Review. Endogenous Antibody Interferences in Immunoassays. Types of Interference. Jane F. Emerson, MD, PhD, 1* Keane K.Y. Lai, MD 1,2 ABSTRACT

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1 Endogenous Antibody Interferences in Immunoassays Jane F. Emerson, MD, PhD, 1* Keane K.Y. Lai, MD 1,2 ABSTRACT The potential for interfering substances to cause inaccurate laboratory results that may cause significant adverse effects on patient care is well known. Immunoassays are subject to interferences that are not readily detectable prior to analysis, but may cause erroneous results. detect interference, and approaches used to resolve discrepancies between laboratory results and the clinical picture. Keywords: immunoassay, interference, heterophile antibody, antianimal antibody, autoantibody Learning Objectives After reading this article, readers should understand potential causes of interference that are specific to immunoassays, methods used to Immunoassays are subject to interferences that are not readily detectable prior to analysis but may cause erroneous results. There are numerous reports in the scientific literature of patients receiving inappropriate medical treatment based on incorrect results of immunoassays. One case illustrating the impact of an immunoassay interference was reported in Lancet 1 in the year In this case a repeated falsely high human chorionic gonadotropin (hcg) result, prompted a young woman to undergo a hysterectomy, chemotherapy, radiotherapy, and a partial pneumonectomy before the interference was discovered and her diagnosis corrected. The case was similar to a medical malpractice case reported in the Seattle Post-Intelligencer newspaper in 2001, in which a woman was awarded $15.5 million due to DOI: /LMMURCFQHKSB5YEC Abbreviations hcg, human chorionic gonadotropin; HAAAs, human antianimal antibodies; HAMAs, human antimouse antibodies; TSH, thyroidstimulating hormone; CLSI, Clinical and Laboratory Standards Institute; CK-MB, creatine kinase muscle and brain; LC-MS/MS, liquid chromatography tandem mass spectrometry; FSH, folliclestimulating hormone; AFP, alpha-fetoprotein; CA, cancer antigen; CEA, carcinoembryonic antigen; PSA, prostate-specific antigen 1 Department of Pathology, Keck School of Medicine, University of Southern California and 2 Southern California Research Center for ALPD and Cirrhosis, Los Angeles, California *To whom correspondence should be addressed. jane.emerson@usc.edu a misdiagnosis of cancer. In that case, fault was found with the physicians and the hospital laboratory. 2 This article emphasizes the potential impact of laboratory errors in immunoassays, describes the types of interference that are specific to immunoassays, and reviews general approaches to reducing errors and associated negative outcomes in patient care. Types of Interference All laboratory assays are subject to interferences. The effects of hemolysis, lipemia, and bilirubinemia (ie, icterus) on laboratory methods, for instance, are well known. Each of these may affect the analytical measurement; good laboratory practice requires validating assays with regard to potential interferences. Because hemolysis, lipemia, and icterus produce measureable spectrophotometric changes in a serum or plasma specimen, automated chemistry analyzers routinely detect these potentially interfering substances. Thus, specimens for chemical analyses are prospectively evaluated for these potential interferences. 3 Immunoassays are subject to other types of interference. Antigen-antibody interactions are the basis of immunoassays, and compounds or conditions that alter these interactions can interfere with measurement. Endogenous antibodies may bind to, bridge, or block the binding sites on capture and signal antibodies (Figure 1). Winter 2013 Volume 44, Number 1 Lab Medicine 69

2 Figure 1 Schematic of how endogenous antibodies can interfere in immunoassays to produce false-positive or falsenegative results. Capture antibody Endogenous antibody Detection antibody Antigen Antigen True Positive Solid Phase False Positive False Negative As a result, falsely high or falsely low results may occur due to the presence of endogenous antibodies. In the case reports referred to in the first paragraph of this article, the interference was positive and produced a falsely high measurement hcg on which the misdiagnosis of choriocarcinoma was made, a type of cancer characterized by high hcg levels in the absence of pregnancy. The 3 types of endogenous antibodies known to cause interferences in immunoassays are heterophile, antianimal, and autoantibodies. Heterophile antibodies are produced without exposure to specific immunogens and are thus considered to be naturally occurring. 4 Heterophile antibodies generally have low avidity but react across multiple species with the capability of binding to multiple antigens. Heterophile antibodies may react with variable avidity to distinct species; also, reactivity to different species may persist for different periods of time within the same patient. 5-7 Human antianimal antibodies (HAAAs) are produced after acute or chronic exposure to specific antigens and are species specific. 8 HAAAs generally have higher avidity than heterophile antibodies. Human antimouse antibodies (HAMAs) constitute a subset of HAAAs and are the most common of this antibody type. Antianimal antibodies may be produced 7,9 in response to therapeutic agents that include animal-derived monoclonal antibodies, or from exposure to animals occupationally (eg, veterinarians) or to pets. Autoantibodies also may interfere with immunoassays. Examples include antithyroglobulin antibodies that affect thyroglobulin immunoassays and anti-insulin antibodies that interfere with immunoassays for insulin or C-peptide. 6,7,10 High titers of potentially interfering antibodies may occur in patients with recent infections, immunizations, and transfusions. incidence Heterophile antibodies may be present in all patients, 11,12 however the potential for immunoassay interference from heterophile antibodies is less than for autoantibodies or HAAA. The overall prevalence of interfering antibodies depends on the assumptions made about their potential to cause interference. Similarly, the frequency of immunoassay interferences resulting from these antibodies depends on what magnitude of bias in the analytical method constitutes a significant interference. In any case, the frequency of interferences is much lower than would be predicted by the prevalence of potentially interfering antibodies. Improvements in assay formulations have decreased the occurrence of interferences from endogenous antibodies. Blocking reagents (typically mouse or rabbit immunoglobulins) have been added to immunoassay formulations to adsorb interfering antibodies. Thus, although the prevalence of potentially interfering antibodies has been reported to be as high as 40%, 11 the incidence of immunoassay interference is estimated to be less than 2%. 13 However, even with a low incidence 70 Lab Medicine Winter 2013 Volume 44, Number 1

3 of interference, the number of potential errors with serious consequences is still high because the number of immunoassays performed is so large. Although the prevalence of interfering antibodies varies from one patient population to another, the incidence of interference also varies by immunoassay type. Two-site assays are the most susceptible to interference. 14 Table 1 displays some assays that are relatively commonly affected. Detecting Interference Both retroactive and proactive approaches exist for detecting interfering antibodies. Retroactive refers to instances when the laboratory result is questioned because it is inconsistent with the clinical findings or exceeds extreme limits for the analyte. A proactive approach would implement a mechanism or procedure that would detect the presence of interfering antibodies prior to obtaining or reporting the result. Retroactive Approach The retroactive approach has obvious shortcomings. For example, quantitative results that are far beyond expected values will be readily flagged for certain assays such as thyroid studies; however, this is not the case Winter 2013 Volume 44, Number 1 Lab Medicine 71

4 Clinical Laboratory Responsibility The Clinical and Laboratory Standards Institute (CLSI) document, Immunoassay Interference by Endogenous Antibodies; Approved Guideline 19 states that it is the responsibility of the clinical laboratory to: Ensure the personnel performing the assay have the required knowledge of endogenous antibody interference. Contact clinicians when there is suspicion of interference in a patient specimen. Follow up on complaints about clinically inconsistent results. Notify the manufacturer/vendor of assay interference problems. Investigate mismatches between assay results and clinical information in conjunction with the manufacturer/vendor. Inform clinicians regarding the nature of the interfering antibody, including how it affects the immunoassay in question and how it may affect other immunoassay tests ordered on the patient. Certain analytes also appear in urine. Because endogenous antibodies are not usually present in urine, a discrepancy between urine and serum concentrations may suggest interference. A specimen may be preincubated with commercially available blocking reagents (some involve blocking antibodies immobilized on the inside surface of a test tube). Compare values of related analytes if applicable: for example, creatine kinase muscle and brain (CK-MB) and troponin. Many reference laboratories are capable of measuring HAMA. If HAMA interference is suspected, quantitative measurement of serum HAMA concentration may be helpful; however, no immunoassay exists, to our knowledge, that is capable of measuring equally well all types of HAMA found among patients 8 because various assays differ in the types of HAMA detected. 9,19 Also, nonimmunometric methods such as liquid chromatography tandem mass spectrometry (LC-MS/MS) are widely available for analytes such as testosterone, hydroxyvitamin D, 21 and immunosuppressants. 22,23 Table 2 summarizes key concepts to consider when investigating potential interference in an immunoassay. Troubleshooting Interferences Once interference is suspected, most clinical laboratories have the capability to investigate using a number of techniques. A multipronged approach to the investigation of suspected interference is preferable to a strategy involving only one option that may not definitively resolve the discrepancy between clinical and laboratory findings. Obtaining patient history regarding therapy with a monoclonal antibody preparation, animal exposure, or transfusions may be helpful. The linearity of the in-house assay for the specimen in question can be measured, taking care to use appropriate diluents. However, linearity might still be observed even in the presence of interfering antibodies. 6 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 nonimmunometric method. Table 2. Immunoassay Interferences: Key Concepts Detection Retroactive Extremes Clinical picture Disease prevalence Proactive Dilutions Heterophile blocking Antianimal screens Clinician education: alerts to patient history of endogenous antibodies Troubleshooting Dilutions Alternate method Heterophile blocking Measure serum HAMA concentration Test urine, if applicable Test related analytes, if applicable Patient history for therapeutic antibodies, animal exposure, transfusions or autoimmune disease HAMA, human antimouse antibody. 72 Lab Medicine Winter 2013 Volume 44, Number 1

5 Conclusions Laboratory professionals must be mindful of the potential for interference in immunoassays because of the possibility of erroneous results that adversely impact patient care. Knowledge of the incidence and mechanisms of immunoassay interferences, and the recommended troubleshooting methods, is crucial to ensure the overall quality of immunoassay results. Because proactively screening for interference in all specimens is not warranted or recommended, it is important to maintain reliable communication with other health care professionals to detect and minimize the impact of erroneous laboratory results. LM CE credit available for this article. For more information, visit pdf+html. References To read this article online, scan the QR code, ascpjournals.org/content/44/1/69. full.pdf+html 1. Rotmensch S, Cole LA. False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations. 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