Impact of glycosylation on NT-proBNP immunoassay performance
by Dr Aleksandra Havelka
Assays for NT-proBNP form a key part of diagnosing/ruling out, predicting and managing heart failure. CLI chatted to Dr Aleksandra Havelka (Gentian Diagnostics) to find out more about the challenges and limitations of measuring NT-proBNP concentrations, particularly the effects of glycosylation, and what developments are on the horizon to overcome these difficulties.
What is NT-proBNP and why is it useful as a biomarker of heart failure?
B-type natriuretic peptide (BNP; also known as brain natriuretic peptide) and the N-terminal fragment of proBNP (NT-proBNP) are biomarkers that are used to assess cardiac function, particularly in diagnosis and management of heart failure. The prohormone proBNP is secreted by cardiomyocytes in response to ventricular stretch, volume overload and increased pressure in the heart (i.e. cardiac stress and stretch). proBNP is cleaved into BNP and NT-proBNP. BNP is the active hormone that promotes vasodilation, excretion of sodium, excretion of water in order to relieve the heart and reduce cardiac workload. NT-proBNP is the non-active part of proBNP; it has no particular function in the body but it is more stable than BNP. The half-life is approximately 20 minutes for BNP compared to 60–120 minutes for NT-proBNP. The increased stability of NT-proBNP due to resistance to enzymatic degradation, is why it is sometimes preferred as a biomarker for diagnosis and management of heart failure.
NT-proBNP is an early and sensitive indicator of heart failure, capable of detecting the disease before clinical symptoms become apparent. It is a valuable biomarker for early diagnosis of non-acute heart failure in outpatient settings. Besides patients with non-acute heart failure, NT-proBNP is also used to aid in the diagnosis of acute heart failure in patients presenting to the Emergency Department with shortness of breath (dyspnea) when heart failure is suspected as a possible cause. It helps differentiate between heart failure and other conditions with similar symptoms. Hence NT-proBNP is an early and sensitive indicator used to aid in the diagnosis of heart failure, both in the acute and non-acute setting. NT-proBNP is also used to stratify the risk and assess severity of the disease: higher NT-proBNP levels indicate more severe disease and higher risk of hospitalization and greater likelihood of adverse cardiac events, including mortality. Prognostically, persistently elevated NT-proBNP levels indicate a poorer prognosis and higher likelihood of cardiovascular complications.
How are NT-proBNP concentrations normally measured?
Types of assay
NT-proBNP is measured in serum and plasma, commonly by immunoassays where antibodies recognize the NT-proBNP protein. The most common type of immunoassay is electrochemiluminescence immunoassays (ECLIA). Many manufacturers provide the assays on their particular instrument platforms. These assays are sensitive, and offer relatively short turnaround times: approximately 20 minutes for the analysis of NT-proBNP in patient samples. However, the total turnaround time also includes sample collection, transportation to the laboratory, processing, and result reporting. Despite these additional steps, the overall time frame remains efficient and well-suited for clinical use. There are also ELISA assays (enzyme-linked immuno-sorbent assays), which involve more hands-on processing with much longer turnaround time. ELISAs are not used in routine care, they are mainly used in a research environment. Additionally, they are point-of-care assays, some of which enable NT-proBNP to be measured in the Emergency Department and outpatient settings providing faster results. There is also possibility to measure NT-proBNP with mass spectrometry, which is not used routinely in clinical practice but is of interest as it will be discussed later in the context of glycosylation.
Cut-off values
NT-proBNP can be measured in both non-acute and acute settings, with different cut-off values used to rule out heart failure. NT-proBNP levels vary by age and gender, with higher values generally observed in older individuals and women. As a result, some manufacturers and laboratories apply age and gender-specific reference ranges. In general, the rule-out cut-off for heart failure in the outpatient setting is 125 ng/L, while in the acute setting, a higher cut-off of 300 ng/L is used. Additionally, age-dependent rule-in cut-off values are applied, ranging from 450 to 1800 ng/L, and are divided into age groups: <50 years, 450 ng/L; 50–75 years, 900 ng/L; and >75 years, 1800 ng/L. These cut-off values help to improve diagnostic accuracy by accounting for natural variations in NT-proBNP levels across different populations.
What limitations/challenges are there with NT-proBNP assay?
Assay standardization
One limitation is the lack of standardization across existing NT-proBNP assays – there is no international standard for NT-proBNP, leading to variations between assays from different manufacturers. As a result, NT-proBNP measurements may differ across laboratories and instrument platforms, making direct comparison challenging.
Biotin interference
The other challenge is biotin interference. Some assays use the biotin–streptavidin interaction as part of the immunoassay technology, so high concentrations of biotin in the blood can interfere with the assay and result in inaccurate measurement and results.
Glycosylation of NT-proBNP
The glycosylation of NT-proBNP is well known in the research community, with multiple publications describing its occurrence and its impact on the measurement of NT-proBNP. However, this knowledge is not widely recognized in clinical practice, including among cardiologists and other healthcare providers, making it an important topic to discuss further.
There are nine potential glycosylation sites on the NT-proBNP peptide, primarily located in the central region of NT-proBNP. Most commercially available assays use antibodies that target this central region of NT-proBNP. If NT-proBNP is glycosylated at the antibody recognition site, it can hinder antibody binding, making the glycosylated form of NT-proBNP undetectable by these assays. This results in a systematic underestimation of NT-proBNP levels.
The impact of NT-proBNP glycosylation on assay accuracy has been well-documented and confirmed in the literature, highlighting a significant limitation in current NT-proBNP measurements.
Assay availability and cost
Lastly, another important limitation is the availability and cost of NT-proBNP assays. Some laboratories lack access to chemiluminescence immunoassay platforms, as they may only have clinical chemistry analysers, making NT-proBNP measurement unavailable in certain settings. Additionally, NT-proBNP assays are relatively expensive, adding to the financial burden of laboratory testing. These factors can limit accessibility, particularly in resource-constrained environments.
Are there any developments on the horizon that will improve NT-proBNP assay methods?
Many manufacturers have already addressed biotin interference so not all NT-proBNP assays on the market rely on biotin–streptavidin complex in their technology. However, glycosylation remains an unresolved issue, as far as I am aware. At Gentian, we are developing an assay that will not be affected by glycosylation, by using antibodies that avoid targeting the central region of NT-proBNP where glycosylation occurs. In addition, the assay from Gentian will be the first assay on the market based on particle-enhanced turbidimetric immunoassay (PETIA) technology, compatible with clinical chemistry platforms. Unlike traditional immunoassays that require chemiluminescence platforms, PETIA-based NT-proBNP measurement can be performed on routine clinical chemistry analysers, which are present in almost every laboratory. Since these are the same platforms used for common tests like C-reactive protein (CRP), NT-proBNP measurement will become more widely accessible, cost-effective and efficient, , improving both throughput and workflow in the laboratory. This assay will have the advantage of detecting ‘total’ NT-proBNP without being affected by glycosylation of the peptide. Also, of course, the Gentian NT-proBNP assay is entirely free from biotin-related assay interference.
We have confirmed that glycosylation has an impact on other commercially available assays but does not affect our assay. When measuring patient samples with varying NT-proBNP concentrations our results consistently show higher NT-proBNP levels compared to other assays. When performing in vitro deglycosylation (removing glycans from NT-proBNP) and reanalysing the same samples with existing commercial assays, NT-proBNP levels increase post-deglycosylation, confirming that native NT-proBNP measurements are underestimated due to glycosylation interference. Deglycosylation of NT-proBNP has no impact on results obtained with our assay.
To further validate our findings, we have also developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method which is not affected by glycosylation. This method consistently detects higher NT-proBNP levels than commercially available assays, supporting our hypothesis that glycosylation causes underestimation in conventional NT-proBNP testing.
The Gentian NT-proBNP assay will overcome key limitations of existing assays by offering:
• accurate NT-proBNP measurement unaffected by glycosylation;
• no biotin interference; and
• seamless integration into high-throughput clinical chemistry analysers, improving workflow and accessibility.
This innovation represents a significant advancement in NT-proBNP testing, ensuring more reliable diagnostics and better clinical decision-making.
Final comments
Accurately measuring total NT-proBNP levels without interference from glycosylation is crucial, as glycosylation levels are unpredictable and vary between patients. This variability makes it impossible to estimate the percentage of glycosylated NT-proBNP in an individual. The literature suggests that glycosylation levels are higher in patients with low NT-proBNP concentrations and decrease as NT-proBNP levels increases. This is a key area of interest that we plan to investigate further during the clinical evaluation of our assay and in comparisons with other methods.
We believe that our NT-proBNP assay will have a clinical impact because levels of glycosylation are unpredictable and highly variable among patients, and may be more pronounced in certain patient subgroups. Subgroups that may benefit from an assay which is independent of glycosylation are patients with diabetes or impaired kidney function. Additionally, based on our discussion with clinicians, we frequently hear that NT-proBNP levels are unexpectedly low in obese patients. We plan to investigate whether higher glycosylation levels in individuals with elevated BMI could explain these disproportionately low NT-proBNP readings. There is also evidence that Entresto® (sacubitril/valsartan), a heart failure medication increases glycosylation of NT-proBNP. We intend to explore this further by including Entresto®-treated patients in the clinical evaluation of our assay.
By addressing the limitations of glycosylation interference, Gentian NT-proBNP assay has the potential to enhance diagnostic accuracy and clinical decision-making across diverse patient populations. We welcome collaboration with researchers and clinicians to further validate its performance and clinical utility.
The interviewee
Aleksandra Mandic Havelka PhD, Chief Scientific Officer Gentian Diagnostics, Moss, Norway
Email: aleksandra.havelka@gentian.com
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