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Macro-TSH and endogeneous antibody interference in immunoassays Ellen Anckaert, M.D., Ph.D. Laboratorium Hormonologie & Tumormarkers UZ Brussel Slide 2 Antibody Ruthenium Biotin Antibody Antigen Non-competitive immunoassay principle Slide 3 Sandwich complex Non-competitive immunoassay principle Slide 4 Microbeads Non-competitive immunoassay principle Slide 5 Slide 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 Slide 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 Slide 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 Slide 9 Endogeneous antibodies against assay antibodies Heterophilic antibodies Human anti-mouse antibodies (HAMA) Rheumatoid factor EtiologyPoorly defined, no clear immunogen Known antigenic stimulus Auto-antibody SpecificityLow: bind different species Ig HighLow: bind Fc region of different species Ig AffinityLowHighLow TiterLowHighHigh in active rheumatic disease Ig classIgG, IgMIgG, IgA, IgMUsually IgM PrevalenceUp to 40%In 40-70% of patients treated with mouse MAbs 5-10% 70% autoimmune rheumatic disease Slide 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 Slide 11 Competitive immunoassay (example FT4) Slide 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: Highlowextremely low Slide 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 Slide 14 2. Fragmentation of antibodies Use of Fab or F(ab)2 fragments Slide 15 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 ) Slide 16 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% Slide 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 Slide 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 Slide 19 Case report macro-TSH (1) 60 year old man, clinically euthyroid TSH 1 232 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 AbNeg anti-TSH receptor Abs Neg Test with an alternative immunoassay method TSH 2 122mIU/l 1 Vitros 5600, Ortho Clinical Diagnostics; 2 Advia Centaur, Siemens Healthcare Diagnostics Test dilution linearity 3 : TSH 1:1 122mIU/l TSH 1:10165 mIU/l(135% recovery) 3 TSH assay diluent and immunoassay: Advia Centaur Test for antibodies against assay antibodies RF Negative Heterophilic blocking tubesNo interference detected Loh T P, JCEM 2012 Slide 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) * 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 Slide 21 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 Slide 22 TSH measurement by different methods InstrumentManufacturerReference rangeTSH (IU/mL) ElecsysRoche0.5 5.0152.0 CentaurSiemens0.4 4.020.5 LumipulseFuji Rebio0.61 4.68112.4 ArchitectAbbott0.35 4.949.8 Immunoassays display variable reactivity with macro-TSH Sakai, Endocr J 2009 Slide 23 CaseSexAgeThyroid antibody positive Clinical signs/ symptoms TSH (mIU/l)ImmunoassayRef 1F56Anti-TgNo274Elecsys Sakai 2009 2323 F-F- mother newborn ---- No 308 828 Elecsys Halsall 2006 456456 F-FF-F 28 45 23 Neg TRAb No Graves HT 5.1 22 9.7 Elecsys Verhoye 2009 7878 FFFF mother newborn - Neg No 55 103 Elecsys Delfia Rix 2011 9F46NegNo24.5Elecsys Mendoz a 2009 10M60Anti-TPONo232Vitros Loh 2012 11M29---- No40-115RIA Bifulco 1987 12 13 FFFF 53 6 Neg No 1.4 ->100 2.7 ->100 Immunoassay Viera 2006 Overview macro-TSH cases, confirmed by GFC Slide 24 Prevalence of macro-TSH 15/495 TSH > 10 mIU/l (3%): low recovery after PEG precipitation Slide 25 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 Slide 26 Tg antibody interference in Tg immunoassays Anti-Tg interference in Tg IMA is a common problem Slide 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 Slide 28 One step method - Labeled Analog Serum Binding Protein T4 FT4 ++ * Anti - T4 Antibody Bound to Particle + * Separate and Count X * Conjugated Analog Slide 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) Slide 30 Elecsys FT4 FT3 immunoassay (Roche Diagnostics) Slide 31 Protection against ani-Ru antibodies Slide 32 Roche claims increased protection against anti-Ru antibodies in next generation IA Slide 33 Case report 28/8/ 5/2/ 11/9 15/5 Normal values 2013 2013 2012 2012 TSH (mIU/l) 0.5520.3440.5690.5150.27-4.2 FT3 (ng/l)3.25.97.06.22.6-4.4 FT4 (ng/l)12.620.821.219.59.3-17.0 Switch to Elecsys FT3 III and FT4 II Slide 34 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 Slide 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 patients 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