Serum thyroglobulin measurement: analytical challenges and considerations Phillip Monaghan Clinical Biochemist The Christie

Serum thyroglobulin measurement: analytical challenges and considerations Phillip Monaghan Clinical Biochemist The Christie

My home town

Manchester City!

Thyroglobulin (Tg) A large homodimeric iodoglycoprotein Heterogeneous Molecular weight ~660,000 Da Thyroid cells and follicular lumen Involved in synthesis of thyroid hormones under influence of TSH. Functioning thyroid tissue is only source of Tg

Clinical Applications Tg is used as a tumour marker for differentiated thyroid cancer (DTC): Monitoring treatment of DTC Successful treatment reflected by undetectable serum Tg Detect disease recurrence Detectable/rising serum Tg indicating residual or recurrent disease Therefore, require assay stability for long-term clinical follow-up. High negative predictive value (NPV) desirable to optimise frequency of follow up and to reassure patient of absence of disease.

DTC: Tg measurement Tg measurement At presentation with thyroid nodule or nodular goitre. Pre-surgery/radioiodine ablation. Current assays unable to determine source of Tg (normal thyroid tissue, tumour, metastases) A few weeks post ablation of thyroid remnant. ON/Off T4 replacement. Following recombinant human (rhtsh; Thyrogen ) stimulation

DTC: Tg interpretation The Clinician Clinical presentation Nature of thyroid surgery Thyroid hormone replacement therapy Results of imaging The Biochemist Knowledge of surgery, ablation and TSH and previous result (if available) will allow biochemist to assess if result is analytically/clinically plausible Essential that TSH is measured at same time.

rhtsh-stimulated serum Tg Marker of residual or recurrent disease. Widely used cut-off value of 2.0 ng/ml However, this is assay-specific and can vary from 1.0 to 5.0 ng/ml

rhtsh-stimulated serum Tg A single rhtsh Tg >2 ng/ml predicted persistent tumour A single rhtsh Tg <0.5 ng/ml gives an 98% likelihood that patient is free of tumour.

High sensitive Tg Patients with a T4-suppressed serum Tg < 0.1 ng/ml rarely have a rhtsh-stimulated Tg above 2 ng/ml. None of these patients had imaging after rhtsh stimulation suggestive of local recurrence or distant metastasis. Recommended monitoring patients with a T4-suppressed Tg and periodic neck ultrasonography.

How is serum Tg measured?

2-site immunometric assay (IMA) Separation Step Signal Analyte concentration = Analyte = Tracer (signal)

Siemens Immulite 2000 Functional sensitivity = 0.9 ng/ml Conversion factor ng/ml x 1.515 = pmol/l Precision: Inter-batch CV of 5.6% (@ 10mg/mL) Standardisation: CRM 457 (Community Bureau of Reference of the European Commission) Reagent antibodies: anti-tg murine monoclonal anti-tg sheep polyclonal conjugated to ALP

Analytical limitations Assay bias: Many assays now stated to be traceable to CRM 457 Prepared from normal thyroid tissue Tissue sources lack uniform glycosylation and iodination and therefore introduce variability when purified Tg is used in immunoassay. Different assays by different manufacturers use different antibodies targeting different epitopes to quantify the same analyte!!

Assay bias: UKNEQAS 8 different methods n = 27 labs Immulite = 16

Recombinant human Tg Powell, R. et al. Recombinant expression of homodimeric 660 kda human thyroglobulin in soybean seeds: an alternative source of human thyroglobulin. Plant Cell Reports. 2011;30:1327-1338. Hoped to improve inter-assay variability.

Recombinant human Tg However Purified protein standard may not represent molecular population of the Tg present in different patients: Genetic polymorphisms. Genetic variation in post-translational modification pathways. Tissue differences leading to secretion of different isoforms. Therefore epitope variations cause differences in immunoreactivity between assays. Makes comparisons of functional sensitivities and cut-off values problematic.

Analytical limitations (2) Auto-antibodies? Production of abnormal Tg by tumour may be recognised as nonself and elicit an immune response. Up to 10% of normal subjects are positive for Tg auto-antibodies This may increase up to 25% in patients with DTC

Thyroglobulin Auto-antibodies Tg auto-antibodies Sandwich complex Detection antibody Capture antibody The epitopes needed for sandwich formation are sterically protected by Tg auto-antibodies (TgAb)

Auto-antibodies Auto-antibody interference is unpredictable May lead to falsely elevated (usually competitive IA) or falsely reduced (usually IMA) results. For patients with detectable TgAb, serum Tg assay results must be interpreted with caution.

TgAb measurement International reference preparation (IRP 65/093) available. Again, variable specificity and epitope expression in different disease states leads to variation between assays Different cut-off values recommended by different assay manufacturers.

UKNEQAS TgAb

TgAb 26 consensus expert recommendations Algorithm for the care of TgAb-positive DTC patients

Technical recommendations Recommendations 1) TgAb and Tg best assessed using IMA. 2) Use TgAb assay traceable to IRP 65/093. 3) Preferable to use Tg and TgAb assays from the same manufacturer. 4) For longitudinal consistency, consecutive TgAb measurement should be performed in the same lab using the same assay every time.

TgAb as surrogate tumour marker Recommendations 15) TgAb levels do not correlate with tumour load. Changes in TgAb can be used as an imprecise surrogate marker of residual benign or malignant thyroid tissue. 16) Trend in TgAb is more important than the absolute concentration.

Future prospects Are there methods available to overcome TgAb interference in Tg measurement?

Immunoassay TgAb interference Assay bias Lack precision at low end High-dose hook effect Anti-reagent antibody interference (HAMA) SOLUTION?

1) LC-Tandem Mass Spectrometry (LC-MS/MS) Analytical method to separate and quantify according to mass to charge ratio of the analyte of interest. Sensitive Specific

Burgeoning field of proteomics

Hoofnagle A, and Wener MH. J Immunol Methods. 2009; 347(1-2): 3 11.

-ive bias +ive bias

LC-MS/MS Both published methods have functional sensitivity of ~0.5 ng/ml However Sample preparation ~20h Technically demanding: [Protein] span 10-orders of magnitude, e.g. for every 1 peptide from Tg there are 40 million peptides from Albumin! Hardware and technical expertise.

TgAb may lead to faster clearance of Ab-bound Tg which results in inappropriately low serum Tg levels even before blood is drawn!?!

Any alternatives?

Tg mrna Development of a quantitative Tg mrna assay Authors were able to accurately distinguish patients with persistent or recurrent disease from those that were disease-free. Cut-off = 5.51pg Encouraging, but independent confirmation required.

Thank you

Novel Immunoassay Interference Causing Falsely Elevated CA19-9 in an Apparently Healthy Patient.

Outline The Patient Past medical History Biochemistry Incidental CA19-9 Tumour Marker Request Radiological Investigations Investigation of Interference Conclusions Tumour Marker Guidelines

The Patient 45 year old Male Electrical Engineer Presentation Presented to GP in early 2007 Tiredness Diarrhoea 2-3 times/day No evidence of Steatorrhoea or dyspepsia Normal appetite and Steady weight Alcohol Consumption 20 units/week On Lipitor for Hypercholesterolaemia Sertraline for anxiety

Past Medical History Perthes disease in childhood (right hip). Recurred 6-7 years ago. Pleurisy

Biochemistry 28/08/2007 Analyte Sodium Potassium Chloride Bicarbonate Urea Creatinine Anion Gap Albumin Result 140 (135-145 mmol/l) 4.1 (3.5-5.0 mmol/l) 100 (99-109 mmol/l) 21 (22-33 mmol/l) 4.5 (2.5-7.0 mmol/l) 87 (50-130 mmol/l) 23 (6-16 mmol/l) 48 (34-45 g/l) 28/08/2007 Analyte Result ALT 39 (<35 U/L) GGT 31 (<50 U/L) Glucose 4.8 (3.5-6.0 mmol/l) 28/08/2007 Analyte Result Haemoglobin 14.8 (13.3-16.7 g/dl) Platelets 257 (150-400 x 10 9 ) WBC 5.6 (3.5-11.0 x 10 9 ) Total Protein ALP Bilirubin 81 (64-83 g/l) 81 (35-125 U/L) 12 (2-17 μmol/l) 20/09/2007 Analyte AFP Result <2.0 (μg/l)

Tumour Marker CA19-9 CA19-9 is a mucin which reacts with monoclonal antibody 111 6 NS 19-9. Believed to be involved in cell adhesion. Serum CA 19-9 levels may be raised in pancreatic carcinoma (70-100% of cases). In pancreatic cancer, the concentration is associated with adverse outcome. CA19-9 is also raised in biliary, liver, lung, breast cancers and various gastro-intestinal malignancies. Increased CA19-9 levels also occur in benign conditions including hydronephrosis, rheumatoid arthritis and non-malignant gastrointestinal and respiratory diseases. [Bates, 1991, Duffy, 1998].

Incidental CA19-9 Test Incidental Testing (GP Request) gave a CA19-9 result of 659 ku/l (cutoff <41) on Siemens ADVIA Centaur.? Pancreatic Pathology

Radiological Investigations Date Investigation Report 18/04/2007 CT Scan Normal Liver, Spleen, Adrenals and Pancreas 24/05/2007 MRI Scan Normal Liver, Spleen, Adrenals and Pancreas. Normal biliary tree. 24/05/2007 MRCP Normal Pancreatic duct system 11/01/2008 CT Scan Normal. No pancreatic mass, No ascites, No bony lesions, No abdominal adenopathy 12/01/2008 Endoscopic Ultrasonography Normal biliary and Pancreatic systems. No Stones. No complications.

Discrepant CA19-9 Results? During the course of investigation, patient was transferred to specialist at Royal Liverpool Hospital. CA19-9 measured at Royal Liverpool Hospital on the Roche E170 result below cutoff value 9 ku/l (<35)!? Sample sent to Charing Cross Hospital for CA19-9 assay using Brahms KRYPTOR platform 8 ku/l (<33) Ca19-9 Test Results 1800 1500 [Ca19-9] (ku/l) 1200 900 600 300 Centaur E170 Kryptor 0 18/01/2007 28/04/2007 06/08/2007 14/11/2007 22/02/2008 01/06/2008 Date

T.R.A.C.E technology

Investigation of Interference Investigations Initiated 01/05/2008 Serial Doubling Dilutions Polyethylene Glycol Precipitation Lectin Inhibition Test Gel Filtration Chromatography Western blot Analysis

Serial Doubling Dilutions [CA 19-9] (ku/l) 600 500 400 300 200 Patient Serum Control Serum 100 0 1 5 10 20 40 80 160 320 Dilution Factor

Additional Biochemistry Test Result CA19-9 Pre-HBT 350 ku/l (<41) CA19-9 Post-HBT 395 ku/l CA19-9 Post Horse Serum 540 ku/l CA19-9 Post Goat Serum 440 ku/l CA125 32.7 ku/l (<35) CA153 9.9 ku/l (<30) CEA 1.8 μg/l (<15) IgG 10.83 g/l (5.3-16.5) IgA 3.67 g/l (0.8-4.0) IgM 1.05 g/l (0.5-2.0) Rheumatoid Factor 6.3 IU/L (<40) Anti-cardiolipin IgG 8 mplunits/ml (0-11) Anti-cardiolipin IgM 3 mplunits/ml (0-7)

Polyethylene Glycol Precipitation (PEG) PEG removes Ig s and serum macro-complexes from sample Results will establish if nature of interference is Macromolecular. 10000 1000 [CA 19-9] (ku/l) 100 10 % recovery 140 120 100 80 60 40 20 1 Untreated PEG treated 0 The Christie Other patients NHS Foundation Case patient Trust

Lectin Inhibition Test (the theory) Lectin binds sialic acid residues (as used for ALP isoenzymes) CA19-9 epitope recognised by antibody (1116-NS-19-9) is sialylated lacto-n-fucopentaose II) PDB = 2CWG Wright C S, Jaeger J. J. Mol. Biol. (1993) July 20;232(2):620-38.

Theoretically, lectin will bind CA19-9 epitope and INHIBIT immunoassay sandwich complex resulting in [CA19-9] immunoassay result.

Lectin Inhibition Test 100000 120 10000 100 [CA 19-9] (ku/l) 1000 100 10 % recovery 80 60 40 1 Untreated Lectin treated 20 0 Other patients Case patient

Gel Filtration Chromatography Separation Based on Molecular Weight. Used Sephacryl S-400 matrix with broad separation range (20-8000 KDa). Larger compounds elute early Smaller compounds elute later

Gel Filtration Results Experiment highlighted longer elution profile for case patient serum Interfering compound is distinct from monomeric CA19-9 and has a LOWER molecular weight 120 100 Control Serum Patient Serum [CA 19-9] (%) 80 60 40 20 V 0 V t 0 0 5 10 14 16.5 19 21.5 24 26.5 29 31.5 34 36.5 39 41.5 44 Elution Volume (ml)

Western Blot Analysis Principles ELECTROPHORESIS Roche Siemens (CA19-9)

Western Blot Results CA19-9 Control Roche Siemens Both antibodies showed equal specificity for CA19-9 CA19-9 Signal

Conclusions HBT s ineffective: unlikely heterophile Precipitated by PEG: macromolecule (protein) Does not bind Lectin: does not possess CA19-9 epitope Interference separated from monomeric CA19-9 by GF: Distinct entity of lower molecular weight Equal antibody Specificity:

The Centaur Immunoassay? Interference reported to MHRA Serum sent to Siemens for analysis

And the Patient? Diagnosed with diverticular disease Advised to continue Lipitor and Sertraline. Dietary modifications recommended. Now fit and well. Reassured that the multiplicity of radiological investigations confirmed there is no pancreatic lesion and the inconsistent results were due to methodological issues.

Lessons Learnt This case highlights an important issue concerning the potential pitfalls encountered in the use of tumour markers as a screening tool.

Tumour Marker Guidelines: No serum marker in current use is specific for malignancy. Markers rarely elevated in patients with early malignancy. No cancer marker has absolute organ specificity (ex PSA) No marker is elevated in 100% of patients with a particular malignancy (ex hcg, choriocarcinoma). Reference ranges for cancer markers are not well defined. Changes in levels over time are likely to be more clinically useful than single result. Many tumour markers lack International Reference Preparations, different assay kits may give different results for the same sera. State assay used on report form.

Acknowledgements Ravinder Sodi (Senior Biochemist, Royal Liverpool) Dr Maureen Leonard (Consultant Biochemist, APH) Thank you