Abu Dhabi, 26 January 2017 - National Reference Laboratory (NRL), part of Mubadala’s network of world-class healthcare providers and managed by LabCorp^®, a world leading life sciences company, providing comprehensive clinical laboratory services, are demonstrating their commitment to continuing medical education for laboratory and other healthcare professionals by announcing their participation in a number of leading healthcare events in the first half of 2017, including two of the largest healthcare events in the world, Arab Health and Medlab.

Approximately 70 percent of medical decisions are influenced by laboratory tests, ordered to diagnose, treat, manage, and monitor a patient’s condition, requiring high levels of education and knowledge in order to ensure high quality of services, improved patients outcomes and reducing the unnecessary costs in the industry.

“In a region that faces unique healthcare challenges and where laboratory testing is a crucial plank of healthcare outcomes, the continuing education of current and future healthcare professionals in the science of laboratory medicine is a core theme of our company’s mission”, said Abdul Hamid Oubeisi, CEO of NRL. “In 2016 alone, we organized seven seminars for an impressive 400 delegates, with our medical and scientific team very active in speaking at conferences across the region in a bid to share their scientific findings, experiences and best practices in laboratory medicine, providing valuable knowledge for the attendees.”

“The continuing innovation and advancements in laboratory testing and technology are remarkable and it is our mission and duty to educate the laboratory professionals, physicians and patients about these exciting changes.  These advancements will only serve to facilitate faster diagnostic and more precise treatment decisions, which should ultimately increase the quality of care for our patients. As a partner of LabCorp, the world’s leading healthcare diagnostics company, and having one of the largest teams of locally-present laboratory experts, NRL is in the best position to assume its role as a thought leader in the region”, said Dr. Basel Altrabulsi, Chief Medical Officer of NRL.

As the largest College of American Pathologists (CAP)-accredited referral laboratory network in the Middle East, NRL is a thought leader in this field, and stressing the importance of accreditation and international standards in medical laboratory services is something NRL’s team continually communicate to the wider healthcare community. During 2016, the quality assurance team of NRL has successfully completed twelve accreditation inspections across the network of NRL laboratories, including nine first time accreditations, which stands as a remarkable and a unique accomplishment in the region. For the first time in the region, on 7 February, NRL will organize an educational event with CLSI, the leader in the development and implementation of clinical laboratory testing standards worldwide. This joint effort will further strengthen the capacities of NRL to deliver best in class, continuing medical education for laboratory professionals in the region.

NRL’s Director of Quality Assurance, Faisal Ibrahim, is giving a talk on ‘Managing Quality and Accreditation in a Large Laboratory Network’ during the first day of the Medlab Congress, running from 6-9 February in Dubai. Laboratory Accreditation will be the topic of one of the workshops  hosted by NRL at Medlab on 8 February, which will include other eminent experts in the field, such as Paul Stennett – CEO of UKAS (UK), Glen Fine, CEO of Clinical and Laboratories Standards Institute (CLSI) (USA), Dr. Maadh Aldouri, Director of International affairs at the UK’s Royal College of Pathologists (UK) and Dr. Laila Abdel Wareth – Chief of Clinical Pathology and Laboratory Medicine Institute – Cleveland Clinics Abu Dhabi (UAE).

During Medlab, on 9 February, NRL, in cooperation with their partner LabCorp from the USA, is hosting a workshop of Coagulation Reference Testing, led by Michael Taylor, M.S., Associate Vice President of Laboratory Corporation of America - Colorado Coagulation. Special Coagulation is one of NRL’s Centers of Excellence that the company is working to establish during 2017.

Dr. Basel Altrabulsi, NRL’s Chief Medical Officer, will also be speaking at the Medlab Conference, giving a lecture on ‘Managing the anatomic pathology laboratory’ and ‘Update on molecular tests and their value in evaluating challenging lesions of thyroid FNAs; Dr. Frank Ryan, Technical Director will talk about ‘Laboratory screening and diagnosis of gestational diabetes mellitus’; Gagan Goel, IT Director will educate visitors on ‘Standardization of lab compendium and its management to obtain Computable Semantic Interoperability’; and Dr. Shereen Atef, Consultant Clinical Pathologist will be discussing ‘The evolving use of serum free light chain assays in clinical laboratory.’

For more information go to: http://www.medlabme.com/

Other events attended by NRL experts:

-       OBS/GYNE Exhibition & Conference – Dubai, 19 - 21 April 2017: http://www.obs-gyne.com/

Dr. Basel Altrabulsi, Chief Medical Officer

April 20TH 2017, Tumor Board – Case Discussion: Endometrial Cancers

ENDS

About National Reference Laboratory

National Reference Laboratory is a Mubadala Company created in partnership with and managed by Laboratory Corporation of America® Holdings (LabCorp®), a world leading life sciences company, providing comprehensive clinical laboratory services.

National Reference Laboratory’s vision is to increase the spectrum, coverage and overall efficiency of laboratory testing, to implement international best practice reference laboratory processes and to set the benchmark for quality standards in the region. Together with the significant resources of LabCorp, National Reference Laboratory offers a comprehensive menu of more than 4,700 tests, providing a complete solution for all clinical testing needs in an efficient and high-quality environment that reduces both turnaround time and logistics-related costs, compared with other laboratories. Through its commitment to a comprehensive Quality Management System (QMS), investment in state-of-the-art technologies, superior connectivity solutions, advanced logistics systems and the engagement of highly-skilled and experienced employees National Reference Laboratory has attained and maintained a position as a trusted resource for all healthcare providers and patients in the region.

NRL owns two laboratories, one in Abu Dhabi and one in Dubai and manages the laboratory of Healthpoint in Abu Dhabi as well as the laboratories of all Imperial College London Diabetes Centre’s branches in Abu Dhabi and Al Ain. In partnership with Cleveland Clinic Abu Dhabi (CCAD), NRL jointly manages the Anatomic Pathology laboratory and is responsible for all CCAD laboratory referral testing.

NRL’s network recently expanded to include two additional laboratories, each located on our client’s premises and managed by NRL: Etihad Airways Medical Centre (Abu Dhabi) and Valiant Clinic (Dubai), a premium outpatient clinic managed and operated by Houston Methodist Global Health Care Services and brought to Dubai by Meraas. This brings the total number of laboratories managed by NRL to nine.

For more info, please visit www.nrl.ae. 

About LabCorp

Laboratory Corporation of America® Holdings, an S&P 500 company, is the world’s leading healthcare diagnostics company, providing comprehensive clinical laboratory services through LabCorp Diagnostics, and end-to-end drug development support through Covance Drug Development. LabCorp is a pioneer in commercializing new diagnostic technologies and is improving people’s health by delivering the combination of world-class diagnostics, drug development and knowledge services. With combined revenue pro forma for the acquisition of Covance in excess of $8.5 billion in 2015 and more than 50,000 employees in over 60 countries, LabCorp offers innovative solutions to healthcare stakeholders. LabCorp clients include physicians, patients and consumers, biopharmaceutical companies, government agencies, managed care organizations, hospitals, and clinical labs. To learn more about Covance Drug Development, visit www.covance.com. To learn more about LabCorp and LabCorp Diagnostics, visit www.labcorp.com.

Arboviruses, or arthropod-borne viruses, are on the march globally. Increased urbanisation and international travel facilitate the spread of mosquito vectors and hence the viral diseases they carry. Zika virus (ZIKV) is currently spreading uncontrollably in the Americas, while dengue virus (DENV) and chikungunya virus (CHIKV) have already become firmly established in most tropical and also many non-tropical regions.

ZIKV, DENV and CHIKV infections are difficult to tell apart, as they manifest with similar clinical symptoms of fever, exanthema and arthralgia and are epidemic in much the same geographic regions. Therefore, laboratory analyses play an important role in differential diagnostics. Serological tests provide a longer diagnostic window than other methods such as direct detection, and are suitable for diagnosing acute infections as well as for disease surveillance. ELISA and indirect immunofluorescence test (IIFT) systems based on optimised antigens enable sensitive and specific detection of anti-ZIKV, DENV and CHIKV antibodies in patient serum or plasma samples.

Mosquito Vectors

Among the most important arthropod disease vectors in tropical regions are the species Aedes aegypti (yellow fever mosquito) and Aedes albopticus (tiger mosquito). These mosquitoes are active day and night and thrive in urban areas, making it difficult for humans to avoid bites. Their larvae multiply in open water reservoirs such as wells, cisterns and cloacae, as well as in small containers where rainwater collects.

Viruses spread by Aedes mosquitoes include members of the family of togaviruses and the family of flaviviruses. Due to their dramatic spread, ZIKV, DENV and CHIKV are among the most significant arboviruses. They are responsible for outbreaks of febrile infectious disease in Asia, Africa and the Americas, and it is feared that they could spread to other regions, including Europe.Aedes albopictus has already established itself in more than twelve southern and central European countries. Arboviruses are also reported to be endemic in the MENA region, although their epidemiology is not well characterised.

There are no antiviral therapeutics for these sometimes life-threatening diseases, and patients with complications have to undergo intensive medical treatment. A vaccine against DENV is in development, but vaccines against ZIKV and CHIKV are unlikely in the near future. The current most effective prophylaxis is individual protection from mosquito bites.

Zika Virus

ZIKV is a member of the family of flaviviruses and was first discovered in the Zika forest in Uganda in 1947. Until 2015 Zika fever was considered an obscure tropical disease, with sporadic outbreaks in countries in Africa and Asia and more recently on Pacific islands. In March 2015 first infections were reported in Brazil, and the virus has since spread rapidly throughout South America, Central America and the Caribbean.

In most cases the disease symptoms are mild. After an incubation period of five to 10 days, a flu-like illness develops with fever, rash, arthralgia, myalgia, headache and conjunctivitis. Zika virus infection has been linked to complications including congenital malformations, in particular microcephaly, and neurological conditions such as Guillain-Barré syndrome.

Chikungunya Virus

CHIKV belongs to the family of togaviruses. Chikungunya fever was first reported in Tanzania in 1952. The virus is now present in over 60 countries in Asia, Africa, Europe and the Americas.

The term chikungunya derives from the Makonde language and means “to become contorted”, referring to the severe joint and muscle pains which occur in 70% to 99% of cases and result in a stooped posture. A rapidly rising high fever and further symptoms such as lymph node swelling, rash, punctual bleeding of the skin (petechia), bleeding of the nose or gums, headache, fatigue and inflammation of the eyes also occur.

Chikungunya fever subsides after around 10 days, generally without any lasting damage. In approximately 10% of patients the joint pains persist for more than three weeks or even months and years. In some cases complications such as accompanying hepatitis, meningitis, encephalitis or meningoencephalitis occur.

Dengue Viruses

DENV belong to the flavivirus family and comprise four different serotypes (DENV 1 to DENV 4). DENV are distributed in Latin America, Central Africa, India, South East Asia, in some parts of the Pacific islands and the Eastern Mediterranean. The virus is also regularly introduced to Europe. According to estimates of the WHO, there are around 50-100 million cases of dengue fever every year.

The disease initially manifests with flu-like symptoms such as high fever, severe headache, muscle and joint pains, exanthema and lymph node swelling. Severe complications in the form of dengue haemorrhagic fever or dengue shock syndrome occur in up to 1% of patients. The risk of haemorrhagic fever significantly increases after a second infection with a different DENV serotype. Severe dengue results in about half a million hospitalisations per year and around 20,000 deaths, many of them in children.

Acute Diagnostics

The most important laboratory methods for acute arboviral diagnostics are direct virus detection, serological tests and, for DENV, detection of the highly specific early antigen NS1. Direct detection by RT-PCR provides reliable identification of the infecting virus, but due to the short viraemic phase it is only effective within the first week after onset of symptoms in ZIKV, DENV and CHIKV infections (Figure 1). Thus, RT-PCR may already be negative by the time a patient consults a doctor. DENV NS1 antigen is detectable at the onset of clinical symptoms in both first and re-infections with DENV and remains detectable past the viraemic phase. NS1 detection serves as a first-line screening test for dengue and helps to minimise the diagnostic gap between RT-PCR and antibody positivity.

Serological methods are effective from soon after clinical onset to beyond convalescence. Antibodies against ZIKV, CHIKV and DENV appear around day four to seven after symptom onset. Acute infections are generally characterised by the occurrence of IgM, with IgG appearing at the same time or shortly thereafter. IgM antibodies reach their peak after two to three weeks and remain detectable for a few months, while IgG antibodies persist life-long. The detection of specific IgM antibodies or a significant rise in the specific IgG titer in a pair of samples taken seven to 10 days apart is evidence of an acute infection. Isolated positive IgG findings may indicate past contact with the virus. In secondary flavivirus infection, including DENV infection with a different serotype, IgM antibodies may be delayed, of reduced intensity or not detectable at all. In these cases a more than tenfold increase in the IgG concentration is observed.

Serological Applications

In addition to their application in acute diagnostics, serological methods are also useful for studying the long-term consequences of infection. For example, ZIKV serological investigations may help to establish if the dramatic rise in cases of microcephaly and Guillain-Barré syndrome observed in Zika-affected regions is a consequence of Zika virus infection. If the link to congenital malformations is confirmed, Zika virus serology could play an important future role in prenatal diagnostics. Pregnant women with serological evidence of an infection could be offered intense prenatal monitoring, while seronegative women may be spared unnecessary worry.

Serology is also useful for screening donated blood. Travellers returning from ZIKV, DENV and CHIKV endemic regions to non-affected countries should defer donation of blood for a few weeks or be confirmed as negative by PCR, depending on the respective organisation’s recommendations. After this time, serological testing can verify the safety of donated blood products.

A further, critical role for serological studies is to monitor the growing epidemiological reach of arboviruses. As ZIKV, DENV and CHIKV are expected to continue to spread around the globe, knowledge about emerging endemic regions is valuable for providing effective patient diagnostics.

ELISA

ELISAs provide fully automated measurement of antibody titers, and are a cost-effective method for screening large numbers of samples. They can be used to determined IgM or IgG antibodies.

Newly developed Anti-ZIKV ELISAs are based on a recombinant non-structural viral protein (NS1) from Zika virus. This highly specific antigen avoids cross-reactivity with other flaviviruses. In a first study with these tests, the IgM and IgG ELISAs showed 100% specificity in clinically and serologically characterised samples. Furthermore, the combination of IgM and IgG ELISAs ensures highest sensitivity of 97%. Data from well-characterised serum panels indicate that there is no cross-reactivity with flaviviruses like dengue, West Nile, yellow fever, tick-borne encephalitis and Japanese encephalitis viruses.

Anti-DENV ELISAs are based on highly purified virus particles of serotype 2. Due to the high structural similarity between DENV 1 to 4, use of one serotype is sufficient to detect antibodies against all four virus types. In clinically characterised sera the IgM and IgG ELISAs demonstrated 100% sensitivity and 99% specificity. They also showed very good correlation with other serological assays. Due to use of whole antigen, cross reactions with other flavivirus antibodies cannot, however, be excluded.

Anti-CHIKV ELISAs utilise a virus-specific structural protein as the antigenic substrate. In clinically characterised samples the IgM ELISA showed 100% sensitivity for detecting acute infections. In a published comparative study the Anti-CHIKV IgM and IgG ELISAs demonstrated the highest sensitivities of all diagnostic tests used, as well as high specificities. Further studies have confirmed the excellent overall agreement of the ELISAs with other serological assays.

Indirect Immunofluorescence

Antibody detection by IIFT is based on cells infected with the corresponding virus, which provide highly sensitive diagnostics. Positive and negative results are evaluated by fluorescence microscopy (Figure 2). In clinically characterised samples the IIFT substrates yielded sensitivities of 96% to 99% and specificities of 95% to 100% for the different parameters.

The Arboviral Fever Mosaic 2 consists of a combination of six substrates of cells infected with ZIKV, CHIKV and DENV serotypes 1 to 4, which are incubated in parallel. The mosaic can help in differential diagnostic delimitation of ZIKV, DENV and CHIKV infections. Due to the use of whole virus particles, cross-reactivity between antibodies against different flaviviruses, originating either from infections or vaccinations such as yellow fever, should be taken into account. Whilst there is generally no or only low-grade cross reactivity in a primary flavivirus infection, in a secondary flavivirus infection, for example a ZIKV infection following a DENV infection or vice versa, high-grade cross-reactivity is typical. The cross-reactivity is stronger for IgG than for IgM antibodies. Titration of the patient sample on this mosaic slide may enable determination of a dominant end-point titer for the virus causing the infection.

Perspectives

The dramatic global rise of ZIKV, DENV and CHIKV has placed billions of inhabitants and travellers at risk of infection. Moreover, it is anticipated that these febrile diseases will continue to spread into previously unaffected areas. Current strategies for managing outbreaks are focused on controlling local mosquito populations and avoiding personal exposure to bites. Vaccine research is also a priority. For persons who nevertheless become infected, efficient differential diagnostics by means of laboratory methods are indispensable. Molecular testing and serology form the backbone of diagnostic strategies. Serological analyses are, furthermore, crucial for monitoring the epidemiological reach and the long-term consequences of these diseases. The anti-ZIKV tests described here are the first commercially available assays for serological detection of anti-ZIKV antibodies. It is hoped that along with established anti-DENV and anti-CHIKV assays, they will help to combat these increasingly threatening diseases.

Allergies are a major health and socioeconomic burden worldwide, but especially in industrialised nations. For example, more than 40% of the population in Europe now suffers from at least one form of allergy. About 70% of these allergic patients are polysensitised. Children are frequently affected by atopic dermatitis, allergic rhinitis and allergic asthma. Precise in vitro diagnostics complement conventional diagnostics and are essential for optimal patient management and efficient treatment. Multiplex systems streamline the procedure by delivering a comprehensive and detailed patient profile in a single test. In particular, immunoblots containing optimised combinations of relevant allergens provide efficient multiparameter detection of specific IgE antibodies in different indications

Molecular Allergology: From Extracts to Components

Molecular allergology is a state-of-the-art approach to allergy diagnostics, whereby defined single allergen components are used for detection of specific IgE in place of traditionally used allergen extracts (Figure 1). The molecular components are highly purified proteins, which are either isolated directly from the allergen source or produced recombinantly. They provide a higher level of standardisation than allergen extracts and enable highly differentiated diagnostics. Molecular allergology systems are a powerful diagnostic tool as they can pinpoint the precise trigger of the allergy, thus facilitating risk assessment and therapy decisions. 

Identification of Cross Reactions

Before embarking on therapy it is critical to establish the exact trigger of the allergy. However, it is common for patients to show multiple reactions in clinical tests such as skin prick tests and in extract-based antibody assays. These may be due to an actual polysensitisation or a monosensitisation with cross reactions. The latter involves an initial reaction against a single source, but the IgE antibodies can also bind to structurally related allergens from other sources, potentially inducing an immune response and a positive in vitro test result. Analysis of the individual components of the allergen sources and the most important panallergens enables the precise trigger of the allergy symptoms to be determined quickly and accurately. 

For example, a pollen-allergic patient who has a confirmed sensitisation to Bet v1 from birch pollen will probably also react to the homologue Cor a1 from hazel pollen. But a Bet v1-induced cross reaction with grasses is unlikely, since there is no Bet v1 homologue in grass pollens. If a birch-pollen allergic patient nevertheless reacts to grasses, this could in turn be because birch and grass pollens also contain ubiquitous allergens (panallergens) which can also lead to cross reactions, for example Bet v4 from birch and Phl p7 from timothy grass (Figure 2). Bet v1 homologues are, moreover, found not just in tree pollen but also in foods. Thus, Bet v1-specific antibodies can also cross react with Ara h8 from peanut. If this causes symptoms it is known as a birch pollen-associated food allergy.

Selection of Suitable Therapy

Depending on the trigger, allergies can be treated by avoidance of the allergen, if possible, or by specific immunotherapy (SIT). SIT has a high chance of success when the patient is primarily sensitised to the major components of the allergen extract, so called if more than 50% of patients sensitised to the allergen source react to these components. Only molecular allergy diagnostics can deliver this in-depth information. The optimal treatment can then be selected, and the patient is spared the stress of unnecessary allergen avoidance or ineffective SIT. 

Risk Assessment and Risk Management

Components from different protein families elicit symptoms of varying severity. Thus, molecular profiling can establish if a patient has a low or high risk of severe systemic reactions such as anaphylactic shock. Patients at risk of life-threatening reactions can then be advised on avoidance of the allergen and on appropriate measures to take in an emergency situation. For example, if a patient is sensitised to allergens from the family of profilins, then mild symptoms are generally to be expected. In contrast, patients who are sensitised to allergens belonging to the family of storage proteins have a high risk of systemic reactions.

Furthermore, families of proteins differ in their heat stability, which plays an important role in food allergies. Heat-labile allergen components (profilins, PR10 proteins) in foods are generally denatured by cooking processes, thus reducing the risk of a reaction.

Molecular Allergy Diagnostics of Childhood Allergies

Sensitisations to peanut, milk and egg are the most common allergies in childhood. Peanut allergy in particular can have serious consequences, often triggering anaphylaxis. Accurate diagnosis of allergies in infants and children is important for assessing the risk of systemic reactions, evaluating the chances of tolerance induction and establishing the necessity of dietary restriction. A paediatric profile (defined partial allergen diagnostics, DPA-Dx, Figure 3) containing the most important allergen components from these three sources provides fast and efficient screening of these sensitisations and is the optimal diagnostic tool to supplement the results from anamnesis and skin testing.

The characteristics and impact of the allergen components of the paediatric profile are explained briefly in the following sections.

Egg: In hen’s egg allergy, Gal d1 (ovomucoid) is the main allergen and serves as an indicator for the severity of the allergic reaction. Sensitisations to the heat-sensitive components Gal d2 (ovalbumin), Gal d3 (conalbumin) and Gal d4 (lysozyme) are associated only with consumption of raw or slightly cooked eggs. However, ovalbumin is used in vaccines and lysozyme is used as a preserving agent, so patients with these sensitisations may also exhibit reactions to pharmaceutical or food products containing the corresponding component.

Milk: A reaction to Bos d8 (casein) indicates a strong allergy to milk and milk products. Casein is frequently used as an additive, thus a sensitisation can also cause intolerance to a wide variety of foods such as chocolates or potato chips. The components Bos d (lactoferrin), Bos d4, Bos d5 and Bos d6 are heat sensitive, and sensitisations to them are mainly associated with reactions to fresh milk. Antibodies against Bos d6 (bovine serum albumin) may in addition cause a reaction to beef.

Peanut: The peanut section of the profile can distinguish a primary peanut sensitisation from a cross reaction with birch pollen and establish the risk for the patient. IgE antibodies against the seed storage proteins Ara h1, Ara h2 and Ara h3 and the lipid transfer protein Ara h9 carry a high risk of a systemic reaction. The severity of the allergy is, moreover, greater when multiple high-risk components are involved. A reaction with Bet v1 on the other hand indicates a cross reaction from a birch pollen allergy.

Case Report Peanut Allergy: The Same, But Different

Two patients individually attend an allergologist with unspecific symptoms, namely prickling in the mouth, eczema, nausea and rhinoconjunctivitis. After detailed anamnesis the allergologist performs a screening test for IgE antibodies against food allergens. A sensitisation to peanut is diagnosed in both patients. In order to assess the risk of a severe systemic reaction and anaphylactic shock, molecular allergy diagnostics are used for the next step, for example using the paediatrics profile (Figure 4).

Patient 1 has no reaction to the peanut-specific allergen components Ara h1, h2, h3 and h9, but is positive for Bet v1 of birch pollen (Figure 4 A). This patient has a primary sensitisation to Bet v1 from birch, and therefore most likely has a birch pollen-associated food allergy due to a cross reaction. Since the patient does not have a true peanut allergy, the risk of life-threatening reactions is low and a strict peanut-free diet is not absolutely necessary. If the birch pollen allergy is a burden for the patient, the recommended therapy would be SIT against birch pollen, which has a high probability of success since the patient is sensitised to the major allergen Bet v1. Since the peanut symptoms are due to a cross reaction, it is likely that the pollen-associated food allergy will also be alleviated by the SIT. 

Patient 2 has positive reactions to the peanut-specific allergen components Ara h1, h2, h3 and h9, but is negative for Bet v1 of birch pollen (Figure 4 B). The patient thus has a true peanut-associated food allergy with a high risk of a systemic reaction, since the sensitisation is directed against several storage proteins. Even traces of peanut could trigger a severe systemic reaction. The patient must therefore strictly avoid the allergen source and should always carry an emergency set.

For patients such as these, only molecular allergy diagnostics are able to pinpoint the exact trigger of the allergic symptoms and clarify the associated risk.

Optimal Strategy

Multiplex molecular allergy tests are an indispensable tool for deciding on the optimal strategy for patient management. The tests allow differentiation between cross reactions and multiple sensitisations, which is particularly important for advising patients on the risk of severe allergic reactions. Precise identification of the allergy trigger, moreover, allows selection of patients for SIT who are most likely to benefit from it, thus improving prognosis. Targeted therapy can spare patients the burden of multiple therapies or unnecessary lifestyle changes involved in allergen avoidance. Thus, molecular allergology provides the bedrock for improved patient care and a better quality of life for allergy sufferers.

In a recent news article, researchers reported the discovery of a drug-resistant “superbug” for the first time in the United States. The bacterium has genetic changes that made it resistant to a last-ditch antibiotic called colistin. The biggest fear is when this resistant gene gets transmitted from one species of bacteria to another. Increasingly, worldwide emergence of antimicrobial resistance has become a threat to patient safety in the healthcare settings, both locally and globally. The increasing prevalence of multi-drug resistant organisms resulted in the loss of therapeutic efficacy for the antibiotics that were previously thought to be effective. Rising rates of infection due to multi-drug resistant organisms (MDROs) not only limit therapeutic options, but it also has substantial impact on patients and cost of care. More recently, the World Health Organization (WHO) reported that resistance to common bacteria has reached alarming levels in many parts of the world and member states are urged to coordinate and strengthen efforts towards global antimicrobial surveillance. In order to tackle the situation, multi-pronged approach has been suggested – integration of Antimicrobial Stewardship Program, rapid diagnostics, education, infection control policies, surveillance and policies for antibiotic use in farming.

Antimicrobial Stewardship Program (ASP)

Globally, effective ASP has shown to reduce antibiotic use by 20 – 40%, decreased the incidence of healthcare associated infections such as Clostridium difficile, Methicillin-resistant Staphylococcus aureus (MRSA) et cetera, reduced cost of care and improved overall patients’ outcomes. In a small nation like Singapore, ASP was implemented in 2011 across various public hospitals. Singapore General Hospital (SGH), the nation’s largest acute–tertiary care hospital with a capacity of 1,700 beds, holds the largest ASP team in Singapore. The team comprises of 15 infectious diseases (ID) trained clinical pharmacists, an infection control director, ID physicians as well as microbiologists. Hospital–wide audit of patients initiated on broad spectrum intravenous antibiotics were subjected to a two–stage prospective audit with immediate and concurrent feedback (Figure 1). A recent study conducted locally also revealed that ASP interventions were safe and associated with significant reduction in length of hospital stay and infection-related re-admissions. Diagnostic (viral or bacterial cause) or prognostic (life-threatening or self-limiting infection) uncertainty makes it difficult for physicians to know exactly when to provide and when to withhold antibiotic treatment. More often than not, broad spectrum antibiotics or combinations of antibiotics are prescribed upfront with the mentality of “better be safe than sorry”. This common mindset of physicians is not unfounded, as studies have demonstrated that in patients with septic shock, every hour of delay in appropriate antibiotics (in the first six hours) have led to a 7.6% reduction in survival. As the ASP team and prescribing physicians depend on information and guidance from the clinical microbiology laboratory to optimise therapy, rapid diagnostics is a vital component to patient care and success of ASPs.

Role of Rapid Diagnostics and ASP

Rapid microbiological tests allows for timely antimicrobial optimisation and this in turn led to decreased mortality, shortened hospital stay and lower hospitalisation costs.Standard techniques for identification of organisms are based on phenotypic methods, which require at least 48 hours for final results, as compared to rapid diagnostic tests, which provide final results within hours of growth (Figure 2). Various rapid diagnostic tests include – polymerase chain reaction (PCR) assays, multiplex PCR, nanoparticle probe technology (nucleic acid extraction and PCR amplification) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). With rapid diagnostics, ASP can play an important role as the ‘middle man’ between clinical microbiology laboratory and prescribing physicians. Microbiological results are conveyed to the physicians via the ASP to ensure accurate interpretation and selection of appropriate antibiotics. Furthermore, ASP also works hand in hand with clinical microbiologists to review microbiological reporting procedures, especially with the introduction of new diagnostic test. The ASP team reviews patients on a daily basis and is also part of the multi-disciplinary team doing daily ward rounds. As such, the ASP team is able to provide feedback on how the reported data will likely be interpreted and allow for selective reporting of microbiological results. In various case reports, selective reporting has demonstrated a significant improvement in the appropriateness of antibiotic prescription and majority of physicians involved in the studies have also reported that selective reporting made their antibiotic choice easier. In addition, the ASP team is pivotal in timely communication of microbiological results to physicians to guide them on the selection of appropriate antibiotics – antibiotic chosen must be able to penetrate to the site of infection sufficiently, and the ASP team can also recommend the right dose, right route of administration and required monitoring parameters.

Surveillance

Clinical microbiology laboratories worldwide conduct surveillance on local antimicrobial resistance trends among microbial pathogens. Cumulating antimicrobial resistance data aids in the development of institution–specific antibiogram. Antibiograms are critical in the development of institutional antibiotic guidelines, to guide physicians on prescribing the appropriate empiric antibiotic therapy that is broad enough to cover for the likely causative pathogens. Henceforth, in SGH, the ASP team works closely with the clinical microbiologists to develop antibiotic guidelines. Additionally, the ASP team audits the use of antibiotics to reduce the unnecessarily broad empiric therapy used. Local antibiograms also provide important information to decide if an antimicrobial should or should not be included in the institution’s formulary.

Moving forward

The blanket use of antibiotics plays a major role in fuelling the selection of resistant organisms. To combat rising antimicrobial resistance, effective communication between the ASP team, clinical microbiologists and physicians is essential. The ASP team serves as important messengers to educate physicians on the need to change their inappropriate mindset that ‘bigger is always better’. Overprescribing antibiotics comes with great implications that may not be apparent but growing evidence has demonstrated otherwise. The effort to combat antimicrobial resistance is not a lone effort, it requires the collaboration of all players – ASP team, microbiologist and prescribing physicians. It is important to remember that laboratory medicine is not cumbersome and antibiotics are not quick solutions!

References

 References available on request

Over the recent years there has been a dramatic increase in 25-OH-D requests, prompting many laboratories to consider the use of automated immunoassays. In this article, the two major techniques that are used for measuring vitamin D will be discussed and compared (binding assay and chemical assay techniques).

Dramatic Increase in Vitamin D Testing

Vitamin D was first recognised as a very important component of the diet back in the late 1800s when rickets was initially described. Presently, rickets has been eradicated from most developed countries. However, it is still a very common problem in areas of the world where food is scarce.

The recent dramatic increase in vitamin D testing is primarily due to two causes. First, there has been a marked rise in vitamin D deficiency throughout the world. The second reason is that vitamin D has increasingly being used as general health marker and several diseases were linked to vitamin D deficiency.

Metabolism

What we commonly refer to as vitamin D actually comes in two different forms: vitamin D2 and vitamin D3. Vitamin D2 is also known as ergocalciferol, calciferol, or just vitamin D. Vitamin D3 is also known as cholecalciferol (it derives from cholesterol). There are two main ways via which vitamin D gets into the body: through the skin and through diet. In the intestine, either previtamin D or vitamin D is absorbed and trapped in the chylomicron molecules. In the skin, under the effect of UV rays of sunlight, 7-dehydrocholesterol is converted to cholecalciferol (Vitamin D3). Vitamin D from the two sources is subjected to hydroxylation in the liver to form 25-OH-D.The hydroxylated vitamin is then alpha hydroxylated in the kidney to form 1,25(OH)₂vitamin D, the active form of vitamin D. 1,25 (OH)₂ vitamin D increases the absorption of calcium and phosphorous in the intestine. It also interacts with the parathyroid gland as feedback in the production of parathyroid hormone, therefore acting as a regulator of new bone formation. Vitamin D is also being recognised as a very important player in the signal transduction mechanisms in several organs like the brain, prostate, breast and colon tissue, as well as the immune cells. The cells in these organs have vitamin D receptors and respond to 1, 25 (OH)₂ vitamin D.

In the circulation, vitamin D is transported by the vitamin D - binding protein, which belongs to the albumin and alpha-fetoprotein gene family. The concentration of vitamin D - binding protein in the plasma greatly exceeds that of 25-OH-D (9 nM versus 50 nM), with less than 5% of available binding sites being occupied.

Measurement of 25-OH-D

The analytical measurement of vitamin D is performed for two major reasons: to determine the nutritional status of vitamin D, and to monitor its therapeutic level. As mentioned before, there are two different types of vitamin D. To adequately monitor therapy, we need to be aware of which vitamin D entity is the one measured in the different assays. Specifically, if an immunoassay or protein-binding assay is to be used, is the antibody reacting equally with both types of vitamin D? The answer to this question is that if the intention of measuring vitamin D is to monitor vitamin D2 therapy, then the assay must measure vitamin D2.

The assays currently available in the market (US and EU) can be classified as binding assays and chemical assays. Chemiluminescence immunoassays (CLIA), radioimmunoassay (RIA), and binding protein assay belong to the binding assays group, while chemical assays include high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The specificity and accuracy of these methods are very variable. Both RIA and CLIA are immunoassays in which the accuracy of the method will depend on the specificity of the antibody used (how well the antibody recognizes D2 and D3). The binding assays are affected by the matrix effects due to the tight binding of the vitamin D - binding protein to vitamin D. Currently, automated immunoassays are very popular and practical for the clinical laboratory.

The first automated vitamin D assay was based on Competitive-Protein Binding Assays (CPBA) for the Nicholis Advantage analyzer. It has the advantages of being inexpensive, can be performed on small sample size, and is co-specific for 25-OH-D2 and 25-OH-D3. This assay underestimated 25-OH-D at low levels and overestimated it at high levels. Immunoassay methods were first reported in the 1980s with a radioimmunoassay (RIA). This assay formed the basis for a subsequent chemiluminescent detection – based system. The Radioimmunoassay (RIA) requires a small sample size and the incorporation of Iodine – 125 as a tracer. It is not subjected to nonspecific interference, and in addition to being rapid it is inexpensive and accurate. Nevertheless, it still requires the use of radionuclides, and some RIA assays discriminate between 25-OH-D2 and 25-OH-D3.

Chemical assays have been originally more technically involved but are also now able to accommodate a large number of tests per day. Chemical methods (HPLC and LC-MS/MS) can report vitamin D2 and D3 independently. Ultraviolet quantitation following HPLC is a very stable, repeatable assay, and provides separate quantitation of 25-OH-D2 and 25-OH-D3. Nevertheless, it requires a larger sample size, needs a preparation step before chromatography and sometimes is subject to interferences with other compounds measured in the ultraviolet spectrum. Also, this assay requires a high level of technical expertise.

LC-MS/MS has been referred to as a “Gold Standard” technique for 25-OH-D3, although results can also be erroneous. This technique requires the skills of an experienced analyst. Another caveat with LC-MS/MS is the presence of the 25-OH-D2 and C3 epimers of vitamin D3 in pediatric specimens. If the assay is not optimised, vitamin D2/D3 result may be higher than expected in the pediatric population due to this epimer. Publications have shown that the C3 epimer may be present in adults as well.

Standardization and External Quality Control Assessment:

With the availability of many vitamin D assays, differences in the reported 25-OH-D values for the same samples were observed among different assays.

These differences could impact the classification of patients’ vitamin D status and therefore affect the clinical management of some patients. The question is if it is appropriate to have a clinical decision limit without assay standardization. To address this issue, the Vitamin D Standardization Program (VDSP), an initiative of the National Institutes of Health Office of Dietary Supplements (NIH ODS), was launched in 2010 in collaboration with the National Institutes of Health, the Center for Disease Control and Prevention (CDC), the National Institute for Standards and Technology (NIST), and Ghent University in Belgium.

Following, the CDC has introduced a Vitamin D Standardization-Certification Program to ensure reliable clinical measurement of vitamin D. It was recommended that all assay manufacturers should participate in the CDC’s Standardization-Certification Program. This is especially important for the in-house reference method of the manufacturers and for the assay measurement systems as they are being developed. The primary steps to standardization are as follows (fig.1):

(1) develop a reference system,

(2) establish metrological traceability and

(3) verify “end-user” test performance.

When participants pass four consecutive surveys, they are awarded a certification for one year, which can be renewed annually.

Controversies Regarding Vitamin D Testing

Past: Over the past decade, a big number of studies linking low vitamin D levels to cancer, heart disease, diabetes, and other diseases haveled many doctors to routinely test vitamin D levels for their healthy patients. Consequently, laboratory professionals are confronted with the challenge of helping clinicians navigate the complexities of vitamin D assays.

The current evidence suggest that the main beneficial effects of vitamin D supplementation relate to musculoskeletal, rather than extraskeletal. Moreover, the exponential increase in vitamin D testing and supplements used in the past few years, have raised justifiable concerns if many vitamin D measurements are being undertaken without evidence-supported indications and if many individuals are being supplemented with little evidence of the benefit.

Present: In response to these concerns in 2013, the Royal College of Pathologists of Australasia (RCPA) published a position statement to clarify the role of vitamin D testing in the context of vitamin D deficiency, with guidelines about who should be tested and when repeat testing should be performed. Also, the U.S. Preventive Services Task Force (USPSTF) published a new recommendation in November 2014, which stated that there is no practical reason for most people to get a vitamin D test. Testing for vitamin D might be indicated in patients with osteoporosis or other bone-related problems, conditions that affect absorption of fat, including celiac disease or weight-loss surgery or who are taking medications that interfere with vitamin D activity, including anticonvulsants and glucocorticoids.

References

References are available on request

Since the announcement of the first working draft of the Human Genome on June 26, 2000, then President of the United States, Mr Bill Clinton had declared, “Genome Science will have a real impact on our lives - and even more, on the lives of our children. It will revolutionise the diagnosis, prevention and treatment of most, if not all, human diseases.” Indeed, the diagnosis, prevention and treatment of genetic diseases since then have seen a remarkable progress in the development of personalised medicine, such as the development of imatinib (Gleevec), a tyrosine-kinase inhibitor used to treat multiple cancers, most notably chronic myelogenous leukaemia (CML) in a targeted manner. The drug has doubled the five-year survival rates of CML patients.

So it is surprising that it was just 60 years ago that the correct number of human chromosomes was identified. Working on human embryonic lung fibroblasts and a modified protocol, Tjio and Levan demonstrated that the number of chromosomes in man was 46 when it was previously thought to be 48. However, the metaphase preparations then were solid stained, so that it was not possible to distinguish the chromosomes beyond classifying them into broad chromosome groupings. The banding era followed in the 70’s. In 1971, Caspersson and his colleagues at the Karolinska Institutet, Sweden, elicited the first chromosome banding technique, Q-banding, allowing all human chromosomes to be identified for the first time. The more widely-used G-banding technique, still employed today, followed shortly, and this enabled Janet Rowley in 1973 to recognise that the Philadelphia chromosome in CML was not a deleted chromosome 21 but was a result of a reciprocal translocation between chromosomes nine and 22. This was the first recognised chromosomal aberration associated with a malignancy and it heralded the discovery of many more recurrent rearrangements in cancers.

Karyotyping Analysis

G-banded karyotyping is still by large the gold standard in the evaluation of chromosomal abnormalities. It provides for a genome-wide view of all the chromosomes in the metaphase cell with information on the numerical and structural rearrangements in the clonally abnormal cell. Many of the aberrations have prognostic value, which guides the clinician in patient management, such as the type of treatment to administer. These recurrent rearrangements help stratify patients into favourable, intermediate and poor prognosis groupings and move away from standard chemotherapy regimens toward personalised medicine with superior overall survival and progression-free survival rates.

The technique has severe drawbacks though, such as the need for freshly acquired viable tissues, is tedious and labour intensive, is dependent on mitotically active cells, and suffers from a low abnormality detection rate. Consequently, chromosomal analysis cannot be done on frozen or formalin-fixed paraffin embedded (FFPE) archived samples. Karyotyping necessitates a period of cell culture and this translates to a longer result turnaround time. Even with fresh samples, at times normal haematopoietic cells divide preferentially over tumour cells so that a less than helpful normal result is obtained while the clonally abnormal karyotype is missed. In particular, aberrant plasma cells in plasma cell dyscrasias like multiple myeloma are notoriously indolent in tissue cultures so the detection rate of clonal abnormalities is reportedly around just 30%. Subtle rearrangements like small deletions or amplifications, or translocations involving the terminal regions, can be missed as the chromosomal resolution of haematological samples is at best around 400 bands per haploid set (BPHS) or about 20 Mb while genes are in the order of kb in size.

Fluorescence in Situ Hybridization (FISH)

To circumvent many of these limitations, fluorescence in situ hybridization techniques were introduced to the diagnostic laboratory. The FISH era in clinical applications began in the 1990s and quickly proved its value in eliciting answers to specific questions. Whereas karyotyping requires a period of cell culture, FISH is a rapid and highly specific assay that is independent of cell culture and which can be applied to both metaphase and interphase cells. It has a high resolution of 100kb or less. In the context of multiple myeloma, FISH can reportedly increase the detection rate to 67-78%. This is over and above the aforementioned karyotyping detection rate of 30%. Additional techniques can be employed to increase the detection rate. This may be accomplished by either identifying the plasma cells during FISH analysis utilising cytoplasmic immunoglobulin-FISH (cIg-FISH) for a targeted analysis or purifying the population of plasma cells through the use of magnetic cell-sorting procedures on CD138+ plasma cells. With such techniques, the abnormality detection rate can be dramatically increased to over 90%.

FISH can also interrogate FFPE specimens to detect gene-specific abnormalities. Depending on the probe construction type (dual fusion, break apart, or locus specific), specific known translocations including cryptic rearrangements, copy gains and losses can be elucidated. Patients with adenocarcinoma type non-small cell lung cancer who are anaplastic lymphoma kinase (ALK) gene-rearranged are highly sensitive to therapy with an ALK-targeted small molecule tyrosine kinase inhibitor such as crizotinib. FISH is considered the gold standard method for detecting ALK rearrangements.

Despite its wide range of applications, the FISH technique suffers from being too specific in that only a few probes can be applied to a single assay. This is due to the limited number of fluorophores available and consequently only a few genes can be detected per assay. The consequence is an exponential increase in costs when a number of tests on different genes / loci need to be performed. Moreover, there also needs to be fore-knowledge of the disease type in order to decide on the type of FISH probes to use.

Chromosome Microarray Analysis (CMA)

Clinical applications of microarray technology began in the new millennium with the International Standards for Cytogenomic Arrays (ISCA) Consortium and the American College of Medical Genetics releasing a consensus statement in 2010 that CMA should be a 1^st tier clinical diagnostic test (ahead of karyotyping) for individuals with developmental disabilities or congenital anomalies, including developmental delay, intellectual disability, autism spectral disorders, or multiple congenital abnormalities. This is because CMA combines the genome-wide view of the DNA by karyotyping with the specificity of FISH, enabling the detection of multiple gains and losses of DNA across the entire genome in a single assay, including cryptic ones. It has the added advantage of being totally independent of cell culture or even of viable cells. When single nucleotide polymorphisms (SNPs) are incorporated into the chip, the assay can detect copy-neutral loss of heterozygosity, a common feature in cancer. The technique is however unable to detect balanced rearrangements like reciprocal translocations which are seen frequently in haematological disorders, as well as being unable to detect other chromosomal abnormalities when the abnormal cell population is below 20%.

The cancer consortium equivalent, the Cancer Cytogenomic Microarray Consortium, later renamed the Cancer Genomics Consortium (CGC), had hoped to quickly leverage on the technology and apply it to cancers. Unfortunately, it has taken some time for the medical oncology community to espouse the technique, which until now has been reliant largely upon karyotyping and FISH. In the context of multiple myeloma, CMA performed on an enriched population of CD138+ plasma cells can detect close to 100% of the clonal abnormalities. CMA when combined with karyotyping or FISH becomes a very powerful diagnostic tool. With the recent 2015 CGC publication in Cancer Genetics conclusively demonstrating the clinical utility of CMA on haematological disorders and renal epithelial tumours, perhaps the test might soon find more support from clinicians.

Next Generation Sequencing (NGS)

Hailed as the next breakthrough in genomics testing and a tool for precision medicine, NGS is possibly one of the most significant technological innovations in diagnostic pathology over the past few decades. The technology allows massively parallel sequencing of millions of DNA templates and is thus largely poised to replace the existing Sanger sequencing or PCR-based assays for genetic testing. Compared to Sanger sequencing, NGS has increased the throughput of DNA sequencing by >500,000 times, while at the same time drastically lowering the costs of sequencing. To put into perspective, the Human Genome Project took 13 years to complete at the cost of US$3 billion to sequence the first human genome by Sanger sequencing. Using NGS, sequencing a human genome costs an average of between one to two thousand dollars nowadays and only takes 1-2 days.

Precision Medicine

There is no doubt that the diagnosis and prognosis of cancers will become increasingly more dependent on the genomic profiling of individual tumours, which will then drive precision medicine to further reduce cancer mortality rates. The continual discovery of new biomarkers will open avenues for the development of the next generation of small molecule targeted therapies that proffer maximal therapeutic effect with minimal side effects. As cancer is a genetic disease driven by heritable or somatic mutations, newer DNA sequencing technologies will continue to play a significant role in the detection of driver mutations and consequently, the management and treatment of the disease. Diagnostic laboratories must be quick to adopt such newer techniques in the ever-changing molecular diagnostics landscape.

Angelman syndrome patients often exhibit severe intellectual and developmental disabilities; sleep disturbances, seizures, jerky movements (especially hand-flapping), frequent laughter or smiling, and usually a happy demeanor. Alternatively, Prader-Willi syndrome patients often have low muscle tone, short stature, cognitive disabilities, behavior problems, and a chronic feeling of hunger that can lead to excessive eating and life-threatening obesity.

Even though the symptoms of each disease are strikingly different, the disorders are an outcome of aberrations in the same region. In about 1% of PWS patients and 2–4% of AS patients, the disease is due to aberrant imprinting or gene silencing, or both. Further, some of these patients were found to have a mosaic methylation pattern of the Prader-Willi/Angelman syndrome critical region.

To further understand the mechanism of pathogenesis and phenotype of patients with mosaic methylation pattern, Mayo Clinic researchers studied 10 patients with mosaic methylation pattern tested between June 2007 and June 2013 at the Mayo Clinic Clinical Molecular Genetics and Clinical Cytogenetics Laboratories. All patients were younger, ranging from two to 11 years old. The study was published in Molecular Cytogenetics.

All of the cases, except for two, had normal copy number and heterozygosity of chromosome 15 as detected by chromosomal microarray (CMA). In the other two cases, absence of heterozygosity was identified. According to Umut Aypar, Ph.D., FACMG, Co-Director of the Mayo Clinic Cytogenetics Laboratory and first author on this paper, these cases were of particular interest to the team.

Case 1 was a four-year-old boy with mild developmental delays, mild hand tremors, and clumsiness. CMA testing revealed absence of heterozygosity spanning the entire chromosome 15, suggesting uniparental isodisomy 15.

“Case 1 was unique because even though the CMA testing suggested uniparental isodisomy 15, the patient had no definitive phenotypic features of Prader-Willi or Angelman syndrome,” said Dr. Aypar. “Further, methylation testing showed a mosaic methylation pattern.”

After the test results were received, researchers obtained additional clinical information, which showed that the patient was much less severely affected than expected for Angelman syndrome.

“While the patient had features similar to those of Angelman syndrome, such as essential tremors and clumsiness (ataxia), he had no seizures, dysmorphic features, or obesity. These findings weren’t traditional with previous research,” added Dr. Aypar.

To identify the difference, researchers identified nine additional previously tested patients with a similar mosaic methylation pattern. Researchers utilised CMA to test whether patients with mosaic methylation were more likely to have uniparental disomy of chromosome 15.

“Of the nine patients, only one (case two, which was an 11-year-old girl with developmental delay, speech delay, and hypotonia) had regions of absence of heterozygosity on chromosome 15,” said Dr. Aypar. “However, this patient had numerous other regions of absence of heterozygosity on multiple chromosomes, suggesting consanguinity.”

Based on the results, patients with mosaic methylation patterns don’t always have absence of heterozygosity spanning the entire chromosome 15. They tend to have milder or atypical Angelman syndrome, and the majority have features that resemble Prader-Willi syndrome.

“We recommend that quantitative methylation analysis be performed for cases of atypical Prader-Willi syndrome or Angelman syndrome,” said Dr. Aypar. “It is also important to follow up with methylation testing when whole-chromosome isodisomy is detected.”

 The article was used with permission of Mayo Foundation for Medical Education and Research. All rights reserved.

The discovery of the circulation of blood by William Harvey and subsequent development of procedures to withdraw blood from a patient's vein for therapeutic purposes have enabled physicians to utilise blood to detect and monitor disease. Today, laboratory medicine remains an integral component of patient care. An estimated 60-70% of medical decisions are based on the results from laboratory testing. Therefore, timely receipt of test results may enable more rapid diagnosis and treatment, which can impact patient outcomes. Yet, laboratory test turnaround time (TAT) has been cited as a primary source of dissatisfaction among physicians and nurses. In surveys conducted by the College of American Pathologists involving physicians and nurses from 138 and 182 institutions, respectively, satisfaction with TAT received below average ratings. Many physicians believed that laboratory TAT caused delays in treatment in the Emergency Department (ED) (42.9%) as well as increased the length of stay in the ED more than 50% of the time (61.4%).

As such, improving turnaround time has become a key barometer of laboratory performance and an addition to quality improvement initiatives in hospitals and institutions. To meet these objectives, laboratories may consider utilising plasma instead of serum for clinical chemistry testing.

Why Plasma?

While serum has typically been used for clinical chemistry testing due to the ability to test a wide range of assays, it may compromise the time to test receipt due to the required clotting (generally ranging from 30-60 minutes). The clotting time for patients on anticoagulant therapy may be longer. Serum is also subject to latent fibrin formation when clotting is inadequate or may be present in samples from patients receiving anticoagulant or thrombolytic therapy. Inadequate tube mixing or incomplete or delayed clotting may cause fibrin, which may range from thin strands to large cloud-like masses. It may also contribute to obstruction of the sample probe in automated instruments and subsequent instrument downtime.

Plasma offers distinct advantages over serum. Plasma—the liquid component of blood—contains blood cells and anticoagulant following centrifugation of whole blood. Heparin is the most commonly used anticoagulant in plasma, which acts primarily through a complex that it forms with anti-thrombin III, a protein that helps to control blood clotting. It also prevents the formation of fibrin from fibrinogen.

Conversely, clotting is not required for plasma, enabling plasma to be centrifuged upon receipt of the specimen in the laboratory. Specimens can be processed more quickly, shortening the turnaround time for test results. There is a potentially higher sample volume yield with plasma, with approximately 15-20% more plasma obtainable from whole blood than with serum.This helps laboratories to adhere to ISO standard 15189, in which laboratories should periodically review sample volume requirements to ensure that excessive amounts of blood samples are not collected.

In addition, interference due to coagulation is eliminated, as coagulation post centrifugation does not occur in plasma.There is also a lower risk of haemolysis and thrombocytolysis. In a healthy population, free haemoglobin is about 10 times less concentrated in plasma than in serum.In anticoagulated blood, there is no obstruction to upward gel movement; therefore, the time required for gel to complete its upward course is generally shorter with plasma tubes. This may result in more reproducible gel barrier formation. 

Most significantly, it is imperative that the in vivo state of a constituent remains unchanged after withdrawal from the body fluid of a patient. Constituents in plasma are more accurately representative of the in vivo status of the patient than those in serum.

Assay Compatibility – the “True” In Vivo State

Generally, most assays in clinical chemistry are compatible with both serum and heparin plasma, enabling the same reference ranges to be used. However, for certain assays or test methods, either serum or plasma may not be acceptable or differences in results obtained in plasma specimens may warrant a change in reference range.For instance, glucose concentrations were noted to be 5% higher in serum than plasma as a result of fluid shift from erythrocytes to plasma due to anticoagulants.In addition, potassium and phosphorus levels may be increased in serum due to release from cells/platelets during the clotting process.Pseudohyperkalemia has been found over the level of 5.5 mmol/L in patients with essential thrombocythemia and serious thrombocytosis. This appears to have been corrected when measured in plasma. Insufficient clotting of serum specimens and fibrin formation within the analyzer reaction vessel may lead to erroneous follicle stimulating hormone results,with the presence of microclots shown to impact lactate dehydrogenase.

The faster processing time with heparinized plasma samples is preferable when urgent critical decisions are based on STAT test results (e.g. for patients suspected to have an acute myocardial infarction). In clinical studies, cardiac markers Troponin T and Troponin I have shown clinically equivalent or clinically acceptable values in both serum and plasma, although one study showed falsely elevated Troponin I due to fibrin in serum samples.Both Creatine Kinase-MB and Myoglobin have demonstrated clinically equivalent results using both specimen types.

While the benefits of plasma have been discussed, it is important for laboratories to consider the limitations. The presence of anticoagulants may interfere with some analytical methods. A slight increase in total protein may be seen in plasma as a result of fibrinogen. Differences in some enzymes (lactate dehydrogenase, alkaline phosphatase, asparate aminotransferase) may be present in plasma. In addition, high levels of lithium and sodium may be observed due to contamination with cations from the anticoagulants.For samples collected in plasma gel tubes, rapid gel barrier movement may trap cellular debris and platelets in the plasma compartment prior to complete separation, which may compromise sample purity. It is important to note that variations may depend on specimen handling and processing as well as the assay platform and manufacturer.

Accurate Testing

Plasma specimens offer the best opportunity for achieving desired turnaround time, which may help laboratories in their performance improvement goals. Faster turnaround of results is particularly vital for STAT testing, in which rapid decisions are necessary for critically ill patients. Additionally, plasma more accurately reflects the patient’s in-vivo state and provides a higher volume yield from the sample.

While standardising the laboratory for one sample type may be desirable, it may not always be practical. Therefore, laboratory professionals should assess each specimen type to determine the most suitable for a particular clinical setting or patient population. It is also important to follow established protocols in the laboratory and the appropriate reference ranges for each assay.

REFERENCES

References available on request – magazine@informa.com

The highest prevalence of this disease was observed in patients from English, African- American, Indian, German, Korean, Japanese, Portuguese, Arab and Iranian origins. A 25-year-old girl with history of prolonged aPTT was analyzed and tested by using Factor deficient plasmas (FVIII, IX, XI and XII). She was found to be having low levels of FXI. Percentage activity of FXI was found to be 45.6% of normal.

Case Study

A 25-year-old female, Sri Lankan in origin was presented in emergency to Al Qurayat General hospital, with a complain of low back pain. She was diagnosed as a case of renal calculus and lithotripsy was planned. Her blood sample was sent to Central laboratory of Al Qurayat General hospital for routine blood tests. Her aPTT (activated partial thromboplastin time) was found prolonged and procedure was delayed to rule out the cause for prolonged aPTT.

The patient presented with a complain of right lumber region pain for last two months along with burning micturition. She also complained of gross hematuria three days back. There was history of nosebleed (epistaxis) two weeks back. The patient also complained of mild bleeding from gums every now and then during the process of cleaning teeth for a long period of time. The age of menarche was 13 years and the patient experienced irregular menstruation since the age of 16 with alterative periods of amenorrhea and Menorrhagia. The patient also complained of changing 4-6 pads per day. Patient also complained of joint swelling after excessive walk when back in Sri Lanka. The most common joints involved were reported as knee joints followed by ankle joints. Patient has history of appendectomy in Sri Lanka. There is no history of excessive blood loss during the operation. There is no history of bruising. No history of smoking. Alcohol and drug abuse was reported. The patient has two siblings who are in good health without such sign and symptoms.

On general physical examination no petechiae and bruising was observed. Liver, spleen and lymph nodes were found to be normal. No other skeletal abnormality was found.

MATERIAL AND METHODS

Principle of the method

Plasma deficient in any of the factors comprising the intrinsic pathway will result in a prolonged partial thromboplastin time (APTT). Coagulation factor deficient plasma can be used to confirm a factor deficiency, in general, and to identify the coagulation factor in patient plasma. A mixture of the respective factor deficient plasma and the patient plasma is tested in the APTT assay. Patient plasma deficient in the specific factor will not be able to compensate for the absence of the factor in the corresponding coagulation factor deficient plasma and therefore result in the prolonged APTT.

Materials Used - Factor VIII, IX, XI and XII deficient plasma; APTT reagent; Calcium chloride solution.

COMPOSITION

Coagulation factor deficient plasmas are lyophilized human plasmas with a residual factor VIII, IX, XI and XII activity <1. The deficient plasmas are manufactured by immunoadsorption from normal plasma and are free from antigen of respective factor. Fibrinogen is present at a quantity of at least 1g/L and the remaining coagulation factors are present in an activity greater than 40% of the normal. The plasma contains mannitol 20g/L as a stabilizer.

PREPARATION OF THE REAGENTS

Deficient plasmas: We dissolved the contents of vial with 1ml of distilled or deionized water. Before use we let it stand for at least 15 minutes at 15-25C, swivelled carefully to mix and avoided the foam formation.

RESULTS

The sample of the patient was received in citrated vial. The test for Prothrombin time (PT) and activated partial thromboplastin time was performed by using Stago ST Art. The mixing study was done and the value for aPTT after correction was found to be 42.7seconds..

Patient’s sample was then tested with Factor VIII, IX, XI and XII deficient plasmas, LA1 and LA2 antibodies.

Discussion

Hemophilia C is a rare inherited autosomal recessive disorder characterised by the low level of FXI in the blood. FXI has a pivotal role in coagulation pathway, after activated by thrombin can bypass initial contact reactions. FXI also plays an important role in generating the thrombin following its initial formation by the tissue factor-factor VII pathway.

This patient had a history of chronic raised aPTT with some associated clinical problems such as on and off nose bleeds, bleeding from the gums which occurred most often while cleaning the teeth, Menorrhagia followed by amenorrhea since the onset of menarche, hemarthrosis which the patient often experienced after a long walk. There is also history of hematuria after the patient diagnosed as having renal calculus.

Coagulation factor studies were done in the Central Laboratory of Al Qurayyat General hospital to rule out the cause of prolonged aPTT. Initially aPTT was found grossly prolonged and was found to be 88.6 sec against the normal control aPTT of 32.3 sec. To find out this prolongation in aPTT is due the clotting actor deficiency or due to the presence of an inhibitor mixing study was done by mixing patient plasma with normal plasma in the ratio of 50/50. The result which we obtained was 37.5 sec. This result excluded the presence of any inhibitor and suggested that there is any intrinsic factor deficiency.

For the purpose of finding the exact coagulation factor of which the patient is deficient of we tested patient’s plasma with Factor VIII, IX, XI, XII deficient plasmas. The aPTT was found to be in near normal range with all the factors except FXI.

The percentage normal activity of the factors was calculated by testing the Factor deficient plasmas with the normal plasma. % activity of FXI in patient plasma was calculated as 45.6%. Thus the patient is found to be having less than 50% activity of normal FXI.

References

Automation has become an essential part of today’s lab as it allows for high volumes of testing at a much faster rate. As a result, pathologists are able to make steady progress by using new tools, instruments and systems that can prepare samples as well as run experiments, and analyse results. Automation offers streamlined solutions for lab procedures and has a number of benefits such as increased productivity, reduced errors, improved workflow coverage and enhanced data quality. Further, automation solutions have now started to extend their footprints into other areas of the lab such as microbiology and molecular diagnostics.

Dr. Basel Altrabulsi, Medical Director, National Reference Laboratory (NRL), Abu Dhabi, says that even though in the past, local, academic, and specialised testing laboratories processed most tests, nowadays centralised testing uses advanced technology as well as global operations to concentrate clinical tests in a single, central laboratory. He adds, “the central lab core is consistency.”

The role of central labs has become quite critical to efficiently managing patients and labs are able to deliver quality data, speed and flexibility to complete projects on time and on budget. Central lab testing offers ‘combinable data’, generated from the same analytic method platform to correlate and standardise results. The end product will therefore be similar regardless of the facility it came from.

Dr. Basel stresses that even though there is resistance to the central model, centralized laboratories improve the speed of reporting results, something that enhances patient safety, while keeping consistency in analysis around the world. As a result of this, laboratory automation systems have gained prominence in developing markets as it enables them to maximise laboratory operations and reduce expenses. Centralised laboratories ensure that hospitals and clinics are able to operate more efficiently as they no longer need to ensure they have a fully equipped and staffed laboratory on site.

An initiative of Mubadala Healthcare, NRL was founded in partnership with and operated by the Laboratory Corporation of America (LabCorp). Its aim is to increase the coverage and overall efficiency of laboratory testing, and implement international best practice testing processes. NRL has deployed lab automation systems to achieve these goals. Its partner, LabCorp, is one of the largest clinical laboratories in the world and is also a pioneer of genomic testing using polymerase chain reaction (PCR) technology. This partnership allows NRL access to LabCorp’s extensive test menu including all esoteric testing and allows NRL to “tap into their knowledge and experience in the field of clinical pathology” which Dr Altrabulsi explains, allows them to deliver the best care to their patients. The lab has also partnered with Integrated Genetics, a LabCorp Speciality Testing Group, which is a leading provider of reproductive genetic testing services.

The automation movement

“Laboratory testing” says Dr Altrabulsi, “has grown from a manual, ‘hands-on’ process providing a simple test menu to an instrument-centric, high-volume clinical engine inside the modern healthcare enterprise.” Lab automation plays an essential part in performing those actions that require high precision and levels of accuracy and has proved extremely useful in helping eliminate human error. Most of the tasks executed in a clinical lab today, from micro plate handling to liquid handling, are achieved through lab automation and these processes handle routine tasks that were once performed by pathologists and lab technicians, and help in achieving a fully traceable and standardized sample processing. Further, the convergence of the different disciplines of laboratory medicine is driving the movement forward and will allow lab automation to expand as well as connect to all the parts of the lab.

While the fields of clinical chemistry and haematology were the first to be automated, areas such as molecular diagnostics and anatomic pathology are most likely to follow soon. Microbiology recently joined the movement with the arrival of fully automated microbiology platforms that automate the entire work flow with one system.

Eliminating human error 

Lab automation is increasingly playing an integral role in making sure patients receive the right treatment and eliminates the risk of human errors. Its major advantage is the fact that it has helped scientists and technicians achieve complex processes in comparatively little time with lower operating costs. Automated systems also have the added advantage of a low error rate, which is important for ensuring that the right diagnosis is made in a timely manner. Some of the factors that have contributed to the steady growth of lab automation include shortage of skilled lab personnel and cost effectiveness, among others. 

As technology changes, some see more advanced bedside testing devices as a challenge to the primacy of the lab in the diagnostic process. Dr Altrabulsi however, sees a role for the lab in supporting all improvements in patient care, including point of care (POC) testing and bedside tests. POC tests are designed to be used at the site of patient care and can be performed outside the physical facilities of a lab. Although a potential threat to the future of labs, POC testing increases the likelihood that the patient, physician, and care team will receive the results immediately, which allows for quicker decisions to be made.

Genetics and future trends

Another advancement is that of genomic technologies that are reaching the point of being able to detect genetic variation in patients with high accuracy and reduced cost, “offering the promise of fundamentally altering the way medicine is practiced”. This has caused the emergence of major analytical and interpretative challenges, ranging from the validation of large numbers of genomic changes in a patient, to the economic feasibility of this approach and its deployment in standard care, to managing the terabytes of data that accompany a single sequenced genome.

“Until recently, genetic data did not drive diagnosis but had a primarily confirmatory role” explains Dr Altrabulsi, citing genomics as a driving force in the future of diagnostic medicine. One of the major challenges facing pathology today is the conversion of pathogenic genetic data into a primary diagnostic tool. This is where lab automation comes into play along with a combination of clinical observation and biometric data, and can assist in shaping clinical decisions and long-term management in a more proactive way. 

Sharing his vision for the future, Dr. Altrabulsi explains that there are many factors that will have an impact on laboratory medicine in the near future and these have the potential to make patient care "more efficient and less expensive". Some of the factors include globalisation of laboratory medicine, technological advances such as POC, automation robotics and integrated diagnostics. Advancements could also include integration of molecular diagnostics into daily testing as well as the integration of pharmacogenomics into the field of cancer management and making non-invasive testing more prevalent.