitonin is the second example. Procalcitonin is a test that can be used in the investigation of sepsis and guide the use of antibiotics. Procalcitonin was not a test available in our hospital before the COVID-19 pandemic. Procalcitonin is not increased in the majority of adult patients with COVID-19; however, an elevated procalcitonin may suggest superimposed bacterial infection and be used to guide treatment of these patients and improve patient outcomes. Early in the COVID-19 pandemic we were approached by our Intensive Care Unit (ITU) and Microbiology consultants who requested that procalcitonin be available for our COVID-19 patients in ITU to guide their antibiotic treatment. We implemented procalcitonin in less than 4|weeks with help from our instrument manufacturer, external quality assessment providers and other Scottish hospitals who provided anonymized patient serum with known values so that we could verify our assay as quickly as possible. We are now in the process of putting together a business case and following the evidence base which will determine whether we continue to offer the procalcitonin test. How would you usually go about adopting a new test?
As highlighted in the two examples above, we must agree a clinical need for a test and then liaise with the users of the service to find out how the test should be implemented into the patient-care pathway. Once we have worked out the clinical utility of the test, then we can carry out the laboratory verification of the test and the laboratory workflow. Verification is very straightforward. For example, the between-batch and within-batch precision, accuracy, linearity on dilution, interferences and sample stability for a test need to be evaluated. The implementation of the test then must be followed by an audit which shows that the test is being used as intended and giving the benefits predicted. If not, the test may need to be withdrawn. The hardest part of the entire process is agreeing how a test is going to be used and fitting it in to the patient-care pathway. In the situation of the COVID-19 pandemic, we have a new disease, caused by a new virus, and new tests that have been created very quickly. How do you start to use a new test in these circumstances – are there any differences in procedure?
There is no difference in the steps that need to be performed we just need to be able to do everything in a much shorter time frame. That is actually much easier than it sounds. In the NHS, the laboratories from different parts of the country are great about helping other laboratories. We regularly share protocols, data and learning. If a new test is released we’ll contact another laboratory and they’ll share their local experience and any problems they have had with the test.
For procalcitonin implementation I contacted the laboratory in Dundee, UK, and they helped us out by lending us kits and reagents, sending us anonymized patient serum with known procalcitonin values, and sharing their data and verification protocols. This allowed us to complete verification incredibly quickly. We will still have to gather the data and evaluate whether the test is providing the benefit that we predicted when we established the clinical need. What are the challenges regarding validation, reference levels, results interpretation and reporting?
Verifying tests is straightforward as we are always evaluating tests in clinical laboratories so are very experienced. Results interpretation can be quite difficult. If we need clinicians to change patient management based on a result then we have to provide them with very clear local guidance on what we want them to do with a result. This might be different from the action they would take in another hospital with different patient pathways, different pressures on patient turnaround times, and different diagnostic facilities. This is where good working relationships with users of the service are key to test implementation. If you just implement a new test without working out where it fits in the patient pathway, it doesn’t matter how great the test is, as it is unlikely to be used well and may not improve patient care. What do you have to think about in terms of logistics?
Many laboratories are understaffed due to a combination of unfilled vacancies and staff on long-term absence. The additional work involved in verifying and implementing a new test does put pressure on staff. However, NHS laboratory staff are highly trained and dedicated. When the staff know how a test is going to be used and the benefit to the local community, they support the implementation and the extra work involved.
Biocontainment and staff safety have been important considerations during the COVID-19 pandemic. We had to adhere to government guidance in the transport, analysis and disposal of samples from patients with suspected COVID-19. This changed laboratory workflows and slowed us down, creating longer turnaround times.
Logistics are a serious consideration for us owing to our geography. Reagent shortages or delays in deliveries have a big impact on small laboratories as they can’t store much surplus reagent stocks because of expiry dates. Unexpected overuse or underuse of a new test can be quite challenging and leave the laboratory short of tests or with expired, wasted kits. There are also several times during the year when the roads are impassable between our central and rural laboratories. We have been down to single numbers of tests remaining several times over the last few years or had failed delivery from manufacturers in winter. There was also a shortage of procalcitonin reagent as there was such a surge in the use of the test during the COVID-19 pandemic. Again, working closely with users of our laboratory services has enabled us to rationalize the use of the test until the global shortage of reagent ended. On a number of occasions we have also shared reagents with other Scottish laboratories to ensure that none of the laboratories were left without reagents. What has been learnt from the current coronavirus situation about diagnostic testing during a pandemic that would help to improve the process in future?
The coronavirus pandemic has shown how robust the infrastructure of the NHS is in Scotland and how adaptable laboratories can be when required. The laboratories really pulled together and worked towards a common goal delivering testing to COVID patients and non-COVID patients during a crisis. The two things that made this possible were: (1) Having a very clear goal – delivery of a service with new testing during a pandemic; and (2) Finances changes which needed to be made to deliver the service got rapid financial approval. How do we take these lessons learned and apply it to the routine delivery of laboratory services? Finance will always be a limiting factor – as it should be! Healthcare is expensive and it is up to us as healthcare professionals to deliver a cost-effective and affordable service. In contrast, having a clear goal, is definitely something that we could do better in the future. In the case of the pandemic, laboratories found different solutions based on local geography, resources and incidence of COVID. The changes made by laboratories in the remote Highlands and Islands were similar, but different than those made by laboratories in major cities. The staff that delivered the service found the best solutions to the goals set by the government – that is the real lesson we need to take away. We need to give very clear goals to services and let local expertise and knowledge drive the changes to solve the problem. The expert Heidi Mendoza BSc MSc PhD RCPath Blood Sciences Department, Raigmore Hospital, Inverness IV2 3UJ, UK E-mail: heidi.mendoza@nhs.net
https://clinlabint.com/wp-content/uploads/sites/2/2020/06/clinlab-logo.png003wmediahttps://clinlabint.com/wp-content/uploads/sites/2/2020/06/clinlab-logo.png3wmedia2020-08-26 09:31:372021-01-08 11:07:48Introducing new tests to a laboratory’s repertoire
LGC has acquired The Native Antigen Company (NAC), one of the world’s leading suppliers of high quality infectious disease antigens and antibodies.
NAC is a developer, manufacturer and supplier of critical reagents to the in vitro diagnostic (IVD), pharmaceutical and academic sectors. It offers a comprehensive portfolio of native and recombinant infectious disease antigens and related products including pathogen receptors, virus-like particles and antibodies for use in immunoassay applications, vaccine development and quality control solutions. NAC was one of the first companies globally to offer antigens for SARS-COV-2 and continues to play an important role in supporting the global response to the COVID-19 pandemic.
The acquisition strengthens LGC’s existing product offering to the IVD sector, which includes a range of quality assurance tools, immunoassay reagents and disease state plasma as well as probes and primers for molecular diagnostics.
“NAC is a natural fit with our clinical diagnostics business and will enable us to provide an expanded portfolio of critical reagents to our customers. NAC’s focus on infectious disease is highly complementary with our existing offer to this segment comprising controls, reference materials, MDx tools and other components,” said Michael Sweatt, Executive Vice President and General Manager, Clinical Diagnostics, LGC.
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Avacta Group, the developer of Affimer biotherapeutics and reagents, has entered into a collaboration with Integumen to evaluate recently generated Affimer reagents that bind the SARS-COV-2 spike protein for the detection of the coronavirus in waste water, to provide a real-time alert system to warn of localised COVID-19 outbreaks.
Over 60 percent of COVID-19 positive patients had gastrointestinal symptoms, such as diarrhoea, nausea and vomiting, and the SARS-COV-2 virus was found in their faecal samples. Sampling waste water from households may therefore provide an early warning system for localised outbreaks in communities.
Recently, Avacta announced that it had generated a number of highly specific Affimer reagents that detect the SARS-COV-2 virus spike protein for use in diagnostic tests and in neutralising therapies.
The collaboration with Integumen, announced 13 July, aims to evaluate some of these Affimer reagents in next-generation sensors, based on the real-time bacteria detection and alert system1 developed by Rinocloud, a subsidiary of Integumen, with the aim of integrating these sensors into Modern Water’s Microtox water contamination system to detect the coronavirus. The award-winning Microtox system, which can detect the presence of contaminating bacteria, virus and toxins, is distributed by Modern Water and has a global footprint of over 3,000 installations. The proposed Affimer sensors would be consumable items to be replaced on a roughly monthly basis.
Once initial testing of the Affimer reagents is completed over the next few weeks, validation of the sensors will be carried out using SARS-COV-2 virus samples in a containment level 3 laboratory at the University of Aberdeen. Upon successful completion of this evaluation, Integumen and Avacta will enter into a supply agreement to allow Integumen to manufacture and commercialise the waste water detection sensors globally by retrofitting into Microtox systems.
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Byondis B.V. (formerly Synthon Biopharmaceuticals) announced that Quantum Leap Healthcare Collaborative (Quantum Leap) selected the company’s investigational antibody-drug conjugate (ADC) SYD985 ([vic-]trastuzumab duocarmazine) for a new investigational treatment arm in its ongoing I-SPY 2 TRIAL for neoadjuvant treatment of locally advanced breast cancer. This treatment arm will focus on treatment for HER2-low early-stage breast cancer.
The I-SPY 2 TRIAL (Investigation of Serial studies to Predict Your Therapeutic Response with Imaging And moLecular analysis) is a standing Phase II randomized, controlled, multicentre study aimed at rapidly screening and identifying promising treatments in specific subgroups of women with newly-diagnosed, high-risk, locally advanced breast cancer (Stage II/III). Quantum Leap, sponsor of the I-SPY 2 TRIAL, leads a pre-competitive consortium that includes the U.S. Food & Drug Administration (FDA), industry, patient advocates, philanthropic sponsors, and clinicians from 16 major U.S. cancer research centres.
The new I-SPY 2 treatment arm will evaluate SYD985 against standard of care therapy in Stage II/III early-stage, high-risk breast cancer patients, with a focus on tumours with heterogeneous and low HER2 expression. Byondis will supply the investigational drug and provide financial and regulatory support. Quantum Leap, as sponsor, will provide the clinical sites and clinical expertise.
SYD985 is Byondis’ most advanced ADC, targeting a range of HER2-positive cancers such as metastatic breast cancer (MBC) and endometrial cancer. The company is currently conducting a Phase III study of SYD985 (TULIP or SYD985.002) to compare its efficacy and safety to physician’s choice treatment in patients with HER2-positive unresectable locally advanced or metastatic breast cancer. Previously, the FDA granted fast track designation for SYD985 based on promising data from heavily pre-treated last-line HER2-positive MBC patients participating in a two-part Phase I clinical trial (SYD985.001).
SYD985 uses Byondis’ unique, proprietary linker-drug (LD) technology. Although marketed ADCs have improved therapeutic indices compared to classical non-targeted chemotherapeutic agents, there is still room for improvement.
SYD985 is comprised of the monoclonal antibody trastuzumab and a cleavable linker-drug called valine-citrulline-seco-DUocarmycin-hydroxyBenzamide-Azaindole (vc-seco-DUBA). The antibody part of SYD985 binds to HER2 on the surface of the cancer cell and the ADC is internalized by the cell. After proteolytic cleavage of the linker, the inactive cytotoxin is activated and DNA damage is induced, resulting in tumour cell death. SYD985 can be considered a form of targeted chemotherapy.
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Dante Labs, a pioneer and leader in genomic testing, Cambridge Cancer Genomics (CCG.ai), a software developer specialising in data-driven precision oncology, and Nonacus, a provider of genetic testing products for precision medicine and liquid biopsy, have signed a collaboration agreement.
In a joint statement they said the partnership aims to build the most comprehensive and patient-centric tumour profiling service enabling improved cancer patient management, treatment and monitoring. By combining Dante Labs’ experience and capacity in delivering a sequencing service for both solid tumour and cell free circulating tumour DNA from liquid biopsies, Nonacus’ sensitive targeted pan-cancer NGS libraries, and CCG.ai’s industry leading AI powered software platform, OncOS, the companies will enable precision oncology at scale.
Improving outcomes for cancer patients means ensuring they have the right drug, at the right time to beat their cancer. This means understanding the molecular profile of the individual cancer and using that data to recommend treatments or clinical trials. Oncologists and clinical researchers will be able to send samples for processing to Dante Labs, who will use library preparation kits from Nonacus and software from CCG.ai to create a sample to report solution. If there are actionable mutations, the report will recommend the right treatments for those mutations, if there are novel or unactionable mutations, the software will also be able to match possible clinical trials. Chris Sale, CEO of Nonacus, said: “Long turn-around time and lack of clinically oriented analysis are the main obstacles to fully deliver the potential of cancer genomics to patients. This partnership will provide the flexibility and accuracy that oncology professionals need to integrate cancer genomics into the care of their patients. The COVID pandemic has increased the backlog of genetic testing for cancer, potentially leaving many suspected cancers unconfirmed and treatments delayed. Dante Labs are one of the biggest clinical sequencing hub in Europe able to process large numbers of samples in high throughput. It is our hope that by combining AI software from CCG.ai and our library preparation kits, together we will be able to process samples and provide bioinformatic analysis critical to determining the best treatment path for patients.”
https://clinlabint.com/wp-content/uploads/sites/2/2020/08/Dr_Chrs_Sale.jpg4803843wmediahttps://clinlabint.com/wp-content/uploads/sites/2/2020/06/clinlab-logo.png3wmedia2020-08-26 09:31:372021-01-08 11:07:47Dante Labs, Cambridge Cancer Genomics and Nonacus collaborate to provide precision oncology at scale
Start Codon, a new model of life science and healthcare business accelerator, has announced its first cohort of start-up companies. Start Codon aims to minimise risk and translate early stage research into successful start-ups, ready for funding and partnership. Start Codon has worked closely with four life science and healthcare companies that were enrolled into the programme in February this year.
They are:
Enhanc3D Genomics, a functional genomics spin-out of the Babraham Institute (Cambridge, UK) whose platform technology links non-coding sequence variants to their target genes in order to identify novel therapeutic targets
Drishti Discoveries, a start-up leveraging a proprietary gene silencing technology to develop therapies for rare inherited diseases
Spirea, a spin-out from the University of Cambridge, who is developing the next generation of antibody drug conjugate cancer therapeutics which carry more drug payload to tumour cells, resulting in greater efficacy, tolerability and the ability to treat more cancer patients
Semarion, a University of Cambridge spin-out, who is revolutionising cell-based assays for drug discovery and life science through its proprietary SemaCyte microcarrier platform, which leverages novel materials physics for assay miniaturisation, multiplexing, and automation
Start Codon plans to invest in and support up to 50 start-up companies over the next five years. The accelerator is now accepting applications for its second and third cohorts of companies. Early stage start-up companies in the life sciences and healthcare space are invited to apply via https://startcodon.co/application-form
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The ReactoMate DATUM from Asynt is a high quality, dual-rod stainless steel and aluminium support system built to ensure the stability and safety of your lab reactor. Sturdy, yet compact, the ReactoMate DATUM support system can accommodate a wide range of reaction vessels from 100 mL up to 5000 mL.
Designed with user-friendliness in mind, the ReactoMate DATUM support system incorporates a suite of innovative features.
Changing a vessel supported by the ReactoMate DATUM is as simple as “Clip & Click”. The novel neck clamp allows fast changeover between reactor vessel sizes thereby enabling simple reaction scale-up, whilst the ingenious mounting mechanism ensures excellent stability and alignment every time.
The Reactomate DATUM support system is fully compatible with all leading brands of overhead stirrers and circulator heating/cooling systems. Designed by chemists for chemists, low-friction polymer bearings line both the overhead stirrer alignment chuck and the neck support to ensure smooth and easy operation.
Ideally suited for use within a benchtop fume hood, adjustable feet allow you to level the ReactoMate DATUM support system ensuring stability and security while you work. Each DATUM system is also supplied with a moulded drip tray that fits perfectly within the base of the support, for safely catching any drips and spills from the reaction vessel during draining.
With a wide range of accessories and upgrades available, including drain manifolds and automation packages, the ReactoMate DATUM support system is the perfect all-rounder for laboratory scale reactions.
For more information, visit: www.asynt.com/product/reactomate-datum
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Technology update – Pushing the ‘norms’ of conventional high-complexity clinical cytometry
, /in E-News /by 3wmediaby Dr Carsten Lange
Flow cytometry is a powerful technique for the detailed analysis of complex populations which, over the last two decades, has evolved from a staple technique of the research laboratory into an essential part of the modern clinical laboratory.
Some of the current ‘norms’ for clinical flow cytometry include its critical use for phenotyping hematological malignancies, as well as playing a vital role – along with other testing methods – in diagnosing disease, informing treatment plans and monitoring patients. Only time can tell how this powerful analytical technology will contribute to the clinical lab of the future. We can, however, anticipate that it will only continue to increase in importance, based on technical innovations that have driven the evolution of flow cytometry over the last decade.
In addition to today’s applications in disease diagnostics, the power of this technology continues to be used in cell biology research and pharmaceutical discovery. This evolution has been made possible by a higher number of analytical parameters to measure cells in suspension. The first cytometers were systems capable of merely three or four parameters, using a single laser and four detectors, and were the size of a small car. Today, however, flow cytometers (including cell sorters) can analyse more than 30 parameters, and new technology in benchtop analysers can deliver exponentially better performance in a smaller footprint.
Shifting paradigms
This paradigm shift, toward higher performance in a small instrument, is driven by clinical laboratories that want to capture the power of flow cytometric analysis, but don’t want to invest a significant amount of time in learning the instrumentation. The democratization of flow cytometry is enabled by key advances in technology. Advantage is being taken of prominent concepts in other scientific fields, such as the telecommunications industry, to allow the subsystems to be miniaturized while at the same time providing even better performance. These compact high-performance systems not only deliver better performance than historically expensive systems, but they are also easy to set up, operate and maintain, enabling a greater number of clinical laboratories to maximize the power of flow cytometry.
The power to see more
Performance of flow cytometers is typically measured by their capacity to resolve and their sensitivity to detect dim and/or rare populations. In this regard, efficient light management for optimal excitation and emission of fluorochrome-tagged cells is critical to performance.
With conventional flow cytometers, laser excitation sources are optimized by shaping and focusing light through a series of lenses and filters onto a flow cell where cells are hydrodynamically focused. However, newer flow cytometers use unique laser designs that are focused onto a flow cell with integrated optics. These systems can ensure increased excitation of the dyes not only on (and within) cells, but also increased collection of the emitted light for integration and measurement. When designing a compact clinical cytometer, the use of fibre optics to carry light is an efficient way of transmission, providing flexibility in laying out system components. These cables capture emitted light to deliver it onto a unique detector array, reducing crosstalk between channels, which improves performance.
Another recent development is a key concept borrowed from the telecommunications industry, the wavelength division multiplexer (WDM), which is used for light detection and measurement. Wavelength division multiplexing is a method used to deconstruct and measure multiple wavelengths of light as signals that relate to analytical parameters. The detectors used to measure each parameter are avalanche photodiodes (APDs), which are highly sensitive semiconductor devices. By contrast, conventional clinical cytometers to date have (and continue to use) photomultiplier tubes (PMTs). The major advantages of using APDs over PMTs include but are not limited to:
Shows the WDM of the first commercially available clinical cytometer to use compact APDs which reduce the overall instrument footprint (DxFLEX, Beckman Coulter). Each WDM contains optical and detector components to selectively measure specific wavelengths. This improves light collection for higher sensitivity to detect dim populations.
The WDM’s innovative and simple design uses a single bandpass filter to select the various colours of light. This contrasts with traditional clinical cytometers, which use a series of dichroic steering filters and bandpass filters that bounce the light along an array, leading to successively less available light, resulting in diminishing light collection efficiency, and ultimately compromising fluorescence sensitivity and resolution.
Simplifying high complexity
Leveraging the linearity of detection systems that use APDs in the operation of the cytometer can be dramatically simplified owing to the predictability of the signals. The linear gain and the normalization performed during the daily quality control routine takes care of the relative variations during instrument set-up commonly seen in instruments. Further, setting up a highcomplexity assay is simplified by using a software gain-only adjustment. The linearity of gain adjustment also simplifies the typically arduous task of spectral compensation which has been the barrier for many to push to a higher number of colours/ parameters. To maximize the benefit of the APD linearity, new software algorithms have been developed that facilitate set-up and analysis of high-complexity experiments by simplifying compensation.
It is now possible to create a compensation library that stores the APD gain settings and spectral spill-over coefficients for every parameter and multicolour combination. This allows users to make a virtual spectral compensation matrix selecting various single colours from the library. In addition, the library can intelligently adjust the compensation values when gains are adjusted owing to the predictive responses of linear APDs. The result is a dramatically simplified and intuitive method of setting up high-complexity applications.
The size factor
For most cytometers, measuring size of particles less than 300|nm is difficult because they deliver relative sizing information using forward scattered light from the 488|nm blue laser. For these systems, particles of less than 1|mm (1000|nm) usually fall below the noise threshold of the laser and detector subsystems. In contrast, newer systems use principles of Mie scattering, which predicts that with lower wavelengths of excitation there will be an increased amount of scattered light and improved resolution.
Therefore, measuring scattered light from a shorter-wavelength 405|nm violet laser versus a longer-wavelength 488|nm blue laser will allow the system to resolve smaller particles. The use of the violet side scatter parameter enables systems to detect particles of less than 0.2|mm (200|nm) in size, enabling excellent resolution of microparticles.
The future is now
Combining powerful performance and innovative design and technology, it is possible to deliver a compact, easy-to-use flow cytometer. Pushing the ‘norms’ of conventional flow cytometry, today’s – and tomorrow’s – cytometers simplify high-complexity applications in the clinical laboratory, as well as a deeper understanding in the frontier applications of hematopoietic cancers. Flow cytometry remains a powerful tool for interrogating complex questions. Today’s clinical laboratories want to harness that power and are demanding smaller and more powerful flow cytometers that are more affordable and easier to use. Using innovation, engineers can deliver solutions to meet the challenge.
The author
Carsten Lange PhD
Beckman Coulter GmbH, 47807 Krefeld, Germany
E-mail: clange@beckman.com DxFLEX flow cytometer: https://www.mybeckman.uk/flow-cytometry/instruments/dxflex
Erythrocyte sedimentation rate: getting the most out of this test
, /in E-News, Editors' Picks /by 3wmediaby Peter Murphy
It was first noticed that the rate of erythrocyte sedimentation changed owing to illness in the 1700s. The use of this attribute as a measure of inflammatory activity due to underlying disease was formalized into a test in the early 1900s and what has become known as the Westergren test has again recently been proposed to be the reference method for measuring erythrocyte sedimentation rate, which is still a commonly used hematology test today. This article allows you to understand why it is used, how the results are affected by physiological factors and how to perform it to obtain useful and reliable results.
Using erythrocyte sedimentation rate measurement to indicate inflammation
Explaining erythrocyte sedimentation rate measurement
The erythrocyte sedimentation rate (ESR) is a general condition indicator and serves as a guide to determine diagnosis and treatment follow-up of different autoimmune diseases, acute and chronic infections and tumours. ESR is the speed at which erythrocytes settle in a tube and provides medical practitioners with valuable information for the diagnosis of their patients. Normal-sized erythrocytes are negatively charged and repel each other, which limits their sedimentation rate. Erythrocytes that form clumps fall faster than small ones, so factors that increase aggregation will increase sedimentation. This increased sedimentation indicates health problems, resulting in a need for additional tests.
Applications of ESR measurement
There’s a long list of conditions for which ESR can be used to assist in making a correct diagnosis or managing the care of a patient: autoimmune diseases such as rheumatoid arthritis, temporal arteritis and polymyalgia rheumatica are well known examples, as is multiple myeloma. When the presence of inflammation is suspected, ESR is a simple and cost-effective way of confirming this. Moreover, for patients with a known condition, the ESR test can provide useful information into the overall effectiveness of their treatment.
The Westergren method
The discovery of the ESR dates back to 1794, but in the 1920s, pathologist Robert Fåhraeus and Alf Westergren developed ESR measurement as we know it. To this day, the so-called Westergren method is recognized as the gold standard, among others by the Erythrocyte sedimentation rate: getting the most out of this test by Peter Murphy It was first noticed that the rate of erythrocyte sedimentation changed owing to illness in the 1700s. The use of this attribute as a measure of inflammatory activity due to underlying disease was formalized into a test in the early 1900s and what has become known as the Westergren test has again recently been proposed to be the reference method for measuring erythrocyte sedimentation rate, which is still a commonly used hematology test today. This article allows you to understand why it is used, how the results are affected by physiological factors and how to perform it to obtain useful and reliable results. Hematology and Flow Cytometry June 2020 13 | Clinical and Laboratory Standards Institute (CLSI). In 2017, the International Council for Standardization in Hematology (ICSH) reconfirmed the Westergren method as the reference method for ESR measurement. The Westergren method owes its popularity to the fact that it’s a simple and inexpensive first-line test, providing valuable information to GPs in the investigation of inflammation after only 60 (or even 30) minutes.
Critical factors of a reliable ESR test
Although the Westergren method may be the gold standard, many factors can meddle with its reliability. Therefore, always keep in mind the following requirements:
Can we speed up ESR measurement?
In the original Westergren method, the ESR is read after 60|minutes. You can imagine this puts practical limitations on the workflow in clinical laboratories. A laboratory investigation, however, showed that 30-minute ESR readings correlate highly with the corresponding 60-minute ESR readings, which is why today most laboratories perform 30-minute ESR readings and then extrapolate them to derive the 60-minute ESR result. There are Westergren alternatives that claim to measure ESR after only 20|seconds, but as it takes at least 10|minutes before sedimentation starts at a constant rate, these tests risk leading to a number of false negatives.
Why speeding up ESR measurement is not a good idea
The Westergren method and faster alternatives
As mentioned above, the 30-minute version of the Westergren test has become the standard in most hospitals and laboratories. However, even though 30|minutes can be regarded as a short time frame, some companies have worked on Westergren alternatives that can be read after mere minutes or even seconds. A major step forward, or so it seems.
What’s the deal with fast ESR measurement methods?
There are several conditions that ESR methods should comply with in order for them to be reliable. For example, test tubes must be held in vertical position, and the blood must be thoroughly mixed and diluted. Still the most important condition of all doesn’t revolve around equipment; it revolves around time. It takes approximately 10|minutes before red blood cell sedimentation starts at a constant rate. This means that ESR readings after 20|seconds do not actually measure sedimentation but calculate a mathematically derived ESR. This, in turn, leads to ESR readings that don’t correlate with the Westergren standard, leading to a number of false negatives. So, in their attempt to speed up the diagnosis of patients, laboratories that use Westergren alternatives risk overlooking important signs of disease.
Speed or reliability?
Healthcare and in vitro diagnostics are being improved daily and theories are constantly evolving. This makes it hard to determine which ESR method is the right one to choose. The choice is even harder when you consider that ESR alternatives are comparable to the Westergren method, as long as you treat healthy people under Erythrocyte sedimentation rate test normal circumstances. It’s when people are ill that the results start to deviate. This is why our advice is to always choose a method that adheres closely to the Westergren method [such as automated ESR analysers Starrsed (RR Mechatronics), MixRate and Excyte (ELITech)]. Westergren has always been the method of choice in fundamental studies, meaning that ESR is essentially based on this procedure. Moreover, the Westergren method is recommended by the CLSI and reconfirmed as the gold standard by ICSH, two organizations that inform healthcare professionals on state of the art technologies for in vitro diagnostic testing.
Not everything can be rushed
Moving forward is part of human nature; it’s why we’re always so busy making things better, faster and more comfortable. But in the case of ESR measurement, we simply have to face the fact that not everything can be rushed. We may be able to speed up the way we live, work and travel; we cannot force red blood cells to settle faster than they do. What we can do, is make ESR measurement tests as reliable as possible and have them help us improve diagnostics and save lives.
Physiological and clinical factors that influence ESR values
In the investigation of inflammation, ESR measurement is often the first-line test of choice as it’s simple, inexpensive and – if based on the Westergren method – reliable, reproducible and sensitive. But as is the case with every test, there are physiological and clinical factors that may influence ESR results. In this section, we’ll tell you more about them. However, when reading about factors that influence ESR results, please keep in mind that much, if not all of this information, is based on studies undertaken with the Westergren gold standard ESR method only. This is mainly due to the fact that the Westergren ESR method has been almost universally used to investigate the clinical utility of the test in a range of disease states, with much of this work published in peer reviewed journals. As a result, there’s a deep body of knowledge that describes the impact of disease, the limitations and sources of interference with the Westergren ESR. As the Westergren method for ESR measures a physical process under a defined set of conditions, this expansive body of knowledge cannot simply be ‘transferred’ to estimations of ESR by methods that use centrifugation or optical rheology.
What’s normal in ESR?
Before discussing the factors that influence ESR results, first we should answer the question: what is normal? When patients suffer from a condition that causes inflammation, their erythrocytes form clumps which makes them settle faster than they would in the absence of an inflammatory response. However, ‘faster’ is a relative term, and what’s ‘normal’ changes based on sex and age category.
Physiological and clinical factors that increase ESR
The most obvious explanation for increased ESR is inflammation. During acute phase reactions, macromolecular plasma proteins, particularly fibrinogen, are produced that decrease the negative charges between erythrocytes and thereby encourage the formation of cell clumps. And as cell clumps settle faster, this increases ESR. Inflammation indicates a physical problem, meaning additional tests and follow-up are needed. However, there are other factors that increase ESR but don’t necessarily come with inflammation. For example, ESR values are higher for women than for men and increase progressively with age. Pregnancy also increases ESR, which means you’ll be dealing with ESR results above average. In anemia, the number of red blood cells is reduced, which increases so-called rouleaux formation so that the cells fall faster. This effect is strengthened by the reduced hematocrit, which affects the speed of the upward plasma current. Another factor that increases ESR revolves around high protein concentrations. And in macrocytosis, erythrocytes have a shape with a small surface-to-volume ratio, which leads to a higher sedimentation rate.
Physiological and clinical factors that decrease ESR
Apart from factors that increase ESR, medical practitioners and laboratory scientists should also consider the factors that decrease ESR. This is especially important as decreased ESR results may lead to missed diagnoses, whereas increased ESR results either lead to the right follow-up or false positives. Polycythemia, caused by increased numbers of red blood cells or by a decrease in plasma volume, artificially lowers ESR. Red blood cell abnormalities also affect aggregation, rouleaux formation and therefore sedimentation rate. Another cause of a low ESR is a decrease in plasma proteins, especially of fibrinogen and paraproteins.
The four factors that determine ESR reliability (dos and don’ts)
As with any test, the reliability of ESR measurements stands or falls with proper implementation. When not reliably performed, the nonspecific indicator for inflammation may point in the wrong direction, and result in either a false positive or a false negative. This may lead to the initiation of unnecessary investigations or worse: the overlooking of serious problems that actually needed follow-up. In this section, we discuss some do’s and don’ts when performing ESR measurement, to guarantee ESR reliability.
Factor 1: blood collection
Do: make sure you mix and dilute the sample 4:1 using a sodium citrate solution. If you adhere to these practices, you standardize the way you handle the blood samples, and therefore their suitability for ESR.
Don’t: leave the sample for too long before testing. We can imagine you’re pretty busy, and that you can’t do everything at the same time. However, when it comes to blood collection for ESR tests, some speed is required. After four hours, the results won’t be as accurate as before, which may negatively impact the reliability of the result. We therefore recommend performing the test within these four hours. If you really can’t make it in time, 24|hours is the max, but only if the sample is stored at 4|°C.
Factor 2: tube handling
Do: hold the tube vertically. A tube that is not held completely vertical can lead to increased sedimentation rates and is one of the technical factors that can affect ESR readings. And as we discussed in the previous paragraph, temperature is a factor too. Therefore, always place the tube in a stable and vertical position and at a constant temperature.
Don’t: expose the sample to vibrations, draft and sunlight, as all of these factors can have a strong influence on the final result obtained.
Factor 3: result reading
Do: wait 30|minutes. This is a very important one. Before reading ESR results, you should always wait 30|minutes. There are ESR testing methods that claim to show reliable results within only 20|seconds, but as it takes 10|minutes before sedimentation starts at a constant rate, these tests do not actually measure sedimentation. In fact, they calculate a mathematically derived ESR, leading to a number of false negatives.
Don’t: include the buffy coat (which is made up of leukocytes) in the erythrocyte column.
Factor 4: test quality
Do: go with an automated ESR test. They provide you with more reliable results, not least because they can correct hazy results. Moreover, automated ESR tests have a higher throughput compared to manual tests and minimize human contact with the tubes, which helps you reduce operations costs and minimize occupational health and safety risks.
Don’t: choose an ESR test that deviates from the Westergren standard. This method has always been the method of choice in fundamental studies, meaning that ESR is essentially based on this procedure. ESR tests that deviate from the Westergren will logically provide you with different ESR values, meaning they can lead you in the wrong direction. This is why the Westergren method is recom-mended by the CLSI and reconfirmed as the gold standard by ICSH.
ESR test as a reliable tool
If you keep these dos and don’ts in mind, you’re well on your way to making the ESR test a reliable tool that’s going to help you diagnose patients fast and error-free.
The author
Peter Murphy MBA(TechMgt), MAACB, BSc, GradDipEd
ELITech Group, Braeside, Victoria 3195, Australia
E-mail: p.murphy@elitechgroup.com
Flow cytometry: a critical technique in combating leishmaniasis
, /in E-News /by 3wmediaby Professor Paul Kaye
Leishmaniasis is classified as a neglected tropical disease. It is the cause of a huge health burden and is common in Asia, Africa, South and Central America, and even southern Europe. This article discusses how flow cytometry can help to evaluate diagnosis, monitor the effects of therapy and help in the creation of a vaccine.
Background
The leishmaniases are a family of devastating diseases, affecting a great many people across the globe and presenting a significant risk to both public health and socioeconomic development. The leishmaniases are vector-borne diseases, caused by infection with one of 20 species of the parasitic protozoan Leishmania (Fig. 1), transmitted through the bite of the infected female phlebotomine sand fly.
They can be broadly classified as tegumentary leishmaniases (TLs), affecting the skin and mucosa, and visceral leishmaniasis (VL), affecting internal organs. Whereas VL is responsible for over 20¦000 deaths per year, TL are non-life-threatening, chronic and potentially disfiguring, and account for around two-thirds of the global disease burden.
Within TL, there are three subtypes: self-healing lesions at the location of sand fly bite (cutaneous leishmaniasis; CL), lesions that spread from the original skin lesion to the mucosae (mucosal leishmaniasis; ML), and those which spread uncontrolled across the body (disseminated or diffuse cutaneous leishmaniasis; DCL). VL, also known as kala azar, involves major organs including the spleen, liver and bone marrow. In addition, patients recovering from VL after drug treatment often develop post kala-azar dermal leishmaniasis (PKDL), a chronic skin condition, characterized by nodular or macular lesions beginning on the face and spreading to the trunk and arms. As it may develop in up to half of patients previously treated and apparently cured from VL, it is thought that PKDL plays a central role in community transmission of VL.
The World Health Organization designates leishmaniasis as a neglected tropical disease (NTD), which together affect more than one|billion people across 149 countries worldwide; true prevalence may be even higher. Disproportionately, NTDs affect the poorest, malnourished individuals, and contribute to a vicious circle of poverty and disease. The significant physical marks, including ulcers, often left in the wake of the TLs may have an impact on mental health and perpetuate social stigma associated with the diseases [5]. There are over 1|million new cases of TL and 0.5|million new cases of VL each year, which together account for the loss of approximately 2.4|million disability-adjusted life years.
Treatment challenges
Leishmaniasis treatment can be quite difficult since at-risk populations may lack access to healthcare, and the limited battery of drugs has been increasingly compromised by resistance. Additionally, because the parasites in question are eukaryotic, they are not dissimilar from human cells, so the medication is also liable to be harmful – even fatal – to host as well as to pathogen.
Although the burden of VL in South Asia has been reduced with single-dose liposomal amphotericin B, the drug is less effective in other geographic locations, namely East Africa. Various drug combinations have been tested, unsuccessfully, and new chemical entities and immune-modulators are in early stages of development and as yet untested in the field. Unfortunately, little has changed in the treatment for CL for the past 50|years.
No vaccines are currently approved for any form of human leishmaniasis, although vaccines for canine VL have reached the market. Barriers to vaccine development include the limited investment in leishmaniases R&D and the high costs involved in bringing new products to those that need them.
Current work
My work on leishmaniasis has taken a holistic view, rooted in the immunology of the host-parasite interaction, but employing tools and approaches that span many disciplines: mathematics, ecology, vector biology and most recently neuroscience. Thirty years of discovery science has led to the development of a candidate for a therapeutic vaccine for PKDL, the mysterious sequela to VL [6]. ‘Therapeutic’ vaccines are given after an individual is infected with a pathogen and are designed to enhance our immune system and help eliminate the infection.
With colleagues from Sudan, we are in the midst of a phase IIb clinical trial funded by the Wellcome Trust, evaluating the efficacy of this therapeutic vaccine in Sudanese patients with persistent PKDL.
However, the research has been a long time in the making and has a long way to go. To continue to make progress, we linked with colleagues in Ethiopia, Kenya and Uganda and at the European Vaccine Initiative (http://www.euvaccine.eu/) in Germany, to develop a new research consortium to evaluate the immune status of people suffering from leishmaniasis. For example, using flow cytometry for blood and multiplexed immunohistochemistry for tissue biopsies, we can enumerate the proportions of lymphocytes, monocytes and neutrophils based on surface marker expression (e.g. CD3, CD19, CD14, CD16), and characterize their function, for instance by expression of cytokines (e.g. interferon-gamma) or other cell surface proteins that define function state. To support this endeavour, we recently received a grant from the European & Developing Countries Clinical Trials Partnership (EDCTP) that will allow us to not only extend our vaccine programme in Sudan [9] but also to address other important research challenges.
To develop vaccines and indeed new drugs, we often need tools capable of performing in-depth comparisons of how the body’s immune system is coping with the infection when a patient is first admitted to hospital and how it changes as the patient undergoes treatment and is hopefully cured. For example, recent evidence suggests that during infection, T lymphocytes may become ‘exhausted’ and unable to fight infection and the exhausted state can be identified by expression of surface molecules such as programmed cell death protein|1 (PD-1) and lymphocyte activation gene 3 protein (LAG-3). It is important to know if exhaustion can be reversed following treatment or whether we need to stimulate new populations of T lymphocytes. By understanding these nuanced changes in immune cells in our blood, we can design ways to improve how vaccines and drugs work in concert with immune cells, and understand why some patients might relapse from their disease or develop PKDL. Flow cytometry is a central tool for immunologists and plays a critical role in uncovering mechanisms of immunity and in assessing how well vaccines work and biomarkers of drug response. It uses antibodies that recognize specific molecules or markers on the surface or inside immune cells, such as those mentioned above, that help us predict their function. These antibodies are fluorescently labelled and the fluorescent signal can be detected by exposing each cell individually to laser light as they pass through a small aperture, the essence of flow cytometry.
For flow cytometry to be beneficial in this project, we needed to purchase five new flow cytometers that could meet exacting standards. They needed to be sufficiently sensitive to identify rare cell populations, often with low levels of surface marker expression. For multicentre research projects, reproducibility of data between sites is essential. Hence, we needed excellent inter-machine reproducibility and the Figure 2. Initial training course with recently appointed flow managers (Credit: Dr Karen Hogg, University of York) | 10 manufacturer had to be able to provide service support across the region. In our search for the right flow cytometer to support the consortium, we settled upon the CytoFLEX, Beckman Coulter Life Sciences’ research flow cytometer, which uses avalanche photodiode detection to arrive at the required level of sensitivity. With assistance from Beckman Coulter, we devised and have run initial training courses with a group of recently appointed flow managers from each partner country, to share standard operating procedures, develop high-level data analysis strategies as well as to provide instruction in routine instrument maintenance.
Beckman Coulter also provides another important aid to reducing errors in flow cytometry for multisite projects such as this, namely freeze-dried antibody cocktails (DURAClone panels) [10], that allow highly multiplexed phenotyping of small volumes of blood added directly to a single tube. Particularly for investigators in remote locations, the use of dry, preformulated reagents, rather than liquid (‘wet’) antibodies, removes the need for a cold chain. Equally importantly, staining of cells when manual mixing of 15 or 16 antibodies is required can introduce data inconsistencies when conducted by different individuals and at different locations.
Together, these innovations have allowed us to establish a new network for flow cytometry in East Africa that will allow us to identify and functionally characterize and identify the types of immune cells present in the blood during these devastating diseases. We will match this data with similar multiplexed techniques in pathology to compare blood immune cell profiles with those of cells found in the skin, to give a more complete picture of the host response to infection before and after treatment or vaccination.
Future Directions
As mentioned, we are currently in the midst of an efficacy trial of our therapeutic vaccine, ChAd63-KH. The technology we are using is similar to that being used by researchers at the university of Oxford to develop a coronavirus vaccine. In short, we introduce two genes from Leishmania parasites (KMP-11 and HASPB1) into a well-studied chimpanzee adenovirus (ChAd63 viral vector). After vaccination with this vaccine, host cells become infected with the virus and express the Leishmania proteins in a way that can be recognized efficiently by the immune system. We are particularly interested in how well this vaccine can generate T|cells to fight the infection.
With the first of our clinical objectives now well underway – the ongoing therapeutic clinical trial in patients with PKDL will be completed in mid-2021 – we have two additional goals. The next, funded by EDCTP, is to start a new clinical trial to determine whether the vaccine can prevent progression from VL to PKDL. And finally, we hope to develop a human challenge model of leishmaniasis to test the vaccine for its ability to protect against infection by different forms of parasite. This would open the way to the development of a cost-effective prophylactic vaccine to prevent these diseases occurring in vulnerable populations across the world.
Our research also has larger ambitions for the long term. Our East African partners are also linked together through their work on leishmaniasis in drug development, as members of the Leishmaniasis East Africa Platform group, established to help coordinate drug development activities in the region by the Drugs for Neglected Diseases Partnership. Central questions about why the disease varies between countries are being addressed, and the increased capacity for flow cytometry will additionally support patient monitoring during drug trials conducted by DNDi or other groups. Indeed, through the capacity building this project provides, we hope this project will extend its reach beyond leishmaniasis, providing muchneeded support for research on other neglected diseases of poverty that affect people in the region, including bacterial, fungal, other parasitic and viral diseases. By continuing to demonstrate the analytical power of flow cytometry and its role in helping design much-needed therapies, we hope to open up additional discovery research possibilities for colleagues in Africa and around the world.
The research described in this article is part of the EDCTP2 programme supported by the European Union (grant number RIA2016V-1640; PREV_PKDL; https://www.prevpkdl.eu).
The author
Paul Kaye PhD, FRCPath, FMedSci
Hull York Medical School, University of York, York, UK
E-mail: paul.kaye@york.ac.uk
Introducing new tests to a laboratory’s repertoire
, /in Corona News, E-News /by 3wmediaExpert opinions from Dr Heidi Mendoza
There are many assessments to make when adding a new test to a lab’s collection. Dr Heidi Mendoza, acting consultant clinical biochemist at Raigmore Hospital, Inverness, UK, shares her experiences and observations of doing exactly that in both ordinary circumstances and during a pandemic, as well as having to contend with the geographic challenges imposed by the nature of life in the Scottish Highlands.
Can you provide a little background about yourself and where you work, please?
I am a clinical biochemist based in Raigmore Hospital, which is a small hospital in the Scottish Highlands. In my current role I provide clinical advice and interpretation for biochemistry tests for general practitioner (GP) practices and three hospitals across the Highlands. Working in the Highlands is incredibly rewarding, but also very challenging! It can take between 2 and 6|hours to travel between hospitals and our patients may have to travel by plane or boat to be seen, with journey times of +12|hours depending on where they live. It really puts the laboratories under pressure to get it right for the patient. Repeat testing isn’t as simple or straightforward as it would be in a city and we have to have excellent systems in place for reporting critical results and getting patients into hospital or transferring them between hospitals. Getting the right test, in the right place, with the right turnaround time is really important for our patients and for our clinicians.
What are the usual circumstances in which you would think about bringing a new test into the lab’s repertoire?
Any new test is a cost pressure on our National Health Service (NHS) and can only be brought in when it demonstrates clear benefits for patients. We have brought in two new tests in the last 12|months that are good examples of the different ways we can bring in new tests to our laboratory.
The first test is the NT-proB-type natriuretic peptide (NTproBNP) test. NTproBNP is used to investigate patients with suspected heart failure and the results can be used to determine whether a patient needs an echocardiogram (ECHO) or not. If they do need an ECHO the NTproBNP result can be used to split patients into those who need urgent ECHO (2|weeks) or routine ECHO (6|weeks). In theory this is a perfect test to implement as it will benefit patients and is cost-effective with respect to the more expensive ECHO investigation. However, NTproBNP has been implemented in other hospitals without reducing ECHO waiting times or the number of ECHOs performed! To ensure that this didn’t happen in our service, I spent 6|months before implementation of the test liaising with cardiologists and GP representatives from across the Highland region. We changed the ECHO referral pathway to include NTproBNP and created useful guidance for GPs on when to, and importantly when not to, request NTproBNP. We implemented the test just under 1|year ago and have seen a positive effect on ECHO referrals. We will still have to attend a 1|year post-implementation review with the Hospital Board to present our audit data and show that investment in the service by introducing a new test has benefited patients and other areas of the service.
Procalcitonin is the second example. Procalcitonin is a test that can be used in the investigation of sepsis and guide the use of antibiotics. Procalcitonin was not a test available in our hospital before the COVID-19 pandemic. Procalcitonin is not increased in the majority of adult patients with COVID-19; however, an elevated procalcitonin may suggest superimposed bacterial infection and be used to guide treatment of these patients and improve patient outcomes. Early in the COVID-19 pandemic we were approached by our Intensive Care Unit (ITU) and Microbiology consultants who requested that procalcitonin be available for our COVID-19 patients in ITU to guide their antibiotic treatment. We implemented procalcitonin in less than 4|weeks with help from our instrument manufacturer, external quality assessment providers and other Scottish hospitals who provided anonymized patient serum with known values so that we could verify our assay as quickly as possible. We are now in the process of putting together a business case and following the evidence base which will determine whether we continue to offer the procalcitonin test.
How would you usually go about adopting a new test?
As highlighted in the two examples above, we must agree a clinical need for a test and then liaise with the users of the service to find out how the test should be implemented into the patient-care pathway. Once we have worked out the clinical utility of the test, then we can carry out the laboratory verification of the test and the laboratory workflow. Verification is very straightforward. For example, the between-batch and within-batch precision, accuracy, linearity on dilution, interferences and sample stability for a test need to be evaluated. The implementation of the test then must be followed by an audit which shows that the test is being used as intended and giving the benefits predicted. If not, the test may need to be withdrawn. The hardest part of the entire process is agreeing how a test is going to be used and fitting it in to the patient-care pathway.
In the situation of the COVID-19 pandemic, we have a new disease, caused by a new virus, and new tests that have been created very quickly. How do you start to use a new test in these circumstances – are there any differences in procedure?
There is no difference in the steps that need to be performed we just need to be able to do everything in a much shorter time frame. That is actually much easier than it sounds. In the NHS, the laboratories from different parts of the country are great about helping other laboratories. We regularly share protocols, data and learning. If a new test is released we’ll contact another laboratory and they’ll share their local experience and any problems they have had with the test.
For procalcitonin implementation I contacted the laboratory in Dundee, UK, and they helped us out by lending us kits and reagents, sending us anonymized patient serum with known procalcitonin values, and sharing their data and verification protocols. This allowed us to complete verification incredibly quickly. We will still have to gather the data and evaluate whether the test is providing the benefit that we predicted when we established the clinical need.
What are the challenges regarding validation, reference levels, results interpretation and reporting?
Verifying tests is straightforward as we are always evaluating tests in clinical laboratories so are very experienced. Results interpretation can be quite difficult. If we need clinicians to change patient management based on a result then we have to provide them with very clear local guidance on what we want them to do with a result. This might be different from the action they would take in another hospital with different patient pathways, different pressures on patient turnaround times, and different diagnostic facilities. This is where good working relationships with users of the service are key to test implementation. If you just implement a new test without working out where it fits in the patient pathway, it doesn’t matter how great the test is, as it is unlikely to be used well and may not improve patient care.
What do you have to think about in terms of logistics?
Many laboratories are understaffed due to a combination of unfilled vacancies and staff on long-term absence. The additional work involved in verifying and implementing a new test does put pressure on staff. However, NHS laboratory staff are highly trained and dedicated. When the staff know how a test is going to be used and the benefit to the local community, they support the implementation and the extra work involved.
Biocontainment and staff safety have been important considerations during the COVID-19 pandemic. We had to adhere to government guidance in the transport, analysis and disposal of samples from patients with suspected COVID-19. This changed laboratory workflows and slowed us down, creating longer turnaround times.
Logistics are a serious consideration for us owing to our geography. Reagent shortages or delays in deliveries have a big impact on small laboratories as they can’t store much surplus reagent stocks because of expiry dates. Unexpected overuse or underuse of a new test can be quite challenging and leave the laboratory short of tests or with expired, wasted kits. There are also several times during the year when the roads are impassable between our central and rural laboratories. We have been down to single numbers of tests remaining several times over the last few years or had failed delivery from manufacturers in winter. There was also a shortage of procalcitonin reagent as there was such a surge in the use of the test during the COVID-19 pandemic. Again, working closely with users of our laboratory services has enabled us to rationalize the use of the test until the global shortage of reagent ended. On a number of occasions we have also shared reagents with other Scottish laboratories to ensure that none of the laboratories were left without reagents.
What has been learnt from the current coronavirus situation about diagnostic testing during a pandemic that would help to improve the process in future?
The coronavirus pandemic has shown how robust the infrastructure of the NHS is in Scotland and how adaptable laboratories can be when required. The laboratories really pulled together and worked towards a common goal delivering testing to COVID patients and non-COVID patients during a crisis. The two things that made this possible were: (1) Having a very clear goal – delivery of a service with new testing during a pandemic; and (2) Finances changes which needed to be made to deliver the service got rapid financial approval. How do we take these lessons learned and apply it to the routine delivery of laboratory services? Finance will always be a limiting factor – as it should be! Healthcare is expensive and it is up to us as healthcare professionals to deliver a cost-effective and affordable service. In contrast, having a clear goal, is definitely something that we could do better in the future. In the case of the pandemic, laboratories found different solutions based on local geography, resources and incidence of COVID. The changes made by laboratories in the remote Highlands and Islands were similar, but different than those made by laboratories in major cities. The staff that delivered the service found the best solutions to the goals set by the government – that is the real lesson we need to take away. We need to give very clear goals to services and let local expertise and knowledge drive the changes to solve the problem.
The expert
Heidi Mendoza BSc MSc PhD RCPath
Blood Sciences Department, Raigmore Hospital, Inverness IV2 3UJ, UK
E-mail: heidi.mendoza@nhs.net
LGC acquires The Native Antigen Company
, /in Corona News, E-News /by 3wmediaLGC has acquired The Native Antigen Company (NAC), one of the world’s leading suppliers of high quality infectious disease antigens and antibodies.
NAC is a developer, manufacturer and supplier of critical reagents to the in vitro diagnostic (IVD), pharmaceutical and academic sectors. It offers a comprehensive portfolio of native and recombinant infectious disease antigens and related products including pathogen receptors, virus-like particles and antibodies for use in immunoassay applications, vaccine development and quality control solutions. NAC was one of the first companies globally to offer antigens for SARS-COV-2 and continues to play an important role in supporting the global response to the COVID-19 pandemic.
The acquisition strengthens LGC’s existing product offering to the IVD sector, which includes a range of quality assurance tools, immunoassay reagents and disease state plasma as well as probes and primers for molecular diagnostics.
“NAC is a natural fit with our clinical diagnostics business and will enable us to provide an expanded portfolio of critical reagents to our customers. NAC’s focus on infectious disease is highly complementary with our existing offer to this segment comprising controls, reference materials, MDx tools and other components,” said Michael Sweatt, Executive Vice President and General Manager, Clinical Diagnostics, LGC.
Avacta, Integumen collaborate for detection of SARS-COV-2 in waste water
, /in Corona News, E-News /by 3wmediaAvacta Group, the developer of Affimer biotherapeutics and reagents, has entered into a collaboration with Integumen to evaluate recently generated Affimer reagents that bind the SARS-COV-2 spike protein for the detection of the coronavirus in waste water, to provide a real-time alert system to warn of localised COVID-19 outbreaks.
Over 60 percent of COVID-19 positive patients had gastrointestinal symptoms, such as diarrhoea, nausea and vomiting, and the SARS-COV-2 virus was found in their faecal samples. Sampling waste water from households may therefore provide an early warning system for localised outbreaks in communities.
Recently, Avacta announced that it had generated a number of highly specific Affimer reagents that detect the SARS-COV-2 virus spike protein for use in diagnostic tests and in neutralising therapies.
The collaboration with Integumen, announced 13 July, aims to evaluate some of these Affimer reagents in next-generation sensors, based on the real-time bacteria detection and alert system1 developed by Rinocloud, a subsidiary of Integumen, with the aim of integrating these sensors into Modern Water’s Microtox water contamination system to detect the coronavirus. The award-winning Microtox system, which can detect the presence of contaminating bacteria, virus and toxins, is distributed by Modern Water and has a global footprint of over 3,000 installations. The proposed Affimer sensors would be consumable items to be replaced on a roughly monthly basis.
Once initial testing of the Affimer reagents is completed over the next few weeks, validation of the sensors will be carried out using SARS-COV-2 virus samples in a containment level 3 laboratory at the University of Aberdeen. Upon successful completion of this evaluation, Integumen and Avacta will enter into a supply agreement to allow Integumen to manufacture and commercialise the waste water detection sensors globally by retrofitting into Microtox systems.
Byondis investigational breast cancer drug selected for trial
, /in E-News /by 3wmediaByondis B.V. (formerly Synthon Biopharmaceuticals) announced that Quantum Leap Healthcare Collaborative (Quantum Leap) selected the company’s investigational antibody-drug conjugate (ADC) SYD985 ([vic-]trastuzumab duocarmazine) for a new investigational treatment arm in its ongoing I-SPY 2 TRIAL for neoadjuvant treatment of locally advanced breast cancer. This treatment arm will focus on treatment for HER2-low early-stage breast cancer.
The I-SPY 2 TRIAL (Investigation of Serial studies to Predict Your Therapeutic Response with Imaging And moLecular analysis) is a standing Phase II randomized, controlled, multicentre study aimed at rapidly screening and identifying promising treatments in specific subgroups of women with newly-diagnosed, high-risk, locally advanced breast cancer (Stage II/III). Quantum Leap, sponsor of the I-SPY 2 TRIAL, leads a pre-competitive consortium that includes the U.S. Food & Drug Administration (FDA), industry, patient advocates, philanthropic sponsors, and clinicians from 16 major U.S. cancer research centres.
The new I-SPY 2 treatment arm will evaluate SYD985 against standard of care therapy in Stage II/III early-stage, high-risk breast cancer patients, with a focus on tumours with heterogeneous and low HER2 expression. Byondis will supply the investigational drug and provide financial and regulatory support. Quantum Leap, as sponsor, will provide the clinical sites and clinical expertise.
SYD985 is Byondis’ most advanced ADC, targeting a range of HER2-positive cancers such as metastatic breast cancer (MBC) and endometrial cancer. The company is currently conducting a Phase III study of SYD985 (TULIP or SYD985.002) to compare its efficacy and safety to physician’s choice treatment in patients with HER2-positive unresectable locally advanced or metastatic breast cancer. Previously, the FDA granted fast track designation for SYD985 based on promising data from heavily pre-treated last-line HER2-positive MBC patients participating in a two-part Phase I clinical trial (SYD985.001).
SYD985 uses Byondis’ unique, proprietary linker-drug (LD) technology. Although marketed ADCs have improved therapeutic indices compared to classical non-targeted chemotherapeutic agents, there is still room for improvement.
SYD985 is comprised of the monoclonal antibody trastuzumab and a cleavable linker-drug called valine-citrulline-seco-DUocarmycin-hydroxyBenzamide-Azaindole (vc-seco-DUBA). The antibody part of SYD985 binds to HER2 on the surface of the cancer cell and the ADC is internalized by the cell. After proteolytic cleavage of the linker, the inactive cytotoxin is activated and DNA damage is induced, resulting in tumour cell death. SYD985 can be considered a form of targeted chemotherapy.
Dante Labs, Cambridge Cancer Genomics and Nonacus collaborate to provide precision oncology at scale
, /in Corona News, E-News /by 3wmediaDante Labs, a pioneer and leader in genomic testing, Cambridge Cancer Genomics (CCG.ai), a software developer specialising in data-driven precision oncology, and Nonacus, a provider of genetic testing products for precision medicine and liquid biopsy, have signed a collaboration agreement.
In a joint statement they said the partnership aims to build the most comprehensive and patient-centric tumour profiling service enabling improved cancer patient management, treatment and monitoring. By combining Dante Labs’ experience and capacity in delivering a sequencing service for both solid tumour and cell free circulating tumour DNA from liquid biopsies, Nonacus’ sensitive targeted pan-cancer NGS libraries, and CCG.ai’s industry leading AI powered software platform, OncOS, the companies will enable precision oncology at scale.
Improving outcomes for cancer patients means ensuring they have the right drug, at the right time to beat their cancer. This means understanding the molecular profile of the individual cancer and using that data to recommend treatments or clinical trials. Oncologists and clinical researchers will be able to send samples for processing to Dante Labs, who will use library preparation kits from Nonacus and software from CCG.ai to create a sample to report solution. If there are actionable mutations, the report will recommend the right treatments for those mutations, if there are novel or unactionable mutations, the software will also be able to match possible clinical trials.
Chris Sale, CEO of Nonacus, said: “Long turn-around time and lack of clinically oriented analysis are the main obstacles to fully deliver the potential of cancer genomics to patients. This partnership will provide the flexibility and accuracy that oncology professionals need to integrate cancer genomics into the care of their patients. The COVID pandemic has increased the backlog of genetic testing for cancer, potentially leaving many suspected cancers unconfirmed and treatments delayed. Dante Labs are one of the biggest clinical sequencing hub in Europe able to process large numbers of samples in high throughput. It is our hope that by combining AI software from CCG.ai and our library preparation kits, together we will be able to process samples and provide bioinformatic analysis critical to determining the best treatment path for patients.”
Start Codon accelerator showcases first cohort of start-up life science companies
, /in E-News /by 3wmediaStart Codon, a new model of life science and healthcare business accelerator, has announced its first cohort of start-up companies. Start Codon aims to minimise risk and translate early stage research into successful start-ups, ready for funding and partnership. Start Codon has worked closely with four life science and healthcare companies that were enrolled into the programme in February this year.
They are:
Start Codon plans to invest in and support up to 50 start-up companies over the next five years. The accelerator is now accepting applications for its second and third cohorts of companies. Early stage start-up companies in the life sciences and healthcare space are invited to apply via https://startcodon.co/application-form
ReactoMate DATUM — a user-friendly support system for laboratory scale reactions
, /in E-News /by 3wmediaThe ReactoMate DATUM from Asynt is a high quality, dual-rod stainless steel and aluminium support system built to ensure the stability and safety of your lab reactor. Sturdy, yet compact, the ReactoMate DATUM support system can accommodate a wide range of reaction vessels from 100 mL up to 5000 mL.
Designed with user-friendliness in mind, the ReactoMate DATUM support system incorporates a suite of innovative features.
Changing a vessel supported by the ReactoMate DATUM is as simple as “Clip & Click”. The novel neck clamp allows fast changeover between reactor vessel sizes thereby enabling simple reaction scale-up, whilst the ingenious mounting mechanism ensures excellent stability and alignment every time.
The Reactomate DATUM support system is fully compatible with all leading brands of overhead stirrers and circulator heating/cooling systems. Designed by chemists for chemists, low-friction polymer bearings line both the overhead stirrer alignment chuck and the neck support to ensure smooth and easy operation.
Ideally suited for use within a benchtop fume hood, adjustable feet allow you to level the ReactoMate DATUM support system ensuring stability and security while you work. Each DATUM system is also supplied with a moulded drip tray that fits perfectly within the base of the support, for safely catching any drips and spills from the reaction vessel during draining.
With a wide range of accessories and upgrades available, including drain manifolds and automation packages, the ReactoMate DATUM support system is the perfect all-rounder for laboratory scale reactions.
For more information, visit: www.asynt.com/product/reactomate-datum