Dr. Ellen Anckaert Dienst Klinische Chemie en...
Transcript of Dr. Ellen Anckaert Dienst Klinische Chemie en...
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!