|Year : 2019 | Volume
| Issue : 4 | Page : 158-164
An overview on interference in clinical immunoassays: A cause for concern
Department of Laboratory Medicine, Pure Health, Al Qassimi Women and Children's Hospital, Sharjah, UAE
|Date of Submission||05-Jan-2019|
|Date of Acceptance||07-Apr-2019|
|Date of Web Publication||11-Nov-2019|
Laboratory Medicine, Pure Health, Al Qassimi Women and Children's Hospital, Sharjah
Source of Support: None, Conflict of Interest: None
Immunoassays are rapid, simple, cost-effective, robust and sensitive laboratory techniques that are extensively used in many important areas of clinical medicine such as diagnosis, prevention, treatment and management of diseases. Universally, it is one of the most extensively used in vitro diagnostic testing methods, and its demand has been increasing exponentially over the past four decades. Routinely, these methods can quantify minute amounts of diverse analytes such as hormones, antibodies, proteins and drugs present in highly complex biological fluids, such as serum, urine, sweat, meconium and cerebrospinal fluid. Immunoassays are successful techniques that can detect up to picomolar concentrations using antigen–antibody reactions without the need of prior extraction. Although immunoassays are useful techniques that help physicians to take quick decisions, they too are subjected to interference from both exogenous and endogenous sources, resulting in false-positive and false-negative results. When the immunoassay results do not appear to fit the clinical picture, it becomes a great challenge to a clinical biochemist, alarms clinicians and confounds immunoassay manufacturers. Sometimes, these results can mislead or miss a diagnosis, resulting in unnecessary investigations and mental trauma to the patient and families. Hence, it is very important for one to be aware of the limitations of this immunoassay, to have knowledge about them and to take necessary actions or precautions to get the right result. There is no single procedure that can rule out all interferences. Furthermore, it is difficult for the laboratorians to identify antibody interference with immunoassay, and thus, physicians should be encouraged to communicate specifically with the laboratory about discordance between results and clinical findings. If there is any suspicion of discordance between the clinical and the laboratory data, an attempt should be made to reconcile the difference. Procedures should be put in place when interference is suspected. Constant communication is required between physician and laboratory about unexpected immunoassay results. Manufacturers can also be communicated to identify the presence of interfering antibody.
Keywords: Biotin interference, heterophilic antibodies, hook effect, immunoassay
|How to cite this article:|
Sequeira S. An overview on interference in clinical immunoassays: A cause for concern. Hamdan Med J 2019;12:158-64
| Introduction|| |
Immunoassays are rapid, simple, cost-effective, robust and sensitive laboratory techniques that are extensively used in many important areas of clinical medicine, such as diagnosis, prevention, treatment and management of diseases. Universally, it is one of the most extensively used in vitro diagnostic testing methods, and its demand has been increasing exponentially over the past four decades. Routinely, this method can quantify minute amounts of diverse analytes such as hormones, antibodies, proteins and drugs present in highly complex biological fluids, such as serum, urine, sweat, meconium and cerebrospinal fluid.
Immunoassays are successful techniques that allow high sensitivity, detecting up to picomolar concentrations with the use of antigen–antibodies immune reaction without the need for prior extraction. They use a range of various mammalian antibodies, for example, monoclonal (mouse) or polyclonal (goat, sheep and rabbit) in their reaction. Most of the methods are highly specific and able to measure a wide range of analytes and are based on simple photo-, fluoro- or lumino-metric detection.
| Interference in Immunoassay: an Underestimated Problem|| |
Although immunoassays are useful techniques that help physicians to take quick decisions, they too are subjected to interference from both exogenous and endogenous sources, resulting in false-positive and false-negative results. They have an analytical error rate of 0.4%–4%, which is much higher than the other routine tests such as liver function test and renal function test. When the immunoassay results do not appear to fit the clinical picture, it becomes a great challenge to a clinical biochemist, alarms clinicians and confounds immunoassay manufacturers. Sometimes, these results can mislead or miss a diagnosis, resulting in unnecessary investigations and mental trauma to the patient and families. Hence, it is very important for one to be aware of the limitations of this immunoassay, to have knowledge about them and to take necessary actions or precautions to get the right result.
There are numerous publications, case studies and articles describing situations, where immunoassays have reported incorrect results and have had adverse effects on patients. One of the earliest immunoassay interferences reported was in 1992, a well-known case of false-positive human chorionic gonadotropin (hCG) result that had led to unnecessary surgery and inappropriate chemotherapy of the patient, as the patient was thought to have post-gestational choriocarcinoma or trophoblast disease based on their persistently increased hCG results. Some most recent reported interference has also been documented, where unnecessary procedures or follow-up testing have been done due to false-positive/false-negative results, for example, misdiagnosis of Grave's disease with apparent severe hyperthyroidism in a patient taking mega doses of biotin.
| Types of Interferences|| |
Assay interference can be exogenous, which is not associated with the properties of the individual specimen and may reflect a system failure, for example, blockage of probes and inadequate mixing of reagents, or can be endogenous interferences (a) that are specimen dependent caused by interaction between components in the sample such as normal serum components in excess (lipids, haemoglobin and hyperbilirubinaemia.); (b) anti-analyte and anti-reagent antibodies such as heterophile, anti-animal and autoantibodies; (c) high-dose hooking and (d) high intake of biotin.
| Interference from Excess of Normal Serum Components|| |
Interference from haemolysed and icteric samples
Although haemolysis causes much less interference with immunoassays when compared to spectrophotometric techniques, haemolysis may be unacceptable for immunoassays of relatively labile analytes such as cTnT, insulin, glucagon, calcitonin, parathyroid hormone, Adrenocorticotropic Hormone and gastrin, due to the release of proteolytic enzymes from erythrocytes that degrade these analytes. Haemolysis may also interfere with some signal generation steps of different types of immunoassays. Grossly, haemolysed specimens should not be used.
Excess bilirubin can also affect many different types of assays, including immunoassays. One such analyte is cTnI, which shows a statistically significant decrease when measured using microparticle enzyme immunoassay technique.
Interference from lipaemic sample
Lipaemia can also non-specifically interfere in immunoassays. Lipoproteins can interfere with antigen–antibody reaction by blocking binding sites on antibodies. This can happen even when antibodies are bound to a solid surface. Depending on the nature of the reaction, the interference can cause both falsely elevated and falsely decreased results. Lipaemia has been shown to cause a negative interference at elevated levels (>22.5 g/L) in electrochemiluminescence immunoassay for testosterone. Lipaemia can interfere with some immunoassays, especially those using nephelometry and turbidimetry endpoints (e.g. apolipoprotein B and haptoglobin assay). Interferences by non-esterified fatty acids have been well documented for free thyroxine assays. Non-esterified fatty acids compete with thyroxine and its derivatives used as labels for endogenous protein-binding sites and depending on the assay format may cause either falsely high or falsely low free thyroxine values.
| Minimising Risk of Interference in Immunoassay from Normal Serum Components|| |
Ideally, specimens should be collected from individuals following an overnight fast to reduce the immunoassay interference from lipids. Turbidity in a lipaemic sample can be visually detected. Ultracentrifugation could be used to remove excess lipids; alternatively, enzymatic cleavage by lipase may be used to treat lipaemic samples before the analysis. Manufacturer's advice should always be followed. Documented procedures for treating specimens identified as haemolysed or lipaemic should be available in all laboratories. Haemolysis is also readily detectable by visual examination before the analysis. Automated detection of haemolysis, icterus and lipaemia is also possible.
| Interference from Endogenous Antibodies|| |
The three main groups of endogenous antibodies known to cause interference in immunoassays are heterophile, anti-animal and autoantibodies. Endogenous antibodies may bind to, or bridge, or block the binding sites on capture and signal antibodies generating incorrect results.
Origins of interfering antibodies
Endogenous antibodies are those that originate within our body. These endogenous antibodies could be naturally occurring or may be associated with occupational exposure to foreign proteins, blood transfusion, multiparity, contact with animals, exposure to endogenous molecules or specific immunogens that lead to autoimmune diseases, such as celiac disease (where the endogenous autoantibodies attack the small intestine) and type 1 diabetes (where they attack the pancreas).
Heterophile antibodies are endogenous antibodies that can bind to assay antibodies. They are produced without exposure or an unknown exposure to specific immunogens  and are thus considered to be naturally occurring. They generally have low avidity but react across multiple species with the capability of binding to multiple antigens. False results can occur when the heterophilic antibodies in the patient sample cross-link the assay antibodies, even in the complete absence of the analyte, thus mimicking the analyte the assay was intended to measure. They tend to interfere in both formats the competitive [Figure 1]b and non-competitive [Figure 2]b and [Figure 2]c however more common in the sandwich or non-competitive. Heterophile antibodies may react with variable avidity to distinct species; furthermore, reactivity to different species may persist for different periods of time within the same patient. Studies have shown that heterophilic antibodies tend to have a greater affinity to the Fc-region of mouse IgG1 antibodies, the most common isotype used in immunoassays, but little or no affinity for F(ab')2-fragments, mouse antibodies of other isotypes or other animal antibodies.
|Figure 1: Mechanism of interference from endogenous antibodies in a competitive immunoassay. (a) Competitive immunoassay without any interference resulting in a true positive or true negative result. (b) Cross-linking of capture and labelled analyte analogue in the presence of endogenous antibody, resulting in false-positive or false-negative result|
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|Figure 2: Mechanism of interference from endogenous antibodies in a sandwich or non-competitive immunoassay. (a) In the absence of interfering antibodies, capture antibody and labelled antibody target, two different epitopes on the analyte giving a true positive result; (b) Presence of interfering antibody can result in cross-linking of capture and labelled (detection) antibody giving a false-positive result; (c) Endogenous antibodies can bind to either capture or labelled antibody only, thus reducing sandwich formation giving a false-positive result|
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Human anti-animal antibodies (HAAAs) are specific and interact strongly with assay antibodies. They are produced after acute or chronic exposure (known exposure) to specific antigens and are species specific  having a higher avidity than heterophile antibodies. They are generally produced in response to animal antibodies such as animal-derived monoclonal antibodies that are injected for diagnostic or therapeutic purposes or from exposure to animals occupationally (e.g. veterinarians) or to pets. One of the most common types of HAAAs is the human anti-mouse antibodies (HAMAs) derived from mice. These antibodies can create serious analytical problems as they can be present in higher concentrations and have high affinity. Interfering antibodies can be specific to the assay reagents (anti-animal antibodies) or the analyte (autoantibodies if to an endogenous molecule). Therapeutic antibodies are therapeutically administered antibodies or their fragments, such as Digibind, which detoxifies digitalis toxicity. Therapeutic antibodies will interfere in immunoassays until excreted by the kidneys.
Autoantibodies are endogenous antibodies that are mostly found in individuals with autoimmune disorders and can cause cross-reactivity to assay antibodies and interfere with immunoassays reactions; for example, patients with thyroid disease have antithyroid antibodies. Another most common anti-self-antibody is the rheumatoid factor, which is a patient antibody (usually IgM) with an affinity to the Fc-region of the patient's own IgG antibodies. Rheumatoid  factor is found in 70% of patients with rheumatoid arthritis and 5%–10% of the general population. Cross-reactivity to assay antibodies occurs as there is significant homology between Fc-domains in human antibodies and Fc-domains in antibodies from several animal species. Other examples of autoantibodies include antithyroglobulin antibodies that can affect thyroglobulin immunoassays and anti-insulin antibodies that interfere with immunoassays for insulin or C-peptide. High titres of potentially interfering antibodies may occur in patients with recent infections, immunisations and transfusions 
| Types of Immunoassay Formats|| |
The measurement of the analyte by immunoassay is achieved using two basic types of methods, that is, either a competitive immunoassay or a non-competitive immunoassay format.
Mechanism of interference in competitive immunoassay
In the competitive-binding immunoassay [Figure 1]a, the unlabelled ligand (or antigen of interest) competes with labelled ligand for binding to a specific binding protein (often an antibody). In the presence of circulating interfering endogenous antibody that cross-links captured and labelled analyte analogues, false-negative or false-positive results can occur [Figure 1]b.
Mechanism of interference in non-competitive immunoassay
The non-competitive two-site immunometric assay, commonly known as the 'sandwich' immunoassay [Figure 2]a, uses two antibodies; a capture antibody and a labelled or detection antibody that is coupled to a signalling agent, both targeting different epitopes on the ligand or antigen or analyte of interest. This format generally provides the highest level of assay sensitivity and specificity.
However, in the presence of circulating interfering antibody (endogenous, e.g., heterophilic antibody), the interfering antibody cross-links the capture and detection antibody in the absence of the analyte or antigen, thus giving a false-positive result [Figure 2]b. As two-site immunoassays are widely used, this type of interference is the most commonly seen. Endogenous interfering antibodies can also interfere with binding to either capture or labelled antibody only, thus reducing sandwich formation, giving a false-positive result [Figure 2]c.
Troubleshooting interferences for endogenous antibodies
There is no single procedure that can rule out all interferences. Furthermore, it is difficult for the laboratorians to identify antibody interference with immunoassay, and thus, physicians should be encouraged to communicate specifically with the laboratory about discordance between results and clinical findings. If there is any suspicion of discordance between the clinical and the laboratory data, an attempt should be made to reconcile the difference. Procedures should be put in place when interference is suspected. Constant communication is required between physician and laboratory about unexpected immunoassay results. Manufacturers can also be communicated to identify the presence of interfering antibody.
- When an interference is suspected, a detailed history should be obtained regarding any past exposure with a monoclonal antibody preparation, animal exposure or transfusions that might have been used for therapeutic or diagnostic purpose
- Unexpected immunoassay results should first be analysed by the same method to exclude analytical errors such as pipetting inaccuracies, inefficient wash, tracer aggregates or other contaminants. Some samples with heterophilic antibody interference can give varying results with repeated testing, and great variation can increase the suspicion of interference. However, lack of variation upon retesting does not exclude interference
- The specimen may be sent to another laboratory for retesting using an alternate method, such as an immunoassay that uses a different technology (homogeneous vs. heterogeneous; competitive vs. non-competitive) and a different reagent antibody source than the original immunoassay suspected to display the interference or a non-immunometric method. The sample should also be analysed for different analytes using a similar format, as interference may be present in several assays
- When the sample is suspected for interference, especially of heterophile antibodies blocking reagents, especially the mouse or rabbit immunoglobulins can be added to the immunoassay formulations to absorb the interfering antibodies before reanalysis. The samples could also be preincubated with commercially available test tubes to which blocking antibodies can be immobilised on the inner surface of those special tubes. Addition of blocking reagents can neutralise the heterophile antibodies and prevent interference
- When there is a suspicion of interference, the sample can be diluted to check for linearity. The presence of interfering antibodies results in non-linearity of results in dilution, due to steric effect and heterogenic nature of the interfering antibody. The samples should be diluted using appropriate diluents mentioned in the kit insert for the analyte to avoid misinterpretation of results caused by matrix and other effects.
| High-Dose Hooking|| |
The hook effect, also known as prozone effect, is a type of interference caused by excessively high concentrations of a particular analyte or antibody simultaneously saturating both capture and detector antibodies, resulting in false negatives or inaccurately low results. High-dose hooking is most commonly seen in one-step (sandwich) immunoassays and nephelometric assays. Ideally, as concentrations of analyte in plasma or serum increase, the response from sandwich immunoassays increases as well. However, as the concentration of analyte increases or exceeds a certain point, the analyte molecules saturate out all reagent antibodies, thus resulting a decline in signal or rather the signals begin to plateau with high concentrations of analyte due to limiting amounts of reagent immunoglobulins [Figure 3]. This leads to falsely decreased results and potential misdiagnosis. Many assays have been reported to suffer from the hook effect in patients with high analyte values and include assays for prolactin, serum-free light chains  and , PSA.
|Figure 3: Mechanism of high-dose hook effect in sandwich immunoassay. As the analyte concentration increases the capture antibody and labelled antibody gets saturated with the analyte, thus decreasing the analyte sandwich formation. This results in an inappropriately low signal that causes erroneous low or normal result in spite of the sample containing elevated analyte concentration|
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Troubleshooting interferences for high-dose hooking
Diluting the samples and reanalysing them, especially for those analytes with extremely high concentrations, are the most common methods to overcome interference, especially the hooks' effect. The samples should be diluted using appropriate diluents mentioned in the kit insert for the analyte.
| Biotin Interference With Immunoassay|| |
Biotin belongs to the family of B-complex vitamins and is also known as Vitamin B7 or Vitamin H. It acts as a coenzyme for carboxylase reactions in gluconeogenesis, fatty acid synthesis and amino acid catabolism. Biotin is present in high content in dietary food, recommended in high doses in patients with multiple sclerosis, mitochondrial diseases, certain inborn metabolic disorders and neurological defects. For the past few years, it has also been used as a beauty supplement and easily available over the counters, and there are no records of the actual amounts that are being ingested. Even in large doses, biotin is considered non-toxic and is unlikely to cause any side effects. This high concentration of biotin in the serum or plasma can significantly interfere with those immunoassays reactions that use biotin-streptavidin binding in their assay, thereby leading to either falsely decreased or falsely increased results depending on the assay, resulting in a patient being misdiagnosed or treated for a wrong disease.
In general, streptavidin and biotin form a strong, highly specific and stable bond with each other. Streptavidin has an extraordinarily high affinity for biotin (dissociation constant [Kd] of 10−14 mol/L) and binds with biotin under a wide variety of chemical conditions. This system has been used for many years and allows the development of sensitive, specific and accurate immunoassay. One main reason for using biotin in immunoassay reactions is that it is a small molecule that can be attached by covalent bond to a variety of targets from large proteins such as antibodies to tiny steroid hormones with minimum effect on its biological activity.
Mechanism of biotin interference in sandwich immunoassay or non-competitive immunoassay
The two most widely used immunoassay formats are the non-competitive sandwich immunoassay and the competitive immunoassay. In a non-competitive sandwich immunoassay, the patient specimen that contains the analyte of interest, the detection or labelled antibodies and the biotinylates capture antibodies is added to a reaction vessel with a streptavidin-coated magnetic particles or solid phase. The biotinylated antibody will bind to the streptavidin-coated magnetic particles. The analyte of interest will be sandwiched between the biotinylated and detection or labelled antibody. The more analyte present in the sample, the more 'sandwich' complexes are formed and captured and the stronger the signal.
However, if there is an excess biotin in the sample, the biotin will bind to the streptavidin sites, preventing the complex formation between biotinylated antibody, labelled or detection antibody and analyte, leading to false-low result. The labelled antibody will bind to the analyte of interest, but without being bound to the solid phase and hence will be washed off, resulting in a signal that is falsely decreased, which will lead to a falsely decreased result [Figure 4]. Some of the assays that can get affected are thyroid-stimulating hormone, pituitary glycoprotein hormones, human chorionic gonadotropin, parathyroid hormone, insulin-like growth factor-1, insulin, thyroglobulin, C-peptide, ferritin, N-terminal pro b-type natriuretic peptide, prolactin and prostate-specific antigen.
|Figure 4: Mechanism of interference from Biotin in Sandwich immunoassay or Non-Competitive immunoassay. (a) Noncompetitive immunoassay without any interference from Biotin result in a true positive result; (b) High levels of biotin bind to the streptavidin sites preventing the complex formation between biotinylated antibody, labelled antibody and analyte leading to false low result|
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Mechanism of biotin interference in competitive immunoassay
In a competitive immunoassay, the patient specimen containing the analyte of interest, labelled analyte and biotinylated antibodies is added to a reaction vessel with a streptavidin-coated magnetic particles or solid phase. The biotinylated antibody will bind to the streptavidin, which is attached to the solid phase. The analyte of interest will compete with the labelled analyte for binding to the biotinylated antibodies. Residual specimen and unbound analyte (labelled or endogenous) will be washed off. The remaining signal will be inversely proportional to the concentration of analyte.
However, if there is an excess biotin in the specimen, the biotin will bind to the streptavidin sites, blocking the biotinylated antibody and hence the analyte of interest. The biotinylated antibody will bind to the analyte of interest, but without being bound to the solid phase, it will be washed off, resulting in a signal that is falsely decreased, which will lead to a falsely increased result [Figure 5]. Some of the assays , that can get affected are 25 hydroxyvitamin D, free triiodothyronine (T3), free thyroxine (T4), total T3, total T4 and cortisol.
|Figure 5: Mechanism of interference from Biotin in Competitive immunoassay (a) Competitive immunoassay without any interference from Biotin resulting in a true positive or true negative result; (b) High levels of biotin binds to the streptavidin sites preventing the complex formation between biotinylated antibody and labelled analyte analogue, leading to false high result|
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| Overcoming Biotin Interference|| |
The increasing consumption of biotin megadose has increased the risk of laboratories reporting erroneous results that could lead to misdiagnosis; thus, it is critical that laboratorians and clinicians are aware of biotin interference so that misdiagnosis and inappropriate treatment can be prevented.
- During clinical history documentation, the patient should be specifically asked for now and past prescribed and over-the-counter medicines and multivitamin supplementations, especially for biotin. If yes, the laboratory should be alerted to the presence of biotin in a patient sample 
- Increasing physician's awareness of the limitations of immunoassay testing is very important
- When results do not appear to be in consensus with the clinical picture, the possibility of interference should always be considered
- If there is any doubt about a result, clinical staff should be encouraged to contact the laboratory
- The laboratorians should become familiar with the availability of alternative assays that are free of biotin–streptavidin components to allow troubleshooting of doubtful results
- Awareness of biotin interference with the test should be increased among the doctors, patients, laboratorians and phlebotomist. Physicians and other practitioners should inquire and advise patients to abstain from biotin intake for a few days before blood draw
- The physician should know the kind of patients and patient population, they are dealing with. Although biotin has a very rapid elimination half-life of about 2 h, theoretically, most of it should clear from the body within 4–5 h. However, pharmacokinetic studies of a patient ingesting megadose (30 mg) of biotin revealed that its interfering effects on laboratory tests persisted for up to 24 h
- For patients on a mega dose of biotin, this finding means that it is prudent to stop taking biotin for at least 2 days before blood draws.
| Conclusion|| |
Awareness on interference in immunoassays is slowly increasing among the clinicians and laboratorians. Due to their ease in measuring large number of serum analytes such as hormones, drugs, tumour markers and other analytes, they have been used extensively in clinical medicine. Some of the advantages of using this technology are they can be easily automated, they have high throughput, good sensitivity and precision, require small volumes of samples and also can be quickly measured. While these immunoassays offer a number of advantages, they also have a number of disadvantages too and subject to interference from both exogenous and endogenous sources, resulting in false-positive and false-negative results.
When the immunoassay results do not appear to fit the clinical picture, it becomes a great challenge to the clinical biochemist, alarms clinicians and confounds immunoassay manufacturers. Sometimes, these results can mislead or miss a diagnosis resulting in unnecessary investigations and mental trauma to the patient and families. Hence, it is very important to be retroactive and proactive in detecting these interferences, to have knowledge about them and to take necessary actions or precautions to get the right result. A proper process needs to be in place in order to make both laboratories and physicians aware of the potential for immunoassay interference, which can lead to clinical misinterpretation. When the result does not appear to fit the clinical picture, an attempt should be made by the clinician and the laboratory to reconcile the difference. Constant communication is required between physician and laboratory about unexpected immunoassay results. Physicians should be encouraged to communicate specifically with the laboratory about discordance between results and clinical findings.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]