2. 404 The Immunoassay Handbook
TABLE 1 An A–Z of Sources of Interferences in Immunoassays
(Boscato et al., 1989; Boscato and Stuart, 1986, 1988; Braunstein,
2002; Diamandis, 2004; Ismail and Barth, 2001; Ismail et al.,
2002b; Itoh and Yamaguchi, 1995; Jones, 2002; Kohse and Wisser,
1990; Kricka, 1999, 2000; Kricka et al., 1990; Kroll and Elin, 1994;
Levinson, 1992; Tate and Ward, 2004; Van Kroonenburgh and
Pauwels, 1988; Weber et al., 1990)
Anti-animal antibodies (cow, goat, horse, mouse, pig, rabbit, sheep, rat)
High-dose hook effect
Human anti-animal antibodies
Insufﬁcient sample volume
Partially ﬁlled collection tubes
FIGURE 1 Sandwich immunoassay for a true antigen-positive (a) and
the mechanism of false-positive (b) and false-negative (c) interference.
TABLE 2 Pharmaceutical and Experimental Drug Agents
Derived from Animal Sources
Animal Source Agent
Chicken Hyaluronic acid
Horse Anti-thymocyte globulin, Premarin
Malayan pit viper Ancrod
Mouse Monoclonal antibody therapeutic and
Pig Factor VIII, insulin, heparin
Rat Monoclonal antibody therapeutic agents
TABLE 3 Therapeutic Monoclonal Antibodies
Name) Treatment Indication (Target)
Murine antibodies (sufﬁx—omab)
Non-Hodgkin lymphoma (CD20)
Transplant rejection (T cell CD3
Tositumomab (Bexxar) Non-Hodgkin lymphoma (CD20)
Chimeric Mouse/human (sufﬁxes—ximab)
Abciximab (ReoPro) Cardiovascular disease (glycoprotein
Cetuximab (Erbitux) Colorectal cancer, head and neck
cancer (epidermal growth factor
Inﬂiximab (Remicade) Autoimmune disorders (TNF-α
Non-Hodgkin lymphoma (CD20)
Basiliximab (Simulect) Transplant rejection (CD25)
Humanized antibodies from mouse (sufﬁx—zumab)
Bevacizumab (Avastin) Colorectal cancer, age-related macular
degeneration (vascular endothelial
Crohn’s disease (TNF-α signaling)
Daclizumab (Zenapax) Transplant rejection
Eculizumab (Soliris) Paroxysmal nocturnal hemoglobinuria
Efalizumab (Raptiva) Psoriasis (CD11a)
Gemtuzumab (Mylotarg) Acute myelogenous leukemia (CD33)
Natalizumab (Tysabri) Multiple sclerosis and Crohn’s disease
Omalizumab (Xolair) Mainly allergy-related asthma (IgE)
Palivizumab (Synagis) Respiratory syncytial virus (RSV F
Trastuzumab (Herceptin) Breast cancer
Ranibizumab (Lucentis) Macular degeneration (vascular
endothelial growth factor A)
Humanized antibodies from rat
Alemtuzumab (Campath) Chronic lymphocytic leukemia (CD52)
Ertumaxomab (Rexomun) Breast cancers (CD3E)
3. 405CHAPTER 5.3 Interferences in Immunoassay
CK-MB values (range 10–1000µg/L in 1008 blood donors)
to <3µg/L following treatment with nonimmune mouse
serum (Thompson et al., 1986). Another study demon-
strated interfering antibodies in a set of 40 samples evalu-
ated by four immunoassays (thyroid-stimulating hormone
(TSH), prostate-speciﬁc antigen (PSA), β-human chori-
onic gonadotropin (β-hCG), and cortisol) (Emerson et al.,
2003). Three different interference assessment methods
revealed an interfering antibody in 7.5–16.2% of samples
tested in a competitive immunoassay (cortisol) and 0–83.6%
of samples tested in sandwich immunoassays (Table 4).
However, less than 11% of the test interferences were clin-
ically signiﬁcant (changing the result into or out of the ref-
erence interval). Based on heterophile blocking reagent
(HBR) criterion, only 2.9% of tests had interference. Based
on serial dilution criterion, only 10.8% of tests had interfer-
ence. A large study involving testing for TSH and gonado-
tropins found a signiﬁcant interference in samples from 28
of the 5310 patients tested (Ismail et al., 2002a). Another
broad based investigation examined results on samples from
10 donors tested for 74 analytes in 66 laboratories in 10
countries and found that 8.7% of the 3445 results were false
positives due to interference (Marks, 2002).
Human anti-animal antibody responses can be of differ-
ent classes (IgG, IgA, IgM, or rarely IgE) (Chatenoud et al.,
1986; Frodin et al., 1992; Iitaka et al., 1991; McCarthy
et al., 1988), and they can have anti-idiotype, anti-isotype,
or anti-anti-idiotype speciﬁcity (Frodin et al., 1992; Reins-
berg, 1995). Generally, isotype antibodies are more com-
mon than idiotype antibodies (Lind et al., 1991), but some
instances of exclusively anti-idiotype antibodies have been
reported (Legouffe et al., 1994). The magnitude and dura-
tion of a HAMA response show great variability. The
reported serum concentrations range from µg/L to g/L
(Ledermann et al., 1988; Moseley et al., 1988) and can per-
sist in blood from weeks to months after exposure to mouse
immunoglobulin (Baum et al., 1994; Sharma et al., 1992).
Circulating antibodies with speciﬁcities for a range of
animal immunoglobulins have been reported; anti-rabbit
(IgG, IgA, and IgM) (Hiemstra et al., 1988) and anti-goat
antibodies (Larsson et al., 1981; Vandalem et al., 1980) are
particularly important because these animals are sources of
antisera for immunoassay reagents. For example, human
anti-rabbit antibodies (HARA) and human anti-horse anti-
bodies can arise following treatment with the immunosup-
pressant agents, rabbit and equine anti-thymocyte globulin
(ATG), respectively (Hiemstra et al., 1988; Harkiss, 1984).
Other anti-animal antibodies causing interferences include
anti-sheep antibody (Hunter and Budd, 1980) and anti-cow
antibody (Kwong and Teale, 1994). In addition, human
anti-chimeric antibodies have been detected in patients
treated with chimeric antibodies (Aﬁf et al., 2010; Buist et al.,
1995; Goto et al., 2009).
Not all animal-derived agents lead to antibody produc-
tion. Administration of Digibind™, a sheep anti-digoxin-Fab
used to treat digoxin poisoning, has not been reported to
lead to the formation of anti-sheep antibodies. Also, not all
antibodies cause interferences. Human anti-porcine anti-
bodies are formed in hemophiliacs treated with porcine fac-
tor VIII, but no assay interferences have been reported
(Morrison et al., 1993).
Another issue is that sequence homology between IgG
molecules from different animal species can lead to cross-
reactivity between anti-animal antibodies and animal
immunoglobulins. This has been illustrated by a study in
which interfering antibodies could be blocked with similar
efﬁcacy by mouse IgG1, mouse IgG2a, rat IgG and by
mouse, goat, or sheep serum (Sampson et al., 1994).
Another study showed that positive interferences in a two-
site CK-MB assay could be reduced by 80% using nonim-
mune animal sera in the order of effectiveness of mouse>
sheep=cow>rat>guinea pig>rabbit cat=dog (pigeon
sera had no effect) (Thompson et al., 1986).
The FDA has recognized the problems that can arise
from anti-animal antibodies and requires that the package
insert of an in vitro diagnostic device state the following as
a limitation: “As with any assay employing mouse anti-
bodies, the possibility exists for interference by human
TABLE 4 Screening for Immunoassay Interferences Using Three Methods (Emerson et al., 2003)
Interference Test ICON HBR Pretreatment Serial Dilutions
Deﬁnition of interference Positive color reaction on
negative control zone
Signiﬁcant discrepancy between
result on a 1:1 dilution before and
after HBR treatment
Signiﬁcant discrepancy between
result on a 1:1 dilution and any
Test % Positive % Positive % Positive
Cortisol (polyclonal goat anti-rabbit
capture and polyclonal rabbit conjugate)
7.5% 11.1% 16.2%
Test % Positive % Positive % Positive
TSH (polyclonal goat anti-mouse:
monoclonal mouse capture and
polyclonal goat conjugate)
5% 55.6% 38.2%
PSA (monoclonal mouse capture and
monoclonal mouse conjugate)
0% 17.5% 55%
β-hCG (polyclonal goat anti-mouse:
monoclonal mouse capture and
polyclonal rabbit conjugate)
0% 73% 83.8%
4. 406 The Immunoassay Handbook
anti-mouse antibodies (HAMA) in the sample” (Services
FDA US DoH, 1996). A subsequent FDA recommenda-
tion stated: “If the assay kit employs mouse monoclonal
antibodies, include a warning that specimens from patients
who have received preparations of mouse monoclonal
antibodies for diagnosis or therapy may contain human
anti-mouse antibodies (HAMA) and may show either
falsely elevated or depressed values when tested” (Services
FDA US DoH, 1996).
Circulating anti-animal antibodies are not just a prob-
lem for humans. In the veterinary world, canine anti-
mouse antibodies (CAMAs) were found after treatment
with mouse monoclonal agents (e.g., mouse monoclonal
antibody 231 for lymphoma), and low levels of preexisting
CAMA were found in all of the dogs tested (Jeglum, 2009).
False-positive test results have also been seen in tests
designed to detect feline leukemia virus infection in cats;
this was attributed to the presence of circulating antibodies
directed against mouse immunoglobulins. This study esti-
mated that 0.14–0.57% of cats had anti-mouse antibodies
(Lopez and Jacobson, 1989).
Antibodies to microorganisms can also be a source of
interference. Escherichia coli septicemia, in a patient with a
restricted IgM lambda paraprotein, has been associated
with increased false-positive immunoassay test results for
cardiac troponin I, thyrotropin, hCG, alpha-fetoprotein,
and CA-125. The falsely positive results could be normal-
ized (and the IgM lambda paraprotein removed) by incu-
bation with irrelevant murine monoclonal antibodies or
with formalin-killed E. coli from the patient’s infection,
thus indicating that the IgM lambda antibody response
produced an antibody that had anti-immunoglobulin
activity and caused the falsely increased assay results
(Covinsky et al., 2000).
Heterophile antibodies are antibodies produced against
poorly deﬁned antigens, and these are generally weak anti-
bodies with multi-speciﬁc activities. This is in contrast to
strong human anti-animal antibodies produced against
well-deﬁned antigens as a result of treatment with animal
immunoglobulins (Kaplan and Levinson, 1999). Hetero-
phile antibodies can bridge capture and conjugate antibod-
ies in sandwich assays but cannot compete well with the
high-afﬁnity antigens in competitive binding assays. It has
been suggested that antibodies should be called hetero-
phile when: “there is no history of medicinal treatment
with animal immunoglobulins or other well-deﬁned immu-
nogens and the interfering antibodies can be shown to be
multi-speciﬁc (react with immunoglobulins from two or
more species) or exhibit natural rheumatoid factor activity”
(Kaplan and Levinson, 1999).
Antibodies to Assay Reagents
The label in an enzyme conjugate can also be the target for
circulating antibodies. The observation of an interference
associated only with assay systems which employed horse-
radish peroxidase but not 125I as a label was suggestive of
speciﬁcity of the interferent for the peroxidase label (John
et al., 1989). Circulating antibodies that recognize an epit-
ope present only on the monoclonal antibody–enzyme
conjugate have been implicated in a positive interference
in a whole-blood tacrolimus immunoassay. Immunoab-
sorption studies showed that the interferent bound to the
conjugate of antibody and beta-galactosidase but not to
the unconjugated antibody or beta-galactosidase (Parikh
et al., 2010).
Likewise, the ruthenium (Ru) chelate label in an electro-
chemiluminescent immunoassay can be the target of inter-
fering antibodies. Anti-Ru antibodies have been found
in some sera that lead to falsely elevated fT3 results
(Elecsys® system). The antibody binds to ruthenylated
anti-T3 antibody but not the unlabeled antibody. Interest-
ingly, this antibody did not interfere with the Elecsys free
thyroxine or TSH assays both of which utilize Ru-labeled
antibody reagents, and it is thought that the fT3 assay is
more prone to interference because it uses low concentra-
tion of Ru-labeled antibodies (Sapin et al., 2007). In the
context of electrochemiluminescent assays, interference
possibly due to a circulating anti-streptavidin antibody has
been thought to be the cause of falsely high serum vitamin
D results (Khieng and Stevens, 2010).
Autoantibodies and Anti-analyte
Rheumatoid factor (RF) is a circulating IgM autoantibody
found in the serum of most patients with rheumatoid
arthritis and in other related and unrelated diseases. It is a
known interferent in immunoassays (Despres and Grant,
1998; Hamilton, 1989; Larsson et al., 1991) and in a sand-
wich assay interferes by interacting with the Fc region of
IgG reagent antibodies. RF can cause false-negative inter-
ference by blocking the capture antibody or can cause
false-positive interference by bridging capture antibody
and antibody conjugate (Larsson et al., 1991; Selby, 1999).
In one study, positive interference by RF in a cardiac tro-
ponin I microparticle enzyme immunoassay was elimi-
nated by pretreatment with polyclonal antiserum against
RF (Dasgupta et al., 1999a).
Anti-insulin antibodies can cause falsely low values of
insulin by binding insulin such that it is not able to bind to
the assay antibody reagents (Kim et al., 2011), however,
removal of such antibodies by precipitation can eliminate
this type of interference (Nishii et al., 2010).
Various studies have demonstrated the presence of anti-
troponin antibodies in different patient groups, including
the coprevalence of autoantibodies to cTnI and cTnT in
blood donors (Adamczyk et al., 2010; Nussinovitch and
Shoenfeld, 2010; Ruchala et al., 2007). These antibodies
masked the release of troponin and thus led to false nega-
tive test results (Eriksson et al., 2005). Alternatively, persis-
tently elevated levels of cTnI have been reported in a
patient with cardiac disease, and these were ascribed to a
circulating complex of cTnI and IgG (Plebani et al., 2002).
Macroprolactin is a complex of prolactin and anti-
prolactin, and this circulating complex can cause apparent
hyperprolactinemia. However, macroprolactin interfer-
ence can be eliminated in prolactin immunoassays by
either ultracentrifugation or polyethylene glycol (PEG)
precipitation (Beltran et al., 2008; Quinn et al., 2006).
5. 407CHAPTER 5.3 Interferences in Immunoassay
Paraproteins and Immune Complexes
The presence of paraproteins has been associated with
immunoassay interferences. For example, elevated serum
TSH has been reported in a patient with paraproteins
(IgG kappa and IgM lambda) that could not be blocked in
a heterophile antibody blocking tube. The interference
was speciﬁc to the UniCel® DxC 880i (no interference on
the Architect® i2000SR®) and only normalized after the
disappearance of the monoclonal bands (Imperiali et al.,
2010). Another way in which a paraprotein can cause
apparently aberrant hormone levels is by providing addi-
tional hormone-binding capacity. Increased total thyrox-
ine (T4) and triiodothyronine (T3) have also been reported
due to an IgA-lambda-secreting multiple myeloma. The
paraprotein bound both T4 and T3 and thus acted as an
additional thyroid hormone-binding protein thus increas-
ing the serum concentration of these hormones rather
than interfering in the radioimmunoassay (RIA) itself
(Cissewski et al., 1993).
In some cases of paraproteinemia, the concentration of
the paraprotein is so high as to signiﬁcantly increase serum
viscosity, and this can compromise the accurate dispensing
of the sample. A sample that is hyperviscous due to a poly-
clonal gammopathy has also been shown to cause falsely
elevated T4 values in RIAs in a method speciﬁc manner
(Tamagna et al., 1979).
Hyperviscous samples due to circulating immune com-
plexes can interfere in nephelometric methods (e.g., IgM,
IgA) as a result of precipitation of the complexes by PEG
in the reaction mixture. Interference was also found in
radial immunodiffusion methods because of failure of
immunoglobulins to migrate as a result of molecular inter-
actions (Levinson et al., 1988).
DRUGS, HERBAL REMEDIES, BLOOD
SUBSTITUTES, AND IMAGING AGENTS
Pharmaceutical preparations administered to patients for
therapeutic or diagnostic purposes (e.g., antibody-based
imaging agents) can be the source of a wide range of test
interferences including interferences in immunoassays.
A drug may alter the concentration of an analyte by an
in vivo action, and this may be the desired or undesired
effect of the drug (in vivo interference). Much more com-
monly encountered is an in vitro interference ascribable to
an effect of a drug or its metabolite(s) on an analytical reac-
tion. The panoply of drug interferences is cataloged
Some drugs cross-react with antibody reagents, e.g.,
ﬂudrocortisone derivatives cross-react to produce false-
positive cortisol results (Berthod and Rey, 1988), in others,
a metabolite cross-reacts and complicates measurement of
the drug as in the case of cyclosporin A metabolites in
immunoassays for cyclosporin A (Steimer, 1999). Other
drugs, such as dipyrone have also been shown to interfere
in immunoassays that use a peroxidase label (Gascon-
Roche et al., 1995). An interesting example of drug inter-
ference is provided by Digibind, a digoxin-Fab antibody
used in the treatment of digoxin overdose. In vitro it has
been shown to cause a negative interference in the mea-
surement of total digitoxin concentrations by both ﬂuores-
cence polarization immunoassays and to a greater extent in
chemiluminescent immunoassays via direct binding to
digitoxin (Digibind neutralized both digitoxin and digi-
toxigenin in vitro) (Dasgupta et al., 1999b).
A more recent concern has been interferences caused by
herbal remedies, especially in digoxin immunoassays,
which can be inﬂuenced by the Chinese medicines Chan Su
or Dan Shen (Dasgupta and Bernard, 2006). Ingestion of St
John’s wort is associated with abnormally low levels of
cyclosporine, digoxin, and theophylline (Dasgupta, 2003).
Contamination of a Chinese medicine with Western drugs
(e.g., phenytoin) is yet another source of false-positive
results (Dasgupta and Bernard, 2006).
Blood substitutes based on polymerized hemoglobin or
perﬂuorocarbon emulsions (e.g., Perﬂubron) are in devel-
opment as temporary oxygen carriers. A deleterious con-
sequence of the administration of such agents is that serum
and plasma samples from patients receiving hemoglobin-
based blood substitutes are red in color, whereas those
from patients receiving perﬂuorocarbon agents appear to
be lipemic. Although some immunoassays are unaffected
by the presence of a polymerized hemoglobin-based blood
substitute, others show positive (e.g., AxSym® gentamycin
assay) and negative interferences (e.g., Axsym vancomycin
and Stratus® CK-MB assay). However, in these studies, no
immunoassay interferences were found due to the pres-
ence of the perﬂuorocarbon-based blood substitute (Ma
et al., 1997).
Studies with the hemoglobin-based oxygen carrier
HBOC-201 (glutaraldehyde-polymerized bovine hemo-
globin) revealed no signiﬁcant interferences produced
by the presence of this blood substitute (in vitro
concentrations—60g/L) in various therapeutic drug
immunoassays (Callas et al., 1997). In contrast, studies
with Hemospan®, a PEG-conjugated human hemoglobin,
showed a positive interference in a cTnI immunoassay
(Beckman Access® II method) (Cameron et al., 2009).
In vitro studies with a series of 12 different contrast agents
used in coronary angiography revealed false-positive results
for all agents tested using an Opus Magnum™ cTnI assay,
but only one contrast medium (Lipiodol®; poppy-seed oil)
gave a positive result with the ACCESS cTnI assay (Lin
et al., 2006). A separate study demonstrated that both
iodine-based radiopaque contrast agents (Ioversol 350,
Iopamidol 370, Iomeprol 300, Iomeprol 400, Iohexol 300)
and gadolinium-based contrast reagents (gadopentetic
acid) interfere with immunoradiometric assays for carcino-
embryonic antigen (CEA), CA-130, and tissue polypeptide
antigen (Watanabe et al., 1998). However, in a study target-
ing interference by gadolinium-based magnetic resonance
contrast agents, no immunoassay interference was seen on
multiple immunoassay analyzers (Proctor et al., 2004).
6. 408 The Immunoassay Handbook
ICTERUS, HEMOLYSIS, LIPEMIA, AND
OTHER SAMPLE MATRIX COMPONENTS
Elevated levels of bilirubin (40 mg/dL) have been shown
to cause a statistically signiﬁcant decrease in cTnI con-
centrations (but not CK-MB assays) measured using
microparticle enzyme immunoassays, but the mecha-
nism of this interference is unclear (Dasgupta et al.,
A negative interference due to hemolysis or elevated
hemoglobin has been observed in a cTnT immunoassay.
In addition, proteases released from red blood cells during
hemolysis contribute to this effect by degrading cTnT, as
evidenced by the reduction in the negative interference by
a protease inhibitor (pepstatin A) (Sodi et al., 2006).
Dilution of the sample usually limits any adverse effects
of lipemia, but nevertheless, it has been shown to cause a
negative interference at elevated levels (>22.5g/L) in elec-
trochemiluminescent immunoassay for testosterone (Owen
et al., 2010). Also, lipemia is a source of interference in
nephelometric immunoassays (e.g., apolipoprotein B, hap-
toglobin assay) (Bossuyt and Blanckaert, 1999; Vander
Heiden et al., 1983).
Other components of the sample matrix can produce
positive interferences. This has been observed for samples
spiked with alkaline phosphatase in a Stratus ﬂuorimetric
EIA for cTnI (Dasgupta et al., 2001). This type of assay is
based on radial diffusion of sample and reagents away from
a central application zone that contains the immobilized
capture antibody. High levels of endogenous alkaline
phosphatase in a sample may not wash away from the cen-
tral zone and thus mimic captured alkaline phosphatase
conjugate, and this may lead to a false-positive result
(Dasgupta et al., 2001). Fibrinogen can also be the cause of
assay interferences when using plasma samples, but the
interference can be eliminated by thermal coagulation
(Allner, 1985). Tiny ﬁbrin strands due to incomplete sepa-
ration of serum can interfere with an immunoassay; how-
(Nosanchuk et al., 1999).
Another interesting example of assay interference arises
from matrix instability. Positive interference in a phenyt-
oin RIA was previously encountered in liquid control sera
that had been shipped internationally (>2 weeks shipping
time) (Wild, 1982). The interference was due to the release
of nonesteriﬁed fatty acids in the control sera during trans-
port. It was surmised that the nonesteriﬁed fatty acids dis-
placed phenytoin that nonspeciﬁcally bound to serum
proteins. Nonesteriﬁed fatty acids made more phenytoin
available to compete with labeled phenytoin for antibody
binding sites; thus resulting in a falsely positive increase in
False positives can also arise as a result of contamination or
from carryover during sampling on an automatic analyzer.
Good laboratory practice usually eliminates contamina-
tion of specimens. Generally, laundry systems on immu-
noassay analyzers are optimized so that carryover due to
successive transfer of residual sample on a dispensing
probe is extremely low (e.g., <0.001%) (Matsushita et al.,
1996). A further issue speciﬁc to the sample type is that the
presence of ﬁbrin in serum samples leads to false-positive
test results in immunoassays for cTnI concentrations
(AxSym® analyzer method) (Kazmierczak et al., 2005;
McClennen et al., 2003).
BLOOD COLLECTION TUBES
A blood collection tube is a complex device that is fabri-
cated from multiple components made of different
materials that can interact with the components of a
specimen or shed interfering material into the specimen.
In the past, this has been a particular issue for therapeu-
tic drug monitoring due to drug adsorption to the sepa-
rator gel (Bowen et al., 2010), also the different
preservatives present in collection tubes and storage
conditions can impact test results (Tate and Ward, 2004;
Bowen et al., 2010; Evans et al., 2001). Likewise, silicon-
ized plastic tubes have been shown to cause false-nega-
tive results in ACTH RIAs and false-positive results in a
C-reactive protein immunoassay (Chang et al., 2003;
Galligan et al., 1996).
More recently, immunoassay platform-dependent posi-
tive interferences in competitive assays, especially total T3,
have been identiﬁed and attributed in part to a component
of serum separator tubes, the organosilicone surfactant
Silwet® L-720 (Bowen et al., 2005, 2007). This surfactant
was shown to displace capture antibody from the solid sup-
port, and thus in an assay, bound label would be lost, and
this would mimic high concentrations of the analyte.
Indeed, there is a cocktail of surfactants in each collection
tube, and there is a potential for each of these additives to
interfere with an immunoassay.
This particular example of tube additives causing inter-
ference is an important reminder that controls for an assay
operate at their best when they are treated exactly the same
as a patient sample. The standard practice for most labora-
tories with automated immunoassays is to have the control
reagents stored within the machine where it is directly
sampled by the machine when necessary. In this type of
process, the control reagents do not come into contact
with the collection tubes, which in the case of collection
tube additives was a missed opportunity to detect interfer-
ence (Kricka et al., 2005).
CROSS-REACTIVITY OF ANTIBODY
In the early days of immunoassays, some antisera were
cross-reactive, and this caused false positives (e.g., lutein-
izing hormone [LH] in hCG assays) (Thomas and Segers,
1985). Nowadays, highly speciﬁc antibodies with low
cross-reactivity predominate; cross-reactivity can still be a
problem in some assays due to the presence of structurally
similar substances present in the sample (e.g., drugs or
drug metabolites, or hormones that share a subunit)
(Steimer, 1999; Datta et al., 1996). The problem of cross-
reactivity is speciﬁcally addressed in immunoassay package
inserts in the SPECIFIC PERFORMANCE CHARACTERISTICS—
SPECIFICITY section. This section states the cross-reactivity
of the assay antibody with relevant candidate interferents.
7. 409CHAPTER 5.3 Interferences in Immunoassay
For example in the case of hCG assays, the FDA has rec-
ommended that “speciﬁcity studies be performed on speci-
mens with high physiological concentrations of luteinizing
hormone (LH), follicle stimulating hormone (FSH), and
thyroid stimulating hormone (TSH). High levels of LH
should not signiﬁcantly cross-react with the hCG antibody
used. Similar studies may also be performed with human
placental lactogen (hPL) and human growth hormone
(hGH). Spiking of samples may be necessary” (Services
FDA US DoH, 1996).
HIGH-DOSE HOOK EFFECTS
Another source of interference in the form of a false nega-
tive is attributable to a “high-dose hook effect.” In this
situation, a high concentration of an analyte gives similar
response to that of a much lower concentration due to
saturation of the capture antibody and the antibody conju-
gate by the high concentration of analyte (Akamatsu et al.,
2006). A similar effect can be seen in both qualitative and
quantitative hCG assays due the presence of hCGβ, a sub-
unit of the analyte (Grenache et al., 2010; Gronowski et al.,
2009). In some assays that do not detect hCGβ, falsely
decreased results can also occur because the competing
hCGβ molecule saturates just one of the antibodies
(Grenache et al., 2010).
Strategies to Identify
Potential Cases of Assay
There have been many strategies proposed for the detec-
tion of assay interference within the context of the clinical
laboratory. Many of these methods are technical tests,
measurements, or treatments that can be performed in the
laboratory, but a powerful tool for discovering and con-
ﬁrming the presence of assay interference is recognition of
the clinical context of the test. Indeed, in many cases of
medical misadventure and poor patient outcome, the clini-
cian ordering the test does not initially believe the patient’s
test result. But because they are not aware of immunoassay
interference, they repeat the erroneous test enough times
to convince themselves of the test result’s clinical signiﬁ-
cance. Sometimes, the tragic consequence is unnecessary
surgery, chemotherapy, and/or sterilization.
In the ideal world, the clinician faced with a test result
that does not ﬁt with the patient’s presentation will con-
sult the laboratory. Whenever a clinician (or patient)
contacts the laboratory with doubt about an immunoas-
say result, this is an opportunity to consider the possibil-
ity of assay interference. If this opportunity is missed,
then there is little use for knowing the myriad of meth-
ods available for evaluating and proving immunoassay
In addition to increasing awareness of immunoassay
interference among clinical colleagues, the laboratory can
identify potential cases of interference by examining the
consistency of test results with other laboratory values and
evaluating the probability of the test result in the context
of the prevalence of disease (Bayesian analysis).
There is not always a proper venue to properly educate
clinical colleagues to the possibility of immunoassay inter-
ference. The usual opportunities to educate clinicians
within the context of a hospital laboratory may be through
participation in teaching rounds, clinical conferences, lab-
oratory newsletters, and notiﬁcations through electronic
medical record and laboratory test ordering systems.
Another opportunity is to educate colleagues in anatomic/
surgical pathology that may interact regularly and closely
with clinicians on speciﬁc cases. In our experience, this is
particularly effective in the setting of oncology conferences
where immunoassays for tumor markers are routinely dis-
cussed. A surgical pathologist that is present to discuss his-
tologic ﬁndings may also seize the opportunity to mention
the possibility of immunoassay interference when the discus-
sion arises of an unexpected serum tumor marker result.
EXAMINATION OF PLAUSIBILITY
OF TEST RESULTS
A comprehensive interpretation of immunoassay results
can be performed in the context of additional laboratory
testing or in the context of statistical probability based on
disease prevalence. These types of tools can be used in
quality assurance audits or can be potentially automated
within the laboratory information system.
Regarding the interpretation of test results in the con-
text of all available laboratory data, a recent study of para-
thyroid hormone (PTH) identiﬁed potential cases of
heterophile antibodies by excluding the cases that had a
“clinically plausible reason” as determined by laboratory
values (Cavalier et al., 2008). This study interpreted
PTH in combination with serum 25-hydroxyvitamin D
(25VTD), ionized calcium, and estimated glomerular ﬁl-
tration rate (eGFR). A rule was set that ﬂagged an elevated
PTH as suspicious when 25VTD, ionized calcium, and
eGFR from the same patient were all within their respec-
tive normal reference ranges. Based on this rule, 9% of
samples with elevated PTH were deemed suspicious and
were treated in heterophile antibody blocking tubes
(Scantibodies, Santee, CA). Of these suspicious samples,
40% had a decrease in PTH after treatment with blocking
tubes; half of these samples with a decrease had a ﬁnal
value that fell into the normal reference range. The
remaining 60% of suspicious samples (unchanged by het-
erophile blocking) were then examined for the presence of
RF; if the sample was RF positive, then the sample was
treated with an RF precipitating reagent (IBL, Hamburg,
Germany), and this revealed that 24% of heterophile
negative samples had interference attributable to RF.
This method of identifying suspicious samples has the
potential for wider implementation in laboratories with
sophisticated laboratory information systems. Indeed, as
a matter of routine quality assurance, rules can be written
into information systems to ﬂag suspicious immunoassay
results that are not plausible based on the context of
other laboratory test results. In addition to PTH, thyroid
function testing and other hormone immunoassays are
usually part of a multi-analyte or multiparameter analysis
and would be amenable to this identiﬁcation strategy.
Based on the identiﬁcation of suspicious cases, a more
8. 410 The Immunoassay Handbook
intensive chart review or laboratory investigation could
Another powerful tool in the identiﬁcation of immuno-
assay interference is to determine the probability of a test
result based on the disease prevalence. Previous studies
have examined the utility of statistical methods in uncover-
ing laboratory errors (Le et al., 2011; Oosterhuis et al.,
2000). A recent study applied the statistical method of
Bayesian analysis to identify immunoassay interference
(Ismail and Ismail, 2011). The authors of this study dem-
onstrated the potential of Bayesian analysis in identifying
interference with various analytes including TSH, PSA,
hCG, and troponin. In the example of PSA, the starting
point of analysis is the recognition of prostate cancer prev-
alence of approximately 1 in 5000 (0.02%) among asymp-
tomatic 50-year-old men. Using the 0.4% false-positive
rate of immunoassays reported in the literature, the calcu-
lated probability of a false-positive test is approximately
95%. With increasing age, the prevalence of disease
increases, and therefore, the false-positive rate decreases.
Thus, elevations of PSA at a younger age are more suspi-
cious than at an older age. In the example of TSH, sub-
clinical hypothyroidism has a prevalence of 1% in young
adults and children compared to a prevalence of 17% in
elderly females. Again, assuming a false-positive immuno-
assay interference of 0.4%, a raised TSH in elderly female
patients would be false in 2% of cases compared to 30% of
cases in young adults or children.
Reexamining all immunoassay results in a clinical labo-
ratory is not practical, but developing a targeted approach
that can be automated within the laboratory information
system may provide an additional level of security in the
identiﬁcation of immunoassay interference.
Strategies to Prove the
Presence of Interference
Once a potential interference has been identiﬁed, there are
several methods of proving that the test result is due to
interference (Sturgeon and Viljoen, 2011).
The simplest and ﬁrst check for an immunoassay assay result
that is outside the reference range is to perform a linear
dilution. Immunoassay interferences including heterophile
antibodies do not typically dilute linearly. Dilution ratios of
1:5 and 1:10 are commonly used for such studies, and
Table 5 (Landau-Levine et al., 1999; Pan and Wang, 2011;
Zhu et al., 2008) shows some examples of results of dilution
studies for specimens containing an interferent.
TEST BY ANOTHER METHOD
Immunoassay interferences can be speciﬁc to the antibody
clones used in the assay. When interference is suspected,
the sample should be tested by other methods. The most
important consideration is that the alternative method
must use different antibody clones. It is common to ﬁnd
immunoassay reagents from different manufacturers that
use the same antibody clones for a given analyte. Further-
more, if the sample is sent to a third party laboratory to be
tested, the speciﬁc method and antibody clones used
should once again be veriﬁed.
Since urine is free of immunoglobulins, examining urine
for the analyte of interest may be more reliable. For
TABLE 5 Examples of Dilution and Blocking Studies for Specimens Containing Interferents: Patient A: Positive Interference due to HAMA.
Patient B: Positive Interference and Use of HBT. Patient C: Positive Interference and Use of IgG Mixture as Blocker
for Dilution Comments
Patient A (Pan and
BNP assay (pg/mL)
Untreated 1 1551 Does not dilute linearly. HAMA assay was
positive (40.5ug/L) thus conﬁrming HAMA as
Untreated 2 1061
Untreated 5 247
Untreated 10 31.9
Untreated 100 2.56
Patient B (Zhu
et al., 2008)
Untreated 1 19.99 Initial result differed from TnI test performed on
patient by another method
Untreated Diluted 1.46 Does not dilute linearly (93% decrease)
Treated in a HBT 1 0.03 Reduction in test value after incubation in HBT
indicates a heterophile antibody interference
Patient C (Landau-Levine
et al., 1999)
Untreated 1 9.9 Initial result differed from TSH test performed
by another laboratory
+Mouse and goat IgG 1 1.8 Reduction in test value after incubation with IgG
mixture indicates an interference, conﬁrmed as
HAMA by assay (44 µg/L)
9. 411CHAPTER 5.3 Interferences in Immunoassay
example, with β-hCG, the serum test is susceptible to
interference, but the urine is not. This property of urine is
the basis of a strategy recommended by American
Congress of Obstetricians and Gynecologists (ACOG) in
the case of false-positive serum hCG test results (ACOG.
TREAT THE SAMPLE TO REMOVE
There are several chemical treatments and multiple com-
mercially manufactured reagents available to remove or
block heterophile antibodies from a sample. Strategies that
involve relatively mild conditions to remove interferents
include PEG precipitation and exposure to immobilized
Protein A or Protein G. For more stable analytes such as
CEA, acid extraction or heat inactivation can be used to
remove interferents (Primus et al., 1988). However, a par-
ticularly convenient method is to use a blocking tube—
e.g., a Heterophilic Blocking Tube (HBT) or a Nonspeciﬁc
Antibody Blocking Tube (NABT) (Scantibodies Labora-
tories, Inc. Santee, CA) (http://www.scantibodies.com/
blockers.html). A sample of the specimen is incubated in
the tube and the reagents contained in a lyophilized pellet in
the tube bind to any interferents in the sample. This type of
pretreatment procedure is only intended to conﬁrm the
original assay result or show the result to be incorrect due to
the presence of an interferent—it cannot be used to gener-
ate a reportable result. Another nonspeciﬁc sample pretreat-
ment procedure is to add animal immunoglobulin or
nonimmune serum. This may not always be successful, and
in one case, blocking of the interference required the spe-
ciﬁc monoclonal antibody that had been infused into the
patient and also required prolonged incubation with high
concentrations of the antibody (Kricka et al., 1990). There is
also a range of specially formulated blockers from a number
of commercial sources (see BLOCKERS AND TEST PANELS).
Measures to Prevent
and Existing Regulatory
The time to identify an immunoassay’s susceptibility to
interference is during the development and validation of
the test prior to clinical use. As well as checking the immu-
noassay reagents for interference, preanalytical variables
should also be examined. These variables may include
drugs the patient may be taking that will be present in the
serum, the type of anticoagulant the sample will be col-
lected with, and ﬁnally the type of tube that is used for
collection (see BLOOD COLLECTION TUBES).
BLOCKERS AND TEST PANELS
Many commercial assays include blocking agents, and
there are panels of samples that are positive for interferent
(e.g., heterophile antibodies, RF) that can be used to assess
and validate the effectiveness of the blocking agent chosen
for an assay.
As exempliﬁed by a recent study of a cytokine immuno-
assay targeted at patients with autoimmune disease, when
targeting a patient population with a high potential for
interference, multiple heterophile antibody blocking
reagents can be surveyed to ﬁnd the best reagent to elimi-
nate interference (DeForge et al., 2010). In addition to
identifying a blocking reagent during the test validation,
the various blocking reagents can be examined to see if
they are compatible with the test (e.g., examine whether
the blocking reagents cause interference).
Different formulations of blocking agent are available
from several companies including Scantibodies Labora-
tories, Inc. (Santee, CA), Bioreclamation, LLC (Hicks-
ville, NY), Millipore (Danvers, MA), IBL International
(Hamburg, Germany), and Meridian Life Science Inc.
REGULATIONS AND STANDARDS
The general concept of analytical interference in the clini-
cal laboratory has been well recognized for several decades.
For immunoassays, laboratories have been compelled by
federal law (CLIA ‘88) to check for common interferences
such as lipemia, hemolysis, and icterus during the validation
of a new test (Medicare, 1992). Immunoassay interference
must also be determined by the clinical assay manufacturer
and is a standard requirement for the clearance or approval
of a new immunoassay by the U.S. Food and Drug Admin-
istration. However, there is minimal regulatory guidance
on the appropriate approach to interference from endoge-
nous antibodies. There is a recent comprehensive CLSI
document, which is recognized by the FDA as a complete
standard (Clin. Lab. Stds. Inst., 2008).
The CLIA ‘88 requirements for assessing the effect of
interference on clinical laboratory tests are extended by
organizations with deemed status for the accreditation and
inspection of clinical laboratories in the United States (e.g.,
College of American Pathologists, Joint Commission).
Clinical Consequences of
The following clinical cases illustrate some dire conse-
quences of immunoassay interferences.
There are now many documented case reports and case
series of clinically signiﬁcant immunoassay interference.
However, one case in particular gained tremendous media
attention in the United States because of the clinical con-
sequences, timeline to resolution, and eventual economic
judgment through the judicial system. Due to a falsely
positive serum hCG, Jennifer Rufer was subjected to doz-
ens of clinical tests, radiological imaging, multiple doses of
chemotherapy, lung surgery, and hysterectomy (Rufer,
Ms Rufer initially presented with abdominal pain and
vaginal bleeding and was diagnosed as having an ectopic
pregnancy by an obstetrician–gynecologist. She had an
elevated serum hCG determined by a commercial
10. 412 The Immunoassay Handbook
laboratory; this was consistent with ectopic pregnancy, and
the patient was treated with low-dose chemotherapy.
However, the hCG did not decrease with treatment and so
she was referred to a gynecologic oncologist at a Univer-
sity hospital. She continued to have a persistently elevated
serum hCG determined by the University hospital labora-
tory; this resulted in her being diagnosed with gestational
trophoblastic disease and she was subjected to higher doses
of chemotherapy, hysterectomy, and lung surgery for sus-
picion of metastatic disease.
Unfortunately, both the commercial laboratory and
University hospital were using hCG assays from the same
manufacturer. The laboratory director at the University
hospital conducted an extensive investigation of the test by
examining different batches of reagent and performing
dilution studies on the patient’s sample. Ms Rufer’s sample
was the only sample, out of 50 that were tested, that failed
the dilution analysis. This result was reported to the man-
ufacturer, but the manufacturer did not subsequently
respond to the clinical laboratory’s request for assistance
in interpretation. The possibility of falsely elevated hCG
due to interference was not well known in the medical lit-
erature at that time. The package insert from the manufac-
turer stated the following limitations:
1. For diagnostic purposes, the hCG results should be
used in conjunction with other data: e.g., symptoms,
results of other tests, clinical impressions.
2. Elevated hCG levels have been associated with
some abnormal physiological states and should be
considered if consistent with clinical evidence.
3. Specimens from patients who have received prepa-
rations of mouse monoclonal antibodies for diagno-
sis or therapy may contain HAMAs. Such specimens
may show either falsely elevated or depressed values
when tested with assay kits which employ mouse
4. Infrequently, hCG levels may appear consistently
elevated due to the presence of heterophilic anti-
bodies or to nonspeciﬁc protein binding. If the hCG
level is inconsistent with clinical evidence, results
should be conﬁrmed by an alternate hCG method.
Finally, the University laboratory sent the sample to two
other clinical laboratories: one that used a different manu-
facturer found a normal result; the second used the same
manufacturer as the University laboratory and similarly
found an elevated level. The manufacturer performed its
own internal assessment and initially concluded that the
assay was performing as intended. The University hospital
physicians eventually determined that Ms Rufer’s elevated
serum hCG was due to interference and her medical treat-
ments had all been unnecessary, Ms Rufer sued both the
University hospital and the manufacturer of the hCG test.
The University hospital was sued for malpractice, and the
manufacturer of the test was sued for product liability. The
product liability was because it was believed that the man-
ufacturer had prior knowledge and had not warned physi-
cians that their test could produce false-positive results
leading to misdiagnosis and unnecessary treatment for
gestational trophoblastic disease. The package insert was
found to be inadequate because it did not specify that het-
erophile elevations of the assay could actually be false,
phantom, or otherwise analytically in doubt. Furthermore,
it was found that the manufacturer had received over 40
complaints of false-positive results including multiple
cases similar to Ms Rufer’s with unnecessary chemother-
apy and surgery. In the fall of the same year that Ms Rufer
was initially diagnosed, the manufacturer had drafted, but
never sent out, a general letter to physicians recommend-
ing a urine test to conﬁrm serum hCG results that were in
doubt. A jury trial awarded Ms Rufer and her husband $16
million in damages with 50% of the fault placed on the
The Rufer case has created heightened awareness among
clinicians, laboratorians, and test manufacturers of immu-
noassay interference by endogenous antibodies. Some
important lessons include:
1. Immunoassay should not be used as a sole diagnos-
tic tool; it must be used in conjunction with other
clinical ﬁndings, imaging studies, or laboratory
2. False immunoassay results can have catastrophic
3. Concerns or doubts by clinicians of test results
must be treated with the utmost respect and be
4. Laboratories cannot place the responsibility of
investigation solely on the test manufacturer.
5. Laboratories in the United States potentially hold
equal liability with the manufacturers of the tests
MALE UROLOGIC ONCOLOGY PATIENTS
WITH HETEROPHILE INTERFERENCE
In a series of cases reported in 2000, it was observed that
serum markers of testicular cancer can be falsely elevated
in the absence of active disease (Morris and Bosl, 2000);
however, this series did not identify the mechanism of false
positivity, and heterophile antibodies were not described
as a possible etiology. Subsequently, there have been sev-
eral cases of heterophile antibodies with clinical signiﬁ-
cance in urologic oncology patients.
In one case, a 39-year-old male with stage IIB metastatic
malignant germ cell tumor (GCT) and elevated serum
hCG underwent orchiectomy with three cycles of combi-
nation chemotherapy (Ballieux et al., 2008). Serum hCG
initially normalized, but then 2 months later, hCG was
elevated. Patient underwent another operation for retro-
peritoneal lymph node detection which was negative for
tumor. Serum hCG became further elevated and a chest
CT-scan revealed enlarged mediastinal lymph nodes. The
patient was then treated with second and third-line che-
motherapy which did not normalize his serum hCG.
Reanalysis revealed that the serum samples prior to the
ﬁrst round of chemotherapy had conﬁrmed elevated serum
hCG; however, all subsequent serum hCG elevations were
found to be false positives.
Additional cases of heterophile interference in male
patients include a 44-year-old man with an orchiectomy for
low-stage nonseminomatous GCT who has a persistently
elevated hCG after chemotherapy (Gallagher et al., 2010),
and a 43-year-old man with orchiectomy for low-stage
11. 413CHAPTER 5.3 Interferences in Immunoassay
seminomatous GCT who had a falsely elevated hCG at his
routine 5-year follow-up (Trojan et al., 2004).
THE ANIMALS WE LOVE
Human Anti-rabbit Antibody (HARA)
A bad outcome from anti-rabbit antibodies is illustrated by
a case of a woman in her late twenties who presented with
infertility and amenorrhea (Berglund and Holmberg, 1989).
High serum FSH values were noted, and this led to a series
of unnecessary diagnostic procedures, including laparos-
copy, laparotomy, and an ovarian biopsy. After a few
months, the patient’s regular menstrual cycle resumed
spontaneously. However, her FSH levels remained elevated.
Reanalysis of her samples with a goat antibody-based assay
gave normal values for FSH.
A better outcome from anti-rabbit antibodies is illus-
trated by a case of a 52-year-old woman who was referred
for further investigation because of a 9-year history of per-
sistently raised fasting concentrations of gut hormones.
Irritable bowel syndrome had been diagnosed 16 year pre-
viously (Park et al., 2003).
Previous investigations included computed tomography
scan of the abdomen, magnetic resonance imaging of the
pancreas, and an octreotide scan. All of these imaging
studies were normal; therefore, the abnormal blood test
results were attributed to hyperplasia of pancreatic islet
cells. The only drug she was receiving was estrogen from
an estrogen implant. Two yearly magnetic resonance
imaging studies were normal, but there was a suggestion of
hyperplasia of the pancreatic islet cells.
On referral, many of the patient’s fasting gut hormone
concentrations were still elevated (vasoactive intestinal
polypeptide 120pmol/L [normal, <30], pancreatic poly-
peptide 72pmol/L [<300], gastrin 125pmol/L [<30],
glucagon 63pmol/L [<50], somatostatin 103pmol/L
[<150], and neurotensin 101pmol/L [<100]). Imaging of
the abdomen and an octreotide scan were normal, and the
patient was scheduled for pancreatic angiography with cal-
cium stimulation to ascertain whether she had abnormal
functioning islet cells.
Because it is unusual for a neuroendocrine tumor to
secrete more than one hormone, the possibility of interfer-
ing antibodies was considered for this patient. A more
detailed clinical history from the patient revealed that she
and her husband had kept large numbers of pet rabbits (as
many as 80 at one time) and that she had a presumed rabbit
induced allergic rhinitis. A further link to rabbits was that
her husband was an ofﬁcer of the British Rabbit Council.
The RIAs used to measure gut hormones in this patient
were all based on rabbit antibodies. The gastrin assay was
selected for blocking studies, which were performed by
adding small concentrations of nonimmune rabbit serum
to the gastrin assay buffer. The original gastrin result was
elevated at 95pmol/L (no rabbit serum added). Addition of
0.5 and 1% of rabbit serum reduced the gastrin result to
<20pmol/L, which is in the normal range (<30pmol/L).
The angiography was canceled, and the patient was dis-
charged with the diagnosis of presumed irritable bowel
syndrome with heterophilic (rabbit) antibodies interfering
with the various gut hormone assays.
Human Anti-goat Antibody (HAGA)
(Alvarez and Scott, 1993)
An 84-year-old woman had a discordant, elevated creatine
kinase (CK) isoenzyme by immunoassay (Stratus CK-MB
assay result, 12–15fg/L) and by electrophoresis (>95% MM
isoenzyme, no detectable MB). Mouse IgG was added to
test for HAMA and had no effect, indicating that the sam-
ple probably did not contain HAMA. However, the addi-
tion of normal goat serum reduced the measured CK-MB
to 1.7fg/L. This suggested that a human anti-goat antibody
was the most likely cause of the interference. The Stratus
CK-MB assay includes goat IgG as a component of the
anti-CK-MB-alkaline phosphatase conjugate reagent.
When a CK-MB conjugate that did not contain goat IgG
was utilized, the interference similarly disappeared. It was
surmised that the interfering antibody reacted with the
goat IgG, and the resulting complex caused unreacted
conjugate to be retained in the central measurement
zone of the radial diffusion device used in the Stratus
THE DRUGS WE TAKE (PARIKH ET AL.,
A 35-year-old man with end-stage renal disease received a
cadaveric renal transplant. According to protocol, on the
day of transplantation, his immune suppression was initi-
ated by rabbit-ATG; on the day after transplantation, he
continued immunosuppression by mycophenolate mofetil,
prednisone, and tacrolimus. Twelve hours after his ﬁrst
1mg dose of tacrolimus, his whole-blood concentration
was determined to be 24.4ng/mL by a Siemens Dimen-
sion® RxL immunoassay. This tacrolimus level was
elevated since the target whole-blood concentration is
5–15ng/mL after transplantation. The patient continued
to do well clinically without signs or symptoms of drug
toxicity or rejection and had repeat measurements of
tacrolimus. Tacrolimus concentrations remained elevated
at 24.0 and 23.6ng/mL on the third and fourth days after
transplantation, respectively. The patient was assessed for
RF, but this was negative. After obtaining the elevated
tacrolimus level on the fourth day of transplantation, the
clinical laboratory had the timely insight to send a blood
sample to a reference laboratory for tacrolimus measure-
ment by liquid chromatography–tandem mass spectrome-
try (LC–MS/MS). The reference laboratory determined
the tacrolimus level to be <2ng/mL. Therefore, the
patient’s tacrolimus dosage was increased to 2mg twice a
day. The patient continued to have tacrolimus levels per-
formed on LC–MS/MS, and these levels began to fall
within the therapeutic range.
The patient’s sera were investigated for interference by
HAMA, but treatment by HBR (Scantibodies Lab. Inc) or
irrelevant murine monoclonal antibodies did not remove
the interference. The sera were then investigated for anti-
bodies directed against the beta-galactosidase label by
examining the sera’s ability to interfere in the Siemens
RxL cyclosporine A immunoassay that utilizes the identi-
cal beta-galactosidase conjugate. The patient’s sera dem-
onstrated no interference on the Siemens cyclosporine
assay. Subsequent investigation revealed the interfering
antibody to be directed against an epitope generated by
12. 414 The Immunoassay Handbook
the conjugation of the anti-tacrolimus monoclonal anti-
body to the beta-galactosidase reporter.
This case illustrates how interfering antibodies can defy
the standard methods of investigating for assay interfer-
ence by endogenous antibodies. The authors of this case
report suggest a potential solution for uncovering interfer-
ence in immunoassays used for therapeutic drug monitor-
ing. They recommend performing the ﬁrst measurement
of the drug of interest before initiating immunosuppres-
sion. Thus, the false-positive detection by interference can
be immediately identiﬁed.
Immunoassay is a powerful technology, however, it should
be remembered that it is an estimate for biological pro-
cesses. Caution should be taken by treating physicians
when the result of an immunoassay does not ﬁt the overall
clinical picture of a patient. As laboratorians, our role is to
be vigilant to the concerns of clinicians and patients. We
are not only responsible for identifying immunoassay
interference in the context of extreme results, but we need
to develop processes for identifying immunoassay interfer-
ence so that potentially disastrously erroneous results are
not reported on patients.
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