Point-of-care (POC) testing has the potential to provide results in a much shorter time than centralized lab testing, allowing faster implementation of appropriate treatment. This article discusses the technological developments in POC tests, considerations for the implementation of a POC system, as well as how to ensure accurate and reliable results.
A new type of blood test for breast cancer could help avoid thousands of unnecessary surgeries and otherwise precisely monitor disease progression, according to a study led by the Translational Genomics Research Institute (TGen) and Mayo Clinic in Arizona.
The study suggests that the test called TARDIS — TARgeted DIgital Sequencing — is as much as 100 times more sensitive than other blood-based cancer monitoring tests.
TARDIS is a “liquid biopsy” that specifically identifies and quantifies small fragments of cancer DNA circulating in the patient’s bloodstream, known as circulating tumour DNA (ctDNA). According to the study, TARDIS detected ctDNA in as low as 2 parts per 100,000 in patient blood.
“By precisely measuring ctDNA, this test can detect the presence of residual cancer, and inform physicians if cancer has been successfully eradicated by treatment,” said Muhammed Murtaza, M.B.B.S., Ph.D., Assistant Professor and Co-Director of TGen’s Center for Noninvasive Diagnostics. He also holds a joint appointment on the Research Faculty at Mayo Clinic in Arizona, and is one of the study’s senior authors.
For example, Dr. Murtaza explained, TARDIS is precise enough to tell if early stage breast cancer patients have responded well to pre-operative drug therapy. It is more sensitive than the current method of determining response to drug therapy using imaging.
“This has enormous implications for women with breast cancer. This test could help plan the timing and extent of surgical resection and radiation therapy after patients have received pre-operative therapy,” said Dr. Barbara A. Pockaj, M.D., a surgical oncologist who specializes in breast and melanoma cancer patients at Mayo Clinic in Arizona, and is the study’s other senior author. Dr. Pockaj is the Michael M. Eisenberg professor of surgery and the chair of the Breast Cancer Interest Group (BIG), a collaboration between researchers at Mayo, TGen and ASU.
Unlike traditional biopsies, which only produce results from one place at one time, liquid biopsies use a simple blood draw, and so could safely be performed repeatedly, as often as needed, to detect a patient’s disease status.
“TARDIS is a game changer for response monitoring and residual disease detection in early breast cancer treated with curative intent. The sensitivity and specificity of patient-specific TARDIS panels will allow us to tell very early, probably after one cycle, whether neo-adjuvant (before surgery) therapy is working and will also enable detecting micro-metastatic disease and risk-adapted treatment after completing neo-adjuvant therapy,” said Dr. Caldas, who also is Senior Group Leader at the Cancer Research UK Cambridge Institute, and one of the study’s contributing authors.
Following further clinical testing and trials, TARDIS could someday be routinely used for monitoring patients during cancer treatment, and discovering when patients are essentially cured and cancer free.
“The results of these tests could be used to individualize cancer therapy avoiding overtreatment in some cases and under treatment in others,” Dr. Murtaza said. “The central premise of our research is whether we can develop a blood test that can tell patients who have been completely cured apart from patients who have residual disease. We wondered whether we can see clearance of ctDNA from blood in patients who respond well to pre-surgical treatment.”
Current tests and imaging lack the sensitivity needed to make this determination.
“Fragments of ctDNA shed into blood by tumours carry the same cancer-specific mutations as the tumour cells, giving us a way to measure the tumour,” said Bradon McDonald, a computational scientist in Dr. Murtaza’s lab, and the study’s first author.
“The problem is that ctDNA levels can be so low in non-metastatic cancer patients, there are often just not enough fragments of ctDNA in a single blood sample to reliably detect any one mutation. This is especially true in the residual disease setting, when there is no obvious tumour left during or after treatment,” McDonald said. “So, instead of focusing on a single mutation from every patient, we decided to integrate the results of dozens of mutations from each patient.”
Translational Genomics Research Institutewww.tgen.org/news/2019/august/07/new-ctdna-blood-test-for-cancer/
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:
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
Researchers at Karolinska Institutet have developed a method based on artificial intelligence for histopathological diagnosis and grading of prostate cancer. The AI-system has the potential to solve one of the bottlenecks in today’s prostate cancer histopathology by providing more accurate diagnosis and better treatment decisions. The study shows that the AI-system is as good at identifying and grading prostate cancer as world-leading uro-pathologists.
“Our results show that it is possible to train an AI-system to detect and grade prostate cancer on the same level as leading experts,” says Martin Eklund, associate professor at the Department of Medical Epidemiology and Biostatistics at Karolinska Institutet who led the study. “This has the potential to significantly reduce the workload of uro-pathologists and allow them to focus on the most difficult cases.”
A problem in today’s prostate pathology is that there is a certain degree of subjectivity in the assessments of the biopsies. Different pathologists can reach different conclusions even though they are studying the same samples. This leads to a clinical problem where the doctors must pick treatment based on ambiguous information. In this context, the researchers see significant potential to use the AI-technology to increase the reproducibility of the pathological assessments.
To train and test the AI system, the researchers digitized more than 8000 biopsies taken from some 1200 Swedish men in the ages of 50–69 to high-resolution images using digital pathology scanners. About 6,600 of the samples were used to train the AI system to spot the difference between biopsies with or without cancer. The remaining samples, and additional sets of samples collected from other labs, were used to test the AI system. Its results were also compared against the assessments of 23 world-leading uro-pathologists. The study was conducted in collaboration with researchers at Tampere University in Finland.
The findings showed that the AI-system was almost near-perfect in determining whether a sample contained cancer or not, as well as in estimating the length of the cancer tumour in the biopsy. When it comes to determining the severity of the prostate cancer, the so-called Gleason score, the AI system was on par with the international experts.
“When it comes to grading the severity of the prostate cancer, the AI is in the same range as international experts, which is very impressive, and when it comes to diagnostics, to determine whether or not it is cancer, the AI is simply outstanding,” says Lars Egevad, professor in pathology at Karolinska Institutet and co-author of the study.
The initial findings are promising but more validation is needed before the AI system may be rolled out broadly in clinical practice, according to the researchers. That is why a multicenter study spanning nine European countries is currently underway with completion slated by the end of 2020. That study, which is financed by EIT Health, aims to train the AI-system to recognize cancer in biopsies taken from different laboratories, with different types of digital scanners and with very rare growth patterns. In addition, a randomized study starting in 2020 will examine how the AI-model may be implemented in Sweden’s health care system.
“AI-based evaluation of prostate cancer biopsies could revolutionize future health care,” says Henrik Grönberg, professor in cancer epidemiology at Karolinska Institutet and head of the Prostate Cancer Center at St Göran Hospital in Stockholm. “It has the potential to improve the diagnostic quality, and thereby secure a more equitable care at a lower cost.” Karolinska Institute
AESKU.GROUP recently announced completed acquisition of immunofluorescence assay (IFA) specialist MBL BION from MBL Intl., securing their position as the leading provider of IFA solutions to clinical laboratories. The acquisition increases AESKU.GROUP’s addressable market and strengthens its market position.
AESKU.GROUP has a track record of developing, producing, and marketing innovative diagnostic assays and automated systems, and has a global network of proven distribution partners. A significant factor in their success is complete solutions for clinical laboratories, including all-in-one testing and reading automation with the SQII for enzyme immunoassay (ELISA), HELIA® for line immunoassay (LIA), and HELIOS®, which was the first all-in-one system on the market for IFA. This innovation, along with the confirmation of IFA as the gold-standard for autoimmunity screening in clinical guidelines, has led to over 400 HELIOS placements worldwide and in-creased demand for AESKU IFA reagents.
MBL Bion has over 40 years’ experience manufacturing IFA products of the highest quality and is a leading provider of autoimmunity and infectious disease testing slides. They have a strong market presence in the Americas, and a centre of manufacturing excellence in Des Plaines, IL, USA. Adding MBL Bion’s manufacturing capacity and broad range of reagents lets AESKU.GROUP help laboratories expand their IFA testing with the most extensive range of IFA products available on all-in-one automation combined with a unique quality control portfolio for the diagnosis of autoimmune and infectious diseases.
AESKU.GROUP CEO Dr. Torsten Matthias was delighted with the acquisition, saying, “We have found true synergy. MBL BION’s operations and product portfolio fit perfectly with the AESKU.GROUP. Between the expansion of our IFA manufacturing in Buffalo, NY, and MBL BION’s Des Plaines operations, AESKU can speed order processing and delivery times. Furthermore, the high quality of their HEp-2 cells and extensive infectious disease testing slides add incredible value to our new HELIOS ‘HTC’ humidity and temperature control module. For the first time, clinical laboratories can access the highest level of automation and environmental control for both auto-
immunity and infectious serology testing.”
UK-based Avacta Group, the developer of Affimer biotherapeutics and reagents, will collaborate with US-based Adeptrix to develop a high throughput Covid -19 antigen test using Adeptrix’s proprietary bead-assisted mass spectrometry (BAMS) platform.
The Affimer-based BAMS coronavirus antigen test that will provide clinicians with a significant expansion of the available testing capacity for Covid-19 infection in hospitals.
Adeptrix’s novel BAMS platform combines enrichment of the sample to improve sensitivity with the power of mass-spectrometry to improve specificity. Hundreds of samples per day can be analysed by a single technician using BAMS, exceeding the capacity of single PCR machine, making BAMS a very attractive high throughput technique for Covid-19 screening in the clinical setting.
The diagnostic test will allow hospitals around the world to utilise their existing installed base of mass spectrometers that are not currently used for Covid-19 testing, thus contributing significantly to the increase in global testing capacity. Avacta’s recently developed Affimer reagents that bind the SARS-COV-2 spike protein will be used to provide the capture and enrichment of the virus particle from the sample which could be saliva, nasopharyngeal swabs or serum.
The companies are aiming to have a BAMS test ready for clinical validation, regulatory approval and manufacturing in June. Adeptrix and Avacta are already in discussion with large-scale manufacturing partners to rapidly deploy this new high throughput test.
Dr Alastair Smith, Chief Executive Officer of Avacta Group, commented: “We believe that the BAMS test will be hugely attractive as an adjunct to PCR testing because it uses laboratory equipment that is already in hospital labs but not currently used for Covid-19 testing so it provides incremental testing capacity.
I have made it clear that we intend to partner the SARS-COV-2 spike protein Affimer reagents with several select companies to support antigen test development on multiple diagnostic test platforms. This will contribute most effectively to the urgent need to increase antigen testing capacity globally and maximise the commercial return to Avacta. Adeptrix is one example of this and other discussions are underway. I look forward very much to further updating the market in the near future.”
Dr. Jeffrey C. Silva, Director of Product Development, Adeptrix Corporation commented: “Mass spectrometry can enhance the diagnostic utility of immunoassays, as it is capable of monitoring both existing and emerging viral strains by accurately measuring the molecular components of the virus. BAMS provides an ideal multiplexing platform to obtain higher specificity for monitoring Covid-19 infection.”
Labs working to combat Covid-19 will benefit from this initiative, as CytoSMART aims to reduce the huge workload currently facing researchers on projects vital to controlling the disease.
CytoSMART’s unique and compact live-cell microscope films living cell cultures without disturbing their growth or behaviour. The device operates from inside cell culture incubators and is accessible from an online environment. This enables researchers to analyse their cell cultures remotely and assess e.g. the cytopathic effect, which is caused by virus replication. Using the CytoSMART Lux2, researchers will know when to take action for the next step and harvest the virus.
“We aim to do our part to assist researchers in minimizing the time they have to spend in high-contamination labs, by providing them with remote video access to evaluate the status of their cell cultures. The video data is used to remotely monitor the cytopathic effect, this way researchers know when it’s the right time to harvest the virus.” – Joffry Maltha, CEO at CytoSMART Technologies.
According to guidelines by the CDC and the WHO, isolation and characterization of Covid-19 should be performed in BSL-3 laboratories. Performing research in Biosafety Level 3 and 4 laboratories (BSL-3 or BSL-4) means working in a highly controlled area. Many precautionary measures must be taken to ensure the safety of researchers and help prevent the diseases they are working with from spreading outside the lab. Removing and replacing the protective clothing and apparatus can be time consuming and expensive, so entering the lab should ideally only occur when absolutely necessary.
Maltha commented: “We need to help scientists who are working in BSL-3 and BSL-4 laboratories to combat Covid-19. We know that our system can help researchers in monitoring cell growth and deciding when they need to go to the high containment labs and run further experiments.
National Institutes of Health (NIH) scientists have developed an ultrasensitive new test to detect abnormal forms of the protein tau associated with uncommon types of neurodegenerative diseases called tauopathies. This advance gives them hope of using cerebrospinal fluid, or CSF – an accessible patient sample – to diagnose these and perhaps other, more common neurological diseases, such as Alzheimer’s disease.
Scientists have linked the abnormal deposition of tau in the brain to at least 25 different neurodegenerative diseases. However, to accurately diagnose these diseases, brain tissue often must be analysed after the patient has died. For their study, the researchers used the same test concept they developed when using postmortem brain tissue samples to detect the abnormal tau types associated with Pick disease, Alzheimer’s disease and chronic traumatic encephalopathy (CTE). They adapted the test to use CSF for the detection of abnormal tau of progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and other less common tauopathies.
They detected abnormal tau in CSF from both living and deceased patients. In one case, the test led to a corrected diagnosis in a patient who had died from CBD, but who was initially diagnosed with PSP. The new test is called 4R RT-QuIC – which stands for 4-repeat tau protein amplified in a real-time, quaking-induced conversion process.
The researchers plan to continue evaluating the clinical performance of 4R RT-QuIC by analysing larger sets of CSF samples. One focus will be to compare test results from tauopathy patients who agree to provide CSF samples both before and after death. The scientists hope this type of evaluation will help them better understand how abnormal tau in CSF evolves during brain disease.
NIHwww.niaid.nih.gov/news-events/nih-scientists-develop-test-uncommon-brain-diseases
New research led by scientists at the Medical Research Council (MRC) Toxicology Unit and University of Leicester suggests that, by analysing levels of tumour-derived DNA in the blood, the early detection of lung cancer could be improved.
The study found that, in preliminary tests using mice, a blood test could measure the circulating levels of DNA in the blood which cancer cells shed as they grow and multiply, and could even predict the presence of tumours in the lungs before they became cancerous.
Lung cancer is the number one cause of cancer-related death around the world, partly due to the difficulties in detecting the disease at an early stage. By the time lung cancer is diagnosed, it has often spread to other parts of the body making it much more difficult to treat, which is why improved diagnosis at an earlier stage is key to beating the disease.
The scientists at the University of Leicester alongside the MRC Toxicology Unit, now part of the University of Cambridge, used mice with a mutation in a gene called KRAS to model the pre-cancerous stages of lung cancer.
The researchers took regular computed tomography (CT) scans to monitor the development of small pre-cancerous lung tumours in the mice. To determine whether circulating DNA could be used to detect the tumours before they became malignant, blood samples were taken along with the CT scans at different time intervals.
The team found that the mice developing cancerous lung tumours had higher levels of circulating DNA compared with healthy mice, and that the levels of DNA released by the cancerous tumours into the blood of the mice correlated with the size of the tumours seen on the CT scans. The circulating DNA was then analysed for the presence of the precise KRAS mutation that caused the tumours to develop. The researchers found that, significantly, in later stages of tumour development where tumours were still pre-cancerous, the KRAS mutation could still be detected in circulating DNA.
Professor Catrin Pritchard, Deputy Director of Leicester Cancer Research Centre and co-author of the study said: “These findings are promising as they show that we may be able to detect premalignant lung cancer from a patient’s circulating DNA using a simple blood test.”
Professor Jacqui Shaw, Professor of Translational Cancer Genetics and Director of the Leicester Precision Medicine Institute at the University of Leicester said: “This was an investigational study in mice and more work is needed before it can be translated to humans. Future studies will need to be conducted using mice bearing pre-cancerous lesions in other tissues as well as studies using samples from humans bearing suspicious lung lesions.”
University of Leicester
https://tinyurl.com/yy4f6hdd
Concarlo Holdings has received a US patent for IpY, a novel therapeutic peptide that addresses drug-resistant breast cancer by targeting a unique cellular pathway — p27Kip1. The patent is the latest step in Concarlo’s journey to commercialize revolutionary medicines for metastatic breast cancer.
Concarlo has also announced that a new provisional patent application has been filed for modified versions of the therapeutic peptide that are believed to exhibit enhanced bioavailability. Concarlo is a Brooklyn, New York-based biotechnology innovator dedicated to developing sophisticated, targeted therapies and diagnostics in the oncology space. The IpY technology is the first to address the high incidence of drug-refractory disease that develops with currently available CDK4 inhibitor (CDK4i) treatments. Such a solution has the potential to drastically increase overall survival of breast cancer patients.
The recent introduction of CDK4i drugs, a class of medicines that directly targets the CDK4/6 pathway implicated in many malignancies, has had a significant impact on the way in which metastatic breast cancer is managed. However, such therapeutics are associated with patients transitioning to a treatment-resistant form of the condition, despite initial extended periods of remission. Backed by more than 20 years of research and development expertise, Concarlo has developed IpY and a companion diagnostic, ApY, to effectively overcome the issue of CDK4i resistance and roll out a more targeted treatment approach for optimized patient outcomes.
“Despite the clinical efficacy of CDK4 inhibitors, we’re seeing that primary or secondary resistance to therapy is presenting a significant challenge to overall survival,” said Dr. Dominique Bridon, Chief Development Officer at Concarlo. “With the IpY technology and its unique mechanism of action, we’re effectively targeting CDK4 while simultaneously inhibiting another target — CDK2 — which has been found to be a key molecular player in the development of drug resistance. In doing so, we are the first company to successfully address the CDK4i resistance issue to provide long-term durable tumour arrest. Combined with its highly specific targeting and low toxicity profile, the positive impact of this drug on the breast cancer treatment landscape is hard to understate.”
Concarlo was formed in 2016 and is supported by a team of internationally renowned experts forming its Scientific Advisory Board. To date, the company has raised more than $3.1 million to support the development, improvement, and commercialization of its IpY and ApY technologies to bring a precision medicine approach to breast cancer management. The newly issued patent for IpY and the provisional patent application for modified versions of the peptide are the first key milestones in Concarlo’s plan to build an extensive patent estate to maintain market exclusivity for its clinically relevant therapeutics.
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