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concentrations characteristic of patients
with metastatic disease. These high Tg levels
overwhelm the assay’s reagent binding
capacities, which yields inappropriately low
values. Although adoption of a two-step
assay design has reduced this problem, a
hook effect still can occur when measuring
saline wash-outs of the needles used to biopsy
metastatic lymph nodes. In such cases,
Tg levels often exceed 10,000 µg/L. Because
of these challenges, laboratories need to use
linearity studies to check the high range for
hooking and the potential for carry-over
contamination of specimens.
Countering Interferences
Unfortunately, the Tg IMA methodology
favored by most laboratories is more prone
to interferences from both human antimouse
antibody (HAMA) and TgAb than
is Tg RIA methodology (3,12,13). HAMA
interference usually results in a falsely high
serum Tg that may prompt unnecessary
imaging or radioiodine treatment for presumed
disease. In contrast, TgAb interference
causes falsely low or undetectable
serum Tg that can have more serious consequences
because it can mask the presence
of disease. Whereas contemporary IMA
methods routinely include blocker reagents
to minimize HAMA interference to around
0.5%, currently there are no effective measures
to overcome TgAb interference encountered
with Tg IMA methodology (8).
12 CliniCal laboratory news July 2011
HAMA in the specimen can interact
with one of the monoclonal antibody reagents
to create a false signal that simulates
the presence of a high antigen (Tg) concentration
(12). Rarely, HAMA can block the
participation of the antibody reagents and
cause a falsely low Tg value (13). Physicians
should suspect HAMA when the serum Tg
level appears inappropriate in the context
of the patient’s clinical status, or fails to
respond appropriately to changes in TSH.
An example would be when Tg levels rise
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so for the approximately 20% of DTC patients
with detectable TgAb, the TgAb concentration
can be used to monitor changes
in tumor mass in preference to measuring
Tg by IMA (14). Specifically, when the antigenic
stimulus is removed by thyroidectomy,
TgAb concentrations typically decline
by approximately 50% within the first year
and eventually disappear within a median
of 3 years when patients are rendered disease-free
(14). Conversely, TgAb concentrations
rise in response to increased antigen
concentrations following second surgeries,
fine needle biopsy, or radioiodine therapy
as well as with recurrence. Serial monitoring
of TgAb concentrations can overcome
the problem of unreliable Tg IMA measurements.
TgAb Interference in IMA
TgAb interference with Tg IMA measurements
can cause Tg underestimation and
the reporting of falsely low or undetectable
values that can mask the presence of
disease (3,6). Although RIA methods tend
to be more resistant to TgAb interference,
they too can produce falsely low or high
values, depending on the characteristics
of the assay reagents and the endogenous
TgAb in the specimen. Therefore, reliable
TgAb detection is critical for authenticating
all Tg measurements. Although the
propensity for interference is related to the
TgAb concentration, high TgAb levels do
not necessarily produce interference, and in
some cases, low TgAb concentrations may
profoundly interfere (3).
Manufacturer-recommended assay cutoffs
for detecting TgAb are typically set in
the detectable range and relate to the diagnosis
of autoimmune thyroid disease, not
the detection of interfering TgAb. Inappropriate
cutoffs, together with differences in
assay sensitivity and specificity, cause some
specimens to be classified as TgAb-positive
by one method and TgAb-negative by another
(5). Laboratorians should be aware
that any TgAb detected above the analytic
sensitivity limit has the potential to interfere
with Tg measurement. Notably, these
between-method disparities exist despite
the methods’ purported standardization
with the same international reference
preparation (WHO 1st IRP 65/93). TgAb
method-related disparities are difficult to
eliminate because they reflect patient-related
TgAb heterogeneity compounded by
differences in assay sensitivity and specificity
(5).
Labs on the Front Lines
Given the importance of life-long postoperative
monitoring of DTC patients, labs
have a vital responsibility to ensure that Tg
measurements are as accurate as possible,
and that they keep abreast of and address
the analytical limitations of their Tg assay
methods. Ongoing dialogue with endocrinologists
and oncologists likewise is essential,
so these clinicians can be well-informed
of any method changes and confer readily
with laboratorians about any discrepant
results. CLN
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Carole Spencer, MT, PhD,
FACB, is a professor of
medicine in the Department
of Medicine at the University
of Southern California.
Email: cspencer@usc.edu