Strange thyroid function tests: biological pitfall or real pathology? Fleur Wolff, LHUB-ULB, September 2017
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1 Strange thyroid function tests: biological pitfall or real pathology? Fleur Wolff, LHUB-ULB, September 2017
2 Introduction o Immunoassays widely used in laboratory practice High throughput measurement Rapidity Good sensitivity Vulnerability to interferences (causing false positive or negative results)
3 Introduction o Analytical interferences Stress Inappropriate treatment (supplementation therapy) Unnecessary investigations
4 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)
5 Case report N 1 o Thyroid function tests: Parameters Unity Concentration Reference value TSH mu/l > T4L pmol/l T3L pmol/l Anti-Tg ku/l 11 <115 Thyroglobulin µg/l 37 <55
6 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
7 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)
8 Macro-TSH Detection of Macro-TSH with immunoassay o Variable detectability due to the affinity of the antibodies used Hattori N, 2016
9 Macro-TSH Identification o Nonlinearity at inscreasing dilution factor o No changes of TSH concentration after serum s treatment with heterophile blocking tubes
10 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
11 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
12 Case report N 2 o Newborn (Girl, Date of birth: 6/01/2017) o Inherited metabolic disease suspected (6/01/ /01/2017) Severe hypoglycemia High lactate levels Elevated transaminase levels
13 Case report N 2 o Thyroid function tests (19/01/2017) Parameters Unity Concentration Reference value TSH mu/l < T4L pmol/l > T3L pmol/l 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?
14 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)
15 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)
16 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
17 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 )
18 Biotin Piketty ML, 2016
19 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)
20 Biotin interference o TSH (Roche ) o T3L (Roche )
21 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)
22 Case report n 2 o Sample analyzed on another analytical platform (Centaur )- 20/01/2017 Parameters Unity Concentration Reference value TSH mu/l T4L pmol/l o Sample undertaken 48h after stopping biotin (Roche )- 23/01/2017 Parameters Unity Concentration Reference value TSH mui/l T4L pmol/l
23 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
24 Biotin o TSH (Centaur )
25 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
26 Biotin o Validation of a simple method to remove biotin interference Magnetic microparticles coated with streptavidin (Sigma-Aldrich ) Piketty et al, 2017
27 Biotin o Biochemical parameters (Roche ) before and after the procedure Piketty et al, 2017
28 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)
29 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
30 Case report N 3 o Thyroid function tests (Tg: Immulite-Siemens) Param. Unity Reference value Oct April 2006 Oct Jully 2007 Oct Oct Nov th Dec TSH mu/l T4L pmol/l <6 < ACAT ku/l < <11 <11 13 <11 <11 <11 Tg µg/l < <0.5 <
31 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 th Dec TSH mu/l 6.11 >99 23th Dec T4L pmol/l ACAT ku/l <11 <11 <11 Tg µg/l
32 Case report N 3 o Thyroid function tests after serum treatment with heterophile blocking tubes Parameters Unity Nov th Dec Tg µg/l <0.5 <0.5 o Measurement by another analytical platform (Roche) Parameters Unity Nov th Dec Tg µg/l <0.1 <0.1
33 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 %) (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)
34 Heterophilic antibodies Mechanism of interference o May induce false positive or false negative interference False positive False negative Censi S, 2016
35 Heterophilic antibodies Identification o Non linearity after dilution o HBT: specific but sometimes not sensitive o PEG precipitation
36 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
37 Case report N 4 8 years o Thyroid function tests requested by the pediatrician o None clinical information available
38 Case report N 4 o Thyroid function tests (11/8/2016) Parameters Unity Concentration Reference value TSH mu/l T4L pmol/l >
39 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 T4L pmol/l
40 Antistreptavidin antibodies Introduction o Same mechanism of interference compared with biotin But interference endogeneous and therefore not transient Rulander N, 2013
41 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)
42 Antistreptavidin antibodies o Case reports Piketty M, 2017
43 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
44 Take home messages o Be carefull o But don t be paranoid, interferences remain occasional THANK YOU!
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