Review

Endogenous Antibody Interferences

in Immunoassays
Jane F. Emerson, MD, PhD,1* Keane K.Y. Lai, MD1,2

ABSTRACT detect interference, and approaches used to resolve discrepancies

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.
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 Lancet1 in the
year 2000. 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: 10.1309/LMMURCFQHKSB5YEC
Abbreviations
hCG, human chorionic gonadotropin; HAAAs, human antianimal
antibodies; HAMAs, human antimouse antibodies; TSH, thyroid-
stimulating hormone; CLSI, Clinical and Laboratory Standards
Institute; CK-MB, creatine kinase–muscle and brain; LC-MS/MS,
liquid chromatography–tandem mass spectrometry; FSH, follicle-
stimulating 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 2Southern California Research Center for ALPD
and Cirrhosis, Los Angeles, California
*To whom correspondence should be addressed.
E-mail: 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).

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 Review
Figure 1
Schematic of how endogenous antibodies can interfere
in immunoassays to produce false-positive or false-
negative results.
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
produced7,9 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
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Capture antibody
Endogenous antibody
Detection antibody
Antigen
Antigen
Solid Phase
True Positive False Positive False Negative
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
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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
for most tumor-marker assays. Thyroid-stimulating
hormone (TSH) concentrations characteristic of thyroid
disease, for example, vary to a far lesser degree than
hCG concentrations in pregnancy or in certain cancers.
Therefore, use of thresholds based on physiologically
unreasonable results to alert technologists of a potential
interference is not practical in all immunoassays.
Cases in which the results are erroneously normal, or
abnormal but not implausible, are more difficult to detect.
Retroactive detection of false positive or false negative
results usually requires knowledge of the clinical picture.
Laboratories do not typically receive sufficient clinical
information to determine whether the laboratory result is
consistent with the patient’s condition. Also, the clinical
picture is often complex, rendering interpretation of
the result in clinical context beyond the scope of the
personnel responsible for reviewing and releasing the test
results. Thus, retrospective detection of immunoassay
interferences relies heavily on healthcare providers
who may have limited awareness of the potential for
immunoassay interference.
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An algorithm has been suggested15 that would compare
immunoassay results to the prevalence of disease. In this
approach results that are positive for a disease of low
prevalence, or negative for a disease of high prevalence,
are considered suspect.
Proactive Approach
Various methods have been proposed to detect or
block the effects of interfering antibodies. Suggested
approaches include looking for discrepancies among
alternative methods of measurement,6,9,16 serial dilutions to
reveal nonlinearity,6,9,16 screening for antimouse antibodies
with the Tandem ICON ImmunoConcentration human
chorionic gonadotropin assay9,16 (Hybritech Inc, San Diego,
CA) or pretreating specimens with blocking reagents.16-18
A study16 investigating the general usefulness of some of
these approaches concluded that introducing a protocol
to prescreen all samples for the presence of endogenous
interfering antibodies is not warranted because the
approaches were associated with too low an event rate to
justify routine implementation or with too high a prevalence
and were too nonspecific to be useful.16
Another proactive approach would be to request pertinent
patient history, such as previous treatment with monoclonal
antibody preparations or a history of erroneous laboratory
results. This approach would be difficult to implement and
is likely to be an ineffective strategy because it is unknown
how many cases of interference could be predicted with this
type of information.
Table 1. Immunoassays Commonly Affected
by Endogenous Antibodies
Endocrine Substances Tumor Markers Other
Cortisol16 AFP26 CK-MB9
Estradiol9 CA 1259 Ferritin29
Free thyroxine9 CA 15-327 Hepatitis B surface
FSH9 CA 19-928   antigen9
LH CEA
9 9
Troponin I9
Progesterone9 hCG9
Prolactin9 PSA16
Testosterone24
Thyroglobulin25
Thyroxine9
Triiodothyronine9
TSH9
FSH, follicle-stimulating hormone; LH, luteinizing hormone; TSH, thyroid-
stimulating hormone; AFP, alpha-fetoprotein; CA, cancer antigen; CEA,
carcinoembryonic antigen; hCG, human chorionic gonadotropin; PSA, prostate-
specific antigen; CK-MB, creatine kinase–muscle and brain.
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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.
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.
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• 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 patients8 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, 20
25-hydroxyvitamin D, 21 and immunosuppressants. 22,23
Table 2 summarizes key concepts to consider when
investigating potential interference in an immunoassay.
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.
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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
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