National Health and Nutrition Examination Survey

Introduction

It has been proposed that racial/ethnic variation in prostate cancer incidence may be, in part, due to racial/ethnic variation in sex steroid hormone levels. However, it remains unclear whether in the US population circulating concentrations of sex steroid hormones vary by race/ethnicity. To address this, concentrations of testosterone, sex hormone binding globulin, androstanediol glucuronide (a metabolite of dihydrotestosterone) and estradiol were measured in stored serum specimens from men examined in the morning sample of the first phase of NHANES III (1988-1994). This data file contains results of the testing of 1637 males age 12 or more years who participated in the morning examination of phase 1 of NHANES III and for whom serum was still available in the repository. Data Documentation for each of these four components is given in sections below.

Testosterone

The androgen testosterone (17β -hydroxyandrostenone) has a molecular weight of 288 daltons. In men, testosterone is synthesized almost exclusively by the Leydig cells of the testes. The secretion of testosterone is regulated by luteinizing hormone (LH), and is subject to negative feedback via the pituitary and hypothalamus. Testosterone promotes the development of the secondary sex characteristics in men and serves to maintain the function of the prostate and seminal vesicles. Most of the circulating testosterone is bound to carrier proteins (SHBG = sex hormone-binding globulin). In women, small quantities of testosterone are formed in the ovaries. In physiological concentrations, androgens have no specific effects in women. Increased production of testosterone in women can cause virilization (depending on the increase). The determination of testosterone in women is helpful in the diagnosis of androgenic syndrome (AGS), polycystic ovaries (Stein-Leventhal syndrome) and when an ovarian tumor, adrenal tumor, adrenal hyperplasia or ovarian insufficiency is suspected. Testosterone is determined in men when reduced testosterone production is suspected, e.g. in hypogonadism, estrogen therapy, chromosome aberrations (as in the Klinefelter’s syndrome) and liver cirrhosis.

Elecsys Testosterone is based on a competitive test principle using a monoclonal antibody specifically directed against testosterone. Endogenous testosterone released from the sample by ANS (8-anilino-1-naphthalene sulfonic acid) and norgestrel competes with the added testosterone derivative labeled with ruthenium complex for the binding sites on the biotinylated antibody.

Sex hormone-binding globulin (SHBG)

Sex hormone-binding globulin (SHBG) is the blood transport protein for testosterone and estradiol. It is a large glycoprotein with a molecular weight of about 95 kD, and exists as a homodimer composed of two identical subunits. Each subunit contains two disulfide bridges. Planar C18 and C19 steroids with a 17α -hydroxyl group bind particularly well, whereas C19 17-ketosteroids such as dehydroepiandrosterone (DHEA) and androstendione do not bind so easily. SHBG has a high binding affinity to dihydrotestosterone (DHT), medium affinity to testosterone and estradiol, and only a low affinity to estrone, DHEA, androstendione, and estriol. SHBG binds reversibly to sexual steroids. Albumin, which exists in far higher concentrations than SHBG, also binds sexual steroids – although with a clearly lower binding affinity (e.g. about 100 times lower for testosterone). SHBG has a half-life of about 7 days and is produced mainly by the liver. Its synthesis and secretion are regulated by estrogen. SHBG serum concentrations depend on the extent, duration, and the kind of estrogen applied, and how regulation takes place. Androgens and gestagens with androgenic residual action have the opposite effect. In the serum SHBG mainly takes over the transportation of steroids and the reduction/regulation of the effect of androgen. Decreased SHBG serum levels are associated with conditions where elevated androgen levels are present or where the effect of androgen on its target organs is excessive. This explains the gender-related differences seen between men and women, especially during puberty.

 Measurement of SHBG can be an important indicator of an excessive/chronic androgenic action where androgen levels are normal, but where clinical symptoms would seem to indicate androgen in excess. SHBG is a useful supplementary parameter in the determination of androgen where a relatively high concentration of free androgen (e.g. testosterone) is suspected.

By calculating the free androgen index (FAI), also called free testosterone index (FTI), from the ratio of total testosterone (TT) to SHBG [% FAI or FTI = (TT/SHBG) * 100], it is possible to calculate the approximate amount of free testosterone (FTc), as there is a direct correlation between FAI and FT. By additionally taking the non-specifically albumin-bound testosterone into account, it is possible to calculate the bioavailable testosterone (BATc), which is the sum of free testosterone and the albumin-bound testosterone fraction, calculated via the association constant to albumin. Only free testosterone is biologically active, and it best indicates the clinical situation of the patient. Free and bioavailable testosterone are also referred to as non-SHBG-bound testosterone and can be obtained by precipitation of the SHBG-bound-testosterone with ammonium sulfate, and by equilibrium dialysis.

 Elevated SHBG levels can be seen in elderly men, and are often found in patients with hyperthyroidism and cirrhosis of the liver. SHBG levels also increase when oral contraceptives or antiepileptic drugs are taken. Pregnant women have markedly higher SHBG serum concentrations due to their increased estrogen production. Decreased SHBG concentrations are often seen with hypothyroidism, polycystic ovarian syndrome (PCOS), obesity, hirsutism, elevated androgen levels, alopecia, and acromegaly.

Elecsys SHBG employs two monoclonal antibodies specifically directed against human SHBG. Cross-reactivity with α 1-fetoprotein (AFP), corticosteroid binding globulin (CBG), DHT, estradiol, fibrinogen, human immunoglobulin A (IgA), human immunoglobulin G (IgG), plasminogen, thyroxine binding globulin (TBG), testosterone, thyroglobulin (Tg), transferrin, and thyrotropin (TSH) is negligible.

Androstanediol glucuronide

5α-Androstane-3α, 17β-diol glucuronide is a C19 steroid and is either abbreviated as 3α Diol G, 5α Diol G or simply, α Diol G. It is produced mainly as a metabolite of testosterone and ihydrotestosterone (DHT). It is largely produced in target peripheral tissues such as the skin, especially around hair follicles. The stimulation by large amounts of 3α Diol G leads to excessive hair formation, notably where hair is not normally present in women.

In recent years the interest in the measurement of this steroid has increased among clinical investigators studying women suffering from idiopathic hirsutism.

Among the steroids known to be precursors for 3α Diol G are dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), dihydrotestosterone (DHT), androstenedione and testosterone. Only 3α Diol G has been shown to increase with hirsutism and decrease with treatment. This correlation has also been demonstrated in patients with polycystic ovarian syndrome (PCO). 3α Diol G determinations have therefore proved to be a useful indicator in a variety of ways including monitoring the progress of treatment of idiopathic hirsutism and women with PCO.

Furthermore, diabetic patients (both men and women) under cyclosporine A therapy have shown increased 3α Diol G levels, a side effect resulting in the appearance of hair in previously hairless areas.

Estrogens

Estrogens are responsible for the development of the secondary female sex characteristics. Together with gestagens they control all the important female reproductive processes. The biologically most active estrogen is 17 β -estradiol. This is a steroid hormone having a molecular weight of 272 daltons. Estrogens are produced primarily in the ovary (follicle, corpus luteum), but small quantities are also formed in the testes and in the adrenal cortex. During pregnancy, estrogens are mainly formed in the placenta. About 98% of estradiol is bound to transport proteins (SHBG = sex hormone binding globulin). Estrogen secretion is biphasic during the menstrual cycle. The determination of estradiol is utilized clinically in the elucidation of fertility disorders in the hypothalamus-pituitary-gonad axis, gynecomastia, estrogen-producing ovarian and testicular tumors and in hyperplasia of the adrenal cortex. Further clinical indications are the monitoring of fertility therapy and determining the time of ovulation within the framework of in vitro fertilization (IVF).

Elecsys Estradiol II employs a competitive test principle using a polyclonal antibody specifically directed against 17 β -estradiol. Endogenous estradiol released from the sample by mesterolone competes with the added estradiol derivative labeled with a ruthenium complex for the binding sites on the biotinylated antibody.  

Testosterone

Immunoassay for the in vitro quantitative determination of testosterone in human serum and plasma.

Specimen collection and preparation
Only the specimens listed below were tested and found acceptable. Serum collected using standard sampling tubes or tubes containing separating gel. Li-, Na-, NH+4 -heparin, K3-EDTA, and sodium fluoride/potassium oxalate plasma. When sodium citrate is used, the results must be corrected by + 10%.

Criterion: Recovery within 90-110% of serum value or slope 0.9-1.1 + intercept within <± 2x analytical sensitivity (LDL) + coefficient of correlation > 0.95.

Stable for 1 week at 2-8°C, 6 months at -20°C. Freeze only once.5,6

Stability of serum obtained with tubes containing separating gel: 48 hours at 2-8°C (note the data provided by the tube manufacturer).

When processing samples in primary tubes, follow the instructions of the tube manufacturer.

Centrifuge samples containing precipitates before performing the assay. Do not use heat-inactivated samples. Do not use samples and controls stabilized with azide.

Ensure the patients’ samples, calibrators, and controls are at ambient temperature (20-25°C) before measurement. Because of possible evaporation effects, samples, calibrators, and controls on the analyzers should be measured within 2 hours.

Sex hormone-binding globulin (SHBG)

 Only the specimens listed below were tested and found acceptable. Serum collected using standard sampling tubes or tubes containing separating gel, or lithium heparin plasma.

Criterion: Recovery within 90-110% of serum value or slope 0.9-1.1 + intercept within <± 2x analytical sensitivity (LDL) + coefficient of correlation > 0.95.

Stable for 3 days at 2-8°C, 1 month at -20°C. Freeze only once.11

When processing samples in primary tubes, follow the instructions of the tube manufacturer.

Centrifuge samples containing precipitates before performing the assay. Do not use heat-inactivated samples. Do not use samples and controls stabilized with azide.

Ensure the patients’ samples, calibrators, and controls are at ambient temperature (20-25°C) before measurement.

Because of possible evaporation effects, samples, calibrators, and controls on the analyzers should be measured within 2 hours.

Androstanediol glucuronide

Approximately 0.2 ml of serum is required per duplicate determination. Collect 4-5 ml of blood into an appropriately labeled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4oC for up to 24 hours or at -10oC or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling. 

Estrogens

Immunoassay for the in vitro quantitative determination of estradiol in human serum and plasma. The electrochemiluminescence immunoassay “ECLIA” is intended for use on the Roche Elecsys 1010/2010 and MODULAR ANALYTICS E170 (Elecsys module) immunoassay analyzers.

Specimen collection and preparation
Only the specimens listed below were tested and found acceptable: Serum collected using standard sampling tubes or tubes containing separating gel. Li-, Na-, NH+4 -heparin, K3-EDTA, sodium citrate, and sodium fluoride/potassium oxalate plasma.

Criterion: Recovery within 90-110% of serum value or slope 0.9-1.1 + intercept within <± 2x analytical sensitivity (LDL) + coefficient of correlation > 0.95.

Stable for 2 days at 2-8°C, 6 months at -20°C. Freeze only once.

When processing samples in primary tubes, follow the instructions of the tube manufacturer.

Centrifuge samples containing precipitates before performing the assay. Do not use heat-inactivated samples. Do not use samples and controls stabilized with azide.

Ensure the patients’ samples, calibrators, and controls are at ambient temperature (20-25°C) before measurement.

Because of possible evaporation effects, samples, calibrators, and controls on the analyzers should be measured within 2 hours.

Testosterone

Test principle
Competition principle. Total duration of assay: 18 minutes.

1st incubation: 50 µL of sample is incubated with a testosterone-specific biotinylated antibody and a testosterone derivative labeled with a ruthenium complex. The binding sites of the labeled antibody become occupied partly by the sample analyte (depending on its concentration) and partly by the ruthenium-labeled hapten to form the respective immunocomplexes.

2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin.

The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell.

Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.

Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode.

Assay
Resuspension of the microparticles takes place automatically before use.

Read in the test-specific parameters via the reagent barcode. If in exceptional cases the barcode cannot be read, enter the 15-digit sequence of numbers.

Elecsys 2010: Bring the cooled reagents to approx. 20°C and
place on the reagent disk (20°C) of the analyzer. Avoid the formation
of foam. The system automatically regulates the temperature of the
reagents and the opening/closing of the bottles. 
 

Calibration
Traceability: This method has been standardized via ID-GC/MS (“Isotope Dilution Gas Chromatography Mass Spectrometry”).

Every testosterone reagent set has a barcoded label containing the specific information for calibration of the particular reagent lot. The predefined master curve is adapted to the analyzer by the use of Elecsys Testosterone CalSet II.

Calibration frequency: Calibration must be performed once per reagent lot using fresh reagent (i.e. not more than 24 hours since the reagent kit was registered on the analyzer). Renewed calibration is recommended as follows:

Elecsys 2010:
after 1 month (28 days) when using the same reagent lot
after 7 days (when using the same reagent kit on the analyzer) 
 

Sex hormone-binding globulin (SHBG)

Test principle
Sandwich principle. Total duration of assay: 18 minutes.

1st incubation: 10 µL of sample, a biotinylated monoclonal SHBG-specific antibody, and a monoclonal SHBG-specific antibody labeled with a ruthenium complex form a sandwich complex.

2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin.

The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell.

Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.

Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode. 

Assay
Resuspension of the microparticles takes place automatically before use. 

Read in the test-specific parameters via the reagent barcode. If in exceptional cases the barcode cannot be read, enter the 15-digit sequence of numbers.

Elecsys 2010: Bring the cooled reagents to approx. 20°C and
place on the reagent disk (20°C) of the analyzer. Avoid the formation
of foam. The system automatically regulates the temperature of the
reagents and the opening/closing of the bottles.

Calibration
Traceability: This method has been standardized against the 1st
International Standard for SHBG from the National Institute for Biological Standards and Control (NIBSC) code 95/560.11

Every SHBG reagent set has a barcoded label containing the specific information for calibration of the particular reagent lot. The predefined master curve is adapted to the analyzer by the use of Elecsys SHBG CalSet.

Calibration frequency: Calibration must be performed once per reagent lot using fresh reagent (i.e. not more than 24 hours since the reagent kit was registered on the analyzer). Renewed calibration is recommended as follows:

Elecsys 2010:
after 1 month (28 days) when using the same reagent lot
after 7 days (when using the same reagent kit on the analyzer)

Androstanediol glucuronide

The principle of the following enzyme immunoassay test follows the typical competitive binding scenario. Competition occurs between an unlabeled antigen (present in standards, control and patient samples) and an enzyme-labeled antigen (conjugate) for a limited number of antibody binding sites on the microwell plate. The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtitre plate reader. The intensity of the color formed is inversely proportional to the concentration of 3α Diol G in the sample. A set of standards is used to plot a standard curve from which the amount of 3α Diol G in patient samples and controls can be directly read. 

Reagents Supplied
1. Rabbit Anti-3α Diol G Antibody Coated Microwell Plate-Break Apart Wells - Ready To Use.
Contents: One 96 well (12x8) polyclonal antibody-coated microwell plate in a resealable pouch with desiccant.
Storage: Refrigerate at 2-8ºC
Stability: 12 months or as indicated on label. 

2. 3α Diol G-Horseradish Peroxidase (HRP) Conjugate Concentrate - Requires Preparation.
Contents: 3α Diol G-HRP conjugate in a protein-based buffer with a non-mercury preservative.
Volume: 300 μl/vial
Storage: Refrigerate at 2-8ºC
Stability: 12 months or as indicated on label.
Preparation: Dilute 1:50 in assay buffer before use (eg. 40 μl of HRP in 2 ml of assay buffer). If the whole plate is to be used dilute 240 μl of HRP in 12ml of assay buffer. Discard any that is left over. 

3. 3α Diol G Calibrators - Ready To Use.
Contents: Six vials containing 3α Diol G in a protein-based buffer with a non-mercury preservative. Prepared by spiking buffer with a defined quantity of 3α Diol G. 

*Listed below are approximate concentrations, please refer to vial labels for exact concentrations. 

Calibrator Concentration Volume/Vial
Calibrator A 0 ng/ml 2.0 ml
Calibrator B 0.25 ng/ml 0.6 ml
Calibrator C 1 ng/ml 0.6 ml
Calibrator D 3 ng/ml 0.6 ml
Calibrator E 10 ng/ml 0.6 ml
Calibrator F 50 ng/ml 0.6 ml
Storage: Refrigerate at 2-8ºC
Stability: 12 months in unopened vials or as indicated on label. Once opened, the standards should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing cycles.

4. Control - Ready To Use.
Contents: One vial containing 3α Diol G in a protein-based buffer with a non-mercury preservative. Prepared by spiking buffer with a defined quantity of 3α Diol G. Refer to vial label for expected value and acceptable range.
Volume: 0.6 ml/vial
Storage: Refrigerate at 2-8 ºC
Stability: 12 months in unopened vial or as indicated on label. Once opened, the control should be used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing cycles.

5. Wash Buffer Concentrate - Requires Preparation.
Contents: One bottle containing buffer with a non-ionic detergent and a non-mercury preservative.
Volume: 50 ml/bottle
Storage: Refrigerate at 2-8ºC
Stability: 12 months or as indicated on label.
Preparation: Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be used dilute 50 ml of the wash buffer concentrate in 450 ml of water.

6. Assay Buffer - Ready To Use*.
Contents: One bottle containing a protein-based buffer with a non-mercury preservative.
Volume: 15 ml/bottle
Storage: Refrigerate at 2-8ºC
Stability: 12 months or as indicated on label.
*Warm to completely dissolve before use.

7. TMB Substrate - Ready To Use.
Contents: One bottle containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or DMSO containing buffer.
Volume: 16 ml/bottle
Storage: Refrigerate at 2-8ºC
Stability: 12 months or as indicated on label.

8. Stopping Solution - Ready To Use.
Contents: One vial containing 1M sulfuric acid.
Volume: 6 ml/vial
Storage: Refrigerate at 2-8ºC
Stability: 12 months or as indicated on label.

Estrogens

Test principle
Competition principle. Total duration of assay: 18 minutes.

1st incubation: By incubating the sample (35 µL) with an estradiol-specific biotinylated antibody, an immunocomplex is formed, the amount of which is dependent upon the analyte concentration in the sample.

2nd incubation: After addition of streptavidin-coated microparticles and an estradiol derivative labeled with a ruthenium complex, the still-vacant sites of the biotinylated antibodies become occupied, with formation of an antibody-hapten complex. The entire complex becomes bound to the solid phase via interaction of biotin and streptavidin.

The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell.

Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.

Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode. 
 

Reagent handling
The reagents in the kit have been assembled into a ready-for-use unit that cannot be separated. All information required for correct operation is read in via the respective reagent barcodes.

Storage and stability
Store at 2-8°C.
Store the Elecsys Estradiol II reagent kit upright in order to ensure complete availability of the microparticles during automatic mixing prior to use.

Assay
Resuspension of the microparticles takes place automatically before use.

Read in the test-specific parameters via the reagent barcode. If in exceptional cases the barcode cannot be read, enter the 15-digit sequence of numbers.

Elecsys 2010: Bring the cooled reagents to approx. 20°C and place on the reagent disk (20°C) of the analyzer. Avoid the formation of foam. The system automatically regulates the temperature of the reagents and the opening/closing of the bottles.

Calibration
Traceability: This method has been standardized via ID-GC/MS (“isotope dilution-gas chromatography/mass spectrometry”).

Every Estradiol II reagent set has a barcoded label containing the specific information for calibration of the particular reagent lot. The predefined master curve is adapted to the analyzer by the use of Elecsys Estradiol II CalSet II.

Calibration frequency: Calibration must be performed once per reagent lot using fresh reagent (i.e. not more than 24 hours since the reagent kit was registered on the analyzer). Renewed calibration is recommended as follows:

Elecsys 2010:
after 1 month (28 days) when using the same reagent lot
after 7 days (when using the same reagent kit on the analyzer)

For all analyzers:
as required: e.g. quality control findings outside the specified limits
Calibration verification: Not necessary. The analyzer’s software automatically checks the validity of the curve and draws attention to any deviations. 
 

Testosterone

Calculation
The analyzer automatically calculates the analyte concentration of
each sample (either in nmol/L, ng/mL or ng/dL).
Conversion factors: nmol/L x 0.288 = ng/mL
ng/mL x 3.47 = nmol/L
ng/mL x 100 = ng/dL

Sex hormone-binding globulin (SHBG)

Calculation
The analyzer automatically calculates the analyte concentration of each sample either in nmol/L, µg/mL or mg/L (selectable).
Conversion factors: nmol/L x 0.095 = µg/mL (mg/L)
µg/mL (mg/L) x 10.53 = nmol/L. 

Androstanediol glucuronide

Results
A. Calculate the mean absorbance for each Standard, Control or unknown.
B. Using a linear-log graph paper, plot the mean absorbance readings for each of the standards along the y-axis versus the androstanediol glucuronide concentrations in ng/mL along the x-axis.
Alternatively, any data reduction software designed for immunoassays could be used. Four-parameter curve-fit is recommended.
C. Draw the best fitting curve through the mean of the duplicate points.
D. Determine the androstanediol glucuronide concentrations of the Controls and unknowns from the standard curve by matching their mean absorbance readings with the corresponding androstanediol glucuronide concentrations. If the absorbance reading for the first standard exceed the limitations of the plate reader, a second reading at 405 nm is needed (reference filter 600 or 620 if available). In this case, proceed to construct a second standard curve as above with the absorbance readings of all Standards at 405 nm. The concentration of the off-scale samples at 450 nm are then read from the new standard curve. The readings at 405 nm should not replace the on-scale readings at 450.
We use Softmax Pro as our data reduction software.

Estrogens

Calculation
The analyzer automatically calculates the analyte concentration of each sample (either in pmol/L, pg/mL, ng/L or additionally in nmol/L with E170).
Conversion factors: pmol/L x 0.273 = pg/mL (ng/L)
pg/mL x 3.67 = pmol/L

Limitations - interference
The assay is unaffected by icterus (bilirubin < 1129 µmol/L or < 66 mg/dL), hemolysis (Hb < 0.621 mmol/L or < 1.0 g/dL), lipemia (Intralipid < 1000 mg/dL), and biotin < 147 nmol/L or < 36 ng/mL.

Criterion: Recovery within ± 10% of initial value.

In patients receiving therapy with high biotin doses (i.e. > 5 mg/day), no sample should be taken until at least 8 hours after the last biotin administration.

No interference was observed from rheumatoid factors up to
a concentration of 1200 IU/mL.

In vitro tests were performed on 18 commonly used pharmaceuticals.
No interference with the assay was found.

The risk of interference from potential immunological interactionsbetween test components and rare sera has been minimized by the inclusion of suitable additives. 

 

Testosterone

Quality control
For quality control, use Elecsys PreciControl Universal 1 and 2.

Controls are run at begin of day, after every 100 samples and at the end of the day, at least once per reagent kit, and after every calibration. The ranges are based on the manufacturer’s given range.

Beginning of day controls must fall within 2 SD to begin the run.  We accept the controls if they fall within 3 SD only if the previous time was within 2 SD.  This criterion applies to both controls within and across runs.

Sex hormone-binding globulin (SHBG)

Quality control
For quality control, use Elecsys PreciControl Universal 1 and 2.
Controls are run at begin of day, after every 100 samples and at the end of the day, at least once per reagent kit, and after every calibration. The ranges are based on the manufacturer’s given range.

Beginning of day controls must fall within 2 SD to begin the run. We accept the controls if they fall within 3 SD only if the previous time was within 2 SD. This criterion applies to both controls within and across runs.

Androstanediol glucuronide

Quality control
Two levels of control are provided with the kit. We run each control on a plate. Controls and samples are done in duplicate. The manufacturer’s range is used as a guideline until we have at least 20 data points to set our own mean and SD.

If both controls are within 2 SD – we accept the data. If one control is within 2 SD and the other within 3 SD – we accept the data. If one or both controls are outside 3 SD – we do not accept the results and repeat the plate.

Estrogens

Quality control
For quality control, use Elecsys PreciControl Universal 1 and 2.

Controls are run at begin of day, after every 100 samples and at the end of the day, at least once per reagent kit, and after every calibration. The ranges are based on the manufacturer’s given range.

Begin of day controls must fall within 2 SD to begin the run. We accept the controls if they fall within 3 SD only if the previous time was within 2 SD. This criterion applies to both controls within and across runs.

Testosterone

Limitations – interference
The assay is unaffected by icterus (bilirubin < 513 µmol/L or < 30 mg/dL), hemolysis (Hb < 1.1 mmol/L or < 1.8 g/dL), lipemia (triglycerides < 22.8 mmol/L or < 2000 mg/dL), and biotin < 123 nmol/L or < 30 ng/mL.

Criterion: Recovery within ± 10% of initial value.

In patients receiving therapy with high biotin doses (i.e. > 5 mg/day), no sample should be taken until at least 8 hours after the last biotin administration.

In vitro tests were performed on 17 commonly used pharmaceuticals.
No interference with the assay was found.

In isolated cases, elevated testosterone levels were seen in samples from female dialysis patients > 70 years old.

The risk of interference from potential immunological interactions between test components and rare sera has been minimized by the inclusion of suitable additives.

In rare cases interference due to extremely high titers of antibodies to ruthenium can occur. Elecsys Testosterone contains additives which minimize these effects.

Extremely high titers of antibodies to streptavidin can occur in isolated cases and cause interference.

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

Measuring range
0.069-52.00 nmol/L or 0.020-15.00 ng/mL (defined by the lower detection limit and the maximum of the master curve). Values below the detection limit are reported as < 0.069 nmol/L or < 0.020 ng/mL. Values above the measuring range are reported as > 52.00 nmol/L or > 15.00 ng/mL.

Analytical sensitivity (lower detection limit)
0.069 nmol/L (0.02 ng/mL)

The detection limit represents the lowest measurable analyte level that can be distinguished from zero. It is calculated as the value lying two standard deviations above that of the lowest standard (master calibrator, standard 1 + 2 SD, within-run precision, n = 21).

Functional sensitivity
0.42 nmol/L (0.12 ng/mL)
The functional sensitivity is the lowest analyte concentration that can be reproducibly measured with a between-run coefficient of variation of less than or equal to 20%.

Sex hormone-binding globulin (SHBG)

Limitations – interference
The assay is unaffected by icterus (bilirubin < 1026 µmol/L or < 60 mg/dL), hemolysis (Hb < 1.8 mmol/L or < 2.9 g/dL), lipemia (Intralipid < 2700 mg/dL), and biotin < 60 ng/mL.

Criterion: Recovery within ± 10% of initial value.

In patients receiving therapy with high biotin doses (i.e. > 5 mg/day), no sample should be taken until at least 8 hours after the last biotin administration. No interference was observed from rheumatoid factors up to a concentration of 1160 IU/mL.

There is no high-dose hook effect at SHBG concentrations up to 1000 nmol/L.
In vitro tests were performed on 16 commonly used pharmaceuticals. No interference with the assay was found.

As with all tests containing monoclonal mouse antibodies, erroneous findings may be obtained from samples taken from patients who have been treated with monoclonal mouse antibodies or have received them for diagnostic purposes.

In rare cases interference due to extremely high titers of antibodies to ruthenium can occur. Elecsys SHBG contains additives which minimize these effects.

Extremely high titers of antibodies to streptavidin can occur in isolated cases and cause interference.

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings. 

Measuring range
0.350-200 nmol/L (defined by the lower detection limit and the maximum of the master curve). Values below the detection limit are reported as < 0.350 nmol/L.

Values above the measuring range are reported as > 200 nmol/L.

Analytical sensitivity (lower detection limit)
0.35 nmol/L

The detection limit represents the lowest measurable analyte level that can be distinguished from zero. It is calculated as the value lying two standard deviations above that of the lowest standard (master calibrator, standard 1 + 2 SD, within-run precision, n = 21).

Method comparison
A comparison of Elecsys SHBG (y) with a commercially available SHBG test (x) using clinical samples gave the following correlations:

Number of samples measured: 109
Passing/Bablok12 Linear regression
y = 1.17x - 3.26 y = 1.15x - 1.82 
r = 0.909 r = 0.981
SD (md68) = 3.24 Sy.x = 4.15

The sample concentrations were between approx. 11.2 and 155 nmol/L.

Analytical specificity
For the monoclonal antibodies used, non detectable cross-reactivities were found for the following substances:

AFP, CBG, DHT, estradiol, fibrinogen, human IgA, human IgG, plasminogen, TBG, testosterone, Tg, transferrin, and TSH.

Androstanediol glucuronide

Specificty (cross reactivity)
The following compounds were tested for cross-reactivity with the Direct 3α Diol G ELISA kit with 3α Diol G crossreacting at 100%.

Sensitivity
The lower detection limit is calculated from the standard curve by determining the resulting concentration of the mean OD of Calibrator A (based on 10 replicate analyses) minus 2 SD. Therefore, the sensitivity of the Direct 3α Diol G ELISA kit is 0.1 ng/ml.

Steroid %Cross Reactivity
3α Diol G 100
Testosterone 0.2
Progesterone 0.16
Androstenedione 0.14
Cortisol 0.05

The following steroids were tested but cross-reacted at less than 0.01%: Corticosterone, Dehydroepiandrosterone, Dihydrotestosterone, Epiandrosterone, 17β-Estradiol and Estrone.

Intra-Assay Precision
Three samples were assayed ten times each on the same calibrator curve. The results were < 8% CV at each level, 0.87, 6.86 and 21.26 ng/mL

Inter-Assay Precision 
Three samples were assayed ten times over a period of four weeks. The results were 10.4, 6.5 and 10.8 %CV at 0.98, 7.05 and 20.92 ng/mL, respectively

Recovery
Spiked samples were prepared by adding defined amounts of 3α Diol G to three serum samples. The results ranged from 88 to 115%.

Linearity
Three serum samples were diluted with calibrator A. The results showed linearity at dilutions of 1:2, 1:4 and 1:8

Expected Normal Values
As for all assays each laboratory should collect data and establish their own range of expected normal values.

Normal Range (ng/ml)
Males 1.53-14.82
Premenopausal 0.22-4.64
Postmenopausal 0.61-3.71
Puberty (Female) 0.51-4.03

Estrogens

Erroneous test results may be obtained from samples taken from patients who have been exposed to vaccines containing rabbit serum or when keeping rabbits as pet animals.

In rare cases, interference due to extremely high titers of antibodies to streptavidin and ruthenium can occur. Elecsys Estradiol II contains additives which minimize these effects.

For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history, clinical examination and other findings.

Measuring range
18.4-15,781 pmol/L (5.00-4300 pg/mL) (defined by the lower detection limit and the maximum of the master curve). Values below the detection limit are reported as < 18.4 pmol/L or < 5.00 pg/mL. Values above the measuring range are reported as > 15,781 pmol/L or > 4300 pg/mL (or up to 78,905 pmol/L or 21,500 pg/mL for 5-fold diluted samples).

Analytical sensitivity (lower detection limit)
18.4 pmol/L (5.0 pg/mL)

The detection limit represents the lowest analyte level that can be distinguished from zero. It is calculated as the value lying two standard deviations above that of the lowest standard (master calibrator, standard 1 + 2 SD, within-run precision, n = 21).

Functional sensitivity
44 pmol/L (12 pg/mL)

The functional sensitivity is the lowest analyte concentration that can be reproducibly measured with a between-run coefficient of variation of  ≤ 20%.

REFERENCES

Testosterone

Bablok W et al. A General Regression Procedure for Method Transformation. J Clin Chem Clin Biochem 1988;26:783-790.

Juhl UM, Rippegather G, Weller J, Zawta B. Important Facts on Reproduction Medicine/Fertility Diagnosis, Questions and Answers. 1994; Boehringer Mannheim, Cat. No. 1322958.

Nieschling E, Behre HM. Testosteron Action, Deficiency, Substitution.
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Runnebaum B, Rabe T. Gynäkologische Endokrinologie und Fortpflanzungsmedizin Springer Verlag 1994; Band 1:36-38,70,116 Band 1:39-40, 520-521, 593-594, 422-423. ISBN 3-540-57345-3, ISBN 3-540-57347-x.

Thienpont L, Verhseghe PG, Van Brussel KA, De Leenheer AP. Estradiol-17-β Quantified in Serum by Isotope Dilution-Gas Chromatography-Mass Spectrometry. ClinChem 1988(34);10:2066-2069.

Tietz NW. Clinical Guide To Laboratory Tests. 3rd ed. Philadelphia, Pa: WB Saunders Co, 1995:578.

Wheeler MJ. The determination of bio-available testosterone.
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Sex hormone-binding globulin (SHBG)

Avvakumov GV, Grishkovskaya I, Muller IA, Hammond GL. Resolution of the human sex hormone-binding globulin dimer interface and evidence for two steroid-binding sites per homodimer. J Biol Chem 2001;276:34453-34457.

Bablok W, et al. A General Regression Procedure for Method Transformation. J Clin Chem Clin Biochem 1988;26:783-790.

Burger HG. Androgen production in women. Fertil Steril 2002;77:3-5.

Burger HG, Davis SR. The role of androgen therapy. Best Pract Res Clin Obstet Gynaecol 2002;16:383-393.

Davis S. Testosterone deficiency in women. J Reprod Med 2001;46:291-306.

Hammond GL, Bochinfuso WP. Sex hormone-binding globulin/androgen-binding protein: steroid-binding and dimerization domains. J Steroid Biochem Molec Biol 1995;53:543-552.

Morley JE, Patrick P, Perry III HM. Evaluation of assays available to measure free testosterone. Metabolism 2002;51:554-609.

Petra PH. The plasma sex steroid binding protein (SBP or SHBG). A critical review of recent developments on the structure, molecular biology and function. J Steroid Biochem Molec Biol 1991;40:735-753.

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Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab 1999;84:3666-3672.

Androstanediol glucuronide

Check, J.H., et al, Falsely elevated steroidal assay levels related to heterophile antibodies against various animal species. Gynecol Obstet Invest 40:139-140, 1995.

Deslypere, J.P., et al., Plasma 5α-Androstane-3α,17β-diol and urinary 5α-Androstane-3α,17β-diol glucuronide, parameters of peripheral androgen action: A comparative study. J. Clin. Endocrinol. Metab. 54/2:386-391, 1982.

Gunther, H.J. and Wilson, J.D., Formation of 5α- Androstane-3α,17β-diol by normal and hypertrophic human prostate. J. Clin. Endocrinol. Metab. 44/1:107-115, 1977.

Moghissi, E., et al., Origin of plasma androstanediol glucuronide in men. J. Clin. Endocrinol. Metab. 59/3:417-421, 1984.

Pang, S., et al., 3α-Androstanediol glucuronide in virilizing congenital adrenal hyperplasia: A useful serum metabolic marker of integrated adrenal androgen secretion. J. Clin. Endocrinol. Metab. 73/1:166-174, 1991.

Reiner, B.J., et al., Serum 3α-Androstanediol glucuronide measurements in sexually mature women with congenital adrenal hyperplasia during therapy. J. Clin. Endocrinol. Metab. 69-105-109, 1989.

Riddick, L., et al., 3α-Androstanediol glucuronide in premature and normal pubarche. J. Clin. Endocrinol. Metab. 72:46-50, 1991.

Scanlon, M.J. et al., Serum androstanediol glucuronide concentrations in normal and hirsute women and patients with thyroid dysfunction. Clin. Endocinol. 29:529-538, 1988.

Vexiau, P., et al., Increase in plasma 5α-Androstane-3α,17β-diol glucuronide as a marker of peripheral androgen action in hirsutism: A side-effect induced by cyclosporine A. J. Steroid Biochem. 35/1:133-137, 1990.

Estrogen

Bablok W, et al. A General Regression Procedure for Method Transformation. J Clin Chem Clin Biochem 1988;26:783-790.

Iqbal MJ, Dalton M, Sawers RS. Binding of testosterone and oestradiol to sex hormone binding globulin, human serum albumin and other plasma proteins: evidence for non-specific binding of oestradiol to sex hormone binding globulin. Clin Science 1983;64:307-314.

Johnson MR, Carter G, Grint C, Lightman SL. Relationship between ovarian steroids, gonadotropin and relaxin during the menstrual cycle. Acta Endocrinol 1993;129/2:121-125.

Juhl UM, Rippegather G, Weller J, Zawta B. Important Facts on Reproduction Medicine/Fertility Diagnosis Questions and Answers. 1994; Boehringer Mannheim, Cat. No. 1322958.

Lichtenberg V, Schulte-Baukloh A, Lindner Ch, Braendle W. Discrepancies between results of serum 17??-Oestradiol E2 determinations carried out using different immunoassay kits in women receiving oestrogen replacement therapy. Lab med 1992;16:412-416.

Runnebaum B, Rabe T. Gynäkologische Endokrinologie und Fortpflanzungsmedizin Springer Verlag 1994;Band 1:83-86,517-524. Band 2:395-400,403-408. ISBN 3-540-57345-3, ISBN 3-540-57347-x.

Thienpont L, Verhseghe PG, Van Brussel KA, De Leenheer AP. Estradiol-17-β Quantified in Serum by Isotope Dilution-Gas Chromatography-Mass Spectrometry. ClinChem 1988(34);10:2066-2069.

Tietz NW. Clinical Guide To Laboratory Tests. 3rd ed. Philadelphia, Pa: WB Saunders Co, 1995:216.