Macro-TSH and endogeneous antibody interference in immunoassays

Ellen Anckaert, M.D., Ph.D.Laboratorium Hormonologie & TumormarkersUZ Brussel

Antibody

Ruthenium

Biotin

Antibody

Antigen

Non-competitive immunoassay principle

Sandwich complex

Non-competitive immunoassay principle

Microbeads

Non-competitive immunoassay principle

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 Heterophilic antibodies

Autoantibodies against thyroid hormones 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

Heterophilicantibodies

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 protectionUse 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

Variable region from mouse IgG

C1 constant region from human IgG

Fc-fragment cleaved off

3. Chimeric Antibodies

Constructed from 2 different species (mouse / human )

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 232 mIU/l (0.45-5 mIU/l) FT4 10 pmol/l (10-23 pmol/l) TPO Ab 496 IU/ml (0-50 IU/ml) Tg Ab Neg anti-TSH receptor Abs Neg

Test with an alternative immunoassay method TSH2 122mIU/l

1 Vitros 5600, Ortho Clinical Diagnostics; 2 Advia Centaur, Siemens Healthcare Diagnostics

Test dilution linearity3: TSH 1:1 122mIU/l TSH 1:10 165 mIU/l (135% recovery)

3 TSH assay diluent and immunoassay: Advia Centaur

Test for antibodies against assay antibodies RF Negative Heterophilic blocking tubes No interference detected

Loh T P, JCEM 2012

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)

* Advia Centaur, PEG recovery in ‘normal’ euthyroid patient serum was 40%

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: 50-179%) Consisitent with clinical euthyroid state Suggests low biological activity of macro-TSH

Loh T P, JCEM 2012

Confirmation of macro-TSH by gel filtration chromatography

Patient serum: TSH peak fraction that approximates the molecular size of IgG (dots).

Patient serum incubated with hypothyroid serum: immunoreactivity of the HMW fraction, confirming excess TSH binding capacity and macro-TSH (trangles).

Loh T P, JCEM 2012

TSH measurement by different methods

Instrument Manufacturer Reference rangeTSH (µIU/mL)

Elecsys Roche 0.5 5.0 152.0Centaur Siemens 0.4 4.0 20.5

Lumipulse Fuji Rebio 0.61 4.68 112.4Architect Abbott 0.35 4.94 9.8

Immunoassays display variable reactivity with macro-TSH

Sakai, Endocr J 2009

Case

Sex Age Thyroid antibody positive

Clinical signs/ symptoms

TSH (mIU/l) Immunoassay

Ref

1 F 56 Anti-Tg No 274 Elecsys Sakai 2009

23

F-

mothernewborn

--

NoNo

308828

ElecsysElecsys

Halsall 2006

456

F-F

284523

NegNegTRAb

NoNoGraves HT

5.1229.7

ElecsysElecsysElecsys

Verhoye 2009

78

FF

mothernewborn

-Neg

NoNo

55103

ElecsysDelfia

Rix 2011

9 F 46 Neg No 24.5 Elecsys Mendoza 2009

10 M 60 Anti-TPO No 232 Vitros Loh 2012

11 M 29 --

No 40-115 RIA Bifulco 1987

1213

FF

536

NegNeg

NoNo

1.4 ->1002.7 ->100

ImmunoassayImmunoassay

Viera 2006

Overview macro-TSH cases, confirmed by GFC

Prevalence of macro-TSH

15/495 TSH > 10 mIU/l (3%): low recovery after PEG precipitation

Tg antibody interference in Tg immunoassays

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

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

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

SerumBindingProtein

T4

FT4

+ + *

Anti - T4 AntibodyBound to Particle

+

*Separateand Count

X*

ConjugatedAnalog

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 protein-dependent 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