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Erroneous augmentation of multiplex assay measurements in patients with rheumatoid arthritis due to heterophilic binding by serum rheumatoid factor.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 4, April 2011, pp 894–903
DOI 10.1002/art.30213
© 2011, American College of Rheumatology
Erroneous Augmentation of Multiplex Assay Measurements in
Patients With Rheumatoid Arthritis Due to Heterophilic
Binding by Serum Rheumatoid Factor
Derrick J. Todd,1 Nicholas Knowlton,2 Michael Amato,1 Mark Barton Frank,2 Peter H. Schur,1
Elena S. Izmailova,3 Ronenn Roubenoff,4 Nancy A. Shadick,1 Michael E. Weinblatt,1
Michael Centola,2 and David M. Lee1
Results. In RA patients with high-titer RF, 69% of
analytes demonstrated at least a 2-fold stronger multiplex signal in non–RF-depleted samples as compared to
RF-depleted samples. This degree of erroneous signal
amplification was less frequent in low-titer RF samples
(17% of analytes; P < 0.0000001). Signal amplification
by heterophilic antibodies was blocked effectively by
HeteroBlock (>150 ␮g/ml). In 35 RA patients, multiplex
signals for 14 of 22 analytes were amplified erroneously
in unblocked samples as compared to blocked samples
(some >100-fold), but only in patients with high-titer
RF (P < 0.002). Two other blocking agents, heterophilic
blocking reagent and immunoglobulin-inhibiting reagent, also blocked heterophilic activity.
Conclusion. All multiplex protein detection platforms we tested exhibited significant confounding by RF
or other heterophilic antibodies. These findings have
broad-reaching implications in the acquisition and interpretation of data derived from multiplex immunoassay testing of RA patient serum and possibly also in
other conditions in which RF or other heterophilic
antibodies may be present. Several available blocking
agents effectively suppressed this erroneous signal amplification in the multiplex platforms tested.
Objective. Serum rheumatoid factor (RF) and
other heterophilic antibodies potentially interfere with
antibody-based immunoassays by nonspecifically binding detection reagents. The purpose of this study was to
assess whether these factors confound multiplex-based
immunoassays, which are used with increasing frequency to measure cytokine and chemokine analytes in
patients with rheumatoid arthritis (RA).
Methods. We performed multiplex immunoassays
using different platforms to measure analyte concentrations in RA patient samples. Samples were depleted of
RF by column-based affinity absorption or were exposed
to agents that block heterophilic binding activity.
Supported in part by Crescendo Bioscience and Millennium
Pharmaceuticals.
1
Derrick J. Todd, MD, PhD, Michael Amato, BS, Peter H.
Schur, MD, Nancy A. Shadick, MD, MPH, Michael E. Weinblatt, MD,
David M. Lee, MD, PhD (current address: Novartis, Basel, Switzerland): Brigham and Women’s Hospital and Harvard Medical School,
Boston, Massachusetts; 2Nicholas Knowlton, MS, Mark Barton Frank,
PhD, Michael Centola, PhD: Oklahoma Medical Research Foundation, Oklahoma City; 3Elena S. Izmailova, PhD: Millennium Pharmaceuticals Inc., Cambridge, Massachusetts; 4Ronenn Roubenoff, MD,
MHS: Biogen Idec, Cambridge, Massachusetts (current address: Novartis Institutes for Biomedical Research, Cambridge, Massachusetts).
Dr. Frank owns stock in Crescendo Bioscience. Dr. Izmailova
owns stock in Takeda. Dr. Shadick has received research grant support
from Biogen Idec, Crescendo Bioscience, and Amgen. Dr. Weinblatt
has received research grant support from Biogen Idec, Crescendo
Bioscience, Millennium Pharmaceuticals, and the Raymond P. Lavietes Foundation, and he has served as a consultant to Biogen Idec
and Crescendo Bioscience. Dr. Centola has served as a consultant to,
and owns stock in, Crescendo Bioscience. Dr. Lee has received
research grant support from Biogen Idec, Crescendo Bioscience, and
Millennium Pharmaceuticals, and is currently employed by Novartis
Pharma AG.
Address correspondence to Derrick J. Todd, MD, PhD, or
David M. Lee, MD, PhD, Brigham and Women’s Hospital, 75 Francis
Street, Boston, MA 02115. E-mail: dtodd1@partners.org or
dlee1@partners.org.
Submitted for publication July 20, 2010; accepted in revised
form December 16, 2010.
Rheumatoid arthritis (RA), the most common
form of autoimmune inflammatory arthritis, is a heterogeneous disease, both in clinical presentation and in
response to therapies. Early effective therapy substantially improves long-term functional status in RA patients (1). Accordingly, considerable recent research has
focused on molecular profiling of patient cohorts in
efforts to define the molecular pathology of RA and to
identify biomarkers useful for disease diagnosis and for
prediction of therapeutic responses (2–5).
894
RHEUMATOID FACTOR INTERFERENCE WITH MULTIPLEX ASSAYS
Multiplex immunoassay methods have emerged
as a powerful high-throughput means of quantifying
pathogenic pathways and identifying candidate biomarkers in disease cohorts (6). These techniques typically use
multiple combinations of analyte-specific antibodies derived from non-human species. Although these methods
have provided substantial insight into the biology of RA,
there are technical reasons to be concerned about the
use of this antibody-based technology in disease states
such as RA, in which there is a high prevalence of serum
rheumatoid factor (RF). RF is an antibody with specificity for antigenic epitopes on Fc portions of IgG. RF
has the potential to interfere with immunoassays by
binding to the antibodies used to detect them, resulting
in analyte-independent signals. Nonspecific effects of
RF may be increased in multiplex immunoassays relative
to monoplex assays because increased antibody concentrations and diversity present increased opportunity for
nonspecific RF binding.
Because of these concerns, we evaluated the
potential of RF-driven interference in several multiplex
immunoassays (both solid-phase and bead-based platforms) in patients with RA. We report herein our
findings of a substantial and idiosyncratic impact of RF
on this methodology as well as a means to suppress such
interference.
PATIENTS AND METHODS
Serum samples and RF testing. Serum samples were
obtained from patients with RA and from healthy volunteer
donor controls. All RA patients were either part of the
Brigham Rheumatoid Arthritis Sequential Study (BRASS)
cohort (7) or met the American College of Rheumatology 1987
criteria for RA (8). Samples were tested for the presence of RF
by either enzyme-linked immunosorbent assay (ELISA; TheraTest Laboratories) or nephelometry, as specified in the
Results section. Samples were then allocated to one of several
experiments in which different multiplex immunoassay platforms were used to assay for the presence of prespecified
cytokine and chemokine analytes.
The study was conducted in accordance with protocols
approved by the Institutional Review Boards of Partners
Healthcare System and Oklahoma Medical Research Foundation.
Column depletion of RF. Column affinity absorption
was used to deplete completely RF from 9 RA patient serum
samples. Baseline RF values were measured by nephelometry
(range 13–680 IU) (9). Each sample was then apportioned into
2 fractions. One fraction was depleted of RF by column affinity
absorption against human IgG-conjugated Sepharose. Serum
samples were diluted 1:1 with Tris buffered saline and incubated for ⬎4 hours at 4°C with an equal volume of IgG
Sepharose 6 Fast Flow (GE Healthcare). The second fraction
was processed in parallel, except that it was mock-depleted of
895
RF by column absorption against unconjugated Sepharose.
With these techniques, all RF-depleted fractions were depleted of detectable RF, as confirmed by nephelometry. In
total, 18 fractions were processed (9 RF-depleted and 9
RF–mock depleted).
Multiplex immunoassay platforms. The following
commercial multiplex immunoassay platforms were used according to the manufacturers’ protocols: RayBiotech (RayBiotech), SearchLight (Thermo Scientific Pierce), and Luminex
(Invitrogen). Luminex assays were performed in house. RayBiotech and SearchLight analyses were performed by the
commercial vendors (RayBiotech and Thermo Scientific
Pierce, respectively). The specific analytes measured are described below.
RF blocking reagents. The following heterophileblocking commercial reagents were used in multiplex assays at
the concentrations described below: HeteroBlock (Omega
Biologicals), heterophilic blocking reagent (HBR; Scantibodies), and immunoglobulin-inhibiting reagent (IIR), which consisted, in part, of antiidiotype antibodies (Bioreclamation).
Statistical analysis. For the column affinity absorption
experiment, we calculated the ratio of analyte concentrations
measured in RF–mock depleted and RF-depleted fractions.
The resultant M:D ratios were not normally distributed and
thus were ranked before applying an analysis of variance
(ANOVA) in which data for the 3 RF tertiles were subjected
to a Kruskal-Wallis test (10,11). For the Luminex platform
experiment performed on sera from 35 RA patients, the mean
fluorescence intensity (MFI) results were likewise not normally distributed and thus were ranked prior to a two-way
ANOVA in which the samples were paired on the blocking
variable. For all statistical calculations, a Bonferroni-corrected
alpha level of 0.05 was considered statistically significant.
RESULTS
Association of strongly positive analyte signals
with RF positivity in a multiplex antibody–based platform using sera from patients with RA. An initial
assessment of RF interference on multiplex immunoassay platforms was done on a highly multiplexed solidphase immunoassay that measures 507 RA diseaserelevant proteins (RayBiotech). Serum samples from 6
RF-positive RA patients, 6 RF-negative RA patients,
and 6 healthy volunteer donor controls were analyzed.
Remarkably, in 5 of 6 RF-positive samples, ⬎70% of all
measured analytes registered a “strongly positive” signal, defined here as an MFI that was at least 10 times
that of the corresponding negative control. Only RA
patients with high-titer serum RF values (⬎100 IU by
ELISA) exhibited this unlikely multiplex profile (Figure
1). These results raised concern that, in RF-positive RA
patients, signals were inappropriately driven by RF.
Erroneous amplification of multiplex immunoassay signals by serum RF. We next tested the effect of RF
depletion on a bead-based multiplex platform (Search-
896
Figure 1. Association of strongly positive multiplex signals with rheumatoid factor (RF) positivity in patients with rheumatoid arthritis
(RA). A solid-phase multiplex immunoassay (RayBiotech) was used to
measure 507 different cytokines and chemokines in serum samples
from 18 subjects: 6 RA patients with high-titer RF, 6 RA patients with
low-titer RF, and 6 healthy control subjects without detectable RF.
The percentages of total analytes that demonstrated a strongly positive
signal, defined as a signal that was at least 10 times greater than that
of the negative control for each sample, are shown at the top.
Representative profiles from an individual patient in each of the 3
groups are shown at the bottom.
Light) in serum samples from 9 RA patients. Baseline
RF values were measured by nephelometry, and serum
samples were stratified into RF-low, RF-mid, and RFhigh tertiles. Each serum sample was then apportioned
into RF-depleted or RF–mock depleted fractions. Each
RF-depleted and RF–mock depleted fraction was then
tested in duplicate for the presence of 16 analytes:
acute-phase serum amyloid A (A-SAA), E-selectin,
intercellular adhesion molecule 1, interferon-␥ (IFN␥),
interleukin-1 receptor antagonist (IL-1Ra), IL-2R, IL-6,
IL-7, matrix metalloproteinase 1 (MMP-1), MMP-3,
MMP-9, RANTES, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, tumor necrosis factor receptor type I (TNFRI), and TNFRII. Three of the 16
analytes (IFN␥, IL-6, and IL-7) were present at levels
TODD ET AL
below the detection threshold in all fractions. Results
for 1 analyte (A-SAA) did not replicate.
For the remaining 12 analytes, the concentrations
measured by multiplex immunoassay were frequently
much greater in RF–mock depleted fractions than in
their corresponding RF-depleted fraction (Figure 2)
(Data on the depletion of serum RF and reduction of
the analyte signal in multiplex-based immunoassays for
the 6 remaining analytes, TIMP-1, RANTES, TNFRI,
TNFRII, IL-1Ra, and IL-2R, are available online at
http://www.brighamandwomens.org/research/labs/
todd/.) Interestingly, RF depletion affected the measured concentration of most, but not all, analytes (e.g.,
MMP-9, E-selectin). Analytes with highest residual serum concentrations after antibody depletion appeared
to be least affected, suggesting that interference effects
are dependent on the relative levels of heterophilic
activity and analyte.
These data were then analyzed for the effects of
RF on multiplex signal amplification in RF–mock depleted fractions. For each of the 12 measurable analytes
per patient, we calculated the ratio of analyte concentrations measured in RF–mock depleted and RFdepleted fractions (M:D ratio). Hence, an M:D ratio of
2 represents a 2-fold increased signal in the RF–mock
depleted fraction. For samples in the RF-high tertile,
M:D ratios were ⬎2 in 69% of measurable analytes, and
M:D ratios were ⬎3 in 25% of measurable analytes.
In contrast, M:D ratios were ⬎2 in only 17% of RF-mid
and RF-low tertiles, and were ⬎3 in only 6% and
0% of RF-mid and RF-low tertiles, respectively (data
available online at http://www.brighamandwomens.org/
research/labs/todd/). The mean ⫾ SEM M:D ratio was
3.41 ⫾ 0.67 for the 36 measurable analytes in the 3
RF-high samples. This was significantly greater than the
mean M:D ratios in the RF-low (1.50 ⫾ 0.08) and the
RF-mid (1.46 ⫾ 0.13) samples, which were not statistically different from one another, although they were
significantly different from the RF-high samples (P ⬍
0.0000001 for each comparison).
Collectively, these multiplex immunoassay data
demonstrate that in patients with RA, many measured
analyte concentrations are erroneously amplified. This
effect is most pronounced in patients with the highest
RF titers, and it is abrogated by RF depletion.
Heterophilic binding and multiplex signal amplification in patients with RA. We next assessed the
confounding activity of RF in the most commonly used
multiplex immunoassay platform (Luminex), assaying
for a set of chemokine analytes commonly studied in RA
(2). In this series, in addition to using the Luminex
RHEUMATOID FACTOR INTERFERENCE WITH MULTIPLEX ASSAYS
897
Figure 2. Reduced analyte signal in multiplex-based immunoassays following depletion of serum rheumatoid factor (RF). Using human Ig–conjugated Sepharose, RF
was successfully depleted (RF-D) from the serum samples of 9 patients with
rheumatoid arthritis. Mock depletion (RF-M) was conducted against Sepharose
beads. Using SearchLight multiplex immunoassay, 12 analytes were reproducibly
detectable. The plots depict the measured concentrations of A, matrix metalloproteinase 1 (MMP-1), B, MMP-3, C, MMP-9, D, tissue inhibitor of metalloproteinases
2 (TIMP-2), E, intercellular adhesion molecule 1 (ICAM-1), and F, E-selectin in
RF–mock depleted and RF-depleted serum fractions (corresponding to the left
y-axis). Values are the mean ⫾ SEM. Data for individual patients are plotted along
the x-axis in order of ascending baseline RF value (solid line, corresponding to the
right y-axis). Plots for the remainder of detectable analytes are available online at
http://www.brighamandwomens.org/research/labs/todd/.
platform in a traditional manner, assays were run such
that only signals from heterophilic interference could be
detected. This was done by using primary and secondary
detection antibodies that were not matched for analyte
binding. In this context, a positive signal could only be
derived from heterophile (RF) activity in each subject’s
serum. Results were compared to the signals obtained
from standard multiplex immunoassays (paired detection antibodies). For these studies, we used a single
heterophilic blocking agent that has been shown to block
RF interference in RA serum samples (HeteroBlock)
(2).
In our initial studies, we sought to optimize use of
HeteroBlock to suppress heterophile activity in RA
patient serum. Samples from 3 RA patients were incubated with 0, 1.5, 15, 150, or 1,500 ␮g/ml of the blocking
agent, and the effects of the blocking reagent were
assessed in a Luminex multiplex assay that included 5
chemokines: macrophage inflammatory protein 1␣
(MIP-1␣), MIP-1␤, monocyte chemotactic protein 1
898
TODD ET AL
Figure 3. Suppression of the multiplex signal amplification driven by heterophilic
binding activity in sera from patients with rheumatoid arthritis (RA). Serum samples
from 3 patients with RA were incubated with 0, 1.5, 15, 150, or 1,500 ␮g/ml of
HeteroBlock and assayed by Luminex multiplex immunoassay for analytes macrophage inflammatory protein 1␣ (MIP-1␣), MIP-1␤, monocyte chemotactic protein 1
(MCP-1), and eotaxin, using either an appropriate antibody–matched bead (solid
symbols) or an inappropriate antibody–mismatched cross-bead secondary reagent
(open symbols). Values are the mean ⫾ SEM.
(MCP-1), eotaxin, and RANTES. RANTES was not
quantifiable in these assays. In the absence of blocking
agent, 2 of the 3 patient samples demonstrated strongly
positive signals for MIP-1␣, MIP-1␤, MCP-1, and eotaxin. In these patients, a substantial fraction of analyte
reactivity was suppressed by the addition of HeteroBlock, suggesting a confounding signal from heterophilic
binding (Figure 3).
To assess this further, we performed parallel
multiplex assays with mismatched primary and secondary detection antibodies, such that only nonspecific
signal from RF and heterophilic antibody activity could
be detected. Signals in mismatched and standard assays
were similar for the 2 patient serum samples with
strongly positive analyte activity, confirming that significant nonspecific signal is observed in serum from these
patients. Furthermore, nonspecific signals in sera from
these 2 patients were suppressed to undetectable only
with an adequate amount of blocking agent (ⱖ150
␮g/ml), a concentration at which true analyte signal
remained readily detectable in MIP-1␣, MCP-1, and
eotaxin (Figure 3).
Interestingly, despite having a high titer of RF,
the third patient sample did not demonstrate nonspecific
signal, and the analyte-specific signal was not altered by
blocking agent. These results confirmed that heterophilic binding activity accounts for erroneous signal
amplification in some, but not all, RA patients.
Association of multiplex signal amplification
with serum RF in patients with RA. Having determined
the optimal concentration of HeteroBlock, we proceeded to assess the reproducibility of RF-associated
aberrant signal amplification, testing for heterophilic
signal amplification in serum samples from a replication
cohort of 35 patients with RA. Serum RF values were
measured by ELISA, and samples were assigned to 1 of
3 groups: RF-negative (IgM, IgA, and IgG RFs all
⬍25 IU), RF-low (any IgM, IgA, or IgG RF ⬎25 IU and
all ⬍100 IU), and RF-high (any IgM, IgA, or IgG RF
⬎100 IU).
Each sample was either incubated with 150 ␮g/ml
of blocking agent (blocked) or vehicle control (unblocked). Multiplex bead-based (Luminex) assays were
used to measure the concentrations of 23 cytokines that
are also commonly analyzed in RA studies, 4 of which
(IL-17, IL-8, IFN␣, and TNF␣) are shown in Figure 4.
RHEUMATOID FACTOR INTERFERENCE WITH MULTIPLEX ASSAYS
899
Figure 4. Association of multiplex signal amplification with serum rheumatoid
factor (RF) in patients with rheumatoid arthritis (RA). Serum samples from 35
patients with RA were incubated with 150 ␮g/ml of HeteroBlock or vehicle control
and assayed for analyte signal by Luminex multiplex-based immunoassays for
interleukin-17 (IL-17), IL-8, interferon-␣ (IFN␣), and tumor necrosis factor ␣
(TNF␣). The results are plotted in 2 dimensions: the mean fluorescence intensity
(MFI) results for blocked samples are on the y-axis, and the MFI results for the
unblocked samples are on the x-axis. Patient samples are color-coded based on RF
status: blue represents RF-negative (IgM, IgA, and IgG RF all ⬍25 IU), green
represents RF-low (any IgM, IgA, or IgG RF ⬎25 and all ⬍100 IU), and red
represents RF-high (any IgM, IgA, or IgG RF ⬎100 IU). The black circle represents
an analyte-spiked healthy serum sample control with no measurable heterophilic
activity. Solid curves represent y ⫽ x. Deviation from this line represents signal
blocked by HeteroBlock. For each plot, P values are the ranked two-way analysis of
variance. Asterisks indicate a significant degree of signal amplification for RFpositive patients, using a cutoff P value of ⬍ 0.002 (Bonferroni correction for
multiple comparisons). Plots for the remainder of detectable analytes are available
online at http://www.brighamandwomens.org/research/labs/todd/.
(Data on the association of multiplex signal amplification with serum RF in RA patients for 18 analytes,
MIP-1 ␣ , MIP-1 ␤ , MCP-1, eotaxin, granulocyte–
macrophage colony-stimulating factor (GM-CSF),
IFN␥, IL-13, IFN␥-inducible 10-kd protein (IP-10),
IL-1Ra, IL-1␤, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10,
IL-12, and IL-15 are available online at http://www.
brighamandwomens.org/research/labs/todd/.) Again,
RANTES values could not be quantified. As a control,
a serum sample without measurable heterophilic activity was “spiked” with recombinant analyte and then
assayed.
In the 7 RF-negative and 11 RF-low patients,
there were very few analytes for which the measured
concentrations differed between blocked and unblocked
samples. In contrast, many of the unblocked samples
from the 17 RF-high patients were markedly influenced
by erroneous signal amplification when measured for
most analytes, some ⬎100-fold. Using ranked 2-way
ANOVA and a cutoff P value of ⬍ 0.002 as a Bonferroni
correction for multiple comparisons, we identified statistically significant differences between unblocked and
blocked RF-high samples when assayed for GM-CSF,
IL-1Ra, IL-1␤, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13,
IL-17, MIP-1␣, MIP-1␤, and TNF␣. Data for IL-17,
IL-8, IFN␣, and TNF␣ are shown in Figure 4; see online
at http://www.brighamandwomens.org/research/labs/
todd/ for the remaining data. There were trends toward
900
TODD ET AL
Figure 5. Successful blockade of heterophilic signal amplification by multiple blocking agents in
patients with rheumatoid arthritis (RA). A, Samples from a patient with RA or a healthy volunteer
donor control were each combined with analyte-spiked serum from a volunteer donor control with
no detectable heterophilic activity, and this mixture was admixed with 0, 40, 400, or 1,600 ␮g/ml of
Scantibodies (SB) heterophilic blocking reagent. MIP-1␣ ⫽ macrophage inflammatory protein 1␣;
IL-8 ⫽ interleukin-8. B, Samples from 3 patients with RA or an analyte-spiked serum from a
volunteer donor with no detectable heterophilic activity were each combined with 0, 200, 400, or
600 ␮g/ml of immunoglobulin-inhibiting reagent (IIR). Values are the mean fluorescence intensity
(MFI) of the analyte (y-axis) for each fraction (x-axis), as measured by Luminex multiplex
immunoassay. Results are representative of other analytes, as detailed in the Results.
significance for IFN␣ (P ⫽ 0.0055) and IL-15 (P ⫽
0.0234). There were no significant differences for eotaxin, IFN␥, IL-8, IL-12, IP-10, and MCP-1. The
“spiked” sample consistently showed no differences in
measured analyte concentrations between blocked and
unblocked samples across all analytes. Therefore, at 150
␮g/ml, the blocking agent itself did not interfere with the
ability of the multiplex immunoassay to detect genuine
analyte concentrations. These data indicate that heterophilic binding activity causes significant signal amplification across many (but not all) analytes, and this effect
was almost exclusively limited to patients with high-titer
RF values.
Effective blockade of heterophilic multiplex signal amplification by other agents. We next assessed the
blocking capabilities of two other commercially available
blocking agents: heterophilic blocking reagent and
immunoglobulin-inhibiting reagent. To test HBR, RA
patient serum was first mixed with a separate analytespiked control (with no heterophilic activity), and aliquots of this were then incubated with 0, 40, 400, or
1,600 ␮g/ml of HBR. Each of the admixtures was then
tested for 23 analytes by bead-based (Luminex) multiplex assays (Figure 5A). Unblocked, unspiked RA pa-
tient serum was used as a control. In addition, serum
from a healthy volunteer donor control was processed in
parallel with the RA patient serum for all assays.
For the RA patient only, many of the analytes
demonstrated signal amplification in unblocked serum
samples, whether spiked or not. Supporting this, HBR
effectively blocked the erroneously amplified signal
down to the correct spiked cytokine concentrations
observed in the volunteer donor control serum, which
showed no inappropriate signal amplification. Figure 5A
shows examples of MIP-1␣ and MIP-1␤, which are
representative of the amplification patterns also observed for IL-1␤, IL-1Ra, IL-2, IL-4, IL-6, IL-7, IL-12,
IL-15, and TNF␣ (data not shown). Again, some analytes, eotaxin, IFN␥, IL-8 (Figure 5A), IL-13, and IP-10,
were not erroneously amplified by heterophilic binding.
Importantly, in the volunteer donor control serum, there
was little if any unblocked signal activity, and there was
no change in the spiked analyte signal despite increasing
concentrations of HBR.
IIR was tested in a separate experiment in which
serum samples from 3 patients with RA and 1 spiked
volunteer donor control sample were each admixed with
0, 200, 400, or 600 ␮g/ml of IIR. These samples were
RHEUMATOID FACTOR INTERFERENCE WITH MULTIPLEX ASSAYS
tested for 23 analytes by multiplex bead-based (Luminex) assays (Figure 5B). All 3 unblocked RA samples
demonstrated varying degrees of heterophilic signal
amplification, which was effectively blocked by IIR (such
as MIP-1␣ and MIP-1␤ in Figure 5B). Comparable
patterns of signal amplification were also observed for
GM-CSF, IFN␣, IL-1␤, IL-1Ra, IL-2, IL-4, IL-6, IL-7,
IL-10, IL-12, IL-15, IL-17, and TNF␣. Again, some
analytes demonstrated no signal amplification: IFN␥,
IL-8 (Figure 5B), and IP-10. Multiplex signals were
unaffected in spiked serum samples from the healthy
volunteer donor control (Figure 5B). Collectively, these
2 experiments confirm that erroneous heterophilic signal
amplification of multiplex-based immunoassays can be
blocked effectively by a number of commercially available agents without apparent interference from the
blocking agent itself.
DISCUSSION
The data from the present study show that RF
heterophilic antibodies adversely affected the ability of
multiplex-based immunoassay platforms to measure serum cytokines and chemokines in RA patients. The
signal augmentation by RF was unpredictable both in its
magnitude and in its correlation with RF titer. The effect
was also unpredictable for any given analyte, since some
analytes were affected disproportionately more than
others. Multiple agents designed to block heterophilic
binding effectively dampened the erroneous signal amplification across all platforms tested. These findings
have broad-reaching implications for the interpretation
of multiplex-based immunoassay results obtained from
patients with RA.
It has previously been recognized that RF and
other heterophilic antibodies can cause erroneous amplification of signal (sometimes called heterophilic interference) in traditional immunoassays such as monoplex
ELISA (12). In these and other sandwich-based immunoassays, heterophilic antibodies in the test sample
bridge the capture and detection antibody reagents (13).
This interaction bypasses the antigen specificity of the
assay and gives rise to erroneously amplified results. A
similar mechanism likely accounts for the RF-associated
signal augmentation that we observed in multiplex immunoassays, although some analytes were much more
adversely affected than others. One likely explanation
for this is that multiplex reagent antibodies are differentially bound by RF as a function of their species of
origin and by their Ig subclass (14), an effect that could
be minimized by manufacturers’ elimination of analyte-
901
specific antibodies that show RF binding susceptibility.
Until then, investigators will need to account for RF
interference through the use of depletion or blocking
protocols.
We used column-based human Ig techniques to
deplete RF and were able to eliminate RF heterophilic
antibody activity (Figure 2). A similar depletion technique using protein L–Sepharose beads has been used
effectively by other investigators (2,15,16). Unfortunately, these RF depletion techniques are laborintensive and costly, which limits their broad applicability.
Several commercially available agents claim to
block heterophilic interference, which provides a more
cost-effective and labor-favorable approach than RF
depletion. We showed here that HeteroBlock, HBR, and
IIR each effectively quenched heterophilic antibody
activity in multiplex-based immunoassay testing of RA
patient serum, without measurably altering the analyte
signal in spiked control samples. Since blocking agents
Figure 6. Methodologic approaches for identifying and addressing
rheumatoid factor (RF) interference in multiplex immunoassays. The
flowchart shows approaches to antibody (Ab)–based multiplex measurements in cohorts of subjects with heterophile activity, such as RF
in patients with rheumatoid arthritis (RA), with reference to the
techniques used in the present study.
902
neither dampened nor amplified true analyte measurement, our findings suggest a method for effectively
dealing with confounding RF when using multiplex
immunoassay methods in RA patients (Figure 6).
Our results are congruent with those of previous
studies by other investigators documenting that heterophilic activity confounds multiplex immunoassay measurements in RA patients (2,15,17). It is noteworthy that
in our cross-bead study, HeteroBlock quenched heterophilic cross-bead binding at concentrations ⱖ150 ␮g/ml
(Figure 3). This amount of HeteroBlock required to
suppress heterophilic activity in our assays was substantially higher than that needed in a previous study (2).
Although this difference may be attributable to our
selection of patients with high RF titers, our results
nonetheless underscore the need for use of appropriate
amounts of heterophile-suppressing reagents in this
disease population.
Multiplex immunoassay methods have been used
in many studies of RA patients in an attempt to improve
our understanding of disease pathophysiology and treatment effects. Indeed, a widespread up-regulation of
serum cytokines and chemokines has been reported
prior to RA disease onset, an effect that also correlated
with RF-positive status (2–5,18). Most of these studies
did not account for heterophilic binding activity by RF
(3–5,18). Multiplex-based immunoassays have also been
used to measure changes in cytokine and chemokine
levels in proof-of-concept clinical trials in RA patients
(5,19–21). Again, most of these studies did not account
for serum RF (5,19,21), which has been shown in
separate studies to decrease in response to pharmacotherapeutic agents (22–25). Overall, our findings raise
concern regarding the veracity of interpreting results
derived from methodologies confounded by RF.
Our findings potentially have generalized applicability in disease states other than RA. Heterophiles
interfere with immunoassay-based measurement of anti–
double-stranded DNA antibodies in patients being
treated with anti-TNF␣ pharmacologic agents for a
variety of disorders, including RA, inflammatory bowel
disease, and seronegative arthritis (17). Highly multiplexed platforms have been used to study cytokine and
chemokine profiles in systemic lupus erythematosus
(26,27), Sjögren’s syndrome (28,29), and hepatitis C
virus infection (30,31). Notably, RF is often readily
detectable in these conditions as well as in other chronic
inflammatory disease states (e.g., subacute bacterial
endocarditis) (32) and in a subset of otherwise healthy
subjects. It is unknown, and largely unaddressed,
whether RF has any contributing effect on multiplex
TODD ET AL
immunoassay signals in these conditions. Furthermore,
our data re-open the question of whether investigators
must account for other non-RF heterophilic antibodies
as potential confounders of multiplex-based immunoassays, especially since heterophilic antibodies are present
in upward of 40% of otherwise healthy subjects (33).
In summary, the data presented herein argue
strongly that investigators must be aware of, and account
for, serum RF heterophilic activity when using multiplex
immunoassays to measure cytokine and chemokine concentrations in patients with RA. In our experiments,
serum RF was associated with erroneous signal amplification of many (but not all) of the analytes assayed. In
some instances, the effect was profound, leading to a
⬎100-fold amplified fluorescence reading. Further, even
the RF-positive subset of patients was affected in an
unpredictable manner. Our results therefore preclude
post hoc correction for RF status and mandate protocols
designed to deplete or block RF heterophilic activity
prior to multiplex immunoassay–based quantitative
methods in patients with RA. These considerations are
crucial, given the ever-expanding search for biomarkers
of RA disease activity and the proliferation of multiplexbased immunoassay techniques used to achieve this end.
ACKNOWLEDGMENTS
We acknowledge Alexander Parker and Michael Pickard for their input on the experimental design.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Todd had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Todd, Knowlton, Frank, Schur, Izmailova, Roubenoff, Shadick, Weinblatt, Centola, Lee.
Acquisition of data. Todd, Knowlton, Amato, Frank, Schur, Izmailova,
Shadick, Weinblatt, Centola, Lee.
Analysis and interpretation of data. Todd, Knowlton, Schur, Izmailova, Roubenoff, Shadick, Weinblatt, Centola, Lee.
ROLE OF THE STUDY SPONSORS
Crescendo Bioscience and Millennium Pharmaceuticals provided financial sponsorship for commercial assay costs. Employees of
these companies participated as coauthors of the manuscript by
contributing to the study design, the acquisition, analysis, and interpretation of the data, and the manuscript preparation. The authors
independently collected the data, interpreted the results, and had the
final decision to submit the manuscript for publication. Crescendo
Bioscience and Millennium Pharmaceuticals reviewed the manuscript
prior to submission, but publication of this article was not contingent
upon approval by these companies.
RHEUMATOID FACTOR INTERFERENCE WITH MULTIPLEX ASSAYS
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