Review Article

Analytical error and interference in immunoassay: minimizing risk

Catharine M Sturgeon1 and Adie Viljoen2

1
Department of Clinical Biochemistry, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh EH16 4SA; 2Department of
Clinical Biochemistry, Lister Hospital, Stevenage, Hertfordshire SG1 4AB, UK
Corresponding author: Catharine M Sturgeon. Email: C.Sturgeon@ed.ac.uk

Abstract
Although generally robust, immunoassays remain vulnerable to occasional analytical errors that may have serious
implications for patient care. Sporadic errors that occur as a result of properties of the specimen are particularly difficult
to detect. They may be due to the presence of cross-reacting substances, antianalyte antibodies or antireagent
antibodies, all of which may lead to erroneously high or low results. Low results may be observed for tumour markers due
to high-dose hooking in the presence of very high analyte concentrations. Erroneous results can occur unexpectedly with
any specimen and there is no practical means of identifying specimens likely to cause problems in immunoassays. The
possibility of interference should always be considered when results do not appear to be in accord with the clinical
picture. Errors can occur in even the best-managed laboratories and their early investigation is always desirable. If there is
any doubt whatsoever about a result, clinical staff should be encouraged to contact the laboratory. Investigations for
possible interference that can be undertaken in most laboratories include testing for linearity on dilution, recovery
experiments, treatment with heterophilic blocking tubes and confirmation using a different method. It may be desirable to
consult specialist laboratories if more complex studies are necessary. Informing clinical and laboratory staff of the ever-
present possibility of unexpected interference, ensuring brief clinical details are available to laboratory staff, and above
all facilitating excellent communication between laboratory and clinical staff are key to minimizing the risk of clinical
mismanagement due to unsuspected interference.

Ann Clin Biochem 2011; 48: 418– 432. DOI: 10.1258/acb.2011.011073

Introduction and calcitonin),9 – 11 troponin12 and possibly thyroid stimulat-

Immunoassay remains the method of choice in the clinical
laboratory for analysis of many analytes, particularly
complex heterogeneous molecules. It is not surprising, since
immunoassays involve the reaction of complex biological
reagents (usually antibodies) with other complex biological
reagents (the analyte) in a variable biological matrix (often
serum or plasma), that they are inherently vulnerable to
different types of interference.1 – 6 However, the frequency
with which such interference occurs – and most importantly,
the proportion of consequently erroneous results that may
significantly and adversely affect clinical management – is
rather difficult to assess.7,8 Serious clinical errors are most
likely to occur when decisions about patient management
are directly linked to laboratory results, alternative means
of corroborating those results are not readily available and
they are not part of a panel, which is often the case for
tumour markers (e.g. human chorionic gonadotropin
[hCG], prostate specific antigen [PSA], CA125, thyroglobulin
This article was prepared at the invitation of the Clinical Sciences
Reviews Committee of the Association for Clinical Biochemistry.
ing hormone (TSH).3 Awareness by laboratorians of the types
of interferences most likely to be encountered for particular
assays and analytes, together with excellent and proactive
communication with clinical staff, is essential to increase
the likelihood of such errors being detected in time to
prevent unwarranted or inappropriate clinical treatment.
Whether the risks of waiting for confirmation of a result
outweigh the risks of failing to take immediate action is a
decision for the clinician, always remembering that having
the wrong result may be worse than having no result.
Exogenous errors, which are not associated with proper-
ties of the individual specimen and which may reflect
system failure (e.g. a blocked probe on an automated analy-
ser), differ from errors due to endogenous interferences,
which are usually specimen-dependent and are much
more difficult to detect. Causes of both types of error are
briefly reviewed here but the main focus of this practically
oriented article is on how to investigate suspected endogen-
ous interferences and minimize the risk of them adversely
affecting clinical management (Box 1).
Although not considered here, it is of course also essential
for laboratory and clinical staff to be well-informed about

Annals of Clinical Biochemistry 2011; 48: 418 –432

 Sturgeon and Viljoen. Immunoassay error and interference
................................................................................................................................................
Box 1 Minimizing risk of interference – key points
1. Substances such as immunoglobulins, other proteins, lipids and
bilirubin present in some blood serum samples can interfere in some
immunoassays to give falsely high or low results.
2. Immunoassays can be designed by careful selection of reagents,
addition of blocking agents and reaction kinetics to minimize, but
probably not eliminate, the effects of such interferents.
3. Falsely high or falsely low results due to interferences endogenous to
the specimen present a particular risk to patient care because they (a)
are not detectable by normal laboratory quality control procedures,
(b) are reproducible within the test system, (c) are often clinically
plausible and (d) are relatively rare.
4. The diagnostics industry has done much to design assays that are
robust to endogenous interferents and laboratories should include
test robustness in their selection criteria for assay platforms.
5. As with all investigative procedures, both laboratory and clinical staff
should maintain a high index of suspicion in inspecting test results,
especially where major clinical interventions are based on test results
alone. Good laboratory-clinical liaison will assist this.
6. Procedures (e.g. use of blocking reagents) are available for the
laboratory to check whether a suspicious result might be due to
endogenous interferents.
how laboratory test results may be influenced by other
factors including pregnancy, severe illness or medication.
Examples include the variable effect of heparin, phenytoin,
frusemide, carbamazepine or salicylate on free thyroid
hormone results,13,14 finasteride and other 5-a-reductase
inhibitors on PSA,9 and increases in binding proteins (e.g.
cortisol binding globulin, sex hormone binding globulin
and thyroxine binding globulin) due to pregnancy or oral con-
traceptives which may affect hormone measurements.6
Laboratories providing testing for drugs of abuse need to be
aware of the effect of potential adulterants that can be added
to urine (e.g. household chemicals such as bleach, table salt,
laundry detergent and vinegar, as well as products containing
nitrite, hydrogen peroxide and peroxidase or glutaraldehyde)
and which may successfully mask illicit drugs present at mod-
erate concentrations.15 Ingestion of poppy seed cake may yield
a positive screening test for opiates while hemp oil exposure
can cause positive results for marijuana.15
Frequency and type of errors occurring
in clinical laboratories
Fewer errors now occur in the analytical phase of analysis
than in the pre- and postanalytical phases, reflecting major
advances in automation and sample handling.16,17 With total
error rates reportedly in the range 0.012–0.6% for laboratory
tests,16 the frequency of errors in the analytical phase,
expressed as a percentage of those occurring in all phases of
analysis, has been estimated as between 7% and 13%.17
These analytical errors include those due to equipment mal-
function and undetected failure in quality control as well as
those classified as exogenous or endogenous.17 Erroneous
results of either type may be inappropriately high or low.
Errors due to exogenous factors
Exogenous errors may be random (e.g. due to variability in
sample pipetting or other manipulations) or systematic (e.g.
419
consistent deviations from the ‘true’ value due to calibration
errors), may affect many results, and should usually be
detected by good internal quality control18 and occasionally
retrospectively by careful examination of external quality
assessment (EQA) results1 (Table 1). It has recently been
suggested that study of the outlier rate is a useful parameter
with which to assess the robustness of assays.19 The effec-
tiveness of such procedures is likely to have improved
with increasing focus on continuous quality improvement
in clinical laboratories, as exemplified by the Clinical
Laboratory Improvement Act in the United States and strin-
gent requirements for laboratory accreditation in the United
Kingdom.
Errors due to endogenous interferences
In contrast, endogenous interferences are almost always
sporadic and specimen-dependent, and hence more difficult
to identify. Moderate interference is generally more difficult
to detect than gross interference, where results are more
likely to arouse clinical suspicion. If unrecognized, interfer-
ence may cause errors in interpretation of results due to the
analytical specificity of the method used (e.g. unintended
detection of cross-reacting substances closely resembling
the analyte being measured), due to the presence of abnor-
mally high concentrations of normal serum constituents
(e.g. haemolysed or lipaemic specimens), or due to for-
mation of antibody or other macromolecular complexes.
Whatever the cause, the identification of endogenous inter-
ferences – many of which are associated with measuring
substances other than those intended – always depends
on the vigilance of well-informed clinical and laboratory
staff (Table 1).
Cross-reacting substances
An understanding of what an individual immunoassay
measures and of its vulnerability to clinically relevant cross-
reacting substances is essential, in order to ensure that the
assay used is appropriate for the clinical application as
well as to minimize the risk of misinterpretation of results.
The specificity of any immunoassay is primarily determined
by the specificity of the antibodies used and the assay
format.1 It is important to remember that cross-reaction
may be either positive or negative as illustrated in
Figure 1. Although cross-reactivity is often quoted as a per-
centage of the assay result of the interferent as compared
with the signal obtained by an identical concentration of
analyte, the concentrations of both analyte and interferent
likely to be encountered are often very different. In order
to provide a more clinically useful means of assessing the
likely degree of interference that would be encountered in
routine practice, it has been suggested that cross-reactivity
should be calculated at the concentrations of the cross-
reactant likely to be encountered in health and disease
and then expressed as the apparent percentage change in
the measured endogenous analyte concentration.20
For many analytes what it is most clinically relevant to
measure is still not well-established, although concerted
420 Annals of Clinical Biochemistry Volume 48 September 2011
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Table 1 Comparison of exogenous and endogenous types of errors

Type of
error Characteristics Possible causes
Exogenous Often associated with change in Incorrect or degraded calibrant
bias of IQC and/or EQA results
Incorrect reconstitution of
freeze-dried controls
Failure to recognize need for
recalibration
Lot-to-lot reagent variation
Faulty pipettes or dispensers
Means of minimizing occurrence and
facilitating recognition
Carefully following manufacturers’ instructions
Implementing clearly defined IQC procedures
Participating in appropriately designed EQA
schemes

Increased imprecision Imprecise pipetting

Mis-sampling due to probe
blockage
Failure of bubble detection
Inadequate mixing of reagents
and/or frozen specimens
Inadequate washing
Contaminants on reaction tubes
Ensuring laboratory staff are well trained, facilitating
early recognition and correction of errors
Establishing good communication with clinical staff
and technical staff in relevant diagnostic
companies

Endogenous Sporadic Normal serum components in Standardized procedures for assessing

Specimen-dependent excess
Difficult to identify Cross-reacting substances
Antianalyte and antireagent
antibodies and other complex
formation
High-dose hooking
haemolysed or lipaemic specimens
Awareness of assay characteristics
Protocols for assessing possible interference
Established protocol for vulnerable analytes (e.g.
tumour markers)

IQC, internal quality control; EQA, external quality assessment

international efforts are being made to address this for ana-
lytes as diverse as insulin, parathyroid hormone (PTH),
thyroid hormones, troponin and vitamin D.21 In a prototype
International Federation of Clinical Chemistry and
Laboratory Medicine (IFCC) project, six International
Reference Reagents for clinically important forms of hCG
were prepared to enable better characterization of what
current hCG immunoassays measure.22 Availability of
these Reference Reagents, together with complementary
antibody mapping studies,23 should facilitate development
of clinically more relevant assays in the future.22 For use
as a tumour marker, for example, immunoassays should
(a)
Correct result
(b)
Positive cross-reaction
Cross-reactant-
two epitopes shared
(c)
Negative cross-reaction
Cross-reactant-
one epitope shared
Figure 1 Cross-reactions in two-site immunoassay. (a) Antibodies bind to
specific analyte – correct result obtained; (b) cross-reactant sharing two epi-
topes in common with analyte – positive cross-reaction; (c) cross-reactant
sharing only one epitope in common with analyte – negative cross-reaction.
(Reprinted with permission from Seth J, Sturgeon CM. Pitfalls in immunoas-
says in endocrinology. Endocrinology and Metabolism In-Service Training and
Continuing Education. 1993;11(4):89–99. # American Association for Clinical
Chemistry, Inc.)
recognize both intact hCG and its free beta-subunit
(hCGb) as some tumours may produce only the subunit.9
In contrast, immunoassays which recognize only intact
hCG are adequate for the diagnosis and monitoring of
pregnancy.
A clearer understanding of what forms of an analyte are
present in physiological specimens will be much facilitated
by the increasing availability of well-validated mass spectro-
metric methods. Already in use in many routine laboratories
for measurement of relatively simple molecules such as
steroids, drugs of abuse and therapeutic drugs, technologi-
cal advances mean that mass spectrometry is increasingly
being applied to study complex heterogeneous molecules.
A recent report describes the identification of new clinical
variants of PTH and their measurement, together with
that of previously identified forms of PTH, using quantitat-
ive mass spectrometric immunoassays.24
While availability of highly specific assays is undoubtedly
necessary to improve understanding of the effect of disease
processes on circulating analyte concentrations, in clinical
practice, use of assays with broader specificity is sometimes
advantageous. Table 2 provides some examples illustrating
the importance of sound understanding of the specificity
of the immunoassay used. For some analytes, observed
cross-reactions may be clinically beneficial (e.g. cross-
reaction of hCG in an assay for luteinising hormone [LH]
may be helpful in identifying unsuspected pregnancy in a
woman with amenorrhoea1 or of recombinant insulin ana-
logues in assays for insulin25) provided the characteristics
of the immunoassay are appreciated by the user, while for
other analytes they highlight a need for immunoassays
with improved specificity (e.g. digoxin, growth hormone
[GH]) or better standardization (e.g. PSA, PTH).
 Sturgeon and Viljoen. Immunoassay error and interference 421
................................................................................................................................................

Table 2 Examples of analytes for which the analytical specificity of the immunoassay method used is likely to affect clinical interpretation
Effect of presence of
cross-reactant on analytical
Analyte Potential cross-reactants result obtained Clinical implications References
Chorionic hCG beta-subunit (hCGb) Potentially higher hCG results Use of an immunoassay recognizing 9
gonadotrophin obtained in immunoassays hCG þ hCGb essential for oncology
(hCG) recognizing hCGß applications as some testicular
cancers may produce only hCGb and
not intact hCG
Digoxin Aldosterone antagonists – e.g. Falsely elevated or lowered digoxin Not possible to assume 70,71
spironolactone, canrenoate concentrations interchangeability of immunoassays.
Analytical interferences in digoxin
immunoassays (both from
digoxin-like immunoreactive
substances and drug metabolites) are
a real problem, likely to become even
more important as lower therapeutic
ranges are recommended.
Confirmation in a physicochemical
method such as HPLC is highly
desirable
Growth hormone GH receptor antagonist – e.g. Falsely elevated or lowered GH Only certain immunoassays can be 72
(GH) Pegvisomant (Somavertw) concentrations used to measure GH in the presence
of Pegvisomant
Insulin Novel insulin analogues – e.g. Differences in cross-reactivity of Knowledge of such differences may be 73
Insulin Lispro (Humalogw), novel insulin analogues are critical for adequate assessment, e.g.
Insulin Detemir (Levemirw) reflected in discordant insulin of factitious hypoglycaemia
concentrations as measured in
different methods
Luteinising hCG Apparently measurable LH in early Failure to appreciate this when using an 74
hormone pregnancy, when LH and FSH are LH assay which cross-reacts with
both suppressed hCG may delay pregnancy diagnosis
in an amenorrhoeic patient
Parathyroid N-truncated fragments – e.g. Different results for patients with Not possible to assume 75
hormone (PTH) 7 –84 PTH chronic renal failure depending on interchangeability of immunoassays.
the assay used Establishment of appropriate
method-specific reference intervals
or cut-off values essential for clinically
effective application of clinical
guidelines
Prostate specific PSA complexed to Measurement of ‘total’ PSA Not possible to assume 26,76
antigen (PSA) a1-antichymotrypsin inhibitor influenced by relative recognition interchangeability of immunoassays.
of free and complexed forms of For non-equimolar assays,
PSA establishment of appropriate
method-specific decision points
essential for screening applications
Testosterone Other steroids – e.g. Potentially higher testosterone Inappropriate clinical referral and 77,78
dehydroepiandrosterone results in immunoassays cross- unnecessary further investigations
sulphate (DHAS) reacting with other steroids.
Most likely to be a problem in direct
non-extraction methods

Encouragingly, the latter is being proactively addressed for
PSA under the auspices of the NHS Cancer Screening
Programme.26 Comparability of PTH results should be simi-
larly improved by calibration of PTH immunoassays in
terms of the recently established International Standard
for PTH(1-84), IS 95/646.27
Lipids, haemoglobin and other serum
constituents
There is considerable literature relating to the effect of excess
concentrations of normal serum constituents on immunoas-
says.20 This has been comprehensively collated by several
authors20,28,29 and is also presented in data sheets for most
commercially available tests. Manufacturers’ advice should
always be followed, and documented procedures for treat-
ing specimens identified as haemolysed or lipaemic
should be available in all laboratories. Turbidity in a lipae-
mic sample can usually be visually detected and, if
required, triglycerides removed from the sample either by
precipitation or by ultracentrifugation before repeating the
analysis on the clarified sample. Haemolysis is also readily
detectable provided specimens are visually examined
before analysis. Automated detection of haemolysis,
icterus and lipaemia is possible: the advantages of this
approach, including a helpful algorithm, have been reported
for 28 analytes including some proteins.30 Immunoassay
results for some analytes (e.g. adrenocorticotrophic
hormone, gastrin, insulin and PTH) are particularly likely
422 Annals of Clinical Biochemistry Volume 48 September 2011
................................................................................................................................................

to be affected adversely by haemolysis20 and a repeat speci-
men should always be requested. The possible effect of
additives in specimen collection tubes should also be
considered.28
Antibody- and antibody-like interferences
Since immunoassays rely on interactions between the
analyte being measured and reagent antibodies to produce
the assay signal, it is not surprising that they are particularly
susceptible to interference from other unrelated antibodies
that may be present in the reaction mixture. It is important
to recognize that interfering antibodies may be present only
transiently in a patient’s serum, and that their characteristics
and reactivity may vary, such that no immunoassay can be
considered to be completely robust to all possible interfer-
ence. Such interference, which again may be positive or
negative, is more likely to occur in two-site ‘sandwich’
assay formats, in which interfering antibodies may either
interact with the analyte to prevent binding or may form
a bridge between the signal and capture antibodies in the
absence of analyte (Figure 2). In a large study of 11,261
serum samples, carcinoembryonic antigen (CEA) was
measured using a two-site, two-step immunometric assay
using mouse monoclonal antibodies.7 The frequency of
interference in these specimens was found to be 4%, a pro-
portion that could be reduced to 0.10% by removing the Fc
fragments from the capture antibody.7 This was reduced
even further by adding heat-treated, non-specific murine
immunoglobulin to the assay buffer. Some manufacturers
of commercial immunoassays do their best to minimize
the risk of such interference, usually by adding non-immune
animal serum samples or other blocking agents to the reac-
tion buffer. While appreciating that no measure can be
relied upon to be effective in removing interference in
every specimen – reflecting the biological variability
described above – more could certainly be done by some
manufacturers to reduce the risk of interference.
The majority of published reports relate to interference
from antireagent antibodies3,8,31 although other complex
formations can occur, as described below.
Interference due to antireagent antibodies
Some patient serum samples contain antibodies capable of
binding to animal immunoglobulins such as those used in
immunoassays, although estimates of the frequency of
such antibodies vary widely (,1% to 80% is suggested in
one review,31 0.4– 4% in another2), reflecting differences in
detection methods and populations studied.31 However,

(a) Normal assay,

correct result
Capture Ab Analyte Labeled Ab

(b) Increased signal,

or high result

Anti-reagent Ab present
binding capture & labeled Ab

(c) Decreased signal,

or low result

Anti-reagent Ab present
blocking either capture or labeled Ab

(d)

Correct result

Suppression of interference by
addition of non-immune IgG

Figure 2 Interference from antireagent antibodies in two-site immunoassays. (a) Interfering antibodies absent – correct result obtained; (b) cross-linking of
capture and labelled antibody in the presence of anti-reagent antibody – incorrectly high result (analyte may or may not be bound as well, depending on
steric factors, but in either case the result will be falsely high); (c) interfering antibodies binding to either capture or labelled antibody only, reducing sandwich
formation – incorrectly low result obtained; (d) effect of adding non-immune animal IgG to reduce interference – correct result obtained. (Reprinted with per-
mission from Seth J, Sturgeon CM. Pitfalls in immunoassays in endocrinology. Endocrinology and Metabolism In-Service Training and Continuing Education.
1993;11(4):89–99. # American Association for Clinical Chemistry, Inc.)

................................................................................................................................................
Table 3 Characteristics of antireagent antibodies1,31
Type of antibody Aetiology
Human Produced in response to a direct antigenic
anti-mouse stimulus, most frequently following
antibody treatment with mouse MAbs either for
(HAMA) imaging or treatment, but also in
subjects working with animals
Heterophilic Poorly defined antibodies developed in
antibody response to no clear immunogen
Rheumatoid Autoantibodies often present in serum
factor samples from patients with rheumatoid
disease
MAb, monoclonal antibody
the mechanism of interference and its severity depend both
on assay design and on the nature of the interfering anti-
body (Table 3). In general, interferences caused by human
anti-mouse antibody (HAMA) are likely to be more pro-
nounced than those due to heterophilic antibody, while
antibodies in serum samples from patients with rheumatoid
disease may not always be of sufficiently high affinity to
cause the bridging observed with HAMA or heterophilic
antibodies (Figure 2).
Interference due to antianalyte antibodies
and other complex formation
Patients with autoimmune disease
Patients with autoimmune disease are likely to have circulat-
ing antibodies that may disturb the binding of analytes to
reagent antibody. Such antibodies may sometimes be
predictable – e.g. circulating insulin antibodies in insulin-
treated patients with diabetes – but may also be unexpected
and can cause major difficulties. These are perhaps most
likely to be observed for thyroid function tests, at least in
part because of the frequency of testing. Unusual combinations
of results such as concomitantly raised or suppressed TSH and
free hormone concentrations or a significantly high or low free
hormone result accompanied by inappropriately ‘normal’ or
‘unresponsive’ TSH should raise suspicion of interference. It
is essential to consider other explanations not associated
with interference. These may include (most frequently) poor
compliance or (more rarely) clinical conditions relating
to hypothalamic–pituitary pathology (e.g. TSH-secreting
tumour), hereditary binding protein abnormalities (e.g. famil-
ial dysalbuminaemic hyperthyroxinaemia [FDH]) and end
organ resistance to either TSH or free hormone.13
Free hormone assays that rely on competition of a thyrox-
ine analogue with unbound thyroxine in the sample can
give spuriously high results, as albumin binding of the
analog is enhanced by the FDH mutation.32 This is less
Sturgeon and Viljoen. Immunoassay error and interference 423
Subjects in whom most likely to
Characteristics be present
Most readily identified antireagent antibodies. Those who have previously
High-affinity antibodies which may be present undergone treatment with MAbs
at high concentrations (.1000 mg/L either for imaging or treatment
reported).
May be of IgG, IgA, IgM or (rarely) IgE class.
May persist long-term
Two general types reported – one detecting an Variable prevalence with variable
epitope present on rabbit Ig only and one analytical relevance
detecting epitopes on goat, cattle, horse
and mouse but not rabbit
Bind to multiple antigenic determinants on the Patients with autoimmune
Fc region of IgG. rheumatic diseases, but can also
May be of IgM, IgG or IgA classes. be seen in patients with
Cause falsely high values in turbidimetric or infections, particularly persistent
latex particle agglutination assays. infections
May interfere in two-site immunoassays, e.g.
by binding to Fc portions of other
immunoglobulins and causing aggregation
likely to cause problems in two-step procedures incorporat-
ing a wash step, but thyroid hormone results can still be
misleading. In patients with suspected FDH, molecular
genetic testing is comparatively simple and provides an
unambiguous diagnosis.33 Similarly, although their preva-
lence is difficult to assess, the presence of antibodies to thy-
roxine or tri-iodothyronine may affect some thyroid hormone
methods, giving high or low results, while apparently not
altering results in others.34 Antibodies to insulin may
cause very high insulin results.35
Subjects with macroprolactinaemia and other
macrocomplexes
In some patients’ serum samples, high molecular mass com-
plexes of prolactin, termed macroprolactin, form a substan-
tial component of the total immunoreactive prolactin
measured by immunoassays.36 Macroprolactins may be
present in the serum of patients with significantly elevated
monomeric prolactin. They have minimal bioactivity
in vivo but are themselves variable and also are recognized
to different extents in current prolactin immunoassays.
Depending on the design of the assay, the antibody speci-
ficity and the nature of the particular macroprolactin, their
presence can lead to erroneously high ‘total’ prolactin
results, with potential for clinical mismanagement. While
some immunoassays are less vulnerable to macroprolactin
interference than others, even these may yield misleadingly
high results in some patients (e.g. 4% for one such immu-
noassay, although 9 of the 16 patients whose serum
samples contained macroprolactin also had raised mono-
meric prolactin).37 Screening for macroprolactin in all
serum specimens with total prolactin concentrations above
an agreed cut-off is therefore widely recommended.
Recent reports suggest that in some patients, similar com-
plexes may be observed for other analytes including cardiac
troponin (after acute myocardial infarction),12,38 follicle
stimulating hormone,39 TSH33,40 and vitamin B12,41 but
further study is required.
424 Annals of Clinical Biochemistry Volume 48 September 2011
................................................................................................................................................
Subjects with differentiated thyroid carcinoma
In patients with treated differentiated thyroid carcinoma,
undetectable serum thyroglobulin concentration following
TSH stimulation is considered the most reliable marker of
cure.11,42 However, the presence of thyroglobulin antibodies
or heterophilic antibodies, respectively, may lead to false
decreases and increases in measured concentration, as has
recently been described in detail.11,42,43 Thyroglobulin anti-
bodies are often present in patients with autoimmune
thyroid disease, with 10% of healthy individuals having
measurable levels.5 Quantification of thyroglobulin by
tandem mass spectrometry may ultimately circumvent the
problem of antibody interference.44 In the meantime, effec-
tive multidisciplinary collaboration is essential to minimize
the risk of error, as has recently been comprehensively
described.43
Identifying results requiring investigation
for possible interference
While considerable information is now available about the
nature of possible endogenous interferences that may be
encountered in clinical specimens, identifying such interfer-
ence still relies almost entirely on a high index of clinical
suspicion, i.e. the perception that a result does not fit the
clinical picture. The random and sporadic nature of most
interferences means that unless a cohort of patients at par-
ticular risk can be identified (as is the case for subjects
with confirmed hyperprolactinaemia whose serum may
contain macroprolactin45), screening for possible interfer-
ence is unlikely to be either feasible or cost-effective.
Interference should always be suspected if results do not
correlate with the clinical picture. This is facilitated by a
good understanding of the limitations of immunoassays in
general as well as of the specific analytical characteristics
of the method used. Laboratory staff are well placed to con-
tribute to this process, which is aided considerably by pro-
vision of brief clinical details on request forms. Clinical
colleagues should always be actively encouraged to
contact the laboratory if they have any doubt about a
result. When clinical staff are the first to suspect that
results may be incorrect, early dialogue is essential so that
relevant specimens can be retrieved before they are dis-
carded and appropriate investigation instituted.
Investigating suspected antibody
or antibody-like interference
The extent of investigations undertaken once interference is
suspected is likely to be influenced to some extent by how
much time and serum is available, but the most critical
consideration should be the possible implications for
patient management. Establishing whether interference is
the cause of a clinically unexpected increase in a tumour
marker such as hCG – where erroneous results can and
have led to unnecessary chemotherapy or other treatment
and also have ongoing implications for serial monitoring –
is much more critical than demonstrating whether an
apparently high LH result in a woman with
perimenopausal symptoms is due to antibody interference.
Failure to identify heterophilic antibody interference in
serum from a patient with a favourable prognosis germ
cell tumour, for example, recently resulted in him receiving
several courses of toxic and unnecessary second- and
third-line chemotherapy.46
Even when resources are limited, once interference is sus-
pected, there are several simple steps that should always be
taken before the need for more specialized investigations is
considered. Possible steps are shown in the flow chart in
Figure 3 and discussed below.
Initial procedures as soon as interference
is suspected
As soon as an erroneous result is suspected, the specimen
identity should be confirmed before repeating the assay to
exclude the possibility of an analytical error. Where
samples are subaliquoted (even by automated procedures),
it is highly desirable to carry out the repeat assay on
serum from the original correctly labelled primary tube. If
the result is confirmed on repeat analysis, relevant clinicians
should be alerted by telephone, all relevant results relating
to the specimen removed from, and the reason flagged in,
the laboratory computer with an explanatory note, and a
further specimen urgently requested. Depending on the
timescale in which this specimen can be acquired, primary
investigations for possible interference should be performed
on the first or second specimen.
Preliminary investigations for suspected
interference
While unfortunately there are no tests that unequivocally
exclude the possibility of interference, several relatively
simple investigations may confirm it (Figure 3).
Confirmation of results in other immunoassay methods
It is usually desirable for the investigating laboratory to send
an aliquot of the specimen to other laboratories for confir-
mation of the result by one or more different methods.
Ideally, at least one of these would depend on an alternative
methodology (e.g. radioimmunoassay), but in practice this is
usually not feasible, except for hCG and thyroglobulin (see
Appendix). Reference methods (e.g. equilibrium dialysis for
free hormones) are clearly desirable, but are well-established
for relatively few analytes. If, after taking account of
method-related differences in bias as reflected in EQA and
other available data, results differ significantly, this pro-
vides convincing evidence of interference although it will
not necessarily identify which result is ‘correct’.
Dilution and recovery studies
Serial dilution and recovery studies can be informative, as
lack of linearity of results on dilution of the specimen (in
the diluent provided by the manufacturer) or low recovery
of known amounts of analyte (e.g. the assay standard)
 Sturgeon and Viljoen. Immunoassay error and interference
................................................................................................................................................
Figure 3 Flow chart showing sequence of investigations that might be undertaken to investigated suspected immunoassay interference
added to the serum being investigated are suggestive of
interference. Determining the limits of acceptability for
such studies is difficult, however, and apparently satisfac-
tory linearity and/or recovery may be observed even in
the presence of an interfering substance. Conversely, some
specimens that are apparently free from interfering sub-
stances do not dilute out linearly, particularly in some of
the more complex tumour marker assays (e.g. CA19-920),
making interpretation of results even more difficult.
Treatment with heterophilic blocking reagents
Many laboratories keep a supply of commercially available
heterophilic antibody blocking tubes, and treating the
specimen with these according to the manufacturers’
instructions is also desirable. The tubes are coated with a
heterophilic blocking reagent (HBR) directed against
human heterophilic antibody, which is claimed to block
interference by steric hindrance.47 Results that differ from
the original result following treatment with these tubes
suggest interference, but obtaining the same result does
not necessarily exclude it.
HBR is also commercially available in solution and is rec-
ommended for use at a concentration of 400 mg/mL.47
Interestingly, it has recently been shown that lower concen-
trations of HBR appear to enhance interactions in a model
system rather than blocking them.48 Whatever the expla-
nation for this observation, which is in accord with some
425
(e
])
other reports, it highlights the need to ensure that a sufficient
concentration of HBR is present to suppress interference. To
address this, some laboratories routinely treat specimens
sequentially with more than one HBR tube when investi-
gating potential interference. If this is done, the analyte
should be measured after each treatment until the same con-
centration is obtained in two consecutive determinations.
Addition of non-immune animal serum
Adding non-specific immunoglobulins to the reaction mixture
may reduce interference if the human antibodies bind to these
instead of binding the assay antibodies.7 Murine and bovine
antibodies reduce interference in the highest percentage of
patient’s samples and also have the highest avidity for hetero-
philic antibodies. Ingestion of bovine IgG in milk has been
suggested to be one of the factors inducing heterophilic anti-
bodies in some subjects, which might explain the effectiveness
of bovine IgG as a blocking agent.7 Polymerized IgG prep-
arations have been shown to be superior to native IgG.49
Trial and error is required in determining the optimal concen-
tration to be added with appropriate controls included.
Polyethylene glycol precipitation
At neutral pH, immunoglobulins are not particularly
soluble and it is relatively easy to precipitate them.20
When investigating possible antibody interference in
426 Annals of Clinical Biochemistry Volume 48 September 2011
................................................................................................................................................
immunoassays, reasonably well-controlled precipitation of
potentially interfering antibodies and other high molecular
weight complexes in serum can be conveniently achieved
using polyethylene glycol (PEG), a polymer of ethylene
oxide. In principle, PEG precipitation can potentially be
applied to any immunoassay in which macro-complex for-
mation is suspected of causing interference,20,50 but the
majority of reports to date describe its use in investigating
the presence of macroprolactin (and large molecular
weight forms of prolactin) in serum samples with raised
total prolactin concentrations.36 A recommended protocol
for PEG precipitation45 is provided in the Appendix.
While PEG precipitation can be helpful, it is nevertheless
a relatively non-specific technique and a proportion of the
free analyte is likely to be co-precipitated with any com-
plexes present.50 The extent to which this occurs has been
shown to be both analyte- and method-dependent,51 and
may also be influenced by the characteristics of the individ-
ual specimen. It has been shown, for example, that the
amount of monomeric prolactin co-precipitated with
serum globulins can be increased in the presence of
increased globulin concentrations, giving the erroneous
impression that macroprolactin is present.52 It has recently
been reported that PEG precipitates almost all IgG and
IgM but only 50% of IgA, which could also lead to false-
negative results.37 Confirmation by gel filtration chromato-
graphy (see below) may be desirable for such specimens.52
In addition, PEG can interfere in some immunoassays.50 It
is therefore essential that the PEG procedure used is vali-
dated for both analyte and method and that the results
are interpreted with caution. It is now also recommended
that the absolute post-PEG concentration of prolactin
should be reported to the clinician together with an appro-
priately derived reference interval.45,53 A similarly rigorous
approach is likely to be desirable if using PEG precipitation
to investigate interference for other analytes.
Confirmation of serum results by testing in urine
(hCG only)
If heterophilic antibody interference is suspected for a
serum hCG result, obtaining a urine specimen for qualitat-
ive or quantitative analysis can be helpful.54 As antibodies
are not filtered into urine, a negative urine test result con-
firms interference in the serum immunoassay, provided
the serum hCG concentration is 50 U/L.54 Misleading
results are most often seen with values ,1000 U/L. (It is
of course essential to ensure that the urine tested is from
the same patient as the serum.)
Sample extraction
When investigating suspected interference in steroid immu-
noassays, particularly for testosterone, extracting the specimen
with diethyl ether, re-suspending in appropriate analyte-free
diluent and re-assaying may be desirable.55 The extraction
step should separate the steroid from any binding proteins as
well as remove water-soluble steroid conjugates. Cortisol
measurements in saliva can also be useful to detect binding
protein problems and/or to confirm serum results.56
Confirmation by mass spectrometry
Mass spectrometry potentially provides an additional means
of investigating possible interference for those analytes for
which methods are sufficiently well-validated and not
reliant on antibody immunoadsorption (e.g. testosterone57
and 25-hydroxy metabolites of vitamins D2 and D3 58). Such
techniques have also successfully been applied to assays of
plasma steroids in newborn infants where significant inter-
ference may occur due to steroids such as dehydroepiandros-
terone sulphate and pregnenolone sulphate secreted by the
fetal adrenal zone.59 It is, however, essential to be aware of
the possible influence of matrix effects and to appreciate
that lower results obtained by mass spectrometry cannot be
assumed to be due to increased specificity.60
More complex investigations to characterize
suspected interference
Once preliminary investigations have confirmed probable
interference, further studies may be desirable for some
specimens (Figure 3). Several relevant procedures are
described briefly here. Depending on local arrangements,
it may be most efficient where possible to refer such speci-
mens for further investigation coordinated by a specialist
referral laboratory or EQA centre (see Appendix).
Gel filtration chromatography
In the context of assay interference, the use of size exclusion
gel filtration chromatography to investigate interference has
probably been most widely used to confirm the presence of
macroprolactin.45 Considered the gold standard procedure
for macroprolactin, the method can in principle be readily
set up in any laboratory. However, it is rather slow, labour-
intensive and costly and is not well-suited to routine use.45
Three peaks of immunoreactive prolactin may be
obtained on gel chromatography of normal serum samples
on SuperdexTM SD-75 (GE Healthcare Life Sciences,
Buckinghamshire, UK): macroprolactin elutes first, followed
by ‘big’ prolactin and finally monomeric prolactin.45 In
serum samples containing macroprolactin, the first
(highest molecular weight) peak is significantly more pro-
minent than in normal serum samples. The procedure
described for macroprolactin (see Appendix) can be
readily adapted for other analytes, if necessary selecting a
gel with different size exclusion properties.
Immunoadsorption chromatography on immobilized
IgG binding proteins
Protein A, Protein G, Protein A/G and Protein L are native
and recombinant proteins of microbial origin that bind to
IgGs from a number of species including human, mouse,
rabbit, goat and bovine.61 Commercially available coupled
to cross-linked beaded agarose and other resins, they
provide an attractive means of investigating potential inter-
ference from IgG antibodies, particularly as elution of
bound antibodies can be achieved at physiological pH.
Protein A and Protein G vary in the strength with which
 Sturgeon and Viljoen. Immunoassay error and interference
................................................................................................................................................
they bind to different IgG antibodies, both between species
and between different antibody subclasses from the same
species, so using both (Protein A/G) is attractive. Neither
Protein A nor Protein G reacts with other classes of immu-
noglobulin (IgA, IgM, etc.) so they are not helpful in identi-
fying interfering antibodies of these classes.
Protein A/G is a gene fusion product that contains four Fc
binding domains from Protein A and two from Protein G.61
As it binds to all human IgG subclasses, it can be particu-
larly helpful in purifying IgG antibodies where the subclass
identity is not known. Chromatography on immobilized
Protein A/G has recently been used successfully as a pre-
treatment procedure to eliminate non-specific assay interfer-
ences in individual serum samples from rheumatoid
arthritis patients62 (see Appendix for sample protocol).
Assessment in an unprotected immunoassay
Commercially available immunoassays are protected
against potential interference by addition of blocking
agents. Consequently, access to pairs of immunoassays,
one of which is protected and one of which is not, is now
limited. However, paired immunoassays are still available
in some specialist laboratories.7 Different results in such
assays provide convincing evidence of interference, but as
with most other approaches, obtaining the same result in
both methods does not necessarily exclude interference.
Assessment in an ‘interference’ or ‘non-sense’ assay
An early ‘interference’ assay for the detection of multivalent
antibody-binding substances in serum used the same anti-
body as both capture and signal antibody and was used to
investigate interference in a two-site immunoradiometric
assay for hCG.63 The method relies on the principle that a
signal will only be obtained in the presence of a substance
that could bind two or more molecules of the one antibody,
which would not be the case for an analyte with only one
antibody-binding site. The same principle can be refined
using a combination of two unrelated assays, such as
solid phase hCG and tracer anti-a-fetoprotein (AFP).63
Non-specific binding must be reduced by using a buffer con-
taining a reasonably high concentration of added protein
(bovine serum albumin and bovine IgG are recommended)
and the analyte must not contain repeating epitopes, so anti-
bodies to mucin antigens such as CA15-3 or CA19-9 would
not be suitable. The essential requirement for a non-sense
assay is that it measures nothing but interference.
Double radial immunodiffusion (Ouchterlony) studies
Referral to a specialist immunology laboratory for investi-
gation using these techniques to identify anti-animal immu-
noreactivity in patient serum may be helpful.
High-dose hook or pro-zone effect
Two-site immunometric assays, particularly those in which
signal and capture antibody are added simultaneously, are
427
vulnerable to the high-dose hook effect at very high
analyte concentration.64,65 Free analyte and bound analyte
compete for the limited number of solid-phase antibody-
binding sites, yielding inappropriately low results which
may even hook back to within the reference interval
(Figure 4). The risk of reporting an incorrectly low result
is greatest for analytes with wide physiological concen-
tration ranges, e.g. AFP, CA125, CEA, hCG, PSA, prolactin
and thyroglobulin. Although manufacturers usually
specify a concentration below which hooking is unlikely to
occur in a particular immunoassay, the only means of com-
pletely eliminating this risk is dilution of all specimens. This
is likely to be both costly and over-cautious, particularly in
the absence of good data as to the frequency with which
such high concentrations may occur.
While it is recommended that laboratories have in place a
defined procedure for minimizing the risk of an unidenti-
fied hook,66 some hooked specimens are probably never
identified.64 In practice, failure to recognize extremely high
AFP and hCG concentrations in conditions that are poten-
tially fatal but curable (e.g. AFP in childhood hepatoblas-
toma and hCG in gestational trophoblastic neoplasia)
constitutes a critical clinical error64 which, at best, is likely
to delay treatment.67 Ensuring that such specimens are
always checked at dilution would therefore seem highly
desirable. In some laboratories, all tumour marker results
above a certain threshold (usually somewhat lower than
the highest standard concentration) are checked at dilution.
This requires a rather empirical assessment of the risk the
laboratory is willing to take of missing an unexpectedly
high result.
As always, provision of relevant clinical information and
good communication with clinical staff facilitates identifi-
cation of specimens that are likely to be problematic and
Excess analyte present
Low result
Antibody sandwiich formation reduced
Figure 4 Mechanism of high-dose hook effect in two-site immunometric
assay. As analyte concentrations increase to very high concentrations, both
capture and signal antibodies become saturated with analyte, decreasing
antibody sandwich formation. (Reprinted with permission from Seth J,
Sturgeon CM. Pitfalls in immunoassays in endocrinology. Endocrinology
and Metabolism In-Service Training and Continuing Education. 1993;11(4):
89–99. # American Association for Clinical Chemistry, Inc.)
428 Annals of Clinical Biochemistry Volume 48 September 2011
................................................................................................................................................
for which additional dilutions would be desirable, for
example, when prolactin is measured for the first time in a
patient presenting with a visual field defect.64
Minimizing risk from interference – a shared
responsibility
Although much progress has undoubtedly been made, it is
clear that the often sporadic nature of many errors and inter-
ferences in immunoassay continues to present a number of
challenges – in identifying erroneous results that may
cause patient harm, in understanding their aetiology and
in reducing their risk of recurrence. It is worth considering
briefly what is required from whom in order to meet the
shared responsibility of ensuring the correct result at the
correct time for the correct patient.
Communication between clinical
and laboratory staff
Ultimately, as has been emphasized throughout this review
and elsewhere,6,50,64 excellent communication and exchange
of information between clinical and laboratory staff is absol-
utely essential to minimize the risk of clinical errors arising
from erroneous analytical results. This should ideally include
active laboratory participation in multidisciplinary team meet-
ings. Provision of relevant clinical information with specimen
requests enables laboratory staff to focus on possible sample-
dependent analytical problems. Clinical staff need to be
well-informed about when immunoassay results may be par-
ticularly vulnerable to interference and always encouraged
to question results that do not seem in accord with the clinical
picture, i.e. to expect the unexpected.6,64 Raising awareness of
the possibility of false-positive or false-negative laboratory
results should be mandatory in post-graduate medical training
(e.g. at Specialist Registrar level in the United Kingdom), par-
ticularly for doctors in specialties which regularly rely on
immunoassay results (e.g. oncology and endocrinology). It is
salutary to note that, in relation to one of the worst reported
cases of antibody interference leading to unwarranted treat-
ment (which included an unnecessary hysterectomy in a
22-year-old), doctors involved had complete confidence in
the misleadingly high hCG test results, having used the
same method without apparent incident for many years.68
During the subsequent court case it was noted that although
the relevant test insert advised that such consistently elevated
hCG concentrations should be confirmed by an alternate
method, this was not appreciated by the doctors involved as
the inserts typically go to the laboratory and not the doctor.68
Communication with patients and recording
of results
Patients for whom there is evidence of endogenous assay
interference should be informed that they are at risk of
future false-positive results and should be encouraged to
explain this whenever they have a blood specimen taken.
This information should also be noted prominently in
patients’ medical records and ideally in the laboratory infor-
mation system. In some cases it may simply not be possible
to report a result and it is necessary to attribute this to tech-
nical difficulties. Recording this is essential.
Communication with diagnostics
manufacturers
Manufacturers have significantly reduced the risk of inter-
ference in many immunoassays by careful selection of anti-
bodies, by use of better blocking reagents and through
generally improved understanding of the complex immuno-
logical mechanisms involved.10 Use of humanized anti-
bodies in which amino acids of the assay antibodies have
been exchanged by antibody engineering, for example,
may alter antigenic sites and reduce interference.5 Since
most interfering antibodies bind to the Fc part of immuno-
globulins, removing or modifying the Fc fragment can
also significantly decrease the likelihood of interference.5,7
Further improvement will become even more important as
increasingly complex therapies involving monoclonal anti-
bodies or drug targeting receptors (e.g. those for epidermal
growth factors) enter clinical practice. In this context it is
encouraging to note that the pharmaceutical sector is
already expending considerable effort in validating
methods to detect and characterize anti-drug antibody
responses in patients.69
Nevertheless, interferences will inevitably continue to
cause problems in occasional specimens. When preliminary
investigations suggest this is likely, results should be dis-
cussed at an early stage with the relevant diagnostics man-
ufacturer. Information about the assay configuration and
the antibodies used can be very helpful in designing
further studies. It would be highly desirable if all manufac-
turers agreed to specify the measures taken to reduce inter-
ference in the information supplied with their kits, as has
previously been recommended.49 Such openness could
enable much more focused and effective investigation of
specimens subject to interference, would undoubtedly
benefit patient care and should be strongly encouraged.
Communication with EQA providers
and specialist laboratories
Managed service contracts and other changes in laboratory
organization, often involving consolidation on platforms
from a single manufacturer, mean that even simple checking
of suspicious results by a different method is becoming
increasingly complex and time-consuming to arrange.
Providers of national EQA schemes are well-placed to coor-
dinate such specimen exchange as part of their educational
and collaborative remit and may in the future wish to
promote this actively as part of their services to participants.
Related to this, whether investigations of suspected inter-
ference are best undertaken in-house or are more efficiently
referred to a specialist referral laboratory or EQA centre is
debatable. A major benefit of retaining at least some
in-house capability, perhaps encompassing the preliminary
 Sturgeon and Viljoen. Immunoassay error and interference
................................................................................................................................................
investigations described above, is that awareness of how to
identify problem specimens as well as the required practical
skills are then actively maintained at local level. For more
complex investigations, referral to a specialist laboratory
may be desirable (see Appendix).
Conclusion
There have been major advances in understanding the
mechanisms by which substances present in the occasional
routine clinical specimen can cause false-positive or false-
negative results in immunoassays, and this understanding
has guided the development of in-house and commercially
available assays with improved reliability. Nevertheless,
clinically misleading results due to endogenous interferents
in the test specimen do continue to occur sporadically. They
present a unique and serious threat to patient care because
they are not detectable by normal laboratory quality
control procedures, are reproducible within the test
system, may be clinically plausible and are relatively rare.
While the first line of defence in countering such threats is
the inclusion of assay robustness in the criteria for choice
of test system, the importance of maintaining a high index
of suspicion in inspecting assay results cannot be overstated.
This is a responsibility of both laboratory and clinical staff,
and requires critical review of the test result in relation to
other clinical features, especially where clinical interven-
tions are planned on the basis of the test result alone.
Procedures are available for the laboratory to check
whether a suspicious result might be due to endogenous
interferents.
DECLARATIONS
Competing interests: None.
Funding: None.
Ethical approval: Not applicable.
Guarantor: CMS.
Contributorship: CMS wrote the draft and AV contributed
to the final manuscript.
Acknowledgements: We would like to thank George Klee,
Kjell Nustad, John Seth and Joanna Sheldon for their
careful reading of the manuscript and most helpful and
much appreciated suggestions.
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(Accepted 14 April 2011)
Appendix
Protocols for investigation of interference
Protocol 1. Use of heterophilic blocking tubes (HBT)
and heterophilic blocking antibodies (HBA)47
Procedure
(1) For each serum or plasma specimen use one HBT tube.
Holding the tube upright, tap it sharply on a hard
surface to bring the blocking reagent to the bottom of
the tube;
(2) Pipette 0.5 mL of serum or plasma specimen into the
bottom of the tube, using a new pipette for each
specimen;
(3) Cap the HBT-tube and invert it gently five times to mix
the specimen with the blocking reagent before incubat-
ing for one hour at room temperature;
(4) Assay the HBT-treated specimen using the same method
as used for the untreated specimen. In some cases it may
be desirable to repeat the treatment with a second HBT
tube;
(5) Pre- and post-treatment results that differ significantly
are consistent with heterophilic antibody interference.
Obtaining the same results for both specimens does
not necessarily exclude interference.
Protocol 2. Procedure for screening for macroprolactin
using PEG precipitation.45
Reagents
(1) Prepare 200 mL of phosphate-buffered saline (PBS;
137 mmol/L sodium chloride, 10 mmol/L sodium phos-
phate, pH 7.4);
(2) Prepare 100 mL of 25% w/v PEG by dissolving 25 g of
PEG 6000 in 80 mL of PBS. When dissolved, make the
volume up to 100 mL with PBS and store at 48C (N.B.
solid PEG should be less than 5 years old and PEG sol-
utions should be used within 2 weeks of preparation);
(3) Equilibrate the PEG solution at room temperature prior
to use.
Procedure
(1) Add 250 mL of PEG solution (25% w/v) to 250 mL of
each specimen in appropriately labelled tubes, vortex
thoroughly and incubate at room temperature for 10 min;
(2) Centrifuge the tubes (14,000g; 5 min);
(3) Decant each supernatant into a second appropriately
labelled tube and measure the prolactin concentrations
within 24 h. (For some immunoassays [e.g. the
Beckman Access and Siemens Immulite methods],
dilution of the supernatant 1 in 5 with PBS is also rec-
ommended.) If the prolactin result is over range or if
431
there is insufficient sample to analyse without dilution,
the specimen can be diluted with PBS;
(4) Multiply the prolactin concentrations by 2 to correct for
the dilution with PEG. Additional multiplication will be
required for diluted specimens.
Protocol 3. Use of gel filtration procedure to confirm
presence of macroprolactin or other antibody
complexes45
Reagents
Prepare TRIS-buffered saline (10 mmol/L TRIS buffer, pH
7.4 containing 130 mmol/L sodium chloride, pH 7.4) and
add sodium azide (3 mmol/L).
Procedure
(1) Use a 60 cm  1.6 cm column of SuperdexTM SD-75;
(2) Apply up to 1.0 mL of serum to the column and elute
with Tris-buffered saline ( pH 7.4);
(3) Collect 60 fractions (1.4 mL; labelled from 1 to 60) and
analyse fractions 20 –60 for immunoreactivity ( prolactin
or other as appropriate) by a method known to cross-
react strongly with macroprolactin (e.g. currently
Tosoh AIA or Perkin Elmer DELFIA);
(4) Determine the relevant proportions of complexed and
monomeric analyte by calculating the area under each
peak;
(5) Compare results with those obtained on chromato-
graphing the monomeric analyte following exactly the
same procedure.
Protocol 4. Affinity chromatography on immobilized
Protein A and related proteins79
Procedure
(1) Set up and equilibrate the column with appropriate
buffers according to the manufacturers’ instructions;
(2) Serum specimens may require clarification by centrifu-
gation or filtration through a filter prior to loading. A
1.0 mL cartridge may be loaded with up to 2.0 mL of
human serum mixed with 4.0 mL of the binding buffer;
(3) Elute any unbound material with 6.0 mL of binding
buffer (approximate flow rate 1 mL/min) and collect
1.0 mL fractions;
(4) Elute bound IgG with 5.0 mL of elution buffer (approxi-
mate flow rate of 0.5 mL/min) and collect 0.5 mL
fractions;
(5) Determine the protein and analyte concentrations in the
eluted fractions in the usual way;
(6) Regenerate the cartridges following the manufacturers’
instructions.
Protocol 5. Non-sense or interference assay80
Procedure
(1) Coat microtitre wells (Cat No. 655001; Greiner Bio-One
Ltd, Stonehouse, UK) with 200 mL of mouse mono-
clonal anti-human hCG antibody (Cat No. AB1961;
Abcam, Cambridge, UK; 1:1000 dilution in
432 Annals of Clinical Biochemistry Volume 48 September 2011
................................................................................................................................................
bicarbonate/carbonate coating buffer [3.03 g
NA2CO3 þ 6.0 g NAHCO3 in 1.0 L distilled water,
adjusted to pH 9.6]);
(2) Incubate overnight at 48C and then wash three times
with wash solution (0.05% v/v Tween-20 in PBS
[1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4 and 4.0 g
NaCl in 0.5 L distilled water, adjusted to pH 7.4]);
(3) Add 200 mL of blocking buffer (1% w/v gelatin in PBS)
and incubate for one hour at room temperature on an
orbital shaker;
(4) Discard blocking buffer;
(5) Add 100 mL of sample. Incubate for two hours on an
orbital shaker at room temperature;
(6) Wash wells three times with wash solution as above;
(7) Add 100 mL of horse radish peroxidase (HRP)-labelled
rabbit polyclonal anti-human hCG antibody (Cat No.
AB30451; Abcam; 1:4000) or HRP-labelled mouse
monoclonal anti-human AFP antibody (Cat No.
AB10072; Abcam; 1:16,000) in blocking buffer and
incubate for one hour on orbital shaker at room
temperature;
(8) Wash wells three times with wash solution as above;
(9) Add 100 mL of tetramethylbenzidine solution (Cat No.
T8665; Sigma-Aldrich Company Ltd, Dorset, UK) and
incubate for 10 min at room temperature;
(10) Add 50 mL of sulphuric acid (1 mol/L);
(11) Using a plate reader, measure absorbance at 450 nm.
(Where suppliers have been mentioned in this review, their
products have been selected solely because they illustrate
the points being made. Their inclusion is not intended in
any way to constitute a recommendation or otherwise.)
Specialist laboratories
Contact details for specialist laboratories with particular
expertise in investigation of samples giving apparently aty-
pical results include the following:
† Human chorionic gonadotrophin (hCG) – Professor M Seckl,
Medical Oncology Department, Charing Cross Hospital,
Fulham Palace Road, London W6 8RF, UK;
† Peptide hormones and tumour markers – Dr C Sturgeon, UK
National External Quality Assessment Service (UK
NEQAS [Edinburgh]), Department of Clinical Biochemistry,
Royal Infirmary, Edinburgh EH16 4SA, UK;
† Specialist immunology – Dr J Sheldon, Protein Reference
Unit, St George’s Hospital, Blackshaw Road, London
SW17 0NH, UK;
† Steroid hormones – Dr J Barth, Department of Clinical
Biochemistry & Immunology, Leeds General Infirmary,
Leeds LS1 3EX, UK;
† Thyroid hormones (including equilibrium dialysis for free T4
and gel filtration chromatography for TSH) – Dr D Halsall,
Department of Clinical Biochemistry, Addenbrooke’s
Hospital, Hill Road, Cambridge CB2 2QQ, UK;
† Thyroglobulin, atypical thyroid hormone results (including
screening for familial dysalbuminaemic hyperthyroxinaemia,
anti-T4 and anti-T3 antibodies) and a-subunit assays – Dr PMS
Clark, Regional Endocrine Laboratory, Department of
Clinical Biochemistry, University Hospital Birmingham
NHS Foundation Trust, Birmingham B29 6JD, UK.