Macro-TSH and endogeneous antibody
interference in immunoassays
Ellen Anckaert, M.D., Ph.D.
Laboratorium Hormonologie & Tumormarkers
UZ Brussel
Non-competitive immunoassay principle
Antibody
Antigen
Biotin
Ruthenium
Antibody
Non-competitive immunoassay principle
Sandwich complex
Non-competitive immunoassay principle
Microbeads
Non-competitive immunoassay principle
Interference
Definition: “interference is the effect of a substance
present in the sample that alters the correct value of
the result for an analyte (Kroll & Elin, 1994)
Immunoassay design determines the sensitivity of the
assay to interference
Antibody interference in thyroid hormone assays

Antibodies against assay antibodies


Autoantibodies against thyroid hormones




Heterophilic antibodies
Anti-TSH (macro-TSH)
Anti-thyroglobulin antibodies
Anti-T4, Anti-T3 antibodies
Antibodies against assay antibody detection
molecules
Interferences due to endogeneous antibodies
against assay antibodies
Possible clinical consequences:
• Misclassification of monitoring results
• Unnecessary follow-up examinations
• False therapy decisions
• Unfavorable patient prognosis
Endogeneous antibodies against assay
antibodies
Heterophilic
antibodies
Human anti-mouse
antibodies (HAMA)
Rheumatoid factor
Etiology
Poorly defined,
no clear immunogen
Known antigenic
stimulus
Auto-antibody
Specificity
Low:
bind different species Ig
High
Low: bind Fc
region of different
species Ig
Affinity
Low
High
Low
Titer
Low
High
High in active
rheumatic disease
Ig class
IgG, IgM
IgG, IgA, IgM
Usually IgM
Prevalence
Up to 40%
In 40-70% of
patients treated
with mouse MAbs
5-10%
70% autoimmune
rheumatic disease
Mechanisms of interference by heterophilic
antibodies
Bridging of capture and
detector antibodies
=> Falsely elevated result
Exclusive binding of capture or detector antibody
only
=> Falsely lowered result
Competitive immunoassay (example FT4)
Assay design: whan can the manufacturer do to
reduce heterophilic antibody interference ?
No protection
Use of blocking proteins
Fragmentation of Antibodies
Use of chimeric MABs
Interference level:
High
low
extremely low
1. Addition of blocking antibodies
Addition of a “blocking agent” of the same species as the assay
antibodies:
- animal serum
- animal immunoglobulin
- aggregated mouse monoclonal IgG (MAK33) to eliminate strong HAMA
interferences, usually therapy induced
2. Fragmentation of antibodies
Use of Fab or
F(ab’)2 fragments
3. Chimeric Antibodies
Constructed from 2 different species (mouse / human )
Fc-fragment cleaved off
Variable region from mouse
IgG
C1 constant region from
human IgG
Heterophilic antibody / HAMA interference

Prevalence of interference depends on the immunoassay (IA)
method

Bjerner 2002 (CEA, 11.261 patient samples)
 unblocked IA
4%
 blocked IA (Fc removal)
0.1%
 blocked IA (Fc removal – MAK33)
0.06%

Boscato 1986 (hCG, 668 healthy subject samples)
 unblocked IA
15%
 blocked IA
0.6%

Ward 1997 (TSH, 21.000 patient samples)
 blocked IA
0.03%

addition of “blocking reagent” reduces interference, but is
no garantee for complete elimination of interference
estimated prevalence: 0.03 – 3%

What can the lab do to detect immunoassay
interference by heterophilic antibodies?
Repeat the analysis with an alternative immunoassay,
preferably using assay antibodies from a different species
Treat the sample with an additional blocking agent
(Heterophilic Blocking Tubes, Scantibodies)
Dilute the sample: non linearity indicates assay interference
A negative interference test does not exclude interference
Macro-TSH



Macro-molecule composed of TSH and anti-TSH
immunoglobulin
Reduced renal clearance leads to accumulation of
macro-TSH
Reduced biological activity
 Patients are clinically euthyroid

Immunoreactivity is variable and reduced compared to
native TSH
 spuriously elevated TSH levels to a variable degree using
different immunoassays
 low recovery of added TSH
Case report macro-TSH (1)

60 year old man, clinically euthyroid
TSH1
FT4
TPO Ab
Tg Ab
anti-TSH receptor Abs






Test with an alternative immunoassay method
TSH2


122mIU/l
Test dilution linearity3:



232 mIU/l
(0.45-5 mIU/l)
10 pmol/l (10-23 pmol/l)
496 IU/ml
(0-50 IU/ml)
Neg
Neg
TSH 1:1
TSH 1:10
122mIU/l
165 mIU/l (135% recovery)
Test for antibodies against assay antibodies


1
3
RF
Heterophilic blocking tubes
Negative
No interference detected
Vitros 5600, Ortho Clinical Diagnostics; 2 Advia Centaur, Siemens Healthcare Diagnostics
TSH assay diluent and immunoassay: Advia Centaur
Loh T P, JCEM 2012
Case report macro-TSH (2)

PEG-precipitation of high molecular weight proteins


Pre-PEG TSH
Post-PEG TSH
122mIU/l
3.9 mIU/l (3.2% recovery)
 Presumable interference:



Testing the presence of excess TSH binding capacity = free
anti-TSH antibody




Macro-TSH = macro-molecule formed between TSH and autoimmune anti-TSH Ig
Heterophilic antibodies undetected by HBT
sequestration of added TSH (hypothyroid serum)
macro-TSH has reduced immunoreactivity compared to native TSH
RESULT: low recovery (85%)
Thyroid stimulating Ig bioassay: 120% (normal: 50-179%)


Consisitent with clinical euthyroid state
Suggests low biological activity of macro-TSH
* Advia Centaur, PEG recovery in ‘normal’ euthyroid patient serum was 40%
Loh T P, JCEM 2012
Confirmation of macro-TSH by gel filtration
chromatography
Patient serum incubated with hypothyroid
serum:  immunoreactivity of the HMW
fraction, confirming excess TSH binding
capacity and macro-TSH (trangles).
Patient serum: TSH peak fraction that
approximates the molecular size of IgG (dots).
Loh T P, JCEM 2012
Immunoassays display variable reactivity
with macro-TSH
TSH measurement by different methods
Instrument
Elecsys
Centaur
Lumipulse
Architect
Manufacturer Reference rangeTSH (µIU/mL)
Roche
Siemens
Fuji Rebio
Abbott
0.5  5.0
0.4  4.0
0.61  4.68
0.35  4.94
152.0
20.5
112.4
9.8
Sakai, Endocr J 2009
Overview macro-TSH cases, confirmed by GFC
Case Sex
Age
Thyroid
antibody
positive
Clinical
signs/
symptoms
TSH (mIU/l)
Immunoassay
Ref
1
F
56
Anti-Tg
No
274
Elecsys
Sakai
2009
2
3
F
-
mother
newborn
-
No
No
308
828
Elecsys
Elecsys
Halsall
2006
4
5
6
F
F
28
45
23
Neg
Neg
TRAb
No
No
Graves HT
5.1
22
9.7
Elecsys
Elecsys
Elecsys
Verhoye
2009
7
8
F
F
mother
newborn
Neg
No
No
55
103
Elecsys
Delfia
Rix
2011
9
F
46
Neg
No
24.5
Elecsys
Mendoz
a 2009
10
M
60
Anti-TPO
No
232
Vitros
Loh
2012
11
M
29
-
No
40-115
RIA
Bifulco
1987
12
13
F
F
53
6
Neg
Neg
No
No
1.4 ->100
2.7 ->100
Immunoassay
Immunoassay
Viera
2006
Prevalence of macro-TSH
15/495 TSH > 10 mIU/l (3%): low recovery after PEG precipitation
Tg antibody interference in Tg immunoassays


Anti-Tg antibody prevalence
 10% general population
 25% in DTC
No Tg method completely free from interference
 underestimation in non-competitive assay
 false elevation is possible in competitive assay

Measurement of Tg in follow-up of DTC: should always be
accompanied by anti-Tg measurement using a sensitive antiTg immunoassay

What can the lab do:
 Confirm by an alternative (competitive) immunoassay
method
 Exogeneous Tg recovery test
 low recovery indicates interference
 normal recovery does not exclude interference
Tg antibody interference in Tg immunoassays
Anti-Tg interference in Tg IMA is a common problem
Interference by endogeneous antibodies in
FT4 – FT3 assays
Anti-T4 and anti-T3 antibodies
 Prevalence depends on the selected population and the
method of detection







20% in autoimmune thyroid disease
6% in non-thyroidal autoimmune disease
0-2% in healthy individuals
women > men
Mostly IgG subclass, mostly polyclonal
Most patients also have anti-Tg and/or anti-microsomal
antibodies
Impact on immunoassay (interference) depends on


the assay format
titer, affinity and specificity of the antibody
One step method - Labeled Analog
+
T4
+
*
FT4
Serum
Binding
Protein
X
Anti - T4 Antibody
Bound to Particle
Conjugated
Analog
*
*
+
Separate
and
Count
Interference by anti-ruthenium antibodies in
Elecsys FT4 – FT3 assays
Anti-Ru antibodies

Mainly in areas with textile industry
 Use of Ru in dying process of clothing
 Ru in environment, clothing or food chain

Estimated frequency of interference in first generation
Elecsys FT3 assay (Roche Diagnostics): 0.2% (Sapin, Clin
Chem Lab Med 2007)
Elecsys FT4 – FT3 immunoassay (Roche
Diagnostics)
Protection against ani-Ru antibodies
Roche claims increased protection against
anti-Ru antibodies in next generation IA
Case report
28/8/
5/2/
11/9
15/5
Normal values
2013
2013
2012
2012
TSH (mIU/l)
0.552
0.344
0.569
0.515
0.27-4.2
FT3 (ng/l)
3.2
5.9
7.0
6.2
2.6-4.4
FT4 (ng/l)
12.6
20.8
21.2
19.5
9.3-17.0
Switch to Elecsys FT3 III and
FT4 II
FT4 immunoassays are all binding proteindependent to some extent
Increased TBG in pregnancy
Genetic abnormalities in binding proteins, drugs that displace FT4
from binding proteins, NTI
Anckaert, Clin Chim Acta 2010
Conclusion

Interference in immunoassays
 uncommon

exception: anti-Tg interference in Tg IMA
 no method is completely free from interference
 often unidentified by the laboratory routine quality assurance
check

Immunoassay results that are incongruent with the patient’s clinical
presentation should be tested for interference

Clinician should be actively encouraged to contact the laboratory in
case of any doubt about a result

In case of confirmed interference patients should be informed
about the presence of interfering substances in their serum