dr. ellen anckaert dienst klinische chemie en...
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Performantie van directe steroid hormoon assays
Dr. Ellen AnckaertDienst Klinische Chemie en Radioimmunologie
Steroid hormone immunoassays
TestosteroneEstradiolProgesterone
Physiological backgroundPreanalytical issuesDirect immunoassay performance
Hypothalamus: pulsatile GnRH
leptin neurotransmitters
+
prolactinCRH cytokinesneurotransmitters
-
95% of circulatingtestosterone
Premenopausal women
ovaries adrenals
testosterone
androstenedionDHEA
DHEAS
25% 25%
peripheralconversion in liver, adipose tissue, skin
50%
Indications for serum testosterone measurements
MenAbnormal pubertyCryptorchidismHypogonadismMonitoring anti-androgen therapy in prostate cancerMonitoring testosteron replacement therapy
Neonates with ambiguous genitalia
WomenPCOSHirsutism / Virilization in girls and women
> 1.5 -2 ng/mL: exclude androgen-secreting tumor
Diurnal variation in serum testosterone
Important: serum sample collection in the morning
Bremner, JCEM 1983
Serum testosterone: pre-analytical issues
MenAt least 2 measurements should be perfomed for diagnosis of hypogonadism
30 to 35 % of men with low values in a one measurement have normalaverage 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)
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,TAT,
↓ accuracy in low range, ↑↑ inter-method CV
Sovent extraction and chromatography
Ether Extraction
Chromatography
•Protein denaturation
•Release of steroid hormone from SHBG
•Elimination of (water-soluble) conjugated metabolites
•Elimination of unconjugatedmetabolites
Extraction and chromatographySample ID-GSMS target
(nmol/L) testoDirect RIA (% ID-GCMS)
Extraction/ Chromatography RIA(% ID-GCMS)
A 1.465 125.8
143.4
110.3
146.8
160.9
131.5
124.9
133.3
139.6
138.4
K 1.935 137.3 95.1
135.7 (13.1)
83.3
B 0.925 -
C 2.285 108.3
D 1.760 118.5
E 1.275 92.7
F 1.050 83.8
G 2.065 103.3
H 1.470 91.0
I 1.045 109.3
J 1.165 115.7
Mean (SD) 100.1 (12.7)
ID-GCMS targetted samples: UK-NEQAS; RIA: UZ Brussel3.47 nmol/l = 1 ng/mL
Performance of direct testosterone immunoassays
Measurement of serum testosterone over a broad range in
50 men55 women11 children
Performance comparison to ID/GC-MS of8 automated immunoassays2 direct RIA
Taieb J, Clin Chem 2003
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%)
Taieb J, Clin Chem 20033.47 nmol/l = 1 ng/mL
UKNEQAS ID-GCMS female testosteronetargetting exercise
Kane, Ann Clin Biochem 20073.47 nmol/l = 1 ng/mL
DHEAS interference in direct testosteroneimmunoassays
Female matrix pool A B C
DHEAS level(µmol/L)
4.5 13.8 24.8
Median testosterone measured (nmol/L) [p value]
Roche E170 Modular 1.50 2.60[p<0.0001]
3.80 [p<0.0001]
Abbott Architect 1.85 2.96 [p<0.0001]
3.99 [p<0.0001]
Roche Elecsys 1.40 2.45 [p<0.0001]
3.50 [p<0.0001]
Beckman Access/Dxl 1.65 2.35 [p<0.0001]
2.99 [p<0.0001]
DPC Immulite2000/2500
1.65 1.66 1.80
Bayer Advia Centaur 1.71 1.79 1.80 [p=0.013]
Middle, Ann Clin Biochem 20073.47 nmol/l = 1 ng/mL
Serum testosterone measurement in neonates
Age n No extraction/Purification and RIA(nmol/l)
Extraction/Purification and RIA(nmol/l)
Male infants
Female infants
Birth-3 weeks3 weeks-5 months
1214
13.97.66
3.547.73
Birth-3 weeks3 weeks-5 months
86
4.820.173
1.460.173
Fuqua, Clin Chem 19953.47 nmol/l = 1 ng/mL
Testosteron assays: precisiePrecion profileTestosterone (LWBA)
0
5
10
15
20
25
30
35
40
45
0 5 10 15 20 25Concentration Testosterone (nmol/l)
Inte
rlab
CV
(%)
Sys B
RIA D
Sys E
RIA F
5 nmol/L = 1.4 ng/mL
Testosterone reference values from proven fertile young men
n = 124, well-defined group of healthy young men with normal reproductive function explicitly verified
Provided bymanufacturer
Sikaris, JCEM 2005
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
Elecsys 2 nd generation testosterone assay
AIM: to improve accuracy in female samples
Calibration against ID-GCMS RPMHigh antibody specificityLower sample volume to reduce interferenceDifferent releasing agentChange in assay buffer
Owen, Clin Chim Acta 2010
Testosterone 2nd generation immunoassay
Total-Run Imprecision
0
1
2
3
4
5
6
7
8
9
10
11
0,1 1,0 10,0 100,0
Testosteron (ng/mL)
CV
(%)
Testosteron ITestosteron II
Functional sensitivity: 0.05 ng/mL
PreciControl 1 & 2 and 5 serum poolsNCCLS protocol: 20 days, 2 runs per day, 2 replicates of each control/pool per run
UZ Brussel data
Testosterone 2nd vs LC-MS/MS in women
Improved correlation of Testosteron II with LC-MS/MS
Brandhorst, Clin Biochem 2011
Testosterone 2nd vs LC-MS/MS in femaledialysis patients
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70
P/B RegressionY = 1.098 * X – 0.029N = 17 r = 0.6606
Elec
sys®
Test
oII
Test
oste
rone
conc
entra
tion
[ng/
mL]
Testosterone concentration [ng/mL]
LC-MS/MS Testosteron II is not accurate
Brandhorst, Clin Biochem 2011
Interference in women and children is noteliminated in 2nd generation testosterone
Confirmation by LC-MSMS: children < 1 year; female values > 1 ng/mL
Testo II: UZ Brussel, LC-MSMS: UZ Gent
Conclusion: testosterone immunoassays
Overestimation in the female matrixvariable and unpredictablepresumably due to interference by mostly unknown cross-reactingsubstances 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 clinicallyrelevant range
Endocrine Society Position Statement (JCEM 2007) calls forstandardization of testosterone immunoassays and welldocumented reference values
De menstruele cyclus
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Menstrual cycle
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
Steroid hormoon secretie in de vrouw
Hypothalamus
GnRH
LH FSH
Adenohypofyse
Theca cel Granulosa cel
Synthese androgenen
Synthese oestrogenen
P450scc
P450c17P450 arom
Ovaria Laat folliculair en luteaal: LH receptor
P450scc
Synthese Progesteron
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 ↑
WHO klassificatie van anovulatie
Indications for E2 measurement
Monitoring follicular growthOvulation inductionSuperovulation 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
Progesterone en follow-up ART
p=0.035
0
5
10
15
20
25
30
Ong
oing
pre
gnan
cyra
te/c
ycle
initi
ated
(%)
Significantly lower ongoing pregnancy rate in rFSH patients with higher progesterone levels at the end of stimulation
26
15
Andersen, Hum Reprod 2006
Progesterone >4nmol/L
Progesterone ≤4nmol/L
(4 nmol/L = 1.3 µg/L)
Serum progesterone in pregnancy
20-25 ng/mL: viable pregnancy5-20 ng/mL: grey zone< 5 ng/mL: non-viable (0.3% viable pregnancy)
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 effectsfrom single donors pooled sera from pregnant womentarget value determined with reference method (ID-GCMS)
6 most frequently used automated methods
Coucke W, Hum Reprod 2007
All concentrations are in pmol/l
Target value
Advia Centaur (n=13)
DPC Immulite (n=25)
Elecsys (n=66)
Access (n=7)
Vitros (n=11)
Vidas (n=18)
198
209
24%
24%
21%
14%
11%
11%
23%
49%
24%
22%
15%
16%
598 14% 11% 7% 18% 11% 7%
778
1841
22%
21%
11%
12%
8%
5%
12%
18%
13%
8%
12%
11%
CV %
Imprecision and bias of E2 immunoassays
E2 precision goals: 150-1000 pmol/L: < 25%; 1000-10.000 pmol/L: <10%, Thienpont L, Clin Chem 1996
198 pmol/L = 54 ng/L
198
209
7 %
-12%
-5 %
-4%
5 %
15%
30 %
22%
15 %
18%
9 %
20%
598 9 % -17 % 7 % 36 % -26% 0 %
778
1841
14 %
-4%
-3 %
-6%
22 %
18%
16 %
-10%
-12 %
2%
10 %
43%
BIAS %
Coucke W, Hum Reprod 2007
All concentrations are in nmol/l
Target value
Advia Centaur (n=13)
DPC Immulite (n=25)
Elecsys (n=66)
Access (n=7)
Vitros (n=11)
Vidas (n=18)
6.2 16% 11% 6% 33% 9% 10%
22.5 8% 10% 7% 18% 9% 12%
24.3 8% 8% 7% 11% 7% 9%
41.5 16% 8% 11% 15% 9% 10%
6.2 64 % 22 % -23 % 81 % -10 % 21 %
22.5 35 % 15 % 12 % 63 % 30 % 47 %
24.3 40% 7% 12% 40% 15% 52%
41.5 145% 9% 67% 20% 73% 75%
Imprecision and bias of P immunoassays6.2 nmol/L = 1.9 µg/L
Coucke W, Hum Reprod 2007
CV %
BIAS %
E2 immunoassays: precisiePrecision profile E2 (LWBA)
0
5
10
15
20
25
30
35
40
0,0 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)
Inte
rlab
CV
(%) Sys A
Sys B Sys CRIA DSys E
Analytical goal: CV < 10%
for E2 > 1000 pmol/l
0.15 nmol/l = 40 pg/ml
Analytical goal: CV < 25% CV < 25% for E2 < 1000 pmol/l for E2 < 1000 pmol/l
P immunoassays: precisiePrecision profile Progesterone(LWBA)
0
5
10
15
20
25
30
35
40
0 10 20 30 40 50 60
Concentration Progesterone (nmol/l)
Inte
rlab
CV
(%)
Sys A Sys BSys CSys E
5 nMol/l = 1.5 ng/ml
E2 immunoassay interference
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!