Scandinavian Journal of Clinical and Laboratory
Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Heterophilic antibody interference affecting
multiple hormone assays: Is it due to rheumatoid
factor?
Shiva Mongolu, Annie E. Armston, Erin Mozley & Azraai Nasruddin
To cite this article: Shiva Mongolu, Annie E. Armston, Erin Mozley & Azraai Nasruddin
(2016): Heterophilic antibody interference affecting multiple hormone assays: Is it due to
rheumatoid factor?, Scandinavian Journal of Clinical and Laboratory Investigation, DOI:
10.3109/00365513.2016.1143113
To link to this article: http://dx.doi.org/10.3109/00365513.2016.1143113
Published online: 29 Feb 2016.
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Download by: [Emory University] Date: 08 March 2016, At: 18:18
SCANDINAVIAN JOURNAL OF CLINICAL & LABORATORY INVESTIGATION, 2016
http://dx.doi.org/10.3109/00365513.2016.1143113
ORIGINAL ARTICLE
Heterophilic antibody interference affecting multiple hormone assays: Is it due
to rheumatoid factor?
Shiva Mongolua, Annie E. Armstonb, Erin Mozleyc and Azraai Nasruddind
a
Department of Diabetes & Endocrinology, Hull Royal Infirmary, Hull; bDepartment of Clinical Biochemistry, University Hospital
Southampton, Southampton; cDepartment of Chemical Pathology, Guys and St. Thomas Hospital, London, UK; dDepartment of
Endocrinology, Putrajaya Hosital, Malaysia
ABSTRACT ARTICLE HISTORY
Assay interference with heterophilic antibodies has been well described in literature. Rheumatoid Received 17 July 2015
factor is known to cause similar interference leading to falsely elevated hormone levels when Revised 4 November 2015
measured by immunometric methods like enzyme-linked immunosorbent assay (ELISA) or Accepted 10 January 2016
multiplex immunoasays (MIA). We report a case of a 60-year-old male patient with a history of Published online
rheumatoid arthritis referred to our endocrine clinic for investigation of hypogonadism and was 26 February 2016
Downloaded by [Emory University] at 18:18 08 March 2016
found to have high serum levels of LH, FSH, SHBG, Prolactin, HCG and TSH. We suspected assay KEY WORDS
interference and further tests were performed. We used Heteroblock tubes and PEG precipitation to Heterophilic antibody inter-
eliminate the interference and the hormone levels post treatment were in the normal range. We ference; immunoassay inter-
believe the interference was caused by high serum levels of rheumatoid factor. Although he was ference; rheumatoid factor;
treated with thyroxine for 3 years, we believe he may have been treated inappropriately as his Free PEG precipitation;
T4 level was always normal despite high TSH due to assay interference. Our case illustrates the immunoassays
phenomenon of heterophilic antibody interference likely due to high levels of rheumatoid factor. It
is essential for clinicians and endocrinologists in particular to be aware of this possibility when
making treatment decisions in these groups of patients.
Background We report a case where elevated levels of TSH, LH,
FSH, HCG and SHBG were found due to possible
Interference in immunoassays is an underestimated
interference by RF, which normalized following removal
problem. Although not generally applicable, the preva-
of antibodies by Heteroblock tubes (Skybio HBT-50).
lence ranges between 0.05 to more than 2% and are
A 60-year-old Caucasian male was referred to our
analyte- and assay-specific [1]. The interference can be
endocrine clinic for investigation of hypogonadism. He
analyte-dependent or -independent. Rheumatoid factor
had a history of rheumatoid arthritis and hypothyroidism
(RF) was first described by Waaler around 1940 and is a
diagnosed 3 years previously which was treated with
combination of auto-antibodies of the types IgM, IgG,
Methotrexate and Levothyroxine. He reported symptoms
IgA, IgD, or IgE [2]. RF is found in plasma of 70–80% of
of reduced libido and hot flushes with normal erectile
patients with rheumatoid arthritis (RA) and some other
connective tissue diseases [3]. RF interference is a major function. His testosterone levels were low on two
problem when measuring samples from RA patients. occasions 11.3 and 8.3 nmol/L, respectively, with raised
Interference from heterophilic antibodies can occur in all gonadotrophins (LH 40.1 iu/L, FSH 63.5 iu/L) and raised
types of immunoassays but is most common in SHBG of 172 suggesting a picture of primary
immunometric type of immunoassays. This is due to hypogonadism.
the ability of the antibodies to bridge between the Prolactin was elevated at 901 mu/L along with
capture and the detection antibodies in the immuno- Oestradiol at 227 pmol/L. On reviewing his previous
metric assay creating a false signal [4]. investigations, it appears that although his TSH was
Rheumatoid factor (RF) especially IgM, has been elevated which could have potentially been due to assay
reported to cause assay interference leading to errone- interference, his free T4 levels had always been in the
ous high levels of Mast cell tryptase (MCT) [5], PSA [6], normal range and thyroxine may have been started
CA 19-9 [7], TSH [8] and free thyroxine (FT4) [9]. inappropriately. His serum rheumatoid factor levels
CONTACT Shiva Mongolu, MRCP (UK) smongolu@gmail.com Consultant Physician & Endocrinologist, Michael White Centre for Diabetes, Endocrinology
and Metabolic Bone Diseases, Brocklehurst Building, Hull Royal Infirmary, Anlaby Road, HU3 2RW, UK
ß 2016 Taylor & Francis
2 S. MONGOLU ET AL.
(measured by ELISA) were very high 757 iu/mL (normal Table 1. Hormone levels before and after use of
range 0–30 iu/mL). Assay interference on other platforms Heteroblock tubes.
was suspected and further testing with heterophile Previous After antibody
Analyte result blocking tubes
blocking tubes (HBT) was performed.
TSH (mU/L) 36.1 0.9
FT4 (pmol/L) 13.6 14.2
SHBG (nmol/L) 206 47
Materials and methods LH (IU/L) 50.4 5.3
FSH (IU/L) 58.7 5.9
At our centre, heterophilic blocking tubes (HBT-50, HCG (IU/L) 78.6 4.2
Skybio) are routinely used to exclude heterophilic
antibody interference in TSH and FT4 measurements The sample from our patient was treated with HBT
on the Beckman Unicel DxI. FSH, LH, SHBG, HCG and and re-measured.
FT4 are two-site immonumetric assays whereas
Progesterone, Oestradiol, Testosterone and FT3 are
competitive binding immune-enzymatic assay. The pri-
Results
mary (PMP bound) antibody for Free T4, TSH, LH, FSH The results (Table 1) indicated that this patient had
and Prolactin was Mouse mono-clonal and secondary heterophilic antibody interference to a number of
antibody was Goat poly-clonal antibody. analytes (LH, FSH, HCG, TSH and SHBG), as they returned
The manufacturer provided a blocking agent (HBT-50, to normal values post-treatment. The results Post-HBT
Downloaded by [Emory University] at 18:18 08 March 2016
Skybio) to reduce the likelihood of heterophile or anti- were confirmed by assaying the sample on a different
animal interference. Skybio’s Heterophilic Blocking tubes platform (Siemens Advia). The Prolactin level measured
contain a unique blocking reagent containing specific previously was 816 mU/L but after PEG precipitation for
binders which inactivate heterophilic antibodies. Once macro-prolactin, the actual level was 196 mU/L. This was
the specific binders have bound to the heterophile confirmed by referral to another laboratory.
antibodies they are no longer able to cause interference. We believe that the likely cause of this interference
The HBT Tube constitutes a sample pre-treatment that was high titre of rheumatoid factor. It is likely that
can be used as a secondary confirmation assay or as the this patient was not hypothyroid and was put on
first step in the initial assay. thyroxine after heterophilic antibodies caused falsely
Sample pre-treatment with HBT Tubes indicated that increased TSH.
there was potential interference. With the exception of
FT3 all assays were robust to dilution recovery (linearity)
Discussion
but only oestradiol and SHBG were tested for spiking
recovery. FT3 was not diluted. Rheumatoid factor (RF) was It is well known that interference in immunoassays is an
measured using Latex agglutination (Rheumajet RF, Biokit) underestimated problem. Analytical interference is
and was free from any interference. Latex reagent used for defined as ‘the effect of a substance present in the
RF assay is a suspension of polystyrene latex particles of sample that alters the correct value of the result,
uniform size coated with human gamma globulin. usually expressed as concentration or activity, for an
To validate the use of HBT to measure a number of analyte’ [10].
additional analytes, 10 samples were anonymized and The interference can be analyte-dependent or -inde-
then analytes measured before and after HBT treatment. pendent. Analyte-dependent interferences are due to
No dilution step was involved. There was no significant interactions between constituents in the sample and one
difference in the results of four analytes pre- and post- or more reagent bodies. These include heterophile
HBT treatment, indicating that HBT are suitable for HCG antibodies, human anti-animal antibodies, autoanalyte
(n ¼10, paired t-test p ¼ 0.2040, Deming regression antibodies, rheumatoid factors and other proteins which
r2 ¼ 1), SHBG (n ¼ 20, p ¼ 0.1006, r2 ¼ 0.9876), LH (n ¼ 10, have structural similarities that cross-react with the
p ¼ 0.4272, r2 ¼ 0.9503) and oestradiol (n ¼ 13, antibody [11]. This can lead to either falsely elevated or
p ¼ 0.9823, r2 ¼ 0.9853). However results of samples falsely low analyte concentration depending on the site
after treatment with HBT revealed some differences for of interference.
progesterone (n ¼ 20, p ¼ 0.2552, r2 ¼ 0.6358), FSH Interfering, endogenous antibodies are called hetero-
(n ¼ 10, p ¼ 0.0035, r2 ¼ 0.9777) and FT3 (n ¼ 30, phile antibodies, when there is no history of medicinal
p ¼ 0.0006, r2 ¼ 0.7354), as there was a small but treatment with animal immunoglobulins or clearly
significant bias. Testosterone cannot be measured after defined immunogen, the antibody reacts with immuno-
treatment of samples for HBT as results pre- and post- globulin from two or more species or has rheumatoid
treatment were widely different. factor activity [12]. In the case of RF, false-positives arise
SCANDINAVIAN JOURNAL OF CLINICAL & LABORATORY INVESTIGATION 3
by binding of RF to the Fc constant domain of antigen- [5] Sargur R, Cowley D, Murng S, Wild G, Green K,
antibody complexes if the detection antibody is labelled Shrimpton A, Egner W. Raised tryptase without anaphyl-
axis or mastocytosis: heterophilic antibody interference
anti-human IgG. in the serum tryptase assay. Clin Exp Immunol
In a screening study conducted using native and pre- 2011;163:339–45.
blocked sera containing high titre of Fc-reactive hetero- [6] Anderson CB, Pyle AL, Woodworth A, Cookson MS,
philic antibodies, it was found that 21 commercial Smith JA Jr, Barocas DA. Spurious elevation of serum
assays, covering 19 different analytes were susceptible PSA after curative treatment for prostate cancer: clinical
consequences and the role of heterophilic antibodies.
to interference [13].
Prostatic Cancer Prostatic Dis 2012;15:182–8.
The suggested solutions to eliminate this interference [7] Berth M, Bosmans E, Everaert J, Dierick J, Schiettecatte J,
are PEG precipitation with polyethylene glycol 6000 (PEG Anckaert E, Delanghe J. Rheumatoid factor interference
6000) [14–17] or protein L [14] or using Heteroblock in the determination of carbohydrate antigen 19-9
tubes [16]. Grasko et al. reported the phenomenon of (CA 19-9). Clin Chem Lab Med 2006;44:1137–9.
[8] Georges A, Charrie A, Raynaud S, Lombard C, Corcuff JB.
heterophilic antibody interference affecting ACTH, not
Thyroxin overdose due to rheumatoid factor interfer-
detectable by proprietary hetorophile blocking reagent ences in thyroid-stimulating hormone assays. Clin Chem
leading to unnecessary and costly investigations in a Lab Med 2011;49:873–5.
patient with Cushing’s syndrome [18]. Our case illus- [9] Norden AGW, Jackson RA, Norden LE, Griffin AJ, Barnes
trates an uncommon occurrence of heterophilic anti- MA, Little JA. Misleading results from immunoassays of
serum free thyroxine in the presence of rheumatoid
body interference possibly caused by high serum levels
Downloaded by [Emory University] at 18:18 08 March 2016
factor. Clin Chem 1997;43:957–62.
of rheumatoid factor affecting multiple hormone assays; [10] Kroll MH, Elin RJ. Interference with clinical laboratory
this, in turn, leading to inappropriate treatment with analyses. Clin Chem 1994;40:1996–2005.
Thyroxine. Gulbahar et al. recently reported a similar [11] Tate J, Ward G. Interferences in immunoassay. The Clin
case of falsely elevated hormone levels due to assay Biochem Rev 2004;25:105–20.
[12] Kaplan IV, Levinson SS. When is a heterophile antibody
interference caused by heterophile antibodies affecting
not a heterophile antibody? When it is an antibody
multiple platforms detected by heterophile antibody against a specific immunogen. Clin Chem 1999;45:
blocking tubes [19]. It is therefore essential for clinicians 616–18.
and endocrinologists in particular to be aware of this [13] Bolstad N, Warren DJ, Bjerner J, Kravdal G, Schwettmann
possibility when making treatment decisions in these L, Olsen KH, Rustad P, Nustad K. Heterophilic antibody
interference in commercial immunoassays; a screening
groups of patients.
study using paired native and pre-blocked sera. Clin
Chem Lab Med 2011;49:2011–6.
Acknowledgements [14] De Jager W, Prakken BJ, Bijlsma JW, Kuis W, Rijkers GT.
Improved multiplex immunoassay performance in human
We are grateful to the laboratory staff at Southampton General
plasma and synovial fluid following removal of interfering
Hospital for their assistance in analysing the samples.
heterophilic antibodies. J Immunol Methods 2005;300:
124–35.
Contributions SM wrote the first draft of the manuscript. All [15] Moore TL, Dorner RW, Sheridan PW, Zuckner J.
the authors reviewed and edited the final version. AA and EM Precipitation of 19S IgM rheumatoid factor-IgG circulat-
performed the analysis in the laboratory with Heteroblock ing immune complexes in patients with juvenile arthritis
tubes. by polyethylene glycol and separation by immobilized
protein A. Clin Exp Immunol 1984;56:247–52.
Disclosure statement [16] Bartels EM, Watjen IF, Andersen EL, Danneskiold-Samsoe
B, Bliddal H, Ribel-Madsen S. Rheumatoid factor and its
The authors report no conflict of interest. The authors alone are interference with cytokine measurements: problems and
responsible for the content and writing of the paper. solutions. Arthritis 2011;2011:741071.
[17] Giovanella L, Ceriani L. Spurious increase in serum
chromogranin A: the role of heterophilic antibodies.
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