Performantie van directe steroid hormoon assays. Dr. Ellen Anckaert Dienst Klinische Chemie en Radioimmunologie
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1 Performantie van directe steroid hormoon assays Dr. Ellen Anckaert Dienst Klinische Chemie en Radioimmunologie
2 Steroid hormone immunoassays Testosterone Estradiol Progesterone Physiological background Preanalytical issues Direct immunoassay performance
3 Hypothalamus: pulsatile GnRH - + prolactin CRH cytokines neurotransmitters leptin neurotransmitters 95% of circulating testosterone
4 Premenopausal women ovaries adrenals androstenedion DHEA DHEAS 50% peripheral conversion in liver, adipose tissue, skin 25% testosterone 25%
5 Indications for serum testosterone measurements Men Abnormal puberty Cryptorchidism Hypogonadism Monitoring anti-androgen therapy in prostate cancer Monitoring testosteron replacement therapy Neonates with ambiguous genitalia Women PCOS Hirsutism / Virilization in girls and women > ng/ml: exclude androgen-secreting tumor
6 Bremner, JCEM 1983 Diurnal variation in serum testosterone Important: serum sample collection in the morning
7 Serum testosterone: pre-analytical issues Men At least 2 measurements should be perfomed for diagnosis of hypogonadism 30 to 35 % of men with low values in a one measurement have normal average testosterone levels over 24h Sample collection at 8 a.m. Effect critical illness: transient decrease during several weeks Women: Early morning testosterone (diurnal variation with peak in morning) Preferably early follicular phase (testosterone increase in the late follicular phase)
8 Evolution of steroid immunoassays Extraction/Chromatography RIA specificity, sensitivity Direct RIA Monoclonal Abs with increased specificity Displacers of binding proteins Non-isotopic automated immunoassay TAT, accuracy in low range, inter-method CV
9 Sovent extraction and chromatography Protein denaturation Release of steroid hormone from SHBG Elimination of (water-soluble) conjugated metabolites Ether Extraction Elimination of unconjugated metabolites Chromatography
10 Extraction and chromatography Sample ID-GSMS target (nmol/l) testo Direct RIA (% ID-GCMS) Extraction/ Chromatography RIA (% ID-GCMS) A B C D E F G H I J K Mean (SD) (13.1) (12.7) 3.47 nmol/l = 1 ng/ml ID-GCMS targetted samples: UK-NEQAS; RIA: UZ Brussel
11 Performance of direct testosterone immunoassays Taieb J, Clin Chem 2003 Measurement of serum testosterone over a broad range in 50 men 55 women 11 children Performance comparison to ID/GC-MS of 8 automated immunoassays 2 direct RIA
12 Performance of direct testosterone immunoassays No method acceptable for women/children: 7/10 immunoassays overestimate (mean bias: 46%) Most methods acceptable in men: some underestimation (mean bias: -12%) 3.47 nmol/l = 1 ng/ml Taieb J, Clin Chem 2003
13 UKNEQAS ID-GCMS female testosterone targetting exercise 3.47 nmol/l = 1 ng/ml Kane, Ann Clin Biochem 2007
14 DHEAS interference in direct testosterone immunoassays Female matrix pool A B C DHEAS level (µmol/l) Median testosterone measured (nmol/l) [p value] Roche E170 Modular [p<0.0001] Abbott Architect [p<0.0001] Roche Elecsys [p<0.0001] Beckman Access/Dxl [p<0.0001] 3.80 [p<0.0001] 3.99 [p<0.0001] 3.50 [p<0.0001] 2.99 [p<0.0001] DPC Immulite 2000/ Bayer Advia Centaur [p=0.013] 3.47 nmol/l = 1 ng/ml Middle, Ann Clin Biochem 2007
15 Serum testosterone measurement in neonates Age n No extraction/ Purification and RIA (nmol/l) Extraction/ Purification and RIA (nmol/l) Male infants Birth-3 weeks weeks-5 months Female infants Birth-3 weeks weeks-5 months nmol/l = 1 ng/ml Fuqua, Clin Chem 1995
16 Testosteron Precion assays: profiletestosterone precisie(lwba) In terlab C V ( % ) Sys B RIA D Sys E RIA F nmol/l = 1.4 ng/ml Concentration Testosterone (nmol/l)
17 Testosterone reference values from proven fertile young men Provided by manufacturer n = 124, well-defined group of healthy young men with normal reproductive function explicitly verified Sikaris, JCEM 2005
18 Testosterone immunoassays: conclusion High between-method variability Calibration differences Matrix effects Different antibody specificity ( crossreactivity) Different effect binding proteins Precision / Sensitivity poor Most systems are acceptable for men No assays acceptable for women/children Some systems are superior to others
19 Elecsys 2 nd generation testosterone assay AIM: to improve accuracy in female samples Calibration against ID-GCMS RPM High antibody specificity Lower sample volume to reduce interference Different releasing agent Change in assay buffer Owen, Clin Chim Acta 2010
20 UZ Brussel data Testosterone 2nd generation immunoassay Total-Run Imprecision CV (%) ,1 1,0 10,0 100,0 Testosteron (ng/ml) Testosteron I Testosteron II Functional sensitivity: 0.05 ng/ml PreciControl 1 & 2 and 5 serum pools NCCLS protocol: 20 days, 2 runs per day, 2 replicates of each control/pool per run
21 Brandhorst, Clin Biochem 2011 Testosterone 2nd vs LC-MS/MS in women Improved correlation of Testosteron II with LC-MS/MS
22 Testosterone 2nd vs LC-MS/MS in female dialysis patients Elecsys Testo II Testosterone concentration [ng/ml] P/B Regression Y = * X N = 17 r = Testosterone concentration [ng/ml] LC-MS/MS Testosteron II is not accurate Brandhorst, Clin Biochem 2011
23 Interference in women and children is not eliminated in 2nd generation testosterone Testo II: UZ Brussel, LC-MSMS: UZ Gent Confirmation by LC-MSMS: children < 1 year; female values > 1 ng/ml
24 Conclusion: testosterone immunoassays Overestimation in the female matrix variable and unpredictable presumably due to interference by mostly unknown cross-reacting substances and inaccurate calibration Some systems are superior to others in terms of precision and accuracy in female samples Manufacturer should provide a comparison with ID-GCMS RPM in a series of single donation patient sera across the clinically relevant range Endocrine Society Position Statement (JCEM 2007) calls for standardization of testosterone immunoassays and well documented reference values
25 De menstruele cyclus Menstrual cycle 2007 UpToDate Licensed to Vrije Universiteit Brussel SupportTag: [WEB C1F9DA1-1540] E2: Reflectie van folliculaire groei dominante follikel: 250 à 300 ng/l PROG: reflectie van de aanwezigheid van een grote mature follikel: 1 à 1.5 µg/l post-ovulatoire follikel 3 µg/l adekwaat corpus luteum (D21) 6 µg/l
26 Steroid hormoon secretie in de vrouw Hypothalamus GnRH Adenohypofyse Ovaria LH FSH Laat folliculair en luteaal: LH receptor P450scc P450c17 Synthese androgenen Theca cel P450 arom Synthese oestrogenen Granulosa cel P450scc Synthese Progesteron
27 WHO klassificatie van anovulatie Hypogonadotroop Hypogonadodisme (WHO I, 5-10%) LH,FSH ; E2 Normogonadotrope anovulatie (WHO II, vooral PCOS: 70-85%) LH > FSH; SHBG androgenen Hypergonadotroop Hypogonadisme (WHO III, 10-30%) LH,FSH ; E2 Hyperprolactinemie (5-10%) Prolactine
28 Indications for E2 measurement Monitoring follicular growth Ovulation induction Superovulation for IVF/ICSI Optimalisation assays for: speed, high troughput, good precision at high concentration level Cycle irregularity / Anovulation / Menopause / Girls / Men Monitoring down-regulatie GnRH analogues Demand high sensitivity assays
29 Progesterone en follow-up ART p=0.035 Ongoing pregnancy rate/cycle initiated (%) Progesterone 4nmol/L Progesterone >4nmol/L (4 nmol/l = 1.3 µg/l) 5 0 Significantly lower ongoing pregnancy rate in rfsh patients with higher progesterone levels at the end of stimulation Andersen, Hum Reprod 2006
30 Serum progesterone in pregnancy ng/ml: viable pregnancy 5-20 ng/ml: grey zone < 5 ng/ml: non-viable (0.3% viable pregnancy)
31 Accuracy and precision of automated E2 and P assays using native serum samples Belgian External Quality Assessment (WIV) Fresh frozen serum samples without additives and preservatives no matrix effects from single donors pooled sera from pregnant women target value determined with reference method (ID-GCMS) 6 most frequently used automated methods Coucke W, Hum Reprod 2007
32 Imprecision and bias of E2 immunoassays All concentrations are in pmol/l 198 pmol/l = 54 ng/l CV % Target value Advia Centaur (n=13) 24% 24% DPC Immulite (n=25) 21% 14% Elecsys (n=66) 11% 11% Access (n=7) 23% 49% Vitros (n=11) 24% 22% Vidas (n=18) 15% 16% % 11% 7% 18% 11% 7% % 11% 8% 12% 13% 12% % 12% 5% 18% 8% 11% E2 precision goals: pmol/l: < 25%; pmol/l: <10%, Thienpont L, Clin Chem 1996 BIAS % % -12% -5 % -4% 5 % 15% 30 % 22% 15 % 18% 9 % 20% % -17 % 7 % 36 % -26% 0 % % -3 % 22 % 16 % -12 % 10 % % -6% 18% -10% 2% 43% Coucke W, Hum Reprod 2007
33 Imprecision and bias of P immunoassays All concentrations are in nmol/l 6.2 nmol/l = 1.9 µg/l Target value Advia Centaur (n=13) DPC Immulite (n=25) Elecsys (n=66) Access (n=7) Vitros (n=11) Vidas (n=18) CV % % 11% 6% 33% 9% 10% % 10% 7% 18% 9% 12% % 8% 7% 11% 7% 9% % 8% 11% 15% 9% 10% BIAS % % 22 % -23 % 81 % -10 % 21 % % 15 % 12 % 63 % 30 % 47 % % 7% 12% 40% 15% 52% % 9% 67% 20% 73% 75% Coucke W, Hum Reprod 2007
34 40 35 E2 immunoassays: Precision precisie profile E2 (LWBA) Analytical goal: CV < 25% for E2 < 1000 pmol/l 0.15 nmol/l = 40 pg/ml 30 Interlab CV (%) Analytical goal: CV < 10% for E2 > 1000 pmol/l Sys A Sys B Sys C RIA D Sys E ,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 2,8 3,0 3,2 3,4 3,6 3,8 4,0 Concentration E2 (nmol/l)
35 P immunoassays: Precision profile precisie Progesterone (LWBA) 40 5 nmol/l = 1.5 ng/ml Interlab CV (%) Sys A Sys B Sys C Sys E Concentration Progesterone (nmol/l)
36 E2 immunoassay interference
37 Conclusion direct E2 and P immunoassays Large inter-method CV caused by calibration antibody specificity effect binding proteins Insufficient sensitivity for E2 < 150 pmol/l (40 pg/ml) for P < 5 nmol/l (1.5 ng/ml) not acceptable in men / children Poor method robustness for some methods Manufacturers should provide a comparison with ID-GCMS Some systems are superior to others!
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