Strange thyroid function tests: biological pitfall or real pathology? Fleur Wolff, LHUB-ULB, September 2017
Introduction o Immunoassays widely used in laboratory practice High throughput measurement Rapidity Good sensitivity Vulnerability to interferences (causing false positive or negative results)
Introduction o Analytical interferences Stress Inappropriate treatment (supplementation therapy) Unnecessary investigations
Case report N 1 o 30 years old o Refered to an endocrinologist for an isolated elevation of TSH (measured on a Siemens platform- TSH>99 mu/l) o Absence of thyroid symptoms except a light tiredness o Thyroid not enlarged or tender o Scintigraphy: normal o High TSH concentration confirmed in our laboratory (using Roche platform)
Case report N 1 o Thyroid function tests: Parameters Unity Concentration Reference value TSH mu/l >99 0.27-4.2 T4L pmol/l 14.2 12-22 T3L pmol/l 4.6 3.1-6.8 Anti-Tg ku/l 11 <115 Thyroglobulin µg/l 37 <55
Case report N 1 o Thyroid function tests after serum treatment with heterophile blocking tubes (HBT) No change o Thyroid function tests after polyethylene glycol (PEG) precipitation: recovery <1% Parameters Unity Concentration Reference value TSH mu/l 0.9 0.27-4.2
Macro-TSH Introduction o Mostly a complex of TSH (28kDa) and IgG (anti-tsh) o MM >150kDa o Decreased renal clearance o Low biological activity o Induces biochemical results similar to that of subclinical hypothyroidism o Prevalence in patients with subclinical hypothyroidism (0.79%; 15/1901) (Hattori N, 2016) o May persist for several years (Hattori N, 2017)
Macro-TSH Detection of Macro-TSH with immunoassay o Variable detectability due to the affinity of the antibodies used Hattori N, 2016
Macro-TSH Identification o Nonlinearity at inscreasing dilution factor o No changes of TSH concentration after serum s treatment with heterophile blocking tubes
Macro-TSH Identification o Recovery <25% after polyethylene glycol precipitation (equal volume of serum and 25% PEG 6000) (Hattori et al, 2016) o Diagnosis confirmed using gel filtration chromatography
Macro-TSH Vertical transfer of anti-tsh antibody: o Elevated TSH results in a euthyroid newborn Measurement the TSH and ft4 of the mother Halsall DJ, 2006
Case report N 2 o Newborn (Girl, Date of birth: 6/01/2017) o Inherited metabolic disease suspected (6/01/2017-19/01/2017) Severe hypoglycemia High lactate levels Elevated transaminase levels
Case report N 2 o Thyroid function tests (19/01/2017) Parameters Unity Concentration Reference value TSH mu/l <0.01 0.72-11 T4L pmol/l >100 11.5-28.3 T3L pmol/l 19.3 3-9.3 Biochemical results in favor of hyperthyroidism o Transplacental passage of maternal stimulatory thyrotropin receptor antibody (TSHR-Ab)? No circulating TSHR-Ab (mother) No history of Grave s disease (mother) o Thyrotropin-receptor activating mutations?
Case report N 2 o Contact with the clinician (20/01/2017) Clinical aspects No clinical manifestations of hyperthyroidism Normal thyroid ultrasonography o Medications! Received high dose of biotine (50 mg/j) (rare inherited disease suspected)
Biotin Introduction o Water-soluble B vitamin (B8, B7, H) o Essential coenzyme implicated in carboxyl transfer Pyruvate carboxylase Propionyl-CoA carboxylase Acetyl-CoA carboxylase Methyl-crotonyl-CoA carboxylase o Daily recommanded intake: 30 µg/day (Piketty ML, 2016) o Clinical protocols Rare inherited metabolic diseases: biotidinase deficiency, propionic acidemia (5 à 30 mg/daily), multiple sclerosis (300 mg/daily; dose>10000 times daily recommanded intake)
Biotin Introduction o Supplementation not necessary (Chun K, 2017) o Self-medication to reduce hair loss, to fortify nails (poly-vitamin complex preparation) Dose > 300 X daily recommanded intake o Biotin intake often not reported No toxicity (even at high dose) Leads to analytical interference in many immunoassays
Biotin Interferences o Biotin can affect immunoassays that use the streptavidin-biotin binding to separate immune complexes o This interference can simulate a coherent hormonal profile Hyperthyroïdy (TSH T4L ) (Elston M, 2016, Kummer S, 2016) Vitamin D intoxication (PTH 25(OH) vitamin D ) (Khieng V, 2010) Adrenal disease (ACTH Cortisol )
Biotin Piketty ML, 2016
Biotin interference Interferences o Sandwich assay format (TSH, Tg, FSH, LH, hcg, insulin, C- peptide, IGF-1) or competitive assay format (small molecules: T4L, T3L, stéroïdes, 25-OH vitamine D)
Biotin interference o TSH (Roche ) o T3L (Roche )
Biotin Excretion o Renal excretion o After a 100 mg dose Maximal interference 1h after the dose (Lam L, 2017) o After a 30 mg dose The interference may persist up to 25h (Wijeratne NG, 2012)
Case report n 2 o Sample analyzed on another analytical platform (Centaur )- 20/01/2017 Parameters Unity Concentration Reference value TSH mu/l 0.19 0.3-4 T4L pmol/l 16.9 9-26 o Sample undertaken 48h after stopping biotin (Roche )- 23/01/2017 Parameters Unity Concentration Reference value TSH mui/l 0.88 0.72-11 T4L pmol/l 18.9 11.5-28.3
Biotin Different impact according to the test and the analytical platform o Design «sandwich» less vulnerable o Design «one» or «two step» with or without washing o Sample volume
Biotin o TSH (Centaur )
Biotin o T4L(Centaur ) The biotinylated polyclonal rabbit anti-t4 antibody is already bound to avidin coupled to paramagnetic beads FT4 Acridinium ester labeled FT4
Biotin o Validation of a simple method to remove biotin interference Magnetic microparticles coated with streptavidin (Sigma-Aldrich ) Piketty et al, 2017
Biotin o Biochemical parameters (Roche ) before and after the procedure Piketty et al, 2017
Biotin Advices: o Inform patients on biotin s effect on tests o If the patient is taking high dose biotin: stop taking biotin at least two days before the venipuncture (Chun K, 2017)
Case report N 3 29 years o Underwent a total thyroidectomy for a differentiated thyroid cancer (DTC) in April 2006 o Radioiodine ablation o Long term follow-up including thyroglobulin (Tg) measurement
Case report N 3 o Thyroid function tests (Tg: Immulite-Siemens) Param. Unity Reference value Oct. 2005 April 2006 Oct. 2006 Jully 2007 Oct. 2015 Oct. 2016 Nov. 2016 19th Dec. 2016 TSH mu/l 0.27-4.2 1.3 2.5 69 97 1.3 0.7 2.6 6.11 T4L pmol/l 12-22 16.7 16.7 <6 <6 15 16.7 15.4 11.6 ACAT ku/l <115 14 11 <11 <11 13 <11 <11 <11 Tg µg/l <55 460 4.1 0.9 <0.5 <0.5 2.1 3.8 2.1
Case report N 3 o PET negative (Dec. 2016) o No increase of Tg concentration after recombinant TSH stimulation (Dec. 2016) Parameters Unity 19th Dec. 2016 21th Dec. 2016 TSH mu/l 6.11 >99 23th Dec. 2016 T4L pmol/l 11.6 12.9 ACAT ku/l <11 <11 <11 Tg µg/l 2.1 1.7 2.1
Case report N 3 o Thyroid function tests after serum treatment with heterophile blocking tubes Parameters Unity Nov. 2016 19th Dec. 2016 Tg µg/l <0.5 <0.5 o Measurement by another analytical platform (Roche) Parameters Unity Nov. 2016 19th Dec. 2016 Tg µg/l <0.1 <0.1
Heterophilic antibodies Introduction o Antibodies causing false results in immunoassays o Human anti-animal antibodies o Very common: 30-40% of the general population (interference estimated at 0.05-0.5%) (Oostendorp M, 2017) o Low affinity antibodies with broad specificities (Bolstad N, 2013) o May persist in the blood several months after exposure to animal Ig o Can arise from iatrogenic (vaccines, blood transfusions) and noniatrogenic causes (vertical transfert across the placenta) (Censi S, 2016)
Heterophilic antibodies Mechanism of interference o May induce false positive or false negative interference False positive False negative Censi S, 2016
Heterophilic antibodies Identification o Non linearity after dilution o HBT: specific but sometimes not sensitive o PEG precipitation
Heterophilic antibodies Precautions taken by manufacturers o Blocking reagents: Small amount of animal serum (mouse or goat) Polyclonal animals IgGs o Reduce interferences but does not always eliminate them completely
Case report N 4 8 years o Thyroid function tests requested by the pediatrician o None clinical information available
Case report N 4 o Thyroid function tests (11/8/2016) Parameters Unity Concentration Reference value TSH mu/l 1.1 0.6-4.84 T4L pmol/l >99 12.5-21.5
Case report N 4 o No significant change was observed after using HBT tubes o Sample analyzed on another analytical platform (Centaur )- 11/08/2016 Parameters Unity Concentration Reference value TSH mu/l 1.4 0.7-5.97 T4L pmol/l 13.8 9-26
Antistreptavidin antibodies Introduction o Same mechanism of interference compared with biotin But interference endogeneous and therefore not transient Rulander N, 2013
Antistreptavidin antibodies Introduction o Seroprevalence unknown o Mechanism leading to immunization unknown o More pronounced effect in competitive assay compared to sandwich assay (Piketty ML, 2017)
Antistreptavidin antibodies o Case reports Piketty M, 2017
Take home messages o Detection of interference requires a good collaboration between laboratory and clinical staff o Interference may affect different endocrines axes o When Interference suspected Serial dilution experiment Polyethyleneglycol precipitation Heterophilic blocking tubes Repeat the measurement on a different analytical platform
Take home messages o Be carefull o But don t be paranoid, interferences remain occasional THANK YOU!