Current mass-spectrometry-based strategies will allow us to understand the molecular phenotypes of disease, which will drastically improve the diagnostic power of new clinical tests. In this interview, Professor Cobbaert [head of the Department of Clinical Chemistry and Laboratory Medicine at the Leiden University Medical Center (LUMC), Leiden, The Netherlands] and Dr Van der Burgt (associate professor at the Center for Proteomics and Metabolomics, LUMC) give us their expert opinions on how a strong collaboration between biomarker researchers, clinicians and medical laboratory specialists is necessary to make the development process more efficient. Professor Cobbaert is driven to innovate the field of laboratory medicine: “The clinical lab will change from a care-relevant to a system-relevant cross-sectoral discipline which will greatly affect the development of the entire healthcare system”.
About us
The clinical chemistry lab at the Leiden University Medical Center (LUMC) works closely together with researchers at the Center for Metabolomics and Proteomics (CPM) to develop new bioanalytical tests. The goal is to contribute to Precision Medicine through improved, molecular characterization of health and disease, for the sake of better patient management and patient outcome.
Christa Cobbaert heads the Department of Clinical Chemistry and Laboratory Medicine at LUMC, which encompasses clinical chemistry, hematology, coagulation and blood transfusion.
“In addition to regular patient diagnostics, our department also has responsibility for the hospital-wide central receipt of patient and research specimens. Our department supports research and biobanking from a large variety of clinical groups that want to use our services. Another core task is training and education of lab specialists and medical technicians.
“Some current numbers? Our routine lab works 24/7, we do about 4000 specimens per day, and produce over 4 million tests per year. We have 180 employees, about 140 full time equivalents. The majority are phlebotomists, who collect blood, and medical technologists, who run the analyses. We have an academic staff encompassing multiple laboratory specialists, who are responsible for the lab policy, lab organization, for state-of-the-art test menus, clinical consulting and post-academic training of lab specialists. Head medical technicians, quality control officers, as well as information and communication technology specialists and administrative personnel are a coaching layer between the academics and the operational co-workers.
“Since we are an academic institute, we are responsible for the traineeship of new lab specialists. We also contribute to the education of medical doctors. Teaching future medical doctors about the targeted use of lab diagnostics is key because approximately 70% of medical decisions in hospitals are based on lab results. We further provide teaching contributions in new disciplines such as clinical technology, and contribute to different Masters programmes.”
Dr Yuri van der Burgt is an associate professor at the CPM. “Trained as a chemist, I did a PhD in bioorganic chemistry and moved to the clinical field. At the LUMC I joined pioneering ‘omics’ research for medical care and patient research. For 50% of my time I work for the clinical chemistry lab, and from that position I bridge to the CPM research aiming for improved biomarker translation. CPM has approximately 50 researchers (PhD students postdocs, senior scientists, assistants and associates) and is headed by Manfred Wuhrer. We explore promising biomarkers that are discovered in basic research and aim to verify their potential for translation to the clinic. Mass-spectrometry (MS)-based omics studies have reported a wide variety of biomarkers or signatures, but only a few of these have been translated into a laboratory test. This limited translation is partly due to the lack of standardized protocols, robustness and reproducibility, but more importantly ill-defined or flawed study designs.”
Cobbaert: “We are happy with the cooperation with CPM because it’s very important to have analytical chemists connected to our lab. Once that lab specialists and clinicians have identified unmet clinical needs, analytical chemists support us with the assay development for molecular phenotyping of disease and health using MS-based technology. Together we attempt to bring promising biomarkers from the research field into the clinical arena. We believe that this collaboration should lead to a more robust and effective pipeline for developing medical tests. We also support research from various clinical groups at the LUMC, especially in the domains of Cardiovascular Diseases, Cancer Diagnosis and Kidney Diseases.”
Improving effectiveness
Van der Burgt: “One of the main activities at CPM is the elucidation of modifications on existing protein biomarkers, with emphasis on glycosylation analysis. As we want to make sure that these biomarkers can be of use for the clinic, we do not only report discoveries, but rather aim for further development of our findings into something clinically useful. Therefore we first make an inventory of the unmet needs from the clinicians, and what is actually needed for improved patient care. Hence, the clinical need guides our -omics research. And it is my task to bring these two worlds together. My goal is not just to publish papers on new discoveries, but to contribute to finding more effective solutions: clinically effective, cost effective and safe tests for patient care.”
Cobbaert: “The current pipeline and the current process of financing research is in my perception a wasteful process because there is insufficient attention to the downstream consequences (utility) of the research findings for patient care. Currently the number of papers and citation indices are rewarded, rather than the impact for patient care. Subsidizers should stimulate the translation and implementation of newly discovered biomarkers by making the funding of translation and implementation research inclusive.
“To counteract this inefficient pipeline from discovery to application researchers, clinicians, biostatisticians and lab specialists should collaborate closely. The clinical needs should be the driver of the test development process, rather than the technological push. Once these needs are identified a more informed decision can be made with regard to priorities: ‘This is what we are setting up together and this is where we go for’. The European Federation of Laboratory Medicine (EFLM) Test Evaluation framework provides guidance and encompasses key elements for creating evidence regarding the clinical and cost-effectiveness of new medical tests.
“Our mantra is that our research efforts should lead to precision diagnostics and clinically effective medical tests. In our collaboration with CPM we aim to contribute to better patient management and patient outcome with a targeted approach. As it is essential to add value to clinical pathways and patient management, we need actionable results for better patient care.”
International initiatives
Cobbaert: “We try to educate stakeholders of the biomarkers-medical test pipeline about the usefulness of the Test Evaluation framework for guiding this development process.
“We have asked ourselves: Why is the process from research to application such a wasteful process? What should we do? Last November we organized a precision diagnostics symposium in which we shared our experiences on quantitative proteomics and proposed our solutions [‘Prime time for precision diagnostics driven by unmet clinical needs’ (LUMC, Leiden, The Netherlands, November 2019)]. We also shared our struggles: developing specific molecular tests for proteins is not an easy road. Several barriers had to be alleviated. And that’s difficult to do, sometimes we failed, sometimes we felt that it doesn’t go quick enough. But we all are dedicated to make it a success together.
“Once a medical test is available, and evidence regarding its clinical utility and value has been generated, medical tests have to be implemented in clinical practice, either as a Lab-Developed-Test (rare) or as a Conformité Européene in vitro diagnostic (CE-IVD) test (often). To be successful, clear guidance should be given to doctors regarding its intended use in the clinical care pathway of interest. As a rule of thumb, the average trajectory from promising biomarker to applied medical test in the clinic takes about 10 years.
“In the current curriculum of medical students limited education is given regarding medical test use, notwithstanding the 70% rule (medical decisions are to a large extent based on lab test results). Laboratory specialists have to demonstrate medical leadership by educating physicians on proper test use.
“Collaborations are necessary to innovate laboratory medicine. We all start to understand the need for cooperation between different areas of expertise. A smooth and fruitful interaction between different types of laboratory specialists (e.g. microbiology, pathology, geneticists, immunologists…), researchers and clinicians should help to overcome the old boundaries.”
Collaboration is key
Van der Burgt: “An example of such a collaboration between CPM and clinical chemistry is our work on glycoprotein markers that we recently presented at the symposium on precision diagnostics, ‘Prime time for precision diagnostics driven by unmet clinical needs’ (LUMC, Leiden, The Netherlands, November 2019).
“Structure refinement of the biomarker for prostate cancer, the prostate specific antigen (PSA) demonstrated the importance of glycosylation for further development. We have worked on PSA at the CPM together with the clinical chemistry lab and in that collaborative effort we have seen that we can add extra information on the PSA test readout. Additionally, we aim to discover novel biomarkers for early detection of cancers. We see an enormous worldwide effort there and the result is hundreds, if not thousands, of new markers without any clinical pre-knowledge or knowledge of urgent clinical needs, it was technology-driven.”
Cobbaert: “It should be the opposite, clinical needs and sustainable and affordable health care should be the drivers of the test menu. In that context, our quantitative proteomics based activities for precision diagnostics are becoming more and more appreciated. To make translational research more effective, the funding agencies should also be concerned that the research they support will be applied in the clinical lab and will improve patient care.”
Paradigm shift
Cobbaert: “In the 20th century, our technology did not enable molecular characterization of disease, at least not in the high-throughput manner that is needed in clinical practice. Now we live in the 21st century and technology and medical insights have evolved. I expect a paradigm shift whereby traditional diagnostic tests will be complemented with precision diagnostic tests which enable Predictive, Preventive, Personalized Medicine, with Participation of the patient.
“As we drill down to the molecular level of health and disease, we should be able to provide more refined diagnoses and treatments. In 10 or 20 years, we may expect to read out a patient’s complete molecular phenotype or ‘proteotype’ and we will be able to monitor changes from a personal baseline.
“To innovate lab medicine and to realize the ambitions for Precision Medicine, we also need to find interoperable information technology (IT)-solutions. To that end, we need strategically thinking people who align the different stakeholders of the test pipeline, strive to improve health and patient care and know how to find advanced technical ,IT and organizational solutions to disclose the billions of data. Standardization of IT and making databases interoperable will be key. Unfortunately, we seem to be very far away from standardized interoperable solutions owing to a very fragmented IT-landscape across and even within health institutions.”

In a new study, a team of researchers led by Charis Eng, M.D., Ph.D., Chair of Cleveland Clinic’s Genomic Medicine Institute, identified a metabolite that may predict whether individuals with PTEN mutations will develop cancer or autism spectrum disorder (ASD).

Germline mutations of the tumour suppressor gene PTEN are associated with a spectrum of rare genetic disorders that increase the risk of certain cancers, cognitive and behavioural deficits, benign growths and tumours (i.e., hamartomas), and macrocephaly. These disorders are referred to collectively as PTEN hamartoma tumour syndrome (PHTS), but clinical manifestations vary greatly among patients and often are difficult to anticipate.

For example, subsets of Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS), two well-defined disorders on the PHTS spectrum, are characterized by either a high risk of certain cancers or ASD. There are functional and structural differences between PTEN mutations associated with ASD and those associated with cancer. However, a biomarker that could proactively determine if a patient with CS/BRRS will develop cancer or ASD has not yet been identified.
Previous studies have established metabolic dysregulation as one of the hallmarks of cancer. Specifically, germline variants in the SDHx genes cause an accumulation of the metabolite succinate, which has been linked to tumorigenesis. Some patients with PTEN mutations have been found to have succinate accumulation despite the lack of SDHx mutations, suggesting that variations in metabolite levels may indicate susceptibility to cancer versus ASD.
To investigate this further, Dr. Eng’s team analyzed the metabolite levels of 511 patients with CS, BRRS, or Cowden-like syndrome compared to controls. The results suggest that certain metabolites are associated with specific mutations and/or clinical features.
In particular, they discovered that decreased levels of fumarate, a metabolite formed from succinate, was more strongly associated with ASD or other developmental disorders compared to cancer in individuals with PTEN mutations. These findings indicate that certain metabolites, such as fumarate, may serve as predictive biomarkers that could distinguish patients who will develop neurodevelopmental disorders from those who will develop cancer.
“By identifying a way to differentiate those with germline PTEN mutations who develop cancer and those who develop autism, this provides clinicians with a MedicalXpress.
MedicalXpressmedicalxpress.com/news/2019-09-cancer-autism-pten-patients.html

Beckman Coulter’s Access SARS-CoV-2 IgG assay is now available in markets accepting the CE Mark, the company said in statement 15 June. It has already shipped tests to more than 400 hospitals, clinics and diagnostics laboratories in the United States and has begun shipping to customers globally. Beckman Coulter has more than 16,000 immunoassay analysers worldwide and has increased manufacturing to deliver more than 30 million tests a month.
Many of Beckman Coulter’s analysers can deliver up to 400 routine tests an hour. The Access SARS-CoV-2 IgG test can also be run on Beckman Coulter’s Access 2 analyser, a compact table-top analyser enabling high-quality serology testing to be carried out in small hospitals and clinics.
The Access SARS-CoV-2 IgG Assay is a qualitative immunoassay that detects IgG antibodies directed to the receptor-binding domain of the spike protein of the novel coronavirus that is driving the ongoing global pandemic. It is believed that these antibodies have the potential to be neutralizing antibodies and may play a role in lasting immunity. The test has a confirmed 99.8% specificity and 100% sensitivity at 18 days post PCR confirmed positive test. The assay uses immobilized virus antigens on magnetic particles to capture IgG antibodies from patient serum or plasma samples and reveals them using labelled anti-IgG antibodies.
Commenting on the assay, Shamiram R. Feinglass, M.D., MPH, Chief Medical Officer, Beckman Coulter, said: “An IgG antibody assay such as the test Beckman Coulter has developed can provide valuable information regarding community levels of immunity that will inform public health decision making and rollout of a vaccine when one does become available. The very high sensitivity and specificity of this assay provides a high positive predictive value, even when the overall incidence of disease is low. Additionally, since our assay can be run on multiple different types of analysers, it can be adapted to a variety of healthcare settings to best meet the needs of each community.”

Before starting cancer treatment with fluoropyrimidine-based chemotherapies, it is highly recommended to check for dihydropyrimidine dehydrogenase (DPD) deficiency by measuring uracilemia (or calculating the dihydrouracil:uracil ratio). This article discusses some of the ways of doing this.
Background
Approved for treatment of humans 60 years ago, fluoropyrimidinebased chemotherapies remain important antineoplastic agents. They are widely used in Europe, for example in France 100¦000 patients are medicated with this group of anticancer drugs.
Indeed, 5-fluorouracil (5-FU) and its oral pre-prodrug capecitabine are the backbone in the treatment of colorectal, pancreatic, gastric, breast, head and neck cancers. They work by interfering with enzymes (principally thymidylate synthase) involved in producing new DNA, thereby blocking the growth of cancer cells. They are administered by injection or by mouth. However, the use of fluoropyrimidines is associated with an important risk of toxicity, mainly due to deficiency of the enzyme involved in its catabolism, dihydropyrimidine dehydrogenase (DPD).
In France, health authorities recommend the determination of uracil concentration to guide dosing of fluoropyrimidines. Numerous liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been proposed but they include complex liquid–liquid or solid-phase extraction procedures.
Prescribers may be unaware that their patients lack functional DPD (encoded by the DPYD gene) and hence cannot break down fluorouracil, resulting in its build-up. This can lead to severe and life-threatening side effects such as neutropenia, neurotoxicity, severe diarrhea and stomatitis.
Up to 15% of patients exhibit a partial deficiency, whereas 0.1–0.5% may have a complete deficiency. Consequently, a 5-FU dose can lead to severe or lethal toxicity, and it is therefore highly recommended to screen for DPD status to determine a safe dose for the patient.
This deficiency may be detected either by genotyping (an approach that explores the polymorphisms of the DPYD gene) or by phenotyping, which consists of measuring uracilemia or calculating the 5,6-dihydrouracil:uracil (UH2:U) ratio.
Brief methodological overview

• The genotyping approach explores four variants known for reducing DPD activity (DPYD*13, DPYD*9A, DPYD*2A, and 2846A>T) and has the advantage of producing a fast and relatively inexpensive response by using automated techniques. Its specificity is very good, but its sensitivity is poor (not all DPD deficiencies are detected by genotyping).
• DPD is essential for converting endogenous U to UH2. Therefore, uracilemia or the UH2:U ratio reflect the level of DPD activity. Measurement of these components is feasible in plasma by liquid chromatography with photodiode array detection (LC-DAD) and LC-MS but requires complex sample preparation with protein precipitation, liquid–liquid extraction (LLE) or solid-phase extraction. Up to now, only analytical methods with multiple manual steps involving centrifugation, filtration and evaporation have been reported. Although results are satisfactory, the methods are time-consuming and tedious.

In genotyping, genes causing the deficiency are focused on, whereas with LC-MS/MS, the activity of DPD is estimated by measuring the ratio of the compounds UH2 and U. The first method looks only at the cause, whereas the second, safer method, looks at the result considering all deficiency cases while reducing toxic risks.
Need for accuracy, reliability and robustness
Proposed threshold values of 16 and 150 ng/mL for uracilemia characterize a partial or complete DPD deficiency, respectively. Inaccurate quantification of these threshold values may totally influence patient care and medical decisions. Analytical methods must therefore be accurate, reliable and robust. Automation is undoubtedly the best solution for reduction of errors while ensuring best reproducibility, robustness and reliability.
In this context, Shimadzu has developed a fully-automated procedure for the measurement of U and UH2 in human plasma. It is known as indirect phenotyping and provides faster testing as well as greater accuracy, safety and standardization. It is a method where the extraction is carried out by a programmable liquid handler directly coupled to a LC-MS/MS system.
The Centre Hospitalier Universitaire de Limoges (CHU Limoges), France, has been involved in proposing a method combining accuracy and time-efficiency. They suggested a new solution based on a novel sample preparation system, coupling an HPLC instrument and a triplequadrupole mass spectrometer.
Extraction is performed by an automated sample preparation system, the Clinical Laboratory Automation Module (CLAM)-2030 (Shimadzu Corporation) coupled to an LC-MS/MS system. Responding to the needs of clinical research sites, the CLAM-2030 provides stable data acquisition, lower running costs and improved work efficiency. It can be connected to four models of triple-quadrupole liquid chromatography mass spectrometers. Once the primary (or secondary) tube is loaded onto the automated system, no further human intervention is required as the CLAM-2030 resulting in high standardization.
The system was used in positive electrospray ionization mode. Acquisition method targeted multiple reaction monitoring (MRM) transitions for uracil, dihydrouracil, uracil-13C, 15N2 and dihydrouracil-13C, 15N2. The workflow procedure is summarized in Figure 1.
The CLAM-2030 targets pharmaceutical and medical departments as well as biological analysis labs. It is a technological key system applied in Shimadzu’s European Innovation Center (EuIC) programme. The EuIC merges the cutting-edge analytical technologies of Shimadzu with game-changing topics and expertise in markets and science covered by opinion leaders, strategic thinkers and scientific experts in order to create new solutions for tomorrow. In France, the CHU University Hospital is a cooperation partner of the EuIC.
The CLAM-2030 module automates everything from the preparation of urine, blood, and other biological samples to measurement via liquid chromatography mass spectrometry (LC-MS). Within a few minutes, the CLAM-2030 preparation module completes the blood-sample preparation process including the addition of reagents, mixing of the solution and the addition of a deproteinization liquid, compared to the 15–20 minutes that this process conventionally takes. Further, if the samples and reagents are placed and positioned in special containers for automatic conveyance to the LC-MS by an autosampler, the module can perform all of the processes automatically, on weekends and overnight.
Quick results
By overlapping sample treatment, a result is obtained every 14 minutes after the first sample. This method is fully validated according to ISO 15189 requirements. The result of the validation study are summarized in Table 1. A 5 ng/mL limit of quantification is obtained for both U and UH2 with good linearity (R² >0.995). At 16 ng/mL (threshold value) the inaccuracy and coefficients of variation were less than 5% for intra- and inter-assay tests, clearly sufficient to avoid misdiagnosing the level of DPD activity.
The method has been applied successfully in 64 consecutive patients tested at the CHU Limoges, and its results were similar to those of a classic LC-MS method (LLE for sample preparation) used routinely until then. For each patient, the same diagnosis (absence or presence of DPD deficiency) was given and the Bland–Altman plot (Fig. 2) shows good agreement between the two methods.
Conclusion
As DPD deficiency screening in patients given fluoropyrimidine-based chemotherapy is now highly recommended, most labs in charge of the measurement of U (and UH2) will or are already facing an increase in this activity. Shimadzu therefore proposes a fully-automated solution ensuring an accurate and robust measurement without requiring precious laboratory staff time. The simplicity of operation and the minimization of user involvement in the sample preparation process will help obtain high throughput for the monitoring of 5-FU and capecitabine treatments.

A new calculator developed by the University of Exeter will help clinicians classify whether a patient has type 1 or type 2 diabetes, ensuring they get the best treatment and reducing complications.

The calculator uses a model that takes into account available data about the patient, as well as blood test results. It can be used to identify if a person is likely to have type 1 diabetes, to reduce misdiagnosis. Former Prime Minister Theresa May was initially diagnosed with type 2 diabetes. Only when tablet treatment failed to work was she re-diagnosed with type 1.

It is often difficult for clinicians to diagnose which type of diabetes a patient has. While blood tests such as antibodies against the cells that make insulin, or a person’s genetic risk of type 1 diabetes may help diagnosis,  these tests do not give a diagnosis on their own, and may be interpreted very differently depending on whether or not a person has other features of type 1 diabetes. The new calculator, currently available in beta format, combines available information from blood tests with a person’s age of diagnosis and BMI for a personalised medicine approach. The calculator was developed by researchers at the universities of Exeter, Oxford and Dundee.

The new calculator will build on the success of a similar calculator previously developed at Exeter, to help clinicians determine whether a patient has the diabetes subtype MODY, caused by a single gene. The online calculator has been used by more than 100,000 people, with more than 9,000 people downloading the calculator phone app Diabetes Diagnostics, which will be updated to include the new calculator. New research recently presented at the European Association for the Study of Diabetes conference in Barcelona has shown that almost half of all referrals sent to the UK diagnostic laboratory for MODY now report using the calculator, and those that report using the calculator have a higher detection rate compared with those that do not.
Dr Angus Jones, of the University of Exeter Medical School, who led the research, said: “The right diagnosis in diabetes is absolutely crucial to getting the best outcomes for patients, as treatment is very different in different types of diabetes. However in some people it can be very difficult to know what type of diabetes they have. Our new calculator can help clinicians by combining different features to give them the probability a person will have type 1 diabetes, and assess whether additional tests are likely to be helpful.”
The new beta format calculator can be accessed here: www.diabetesgenes.org/t1dt2d-prediction-model/
University of Exeterwww.exeter.ac.uk/news/research/title_754422_en.html