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Macro-TSH and endogeneous antibody interference in immunoassays

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Presentation on theme: "Macro-TSH and endogeneous antibody interference in immunoassays"— Presentation transcript:

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

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

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