1. Macro-TSH and endogeneous antibody interference in immunoassays
Ellen Anckaert, M.D., Ph.D.
Laboratorium Hormonologie & Tumormarkers
UZ Brussel

2. Non-competitive immunoassay principle
Antibody
Antigen
Biotin
Ruthenium
Antibody

3. Non-competitive immunoassay principle
Sandwich complex

4. Non-competitive immunoassay principle
Microbeads

5. Non-competitive immunoassay principle

6. 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

7. Antibody interference in thyroid hormone assays
Antibodies against assay antibodies
Heterophilic antibodies
Autoantibodies against thyroid hormones
Anti-TSH (macro-TSH)
Anti-thyroglobulin antibodies
Anti-T4, Anti-T3 antibodies
Antibodies against assay antibody detection molecules

8. 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

9. 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
Titer
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

10. 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

11. Competitive immunoassay (example FT4)

12. 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

13. 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

14. 2. Fragmentation of antibodies
Use of Fab or F(ab’)2 fragments

15. C1 constant region from human IgG
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

16. Heterophilic antibody / HAMA interference
Prevalence of interference depends on the immunoassay (IA) method
Bjerner 2002 (CEA, patient samples)
unblocked IA 4%
blocked IA (Fc removal) %
blocked IA (Fc removal – MAK33) 0.06%
Boscato 1986 (hCG, 668 healthy subject samples)
unblocked IA %
blocked IA %
Ward 1997 (TSH, patient samples)
blocked IA %
addition of “blocking reagent” reduces interference, but is no garantee for complete elimination of interference
estimated prevalence: 0.03 – 3%

17. 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

18. 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

19. Case report macro-TSH (1)
60 year old man, clinically euthyroid
TSH mIU/l ( mIU/l)
FT pmol/l (10-23 pmol/l)
TPO Ab IU/ml (0-50 IU/ml)
Tg Ab Neg
anti-TSH receptor Abs Neg
Test with an alternative immunoassay method
TSH mIU/l
Test dilution linearity3:
TSH 1: mIU/l
TSH 1: mIU/l (135% recovery)
Test for antibodies against assay antibodies
RF Negative
Heterophilic blocking tubes No interference detected
1 Vitros 5600, Ortho Clinical Diagnostics; 2 Advia Centaur, Siemens Healthcare Diagnostics
3 TSH assay diluent and immunoassay: Advia Centaur
Loh T P, JCEM 2012

20. Case report macro-TSH (2)
PEG-precipitation of high molecular weight proteins
Pre-PEG TSH 122mIU/l
Post-PEG TSH 3.9 mIU/l (3.2% recovery)
Presumable interference:
Macro-TSH = macro-molecule formed between TSH and autoimmune anti-TSH Ig
Heterophilic antibodies undetected by HBT
Testing the presence of excess TSH binding capacity = free anti-TSH antibody
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: %)
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

21. 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

22. Immunoassays display variable reactivity with macro-TSH
TSH measurement by different methods
Instrument Manufacturer Reference range TSH (µIU/mL)
Elecsys Roche 0.5 
Centaur Siemens 0.4 
Lumipulse Fuji Rebio 0.61 
Architect Abbott 0.35 
Sakai, Endocr J 2009

23. Overview macro-TSH cases, confirmed by GFC
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 -
mother
newborn
308
828
Halsall 2006 4 5 6 28 45 23
Neg
TRAb
Graves HT
5.1 22
9.7
Verhoye 2009 7 8 55
103
Delfia
Rix 2011 9 46
24.5
Mendoza 2009 10 M 60
Anti-TPO
232
Vitros
Loh 2012 11 29
40-115
RIA
Bifulco 1987 12 13 53
1.4 ->100
2.7 ->100
Viera 2006

24. Prevalence of macro-TSH
15/495 TSH > 10 mIU/l (3%): low recovery after PEG precipitation

25. 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 anti-Tg 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

26. Tg antibody interference in Tg immunoassays
Anti-Tg interference in Tg IMA is a common problem

27. 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

28. One step method - Labeled Analog+ + *
FT4
Conjugated
Analog
Serum
Binding
Protein
Anti - T4 Antibody
Bound to Particle X * *
Separate
and
Count + 13

29. 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)

30. Elecsys FT4 – FT3 immunoassay (Roche Diagnostics)

31. Protection against ani-Ru antibodies

32. Roche claims increased protection against anti-Ru antibodies in next generation IA

33. 28/8/ 5/2/ 11/9 15/5 Normal values Case report 2013 2013 2012 2012
TSH (mIU/l)
FT3 (ng/l)
FT4 (ng/l)
Switch to Elecsys FT3 III and FT4 II

34. FT4 immunoassays are all binding protein-dependent to some extent
Increased TBG in pregnancy
Genetic abnormalities in binding proteins, drugs that displace FT from binding proteins, NTI
Anckaert, Clin Chim Acta 2010

35. 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