by Dr Jacqueline Gosink
Gastrointestinal complaints are very common and can be difficult to diagnose. Among the many causes are genetic deficiencies in digestive enzymes. Molecular genetic analysis of polymorphisms in the patient’s DNA can determine if inborn enzyme deficits are behind the digestive problems, aiding differential diagnostics. Primary lactose intolerance, for example, is associated with polymorphisms in the regulatory region of the lactase gene (LCT), whereas hereditary fructose intolerance (HFI) is caused by mutations in the aldolase B gene (ALDOB). A PCR-based DNA microarray provides parallel determination of the two main lactose intolerance-associated polymorphisms (LCT‑13910C/T and LCT‑22018G/A ) as well as the four HFI-associated mutations (A149P, A174D, N334K and del4E4). The fast and simple determination includes fully automated data evaluation, ensuring highly standardized results.
Lactose intolerance
Primary lactose intolerance is a genetically caused deficiency of lactase, the enzyme responsible for splitting lactose into its constituent sugars glucose and galactose. In affected patients, undigested lactose is fermented in the ileum and large intestine, producing by-products such as short-chain fatty acids, methane and hydrogen, which cause the typical symptoms of abdominal pain, nausea, meteorism and diarrhea. Secondary manifestations include deficiencies, for example of vitamins, and as a result unspecific symptoms such as fatigue, chronic tiredness and depression.
Lactose intolerance represents the natural state in mammals. Lactase activity decreases after weaning and in adulthood is often only a fraction of the activity in infancy. Some humans, however, retain the ability to metabolize lactose into adulthood due to specific genetic variants. The frequency of lactase persistence is around 35% worldwide, although it varies greatly between different population groups. It is prevalent in regions with a long tradition of pastoralism and dairy farming, for example in Europe and in populations of European descent. In large parts of eastern Asia, on the other hand, almost 100% of the population is lactose intolerant.
In addition to the primary genetically caused form of lactose intolerance there is also the secondary acquired form. This develops as a result of damage to the intestine, for example from other gastrointestinal diseases such as Crohn’s disease, coeliac disease, infectious enteritis or injury from abdominal surgery. The two forms need to be distinguished diagnostically because of the need for different treatment regimes. Whereas individuals with primary lactose intolerance must adhere to a lactose-free or low-lactose diet for life or alternatively take lactase supplements, those with secondary lactose intolerance need only restrict their dairy intake until the intestinal epithelium has regenerated through treatment of the underlying cause.
Diagnostics of lactose intolerance
Classic diagnostic tests for lactose intolerance are the hydrogen breath test and blood glucose tests, with which the patient’s ability to metabolize lactose is examined. However, these tests have a low specificity and sensitivity and are influenced by individual factors such as the composition of intestinal flora, colonic pH, gastrointestinal motility and sensitivity to lactose fermentation products. Moreover, they cannot distinguish between the primary and secondary forms of lactose intolerance. Molecular genetic testing complements these methods, enabling verification or exclusion of primary lactose intolerance with high probability, as well as differentiation of the primary and secondary forms. Genetic testing is, moreover, a non-invasive and more comfortable examination, which does not carry the risk of provoking symptoms of lactose intolerance in non-lactase-persistent individuals.
LCT polymorphisms
The main mutations associated with lactase persistence are LCT‑13910C>T and LCT‑22018G>A, which are located in the regulatory region of the lactase gene. According to current knowledge, homozygous carriers of the wild-type variants LCT‑13910CC and LCT‑22018GG develop lactose intolerance, while heterozygous carriers of the variants LCT‑13910CT and LCT‑22018GA only show corresponding symptoms in stress situations or with intestinal infections. Homozygous carriers of the mutant variants LCT‑13910TT and LCT‑22018AA are lactose tolerant as adults. These two polymorphisms are strongly coupled.
Hereditary fructose intolerance
HFI is caused by mutations in the gene for aldolase B, an enzyme essential for fructose metabolism. The mutations result in a reduction or loss in activity or stability of aldolase B, which is responsible for catalysing the breakdown of fructose-1-phosphate (F-1-P) to dihydroxyacetone phosphate and glyceraldehyde. The toxic intermediate F-1-P then accumulates in the body, causing symptoms such as nausea, vomiting and digestive disorders and in the longer term liver damage. HFI is a rare disease, occurring, for example, with a prevalence of 1 in 20¦000 in Europe. It manifests already in childhood, but may remain undiagnosed due to patients’ natural dislike of sweets, fruits and vegetables.
In addition to HFI, intolerance to fructose can also be caused by deficits in the transport of fructose into the enterocytes. This form is known as intestinal fructose intolerance or fructose malabsorption. It is much more common than HFI, occurring with a prevalence of about 30%. It is important to distinguish HFI from fructose malabsorption, because of the resulting difference in dietary requirements. Patients with HFI must completely eliminate fructose and its precursors (e.g. sucrose, sorbitol) from their diet to prevent damage to their organs. Patients with fructose malabsorption, however, should follow a fructose-restricted diet.
Diagnostics of HFI
Intolerance to fructose is usually diagnosed by means of the hydrogen breath test, in which a defined amount of fructose is ingested and then the amount of hydrogen in the exhaled air is measured. In patients with HFI, however, the intake of fructose carries the risk of a severe hypoglycaemic reaction. Therefore, a molecular genetic test for HFI should always be performed before a fructose load test. Early diagnosis of HFI is particularly important to avoid permanent damage to the liver, kidney and small intestine.
ALDOB mutations
In Europe the most frequent mutants associated with HFI are the amino acid substitutions A149P, A174D, N334K (in Human Gene Mutation Database nomenclature) and the deletion del4E4 in the aldolase B gene. For HFI to manifest, both alleles of an individual’s DNA must be affected by a mutation. In homozygous genotypes, the two alleles contain the same mutation (paternal and maternal inheritance). If the two alleles exhibit different mutations, this is referred to as a compound heterozygous HFI genotype.
Parallel genetic analysis
Molecular genetic determination of the polymorphisms associated with lactose intolerance and HFI enable diagnosis of these genetic conditions with high certainty. The EUROArray Lactose/Fructose Intolerance Direct enables simultaneous detection of the lactose-intolerance-associated polymorphisms ‑13910C/T and ‑22018G/A and the HFI-associated mutations A149P, A174D, N334K and del4E4. Thus, the two genetically caused metabolic disorders can be assessed with a single test.
he test can be performed on whole blood samples, eliminating the need for costly and time-consuming DNA isolation. In the test procedure (Fig. 1), the sections of DNA containing the alleles are first amplified by multiplex PCR using highly specific primers. During this process the PCR products are labelled with a fluorescent dye. The PCR mixture is then incubated with a microarray slide containing immobilized DNA probes. The PCR products hybridize with their complementary probes and are subsequently detected via the emission of fluorescence signals. The data is evaluated fully automatically using EUROArrayScan software (Fig. 2), and in the case of positive results, homozygous and heterozygous states are differentiated. Numerous integrated controls ensure high reliability of results, for example, by verifying that there are no other rare mutations in direct proximity to the tested positions which could interfere with the analysis.
Studies on blood donors
The performance of the EUROArray was investigated using 116 precharacterized samples from blood donors in Germany and from quality assessment schemes. The EUROArray revealed a sensitivity of 100% and a specificity of 100% with respect to the reference molecular genetic method.
Conclusions
Diagnosis of gastrointestinal disorders often involves a long and challenging process of diagnostic tests and restrictive diets. Since lactose and fructose are widely consumed in many diets, it is important to consider intolerance to these sugars during the diagnostic work-up. Simple genetic analysis enables primary lactose intolerance and HFI to be confirmed or excluded as the cause of gut problems. The parallel analysis offered by the EUROArray enables especially fast and effective diagnostics. Patients diagnosed with these genetic conditions can promptly adapt their diets to ease their symptoms. If the analysis is negative, the physician can focus on searching for other causes of the digestive complaints. The molecular genetic analysis thus provides valuable support for the gastroenterology clinic.
The author
Jacqueline Gosink PhD
EUROIMMUN AG, 23560 Lubeck, Germany
Pharming Ruconest
, /in Corona News, E-News /by 3wmediaThe Netherlands-based Pharming Group, a specialty pharmaceutical company, have seen encouraging results from it RUCONEST (recombinant human C1 inhibitor) treatment in patients with confirmed COVID-19 infections. The patients were treated under a compassionate use program at the University Hospital Basel, Switzerland.
Dr. Michael Osthoff, University Hospital Basel, Switzerland and the treating physician, said: “Although this is an uncontrolled, small treatment experience, the results demonstrate the potential effectiveness of using RUCONEST as an anti-inflammatory approach to inhibit the complement and contact systems after SARS-CoV-2 infection.”
Four male patients and one female patient (between 53-85 years of age) with COVID-19 and suffering from related severe pneumonia, who did not improve despite standard treatment, including hydroxychloroquine and lopinavir/ritonavir, were administered RUCONEST at an initial dose of 8400 U, followed by 4200 U every 12 hours for three additional doses. No allergic reactions or drug related adverse events were reported.
Following treatment with RUCONEST, fever resolved in four of the five patients within 48 hours, and laboratory markers of inflammation decreased significantly (CRP, IL-6). Soon thereafter, the patients were discharged from the hospital as fully recovered. One patient had increased oxygen requirement and was temporarily transferred to the ICU for intubation, but over the subsequent days recovered and was released from the ICU.
Following these initial results, a multinational, randomized, controlled, investigator-initiated clinical trial with up to 150 patients with confirmed COVID-19 infections, requiring hospitalisation due to significant COVID-19 related symptoms is planned. The study will be led by Dr. Osthoff.
RUCONEST® is commercialised by Pharming in the US and in Europe, and the company holds all other commercialisation rights in other countries not specified below. In some of these other countries distribution is made in association with the HAEi Global Access Program (GAP). RUCONEST is distributed in Argentina, Colombia, Costa Rica, the Dominican Republic, Panama, and Venezuela by Cytobioteck, in South Korea by HyupJin Corporation and in Israel by Kamada.
Pharming’s technology platform includes a unique production process that has proven capable of producing industrial quantities of pure high quality recombinant human proteins in a more economical and less immunogenic way compared with current cell-line based methods.
Roche to launched COVID-19 antibody test in early May
, /in Corona News, E-News /by 3wmediaRoche will develop its Elecsys Anti-SARS-CoV-2 serology test to detect antibodies in people who have been exposed to SARS-CoV-2 and launch it in early May in countries accepting the CE mark. The company says it is actively working with the FDA for an Emergency Use Authorisation.
The Elecsys Anti-SARS-CoV-2 immunoassay is an in vitro test, using human serum and plasma drawn from a blood sample, to detect antibodies and determine the body’s immune reaction to SARS-CoV-2. The test may be used in epidemiological research to help better understand the spread of the disease and may also be used together with molecular tests to aid in the diagnosis of suspected COVID-19 patients. Hospitals and reference laboratories can run the test on Roche’s cobas e analysers, which are widely available in laboratories around the world.
Roche notes that antibody testing is central to help identify people who have been infected by the virus, especially those who may have been infected but did not display symptoms. Additionally, the test can support priority screening of high risk groups, such as healthcare workers, food supply workers who might already have developed a certain level of immunity and can continue serving and/or return to work.
Severin Schwan, CEO Roche Group, said: “Following the launch of our high-volume PCR test in mid-March to detect active infection of the disease, we are now going to launch a new antibody test in early May. Every reliable test on the market serves its purpose for healthcare systems to help us overcome this pandemic. Roche is collaborating closely with health authorities and ramping up production to ensure fast availability of the test globally.”
Thomas Schinecker, CEO Roche Diagnostics, commented: “The antibody test is an important next step in the fight against COVID-19. Roche’s antibody test can be quickly scaled and made broadly available around the world as our instrument infrastructure is already in place.”
Oxford companies join forces to scale up production of Covid-19 reagents
, /in Corona News, E-News /by 3wmediaOXGENE and The Native Antigen Company have joined forces to scale up production of SARS-CoV-2 (COVID-19) reagents by combining OXGENE’s proprietary Adenoviral Protein Machine Technology with The Native Antigen Company’s antigen development expertise. Together, OXGENE and The Native Antigen Company will aim to scale their antigen manufacturing capabilities to deliver high-purity, recombinant proteins for the development of diagnostics and vaccines.
Together they are developing an improved, scalable approach to SARS-CoV-2 antigen manufacture. The Native Antigen Company was one of the first recognised suppliers of SARS-CoV-2 antigens in February 2020, demonstrating their ability to rapidly support the diagnostic and vaccine industries with high-quality infectious disease reagents. OXGENE’s Protein Machine Technology allows for the scalable production of viral proteins in mammalian cells using their proprietary adenoviral expression vector. Through genetic modification, the adenovirus is ‘tricked’ into making SARS-CoV-2 proteins rather than its own, thereby harnessing the innate power of highly scalable viral protein production.
The Native Antigen Company’s recombinant SARS-CoV-2 antigens are produced in mammalian cells to ensure full glycosylation and proper protein folding, both of which are essential for full biological and antigenic activity. The rapid scale up production of SARS-CoV-2 antigens is critical for the development of widely available diagnostic tests.
Unlike the PCR tests that are currently being used, these diagnostics will be able to confirm past infections and determine levels of immunity to SARS-CoV-2. This could be invaluable for disease modelling and public health policy, as true transmission rates and case fatality rates can be determined. These tests could also be instrumental for the diagnosis of healthcare workers who have been exposed to the virus to ensure that they have developed natural immunity before returning to work, and to help measure patient immune responses for the rapid development of a SARS-CoV-2 vaccine.
Dr Ryan Cawood, Chief Executive, OXGENE, said: “Our novel Protein Machine Technology represents a significant development in the rapid and scalable generation of high-quality viral proteins. We’re delighted that by collaborating with The Native Antigen Company, we can take advantage of our technology to support the needs of researchers racing to develop much-needed diagnostics and vaccines against COVID-19.”
This collaboration builds on a long-standing collegiate relationship between the two Oxford-based businesses as they work towards developing more scalable technologies for the diagnosis of disease, and the cost-effective manufacture of high-quality diagnostics and vaccines.
OXGENE and The Native Antigen Company aim to complete the first validation of this new paradigm in protein expression within the next month, which could have a demonstrable impact on the race to develop diagnostic kits and vaccines against this virus.
For further information about The Native Antigen Company’s Coronavirus Antigens, please visit: https://thenativeantigencompany.com/coronavirus-dashboard/
Horizon Discovery releases CRISPR screening for primary human B cells
, /in Corona News, E-News /by 3wmediaCambridge, UK-based Horizon Discovery Group, specializing in the application of gene editing and gene modulation for cell line engineering, has released an arrayed CRISPR knockout screening service for primary human B cells to its cell-based screening services.
The new B cell screening service, the first of its kind in the market, will enable researchers to identify genes that affect the function of B cells and examine how this impacts other immune cell types, particularly in infectious diseases, cancer, and auto-immune disorders, such as COVID-19, Burkitt’s lymphoma and multiple sclerosis respectively.
Primary human cells – cells that are freshly isolated from donors – are known to be difficult to study in the lab. However, working with these cells brings scientists one step closer to healthy or diseased micro-environments, enabling them to better understand disease etiology and therapeutic mechanisms, and thereby advance drug discovery and development programs.
“The interest in harnessing the immune system for effective therapies continues to grow, with the global cell therapy market predicted to reach $8.21bn by 2025. Expanding our services to encompass screening of both primary T and B cells is another example of our commitment to apply decades of gene editing experience in support of drug discovery and development for the treatment of human disease,” said Terry Pizzie, CEO, Horizon Discovery.
Dolomite and Mologic collaborate to scale-up manufacture of nanoparticles for Covid-19 diagnostic tests
, /in Corona News, E-News /by 3wmediaAs the COVID-19 pandemic progresses in countries around the globe, there is an urgent need for rapid and reliable point-of-need detection testing. Dolomite Microfluidics and Mologic are working together to accelerate the development and launch of these tests.
UK-based Mologic is a leading developer of rapid response diagnostic tests for diseases such as malaria and Ebola virus disease. Clients include the Bill & Melinda Gates Foundation, where Mologic is leveraging its core technology through its Centre for Advanced Rapid Diagnostics (CARD) to develop the next generation of ultra-sensitive point-of-care diagnostics which are easy to use andinexpensive to manufacture – critical to the success of many global health programmes. Most recently, Mologic has received UK Government funding to develop and manufacture a high sensitivity test for COVID-19 that generates results within minutes – rather than hours or days – without the need for a laboratory or specialist equipment.
The technology behind these tests involves the use of precisely manufactured nanoparticles. Dolomite specialises in equipment that allows the development and scale-up of precision nanoparticles. This is achieved by using microfluidic technology to retain advanced control of production conditions. Dolomite is working with Mologic to combine ground-breaking diagnostic technology with continuous flow microfluidic manufacturing processes to accelerate the validation and release of Mologic’s COVID-19 diagnostic test.
Dutch company CytoSMART Technologies is to donate 100 mini live-cell imaging systems to researchers in high containment labs worldwide
, /in Corona News, E-News /by 3wmediaLabs 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.
Benefits of molecular biology in clinical diagnostics
, /in Corona News, E-News /by 3wmediaThe advent of molecular biology techniques has revolutionized disease diagnosis. CLI discussed with Dr Chandrasekhar Nair from Molbio Diagnostics the benefit that these techniques have brought and how these technologies are being adapted for point-of-care use for rapid diagnosis and the benefit of rural populations.
What has the impact of molecular biology been on disease diagnosis and treatment?
Accurate and timely diagnosis of infectious diseases is essential for proper medical management of patients. Early detection of the causative agent also enables care providers to intervene in a precise rather than presumptive manner and institute adequate measures to interrupt transmission to the susceptible population in the hospital or community.
The conventional diagnostic model for clinical microbiology has been labour and infrastructure intensive and frequently requires days to weeks before test results are available. Moreover, due to the complexity and length of such testing, this service was usually directed at the hospitalized patient population. Bacterial/viral culture has been – and continues to be – the gold standard for detection. However, time taken for some pathogens to grow, coupled with the difficulty in culturing some pathogens has resulted in a demand for alterna tive techniques that would allow direct pathogen detection in clinical samples rapidly.
The application of engineering techniques to the technological revolution in molecular biology has greatly improved the diagnostic capabilities of modern clinical microbiology laboratories. In particular, rapid techniques for nucleic acid amplification and characterization combined with automation and user-friendly software have significantly broadened the diagnostic arsenal. Among the molecular techniques, applicability of PCR-based methods has gained popularity as it allows for rapid detection of unculturable or fastidious microorganisms directly from clinical samples.
Clinical laboratories are increasingly finding utility of molecular techniques in diagnosis and monitoring of disease conditions. Nucleic acid amplification tests are becoming very popular in the diagnosis and management of viral infections [hepatitis B and V viruses (HBV, HCV), human immunodeficiency virus (HIV), influenza virus, etc] because they allow determination of the viral load. In most cases, they are now considered a reference, or gold standard method for diagnostic practices such as screening donated blood for transfusion-transmitted viruses [cytomegalovirus (CMV), HIV, HCV, etc]. Another important case is the use of molecular tests for the detection of the tuberculosis (TB)-causing bacterium Mycobacterium tuberculosis (MTB). Considering the limited sensitivity of smear microscopy, coupled with the steady rise in drug-resistant MTB, rapid molecular tests appear promising.
What are the challenges of implementing molecular diagnostic techniques in developing countries?
For a long time the field of molecular diagnostics has been limited to the domain of large centralized laboratories because of its dependency on complex and expensive infrastructure, highly skilled manpower and special storage conditions. This investment has also resulted in the need for batch testing to make such facilities affordable. As a result, patients and samples need to travel long distances for a test to be conducted and results are delayed, resulting in a loss of follow-up. These factors have led to a concentration of such facilities in urban centres, and poor reach of molecular diagnostics techniques, particularly in low and middle income countries (LMICs). The poor testing rates in the current COVID-19 pandemic are evidence of such dependence on centralized facilities, limiting the ability to test on demand and take appropriate action.
The lack of timely access to good diagnostics resulting in either delayed or inaccurate diagnosis by other methods has been increasingly resulting in spread of disease and poor treatment outcomes.
How can these challenges be overcome?
We need to increase the reach of molecular diagnostics techniques. Given the economic constraints in LMICs, point-of-care technology (POCT) hold a lot of promise and several major global initiatives are devoted to providing such devices. Facilities for testing that can be deployed, set up and run quickly, at affordable costs, with minimal infrastructure requirements and training are critical to the success of the efforts to increase reach. Mobile data coverage, that exists with reasonable density in LMICs, could also be leveraged for better programme management and hotspot detection.
The success of these technologies also depend on uncompromised performance and adherence to quality standards.
Furthermore, designers of POCT devices need to focus on key user requirements which include: (1) simplicity of use; (2) robustness of reagents and consumables; (3) operator safety; and (4) easy maintainability.
What is Molbio Diagnostics doing to meet these demands?
The Truelab® Real Time Quantitative micro PCR System from Molbio Diagnostics brings PCR technology right to the point of care, at all laboratory and non-laboratory settings, primary centres, in the field, near patient – essentially at all levels of healthcare, thereby decentralizing and democratizing access to molecular diagnostics. With a large and growing menu of assays for infectious diseases, this rapid, portable technology enables early and accurate diagnosis and initiation of correct treatment right at the first point of contact. The platform is infrastructure independent and provides complete end-to-end solution for disease diagnosis. With proven ability to work even at primary health centres and with wireless data transfer capability, this game changing technology brings in a paradigm shift to the global fight in control and management of devastating infectious diseases.
Under the aegis of the Council of Scientific and Industrial Research and New Millennium Indian Technology Leadership Initiative partnership, Bigtec Labs (research and development wing of Molbio Diagnostics Pvt. Ltd.) has developed a portable and battery-operated micro PCR system that has since been extensively validated [under the Department of Biotechnology and Indian Council of Medical Research (DBT & ICMR)]. Bigtec has also developed various tests and nucleic acid preparation devices to facilitate ‘sample to result’ molecular diagnostics in resource limited settings. The micro PCR system has since been launched in India through the parent company, Molbio Diagnostics, which has its manufacturing and marketing base in Goa, India.
The system works on disease specific Truenat™ microchips for conducting a real-time PCR. The sample preparation (extraction and purification) is done on a fully automated, cartridge-based Trueprep® AUTO sample prep device. The purified nucleic acids are further amplified on the Truelab® Real Time Quantitative micro PCR System which enables molecular diagnostics for infectious diseases at the point of care.
This compact battery-operated system has single testing capability and provides sample to result within 1 hour. Hence, it enables same-day reporting and initiation of evidence-based treatment for the patient.It also has real-time data transfer capability (through SMS/email) for immediate reporting of results in emergency cases. Physicians benefit from this technology by having a definitive diagnosis, early in the infection cycle, without patients/samples having to travel extensively to centralized facilities.
The Truelab® Real Time Quantitative micro PCR System from Molbio Diagnostics is a cost-effective and sensitive device that can detect diseases accurately with high specificity. The device is battery-operated and portable. This offers the additional advantage of placing the device in almost any kind of laboratory setting, unlike other devices that require uninterrupted power supply, elaborate infrastructure and air-conditioning.
Considering our platform’s potential to perform molecular diagnostics for infectious diseases at the point of care, India has initiated screening for COVID-19 using the Truenat™ Beta CoV test available on the Truelab® Real Time Quantitative micro PCR System. This will allow same-day testing, reporting, and initiation of patient isolation, if required – thereby reducing the risk of infection spreading while waiting for results.
The successful translation of our innovative concept into a product was made possible by Molbio’s multi-disciplinary workforce – with a constant mission to enable better medicine through precise, faster, cost-effective diagnosis at the point of care; to provide every patient access to the best healthcare through cutting edge technologies. Molbio aims to be a leading global player in the point-of-care diagnostics arena by continuing to innovate and bring new technologies for social betterment.
The company is based in India – how does this affect what you do, how is the clinical lab diagnostics industry developing in India and does it create more chances for you?
In India, we have over between 45¦000–50¦000 in vitro diagnostic laboratories – every one of which uses routine conventional diagnostic methods. Only a handful of them have adopted molecular diagnostic testing for reasons mentioned above. But this is changing with the advent of Molbio’s Truelab® platform, with regular standalone laboratories that were, up to now, outsourcing molecular testing, starting to perform the tests themselves. In the short span of a few years, Molbio has established itself as a company focused on making a significant impact in aiding infectious disease diagnostics worldwide with our extensive testing menu.
Our test range covers infectious diseases such as TB, the entire hepatitis range, High risk HPV, H1N1, along with the recent addition of tests for COVID-19, catering to a large population base and addressing diseases with a very significant global mortality percentages. Our rapid test development for Nipah virus and the leptospirosis-causing Leptospira bacteria show our commitment to neglected tropical diseases. Going forward, Molbio will continue to increase the assay range looking at the needs of the global LMIC geography.
The Truenat™ MTB and MTB-RIF tests have started playing a significant role in India’s mission to becoming TB-free by 2025. We would be happy to partner with other National TB Programmes in achieving sustainable development goals well before 2030.
Our vision has always been ‘innovate to have a real impact’ and hence Molbio will continue to bring in newer POCT platforms so that the benefits of science and technology reach the masses.
The interviewee
Dr Chandrasekhar Nair, BE, PhD, chief technical officer, Molbio Diagnostics
For further information visit Molbio Diagnostics (http://www.molbiodiagnostics.com)
Parallel genetic testing for primary lactose intolerance and hereditary fructose intolerance
, /in E-News /by 3wmediaby Dr Jacqueline Gosink
Gastrointestinal complaints are very common and can be difficult to diagnose. Among the many causes are genetic deficiencies in digestive enzymes. Molecular genetic analysis of polymorphisms in the patient’s DNA can determine if inborn enzyme deficits are behind the digestive problems, aiding differential diagnostics. Primary lactose intolerance, for example, is associated with polymorphisms in the regulatory region of the lactase gene (LCT), whereas hereditary fructose intolerance (HFI) is caused by mutations in the aldolase B gene (ALDOB). A PCR-based DNA microarray provides parallel determination of the two main lactose intolerance-associated polymorphisms (LCT‑13910C/T and LCT‑22018G/A ) as well as the four HFI-associated mutations (A149P, A174D, N334K and del4E4). The fast and simple determination includes fully automated data evaluation, ensuring highly standardized results.
Lactose intolerance
Primary lactose intolerance is a genetically caused deficiency of lactase, the enzyme responsible for splitting lactose into its constituent sugars glucose and galactose. In affected patients, undigested lactose is fermented in the ileum and large intestine, producing by-products such as short-chain fatty acids, methane and hydrogen, which cause the typical symptoms of abdominal pain, nausea, meteorism and diarrhea. Secondary manifestations include deficiencies, for example of vitamins, and as a result unspecific symptoms such as fatigue, chronic tiredness and depression.
Lactose intolerance represents the natural state in mammals. Lactase activity decreases after weaning and in adulthood is often only a fraction of the activity in infancy. Some humans, however, retain the ability to metabolize lactose into adulthood due to specific genetic variants. The frequency of lactase persistence is around 35% worldwide, although it varies greatly between different population groups. It is prevalent in regions with a long tradition of pastoralism and dairy farming, for example in Europe and in populations of European descent. In large parts of eastern Asia, on the other hand, almost 100% of the population is lactose intolerant.
In addition to the primary genetically caused form of lactose intolerance there is also the secondary acquired form. This develops as a result of damage to the intestine, for example from other gastrointestinal diseases such as Crohn’s disease, coeliac disease, infectious enteritis or injury from abdominal surgery. The two forms need to be distinguished diagnostically because of the need for different treatment regimes. Whereas individuals with primary lactose intolerance must adhere to a lactose-free or low-lactose diet for life or alternatively take lactase supplements, those with secondary lactose intolerance need only restrict their dairy intake until the intestinal epithelium has regenerated through treatment of the underlying cause.
Diagnostics of lactose intolerance
Classic diagnostic tests for lactose intolerance are the hydrogen breath test and blood glucose tests, with which the patient’s ability to metabolize lactose is examined. However, these tests have a low specificity and sensitivity and are influenced by individual factors such as the composition of intestinal flora, colonic pH, gastrointestinal motility and sensitivity to lactose fermentation products. Moreover, they cannot distinguish between the primary and secondary forms of lactose intolerance. Molecular genetic testing complements these methods, enabling verification or exclusion of primary lactose intolerance with high probability, as well as differentiation of the primary and secondary forms. Genetic testing is, moreover, a non-invasive and more comfortable examination, which does not carry the risk of provoking symptoms of lactose intolerance in non-lactase-persistent individuals.
LCT polymorphisms
The main mutations associated with lactase persistence are LCT‑13910C>T and LCT‑22018G>A, which are located in the regulatory region of the lactase gene. According to current knowledge, homozygous carriers of the wild-type variants LCT‑13910CC and LCT‑22018GG develop lactose intolerance, while heterozygous carriers of the variants LCT‑13910CT and LCT‑22018GA only show corresponding symptoms in stress situations or with intestinal infections. Homozygous carriers of the mutant variants LCT‑13910TT and LCT‑22018AA are lactose tolerant as adults. These two polymorphisms are strongly coupled.
Hereditary fructose intolerance
HFI is caused by mutations in the gene for aldolase B, an enzyme essential for fructose metabolism. The mutations result in a reduction or loss in activity or stability of aldolase B, which is responsible for catalysing the breakdown of fructose-1-phosphate (F-1-P) to dihydroxyacetone phosphate and glyceraldehyde. The toxic intermediate F-1-P then accumulates in the body, causing symptoms such as nausea, vomiting and digestive disorders and in the longer term liver damage. HFI is a rare disease, occurring, for example, with a prevalence of 1 in 20¦000 in Europe. It manifests already in childhood, but may remain undiagnosed due to patients’ natural dislike of sweets, fruits and vegetables.
In addition to HFI, intolerance to fructose can also be caused by deficits in the transport of fructose into the enterocytes. This form is known as intestinal fructose intolerance or fructose malabsorption. It is much more common than HFI, occurring with a prevalence of about 30%. It is important to distinguish HFI from fructose malabsorption, because of the resulting difference in dietary requirements. Patients with HFI must completely eliminate fructose and its precursors (e.g. sucrose, sorbitol) from their diet to prevent damage to their organs. Patients with fructose malabsorption, however, should follow a fructose-restricted diet.
Diagnostics of HFI
Intolerance to fructose is usually diagnosed by means of the hydrogen breath test, in which a defined amount of fructose is ingested and then the amount of hydrogen in the exhaled air is measured. In patients with HFI, however, the intake of fructose carries the risk of a severe hypoglycaemic reaction. Therefore, a molecular genetic test for HFI should always be performed before a fructose load test. Early diagnosis of HFI is particularly important to avoid permanent damage to the liver, kidney and small intestine.
ALDOB mutations
In Europe the most frequent mutants associated with HFI are the amino acid substitutions A149P, A174D, N334K (in Human Gene Mutation Database nomenclature) and the deletion del4E4 in the aldolase B gene. For HFI to manifest, both alleles of an individual’s DNA must be affected by a mutation. In homozygous genotypes, the two alleles contain the same mutation (paternal and maternal inheritance). If the two alleles exhibit different mutations, this is referred to as a compound heterozygous HFI genotype.
Parallel genetic analysis
Molecular genetic determination of the polymorphisms associated with lactose intolerance and HFI enable diagnosis of these genetic conditions with high certainty. The EUROArray Lactose/Fructose Intolerance Direct enables simultaneous detection of the lactose-intolerance-associated polymorphisms ‑13910C/T and ‑22018G/A and the HFI-associated mutations A149P, A174D, N334K and del4E4. Thus, the two genetically caused metabolic disorders can be assessed with a single test.
he test can be performed on whole blood samples, eliminating the need for costly and time-consuming DNA isolation. In the test procedure (Fig. 1), the sections of DNA containing the alleles are first amplified by multiplex PCR using highly specific primers. During this process the PCR products are labelled with a fluorescent dye. The PCR mixture is then incubated with a microarray slide containing immobilized DNA probes. The PCR products hybridize with their complementary probes and are subsequently detected via the emission of fluorescence signals. The data is evaluated fully automatically using EUROArrayScan software (Fig. 2), and in the case of positive results, homozygous and heterozygous states are differentiated. Numerous integrated controls ensure high reliability of results, for example, by verifying that there are no other rare mutations in direct proximity to the tested positions which could interfere with the analysis.
Studies on blood donors
The performance of the EUROArray was investigated using 116 precharacterized samples from blood donors in Germany and from quality assessment schemes. The EUROArray revealed a sensitivity of 100% and a specificity of 100% with respect to the reference molecular genetic method.
Conclusions
Diagnosis of gastrointestinal disorders often involves a long and challenging process of diagnostic tests and restrictive diets. Since lactose and fructose are widely consumed in many diets, it is important to consider intolerance to these sugars during the diagnostic work-up. Simple genetic analysis enables primary lactose intolerance and HFI to be confirmed or excluded as the cause of gut problems. The parallel analysis offered by the EUROArray enables especially fast and effective diagnostics. Patients diagnosed with these genetic conditions can promptly adapt their diets to ease their symptoms. If the analysis is negative, the physician can focus on searching for other causes of the digestive complaints. The molecular genetic analysis thus provides valuable support for the gastroenterology clinic.
The author
Jacqueline Gosink PhD
EUROIMMUN AG, 23560 Lubeck, Germany
Sensitive and precise multiplex assays enable accurate classification and surveillance of tumours
, /in E-News /by 3wmediaby Prof. Godfrey Grech, Dr Stefan Jellbauer and Dr Hilary Graham
Understanding the molecular characteristics of tumour heterogeneity and the dynamics of progression of disease requires the simultaneous measurement of multiple biomarkers. Of interest, in colorectal cancer, clinical decisions are taken on the basis of staging and grade of the tumour, resulting in highly variable clinical outcomes. Molecular classification using sensitive and precise multiplex assays is required. In this article we shall explain the use of innovative methodologies using signal amplification and bead-based technologies as a solution to this unmet clinical need.
Introduction
Cancer is the leading cause of death globally, accounting for 9.6-million deaths in 2018, with 70% of cancer-related mortality occurring in low- and middle-income countries. In 2017, only 26% of low-income countries provided evidence of full diagnostic services in the public sector, contributing to late-stage presentation [1]. There are various aspects that negatively affect the survival rate of patients, including but not limited to:
(a) highly variable clinical outcome mainly due to lack of molecular classification;
(b) treatment of advanced stage of the disease mainly due to lack of, or reluctance to, screening programmes, resulting in treatment of symptomatic disease that is already in advanced stage;
(c) heterogeneity of the tumours that are undetected using representative biopsies of the tumour at primary diagnostics; and
(d) lack of surveillance of patients to detect early progression of disease and metastasis, mainly due to clinically inaccessible tumour tissue and the need of sensitive technologies to measure early metastatic events.
Colorectal cancer (CRC) represents the second most common cause of cancer-related deaths, with tumour metastasis accounting for the majority of cases. To date, treatment decisions in CRC are based on cancer stage and tumour location, resulting in highly variable clinical outcomes. Only recently, a system of consensus molecular subtype (CMS) was proposed based on gene expression profiling of primary CRC samples [2]. Organoid cultures derived from CRC samples were used in various studies to adapt the CMS signature (CMS1–CMS4) to preclinical models, to study heterogeneity and measure response to therapies. Of interest, the epidermal growth factor receptor (EGFR) and receptor tyrosine-protein kinase erbB-2 (HER2) inhibitors were selective and have a strong inhibitory activity on CMS2, indicating that subtyping provides information on potential first-line treatment [3]. In CRC, copy number variations are associated with the adenoma-to-carcinoma progression, metastatic potential and therapy resistance [4]. Our recent studies using primary and matched metastatic tissue showed that TOP2A (encoding DNA topoisomerase II alpha) and CDX2 (encoding caudal type homeobox 2) gene amplifications are associated with disease progression and metastasis to specific secondary sites. Hence, introducing robust and clinically-friendly molecular assays to enable measurement of multiple biomarkers to assess matched resected material and tumour-derived cells or cell vesicles in blood during therapy and beyond, has become a necessity to overcome this deadly toll. In addition, to support diagnostics in remote countries, the assays should allow measurement in low input, low quality tissue material.
To enable precise future diagnosis and patient classification and surveillance, we developed innovative methodologies (Innoplex assays) measuring expression of multiple marker panels representing the primary tumour heterogeneity and the dynamic changes associated with disease progression. We optimized these Molecular Diagnostics Sensitive and precise multiplex assays enable accurate classification and surveillance of tumours April/May 2020 21 | methodologies for multiplex digitalized readout using various sample sources ranging from archival formalin-fixed paraffinembedded (FFPE) tissues and characterization of gene amplifications in blood-derived exosomes. In this article we summarize the Innoplex assays based on the xMAP Luminex Technology and the Invitrogen QuantiGene™ Plex Assay, the research outputs from the University of Malta in terms of the biomarker panels and the commercialization of the assays through Omnigene Medical Technologies Ltd.
Molecular profiling technology and workflow
The Innoplex multiplex assays are based on two components, namely (a) the integration of the Invitrogen QuantiGene™ Plex Assay (Thermo Fisher Scientific) and the xMAP Luminex technology enabling multiplexing of the technique, and (b) the novel panel of biomarkers developed by the Laboratory of Molecular Oncology at the University of Malta, headed by Professor Godfrey Grech. The technologies and the research output provides the versatility of the assays. To date a breast cancer molecular classification panel and a CRC metastatic panel were developed and are currently being optimized for the clinical workflow by Omnigene Medical Technologies Ltd through the miniaturization and automation of the RNA-bead plex assay.
The Innoplex RNA-bead plex assays use the Quantigene branched- DNA technology that runs on the Luminex xMAP technology. Specific probes are conjugated to paramagnetic microspheres (beads) that are internally infused with specific portions of red and infrared fluorophores, used by the Luminex optics (first laser/ detector) to identify the specific beads known to harbour specific probes. The Quantigene branched-DNA technology builds a molecular scaffold on the specifically bound probe-target complex to amplify the signal that is read by a second laser/LED [5].
The workflow of the assay can be divided into a pre-analytical phase involving the lysis/homogenization of the tissue or cells, and the analytical phase that involves hybridization, pre-amplification and signal amplification with a total hands-on time of 2|h. This is comparable to the time required to prepare a 5-plex quantitative real-time (qRT)-PCR reaction. Increased multiplexing within a reaction will result in an increase in hands-on time for qRT-PCR, while the same 2|h are retained for the Innoplex assays. As shown by Scerri et al. [5], qRT-PCR 40-plex reactions will require 9|h to prepare as compared to the bead-based assay which retains a 2|h workflow. Hence, the bead-based assays have the advantage for high-throughput analysis in multiplex format.
Performance and applications
We have shown in previous studies, using breast cancer patient material, that gene expression can be measured using our RNA-based multiplex assays in FFPE patient archival material that was of low quality and low input [6]. Using a 22-plex assay, inter-run regression analysis using RNA extracted from cell lines performed well with an r2>0.99 in our hands. These assays were also evaluated by other groups using snap-frozen and FFPE tissues derived from patient and xenograft samples. In comparison with the reference methods, the bead-based multiplex assays outperformed the qRT-PCR when using FFPE-tissue-derived RNA, giving reliability coefficients of 99.3–100% as compared to 82.4–95% for qPCR results, indicating a lower assay variance [5].
One main advantage of the Innoplex assays is the direct measurement of gene expression on lysed/homogenized tissues and cells, providing a simplified workflow without RNA extraction, cDNA synthesis and target amplification. In addition, due to its chemistry and use of beads, gene expression can be measured in a multiplex format (up to 80 genes) using low input and low quality material. This enables the use of the assay in remote laboratories, and as detailed below for stained microdissected material and to measure multiple markers in low abundance material, such as blood-derived circulating tumours cells.
Comparison of gene expression data from homogenized and lysed patient tissue derived from either unstained or hematoxylin and eosin (H&E)-stained sections shows a high correlation (r2>0.98). This provides an advantage when studying heterogeneous tumours that are microdissected from H&E stained slides. In fact, using this methodology, an estrogen-receptor-positive tumour was analysed and one of the tumour foci had a more advanced tumour expressing the mesenchymal marker, FN1 (fibronectin). This was only possible by running a 40-plex assay on minimal input material (microdissected from 20|μm section) representing markers for molecular classification, epithelial to mesenchymal transition, and proliferation markers [7]. A recent audit on breast cancer diagnosis, indicates clearly that heterogeneous cases characterized using the bead-based multiplex assays on resection tumour samples are not represented in matched biopsies used for patient diagnosis. In fact, only 3.5% of 97 intra-tumour heterogeneous cases were detected in a cohort of 570 patients at diagnosis. The advantage of the digitalized result of the Innoplex assays is to avoid increasing the workload of pathologists when resected samples are re-analysed to characterize multiple sites within a tumour.
Multiplexing provides both sensitivity and versatility in biomarker validation and was instrumental in our hands to measure gene amplifications in cancer-derived exosomes (tumour-derived vesicles in blood) using plasma from CRC patients. Of interest, these methods have been optimized using cancer cell lines to measure RNA transcripts in cells at low abundance, mimicking the isolation of circulating tumour cells from blood [5]. In this study we show that measurement of transcripts of EPCAM (encoding epithelial cell adhesion molecule), KRT19 (encoding keratin, type I cytoskeletal 19), ERBB2 (encoding HER2) and FN1 maintain a linear signal down to 15 cells or less. In addition, the simple workflow with direct measurement using lysed cells enables this assay to be translated more efficiently to the clinical setting. Absolute quantification of transcripts presents alternative endpoint methods to the Invitrogen QuantiGene™ Plex Assay. Droplet digital PCR (dPCR) and Nanostring’s nCounter® technology are precise and sensitive methods. Multiplexing in dPCR is limiting and RNA studies are hindered by reverse transcription inefficiency. The nCounter® technology requires multiple target enrichment (PCR-based pre-amplification) to measure low input RNA, which introduces amplification bias and risk for false positive results.
Summary
In conclusion, the innovative multiplex assays indicate a shift from reactive medicine (treating patients based on average risks) towards predictive, precise and personalized treatment that takes into account heterogeneity of primary tumour, progression of tumour during therapy and the metastatic surveillance of the individual patient. The versatility of the method allows the development of various assays to support different applications (Figs|1 & 2). Our first innovative methods were developed for the molecular classification of luminal and basal breast cancer and to predict sensitivity to specific therapy in triple-negative breast cancer subtype [8]. As discussed above, the multiplex assays have a wide range of possible applications in the diagnosis of tumours and surveillance of tumours during therapy. The main advantages of these methods include:
(a) implementation of high-throughput analysis which has a positive impact on remote testing and implementation of such assays in patient surveillance and clinical trials;
(b) the digitalized result excludes subjectivity and equivocal interpretation, which are common events in image-based measurements, and also eliminates the need for highly specialized facilities and human resources;
(c) accurate and precise detection of multiple targets in one assay, minimizing the use of precious patient samples; and
(d) enables the measurement of gene expression in heterogeneous tumours and low input / low quality patient material. The method is streamlined with the current pathology laboratory practices resulting in a workflow that is cost-effective and with minimal turnaround time.
The authors
Godfrey Grech*1,2 PhD, Stefan Jelbauer3 PhD, Hilary Graham4 PhD
1 Department of Pathology, Faculty of Medicine & Surgery, University of Malta
2 Scientific Division, Omnigene Medical Technologies Ltd, Malta
3 Thermo Fisher Scientific, Carlsbad, CA 92008, United States
4 Licensed Technologies Group, Luminex Corporation, Austin, Texas
*Corresponding author
E-mail: godfrey.grech@um.edu.mt
Acute pancreatitis biomarkers: to many or too few?
, /in E-News /by 3wmediaby Dr Allison B. Chambliss
The diagnosis of acute pancreatitis has long relied on elevations in serum amylase or lipase. Recent test utilization efforts have called f or the discontinuation of amylase in acute pancreatitis, favouring the higher specificity and longer elevation of lipase. However, neither biomarker correlates with disease severity, and early recognition of severe cases remains a diagnostic challenge.
Introduction to acute pancreatitis
Acute pancreatitis (AP) represents one of the most common gastrointestinal-related causes for hospital admissions. AP refers to an inflammatory condition of the pancreas commonly associated with a severe, rapid onset of abdominal pain. Patients may also experience other non-specific symptoms, including fever, tachycardia, nausea and vomiting. AP may be classified as mild, moderate or severe based on the degree of organ failure and systemic complications, a system referred to as the revised Atlanta classification (Table 1) [1].
The most frequent cause of AP is gallstones, which are hardened deposits of bile. Gallstones may account for 40–70% or more of AP cases, depending on the geographic region [2]. Gallstone pancreatitis typically resolves upon spontaneous or endoscopic removal of the stone. Once recovered, gallstone pancreatitis patients typically undergo cholecystectomy, the surgical removal of the gallbladder, to prevent recurrent AP episodes. Alcohol abuse is typically ranked as the second most frequent cause of AP (25–35% of cases), followed by a variety of other rarer causes such as metabolic abnormalities, drugs and toxins, and trauma.
Treatment for most patients involves supportive care, including fluid resuscitation, pain control and monitoring. Although patients with mild disease may recover within a few days without complications, the most severe cases may involve systemic inflammatory response syndrome with the failure of multiple organs, including acute respiratory failure, shock, and/or renal failure. Rapid diagnosis of AP and assessment of risk for disease severity, both of which rely on laboratory testing, are critical to guide patient management. Recurrent episodes of AP may progress to chronic pancreatitis.
Increases in disease prevalence
The annual incidence of AP is estimated at 20–40 per 100¦000 worldwide [3]. Interestingly, the incidence has increased over the past few decades, particularly in Western countries [4]. One study found an increase of 13.2% in AP-related hospital admissions in 2009–2012 compared to 2002–2005 across the USA [5]. Although these epidemiological trends are not entirely understood, several reasons for the overall increasing incidence of AP have been proposed. One hypothesis is the global epidemic of obesity, which may promote gallstone formation. Increases in alcohol consumption could also play a role in some countries. Other experts suggest that the wider availability and increased frequency of laboratory testing may be major factors. This latter concept is in alignment with the fact that although cases in AP have risen, the mortality rate of the disease has, in fact, declined [5]. Nevertheless, mortality remains high in the severe case category.
Biomarkers for AP
Serum amylase and lipase are well-established as the primary biomarkers for the diagnosis of AP. Both amylase and lipase are digestive enzymes; amylase hydrolyses complex carbohydrates to simple sugars, and lipase catalyses the hydrolysis of triglycerides. Although lipase is synthesized predominantly by the pancreas, amylase is produced both by the pancreas (P-type) and the salivary glands (S-type) and is found in several other organs and tissues. Both enzymes are released into the circulation at the onset of AP, and elevations of both are typically observed within 3-6|h [6, 7]. Multiple clinical societies and guidelines recommend a serum amylase or lipase test result greater than three times the upper reference limit as a diagnostic criterion for AP, in addition to characteristic symptoms and imaging findings [2, 8]. Both biomarkers are widely measured by automated enzymatic methods and are thus commonly found in routine hospital laboratories, permitting rapid diagnoses. Notably, most routine assays do not distinguish between P-type and S-type amylase. This distinction requires the analysis of amylase isoenzymes, which is typically limited to reference laboratories.
Questioning the value of amylase
Serum amylase and lipase are well-established as the primary biomarkers for the diagnosis of AP. Both amylase and lipase are digestive enzymes; amylase hydrolyses complex carbohydrates to simple sugars, and lipase catalyses the hydrolysis of triglycerides. Although lipase is synthesized predominantly by the pancreas, amylase is produced both by the pancreas (P-type) and the salivary glands (S-type) and is found in several other organs and tissues. Both enzymes are released into the circulation at the onset of AP, and elevations of both are typically observed within 3-6|h [6, 7]. Multiple clinical societies and guidelines recommend a serum amylase or lipase test result greater than three times the upper reference limit as a diagnostic criterion for AP, in addition to characteristic symptoms and imaging findings [2, 8]. Both biomarkers are widely measured by automated enzymatic methods and are thus commonly found in routine hospital laboratories, permitting rapid diagnoses. Notably, most routine assays do not distinguish between P-type and S-type amylase. This distinction requires the analysis of amylase isoenzymes, which is typically limited to reference laboratories.
Questioning the value of amylase
In contrast to amylase, lipase is reabsorbed by the tubules of the kidney and is not excreted into the urine. Thus, lipase tends to remain elevated for longer than amylase, which may allow for a longer diagnostic window for AP. This advantage, in addition to lipase’s higher specificity for the pancreas, has led some organizations to recommend lipase over amylase for the diagnosis of AP. The American Board of Internal Medicine Foundation’s Choosing Wisely® campaign, in collaboration with the American Society for Clinical Pathology, has recommended: “Do not test for amylase in cases of suspected acute pancreatitis. Instead, test for lipase” [9].
Despite these recommendations, many hospital laboratories still maintain assays for amylase. We performed a retrospective audit at our institution to determine the ordering patterns of amylase relative to lipase in cases of AP. We found that in a cohort of 438 consecutive patients admitted with AP, lipase was ordered for all patients, while amylase was only ordered for 12% of patients [10]. We observed that most of the amylase orders stemmed from patients with gallstone pancreatitis who were referred for laparoscopic cholecystectomy procedures and who were under the care of the surgical team. We speculated that amylase may have been co-ordered with lipase in this subgroup of patients to check for biomarker normalization. Laparoscopic cholecystectomy is ideally to be performed as early as possible when gallstone AP resolves, and normalization of amylase or lipase may be used to document that resolution. Because amylase is believed to fall more rapidly than lipase after AP, trending amylase over time could possibly allow for a quicker documentation of biomarker normalization. However, our study also showed that there was no significant difference in amylase versus lipase in the time for the biomarker to fall below three times the upper reference limit. These observations led us to further question the added value of amylase relative to lipase alone in the diagnosis and management of AP.
Lipase does have limitations that may preclude it from being the AP biomarker of choice in some cases. Lipase may be elevated in non-pancreatic conditions such as renal insufficiency and cholecystitis (Table 2). Both amylase and lipase may rarely be non-specifically elevated due to complexes with immunoglobulins, termed macroamylasemia and macrolipasemia. Further, amylase may be useful in the workup of other pancreatic diseases and, unlike lipase, can be measured in the urine. Quantitation of amylase in body fluids, such as pancreatic fluid and peritoneal fluid, can aid in the evaluation of pancreatic cysts and pancreatic ascites [11]. For these reasons, many laboratories choose to maintain amylase assays.
An unmet need for biomarkers for AP severity
Although AP may be easily diagnosed with elevations in amylase or lipase, there is an unmet need for biomarkers or algorithms that can specifically identify severe forms of AP early in the disease course. Twenty to thirty percent of AP patients may develop a moderate or severe form of the disease involving single or multiple organ dysfunction or failure and requiring intensive care. Identifying the severe cases early such that treatment may be tailored to minimize complications remains one of the major challenges of AP. Risk factors such as old age and obesity often correlate with disease severity. However, neither amylase nor lipase levels correlate with disease severity, and no other laboratory tests are consistently accurate to predict severity in patients with AP.
In 2019, the World Society of Emergency Surgery (WSES) published guidelines for the management of severe AP [12]. These guidelines indicate that C-reactive protein (CRP), an acute phase reactant synthesized by the liver and a non-specific indicator of inflammation, may have a role as a prognostic factor for severe AP. However, CRP may not reach peak levels for 48 to 72|h, limiting it as an early severity indicator. Specifically, WSES recommended that a CRP result greater than or equal to 150|mg/L on the third day after AP onset could be used as a prognostic factor for severe disease. Elevated or rising blood urea nitrogen, hematocrit, lactate dehydrogenase, and procalcitonin have also demonstrated predictive value for pancreatic necrosis infections.
Other biomarkers have been investigated to distinguish mild from non-mild forms of AP. Interleukin-6 has shown good discriminatory capability in combination with CRP [13]. Resistin is a more recently discovered peptide hormone that was first described as a contributor to insulin resistance (hence the name). Resistin is secreted by adipocytes and may play a role in obesity, hypertriglyceridemia, and inflammatory cytokine reactions. A prospective observational study found that resistin levels were better than CRP for predicting severe AP on the third day and for predicting the development of necrosis [14]. However, more studies are needed before resistin can be recommended as a prognostic indicator, and clinical resistin testing is not widely available. Thus, there still remains a need for prognostic severity biomarkers that rise early (prior to 48|h) in the course of AP.
The authors
Allison B. Chambliss PhD, DABCC
Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA
E-mail: abchambl@usc.edu