Free hormone estimates. Never ending story.

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1 Free hormone estimates Never ending story.

2 Roger Ekins ( ) - saturation analysis - comp. immunoassay - microarray - RIA (T4, B12) - Free T4 (never really succeeded)

3 Free hormone hypothesis

4 Law of mass action testosterone SHBG free testosterone testosterone-shbg complex

5 Problems in DIRECT free hormone assays Ultra low concentrations Various equilibrium-disturbing factors Detergents Preservatives Albumin binding blockers Proteins Diluents Buffer components Temperature ph

6

7 Free T4 by equilibrium dialysis Very hard to quantify

8 Free T4 by equilibrium dialysis Calculate free fraction and multiply by total T4

9 Equilibrium dialysis Physical separation Semi-permeable membrane + dialysis chamber Exceedingly sensitive T4 assay needed Ultrasensitive LC-MS/MS assay Ultrasensitive RIA Picomolar concentration (compared to nanomolar total hormone) Considered gold standard Least influenced by interfering factors and binding proteins BUT Very labor intensive Only available in specialized research laboratories

10 ED based RIA kit

11 Methods employing a specific, high affinity antibody Two step labeled hormone/back-titration methods One step labeled analog methods Labeled antibody methods

12

13 Sandwich assay Two epitopes Excess capture antibody Excess detection antibody Most sensitive immunoassays Large dynamic range High specificity

14

15 Competitive immunoassay Only one epitope One antibody (limited quantity) Competion for antibody Not very sensitive Not very specific Small dynamic range

16 Two step labeled hormone/back-titration methods - Less labor intensive (compared to reference methods) - Automated - No sample preparation - Avoids the contact of serum components with the assay tracer

17 One-step, labeled Hormone-Analog methods Based on hormone analog, non-reactive with binding proteins Poor diagnostic accuracy (analog binds to proteins) Prone to many interferences

18 One-step, labeled Antibody methods Based on labeled antibody, non-reactive with protein bound hormone Better diagnostic accuracy then labeled analog Prone to interferences (abnormal binding proteins, pregnany, DRUGS, NTI)

19 FDH cases

20 70 free T4 ( pmol/l) 60 total T4 (5,1-14,1 µg/dl) P97,5 = 26,6 total T4 30 P97,5 = 14,

21 45 free T3 (3,1-6,8 pmol/l) 500 total T3 ( ng/dl) P97,5 = 8,5 300 P97,5 = 202 total T

22 Maand 2017 TSH nl en FT4 nl TSH nl en FT4 hoog jan feb mrt apr mei jun jul aug sep okt Eindtotaal % TSH > 0,3 and ft4 > 22 Not related tot reagent lots

23

24 CALCULATED free T4 and T3 Total T4 and T3 measurement T3RU (T-uptake) to correct for proteïn binding FTI = T4 x T3RU or free thyroxin index OR measure TBG and calculate T4/TBG ratio

25 ANS TBG

26 Thyroxine Labeled with I-125 = ideal tracer

27 FTI= total T4 x T3RU

28 FTI: advantages and disadvantages PRO CONTRA Total T4 is a more robust assay and preferred method in: Patients using interfering drugs (in free T4 assays) Hospitalised patients Pregnant patients Interferences are possible: Autoantibodies to thyroid hormones can interfere with the assay. Binding protein anomalies: familial dysalbuminemic hyperthyroxinemia Two measurements are needed Not many good T3RU assays available anymore

29 ECLIA TOSOH

30

31 Interferences in free thyroid hormone tests Protein Interferences (paraproteins) Congenital TBG excess or deficiency Pregnancy FFA, drugs Familial Dysalbuminemic and Transthyretin-Associated Hyperthyroxinemias Heterophile Antibodies Anti-Reagent Antibodies (rhutenium, streptavidin) T4 and T3 Autoantibodies Clinicians should warn the laborotory in case of doubt

32 Casus: - vrouw van 17 jaar - aspecifieke klachten van nervositas - huisarts vraagt TSH en vrij T4 TEST Units Normal values Roche e602 Roche Modular Siemens Centaur Abbott Architect TSH miu/l RIA Free T4 pmol/l Free T3 pmol/l Total T4 nmol/l Total T3 nmol/l T-uptake (%) % FTI Anti TPO ku/l < < 3 Anti Tg ku/l < TSI U/L <1.0 CBG mg/l TBG mg/l

33 pt. Leen Antonio (type I DM) 12.5 pmol/l Unicel DxI (Beckman-Coulter) pt. Nele Reynaert (nefrocalcinosis) 27 pmol/l (11-27 pmol/l for 3-11 months)

34 September 26, 2017 Six patients taking 10 mg biotin for 7 days

35 Mechanism of biotin interference

36

37 (up to µg/day)

38

39

40 Carol De Ridder

41 67% of Belgian laboratories at risk!

42 TESTOSTERONE

43 Testosterone REFERENCE methods Testosterone (total): LC-MS/MS Free testosterone: equilibrium dialysis Bioavailable testosterone: differential precipitation (ammonium sulphate)

44 Testosterone PRACTICAL methods Testosterone (total): SHBG: Free testosterone: Bioavailable testosterone: competitive immuno-assay (robust in men) immunometric (sandwich) assay (robust but standardisation???) calculated calculated

45 Vermeulen formula

46 Testosterone

47

48 IMMUNOASSAY Testosterone (total) (ng/dl) data Steven Pauwels n Method Measuring interval MS 15,3 to 117,0 IA 5,0 to 108,2 60 Passing-Bablok fit Fit Y on X 40 Passing-Bablok fit Equation IA = -1, ,8743 MS 20 Parameter Estimate Bootstrap 95% CI Intercept -1,996-9,784 to 3,431 Slope 0,8743 0,7636 to 1, LC-MS/MS

49 serum

50 Free cortisol DIRECT mesurement: equilibrium dialysis + LC-MS/MS CALCULATED: Total Cortisol Cortisol Binding Globulin (transcortin)

51 Serum TOTAL cortisol: LC-MS/MS versus immunoassay 70 Cortisol (total) (µg/dl) ECLIA LC-MS/MS (Gent) y = -0, ,15 x n = 51 r = 0,98

52 Coolens equation for calculation of free cortisol in serum

53 free cortisol: Free serum cortisol = non protein bound cortisol total cortisol = easy to measure analytically (destroy binding proteins) easy to calculate if CBG is measured Free urinary cortisol = non metabolized, unchanged cortisol in urine, > 50% of cortisol is glucuronidated or sulphatated many metabolites present (cross reactivity!) hard to measure accurately

54 Urinary free cortisol: direct measurement on automated analysers

55 Urinary free cortisol: Immunotech RIA 300 Immunotech RIA y = 1.21x HPLC (µg/24h)

56 Cortisol in saliva Timing of collection: Thorough understanding of diurnal cycle Collection at standardized times (23:00) Contamination issues: No meals prior to sample (1 hour) No alcohol during 1 day No dairy products (bovine hormones) No acidic or high sugar foods (low ph) No blood contamination!!!

57

58

59 Desired LoQ = 1 µg/l (2,73 nmol/l) ECLIA LoQ = 2,1 µg/l (5,79 nmol/l)

60

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