Interferences in Immunoassay—Still a Threat

This is the authors' abstract. We don't add key points for this paper.

Few things in life are perfect, and unfortunately this also applies to medical diagnostic procedures such as clinical laboratory testing. In spite of the careful efforts of the developers and manufacturers of clinical laboratory tests and the vigilance of the laboratory staffs that perform the testing, most if not all tests are, on occasion, subject to interferences that lead to false-positive or false-negative results (1)(2). Immunoassay tests are no exception. Recent literature has documented false-positive or false-negative results in immunoassays or reports investigations of specific interferences (3)(4). Sources of interferences include plasma (5), serum proteins (e.g., rheumatoid factor, binding proteins) (6)(7), heterophile and anti-animal antibodies (2)(8)(9), drugs and drug metabolites (10)(11), hemolysis (12), agar (13), and cross-reacting substances (14). In most cases, a significant interference is recognized and flagged, but sometimes the interference is unsuspected and undetected. There now is adequate evidence that bad things can happen when false-positive immunoassay test results go unrecognized. The consequences are that the result is seen as a true positive, signifying underlying disease, and that further diagnostic or therapeutic procedures are initiated. Recent publications have drawn attention to the consequences of false-positive human chorionic gonadotropin (hCG) test results (15)(16). In the series of patients described by Cole et al. (15) and Rotmensch and Cole (16), the false-positive hCG results led to unnecessary surgery and inappropriate chemotherapy. This was because the patients concerned were thought to have postgestational choriocarcinoma or trophoblast disease based on their persistently increased hCG results. In addition, one of the patients developed type I diabetes and lapsed into a coma as a complication of the chemotherapy. Others have documented unnecessary surgical procedures or follow-up testing because of false-positive hCG, follicle-stimulating hormone, or thyroid-stimulating hormone results attributable to circulating antibodies with anti-rabbit specificity (17)(18). A report in this issue of Clinical Chemistry describes some clever laboratory sleuthing that has uncovered yet another cause of false-positive test results in immunoassays—broadly reactive antibodies induced by infection with a gram-negative microorganism (19). A patient with an Escherichia coli septicemia was found to have increased immunoassay results for cardiac troponin I, thyroid-stimulating hormone, hCG, α-fetoprotein, and CA-125, but none of these results was consistent with the clinical findings. Serum protein electrophoresis revealed that the patient also had a restricted IgM λ peak. This was identified as the cause of the falsely increased values in the immunometric assays. The circulating restricted antibody presumably recognized the capture and detection antibodies used in the immunoassays because it could be adsorbed with an irrelevant mouse antibody. It thus formed complexes that were indistinguishable from the complexes formed when the specific antigens bound their respective immobilized capture antibody and detection antibody. This observation may provide a new avenue of inquiry that could lead to novel strategies to block immunoassay interferences. Previously, increased concentrations of antibodies (M-spikes, paraproteins) in serum have been recognized as a source of analytical interference because of precipitation, viscosity, or binding effects (20), and the interference described in this report (19) extends the range of this particular type of interference. Public awareness of the scope and extent of medical errors has been heightened by the recent Institute of Medicine report To err is human (21). Medical errors originating from a false-positive immunoassay result have not received the same notoriety as therapeutic or surgical errors; nevertheless, they continue to be a source of concern, and if unrecognized they can have serious adverse consequences for patients. How can we guard against the false-positive or -negative immunoassay results in the future? Dialogue between the physician and the clinical laboratory over unexpected immunoassay test results is one route to avoiding inappropriate clinical intervention based on an abnormal test result. The laboratory can help in several ways. It can provide up-to-date information on assay performance, and review and discuss with the physician the test limitations as outlined in the test package insert. Further testing can be arranged to confirm the test result (i.e., repeat analysis on the specimen, retest a second specimen, or arrange for analysis by another method). In addition, dilution or blocking studies can be performed to confirm the suspicion of the presence of an interferent. Patient education programs could make patients more knowledgeable about the limitations and pitfalls of immunoassay tests and encourage patients to tell their physicians about possible sources of interferences (e.g., likelihood of having anti-animal antibodies attributable to handling of animals or prior exposure to anti-animal diagnostic or therapeutic agents). Finally, for the manufacturers of immunoassay test kits, there must be agreed guidelines for characterizing the effects of interfering substances (22), continued surveillance for new interfering factors, and refinement of immunoassays to render them interference free.