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

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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!