Understanding Xanthochromia

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Understanding Xanthochromia

Posted on 4th September 2024 at 3:34 pm.

Understanding Xanthochromia

When faced with a potential subarachnoid haemorrhage (SAH), the tools we use to diagnose can quite literally be life-saving. Cerebrospinal fluid (CSF) analysis plays a pivotal role, especially when common diagnostic tools like Computed Tomography (CT) scans might not catch early signs.

Traditionally, xanthochromia detection relied on visual assessment – a method that suffers from inconsistency due to subjective interpretation and lacks uniformity across the industry. Today, spectrophotometry has emerged as the preferred method for its precision and reliability in detecting xanthochromia. To ensure the highest accuracy, this technique requires stringent quality control measures. Here, we discuss xanthochromia and SAH, before introducing our dedicated Xanthochromia true third-party control.

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What is Xanthochromia?

Xanthochromia, derived from the Greek word, ‘xanthos,’ meaning yellow, refers to the yellow, or sometimes pink, discolouration of CSF, primarily due to bilirubin, a by-product of haemoglobin breakdown. Why does this matter? Because it’s a tell-tale sign of bleeding within the brain, often indicating SAH when CT scans don’t. Understanding this can help us catch and treat critical conditions before they worsen. Xanthochromia may also be an indicator of intracerebral haemorrhage, brain tumours, infection, or severe systemic jaundice¹.

Subarachnoid Haemorrhage

SAH is a spontaneous intracranial bleed characterised by significant mortality and morbidity rates. Approximately 12% of patients die before receiving medical attention, 33% within 48 hours, and 50% within 30 days of an SAH. Among the survivors, half suffer from permanent disabilities, with an estimated lifetime cost more than double that of an ischemic stroke². Patients which have displayed symptoms often complain of severe headache, nausea, vomiting, photophobia and/or phonophobia³.

CT scans, particularly non-contrasted CTs of the brain or CT angiograms (CTAs), are often the first line of diagnostic tools for suspected SAH. However, up to 5% of SAH cases may not show any signs of haemorrhage on these scans within the first 24 hours, with this figure rising to 50% by the end of the first week and remaining around 30% by the second week⁴.

In contrast, xanthochromia in the CSF can be detected as early as two hours after a bleed and is observed in over 90% of patients within 12 hours of an SAH event. This detection can persist for up to three to four weeks, offering a critical diagnostic window that imaging alone might miss. The conversion from haem to bilirubin in CSF takes roughly 6 to 12 hours, suggesting that xanthochromia is most reliably identified between 6- and 12-hours post-bleed. More than 75% of patients may still present with xanthochromia at 21 days following an SAH¹.

Pathophysiology explained

A ruptured cerebral aneurysm will begin to leak blood into the CSF. This blood is gradually degraded by macrophages to yield various by-products including oxyhaemoglobin, which is subsequently converted to bilirubin in a process lasting between 6 and 12 hours¹. Crucially, this conversion to bilirubin can only occur in vivo, providing a unique marker for diagnosing subarachnoid haemorrhage when observed in the CSF¹.

The Importance of Accurate Detection

In many parts of the world, including the US, visual detection remains a common initial test for xanthochromia in CSF.

Procedure: Spinning a CSF sample in a centrifuge and comparing the supernatant against a vial of water, held against a white backdrop to detect a yellow or pink tint.
Indication: A change in colour indicates that blood has been present in the spinal fluid for at least two hours, with all patients showing signs by 12 hours post-bleed¹.

However, this method is prone to false positives due to:

Dietary influences: High intake of carotenoids (like carrots and spinach).
Medication: Use of Rifampin.
Medical conditions: Clinical jaundice or high protein levels in CSF, which can be seen in conditions like carcinomatosis and meningitis¹.

Spectrophotometry

Spectrophotometry offers a more precise alternative by measuring light absorption in materials at specific wavelengths:

It can detect the presence of bilirubin, which absorbs light at 440 to 460 nm, a definitive indicator of xanthochromia.
Advantages over visual detection: This method eliminates the interference from other pigments or proteins and can distinguish bilirubin from oxyhaemoglobin, crucial for accurate diagnosis.

Quality control is crucial in spectrophotometry to ensure the accuracy and reliability of xanthochromia tests:

Regular Maintenance: Routine checks and maintenance of the spectrophotometer are fundamental to its operation. This helps in maintaining the instrument’s precision in measuring light absorption at specific wavelengths crucial for detecting bilirubin in CSF.
Calibration: Calibrating the spectrophotometer with known standards is essential. This process adjusts the instrument to measure the absorption accurately, particularly vital given bilirubin’s narrow detection window between 440 and 460 nm.

Implementing these stringent QC measures enhances the diagnostic precision of spectrophotometry, boosting confidence in the results. Such practices ensure that patients are diagnosed accurately and receive timely, appropriate treatment, solidifying the value of advanced diagnostic techniques in medical settings.

Introducing Randox Xanthochromia Controls

Diagnosing SAH swiftly and precisely is critical due to its significant immediate and long-term impacts. To aid precise detection, our Liquid Frozen Xanthochromia Positive & Negative Controls are essential tools for laboratories conducting CSF analysis. Here’s what makes them stand out:

Dedicated Xanthochromia true third-party control with only 2 analytes for limited cross-reactivity – Bilirubin & Oxyhaemoglobin
2-day open vial stability at 2° to 8°C and a 11-week shelf life from date of manufacture when stored at -18ºC to -24ºC.
Liquid frozen control provides suitable matrix in an easy-to-use format.
Consistent, clinically significant values.
Suitable for use with UV spectrophotometers, these controls help monitor bilirubin and oxyhaemoglobin levels effectively.

The Randox Xanthochromia Controls are ideally suited for laboratories, both public and private, as well as researchers who perform CSF analysis. Their use is crucial in ensuring the precision of SAH testing, which contributes to more accurate diagnostics and ultimately leads to better patient outcomes.

Considering the crucial role of accurate xanthochromia detection in diagnosing SAH, isn’t it time to review your lab’s capabilities? Explore how Randox Xanthochromia Controls can enhance your diagnostic processes. For more details on how to get these tools in your lab, contact us at marketing@randox.com.

In the fight against conditions like SAH, every second and every test counts. Equip your lab with Randox Xanthochromia Controls to ensure that your diagnostics are as precise and reliable as possible, helping save lives and improve healthcare outcomes.

References

Lipoprotein (a): Molar or Mass?

Posted on 5th August 2024 at 4:29 pm.

Written by Jason Armstrong

Lipoprotein (a): Molar or Mass?

Lipoprotein (a) was first identified in 1963. However, it’s been in the last decade we’ve seen significant advances in our understanding of this ambiguous molecule and its relationship with cardiovascular disease (CVD) risk.

Lp(a) is a macromolecular lipoprotein complex¹ which is thought to display proatherogenic, proinflammatory² and prothrombotic³ potential and is considered an independent causal risk factor for various types of CVD⁴. These properties provide several mechanisms in which elevated Lp(a) levels may contribute to CVD however the true nature of Lp(a)’s relationship to CVD remains largely enigmatic.

Although some of the first studies failed to find a causal relationship, advances in quantification methods soon led to data which showed this relationship did in fact exist. It has been shown that those with the highest levels of Lp(a) are at a 1.5x increased risk of cardiovascular-related death, 1.6x risk of stroke, and up to 4x risk of heart attack when compared with those with the lowest levels⁵.

Accurate measurement of Lp(a) is crucial in determining CVD risk. Quantification methods which account for the size-related variability of Lp(a) molecules are known to produce less bias when compared with those which do not. The Randox Lp(a) reagent is based on the Denka Seiken method which has been shown to produce minimal size-related bias. This assay has FDA 510(K) clearance, illustrating its reliability and safety. Furthermore, the Randox Lp(a) assay is reported in nmol/L, is traceable to the WHO/IFCC reference material, and provides acceptable bias compared with the Northwest Lipid Metabolism Diabetes Research Laboratory (NLMDRKL) gold standard method.

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Physiology and Genetics

Synthesised mainly in the liver, Lp(a), like Low-density lipoprotein (LDL) cholesterol, is composed of a lipid centre made of cholesteryl esters and triacylglycerols, surrounded by a shell of phospholipids, free cholesterol, and an apoB-100 molecule. The major difference between other LDL cholesterol molecules and Lp(a) is the presence of a polymorphic glycoprotein, apo(a), bound to apoB-100 by a single disulphide bond⁵. It is this apo(a) molecule which contributes to Lp(a)’s pathophysiology.

Individuals may possess two different isoforms of apo(a), one of which will have been passed down from each parent, that are expressed codominantly¹. These isoforms are dependent on the number of KIV₂ repeats they contain². Isoforms with less KIV₂ repeats produce smaller apo(a) isoforms which are found at a higher concentration compared with larger isoforms⁶due to the increased rate at which the smaller molecules can be synthesised⁴. The polymorphisms in KIV₂ repeats account for up to 70% of the variation seen in concentration between individuals, with the remainder being attributed to differences in protein folding, transport, and single nucleotide polymorphisms (SNPs)⁴. SNPs are central in the heterogeneity of apo(a), effecting RNA splicing, nonsense mutations and 5’ untranslated region of the LPA gene resulting in shorter gene translation^4,6.

Mass versus Molar?

The quantification of Lp(a) levels is essential in evaluating CVD risk, yet the units of measurement—mass (mg/dL) versus molar (nmol/L) – play a critical role in the accuracy and reliability of these assessments. Historically, Lp(a) levels have been expressed in mass units (mg/dL), but recent advances advocate for the use of molar units (nmol/L) due to their ability to account for molecular variability⁷.

Mass measurement of Lp(a) quantifies the total mass of Lp(a) particles in a given volume of blood, expressed in milligrams per decilitre (mg/dL). This method has been widely used and aligns with other lipid measurements such as cholesterol and triglycerides⁸. However, it does not account for the significant variability in the size and composition of Lp(a) particles. This can result in an overestimation of Lp(a) concentration in those with large apo(a) isoforms, and conversely underestimation of concentrations in patients with small apo(a) isoforms⁹. Consequently, two individuals with the same mass concentration of Lp(a) may have vastly different particle numbers and sizes, leading to potential discrepancies in risk assessment¹⁰.

In contrast, molar measurement expresses the concentration of Lp(a) particles in terms of their molar quantity, measured in nanomoles per litre (nmol/L). This approach provides a more accurate reflection of the number of Lp(a) particles present, irrespective of their size¹¹. By focusing on particle count rather than mass, molar measurement offers a standardised and minimally biased method that better accounts for the heterogeneity of Lp(a) particles¹².

The conversion between mass and molar units is not straightforward due to the variability in the molecular weight of Lp(a) particles. A commonly used conversion factor is approximately 2.5, meaning 1 mg/dL of Lp(a) is roughly equivalent to 2.5 nmol/L¹³. However, this factor can vary depending on the specific characteristics of the Lp(a) particles in a given sample. For this reason, converting between units is discouraged by various relevant organisations including the European Atherosclerosis (EAS)⁶.

Clinical guidelines and risk assessments have traditionally been based on mass concentrations, but the shift towards molar units is gaining traction. The Randox Lp(a) assay, which reports in nmol/L and is traceable to the WHO/IFCC reference material, exemplifies this trend. This assay not only provides a more accurate measurement but also aligns with the NLMDRKL gold standard method, ensuring minimal size-related bias¹⁴.

The choice between mass and molar measurements has significant clinical implications. Accurate assessment of Lp(a) levels is crucial for identifying individuals at risk of CVD and implementing appropriate interventions. As the understanding of Lp(a) continues to evolve, the adoption of molar measurement is expected to enhance the precision and reliability of Lp(a) testing, ultimately improving patient outcomes.

Randox Lp(a) Reagent

For the reasons above, the European Atherosclerosis Society (EAS) recommends that Lp(a) measurement is of the particles (molar) rather than the total mass, to provide a result with minimal size-related bias.

The Randox Lp(a) assay has FDA 510(K) clearance and is one of the only methodologies on the market that detects the non-variable part of the Lp(a) molecule and therefore suffers minimal size related bias providing more accurate and consistent results. The Randox Lp(a) kit is standardised to the WHO/IFCC reference material, SRM 2B, and is the closest in terms of agreement to the ELISA reference method. We also provide a five-point calibrator with accuracy-based assigned target values which accurately reflects the heterogeneity of isoforms present in the general population. Applications are available for a wide range of biochemistry analysers which details instrument-specific settings for the convenient use of the Randox Lp(a) assay on a variety of systems. Measuring units in nmol/L are available upon request.

Features

Excellent Correlation and Precision: The Randox Lp(a) assay demonstrates excellent performance, evidenced by a correlation coefficient of r=0.995 when compared with other commercially available methods and a within-run precision of less than 2.54%.
510(K) Cleared: Randox’s Lp(a) assay has received FDA 510(k) clearance, signifying its safety and effectiveness and ensuring healthcare professionals can trust its accurate and reliable cardiovascular risk assessments.
Dedicated Five-Point Calibrator Available: Five-point calibrator with accuracy-based assigned target values (in nmol/l) is available, accurately reflecting the heterogeneity of the apo(a) isoforms. Dedicated Lp(a) control is available offering a complete testing package.
WHO/IFCC Reference Material: The Randox Lp(a) assay is reported in nmol/l and traceable to the WHO/IFCC reference material (IFCC SRM 2B) and provides an acceptable bias compared with the Northwest Lipid Metabolism Diabetes Research Laboratory (NLMDRKL) gold standard method.
Applications Available: Applications are available detailing instrument-specific settings for the convenient use of the Randox Lp(a) assay on a wide range of clinical chemistry analysers.
Liquid Ready-To-Use: The Randox Lp(a) assay is available in a liquid ready-to-use format for convenience and ease-of-use.

One study¹⁵ compared 5 commercially available Lp(a) assays on an automated clinical chemistry analyser. The assays tested were manufactured by Diazyme, Kamiya, MedTest, Roche, and Randox. The authors show that all the assays tested met the manufacturers claims for sensitivity, linearity, and precision. However, significant bias was observed in 4 out of 5 assays. The only assay which did not display significant bias was the Randox Lp(a) Assay which is traceable to WHO/IFCC reference material. This report highlights the importance of measuring and reporting Lp(a) in molar concentration rather than in mass units to facilitate standardisation and harmonisation in Lp(a) testing¹⁵.

Conclusions

In conclusion, understanding and accurately measuring Lp(a) is crucial for assessing CVD risk. Despite its enigmatic nature, recent advancements have clarified Lp(a)’s role as a significant independent risk factor for CVD. The shift from mass to molar measurement units is enhancing the precision and reliability of Lp(a) assessments, with the Randox Lp(a) assay leading the way in providing minimal size-related bias and accurate results.

To ensure the most accurate and reliable assessment of your patients’ cardiovascular risk, consider integrating the Randox Lp(a) assay into your diagnostic toolkit. With its FDA 510(k) clearance, traceability to WHO/IFCC reference material, and high precision, the Randox Lp(a) assay is an essential component for any modern clinical laboratory.

Integrate the Randox Lp(a) assay into your practice today to enhance the precision of your cardiovascular risk evaluations.

References

Alzheimer’s Disease, ApoE & Risk Detection

Posted on 22nd July 2024 at 1:42 pm.

Written by Jason Armstrong

Alzheimer's Disease & ApoE Whitepaper Banner (1)

Alzheimer’s Disease, ApoE4 & Risk Detection

Alzheimer’s disease touches all of us, whether directly through an affected loved one or through its frequent presence in the news. Alzheimer’s disease is a progressive neurodegenerative disorder characterised by cognitive decline, memory loss, and functional impairments. It is the most common cause of dementia, affecting millions of individuals worldwide¹ and posing significant challenges to healthcare systems. As the global population ages, the prevalence of Alzheimer’s disease is expected to rise, highlighting the urgent need for effective diagnostic and therapeutic strategies.

The current methods used to diagnose Alzheimer’s disease consist of clinical assessment and supporting neuroimaging techniques which can be expensive and, in some cases, fail to provide a definitive diagnosis at the early stages required to facilitate timely intervention and slow disease progression.

With novel therapeutics which aim to slow the progression of Alzheimer’s Disease achieving approval in the United States and being considered for approval in other countries, diagnostics which can identify those at risk of developing this disease are more important than ever. Biomarkers have emerged as vital tools in the early detection and risk assessment of Alzheimer’s disease. Among these, the apolipoprotein E gene (ApoE) has garnered significant attention. The apolipoprotein E protein (ApoE) exists in three common isoforms: ApoE2, ApoE3, and ApoE4. These isoforms combine to form six common genotypes in the general population. Notably, the presence of the ApoE4 allele is associated with a significantly increased risk of developing Alzheimer’s disease.

The Randox ApoE4 Array marks a significant advancement in Alzheimer’s disease biomarkers. This quick and sensitive blood test allows direct ApoE4 genotyping, eliminating the need for traditional molecular techniques. With its fast and accurate results, healthcare providers can efficiently assess an individual’s genetic risk for Alzheimer’s disease.

In this article, we present a summary of our latest whitepaper: Alzheimer’s Disease, ApoE4 & Risk Detection in which we explore the critical role of ApoE genotyping in Alzheimer’s Disease, the innovative technology behind the Randox ApoE4 Array, and its implications for clinical practice. You can download this whitepaper through the link below.

Download Whitepaper

Apolipoprotein E and Alzheimer’s Risk

The ApoE gene transcribes a 229 amino acid protein which primarily functions to mediate lipid transport in the brain and periphery. ApoE is also involved in immune modulation, synapse regeneration, and the clearance/degradation of amyloid-β, a peptide crucial to the development of Alzheimer’s disease².

There are 3 common isoforms of the human ApoE, differentiated through single nucleotide polymorphisms (SNPs) at amino acid positions 112 and 158²:

These three isoforms combine to produce six common genotypes: E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, and E4/E4. Each genotype is associated with a different level of risk for developing Alzheimer’s disease, with the ApoE4 and ApoE2 isoforms presenting the highest³ and lowest risk⁴ respectively.

The structural differences among ApoE isoforms affect their ability to bind lipids, receptors, and amyloid-β, influencing cognitive decline. ApoE2 and ApoE3 bind effectively to HDL (High density lipoprotein), while ApoE4 binds to VLDL (Very low-density lipoprotein), resulting in poor lipidation and toxic aggregates^1,2. Cholesterol is crucial for brain function, supporting membrane integrity, signal transduction, and amyloid-β regulation. The interaction of ApoE with amyloid-β regulates amyloid plaque formation, influencing Alzheimer’s disease onset. Cholesterol must be converted to 24S-hydroxycholesterol to cross the blood-brain barrier. Poor cholesterol and phospholipid transport by ApoE4 increases the risk of late-onset Alzheimer’s disease in E4 carriers².

Randox ApoE Array

The Randox ApoE4 Array is a rapid and highly sensitive blood test that facilitates direct ApoE4 genotyping without the need for traditional molecular techniques. It measures both total ApoE and ApoE4 protein levels directly from a plasma sample, using the ApoE4/total ApoE ratio to classify the ApoE4 status as negative or positive. Additionally, the array can distinguish between heterozygous and homozygous individuals among ApoE4 positive samples, aiding in the assessment of Alzheimer’s disease risk.

Using Randox proprietary chemiluminescent biochip-sandwich immunoassays, the array provides accurate results within three hours from a minimally invasive plasma sample. This method has shown 100% concordance with genotypes achieved through restriction fragment length polymorphism (RFLP) in two separate centres⁵. Compared to other methods like isoelectric focusing, mass spectrometry, bead-based immunoassay, Sandwich ELISA, and PCR, the Randox ApoE Array offers advantages in speed, simplicity, and automation.

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Clinical Implications

The Randox ApoE Array offers significant benefits for managing Alzheimer’s disease through early detection and personalised treatment plans. This minimally invasive blood test identifies individuals at higher genetic risk for Alzheimer’s Disease enabling:

Timely Interventions: Early identification allows for preventive measures and lifestyle modifications, such as cognitive training and increased physical activity, to delay symptom onset.
Regular Monitoring: High-risk individuals can be monitored for cognitive changes, enabling early intervention for mild cognitive impairment.

Personalised Treatment Plans

Accurate ApoE genotyping supports personalised treatment:

Targeted Therapies: ApoE phenotype informs the selection of therapies, especially for ApoE4 carriers.
Risk Stratification: Patients can be stratified by genetic risk for targeted preventive measures.
Optimised Medication: Genotype information guides medication choices, enhancing efficacy.
Family Counselling: Genotyping aids family counselling, advising on preventive measures and monitoring.

Conclusions

The Randox ApoE Array represents a groundbreaking advancement in the early detection and management of Alzheimer’s disease. By providing a rapid, highly sensitive, and minimally invasive method for ApoE genotyping, it empowers healthcare providers to implement timely interventions and personalised treatment plans. This innovative approach not only enhances the accuracy of Alzheimer’s risk assessment but also supports the development of targeted therapies and optimised medication regimens, ultimately improving patient outcomes.

Early detection of Alzheimer’s disease allows for proactive measures that can delay the onset of symptoms, while personalised treatment strategies tailored to an individual’s genetic profile offer a more effective management approach. Furthermore, the array’s capability to provide real-time insights into a patient’s disease stage makes it an invaluable tool in the fight against Alzheimer’s disease.

For a deeper understanding of the critical role of ApoE genotyping in Alzheimer’s disease and the innovative technology behind the Randox ApoE Array, we invite you to download our comprehensive whitepaper be clicking on the image below. You can also visit our website to access this and other valuable resources and to learn more about the Randox ApoE4 Array.

Alzheimer's Disease, ApoE4 & Risk Detection - Cover

References

Active vs Total Vitamin B12

Posted on 18th July 2024 at 3:06 pm.

Written by Jason Armstrong

Total vs Active B12

Vitamin B12, or cobalamin, is a vital water-soluble vitamin that plays an essential role in myelination initiation and development, cellular energy and fatty acid metabolism. It is a cofactor for enzymes methionine synthase and L-methyl-malonyl-coenzyme A mutase and, in addition to folate, is essential for DNA and protein synthesis. In the UK, up to 6% of adults under 60 have been diagnosed with Vitamin B12 deficiency and figures are much higher in elderly populations¹. Additionally, these data do not consider the high rates of missed diagnosis associated with B12 deficiency, which some reports claim to be as high as 26%². New guidance from the National Institute of Health and Care Excellence (NICE) advise that Active vitamin B12 testing is recommended for some groups of patients. In this article, we’ll look at this essential vitamin, B12 deficiency and the associated complications, compare the biomarkers used to diagnose B12 deficiency, and finally, present the new Acusera Active B12 Control.

Aetiology

Vitamin B12 deficiency can arise due to dietary insufficiency, malabsorption resulting from damage to the small intestine, often caused by conditions like Coeliac disease or Crohn’s disease, or via pernicious anaemia – an autoimmune condition which results in an inability to absorb vitamin B12.

It is a common problem in the elderly population – bodily stores of vitamin B12 can take up to 20 years to become depleted, meaning complications have often already begun before diagnosis occurs. The most common source of vitamin B12 comes from dietary intake of animal products therefore vegetarian dietary requirements are considered a considerable risk factor for vitamin B12 deficiency.

Pathophysiology and Complications

Vitamin B12 deficiency significantly impacts health, affecting various bodily functions, potentially leading to a range of complications. Megaloblastic anaemia is a common complication associated with vitamin B12 deficiency and is characterised by the presence of large red blood cell precursors (megaloblasts) in the bone marrow³. The lack of vitamin B12 results in impaired DNA synthesis and an inhibition of nuclear division. However, cytoplasmic maturation is less effected. This results in asynchronous maturation of the nucleus and cytoplasm in erythrocytes and causes the synthesis of abnormally large megaloblasts. This causes the cessation of DNA synthesis and DNA replication errors, culminating in apoptotic cell death. Common symptoms of megaloblastic anaemia include weakness, shortness of breath, palpitations, tachycardia, Hunter glossitis or splenomegaly³.

Pernicious anaemia is a condition commonly associated by vitamin B12 deficiency. Pernicious anaemia is an autoimmune disorder which affects the gastric mucosa resulting in impaired absorption of vitamin B12. Common symptoms of pernicious anaemia include glossitis, hair loss, dry skin, memory loss, poor concentration, poor sleep, confusion and dizziness, shortness of breath, Diarrhoea, indigestion, loss of appetite, mood swings and suicidal thoughts.

Neurological issues may also arise, including numbness, mobility loss, and memory issues, and in some cases, depression⁴. Additionally, B12 deficiency is linked to increased risks of cardiovascular events⁵, infertility⁶, and autoimmune diseases like multiple sclerosis⁷ and lupus⁸. In children, vitamin B12 deficiency can manifest as failure of brain and overall growth and development, developmental regression, hypotonia, lethargy, hyperirritability, or coma⁹.

Complications and manifestation associated with Vitamin B12 deficiency.

Active B12 as a marker of Deficiency

There are several markers of vitamin B12 deficiency. The most used in clinical practice are total vitamin B12, homocysteine, methylmalonic acid (MMA), and Holotranscobalamin (HoloTC) – also known as Active B12. HoloTC accounts for between 10-30% of total B12 and is the metabolically active form of vitamin B12.

When compared with total B12 quantification, HoloTC measurement has been shown to be a more sensitive and specific biomarker of B12 deficiency, particularly at borderline clinical levels¹⁰, in various cohorts^11,12 including those on vegan diets¹³ – a known risk factor for B12 deficiency. Furthermore, HoloTC was shown to provide the higher diagnostic accuracy in clinical and subclinical B12 deficiency versus Total B12, MMA and homocysteine with significantly higher accuracy in women over 50¹¹ – a population at high risk of B12 deficiency.

In response to the mounting evidence of the superior utility of HoloTC quantification, the National Institute for Health and Care Excellence (NICE) have produced new guidelines recommending either total B12 or HoloTC for the initial testing of suspected vitamin B12 deficiency. These guidelines specify the use of active B12 during pregnancy and suggest that active B12 might provide a more specific assessment in certain clinical contexts.

Acusera Active B12 Control

For the reasons stated above, Randox are proud to present the Acusera Active Vitamin B12 Control. This control is designed for use with in vitro diagnostic assays for the quantitative determination of HoloTC in human serum and plasma and is suitable for use on a variety of analysers. This true third-party control is provided in a liquid ready-to-use format reducing preparation time and has an impressive 30-day open vial stability, helping to minimise waste. Like all Acusera controls, the Active B12 Control is supplied at consistent, clinically relevant levels to ensure the test system is challenged at the critical decision limits used to aid diagnosis. Furthermore, this control is provided with assayed target values for a range of analysers which are available through our new SmartDocs portal.

Summary of Benefits:

Dedicated, HoloTC control.
30-day Open Stability.
2-year shelf life.
Liquid Ready-to-use.
Human Serum Based.
Consistent, clinically significant values.
True third-party controls.
Assayed target values.

Ensure the accuracy of your vitamin B12 testing with Randox’s Acusera Active Vitamin B12 Control. Join the other laboratories around the world who trust Acusera to help deliver reliable, clinically relevant test results. Contact us today at marketing@randox.com to learn more and order your supply of the Acusera Active B12 Control.

References

Bordetella Detection & Species Identification Educational Guide

Posted on 17th July 2024 at 10:05 am.

Written by Jason Armstrong

Bordetella Educational Guide Blog Header

Bordetella Detection and Species Identification with the Vivalytic

Cases of Bordetella infections are rising across Europe. Bordetella species are responsible for whooping cough, or pertussis, which literally means violet cough. Vaccine deployment in the 1940s saw a reduction in the morbidity and mortality associated with these infections and now, healthy adults can be expected to make a full recovery. However, vulnerable populations, such as children, the elderly and the immunocompromised, have been shown to be at increased risk of more severe and long-lasting side effects, including increased risk of mortality.

Traditional methods of identifying Bordetella infections take the form of culture, which can take up to 7 days due to the fastidious and slow-growing nature of these bacteria and provide limited sensitivity^1,2. To provide a faster and more sensitive method for the identification of whooping cough pathogens, Randox, in partnership with Bosch, are proud to introduce the Vivalytic Bordetella Cartridge. This real-time PCR assay allows detection of B. pertussis, B. parapertussis and B. holmesii on the Vivalytic system, a universal, fully automated, cartridge-based platform enabling high-plex and low-plex testing, providing an all-in-one solution for molecular diagnostics.

To help you understand the implications of Bordetella infections and those of the Vivalytic system, we have produced a new educational guide, covering the Bordetella species responsible for whooping cough; the pathophysiology and complications associated with these infections; the Vivalytic platform and the benefits it can bring to your laboratory; and finally, a summary of findings presented at ESCMID 2024 in which the Vivalytic Bordetella cartridge showed excellent results. Here, we present this educational guide and a summary of its contents. You can download this guide for free by clicking the download link below.

Download Educational Guide

The Scale of the Bordetella Problem

The rates of positive identification of Bordetella infection are increasing throughout Europe. In England, between January and March 2024, there were 2793 laboratory confirmed cases of whooping cough causing the deaths of 5 infants, compared with a total of 858 cases in 2023³. A rudimentary projection model estimates that without intervention, whooping cough cases in the England could total over 15,000 cases by the end of 2024. Rising cases are not isolated to the UK – increased rates of diagnosis have also been reported in Denmark, Spain, and Croatia⁴. Increased numbers of infections illustrate the need for novel and rapid diagnostics to identify those who have been infected and help reduce the transmission of these bacteria.

Figure 1. Whooping Cough Cases in the UK: 2023 vs 2024 Initial Projection: This bar chart illustrates the total number of whooping cough cases in the UK for 2023 and the projected number of cases for 2024. The total number of cases in 2023 was 858. For 2024, the confirmed cases from January to March were 2,793. The projection for the remaining quarters of 2024 was based on historical seasonal trends observed from the years 2018, 2019, 2020, and 2023, with projections estimating over 15000 cases by the end of 2024. — Whooping Cough Cases in the UK: 2023 vs 2024 Initial Projection: This bar chart illustrates the total number of whooping cough cases in the UK for 2023 and the projected number of cases for 2024. The total number of cases in 2023 was 858. For 2024, the confirmed cases from January to March were 2,793. The projection for the remaining quarters of 2024 was based on historical seasonal trends observed from the years 2018, 2019, 2020, and 2023.

Bordetella genus

Bacteria of the Bordetella genus are gram-negative coccobacilli⁵ which are important pathogens in human medicine as they colonise the respiratory tract leading to a range of pulmonary and bronchial infections⁶. There are 3 main species associated with whooping cough: of B. pertussis, B. parapertussis (Classical Bordetella) and B. holmesii (pertussis-like disease pathogen).

Pertussis is caused by Classical Bordetella: B. pertussis and B. parapertussis. Despite widespread vaccination cases are rising, partially due to waning immunity. Pertussis is highly contagious and particularly dangerous for infants, who account for most pertussis-related deaths. The disease progresses through three phases: catarrhal (cold-like symptoms), paroxysmal (severe coughing fits), and convalescent (persistent cough). Classical Bordetella species share over 98% DNA sequence similarity and share many crucial virulence factors like toxins adenylate cyclase toxin (ACT), pertussis toxin (PXT), and dermonecrotic toxin⁵ yet there are variations in potential hosts and disease. For example, B. pertussis is an exclusively human pathogen, whereas B. parapertussis can infect both humans and sheep⁶.

Bordetella holmesii causes pertussis-like symptoms but is ofen less severe. Unlike classical Bordetella, B. holmesii can cause bacteraemia, especially in immunocompromised individuals. Accurate diagnosis of B. holmesii remains challenging due to its similarities with other Bordetella species.

Whooping cough can lead to complications such as pneumonia, which may develop if fever persists beyond the catarrhal phase². CNS complications like seizures and encephalopathy occur in less than 2% of cases, often due to hypoxia, hypoglycaemia, toxins, or secondary infections². Bordetella toxins, especially PXT, increase histamine sensitivity and insulin secretion. Infants are especially at risk of bradycardia, hypotension, and cardiac arrest.

Vivalytic Bordetella Cartridge

To enhance the detection and species identification of Bordetella, Randox introduces the Vivalytic Bordetella cartridge. This user-friendly assay is designed to detect B. pertussis, B. parapertussis, and B. holmesii from a single nasopharyngeal swab or aspirate sample. Utilising Real-time PCR, it enables rapid and accurate detection up to four weeks after symptom onset, differentiating between human pathogenic Bordetella species. With a time to result of just 47 minutes, this assay is invaluable for patient diagnosis and the containment of Bordetella, helping to reduce aerogenic transmission.

Summary of Benefits:

Sample Volume – 300μl.
Sample Type – Nasopharyngeal swab sample or aspirates.
Real-time PCR detection.
Time to result – ~47 minutes.
Detection of B. pertussis, B. parapertussis, and B. holmesii.

Bordetella-cartridge — Vivalytic Bordetella Cartridge

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Rapid and Accurate Detection of Whooping Cough in Clinical Samples

Zimmerman, 2024

At the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) 2024 congress, the Vivalytic Bordetella array showed excellent performance, with a 97.7% concordance and a 97.9% positive percent agreement (PPA) with the reference method.⁷ It accurately identified all additional positive samples and maintained over 98% PPA across spiked samples, even at low levels. The system’s invalid result rate was notably low at 0.6%, compared to 2.9% with the BioGX assay⁷.

The conclusions drawn from this investigation are as follows:

The Vivalytic Bordetella cartridge provided excellent concordance with a sensitive reference method and delivered fast and accurate results.
This assay is ideal for both hospital laboratories and outpatient settings, thanks to its user-friendly design and quick turnaround times.
Early identification of infected patients will aid in preventing the spread of re-emerging whooping cough epidemics.

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Conclusion

As Bordetella infections rise across Europe, rapid and accurate detection is crucial. The Vivalytic Bordetella Cartridge offers a fast, reliable solution, identifying B. pertussis, B. parapertussis, and B. holmesii with high accuracy in just 47 minutes. This advanced diagnostic tool can help reduce transmission and manage whooping cough effectively.

Take control of your diagnostic capabilities and ensure the best care for your patients. Download our comprehensive educational guide to learn more about Bordetella infections and the benefits of the Vivalytic system.

For more information on the Vivalytic, the panels mentioned, or any of our products, don’t hesitate to reach out to us at marketing@randox.com.

References

Combating Gastroenteritis – Advanced Diagnostic Techniques for Effective Management

Posted on 24th June 2024 at 9:26 am.

Written by Jason Armstrong

Gastroenteritis, often referred to as stomach flu or a stomach bug, affects millions globally each year with symptoms such as diarrhoea, vomiting, abdominal pain, and fever. It is primarily caused by viral and bacterial infections, with rotavirus, norovirus, and Clostridium difficile being the main culprits.

At Randox, we’re dedicated to improving healthcare worldwide. That’s why we’ve produced an educational guide on gastroenteritis and the latest advancements in diagnostic techniques, including a range of novel gastroenteritis test for the Vivalytic POCT system. In this blog, we’ll look at a few of the key points raised in our latest educational guide. You can download this educational guide by clicking the below.

Download Educational Guide

Why Gastroenteritis Matters

Gastroenteritis can lead to severe dehydration, especially in vulnerable groups like children and the elderly. It spreads mainly through the faecal-oral route, which includes consuming contaminated food and water. Prompt and accurate diagnosis is crucial for effective management.

Key Pathogens

Rotavirus

Rotavirus is a major cause of severe gastroenteritis in children. Highly contagious, it leads to rapid dehydration, making rehydration and supportive care essential. Vaccines like Rotarix and RotaTeq are effective in preventing infections.

Norovirus

Norovirus is responsible for most viral gastroenteritis outbreaks. Extremely contagious, it spreads quickly through direct contact and contaminated food. Symptoms include sudden vomiting and diarrhoea, often leading to dehydration. While there’s no specific treatment, staying hydrated is key.

Clostridium difficile

Clostridium difficile, or C. diff, is a leading cause of antibiotic-associated diarrhoea, particularly in healthcare settings. It produces toxins that cause inflammation and damage to the colon, requiring targeted antibiotic treatment for severe cases.

Advanced Diagnostics: The Vivalytic System

Accurate and timely detection of gastroenteritis pathogens is crucial for effective patient management. The Vivalytic Point of Care Testing (POCT) system, developed by Bosch Healthcare Solutions and Randox Laboratories, offers rapid and reliable diagnostics. This system helps healthcare professionals make quicker decisions, improving patient outcomes.

The Vivalytic Gastroenteritis Panels

The Vivalytic panels detailed in our guide include tests for rotavirus, norovirus, and Clostridium difficile. These panels utilise advanced molecular techniques to provide quick and accurate results, helping to streamline the diagnosis process and enhance patient care. By using these panels, healthcare providers can efficiently identify the specific pathogens responsible for gastroenteritis, allowing for targeted treatment and improved patient outcomes.

Features of the Vivalytic System

The Vivalytic system is user-friendly and efficient. It supports both High-Plex and Low-Plex testing, allowing for the simultaneous detection of multiple pathogens from a single sample. This versatility makes it an invaluable tool for healthcare professionals.

More Info

Conclusion

Gastroenteritis, caused by pathogens like rotavirus, norovirus, and Clostridium difficile, presents significant health challenges. Advanced diagnostic technologies, such as the Vivalytic system, are crucial in managing and controlling this condition. For a comprehensive understanding of gastroenteritis and innovative diagnostic techniques, download our detailed educational guide.

For more information on the Vivalytic, the panels mentioned, or any of our products, don’t hesitate to reach out to us at marketing@randox.com

Gastroenteritis - Vivalytic Educational Guide

Serum Indices – Product Spotlight

Posted on 2nd May 2024 at 9:28 am.

Written by Jason Armstrong

Errors can occur at any point in the pre-analytical, analytical, or post-analytical stages of a diagnostic test. It is general practice for errors in the analytical stage to be identified through quality control procedures. However, pre-analytical errors are often treated with less importance than those in later stages of testing. Interference caused by haemolysis, icterus and lipemia (HIL) are common forms of pre-analytical error which affect assay methods, yielding erroneous results. The Randox Acusera Serum Indices (SI) control is designed to monitor an IVD instrument’s response in the detection of HIL interferences.

HIL interference is not novel and has been historically identified through a series of visual assessments. While haemolytic, icteric and lipemic interference causes a visual change in the sample, these methods are not quantitative and are subject to interpretation by laboratory professionals. Modern analysers have built-in capabilities for the automated detection of HIL interference which can quantitatively or semi-quantitatively measure haemolysis, icterus and lipemia, and provide and an index for each. This data can then be used to determine if a sample should be accepted for testing or rejected due to intrinsic interference.

The pre-analytical phase of laboratory testing includes collection, handling, transportation, storage, and preparation of samples. Even when the highest level of care is taken to ensure that all aspects of the pre-analytical phase are suitable and correct, errors can occur, exhibiting the need for clear and efficient quality control processes.

As part of our Acusera quality control range, Randox has developed the Serum Indices quality control to aid in the detection of the common pre-analytical error’s haemolysis, icterus and lipemia, collectively known as HIL. HIL interference can have disastrous effects on the quantification of many analytes, and it is therefore vital to determine levels of interference to improve laboratory efficiency and reduce the frequency of erroneous results.

The graph below shows the wavelengths at which each of these interferents may affect assays and the table below describes these forms of interference:

Classical determination of HIL interference took the form of a visual assessment. A sample was examined for tell-tale signs of one or more of these types of interference. However, these methods are subject to operator interpretation and lack harmonisation and uniformity across the industry. These signs are detailed in the table and illustrated in the graphic below:

Modern clinical chemistry analysers have onboard HIL detection capabilities which offer objective, semi-qualitative or qualitative analysis of these forms of interference in a more precise and consistent manner. Automation of HIL detection improves laboratory throughput along with test turnaround times and enhances the reportability of the results.

Errors at any stage of the analytical process will result in retesting of the sample. Errors in the pre-analytical phase can have repercussions such as increased cost of repeated sample collection and testing, poor test turnaround times, and more seriously, delayed or incorrect diagnosis causing an exacerbation in the condition of the patient. To add to the adverse outcomes on patients, repeated testing places additional stress on laboratory resources and staff which ultimately affects every aspect of a laboratory’s daily activities.

To correctly analyse HIL interference, absorbance readings at different strategically selected wavelengths supplement the calculation of the interference indices. C56-A recommends laboratories consider several parameters when selecting an HIL interference analysis method:

Before implementing results obtained from any method detecting HIL in patient samples, it is imperative to evaluate the specificity and sensitivity of the method at a minimum of two clinically relevant concentrations. This assessment should encompass the sensitivity of the icterus index to haemoglobin and lipids, the haemolysis index to bilirubin and lipids, and the lipemic index to haemoglobin and bilirubin.

In instances of HIL interference, laboratories bear the responsibility of managing the associated results and samples. It is crucial never to utilise an HIL index for the correction of patient results. Typically, if a sample is determined to be affected by one or more of these interferences, the laboratory should reject the result and appropriately dispose of the sample. Nonetheless, in certain scenarios, threshold values can be established. For instance, haemolysis may exert a lesser impact on samples with elevated analyte concentrations. In such cases, laboratories may opt for a distinct procedure in handling these results compared to those exhibiting haemolytic interference at lower analyte concentrations.

Acusera Serum Indices Control

The Randox Acusera Serum Indices (SI) control is designed to be used to monitor an IVD instrument’s response in the detection of haemolyzed, icteric and lipemic (HIL) samples. This control can be utilised in laboratory interference testing to assist in improving error detection of pre-analytical errors affecting clinical chemistry testing. This control provides a full range of clinically relevant testing levels, including a negative (-) and three positives (+, ++ & +++).

The Randox Control offers a comprehensive solution with 3 levels for each form of interference and a negative control, providing a wider coverage compared to alternatives in the market. Our product is conveniently supplied in a lyophilized format, ensuring an extended shelf-life and ease of storage. Customers appreciate the stability of our control, as it consistently meets the 14-day open stability claims, minimizing waste and optimizing laboratory efficiency.

Typical Values

RIQAS Serum Indices External Quality Assessment

The RIQAS Serum Indices EQA programme is designed for the pre-analytical assessment of Haemolytic, Icteric and Lipemic (HIL) interferences. Available in a bi-monthly format with the option to report either quantitative or semi-quantitative results for the HIL parameters, this programme also provides an assessment on how these interferences impact on up to 25 routine chemistry parameters. This provides invaluable information on whether a correct judgement is being made to report results.

• Lyophilised for enhanced stability
• Human based serum ensuring commutable sample matrix
• Bi-monthly reporting
• HIL parameters include the option of quantitative or semi-quantitative reporting
• Interpretation of chemistry parameter results
• Submit results and view reports online via RIQAS.net

How can Randox help?

It is crucial laboratories test for haemolysis, icterus and lipemia to ensure the accuracy of their test processes are maintained. ISO 15189:2022 promotes the identification and control of non-conformities in the pre-analytical process, therefore, using Randox Serum Indices control and RIQAS Serum Indices EQA will help laboratories fulfil the requirements of the new edition of this standard.

Randox Serum Indices control displays improved consolidation, stability, and commutability to ensure laboratories are equipped to accurately determine pre-analytical interferences. Our Serum Indices control can be used with most major chemistry analysers including Roche, Abbot, Beckman, Ortho, and Siemens. When used in conjunction with Acusera 24.7, this control offers laboratories the ability to compare their HIL results with their peer group and identify potential failures in their pre-analytical process.

Simply send us an email by clicking the link below and we will get in touch!

ENQUIRE MORE HERE

Medical Laboratory Professionals Week 2024

Posted on 15th April 2024 at 4:53 pm.

Written by Jason Armstrong

Med Lab Professional Week 2024 - blog header

Medical Laboratory Professionals Week (MLPW) is recognised every year in the last full week of April. It’s an opportunity to increase the public understanding of, and appreciation for, the hard work of clinical laboratory staff around the world. It’s also an opportunity to inject a little fun into the laboratory. So, this year, we’ve created a Lab Professionals QC Bingo card. Have a go and see how many your laboratory can get!

How many boxes does your lab tick?

If you’re calling Bingo! you must be an Acusera 24.7 customer. If not, keep reading to find out how you can make daily life in your laboratory more straightforward.

What are Medical Laboratory Professionals?

Medicine wouldn’t be where it is today without the work of these laboratory professionals. They’re on the frontline. Around 70% of medical decisions are based on results provided by medical laboratory staff. That’s a lot of pressure on the labs to make sure their results are accurate. Clinical laboratory staff not only perform the tests used to guide diagnosis and disease prevention, but they also check all the tests they use through rigorous quality control (QC) procedures.

This involves testing samples of known values to prove that the test system and its components perform as they should and provide accurate results. To do this, laboratories require QC material. It’s important that what’s in a QC is as similar to what you’d find in a patient sample as possible. This is known as commutability. Good commutability helps limit cross-reactivity in the test and inaccurate results.

It’s also important to make sure the QC material has concentrations of analytes at similar values to those used to make diagnostic decisions. If you wanted to validate the length of the ruler on your desk, it wouldn’t be helpful to set it down on a 100m running track. Similarly, when laboratory professionals want to ensure a test is producing accurate results, they want to test the system at the critical values used to make medical decisions so that they can be confident the results at these values are accurate.

Once lab staff have confirmed the accuracy of their tests, they can begin testing patient samples. For most people, what happens to a sample after it’s taken is a bit of a mystery. MLPW is the perfect opportunity to unravel this a little:

After your sample is collected, it gets sent over to the lab. Even just moving it there needs careful handling to make sure it’s still good for testing when it arrives. Once it’s in the lab, the team checks the equipment to make sure it’s working right and giving accurate results. The QC procedure varies depending on what they’re testing for, but they always make sure their tests are legitimate. Once they’ve checked everything and carried out the tests, a pathologist looks at the results to figure out what’s going on. They use this information to help decide on the best treatment plan for you.

Even this watered-down explanation makes it sound like a lot of work, right? At Randox, we recognise the vital role and dedicated efforts of medical laboratory professionals, and the invaluable contributions they make to society, and we hope that now, you do too.

Acusera 24.7

Bingo! That’s exactly how our customers feel when they realise how much time Acusera 24.7 can save them. Our innovative and intuitive QC data software is cloud-based, allowing you to log in from anywhere in the world to review your QC data.

Along with a wide range of interactive charts, including Levey-Jennings charts, Acusera 24.7 determines measurement uncertainty and sigma metrics for you, saving you the time and stress of manually calculating these tricky statistical analyses. And that’s just the beginning. Acusera 24.7 can link to LIMS for automated data entry, meaning lab staff don’t have to manual type long datasets, unless they want to of course; we also provide both semi-automated data upload and manual data entry options.

Access to a range of reports has never been easier. Acusera 24.7 is particularly useful when gaining or renewing your accreditation, and live peer group QC data, to give additional confidence in the accuracy of your results.

But this article is supposed to be about laboratory professionals, so we won’t bang on about it anymore. We just want everyone to know about Acusera 24.7 so they can get that daily bingo! feeling for themselves. If you want to learn more about our reports, charts, advanced statistical analysis, Acusera 24.7 more generally, or how Acusera 24.7 can help you achieve your accreditation, you can follow the links to the relevant blog post.

Last year, we interviewed two of our laboratory staff, Dean and Meadhbh, to find out what a normal day looked like for them. To find out what a day in the life of a laboratory professional is like, take a look at the interviews here

If you’d like to get in touch with us to discuss the advantages of Acusera 24.7, or you’ve made up your mind and want to get in on the action, reach out to us at marketing@randox.com. We’re always happy to brag about how great Acusera 24.7 is, and how we make life simpler for more and more laboratories every day.

Lp(a) Awareness Day 2024

Posted on 22nd March 2024 at 1:21 pm.

Written by Jason Armstrong

Novel and classical insights into Lp(a) concentration and the effects on various cardiovascular conditions.

Despite advances in understanding and technology, cardiovascular diseases (CVDs) remain a major source of mortality across the world. The World Health Organisation (WHO) estimate that 17.9 million people died due to CVDs in 2019, accounting for around 32% of deaths that year¹. First described in 1963, Lipoprotein(a) (Lp(a)) is a macromolecular lipoprotein complex² which is thought to display proatherogenic, proinflammatory³ and prothrombotic⁴ potential and is considered an independent causal risk factor for various types of CVD⁵. These properties provide several mechanisms in which elevated Lp(a) levels may contribute to CVD however the true nature of Lp(a)s relationship to CVD remains largely enigmatic.

Lp(a) concentrations in plasma are principally regulated by variation in LPA gene and levels remain relatively stable throughout one’s lifetime with lifestyle factors having little effect on their concentration⁶. Due to the highly heritable nature of Lp(a) concentration, those with a family history of Familial Hypercholesterolaemia (FH), elevated LDL-C levels, or Atherosclerotic cardiovascular disease (ASCVD) should be screened, their plasma Lp(a) concentration determined, and their risk of CVD established.

In the last 10 years, there have been many advances in the understanding of this ambiguous lipoprotein which support the causal association with CVD, clarify the established evidence and introduce novel mechanisms of action in relation to Lp(a), shedding light on its obscure pathophysiology. However, there are still diagnostic complications associated with Lp(a) measurement as there is little standardisation in methods of determination⁵.

Physiology and Genetics

Synthesised mainly in the liver, Lp(a), like LDL, is composed of a lipid centre made of cholesteryl esters and triacylglycerols, surrounded by a shell of phospholipids, free cholesterol, and an apoB-100 molecule. The major difference between other LDL molecules and Lp(a) is the presence of a polymorphic glycoprotein, apo(a), bound to apoB-100 by a single disulphide bond⁵. It is this apo(a) molecule which contributes to Lp(a)s pathophysiology.

Apo(a) is thought to have evolved from the plasminogen gene (PLG) around 40 million years ago and shares 78-100% sequence homology within the untranslated and coding regions of the fibrinolytic enzyme². Like plasminogen, apo(a) contains unique domains named kringles⁵. While plasminogen contains 5 different kringle structures (KIto KV), apo(a) has lost KI to KIII and instead contains several forms of KIV, namely, 1 copy of KIV₁and KIV_3-10, 1-40 copies of KIV₂, 1 copy of KV and an inactive protein domain at the carboxyl terminus of the molecule⁷. These hydrophilic subunits are highly polymorphic due to the variation in KIV₂ repeats. Individuals may possess two different isoforms of apo(a) one of which will have been passed down from each parent and are expressed codominantly². These isoforms are dependent on the number of KIV₂ repeats they contain². Isoforms with less KIV₂ repeats produce smaller apo(a) isoforms which are found at a higher concentration compared with larger isoforms⁸due to the increased rate at which the smaller molecules can be synthesised⁵. The polymorphisms in KIV₂ repeats account for up to 70% of the variation seen in concentration between individuals, with the remainder being attributed to differences in protein folding, transport, and single nucleotide polymorphisms (SNPs)⁵. SNPs are central in the heterogeneity of apo(a), effecting RNA splicing, nonsense mutations and 5’ untranslated region of the LPA gene resulting in shorter gene translation^5,8.

Lp(a) Pathophysiology

Lp(a) is thought to contribute to the risk of CVD through multiple mechanisms. Firstly, Lp(a) molecules display all the same atherosclerotic risk as LDL-C molecules due to their similar fundamental composition, for example, their propensity for oxidisation upon entering the vessel wall, and promotion of atherosclerosis through inflammatory and immunogenic mechanisms⁹. However, Lp(a) displays more proatherogenic potential due to the presence of the apo(a) molecule. The structure of apo(a) results in decreased fibrinolysis. Due to its structural similarities, apo(a) competes with plasminogen for binding sites, competitively inhibiting plasminogen, ultimately resulting in reduced fibrinolysis⁹.

Lp(a) is thought to be a preferential carrier of oxidised phospholipids² (OxPLs) which covalently bind to apo(a), increase expression of inflammatory proteins, and stimulate the secretion of IL-8 and monocyte chemoattractant protein-1, enhancing its ability to cross the vessel wall⁹. Some claims require further investigation, however, studies have been carried out which show inhibition of plasminogen activation in the presence of Lp(a)¹⁰. It is this indirect mechanism that Lp(a) is thought to conduct its prothrombotic activity^8,9.

Clinical Evidence

Many studies have been carried out to determine the association of Lp(a) concentration and CVD risk. Studies such as the Copenhagen General Population Study, the Copenhagen City Heart Study, Dallas Heart Study, and Ischemic Heart Disease Studies provide strong evidence for Lp(a) as a causal risk factor for CVD. Data analysis of the Copenhagen General Population Study reveal that 20% of subjects displayed Lp(a) concentrations of more than 42mg/dl, or around 105nmol/L¹¹, which is considered to result in increased risk of atherosclerotic disease⁵. It is important to note, there is no accepted conversion factor for converting Lp(a) concentration from mg/dl to nmol/L due to the variability of apo(a) kringles. The unitage will depend on the assay method used⁵. Another study in a healthcare organisation in Israel showed that Myocardial Infarction (MI) and Coronary Artery Disease was 2.5 times more common in those with high levels of Lp(a) than in the age and sex matched control group³. This study³, along with others^5,6,12 describes a linear relationship between Lp(a) concentration and CVD risk, showing at least a 3-fold increase in ASCVD and MI events in adults with Lp(a) concentrations in the top 1% when compared with those in the with concentrations in the bottom 20%³.

The major variation in Lp(a) concentration seen throughout the population, is further evident between ethnicities and sexes. On average, Caucasian subjects display the lowest Lp(a) concentrations, with Black subjects displaying the highest concentrations⁵. However, the large number of functional variants are consistent across ethnicities suggesting that it is the KIV₂ repeats and SNPs that are the major factors contributing to Lp(a) concentration regardless of ethnicity. Lp(a) concentrations are higher in women than men⁸with levels increasing post-menopause thought to be caused by a decrease in oestrogen³.

Lp(a) Testing and Screening

The European Atherosclerosis Society (EAS) recommend that all adults are tested at least once in their lifetime to identify individuals who have high levels of Lp(a) and therefore high CVD risk. Screening is also recommended in children who have a family history of Ischaemic stroke, premature ASCVD or high Lp(a) levels in the absence of other identifiable risk factors⁸. Testing has been associated with reduced mortality rates. This is thought to be because of increased and intensified therapy for those who are identified as high risk due to high plasma Lp(a) concentration⁶.

There are various assays available for the determination of Lp(a) concentration which vary in accuracy and precision. Many of these assays utilise polyclonal antibodies which recognise different antigenic determinants⁸. Due to the variability in apo(a) structure and KIV repeats, these assays often overestimate the concentration of large isoforms and underestimate concentration of small isoforms when determining the true Lp(a) levels⁹. This variation can be partially nullified by using a calibrator series and by selecting a method which is traceable to WHO/IFCC reference material. This allows laboratories to confidently identify individuals considered high risk but may still prove problematic when patients’ results report closer to the assay thresholds.

One study¹³ compared 5 commercially available Lp(a) assays on an automated clinical chemistry analyser. The assays tested were manufactured by Diazyme, Kamiya, MedTest, Roche, and Randox. The authors show that all the assays tested met the manufacturers claims for sensitivity, linearity, and precision. However, significant bias was observed in 4 out of 5 assays. The only assay which did not display significant bias was the Randox Lp(a) Assay which is traceable to WHO/IFCC reference material. This report highlights the importance of measuring and reporting Lp(a) in molar concentration rather than in mass units to facilitate standardisation and harmonisation in Lp(a) testing¹³.

Current and Emerging Therapies

Statins are one of the most potent treatments for the primary prevention of ASCVD through the reduction of LDL-C concentration. However, recent studies reveal that statins have no effect on Lp(a) concentration³ and others suggest that statin administration can increase Lp(a) concentration by up to 11%^5,9. Nonetheless, EAS do not recommend statin therapy be halted as their strong ameliorative effects on CVD risk are well established and surmount the risk related to increased Lp(a) concentration⁸.

Niacin (Nicotinic acid) is another established treatment for the reduction of CVD events and act by increasing HDL levels. Niacin can reduce Lp(a) concentration though the reduction of gene expression in a dose-dependent manner⁵. However, Niacin therapy has not been proven to have beneficial effects on CVD risk⁸.

A recent metanalysis showed a 26% reduction in serum Lp(a) concentration through treatment with PCSK9 inhibitors. This is thought to be due to a shortage of apoB-100 molecules either because of reduced synthesis or competitive binding with other LDL receptors, resulting in reduced Lp(a) concentration⁵. Several studies show the efficacy of PCSK9 inhibitors in reducing CVD risk, but this is not yet an approved therapy^5,8.

New therapeutic strategies aim to target hepatocytes, the site of apo(a) synthesis, to reduce Lp(a) concentration. Antisense Oligonucleotides (ASOs) inhibit apo(a) mRNA in the nucleus and cytoplasm, ultimately inhibiting Lp(a) secretion⁵ through the cleavage of the sense strand by ribonuclease H1⁹. While still in clinical trials, ASO therapies show promise in the battle to reduce CVD risk with some studies displaying an overall reduction in Lp(a) concentration of more than 80%⁹.

Conclusions

There have been major advances in the understanding of Lp(a) pathophysiology in the last 10 years establishing this macromolecular complex as an independent causal risk factor for various forms of CVD, however, more investigation is required to fully understand the mechanisms responsible for this association. With many national healthcare organisations and the EAS recommending universal testing for Lp(a) in adults, more emphasis should be placed on raising awareness of the importance of Lp(a) screening. Finally, more research is needed into therapies which succeed at lowering Lp(a) concentration. While some therapies are in clinical trials, there are currently no approved therapies that achieve this goal.

The Randox Lp(a) assay is calibrated in nmol/L, traceable to the WHO/IFCC reference material, and displays an excellent correlation coefficient of r=0.995 with when compared with other commercially available methods. To accompany this liquid ready-to-use reagent we also offer a dedicated 5 point calibrator with accuracy-based assigned target values (in nmol/l) is available, accurately reflecting the heterogeneity of the apo(a) isoforms.

For more information on this revolutionary assay, visit randox.com/lipoproteina/ or reach out to us at marketing@randox.com.

References

World Health Organization. Cardiovascular Diseases. World Health Organization. Published June 11, 2021. https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases-(cvds)
Schmidt K, Noureen A, Kronenberg F, Utermann G. Structure, function, and genetics of lipoprotein (a). Journal of Lipid Research. 2016;57(8):1339-1359. doi:https://doi.org/10.1194/jlr.r067314
Zafrir B, Aker A, Saliba W. Extreme lipoprotein(a) in clinical practice: A cross sectional study. International Journal of Cardiology Cardiovascular Risk and Prevention. 2023;16:200173. doi:https://doi.org/10.1016/j.ijcrp.2023.200173
Pino BD, Gorini F, Gaggini M, Landi P, Pingitore A, Vassalle C. Lipoprotein(a), Cardiovascular Events and Sex Differences: A Single Cardiological Unit Experience. Journal of Clinical Medicine. 2023;12(3):764. doi:https://doi.org/10.3390/jcm12030764
Stürzebecher PE, Schorr JJ, Klebs SHG, Laufs U. Trends and consequences of lipoprotein(a) testing: Cross-sectional and longitudinal health insurance claims database analyses. Atherosclerosis. 2023;367:24-33. doi:https://doi.org/10.1016/j.atherosclerosis.2023.01.014
Lampsas S, Xenou M, Oikonomou E, et al. Lipoprotein(a) in Atherosclerotic Diseases: From Pathophysiology to Diagnosis and Treatment. Molecules. 2023;28(3):969. doi:https://doi.org/10.3390/molecules28030969
Vuorio A, Watts GF, Schneider WJ, Tsimikas S, Kovanen PT. Familial hypercholesterolemia and elevated lipoprotein(a): double heritable risk and new therapeutic opportunities. Journal of Internal Medicine. 2019;287(1):2-18. doi:https://doi.org/10.1111/joim.12981
Kronenberg F, Mora S, Stroes ESG, et al. Lipoprotein(a) in atherosclerotic cardiovascular disease and aortic stenosis: a European Atherosclerosis Society consensus statement. European Heart Journal. 2022;43(39):3925-3946. doi:https://doi.org/10.1093/eurheartj/ehac361
Tsimikas S. A Test in Context: Lipoprotein(a): Diagnosis, Prognosis, Controversies, and Emerging Therapies. Journal of the American College of Cardiology. 2017;69(6):692-711. doi:https://doi.org/10.1016/j.jacc.2016.11.042
Boffa MB, Koschinsky ML. Lipoprotein (a): truly a direct prothrombotic factor in cardiovascular disease? Journal of Lipid Research. 2016;57(5):745-757. doi:https://doi.org/10.1194/jlr.r060582
Enkhmaa B, Anuurad E, Berglund L. Lipoprotein (a): impact by ethnicity and environmental and medical conditions. Journal of Lipid Research. 2016;57(7):1111-1125. doi:https://doi.org/10.1194/jlr.r051904
Svilaas T, Klemsdal TO, Bogsrud MP, et al. High levels of lipoprotein(a) – assessment and treatment. Tidsskrift for Den norske legeforening. Published online January 12, 2023. doi:https://doi.org/10.4045/tidsskr.21.0800
Wyness SP, Genzen JR. Performance evaluation of five lipoprotein(a) immunoassays on the Roche cobas c501 chemistry analyzer. Practical Laboratory Medicine. 2021;25:e00218. doi:https://doi.org/10.1016/j.plabm.2021.e00218

World Tuberculosis Day 2024

Posted on 21st March 2024 at 4:03 pm.

Written by Jason Armstrong

Tuberculosis in Brief

When we think of Tuberculosis (TB) we tend to think of an old-timey disease. Doc Holliday, the famous gunslinger, died of consumption, the old-world name for TB. As did Fantine from Victor Hugo’s “Les Misérables” and Nicole Kidman’s, Santine, in the 2001 movie, Moulin Rouge! For the videogame fans out there, you might be familiar with Arthur Morgan from Red Dead Redemption 2 who, depending on how you played the game, may have suffered a similar fate. However, this disease is still prevalent around the world today. TB is a bacterial infection caused by Mycobacterium tuberculosis estimated to infect around 10 million people and is responsible for up to 1.5 million deaths each year¹.

Originally discovered in 1882, M. tuberculosis is an airborne pathogen which primarily affects the lungs but can also affect other parts of the body². TB infection exists in 3 states: latent, subclinical, and active. A latent TB infection is asymptomatic and non-transmissible. Subclinical infections are also asymptomatic but transmissible and will produce a positive culture. Finally, active disease is a transmissible state associated with the symptoms of TB². The World Health Organization (WHO) estimates that around ¼ of the world population is infected with M. tuberculosis³. Up to 15% of those infected with TB will progress to active disease, while those who do not are at a heightened risk of infection throughout the rest of their lives⁴. Compared with some other bacterial diseases, TB is not particularly infectious. An infected individual is estimated to infect between 3-10 people per year². However, subclinical TB infections present a challenge in reducing transmission because asymptomatic individuals may unknowingly spread the disease – over 1/3 of TB infections are never formally diagnosed⁵.

The symptoms of an active TB infection include fever, fatigue, lack of appetite, weight loss, and where the infection effects the lungs, a persistent cough and haemoptysis (coughing up blood). HIV-infection is a major risk factor for TB infection and mortality. Up to 12% of all new cases and 25% of TB deaths occur in HIV-positive persons². Other risk factors for the development of TB are, malnutrition, poor indoor air quality, Type 2 diabetes, excessive alcohol consumption and smoking¹.

TB is present around the world. However, as you might expect from the risk factors, low-to-middle income and developing countries account for a disproportionate number of cases. According to WHO, half of all TB infections are found in 8 countries: Bangladesh, China, India, Indonesia, Nigeria, Pakistan, Philippines, and South Africa.

Without effective treatment, TB will kill and estimated 50% of those infected². Treatment for TB typically involves first-line antibiotics such as isoniazid, rifampicin, pyrazinamide, and ethambutol, with second-line drugs including fluoroquinolones and injectable aminoglycosides⁶. Nonetheless, drug-resistant TB accounts for an inordinately large amount of the global AMR burden which can arise from both transmitted and acquired resistance. Resistant M. tuberculosis strains are classified as monoresistant – those resistant to 1 drug; multi-drug resistant (MDR) – those resistant to 2 or more first line treatments, commonly isoniazid and rifampicin; and extensively drug resistant (XDR) – MDR strains which are also resistant to second line therapies like fluoroquinolones and aminoglycosides⁶.

Global rates of TB have been declining. An estimated 75 million lives have been saved since 2000¹. Furthermore, between 2015 and 2020, TB incidence fell by 13.5%⁷. However, the progress made over the last decade has been compromised by the COVID-19 pandemic, illustrated by a, 18% drop in diagnosis between 2019 and 2020⁷. Explanations for this decline include delayed treatment because of lack of access to public transport and healthcare facilities, disruption of laboratory services, a personal desire to avoid the stigma of disease and misdiagnosis due to the similarities in symptoms between TB and COVID-19.

The theme for World Tuberculosis Day 2024 is “Yes, we can end TB!” The WHO have set targets of an 80% decline in new cases and a 90% drop in TB-related deaths by 2030. Screening and preventative treatments are crucial to achieving these goals. Therefore, novel methods of detection which are quick, inexpensive and include drug resistance identification are needed.

Mycobacterium Tuberculosis EQA

It is important for those carrying out TB testing to ensure their instruments and methods are accurate and effective. External Quality Assessment (EQA) programmes are an essential part of this process. QCMD is an independent international EQA organisation primarily focused on molecular infectious diseases to over 2000 participants in over 100 countries.

QCMD offers 2 programmes for those testing for TB through molecular methods: Mycobacterium tuberculosis DNA and Mycobacterium Tuberculosis Drug Resistance.

Mycobacterium tuberculosis DNA EQA Programme

Mycobacterium Tuberculosis Drug Resistance EQA Programme

Mycobacterium Tuberculosis Quality Controls

Those conducting research into TB infections and new methods of detection, screening and drug resistance profiling need to be confident that the equipment they are using is up to the task. Qnostics is a leading provider of Quality Control solutions for molecular infectious disease testing. Our range comprises hundreds of characterised viral, bacterial, and fungal targets covering a wide range of diseases.

Q Controls

Our range of positive run, whole pathogen, third party controls are designed to monitor assay performance on a routine basis. As true third-party controls, assay drift is detected, monitored, and managed, helping to ensure accurate and reliable results. The use of third-party controls will also help to support ISO 15189:2012 regulatory requirements.

Mycobacterium tuberculosis (MTB) Q Control 01

Target Pathogen – Mycobacterium tuberculosis (MTB)

Matrix – Synthetic Sputum

Stability – Single use control designed to be used immediately minimising the risk of contamination

Shelf Life – Up to 2 years from date of manufacture

Regulatory Status – Research Use Only

Mycobacterium tuberculosis (MTB) Q Control 01

Mycobacterium tuberculosis (MTB) Rifampicin Resistant Q Control

Compatible for use with Cepheid analysers, this whole pathogen positive control is designed to monitor the performance of molecular assays used in the detection of Rifampicin resistant Mycobacterium tuberculosis.

Target Pathogen – Mycobacterium tuberculosis (MTB)

Target Genotype – Rifampicin Resistance

Matrix – Synthetic Sputum

Stability – Single use control designed to be used immediately minimising the risk of contamination

Shelf Life – Up to 2 years from date of manufacture

Regulatory Status – Research Use Only

Mycobacterium tuberculosis (MTB) Rifampicin Resistant Q Control

Mycobacterium tuberculosis (MTB) Evaluation Panel Control

QNOSTICS Evaluation Panels cover a range of genotypes and/or levels, and may be used to evaluate assay characteristics, confirm performance claims, and ultimately ensure the assay is fit for purpose. Evaluation Panels may also be used in the validation of clinical assays and the development of new diagnostic tests.

This dedicated MTB Evaluation Panel comprises 3 targets relating to Mycobacterium tuberculosis for validating a new assay or instrument to ensure that everything is working as expected. High and medium concentrations are provided alongside a negative sample.

Target Pathogens – MTB, M. bovis, Rifampicin (Rif) resistant MTB, Isoniazid (INH) resistant MTB, Negative

Matrix – Synthetic Sputum

Panel Members – 8 (Including a negative)

Stability – Single use. Once thawed, use immediately

Shelf Life – up to 2 years from date of manufacture

Regulatory Status – Research Use Only

Mycobacterium tuberculosis (MTB) Evaluation Panel Control

If you are interested in any of the TB quality control products shown above, or any other products from our wide catalogue of molecular controls and EQA programmes, get in touch with us today at marketing@randox.com. To learn more, see the links below which will take you to the relevant sites and brochures.

QNOSTICS – www.randox.com/molecular-infectious-disease-controls/

QNOSTICS Brochure Download

QCMD – https://www.qcmd.org/

QCMD Brochure Download

References

World Health Organisation. Tuberculosis. Fact Sheets. Published November 7, 2023. Accessed March 21, 2024. https://www.who.int/news-room/fact-sheets/detail/tuberculosis
Pai M, Behr MA, Dowdy D, et al. Tuberculosis. Nat Rev Dis Primers. 2016;2(1):16076. doi:10.1038/nrdp.2016.76
World Health Organisation. Tuberculosis. https://www.who.int/health-topics/tuberculosis.
Andrews JR, Noubary F, Walensky RP, Cerda R, Losina E, Horsburgh CR. Risk of Progression to Active Tuberculosis Following Reinfection With Mycobacterium tuberculosis. Clinical Infectious Diseases. 2012;54(6):784-791. doi:10.1093/cid/cir951
Adigun R, Singh R. Tuberculosis. StatPearls Publishing; 2024.
Liebenberg D, Gordhan BG, Kana BD. Drug resistant tuberculosis: Implications for transmission, diagnosis, and disease management. Front Cell Infect Microbiol. 2022;12. doi:10.3389/fcimb.2022.943545
World Health Organisation. Global Tuberculosis Report 2022.; 2022. Accessed March 21, 2024. https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-tuberculosis-report-2022