Evaluation of Biotin Interference on the Abbott

ARCHITECT Assays
M. Hauptman,1 J. Jaraskweski,1 R. Schneider1
Abbott Laboratories, Diagnostics Division, Abbott Park, IL
1

2017 AACC Annual Meeting, San Diego, California • July 30 - August 3, 2017

Abstract
Background and Relevance: The use of biotin as a interference testing as they contain streptavidin or biotin
supplement has increased significantly in recent years in the assay design.
and many health care professionals do not realize
Materials and Methods: For each of 5 ARCHITECT
their patients are taking high doses. The increase has
assays: (Methotrexate; Active B12; Vitamin D; 2nd
resulted in an increased prevalence of people being
Generation Testosterone; Anti-CCP), sample pools were
exposed to levels much higher than the recommended
created and spiked with concentrations of biotin between
daily dose and as a consequence, inaccurate lab results
30 – 1000 ng/mL. For Active B12, a single sample in the
for assays that utilize the free capture biotin-streptavidin
normal range was utilized. For the other assays, two
methodology. The purpose of this study was to identify
sample pools were used for testing with one negative/
any ARCHITECT assays that may be susceptible to
near the lower end of the assay or medical decision
biotin interference based on assay design and then
point and one positive/near the upper end of the assay
evaluate the performance of these assays with high
or medical decision point. The biotin spiked samples
concentrations of biotin. After a comprehensive review
were tested against a control sample preparation (no
of Abbott’s current on market ARCHITECT clinical
biotin) to determine if there was a statistical difference
chemistry and immunoassay methods, no assays were
between the untreated and biotin containing specimens.
identified that utilize the free capture biotin-streptavidin;
however, 5 assays were identified for subsequent

Results:

Concentration of Untreated % Difference from Untreated

Assay Specimen
Sample Pool Sample Pools (95% Confidence)

Active B12
87.91 U/mL Normal -3.51 to 3.70
(LN 3P24)

Anti-CCP 0.66 U/mL Low (Negative) -3.59 to 7.40

(LN 1P65) 10.70 U/mL High (Positive) -0.15 to 3.80

Methotrexate 0.051 umol/L Low -3.80 to 3.71

(LN 2P49) 0.877 umol/L High 0.36 to 6.15

Testosterone – 6.92 nmol/L Low -4.59 to 0.52

2nd Gen
(LN 2P13) 21.36 nmol/L High -4.84 to 1.15

Vitamin D 25.76 ng/mL Low -0.14 to 8.02

LN 3L52 35.16 ng/mL High -0.35 to 7.32

Conclusions: Five ARCHITECT assays potentially susceptible to biotin interference, based on assay design, were
tested at increasingly high concentrations of biotin. No Abbott ARCHITECT assay evaluated as part of this study
yielded result variability due to biotin interference at concentrations up to 1000ng/mL.

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 Introduction
Biotin is a small water soluble B-complex vitamin that has Purpose
been used by diagnostic companies when developing
A comprehensive review of Abbott assays was performed
laboratory tests. Because it can be covalently coupled
to determine which methodologies utilize biotin and/or
to target proteins (ex. hormones), has minimal effect on
streptavi­din in their reagent formulation and could there­
biologic and antigenic activities, and has an inherently
fore be susceptible to a biotin interference. Through these
strong affinity for streptavidin, immunoassays using a
assessments, no clinical chemistry assays were identified
free-capture methodology based on a biotin-streptavidin
and five immunoassays were identified for additional
interaction have been widely adopted by diagnostic
assessments. Although none of the 5 immunoassays
companies for measuring target analytes across different
use the free capture streptavidin/biotin format, four of
disease states.
the five identified assays utilize a streptavidin/biotin
While recommended levels of biotin are obtained complex in the microparticle component: ARCHITECT
through a balanced diet, in recent years the use of biotin Methotrexate (2P49), ARCHITECT Anti-CCP (1P65),
as a supplement and potential treatment modality has ARCHITECT Active B12 (3P24), and ARCHITECT
increased. This trend has resulted in a suspected increase 2nd Generation Testosterone (2P13), and one uses a
in the prevalence of people exposed to biotin levels. As biotinylated anti-Biotin IgG acridinium labeled conjugate
a consequence, some current on market immunoassays complex: ARCHITECT 25-OH Vitamin D assay (3L52)
that utilize a biotin-streptavidin free-cature methodology
have been associated with case reports describing Interference
diagnostic errors and biotin interference. Interference
How Biotin Supplementation can Impact Biotin—
How Biotin Supplementation can Impact Biotin—Streptavidin Assays
In order to determine if a laboratory method is susceptible Streptavidin Assays
to biotin interference, a comprehensive assessment of Patient sample: Bioantibody Capture
Interference – Free
Biotin inhibits the

the assay formats, including biotin interference testing Biotinylated

Antibody Complex
Analyte and
Biotin
complex reacts
with analyte in
the sample
streptavidin binding
sites ability to capture
targeted analyte

when indicated, should be performed to determine the

level of susceptibility and type of bias that may occur Biotin-

Streptavidin

Streptavidin
Streptavidin

when testing patients. In addition to assessing assay Free Capture

Method

methodologies, education and awareness can help

reduce the risk of diagnostic errors for labs using this Magnetic
Microparticle

MAGNET

MAGNET
assay format. Capture
Method

Finally, not all immunoassay formats are susceptible Magnetic

microparticle
No Interference
from free Biotin

to biotin interference and options currently exist for coated with antibody

alternative testing when a patient reports recent use of Biotinylated

Antibody
Free
Biotin
Target
Analyte
Streptavidin
Binding Sites
Ab Magnetic
microparticle

high dose biotin supplementation or treatment prior to

lab testing.

Materials and Methods

Biotin Stock Preparation for testing. For each mL of sample pool, 0.05 mL was
removed from each of the samples. A control sample was
Stock solutions were prepared by dissolving biotin in PBS
prepared with 0.95 mL of patient sample and 0.05 mL of
to multiple target concentrations (table 1). A 1.0 mg/mL
PBS. The remaining 0.95 mL samples were prepared
stock solution of Biotin in PBS was prepared by adding
with varying final concentrations of biotin ranging from
20 mg of Biotin to a 50 mL conical tube and then adding
30, 50, , 500, and 1000 ng/mL.
20mL of PBS, for a final 1.0 mg/mL concentration. The
stock solution was mixed for a minimum of 30 minutes Testing:
prior to use, and subsequently used for preparing
target biotin concentrations for testing with patient Testing was performed on control and experimental
sample pools. samples in replicates of fifteen on a calibrated
ARCHITECT instrument.
Sample Preparation: % difference between the sample and control pool was
Two patient sample pools for each assay type (Exception: calculated using the following equation:
Active B12) were prepared near the medical decision
(Sample value – Reference Value) / Reference Value x
points. A minimum of 10 mL was prepared per pool.
100 = % Difference
Patient samples were split into separate 1 mL volumes
(or greater if 1 mL was not sufficient to test 15 replicates)

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 Materials and Methods (cont’d)
Table 1. Biotin Stock Target Concentrations
Stock Solution Target Amount of 0.1 ng/ml Amount of PBS to Final Concentration
Stock ID
Concentration (ng/mL) Stock to Use (mL) Use (mL) at Testing (ng/mL)

1 20,000 2.0 8.0 1,000

2 10,000 1.0 9.0 500
3 1,000 0.1 9.9 50
4 600 0.06 9.94 30

Results
ARCHITECT Methotrexate* (List 2P49): ARCHITECT Active B12 (List 3P24):
Spiking Biotin up to 1000 ng/mL into Methotrexate Spiking Biotin up to 1000 ng/mL into Active B12 samples
samples does not impact the concentration value does not impact the concentration value more than
more than +/-10% with a 95% confidence. For the +/-10% with a 95% confidence. For the Sample tested,
Low sample, the concentration % difference (with 95% the concentration % difference (with 95% confidence)
confidence) ranged from -3.80 to 3.71%. For the High ranged from -3.51 to 3.70%.
sample, the concentration % difference (with 95%
confidence) ranged from 0.36 to 6.15%. Table 4. Active B12 Results
Table 2. Methotrexate Results Assay Sample
Biotin
ng/mL
Recovery Difference
%
pmol/L
Biotin Assay Recovery Difference
Assay Sample 0 87.91 NA
ng/mL umol/L ug/mL %
0 0.051 0.023 NA 30 89.86 2.21%
Active B12
30 0.050 0.023 -1.78% 1 50 87.89 -0.02%
(3P24)
1 50 0.050 0.023 -1.50%
500 86.47 -1.64%
500 0.051 0.023 -0.21%
1000 87.71 -0.22%
Methotrexate 1000 0.052 0.024 1.28%
(2P49) 0 0.877 0.398 NA
30 0.911 0.414 3.94% ARCHITECT 2nd Generation
2 50
500
0.909
0.909
0.413
0.413
3.75%
3.70%
Testosterone (List 2P13):
1000 0.896 0.407 2.21% Spiking Biotin up to 1000 ng/mL into Testosterone
samples does not impact the concentration value more
ARCHITECT Anti-CCP (List 1P65): than +/-10% with a 95% confidence. For the Low sample
Spiking Biotin up to 1000 ng/mL into Anti-CCP samples the concentration % difference (with 95% confidence)
does not impact the concentration value more than ranged from -4.59 to 0.52%. For the High sample the
+/-10% with a 95% confidence. For the Low sample concentration % difference (with 95% confidence)
the concentration % difference (with 95% confidence) ranged from -4.84 to 1.15%.
ranged from -3.59 to 7.40%. For the High sample the
concentration % difference (with 95% confidence) Table 5. Testosterone Results
ranged from -0.15 to 3.80%. Biotin Recovery Difference
Assay Sample
ng/mL nmol/L ng/mL %
Table 3. Anti-CCP Results 0 6.92 2.00 NA
30 6.75 1.95 -2.59%
Biotin Assay Recovery Difference
Assay Sample 1 50 6.76 1.95 -2.39%
ng/mL U/mL %
500 6.86 1.98 -0.99%
0 0.66 NA 2nd Generation 1000 6.74 1.94 -2.68%
30 0.68 2.64% Testosterone
(2P13) 0 21.36 6.16 NA
1 50 0.68 2.69%
30 20.85 6.01 -2.37%
500 0.68 2.98%
2 50 20.88 6.02 -2.27%
Anti-CCP 1000 0.67 1.35%
500 21.20 6.11 -0.74%
(1P65) 0 10.70 NA
1000 21.32 6.15 -0.17%
30 10.91 1.98%
2 50 10.94 2.21%
500 10.80 0.93%
1000 10.96 2.40% *Not commercially available in U.S.
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 Results (cont’d)
ARCHITECT Vitamin D (List 3L52): Table 6. Vitamin D Results
Spiking Biotin up to 1000 ng/mL into Vitamin D samples Assay Sample
Biotin Assay Recovery Difference
ng/mL %
does not impact the concentration value more than ng/mL nmol/L
0 25.76 64.40 NA
+/-10% with a 95% confidence. For the Low sample 30 26.03 65.07 1.04%
the concentration % difference (with 95% confidence 1 50 26.32 65.80 2.17%
ranged from -0.14 to 8.02%. For the High sample the 500 26.68 66.69 3.56%
concentration % difference (with 95% confidence) Vitamin D 1000 27.47 68.67 6.63%
(3L52) 0 35.16 87.89 NA
ranged from -0.35 to 7.32%. 30 35.41 88.54 0.73%
2 50 35.42 88.55 0.75%
500 36.28 90.71 3.20%
1000 37.34 93.34 6.20%

Conclusion
A comprehensive review has confirmed that no Abbott Current on-market Abbott assays do not pose a risk
on market immunoassay or clinical chemistry methods for interference in patients taking supplemental or
utilize a free capture biotin-streptavidin methodology. mega-dose prescription levels of biotin.
For the 5 assays tested which contain biotin or Study findings can be applied to Alinity ci-series*,
streptavidin during assay development (preformed), Abbott’s next generation of Clinical Chemistry and
biotin added to the test samples at concentrations Immunoassay Systems due to identical reagent and
up to 1000 ng/mL did not cause interference in the assay formats.
reported target sample concentrations.

References
• Seaborg, E. January 2016: Thyroid Month: Beware of Biotin, Endocrine News, • Piketty , M et al., Biotin interference false biochemical diagnosis of hyperthyroidism
2016. in streptavidin-biotin-based immunoassays: the problem of biotin intake and
related interferences. Clin Chem Lab Med 2016.
• Picketty, M., et al., High-dose biotin therapy leading to false biochemical endocrine
profiles: validation of a simple method to overcome biotin interference., Clin Chem • Trambas, CM. More on Biotin Treatment Mimicking Graves’ Disease. NEJM 2016.
Med Lab, 2017; 55(6): 817 – 825.
• Batista, M., et al., Biotin interference in immunoassays mimicking subclinical
• Paxton A.  Beauty fad’s ugly downside: test interference.  CAP Today, September Graves’ disease and hyperestrogenism: a case series. Clin Chem Lab Med.,
20, 2016. 2017; 55(6): e99 – e103.
• Kummer, S. Biotin Treatment Mimicking Graves’ Disease. NEJM 2016: 375 (7) 704 • Lam, L and Kyle, C., A simple method to detect biotin interference on
– 706. immunoassays., 2017 Clin Chem Lab Med., 2017; 55(6): e104 – e106.
• Minkovshky, A et al., High-Dose Biotin Treatment for Secondary Progressive M.S.
may Interfere with Thyroid Assays., AACE Clin Case Rep., 2016; 2(4): e370 – e373.

5 Steps to Help Reduce

Laboratory Errors Caused by Biotin
1. Raise awareness
2. Know your assays and the impact biotin can have on them
3. Educate your health-care providers and patients
4. Have a contingency plan for acute-care settings
5. Recognize there are laboratory methods that are not
impacted by biotin that can provide an alternative option
for your patients

*Submitted for FDA review.

ARCHITECT and Alinity are trademarks of Abbott Laboratories.
©2017 Abbott Laboratories. ADD-00061737
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